Chalkbrood - Status Today and Hopes for Control' - Golden Rule Honey

Introduction 

fIHALKBROOD is a disease that af- 

U fects bee lrrood and is caused by 

the fungus, Ascosphaera apis. Diseased 

larv'ae becorne mummified (Figure 1), 

and the mummies appear rvhite because 

of the mycelium of the fungus (Figule 

2). Horvever, if n'rycelia of opposite 

sex ( * and -) have rnated, spores are 

forrned u'ithin fruiting bodies (Figure 

3 ), and the murnmies become dark 

gray ol black (Figure 4). Mummies 

are found at the hive entrancc, on the 

l)ottom board, and in cells of infected 

colonies. 

Maassen (1913) in Germany published 

the first observations on chalkbrood 

disease. In 1916, he described 

thc associated fungus and named it 

Pericl,stis apis. Later, in the United 

Statgs, Spiltoir (1955) studied thc life 

cycle, and Sp.iltoir and Olive ( 1955 ) 

rcclassified the fungus and established 

a new gcnus, ,4scosphaera, stnce the 

narttc Pericyslis had been prcviously 

uscd as a gcneric name in the red 

algae. 

Distribution 

Chalkbrood has been reported from 

Er.rrope, including the British Isles, for 

rnany ycars. In 1957, Seal reported 

chalkbrood florn Neu' Zealand. 

Baker and Torchio (1968) published 

the first record of A. apis from the 

United States. Their isolates f r o m 

Utah H'erc associated u'ith the leaf cutter 

bee, Megachile inermis, and the 

soil nesting bee, Anthophora pacilicia. 

Latel Thomas and Luce (1972) repolted 

chalkbrood fronr honey bees in 

California, and llitchcock and Christensen 

(1972) found the disease in 

honey bee larvae in Nebraska and Wyon'ring 

and noted that other occurrences 

l Presented at the Midwest Bee Diseases 

and Pests Clinic, Springfield, Illinois, 

March 29, 19?8. 

468 

Chalkbrood - Status Today and 

Hopes for Control' 

by MARTHA GlttlAlr 

USDA, SEA Bee Reseqrch Loborotory 

200 Eqst Allen Roqd, Tucson, Arizono 85719 

of the disease in California, Minnesota, 

North Dakota, and Montana had 

recently come to their attention. Gilliam 

and Taber (1973) reported chalkbrood 

in Arizona, and Conner (1974) 

iound chalkbrood in Ohio. 

Gochnauer et al. (1972) reportcd 

the first record of chalkbrood in Canada 

in honey bees from British Columbia 

and Saskatcheu'an. Nelson et al. 

(1976) surveyed 5,374 colonies in five 

provinces of Canada in 1975 and found 

that 32/6 of these colonies had mummies 

in the combs. However, 75/6 ot 

the infected colonies had less than 10 

cells rvith chalkbrood. In a similar 

survey conducted in w€-tern Canada 

in 1976, 33% oI the package colonies 

and 20Vo of the wintered colonies had 

chalkbrood disease (Nelson et al., 

1977). Thc disease is norv n'idespread 

in the U. S. and Canada. By 1976, 

chalkbrood had becn detected in 33 of 

the 48 mainland states and Hau'aii and 

in 6 of B Canadian provinces (Menapace 

and Wilson, 1976). 

Etiology 

Unfortunately, little is knou,n of 

the epidemiology and pathogenesis of 

chalkbrood disease. According to Bailey 

(1967), honey bee larvae are most 

susccptible to chalkbrood disease iI they 

ingest spores of A. apis when they are 

3-4 days old and are then chilled 2 

days later after they are sealed in their 

cells to pupate. The spores then gern-rinate 

in the gut of the larvac. Initia11y 

the dead lar-vae are covered rvith 

a u'hite fluffy grorvth of mycelia and 

are slvollcn to the size of the cell. 

Later they dry into a hard, shrunken 

chalk-like lump which may bccome 

gray to black if fruiting bodies are 

forrned. The remains of larvae can be 

found in sealed or unsealed cells. Most 

larvae die in the upright stage. Adult 

bees generally remove the nrurnmies 

after rvhich they can be found at the 

colony entrance and on the bottom 

board. 

There has been controversy concerning 

whether natural infection of. A. apis 

occurs by ingestion of spores rvith food 

or via the body surface from spores on 

combs and cell walls. Gochnauer et 

ai. (1975) postulated that once a colony 

was infected, the spores could remain 

viable on the combs, eventually 

germinate w'hen conditions became favorable, 

and the disease could then 

reappear. They aiso suggested that A. 

aprs rnight sunrive in soil, find its way 

into the food chain of honey bees, and 

be transmitted to larvae via contaminated 

brood food. 

Although the ability of infection to 

spread has been considered to be low 

(Bailey, 1963,7, the spores are quite 

resistant and remain infective f or at 

least 15 years ( Toumanoff, 195 1 ) . 

Maurizio (1934) and Betts (1951) 

thought that the disease was probably 

carried by honey, and therefore one 

should avoid feeding honey from infected 

colonies. Maurizio (1934) found 

the fungus in the intestines of bees 

from an infected colony and stated that 

the organisrn rvintercd in bees and in 

honey. Dc Jong and N,Iorse (1976) 

found ,4. apis in the honey sac contents 

of adult u'orker bees from infected 

colonies and shou,ed that spores 

n'ere passed from bee to bee in food 

exchange. 

Further spread of the disease occurs 

rnostly through drifting of bees from 

infected colonies. Horvever, transfer of 

spores by the beekeeper also occurs 

when contaminated tools and combs 

are interchanged in infected and 

healthy colonies (Barthel, 1971). 

Other possible sources of infection include 

contaminated pollen (Mehr et 

al., 1976), wind, water, queen bees 

(De Jong and Morse, 1976; Moeller 

and Williams, i976), and package bees 

(Nelson et al., 1977 ) . 

American Bee Journal 

'ft

Many factors have been postulated 

to contribute to the development of 

chalkbrood disease in bee colonies (see 

Gilliam, 1978). These include chilling 

of larvae, poor ventilation of colonies, 

partially occupied wintering colonies, 

cool moist weather, high humidity and 

temperatures, excessive hive moisture, 

weak colonies, air pollution, the use of 

antiblotics favoring the growth of fungi, 

colonies suffering from other diseases, 

injured brood, genetic factors, 

watery food, and excessive colony 

manipulation. 

It has been suggested that other insects, 

primarily solitary or wild bees, 

play a part in spreading chalkbrood 

disease. Ascosphaera apis has been 

found in or associated with ieaf cutter 

bees, mason bees, soil nesting bees, 

and alkali bees (Baker and Torchio, 

1968; Batra et al., 1973; Clout, 1956; 

Stephen and Undurraga, 1965). Batra 

et al. ( 1973 ) noted the importance 

of the reservoir of inoculum in the 

nests of wild bees. Some individuals 

of most bee species in an area forage 

on the same crops and thus permit an 

exchange of inoculum via the flowers. 

Moreover, they recovered the same 

Figure 

slrqin of 

ffi 

l. filummified honcy bee loruoc infected with unmsted 

Ascosphoero opis. 

7 

Mummies ore white. 

fungi from the honey stomachs of several 

species of bees. 

Treatment 

No chemotherapeutic agent is registered 

for use against chalkbrood disease 

in the United States. Previously 

the losses caused by the disease were 

not considered serious enough to justify 

research on treatment. Since the adult 

bees generally carry the dead brood 

out of the hive, the disease often disappears 

without intervention from the 

beekeeper. 

Destruction of affected combs or 

sections of combs in severe cases has 

been recommended (Betts, 1951; Gochnauer 

et al., 1975 ). Giauffret et al. 

(1969) and Thomas and Luce (1972) 

found that fumigation with ethylene 

oxide killed A. apis in infected combs. 

Moisture accumulation and poor ventilation 

in hives should probably be 

avoided since cold and damp weather 

has been thought to encourage the 

development of chalkbrood, Zand,er 

(1919) emphasized the importance of 

wrapping colonies during the winter 

and keeping them dry. Seal (1957) 

stated that serious spread of the dis- 

tgffi#i 

W* 

i:ee.{ 

hffiffi, 

#-wi 

Figure 3. Motcd slroin of Ascosphoerp opis showing sporus 

within fruiling bodier. 

July 1978 

ease could be avoided by closing the 

hives for winter and keeping the hives 

clear of long grass to prevent dampness 

and to allow adequate ventilation. 

One also can enlarge the entrance to 

a colony to aid ventilation (Gochnauer 

et al., 1975). 

Seal (1957) recommended strengthening 

badly diseased colonies by adding 

young adult bees and hatching 

brood and feeding sugar syrup. He 

also stated that chalkbrood could be 

avoided by not allowing the bees to 

winter in too large a brood chamber. 

Lunder (1972) suggested the use of 

resistant queens, and Mraz (1973) recommended 

requeening with non-susceptible 

strains. Recently Nelson 

( 1975 ) made crosses between New 

Zealand and California stocks of bees 

and found that the resulting stock was 

less affected by chalkbrood than the 

California stock. 

Several authors have conducted experiments 

on the use of chemicals to 

control chalkbrood. Generally, these 

attempts have not been successful because 

of toxicity of the chemicals to 

the bees, the fact that many of the 

chemicals were not acceptable to the 

Figure 2. Unmqled stroin of Ascosphoero opis. Mycelio only, 

#@'&&* 

Figure 4. Mummificd honey bee lowqc inlectcd with moted 

strsin of Ascosphoero opis. Mummicr ore dork groy to block 

469 

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470

ees, and the time and effort required 

to disinfect all the parts of the hive. 

For exarnple, Elbe and Weide (1961) 

found that 0.7/e thymol prevented the 

growth of A. apis in culture. When 

the thymol solution \{'as sprayed on infected 

combs, the disease disappeared. 

Horvever, it was necessary to spray 

every comb and the inner walls of 

the brood chamber. The bees would 

rot accept 0.7/6 thymol in syrup. Barthel 

( 197 1 ) found that thymol in a 

2/6 solution had a fungistatic effect 

in 20 minutes on ,4. apis in uitro. He 

stated that stimulation of the cleaning 

instinct of bees is the principal control 

measure. 

Dallman (1966) tested the disinfectant 

"Fesia-Form" (formaldehyde base) 

on bee colonies infected with chalkbrood. 

He sprayed the solution on 

brood cornbs, the inside parts of the 

hive, and the flight board. Within a 

u'eek after spraying, the aduit bees 

from severely infected colonies had removed 

the mummies, and no reinfection 

was observed throughout the year. 

He found that a 4Vo solution of "Fesia- 

Form" killed the fungus. Barthel 

(1971) performed ln uilro tests on the 

activity of fungicides agatnst A. apis 

and reported that 4/6 "Fesia-Form" 

killed the spores after 30 minutes. 

Antimycotics and antiseplics have 

been tested for possible use in the 

treatment of chalkbrood. Giauffret and 

Taliercio (1967) reported that arnphotericin 

B \r'as the most effective agent 

of those they tested against A. apis. 

How'ever, it rvas not stable. The antiseptics 

tested $'ere quite stable but 

were more toxic for bees than the 

antimycotics. Moeller and Williams 

( 1976) found that 250 ppm of benom;'l 

in sugar s)'rup appeared to have 

a positive effect in reducing but not 

cornpletely eliminating chalkbrood infection. 

Thomas and Luce (1972) rePorted 

that sorbic acid and methyl parahydroxybenzoate 

inhibited A. apis in 

culture. Later Taber et al. (1975) fed 

sorbic acid and sodium propionate in 

pollen-sugar patties to badly infected 

bee colonies. Chalkbrood disappeared 

seven days after the treatment started. 

There were no symPtoms of toxicitY 

in the bees, and no reduction in sealed 

brood occurred. 

The use of ethylene oxide fumigation 

and the selection of chalkbroodresistant 

bees seem to be the two most 

promising means of controlling the 

disease, although research is required 

to test the effectiveness of ethylene 

oxide against A. apis in bee colonies 

and to select and breed resistant bees. 

470 

Speculation 

The origin of chalkbrood disease in 

the United States is a mystery. At 

least two possibilities exist. One opinion 

is that chalkbrood disease has been 

in this country for many years, but it 

was considered unimportant. Since 

rnany competent microbiologists have 

examined diseased bees in this country 

for many years, it seems unlikely that 

they rvould not have observed chalkbrood 

if it were present. I suspect 

that beekeepers were seeing another 

fungal disease, stonebrood, which also 

produces mummies. Some of the people 

who believe that the disease has 

been present for years also think that 

it recently has become more widespread 

and has increased in severity. 

If this is true, the fungus may have 

mutated or strains of bees may have 

become more susceptible. Indeed, 

strains of A. apis in the United States 

may differ from those found in other 

parts of the world. 

The fungus may have been present 

in solitary or wild bees in this country 

for many years. There is no way 

of knorving this since little work has 

been done on the microflora of these 

bees, which may have served as a reservoir 

of inoculum for honey bees. 

Another vierv is that the fungus lvas 

only recently introduced, perhaps on 

imported pollen. Then the rvidespread 

use of antibiotics to control bee diseases 

may have increased the incidence 

of chalkbrood disease by upsetting the 

normal intestinal micloflora of bees. 

Chalkbrood may be a stress-related 

disease of honey bees since abnormal, 

starved, and confined bees seem more 

susceptible. Also, some bee colonies 

are rnore adept at removing the diseased 

larvae (Nelson et al., 1975; Gilliarn 

et al., 1978). 

These conjectures demonstrate that 

there are many interesting avenues of 

research on chalkbrood disease that 

should be investigated. Particularly, 

the taxonomy and infectivity of strains 

of A. apis and the susceptibility of 

strains of honey bees under various 

nranagement procedures need to be 

examined. 

Research Report 

One of the major obstacles in conducting 

research on chalkbrood disease 

has been the difficulty of inducing infections 

in bees for study. Such infections 

would give information on 

how, when, and why bees succumb to 

chalkbrood and also would allow researchers 

to perform controlled experiments 

on methods of treatment. Thus, 

we have conducted experiments to in- 

fect brood in full-size colonies in the 

apiary and brood maintained in an incubator. 

Details of the experiments 

are in the recent paper by Gilliam 

et al. ( 1978). 

Many attempts, most of which were 

unsuccessful or not reproducible, were 

made to infect brood in apiary colonies 

with ,{. a/is under a variety of conditions 

affecting both the pathogen and 

the host colonies. These included the 

use of inocula prepared from artificial 

cultures of. A. apis vs. suspensions from 

naturally infected larvae, application 

of the inocula in syrup sprays and/or 

in pollen cakes, use of mummies with 

sporulated vs. vegetative growth of the 

pathogen, alteration of the ratio of 

adult worker bees to brood by removal 

of adult bees or frames of brood prior 

to application of the inoculum, and 

appiication of ground mummies in 

dusts or in pollen. 

The most severe infections were produced 

in colonies that had been fed 

uncontaminated pollen to promote 

brood rearing and from which varying 

numbers of adult bees were removed 

before the brood u'as sprayed with a 

suspension of 3 black (sporulated) 

rnummies in 90 ml of 5/p sucrose in 

water. When this treatment was applied 

3 times a day on the first, third, 

and f if th day of the experiment, it 

yielded heavy infections (10/p of the 

brood) by the sixth day. However, 

even this treatment r.l'as less ef fective 

in other colonies that u'ere treated at 

later periods. Thus variation in sus- 

ceptibility of bee colonies may be an 

factor in expression of dis- 

:T::rr"", 

Because of the difficulty of inducing 

infections in apiary colonies, we 

devised techniques for removal of 

brood from combs, surface sterilization 

of brood, maintenance of brood in an 

incubator, and inoculation of brood 

wrth A. apis. 

First we sought to determine whether 

A. apis causes chalkbrood disease or 

is simply a secondary invader of larvae 

killed or injured by other events. Evi 

dence for the latter might be supported 

by mummification of dead larvae. We 

also wished to determine whether 

mummification arises from infection by 

both vegetative and sporulated strains 

and whether A. apis can invade larvae 

through the cuticle and,for through the 

mouth. In addition, we wished to determine 

whether young and old larvae 

are equally susceptible to infection. 

Larvae from uncapped cells were 

collected and divided into two age 

groups, small and large, on the basis 

of size. The small larvae were 3-4 

days old, and the large larvae were 

American Elee Journal

4.5-5.5 days old. Half of the larvae 

were killed by freezing with dry ice. 

Groups of larvae were then inoculated 

rrith mated or unmated strains of l. 

apiJ on the dorsal surface of the segments 

(back) or on the mouthparts. 

No mummies were formed as a result 

of the inoculation of dead larvae 

rtith A. apis. However, A. apis did 

grow on many of these larvae. Thus, 

it appears that A. apis can grow and 

in some instances sporulate on dead 

lan'ae but will not mummify them. 

\fummies were produced by inoculation 

of mouthparts and by inocularion 

on the back. Thus, infection may 

occur through the mouth and through 

the cuticle. We observed the fungus 

grorving from the mouth, the anus, and 

through the cuticle of larvae inocuiated 

on the mouthparts. The fungus appeared 

to grow first aerially at the 

poinr of inoculation on larvae inocu- 

Iared on the back. It t h e n grew 

rhroughout the entire body, and the 

lan'ae became mummified. Both mated 

;rrd unrrated strains of A. apis caused 

mummy formation. Three - 4-day-old 

and 4.5-5.5-day-old larvae u'ere equally 

susceptible to chalkbrood disease. 

Sorne rnummies n ere produced in 

the control (live larvae receiving dead 

-1. apis). These larvae may have had 

nJrural infections nhich r,r'ere inappar'rnt 

until the larvae were incubated. 

Hosever, none of the colonies from 

r'hich these larvae u'ere obtained conraincd 

any larvae that n'ere visibly 

infected tlith A. apis. 

\ext, n'e conducted a similar experirrrent 

to dctermine whether A. apis 

rr.ould grolv on or cause mummificarion 

of eggs; small (3 days old), mediurn 

(4-6 days old), and large larvae 

3-10 days old); prepupae (11 days 

, ,ld, ; pupae n,ith no eye color; and 

pupae u'ith eye color. 

Our results shorved that eggs and 

lupae of the honey bee do not supr),rrt 

thc glouth of the fungus and are 

:rot sr.lsceptible to laboratory infection 

'.rirh A. apis. Larvae and prepupae 

:rre susce ptible, and older lar"vae are 

the nrost susceptible. Also, all threc 

,'f thcsc age grolrps are susceptible to 

t)atural infections. Since the colony 

irorn uhich these insects r.r'ere obtained 

had no visible signs of chalkbrood dist'a,.e, 

,4. a|tis may leside in bee colonies 

r'ithout causing the overt signs of disease 

until the proper conditions exist. 

If chilling is a necessary prerequisite 

for infection u'ith our strain(s) of ,4. 

apis as with European strains, this 

condition should be rnet by our procedures 

for removal of brood from the 

comb and surface sterilization. However, 

in Arizona heavy year-round infections 

of chalkbrood disease have 

been seen in colonies, even when the 

average monthly temperature is 29'C. 

Strains of A. apis found in Arizona 

may thus differ from those reported 

from Europe. This question is now 

being investigated. 

In conclusion, A. apis infection occurs 

through the mouth and through 

the cuticle. Mated and unmated strains 

are infective. Ascosphaera apir grows 

on dead larvae but does not mummify 

them so it is pathogenic to honey bee 

larvae. The fungus also can grow as 

a saprophyte on dead larvae, but as 

a saprophyte, the fungus does not produce 

a mummy. 

N,I!PEEEITCTg 

Ba.iley, Ir. 1963, fnfectious Diseases of 

the Honey-Bee. Land Books, London. 

Batley, L, 1967. The effect of temp€rature 

on the pathogenicity of the fungus, 

Ascosphaera apis, for larvae of the honey 

bee, Apis mellif6ra, In Insect Pathology 

and Microbial Control, ed. P. A. 

van der Laan, pp. 162-167. North IIolland, 

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Baker, G. M. antl P. F. Torchlo. 1968, 

New records of Asco€phaera, apls from 

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Ba,rthel, B. 19?I. Der Kalkbrut auf der 

Spur. Ga,rten tlelntlerzucht. C. Imker 

1O: 12-13. 

Batrar IJ. 8., S. W. f. Batra, atral C. t. 

Bohart. 1973. The mycoflora of domestica.ted 

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Mycol. appl, 49: 13-44. 

3etts, A. D. 1951, The Diseases of Bees: 

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Hickmott, Camberley, England. 

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Connor, L. J. 197/t. Chalk brood is noq/ 

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De Jotlg', D. atrcl B. A. Morse. 19?6. Chalk 

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elds (Maassen ex Claussen) Olive and 

Spiltoir in the honey bee, AplB melluera 

L. in California. ADer. Be€ J, Ul: 88- 

90. 

Tounanoff, C, 1951. Les Maladies des 

Abeilles. [,evue Erancalse Aplc. Nurrt, 

Spec, 68. 

Za,naler, E. 1919, Die Brutkrankheiten und 

Ihre Bekempfung. Handbuch der Bienenkunde 

in Einzeldarstellungen. Vol. 1, 

second edition. Ei, Ulmer, Stuttgart. 

July 1978 Reprinted from July, 1978, American Bee Journal 

47L 

VoI. 118 (7): 468-47L

.behaviour, the crops being grown' the pests ,that affect them and the 

pesticides'useC in their control. But above all they must be expelt in 

'dealing rvith people. They must be unbiased and respect the intelest's of 

all the parties concerned. 

REFERENCLS 

PAI-\IER-JON,E-S. T., FONSTER, I. w'. GRTFFECS, L. A. M. l95i EHect on honeli bees ol 

It'letasyslox applied lrom the ai.r as a spray to chou moeUer- - NetD Zealand Journcl 

o, Sclence and Technology 38'.7 (June 19i?). 

PALNIER-JONFS, T.. FORSTER, I. w. 1963 - E{fect on honey-bees -of 

Pbosdrin 'appU"O as s.prays to ivhite clover. Nero Zealand 

Rese4rch 6.:3 (June-Augusl 1963). 

E 

RESEARCH ON CHALI(BROOD DISEASE OF 

HONEY BEF^S 

Dipterex, Thiodan, and 

Journal of Agricultura.l 

' I$artha GILLIAI\I 

S. TTIBER III 

' 

J. BR.{Y ROSE 

u.s'A' 

fntroduction 

chalkbrood disease of honey bees, Apis nzelliiera L, il thought to 

be caused by the heterothalic fungus, Ascosphoero (Nlaass-en 

-iaur.ett; -api! -ex' 

Ol'ive & Spiltoir- Spores-are formed irithiq fruiting bodies 

only tvhln myeelia oi opposite sex come together. Diseased larvae be- 

*r*miiied-'The niummies are rvhite due to the mycelium of the 

"oiriu 

fungus. Horvever, if fruiting ,bodies are forrned, the murnnies are dark 

gray or ,black. 

" - 

Unfortunately, little is knorvn of the epideTiology_ and pathogenesis 

of chalkbiood diseasz. According (1967), honey bee 

-Io-.BAILEY 

Iarvae are most susceptible to challibrood diseasa if they ingest 

^spores 

ot g. cpis $,hen they- are 3--4 days old and are then chilled 2 d-ays 

later after they are- sealed in their cells to pupate- The spores then 

l*.*i""t" in the gut of the lar:vae. Initiaily-the dead larvae are covered 

ivith a white fiuify grorvth of mycelia and ar:e srvollen to the size of 

the cell. Later they dry into a hard, shrunken chalklike lurnp rvhich tnal' 

U*"or"u gray to ,6t"cti if fruiting bodies are forrned. The remains of 

larvae cJn be found in sealed ,or unseal'eC cells' 

- BARTHEL (1971) and MATUS and SARBAK (i974) stated that 

natural irrfection of A. apis could occur either by ingestion o! spor-es 

iuiifr io"i or via the bodl; surface from spores on c-ornbs and cell rvalls. 

iCjUSSff ,(1962) found that the spores germinated on the surface of 

iu..*u, rvhLreas MAURIZIO (1931t thought- that infection rvas through 

the mouth and not through the 'cutiele or spiracles' 

chalkbrood disease has been reported from Europe for man)'years- 

Horvever, it rvas not reported from fhe UniteC States until BAKER and 

fOnCgfb (ig66) fognd-A. opis'associated rvith a leaf cutter bez, Meg.achile 

inernzis Piovaneher ut d o soil nesting 'bee, Anthophora pacifica 

Cresson, Late THONIT\S and LUCE (L972) reported chalkbrood from honey 

451 

w

,i 

beef in california. ancl IitrcHCoCI( anc CIIRISTENSLN (19?2) found tlre 

disease - in honey bee lan.ae fronr Nebr.aska anc \v)-oming. $ri, firsi 

founcl the clisease in bees from Arizona iu rlay, lgzi lctillalt ana- 

TAB_!R. 1.97-3). The cisease r)or\: appears to be ivides?reacl in b:es in 

the Unitecl States. 

De JONG and IiOR.SE (19?6) noted the clifficultl, that resear-chers 

!1r'e 91t;:r'ienced in. inducing' chailtbrood infections iir bees fo5i;J;.: 

.Since 1972. s'e have been corilucting experinrents on ffr" transrnission of 

4. gPt"- Specificall)'. l'e have been attempting to infect brooC artificiattJ-. 

Such infccticns s'ould gir-e infomratio.t on ttft transmission anC path;genesis 

of the disease.anc rvoulc provide a leprocucible .bioassay.'They 

rvould also allorv us to perform ControlleC expeliments .o1 methoCs df 

treat:rrent such 'as the use of nrolc inhibitors (TABER et al.,lg7i).In this 

Papgr. lrre report the $'ork that rve have conducted on full-size-colonies 

in the apiary and on brood rnaintaineC in an ineubator. 

Attempts to infect apiary ctllonies rvith A. apis 

il{any at,ternpts rvere made to infeot brood in apiary colonies rvith 

Ascosphaera . - 

-apis uncer a r-ariety of conditions itrelting both the 

pathogen and the host colonies. These included the use-of inocula 

prepareC frorn altificial cultures of A. opis vs. suspensions frorn naturally 

infected larvae, ap1:lication of the inocula in syrup sprays ana/or ii 

pollen cakes, use of mummies rvith sporulatec vi. vegeiative g.o."tt, ot 

the pathogen, -alteration of the ratio bf adult rvorkuibe"r to iroocl by 

i'emoval of acult bees or frames of brood prior to application of thl 

inoculurn. and application- of grounc mu,nmies in duifs or in pollen. 

The rncst severe infections were produced in colonies thit had 

been fed uncontaminated pollen to promote ,brood rearing and from 

."-hi"lr-.q.f ing nun'rbers of adult bees ivere removed befor-e i suspension 

of 3 black (sporulated) ,mum,mies in 90 ml of 5olo sucrose in rvatei When 

this treatment rvas applied 3 times a day on the first, thirc. anc fifth 

9oy._of the.experiment, it yieldec heavy infections lttioTo of the broo-1 

by' the sixth day. Hrvever, even this ireatment ruas less effective in 

other colonies that rvere treated et later periods. Thus ryariation in susceptibilit.v 

may be an important factor in'expression of disease. 

Attempts to infect brood in an incubator rvith A. apis 

Beeause of the 'difficulty of inducing infections in apiary colonies. 

rve cleviseC teehniques fol reirovat of broid fr.orn coilbs, iurflce sterilization 

of brooc, maintenance ,of brood in an incubator, anc inoculation 

of brooC n'ith A. apis. No differences in survival rvere noted in larvae 

in the incubator f.ed 25oio sucrose solution. royal jelly, or nothing. Thus. 

suspensions of A. apis for inocula were ,prepared by homogenizing i 

black or spore-bearing rnummies (mated- stiain) oi s rvhite cha'iky 

mtrnrmies (unmatec strain) in 5 .rnt of sterile distilled rvater. ThesL 

susp-ensio;rs rvere qlatec to confinn viability and n'ere also sprayec or: 

conrbs, in apiary colonies. Sorne murnmies rv-ere found in spral'ec cornbs 

after 5 days. The dead A. apfs suspensions used for controls s'ere'obtaiped 

by autoclaving the homogenate. 

452

Attempts to infect small and large larvae from uncapped cells rvitft 

;1. aPis 

Firsl rve soLrght .to determine rvhether A. opis causes chall

Table I 

TRE,.\TIlEN'T CROUPS OF S}IALL AND LARGE. LARI:I'E FROM UNCAPI'I:D CELLS 

uslrD To D!:T}]RfrlNE rro-"iitto"ENIC AND sAPRoPl{YTic PRoPEIlrrEs oF 

ASCOSPHAERA API*C 

Trettlnent 

Large lart'ae a 

"Alive" lalvae and live A' olris 

-do- 

-do- 

-do- ".rtiie" lrrvae and dead l' apis 

._ do -:- 

-do- 

-do- 

"D;i'' larvae and live A' oPis 

-do- 

-do- 

-do- 

'PJaa" larvae and dead A' oPis 

-do- 

-do- 

-do- 

Snroll larroe b 

"Alive" larvae and live J' aPis 

-do- 

-do- 

-do- 

"Alirle" larva': and dead '1' aPis 

-do- 

-Co-. 

-Co- "O"aa" larvae arrd live A' aPis 

*do- 

-do- 

-do- "Da;" larvae and dead oPis 

'1' 

-do- 

-do- 

-do- 

a .:C )rr\'le P3r t35t 

tr l0 lf:'\'ae Psr test 

.1t dr)is, stfain 

1\{ated 

Unnlated 

IIated 

Unmated 

Mated 

Unnrated 

Il1ated 

Unmated 

tr{ated 

Unmated 

Itlated 

Unnrated 

Mated 

Unmated 

Nlated 

Unmated 

NIated 

Unmated 

Ir{ated 

Unmated 

IIated 

Unnrated 

Mated 

Unrnated 

Ivlated 

Unmated 

Iv{ated 

Unmated 

Nfated 

Unn.rated 

Ir{ated 

Unmated 

Slte of infculittton 

Ir{otrtlrParts 

-do-- 

B:rck 

-do- 

Iloutl'rParts 

-do* 

Back 

-do- 

I\Ioutlrlla rts 

-do- 

Back 

-do- 

MouthParts 

-do- 

Back 

-do- 

IlouthParts 

Brck -do- 

-do- NlouthParts 

-do- Back 

-do- MouthParts 

-do- Back 

-do- NlouthParts 

.- do 

Back - 

-do- 

mulntnieslvereprocuce(lbyinoculatiorrof'rrrouthl)arts.andlTby 

murntnies lvere proCucecl bv . inoculatiol -ot^^1::t;l-l]':t:'"ild*n11,,1 

inoculation oI1 the f"a"n-irtt'1 ini'-*ttio" 

"t"l''::1tt,3^":o^:.ri:ur'iltfl'ql 

',f,:" :i'1[?3 i'i" "if, . i'i.T,'' # ii'u;' e;:xlq,t' :T -'l: TiY,i'i;ii:; "+i'; 

:tl? ",T,:';;h'";r':':;li"r" ;i. r"'.'?u-, ii;,"9:t1:*^ ":,T,' 

?llr,ll'.:;3:J"';;"t:;y";$,+I'{Y.3.1.'-i""*f"',,i1#"""i1?:?,i'l "'li::lt"illi;,,"3i 

illi:', ; 5ff'?,'$ :: fi :'ilt: i ; il;^;, ;; ; h;"-"s h 

: *. :l i,:."' 'j: l:3J; 

lTJ"",;:"'ilil:"il.'.';:'";r#:!id-l'^1.";:0,":l,T:"*itll:.1:",::f l 

ili*, :?:' #-^,:"?" fi ' ill, ; ;'; inF ; * 

"'1 ff : lt# ":* #,:.::i:13 1f:j 

B:i"'H::.f :'i t' lLi iil t a-'t'" i "' ;i A o p'-'- .:il:',"1-.::l::::1t'l::; 

" 

,,,u,ioll'3r'-lil'.'on,1i',1'*::';:;:;;;;,'ri?;1.1: .:ll::t"l;ll.:'::::0",t'i'l'l 

black tnutntnies, ri--it:"t" btacti anC l1 u'ere rvhiia' iia. Florvever, of the 

454

Small-alive 

-do- 

-do- 

-do- 

-do- 

-do- 

Small dead 

Large-alive 

-do- 

-do- 

-_do- 

-do- 

-do- 

Large-dead 

-do- 

IJ.IIR VA E EX P ER T ill F4{T A LLY L\T'E CT ED IV I'IH 

InoculaLion 

Site 

l\lorrthParts 

-do- 

Back 

-do- 

MouthParts 

Back 

-do- 

MouthParts 

-do- 

Back 

-do- 

MouthParts 

Back 

MouthParts 

Back 

A. cpis slrai'n 

Live-mated 

Live-untnated 

Live-mated 

Live-unmated 

Dead-nrated 

-do- 

Live-unnrated 

Live-mated 

Lise-unmated 

Live-mated 

Live-unmated 

Dead-unmated 

Dead-mated 

Live-unmated 

-do- 

d ?a-..âr. tr.rl:r,v-$ ri.;\. >r* .af! rr'teHr4 *+ s 

i.e*!t*;frE'iEi+;xlg*:,e+* 

ASCOSP''AERA 4PIS 

I,I!rn:Trie5 

ProCuced 

Table 2 

Llrv'a e 

\r'itrr 

A- opis 

GrO\uth 

Bia.k w[ite !11ry.,tr!- snoto 

mummies produeed by inoculation material prepared from lvhite 

ôf 

mummies, .l.B were',Uii"f.'l"J';;f g"ir"r" itnite.' Thu-s- bv selecting 5 

mummies for produflL" or the inoculu;' ;;;bt'i"till nrixec * and 

- strains to prodti-e'ttr* mateC .trui"r ltttut infected the larvae' Horvever, 

it is interestiiftt'^Jiil n1al^1"*l"t of black and rvhite nlurnmies 

\r,ere formed frcm riaterial rpreparec l;; black mummies' A separation 

of */-. material could harie occutt;;-;;'-perhaps' spomlation lvas in 

solne' rvaY inhibiteC' ^:Lr ^ +. 

SmaII ancl lalge lan'ae were equalll'-suscepttllq l" infection since 

we usecl ,trvice ;H;i;;". i;;; ;t iniail ones' Thi.tv murrln'Iies l\'ere 

orocluced amons ", fi'il:;;; ii;;;J imalt laivae' rhus' 3--l 

iav_ord and 4.5- "ilJ 5.b day-ord larvae io be sus-ceotible to chalk- 

"pp"u. 

br-ood disease. This conjecture is ,"ili|#a'u>'-irt. i""i tnut 5 of the 

small and 6 of the large lan'u" upputl"tfi n;a a natural infection of 

chalkbrooC - :!---.^+ir 

1 

e 

I 

3 

2 

2 

y't 

/i 21 

4 

ll{ 

67 

Nextlr,edevelopedanrethoc]ofsurfacesterilizatiorroflarvaervith 

;;-rrb -g""t"I1.lj"""tt (Jspersillrrs spp' and 

a germicic. t" 

b"lftilirr* spp.)'rvithout ";;1";; harming the larvae' J 

Afterthelarvaelvere\l-ashecl.frorna.combo.ntoasterileltTassjlinR 

torvel, they u'ere'itlait'iaitufrr; pi"ft"J up 'rvith the suction apparatus' 

\\,ashed rvith a r,#;;'=;l"ti;; b1..n"i""r; tior" a prastic squeeze bottle' 

ancl rinsec ,trvice ;itl"r;;til-Jirt1r"a'."ater fro.r a-squ"e'e bottle' They 

rver.e placea or, ,i",.'if""ilg; to Ufoi'th;;" Cry and ivere then put into 

sterile vial caps i" p"lt'i'Jiihes 'by t'eleasing the suction' 

I 

6 

2 

4 

2 

3 

I 

3 

3 

2 

455 

{, 

T-: 

L. 

t: 

3, 

t 

J 

i I,t 

E 

{ 

T 

t- 

)t. 

11. 

tt, 

I t, 

ti- 

li 

tt 

t, 

ti t\ 

ll 

II 

t, 

'I I 

:li 

.r i 

:.1 

'ii :l t 

tf i 

.rl 

.r I 

.:F 

.t I 

,:i ! 

i:i:, 

:.!i. 

!i 

tl 

t. I 

'-j

. Attcrrrlrts to infect cggs, lal'\'ac and pupae rvith rl' 0pis 

,lhen rre desi.qned an exper.im"Ii tJ aeterinit're ivhether 'rl' cpis 

rvould grow otr o.""o.,r" mummifica,i""'"t""g!s; snrall. nredintn' and 

large tartuu , pr",rffiI ;;;;;"i;ith';" -"1'"'-"oio. ; and 1>upae rvith 

"'" "l.TI-rree,tment 

fJilili,#,",;:ili','",.J;:.h:{ff.i: ll?:,1ltti{i:ii{ 

f;'::;;:u"ll,?'.".,11fi1;u' ;,;""'*tri- ster' J iis t * lecl rvater. rr,6i' thev rvere 

bloueci ar"rrl placecl*";;;;; ir,e siae. "f " ;1;;iie vial cap to simulate the 

rrosition occupied ri-iit" ""iri "i-irr"^;;b- S'rali and medium larvae 

i*.ere rvashed frorn uncappcd -combs. Large larvae, plepupas, arrc prrpae 

\\'ere rvashed fro"l -"oni6s af ter l"'"iooTtte the cells' All larvae lvere 

sterilizec u. a"r..iilJ'6uior". erpuu-iu""r""t"uttt"a rvith Roccal on the 

sterile to*,et or, .uiilt i*t^*:"Jioliected. Thel' rvere.then individually 

he*l rvith ro.""p.^'ulri'riir"a trvice'liiin'ttuiir" distilled rvater' Each 

insect rvas placed'i;^;.i"ril" "i"r "up'""J ii""i"a as shorvn in Table 3' 

'i.:. ?obte 3 

T3E:\Tf1ENT GROUPS OF EGGS' LAR'VAE' "\iiD PUPAE INOCULATED 

ASCOSPI'AEIIA APJS 

Treatmelt 

i?f': r. opis-nrated 

Live A. nPis-unmat-eo 

Dea,i -4. oPis-mateo , 

;;;e;: oiris-unmated 

Srnoll loruse - 

Live A. opi's-nratecl 

Live A. oPis-nrated 

Live A. opis-unnrated 

Live 11. aPi's-unmat-eo 

Dead -'1. oPis-maied 

Deal J. aPis-mateo 

;;;e ;' oPi's-unmated 

5;;e ;. oPis-unnrated 

j\fediunr larz'ae 

Live A. oPis-mateo 

l,ive -'1. oPi's-mated 

ili;'; ;. 0pi)--unmated 

;ii; t. oPis-unmated 

Dead A. oPi's-mated 

DeaC A. aPi's-nrated 

"D;;a ;. crPis-unmated 

;;;J A. opi.s-unmated 

Lorge larxae 

Live A. opis-mateq 

Live 4. opis-matecl 

;i;;;. aPi's-unmated 

;i;; ;. oPis-unrrrat-ed 

Deai A. aPis-rnat€d 

" 5 iotto,a Per lre3tment 

456 

BodY 

tsodY 

BodY 

Body 

Site of 

trocrt:aCon 

Back 

IUouthParts 

Back 

1\louthParts 

Back 

I\IouthParts 

Back 

IlouthParts 

Back 

.:."ttotrthParts 

Back 

!{outhParts 

Back 

MoutbParts ' 

Back 

IlouthParts 

Back 

NlouthParts 

Back 

l'toutl'lParts 

tlack 

Treatme:rt 

DeaC --1. aPis-mated - 

Dead ,1. aPis-utrmated 

Dead .rl. aPis-unmated 

PrepuPae 

Live A. oPis-mat€d 

Live J. opis-nrated - 

Live 11. oPis-unmated 

Lir-e A. cPis-unrnated 

Deal l. oPis-mated 

Deal -{' opis-nratecl 

b""a ;. cPis-unmated 

o"ia -e. oPis-unmated 

Pupae-l\-o Ege Color 

Lii'e A. oPis-mated 

Live -1. oPis-mated 

lii'u a- aPis-unmated 

r-i'*'" .r. oPis-trnmated 

Deal A. aPis-ma"ed 

DeaC A. cPis-nrated 

-oiie .1. oPis-unnrated 

6"iJ a. oPis-unnrirted 

PuPae-EUe Color - 

Lii'e '1. oPi's-mated 

Live A. oPis-mated 

;ii';;" opis-unmated 

Lii-; A- aPis-unmated 

Dead A- oPis-tna'ted 

DeaC ,'l' cPis-mated 

"D;;a ;. airis-trnmated 

i."a .'r. oPi's-unrnated 

WITH 

Site of 

I,nocugation 

MouthParts 

Back 

MoutlrPalts 

Back 

IloutlrParts 

Back 

I\lotrthParts . 

Bcck 

IllouthParts 

Eack 

IUouthParts 

Back 

llotlthParts 

Elack 

IlIouthParts 

Back 

IlouthPirrts 

Back 

tr{outhParts 

Bask 

Moutl'rParts 

Back 

I\IouthParts 

Back 

llfouthParts 

Back 

'IlouthParts 

i-l-*iie: I 

r:'rlt+i_ I 

rr.a:u;l

Since rve noted that the mediurn ancl large larvae. prepupae, arnd pupae 

requirel less humidity in the petri clishes than the eggs ancl srn-all 

larvae, rve aCclecl lesi rvater to the filter papers in -clishes containing 

these 'stages- The mummifiecl larvae and prepupae luoked more like 

those f roin natural hive inf ections. possibly b:cause of the lou'er 

hurniditl'. 

All broocl \vas incubated at 25oC ancl obserred for 15 days for 

growth of A. apis and mummification. No grorvth-occurrecl on any-eggs 

6. p.,po". Altei incubation for 7 da1's, several rvith e1'e color had developed 

rvings, legs, antennae. and hair- 

As shorvn on Table 4, 26 mummies were formed. llorvever, 12 pf 

these rvere apparently the result of natural infections of A. apis since 

they rvere in'fne control gr:oups inoculated lvith dead A. rrpis' Of these 

12, 5 rvet'e medilm-sized la1.ae, 5 l-e're large lari-ae' and 2 rvere pre- 

pupae. Therefore, by experimental inoculation, or\e rnummlr was produci., 

the meclium-sized lattae, 8 in the large larvae, and 5 in the 

"a- prepllpae. Thus, more ,mummies .rvere produceC from large larvae though 

few murnrnies rvere produced in any lan'ae on preffil De JONG and 

MORSE (1976) also observed rvide variation in infection rates of larvae 

Table 4 

MUNL\ITESI AND GRO\{TH OF .ASCOSPHAERA .APi.s PRODUCED BY INOCUL-A'TION OF 

EGGS, LARVAE AND PUPA-E WITFI ASCOSF}IAERA APT.S 

Stage 

Small lan'ae 

Iv{editrm la|va 

lledium larva 

Medium larva 

Irfedium larva 

Ir{edium larva 

Itledium larva 

Large larva 

Large larva 

Large larva 

Large larva 

Large lan'a 

Large larva 

Large larv;r 

Prepupae 

Prepnpae 

Preprrpae 

Prepupae 

Preprrp:re 

Preprrpae 

Site o! 

fnoculation 

I\louthparts 

1!Iouthp:rrLs 

Back 

lvlouthparts 

lvtouthparts 

Back 

Back 

I\Iouthparts 

Ir[outhparts 

Back 

Brck 

1\{outhparts 

Back 

Beck 

Blck 

Ilouthparts 

B:.,:k 

Illouthparts 

I\loutlrparts 

Back 

e. dpls strain 

Live-mated 

Live-nrated 

Live-trnmated 

Dead-m:rted 

Dead-unmated 

Dead-mated 

Dead-unmated 

Live-mated 

Live-unmated 

Live-mated 

Live-unmated 

Dead-mr,ted 

Dead-n'rateC 

Dead-unrnated 

Live-mated 

l,ive-mated 

Live-urrmated 

Dead-mated 

Dead-unmated 

Dead-unnrated 

G:o\vth of 

iltum- A. cpis 

inues 

Ju!?u {1;,-too.* 

I - 

N2- 

/t 

I 

I 

I 

I I 

5 

lr 

3 

I I2 

, 

I 

I 

-157 

ril 

i:! 

it 

ti 

r'i 

ti 

I !t' 

i I 

{ !t! 

:.-.t

fed,-l.cpis.Irrthepresenttest.alltllutntniesfouncrverervhite(unnrated) 

to gray (p'obablyi ;",rrliiil""t-"i"i"i "na uttn',ut"d strains)' Othe. than 

the :l]uurtuies. onl| i';;;11 and 1-;;t;;;ized.larva inoculated u'ith 

" live A. apis ,r-o.i.'JO;;:^;;";if-r a. In thcse 2 lar'ae, A' cpfs 

"r "pis. 

grerv fronr tt," n,o'itri'ltt'i=' anC th.r;;gh'tho cuticle' None of the other 

inoculated insects-fieiCed at'ty grorvth of A' apis' 

Eggs, ptpuu,'anC the- snrallest larvae of the honey bee are not 

-i,rfection 

susceptible to tarJritoi-y r'-ittt ,r- apis- Ilteciurn and larrge larvae 

ancl prel:uoo" il;" i;ftu 

"'i"t'!"J&'ibl;;';;A 

'3f" :h" tnost susceptible' 

Also, all three of these age groups *t*ptible natural infections' 

"t:" 'to 

the colony f.or,.'.uti"t, tfiuri inr'u"tJ'*ete obtained had no visible signs 

ofchalkbrooeai*ur"."e"rtupr,a.apisresidesrvithinbeecolonies 

withour causing;;;;J.ii.,, 

oi disease until the prope. conditions 

exist.Itchil}ingisanecessaryprerequisite-fo-rinfectionrvithour 

strain(s)ofA-op;sus*'ithEuropeanstrains(BAILEY'1967)'this'conclition 

should be rnet b,v our p'ot"J*"t for removal of brood fr:om the 

co:t-rb and surface sterillation. Horvever, itt Arizona-rve have noted heavy 

year-round infections of larvae' even rvhen the average monthly 

tetnperaturu, u'J'z!;C' Strains of a' apis found in Arizona may thus 

difter from those ,"p".tJ irom Europe. w" ut. norv investigating this 

question' 

'- Acknorvledgernent 

We thank Dinorah DUNHAI\{ anC Leslie CAPIN for their excellent 

technical assistance rvith brooC in the incubator' 

REFERENCES 

'.'*"L""!;.onlnii; *,fn?".i'ii*1eo'o,:lTg'lfi:i--"??r^::i'::i11i$;tt1it.,ii+#*t'i,+i1"; 

logy ancl rliccoolat^"'co'itrol;' -;a'- P' A' van der LaarL p' rui 

Amsterdan) 

records or Ascosphoera aPis rrom North 

B-\KER. G. M.'ni;"rT;n,TlFftl?-rl3ot" 

- :*" 

B.{RT.i{EL, 8., 19lr' auf der spur' c.arten u' ]Kileinllerzucl" c' lmker' t' 

-'Jtt-*t'*orut 

t'2-13 

DE JONG D.' R" A' IIORSE' l9i6' Cha'lk brbo't : a nelv disease' N' Y' Food Lite Sci" 

- 

9, 12-11 

G'{RY''r\.'*aT;'}T'^fi!f;1"1"?.':;' ,'TjX- t161' - Honev be3 (Apis merriterd r''} 

'rarvae 

ln con- 

c,r""*.. -, l.-:*jn":it"ii;'.U; r:."r',?'i9.i5l':s 

tinous be3 P':'cou( 

r,tcrco"x. {, p,. y;:tigtth{:f ,,1% - 

United Statei' Ar 

}r.\Tusr, F.. l. ?tF,?,"fr: 

an'r diserscs 

.encountered 

cn"'x brood disease or honev bees in the 

d'isease jJl Hungar!')' rrasvat Aua' 

t*,"::""-oithalkbrood' 

uber die *"':o"". (Poric)'stis-ltvhose) der Bienen' Arch' Btenen' 

"";L; 

""ntofrl?a"l;i,'r?'r:rr? 

RoussY, L., 1962 * Nouvette contributisn a l'€tude du Pericystis apis (Ir'\)' Gaz' Aplcole' 

63, r0r-105 

T-{*BER. s. III' 196r - Fcrceps design for trsrLsferring honey bee eggs' J' Eeon' Entotnol" 

51' 21i-258 

T-{BERAS'eII';"1 

J' MrI-Ls' tt:t' A possible coitrol lor chalk brood di-se3se' 

,:,îfi"ff' 

: 

rHNAs. 

S;,,*;",i jk:1.ll";J,Sl ,:otif;:uiJ;r:"i::' T"iS'#"":3""?1""J: tli''t"1lii$ff 

-in'tir. see i.' rrz' ge-90 

458

INTERI{ATIONAL FEDERATION OF BEEKEEPERS' ASSOCIATIONS 

APITIONDI,I\ 

THE XXVIth INTTRNATIONAL 

CONGRESS OF APICULTURE 

ADELAIDE, AUSTRALIA 

October 13-19' 1977 

APIIVIONDIA PUBLISITING IIOUSE 

BUCHAREST' RO}:.C.}{L.1'

RIISEARC]II ON CHAI,I(I}R0OD I)IST]ASN ON }IONII}: BIiTiS 

Intloduction 

Nlrrr'tlrt GILLIr\l\t 

^S. T.\BER III 

J. Bt.t,\v ROSB 

u.s.A. 

Clralkbrood ,ciisease of hopey bees, ;lpis rrtelliJetu L.. is tirought to 

be c'atrsecl b1' the heterothulic fungus, ilscc.nphoet'n npis (i\'Iaassen ex 

Claussen) Olive & Spiltoir. Spores are fornleti t'ithin fruiting bodies 

onll' rvhen m,vce,lia o,f opposite sex cotlte together. Diseased larvae beconte 

ntunlrriifiecl. The unuurmies are rr,hite due to the r-n1-celiutn of the] 

fungus. HowerreL, if fluiting bodies arc folnrcd, tlte nruurrnies are clark 

grav or blac!.:. 

Unfoltunately, Iittle is knorvn o{ the epiderliolog3' and pathogenesis 

of chalkbrood diseas'^. Accolcling to B,\ILEY (1967), honel' bec 

lar-r'ae are rnost .susceptible to challibrood disease if the,v ingest spores 

of. A. apis rvhen they are 3-4 da1's old :rncl are then chilled 2 days 

later after they are sealed in their cells to pupate. 'I'he .spores then 

ger,rninate in lhe gut of the larvae. Initially the 'dead larvae are coYered 

rvith a u'hite fluffy growth of m1'celia ancl ar'e ss'olleu to the size of 

the ,celi. Late.r' they dry' into a hard, shrunlien chalklike lrtn-ip u'hich tnay 

becorne gra1.' to blaCli if fruiting bodies are fornred. llhe leuains of 

larvae can be fortnd ,in sealed or unsealed cells. 

BARTI{EL (1971) and l\IAi[tlS and SARI]'\K (197+) statcd t]rat 

natural infection of A. opis coulci occur eitlter bf ingestion of spores 

u'ith foocl or via the body sulface fronr spore's ot-t cotnbs allcl cell u'al.ls. 

ROUSSY (1962) found that the spores gennirlated on the sut'face of 

la5,ae, \vhereas MAURIZIO (1934) thought that inferctiou rt'as thloug'h 

the .rnoulh ancl not thlough the cutic'le or spilat:lc's. 

Chalhbloorl ,clisease has been repoltecl fl'om Ettrope lol lnany )'ears. 

]Iou,er,e.r'. it'rr.as not reportecl frclnr thr: t-initecl States r"rntil Br\KER ancl 

TOItCillO (l96tl) lor.rnci rt. opis associatccl ri'ith a lcal'c'uttcl llc,:, i\feortt'ltilc 

hternri.s I)r'clvanc:hel arrcl a soil rrestirrg ltet-, ,btlhoTthrtro p

ll 

Sirtce 1972, rr-e har-e been conriucting ex1:o'iments on the trau-rsmission r-rl 

'1. npis. S1:elificalil', u'e have been attelnpting to inlect broor.l altif icialll'. 

Sur'h infectior.rs u'ouki give irrl'cllrnation on the tlansurission arrci pathoger.icsis 

of th.: ilisrilst anrl u'ou1d prortide a relll'odu(:ible bioassa-v. Tltel' 

u'or-rlcl also allou' lrs to perfoml controlled experimcnls on nlcthod,s of 

tleatlrent such as the r-rse of rrrolcl inhibitors (T.\RDR et al., 1975). Iu ti-lis 

llallcr'. u'e repolt the rvor'k that s-e har-e concluctecl or-r luli-size colonies 

in the &pialr. and on blood meriutainecl in au irtcubator. 

Attcmpts to infect apiary colonies rvith A. rr1:is 

\Iany altentpts u,ele urade to infect blood in apiary colonies u'ith 

'lscosphcrerc 

opis uncler a variety of conditions aliiecting bot,h the 

pathogen and ihe host colonies. These included the use of inocula 

preitared from altilicial cultures of rl. cpis \is. suspensions from naturally 

infected lar-vae, application of the inocula in s5'rup sprays and/or in 

pollen cakes, use of rliummies uzi,th sporulaterl vs. vegetative growth of 

the pathogen, alteration of the ratio of adult tl'orker bees to brood by 

irerroval of adult bees or frames of broocl priol to application of the 

inoculurn, and application of ground tnumtnies in dusts or in pollen. 

The most se\:ere infections u.ere produced in colonies that had 

been fed uncontatniuated pollen to pt'omote blood rearing and froul 

u']rich \.ar)'ing numbers of aduit bees t'ere retloved befot'e a suspension 

of ll ;black (sporulated) mumrnies in 90 nl of 5o7o suc,fose in $'ater. When 

this treatment rvas applied 3 times a day on the first, third. and fifth 

da1's of the experirrent, it 3'ietded hear-y infections (100/o of ihe brood) 

by' the sixth day. Hrvever, er,en this treatment rvas less effective in 

other colonies that rvere treated at later periods. Thus variation in susceptibility 

rnay be an impor-tant factor in ,exprcssion of disease. 

Attempts to infect brood in an incubator rvith A. apis 

Because of the difficulty of ir-rducing infections in apiary ,colouies, 

u,e dcvise.d techniques for removal of blood from combs, surface sterilization 

of brood, tnaintenance of brood in an incubator, ancl inoculatiotl 

of brood u'ith A. opis. No differences in sun'irral rvere noted in larvae 

in the iucr,rbator ted 25010 sucrose solution, ro1'al jelly, or nothirrg. Thus, 

suspensions of zl. npis for inocula \\'ere ipr'epared by homogenizing 5 

black or spore-bearing mutntnies (mated strain) or 5 rvhite chalhy 

rnumnries (unmatcd strain) in 5 ,rnl of sterile ,clistilled rvater. These 

suspeltsiolts l\Iel'e plated to ,conlit'tn viability and rvere also sprayed otl 

conrbs in apiaiS colonies. Some rnurnrries \vere found in sprayed courbs 

after 5 days. The dead zl. opis suspensions used for contt'ols rvere obtained 

by autoclaving the homogenate. 

6B

i 

{ 

Attempts to infect small and large larvae from uncapped cells rvith 

A. apis 

First rve sought ,to determine rvhether A. apis causes chalkbroocl 

discrase or is simply a secondar.y invader oI lanrae killecl or irriur:e,cl bl' 

other errents. Evidence for thc latter might be supportecl by grot'th of 

the fungus on dead larwae. We also s'ished to deterrnine rl'hether munrmification 

arises from infection b-v both vegetative and sporulatecl strains 

ancl rvhether A. opis can invade larr.ae through the cuticle ancl/or. per os. 

In acldition, u,e rvished to deternrine g'hetlicr vourlg an,cl olcl lan'ae are 

equally susceptible to infection. 

The u'ater removal technique of ClATl.Y et al. (1.961) u'as ernplol'ed 

rvithout removing any cappings from l,he cells. Thus, the larr.ae rvere 

rvashed from a cornb onto sterile a,bsorbent paller. T'hen each lan-ae 

rvas individlrally picked up on the polished end of a glass tube attachecl 

to a $rater suction devi,ce. while held 'to the glass tube, each sras u.ashed 

3 times u'ith sterile clistilled lvater from a plastic sqlleeze bottle. 

The larva,e rvere ,divicled into trvo age groups. srnall and large, on 

the basis of size. The small }ar.r.ae were 3--4 davs old (average u'eight 

54 mg). ancl the large iar-r,-ae were 4.5-b.5 dal's old (average u.eight 

134 rrng). The treatmen,t groups of larvae are ,shorvn in Table 1. 

Lan'ae in the ((alive" treat'ment groups u,ere indir.idualh' pracecl 

in sterile vial caps in sterile glass petri dishes that contained a piece of 

filter paper kept rnoist u'ith sterile distilled rrater. Eight-l0 larvae 

leceiving the same treatment ,ll'ere placed in the same petri clish. Larvae 

in the ((dead" treatment groups u'ere placed in sterile r,'ierl caps and 

petri dishes and ,were killed r,vitJr dly ice. 

A drop of the approp'iate homogenate \\:as place.d eithe. on the 

mouthparts or ,on the dolsal side of the larr.ae. We confirmeC consulrption 

of the homogenate by direct observation. The clishes rvere placec 

in a 25rC inc,ubator. Letrr.ae u'ere o,bservecl clail1" for 10 da1-s tvith a 

clissecting rni'closcope fol moveurent, fungal grou'th, discoloratiorr, and 

nrunrrnif icaticn. 

'I'he lesults are shon'n in Table 2. Eler-en urLrrnnties u'ere fornrecl 

in control tleatment grollps leceiving cleacl ..1. c7ri.s. These Iarvae rnaliravc 

httcl trtttut'al itrfec'tions u'hich r,r":re inarJ'liralent lrnl.il thc lan'ac t'(:r.e 

inc'r.rbaterl. Iloq'cver'. trorre of the colclnies frou'r ri-hic'h lalr.uc rr-ct'e obtained 

r:ontainec anv larvae thiit u'cre r-isibl.r' iulectecl l-ith ;1. rrpi.s. Nol 

wet-e altv rntturntics for:nil at. the err.rtltrncc or on the bottciurs of these 

colonies. T'hr:r'efore. sorne l>ec r'olonics or lalvae rlav harbor'-1. cpis 

$'ithor.tt sltorving sigrts cll cliscitsc. Snr:rll larviie tirat u'err: natur-allf ilfcctecl 

recluilecl 5'*7 da1's of incubation to lnunrntifr- : tire large l;u.r-ae 

requ irccl 6-B da1's. 

69 

d 

!t 

lj 

ii 

ta 

i1 

1i 

ll 

II 

I 

t! 

I 

lt 

lli 

H 

ll 

it 

It 

It

TRIJ,.\'T\1EN'T CRoLIPS O}' S11,\LI. AND I-.{IIGE LANVAE }'ROXI 

LISED 'TO DE]-I;R]'!\-E TI]E I"\THOGENIC ,.\ND SAPI]OPIIYTIC 

TISCOSP/I4EIt A ApJ.S 

Large lorxae a 

UNCA,I'PED CELLS 

I'JioPtiR'rtiis ()l' 

1'realnr ent /1 (.pis..st.min Sile of inocrrlatiot] 

"A1ite" Iarvae and live A. apis 

*-do* 

-clo- 

-do- 

".-\li'r'e" larrrae and dead A. apis 

-do- 

-do- 

-do- 

"Dead" larvae and live A. apis 

-do- 

-do_. 

-do- 

"Dead" lan'ae and dead A. apis 

*do_ -do- 

*do- 

Small larcae b 

"r\live" larvae and live A. apis 

-do* 

-do* 

-do- 

"llivc" 

ku'vac and dead -1. opis 

-do- 

-do- 

-do* 

''I)eltd" lln'ae and live ,4. opis 

-do- 

-do- 

-do- 

"Dead" larr.ac and dcad r{. clris 

*do- 

-do- 

-clo- 

at :10 ]arva o pel' tost 

b l0 larvae pcr le\t 

NIated 

Unmated 

NIatcd 

I-Trrnlated 

llated 

Ljnmated 

tr{ated 

IJr"rmilted 

N{ated 

Unr-nated 

n{ated 

llnmated 

Mated 

Unmated 

1\{ated 

Unmated 

NIated 

Unrnated 

I,Iated 

Unmated 

NIated 

tinrnated 

IVIated 

Unr-rrated 

llatcd 

flnnriited 

NIated 

LInnr:rted 

llated 

Iir-rmatccl 

\{atc.d 

IJn nratt:d 

l{outlrparts 

-do- 

Back 

-do- 

IVlorrthpalts 

-do- 

Rack 

-do- 

Mouthparts 

-do- 

Back 

-do- 

Morrthparts 

-do- 

Back 

-do- 

llor-rUl-rparts 

-do- 

Back 

-do- 

1\'loutl'rparts 

-do- 

Back 

-donlotrthparts 

-do* 

Back 

-don{outhparts-dollack 

-do* 

'1'abIc 1 

No urutrturies "tr:ere formecl as a rcsult of the inoculation of dearl 

lan'ae l'ith ;1. (rJii.r. IIo\\,e\:€r, :1. opi.s did grolv oll 3ll of these larvae. 

Thus. it arlrpears that ..1. opi.s can grow and in sorne instan,cas sporulate 

on dead larvae but rvill uo,t ututnulify them. 

Forty-three {nurnn'ries \vere formed as a result of inocuration oI 

Irrlvae rr,ilh Iir,'e tl. apis. Both the srnall and large larvae requireds-Z 

70 

:yl

da1.s after inoculation for mummy formation. Tr.ventl'-six of the 

murnmies were prod'uced by inoculation of mouthparts. and 17 by 

inoculation on the ,back. Thus, infe'c'tion n-Iay occur per os and 'through 

the outicle. lVe observed the fungus gr'ou'ing frorn the mouth' the anus, 

ancl through the cuticle of larvae inoculatecl on the rnouthparts. The 

fungus appeared to gro'nr.' first aerially at the point of inoculation on 

Iarvae inoculated on ,the baok. It tllen grew throughout the entire body. 

and the larvae rbecame mumrnified. A11 experimentalll- produced 

mummies were much flatter than those gener-nlly found in bee colonies. 

Both matecl and unmated strains of A. o.pis caused mumtny formation. 

Of the nlunt.lnies prod,ucecl from the material prepared fro;n 

blaol< mumpries, 11 rvere blaok and lL rvere white. Ho$'et'er, of the 

mumrmies produced by inoculation of matel'ial prepared frotrr n'hite 

mummies, 18 g'ere ,blaak and only 3 rvere rvhite. Thus, by selecting 5 

mummies for production of the inoculu,m, we obr.iously mixed * and 

- strains to produce the' matEd strains that infected the lalrrae. I{ou'ever, 

it is interesting that an ,eQllol nul]lher of black and rvhite ntummies 

tvere forlxed from 'material iprepare,d from black lnumll'Iies. r\ separation 

of. * l- ,material could have occurred or, perhaps, sporulation $'as in 

some \vay inhibite'd. 

Small-alive 

-do- 

-do- 

-do- 

-do- 

-do- 

Sn.rall dead 

Large-alive 

-do- 

-do- 

-do- *do- 

-do-* 

Large-dead 

-do- 

Inocub'tion 

Site 

Mouthparts 

-do- 

Ilrtr:k 

-do- 

1\{outlrparts 

Ilack 

-do* 

I\Iorrthpitrts 

-do- 

Back 

-clo- 

Nlottthparts 

Il aclt 

1\{outhparts 

Back 

A, opis StIJain 

Live-mated 

Live-unmated 

Live-nrated 

Live-unmated 

Dead-mated 

-do- 

I-ive-unmated 

Live-mated 

L,ive-unmated 

Live-mated 

Live-ttnmitted 

De:rd-unmated 

Dcad-mated 

Live-unmated 

-do- 

Table 2 

[art'€"e 

truummles With 

Prodnccd A; apjs 

Gro\\'th 

Black wrrite sljr!- spor.t 

Sr"nall and Iarge larvae \vere equall1' susc'eptible to infection since 

\ve used ,tu'ice as malty large lan.ae as srllall ones. Thirtl, urtllnlnies \t:el-e 

producecl among lar"gc lat'vae and 12 alrtoug small lalYae. flhns. 3-.-4 

1 

5 

1 

3 

2 

7 

6 

, 

4 

, 

221 

34 

7 

J 

3 

1 

1 

6 

1-l 

7 

7I

clal'-old ancl -1.5-5.5 day-olcl larr,ae iri)pcar.to be susceptible to chalkbrood 

rlisease. This conjecture is leinfolcccl b)'the fact that 5 of 1hc 

smarll ancl {j oI thc large lan'ae altltalt:nt])'harl a rrutural infct:tion of 

challibrooll. 

Next rr,e tler,'clr>1ted a rnelhoci oi snlface stelilization ol lat'r,ae n:ith 

ir gerrnicicle 1o elinrinate comb conlanrinants (;lspcrgillu.s sPp. and 

Paricillium sltlt,) n'ithout harrrring the lur.r'ae. 

:\ftel tltc.lat'r'ae tt'ere tl'asl-ted fi'onr a conrit onto n slelile NtrassilinR 

{ou-el, thcv t:elc irTclir;irluall-r- picked up n'i1h the suction apparatus. 

n-ttshed t'ith a 1: I250 solution of llr'lccalri from a ltlastic sclllceze bottle. 

arlci I'irlsel tri'ice u'ith sterilc clislillcd rvater flonr a squeeze,bott.le. 1'he1r|c't'e 

lllacecl on stet'ile ilaper to bklt thern clr'f anrl n'ere t,hen put into 

stelile vial caps in petri. dishes ,by leleasing the suction. 

Attcrnpts to infect eggs, Iarvac and pupae rvitlt ;1. apts 

Then r.rre designed an experiment to de,tern-rine rvhether ,zl. cpis 

rvoulcl grow on or cause mumrnificatiou of eggs ; snall, medium, and 

Iarge lan'ae ; prepupae ; pupae n'ith no eye color ; and pupae rvith 

eye 'color. 

ifhe treatment groups are shotn in Table 3. Eggs rvere inclivid,uall5, 

retnoved frorn their ,cells u'ith special forceps (TABER, 1961), rinsed rvith 

Roccal, and lt'ashed trvice rvith sterile distilled rvater. Then they rr'ele 

blotted and placed again.s,t th'e side of a sterjle vial cap to simulate the 

position occupied in the cell of the comb. small and nediu,m larvae 

l-eLe rl'ashed from uncapped combs. Large iarvae, ,prepupae, and pupae 

tl.eLc lvashed from combs ,after uncapping the cells. All larvae rvere 

sterilized as desclibed 'before. Pupae u'ere washed rvith Roccal on the 

sterile tou'el on t'hicir they u'ere ,colle,cted, They t'ere then individualillrelcl 

rl'ith forceps aud rin'sed trvice urith sterile distilled triiter. Each 

insecL rvas placed in a sterile vial cap and treated as shorvn in Table 3. 

Since u'e notecl that 1he rnedium ancl large lalvae, prepupae, and pupae 

required less humidity in the petri dishes than the eggs and small 

larr;ae, rve added less trater to the filter papers in clishes ,containing 

these stages. The nrumrlified lan'ae and prepupae looked more like 

those f rom natural hive infections. possibly because of the lorver 

humidity. 

All bloocl rvas incubaled at 25"C and observed lor 15 da1'5 1ot' 

grorvth of A. apis and .nurnrnification. No grorvth occurred on any eggs 

or llupae. r\lter irlcubation for 7 dal's, sel.eral rvith eye color had devek:pecl 

rvings, Iegs, antenrrae, and hair. 

As shorvn otr Table {, 26 munrrnies rvere forrned. I{orvever, 12 of 

these rvere apparently the result of natural infections of A. opis since 

they rvere in the control groups inoculated rvith .dead A. apis. Of these 

72

TR}}.\'I}II]NT' GROUIJS OF EGGS, L.{R,1/AE, AND PUPAE 

ASCOSPTIAER-A APISA 

Treatlnent 

Eggs 

Live A. apis-m:rted 

l,ive,4. opis-trnnr;rted 

Dead ;1. ripis-mrted 

De:rd 11. npis-unrnated 

Sntoll lcrt'ae 

Live ;1. npis-ntated 

LiYe ll. apis-nrated 

Live,4. npis-rrnmatecl 

Live zt. npis-unnrated 

Dea'd A. opis-mated 

Dea'C ;1. apis-ntated 

Dead ,4. cpis-unmated 

Dead .r1. cpis-unntatecl 

Medium lartae 

Live ;L. apis-mated 

Live A. apis-mated 

Live ,4. apis-unmated 

Lit'e,4. opis-unrnated 

Dead ;l. cpis-rnated 

Dead rl. crpi.s-nrated 

Dead -4. crpis-unrnated 

Dead A. cpis-unnr:rted 

Large lort:ae 

Live A. opis-nratecl 

Live ;1. cpis-matecl 

Live zt. cpis-trnmated 

Live A. opis-r:nmated 

DeaC ,4. npis-rnated 

a 5 insect.s per tt*rtment 

Bociy 

ISody 

Ilody 

Body 

INOCUL.{'TED l\'I'rH 

Table 

Site of 

I.nocu lation Treatment Site of 

Inocu,lation 

Rack 

Xloutlrparts 

Ilack 

Moutlrltarts 

Back 

Mouthparts 

Ilack 

Mouthpalts 

tslck 

Mouthparts 

Back 

l{outlrparts 

Back 

Mouthparts 

Back 

Mouthl;:u.ts 

B:rck 

IVIoUtlrpar.ts 

Ilack 

l,{oullrparts 

Back 

De;r'd l. opis-nratecl 

Deircl -,1. cpi.s-unnt:rted 

Dead,4. apis-unnr:rted 

Prepultae 

Live ;1. opis-materd 

Lir-e .1. apis-mated 

Lite' ;1. cpis-lrnm;rted 

Live ;1. cpi;-unntatecl 

I)ea:l rl. opis-matecl 

Dea,C -.1. apis-nrated 

De:rd,4. opis-unn-rated 

Dead ,zl. apis-unmated 

Pupce-No Eqe Color 

Live ,,1. apis-mated 

Live -;1. apis-mated 

Live .21. cpis-unmated 

Live ,4. apis-unntated 

I)eacl rI. opis-mated 

f)ead -.1. cpis-n-rated 

Dead ;1. cpis-unntatecl 

f)e:rd A. opis-unrnatecl 

Pupae-Eye Color 

Live .1. opis-rnated 

Live .1. cpis-mated 

Live ;1. cpis-unmatecl 

Live :1. apis-rrnntatecl 

De:rd rl. cpis-mated 

Dead .,I. apis-muted 

Dead ,4. apis-unmated 

De'ad A. cpis-unmated 

Motrtlrltarts 

llack 

Mouthparts 

Iiacli 

Moutlrlr;rr-ts 

llacli 

I\{or,r tlrpirrts 

Llack 

Moutlrparts 

tlack 

lUoutltparts 

Back 

1!Ioutl'rparts 

Back 

N{outhlrarts 

Back 

l\{outl-rptrrts 

Back 

Mouthpar.ts 

Bacli 

Mouthirarts 

Back 

l\{outhparts 

Ba,ck 

nlontlrparts 

B:l,ck 

Mou.tl-rparts 

12, 5 $'e|e mecliLur.l-sizeci larvae, 5 u,ere lalge larvae. alrcl 2 1,e1e irle_ 

puPae. 'lhe|efore, b1' exllerimental inoculation, olle nrunpl)' was lll'ocluced 

in the mecliur.n-sizecl larvae, B in the iarge lan.zre. and 5 in the 

ill'ePlU)iie. Thlts. n-iore lrulltllies were llrodLlceal fronl lar.ge larvae though 

fe*r 'u'r.-ries rvere l)rodll(:ed in any ltrr.r-ae on l)rer)aLle. I)e .IoNG a'cl 

MORSE (1976) also observecl rvicle val'iation ir-i infec.tion rirlcs oI lar.\-ae 

lcd "1. api.s. In.the llresent test. all nrunrr.nies loun(l \\'ere \\.llite (r-rqprtricrl) 

to gra)'(pt'obablf il l'ltixtLtre of ,matecl eln(l unn-rirteil strtrins). Othel tlarr 

the trrutntrlies, onlr' l srnall ancl l nterliunr-size(i lar'\.lt inocr,rlatcd u.ith 

li'e 11.. opis siro\\'ed an' g.o1\'t,h of .1. .pis. ln tlrese 2 la.r,ae. .1. 'pi,s 

/i)

gre\\: fror-rl the moutir. anus. and through the cuticle. None of the otirer 

inoculateci insects f ielded an1' grorvth of .,1. apis. 

'I'ahle 4 

}IL"\IfIIF:S .C.\T] CROWTH OF ..1.SCOSPHAEFIA APIS PRODUCEIJ T]V TNOCIJI,ATION OF 

EGGS. L,\R\:AE. AND PUP,\E WITH ,ISCOSPIJAERA APIS 

Slage 

,Snrirll lan'ae 

Xledirrm .l:rn'rr 

llediunr lalr-a 

lleriiunr lan-a 

\Iedium lar.r'a 

lledium larva 

lledium i;irva 

Large Iarta 

I-arge lan'a 

Large larva 

Large lalva 

Large larr-a 

Lalge larva 

Large larva 

Prepupae 

Prepu;rae 

Preptrl:ae 

Prepupae 

I)re1tu1:ae 

Prcpul-lae 

Sitc of 

Inocrr lrt i ()rl 

llouthparts 

1\louthltitlts 

Llack 

Nloutlrparts 

I{outlrptrrts 

B:rtk 

Back 

N{outhparts 

l{outhparts 

Back 

Back 

Mor.rthparts 

B:ltk 

tleck 

Back 

Xlouthpar"ts 

B:;ck 

trIorrthltarts 

X'Ior-rtlrparts 

Ba

differ frorn those repor-te,d ft.om Europe. 

question. 

Acknorvledgement 

We are norv inl.estigating this 

we thanli Dinorah DUNTFIAM and Leslie cApI,N for their excellent 

technical ,assistance with br.ood in the incuibator. 

REFEIiENCES 

B"\rLEY. L.' 196?. - The effect of temperature on the pathogenicity of the fungus, 

AscosphQera (rpis, ror .larvae of tlre h()ne). bec. rrpis melliJera. tn: ",Insect I,athology 

and Microbial Control'., ed. f,.,q.. \.an der Laan, p. 162*167, Norr.h-Holland, 

Amsterclarn 

B-AKER. c. M.' p. F. TORcHto. 1968. - Ne$. records oi t\scosphdeta apis frorn North 

America. MVcologia, 60, tB9_I90 

BARTHEL, ''., I9?1. - Der Kalkbrut aut der spllr. carten u. r.reintierzuchr c. rmker, 4, 

12-13 

DE JONG D., tl. A. MORSE, 1926. - Chalk broocl: a ne\\, disease, A. y. Fo.d Life Sci-, g, 

12-14 

GARY' N. E., R' \v. FIcKEri. R. c. sttEIN, 1$6r. - Hone}' bee lar'ae (Apis nleililera L., 

for bird food. Auicult. Mdg., 67, 27-Bz 

GILLI.{M' .l\4.' S' TABER lII, 1973. * ]\{icroor'Banisms ancl diseases encounterect in continous 

bee production. Amer. Bee J., 715, 222-2ZJ 

t{ITCIICOCK, J. D., M. CHRIS?.ENS.EN, tg72. - Chalk broocl ciisease of honcy bees in the 

United States. Amer, Bee J,, 112, ZlB-Ztg 

MAafUS, F.' I' SAIiB.{K, r9?{. 

toruosA Lapia, 

- 

29, 250-233 

(OccLirence o-t chalkbrood clisease in llungary). MagVar AtIa- 

MAURrzro. A., 193{. 

- 

Auncle, t;,105-1tB 

uber cle Kalkbrut (},eric}'stis-M}'kose) der Bienen. Arch. Bie.nen- 

ROUSSY' L., 1902. - NoLlvetle contribution a I'dttrde du Pericystis apts (MN). Gaz, Apicote, 

6ll, 101-10; 

TAIJER, s. rII, r96t. - Fol'ceps design for transferring honey bee eegs. J. Ecoil. Entontot., 

51, 247-250 

TABEtt S. ttr. R. SACKETT, J. NIIt,t,S, 1175. - A possiblc control tor chalk brood di.sease, 

Atner. tlee J.,7ti, Z0 

THOMAS, G. NI., A. LUCE, 19?2. - An epizootic ol chalk brood.Ascosphaeru (tpis ltaassen 

ex Ctalrsson) Olive atlct Spiltoir in the honey llce. Apis ntelliJera L. in Cdlilornia, 

Anter. Bea J., tt2. 'n'i-Cl. 

I 

i 

I 

I 

i 

i 

J 

I

y" * 

-rrJ 

2' tgrs

t' 

JoURNAL OF TNVL.RTEBRATE pATHoLocy 32,222-223 (1978) 

Recent lsolations of Bacillus pulvifaciens from Powdery Scales of Honey 

Bee, Apis mellifera, Larvael 

Bacillus pulvifaciens was originally described 

by H. Katznelson (J. Bacteriol. 59, 

153-155, 1950). The organism was isolated 

from larval scales of honey bees, Apls 

mellifera, that were dry, powdery, and light 

brown. Limited attempts to infect bee 

colonies with material from the powdery 

scales and culture material did not definitely 

establish whether the organism was a pathogen 

or a saprophyte. Later H. Katznelson 

and C. A. Jamieson (Sci. Agr.32,219-255. 

1952) concluded after more extensive work 

that the bacillus was not pathogenic to the 

honey bee, although J. D. Hitchcock (cited 

by R. E. Gordon, W. C. Haynes, and C. 

Hor-Nay Pang, U. S. Dept. Agr. Handb. 

427, l-283,1973) isolated additional strains 

of B. pulvifaciens frompowdery scales and 

has evidence that the organism is pathogenic 

to honey bees. 

R. E. Gordon et al. (loc. cit.) examined 

seven strain s of B. pulvifaciens , all from one 

source (H. Katznelson), and postulated that 

B. pulvifaciens may form a connection between 

B. larvae and B. laterosporus, organisms 

also associated with honey bees. 

Recently, we isolated B. pulvifaciens 

from larval remains. Thus, the purpose of 

this paper is to describe the larvae from 

which the organisms were isolated and to 

compare the morphological and physiological 

properties of our strains with those 

previously described. 

A brood comb from a moribund colony in 

the apiary of an Arizona beekeeper was 

supplied by Dr. Gordon Waller. Examination 

of the contents of this comb revealed 

many powdery scales that were dry and 

chalky white rather than light brown as de- 

I Mention of a proprietary product or company name 

does not constitute an endorsement of this product by 

the U. S. Department of Agriculture. 

0022-201v78t0322-0222$01.00t0 222 

Copyright O 1978 by Academic hess, Inc. 

All rights of reproduction in any fom reserved. 

scribed by H. Katznelson (loc. cit.). Some 

scales were positioned on the sides of the 

cells, and others were on the bottoms extending 

up one side. All were similar in appearance. 

Another brood sample was received 

from Mrs. Diana Menapace of the 

USDA Bee Laboratory in Laramie, Wyoming. 

This material was from a bee colony 

in Iowa and contained a yellow crumbling 

larva that had been removed from its cell. 

Scales from both sources were streaked 

directly onto plates of nutrient agar (Difco), 

and the plates were incubated at37"C under 

aerobic conditions for 10 days. At that time, 

pure cultures of bacterial colonies that were 

bright orange were obtained. Twenty strains 

from the Arizona sample and one from the 

Iowa sample were maintained on slants of 

nutrient agar for further study. 

Gram-stained slides of the isolates revealed 

ellipsoidal central spores that swelled 

the sporangia. Many spores had stained 

remnants of the sporangia adhering to them, 

which gave the spores a pointed appearance. 

The isolates were then subjected 

to the morphological and biochemical tests 

for the genus Bacillrzs described by R. E. 

Gordon et al. (loc. cit.) except that motility 

was determined in motility test medium 

(BBL) rather than microscopically. 

The results of our tests agree with those 

listed for B. pulvifaciens by R. E. Gordon 

et al. (loc. cit.). All strains were Grampositive; 

formed acid from glucose, mannitol, 

and trehalose; reduced nitrate to nitrite; 

liquified gelatin; and decomposed 

casein. Seven of the strains were motile. 

Also, all but five strains were catalasenegative. 

These five strains gave a delayed 

weak-positive reaction by producing a few 

tiny gas bubbles on a small part of the 

periphery of the streak after lUVo HrO, was 

added to growth on plates of nutrient 

./. 

, 

i.) $;,ti i:$ 

!:i',1. 11;4

agar. The bubbles arose from the creamy 

whitish growth at the periphery that was 

noted first by H. Katznelson (loc. cit.). 

Cultures giving the delayed weak-positive 

reactions were rechecked for purity and 

found to be pure B. pulvifaciens. R. E. 

Gordon et al. (loc. cit.) observed this reaction 

with their strains and stated that it reinforced 

their idea th at B . pulvifociens may be 

a connecting link between catalase-positive 

and catalase-negative species. 

Thus, powdery scales having different appearances 

seem to yield B. pulvifaciens 

strains that have morphological and biochemical 

reactions consistent with those of 

NOTES 

223 

the strains originally isolated by H. Katznelson. 

This does not, however, rule out the 

possibility that strain differences might 

contribute to the variations in color or consistency 

of powdery scales. 

Key Wonos: Bqcillus pulvifaciens; honey 

bee; powdery scale disease. 

U. S. Department of Agriculture 

Agricultural Research Service 

Bee Research Laboratory 

2000 E. Allen Road 

Tucson, Arizona 857ii9 

Received November 16, 1977 

Menrne Grlueu 

DrNones R. DuNHeu

Fungi 

Marthn Gillinm 

Introduction 

Chalkbrood 

History 

Distribution 

Etiology 

Treatment 

Bettsin alaei, the Pollen Mold 

History 

Distribution 

Etiology 

Treatment 

Stonebrood 

History 

Distribution 

Etiology 

Treatment 

Other Diseases 

Melanosis 

Other Molds 

Yeasts 

CHAPTER 5 

ls 

i 

Intrc 

Fu 

centi 

State 

I{or"u 

crca.! 

if th.; 

ChaI 

cl- 

It is 

Spilt 

rvher 

are tl 

Dir 

orvin 

are fr 

Th 

thou, 

7e63) 

7968 

Hi: 

lr{: 

chalk 

and r 

horre 

rvhet 

a det 

the n 

In

Introduction 

Fungi are common saprophytes of bees and combs' Until re- 

."^tlyf fungal diseases of hott"y bees were rare in the United 

States. Stonebrood is still considered to be of minor importance. 

However, the incidence and severity of chalkbrood disease is increasing, 

and this disease may become a major economic problem 

if the trend continues. 

Chalkbrood 

Chalkbrood is a fungal disease that affects onlyhoney bee brood. 

It is caused by Ascoiphaera apis Maassen ex Claussen (Olive et 

Spiltoir), a heterothallic organism in which spores are formed only 

*ih"r, .ny""lia of opposite sex (* and -) come together' Spores 

are theniormed within dark brownish-green fruiting bodies. 

Diseased larvae become mummified' The mummies are white 

orving to the mycelium of the fungus. However, if fruiting bodies 

are fo"rmed, the mummies become dark gray or black (Figure 5'1)' 

The disease is usually transient and not considered serious, although 

endemic infectiron can be persistent and damaging (Bailey 

1963i This disease was not reported in the United States until 

1968 (Baker and Torchio 1'968). 

History 

Maassen (1913) in Germany published the first observations otr 

chalkbrood disease. He later $t1,6) described the associated fungus 

ancl named it Pericystis apis.ln 1912, Priess gave Claussen a moldy 

honey comli and a sla.,i culture of a fungus' He wanted to.know 

rvhetirer the fungus was a pathogen. Claussen (1921) published 

.r detailed pup", on the morphology of the fungus and retained 

the name Pericystis apis. 

In England,'Betts (1912a) describecl a hive fungus, Pericystis 

79

F tbai 

trn 

:: lr" 

ii+,. 

i6i 

iq*, 

iE 

s?" 

rtA' 

kllt 

:.F. 

-l?; 

* 

., *:t 

:i=lii .$i 

-.]*d 

;ir 

.:ei. 

.''ri 

.;g!. 

ii 

:,f 

;i::' 

.:-.) 

, ji:. 

.it: 

:,"tr;:!' 

. lti;: 

..i:i; 

'lii't 

'iirl 

":l-] 

]]e, 

I r,iir, 

.i:-r 

, ,:iil 

;j:, 

, j;.1 

.,- ." ,:t' i: -- 

rt.:r:j i:.-: ;ri.'- - _ .:--::,:r: ::;; 

Figure 5.1 . Chalkbrood in rvorker brood ' (Photo b1' M' V' Snrith) 

hktci. Later in 1919 she examined the chalkbrood funSus and noted 

that it rvas not identicai to Pericqstis aluei' 

subsequently, detailecl mycological observations on Pcricyslis 

npis rvere repo;ted by Varitchak (1933) in France, Maurizio (7934, 

1g35) in s$,itzerland, and Prokschl (1953) in Austria. Moreover, 

N4aurizio (7934,1935) demonstrated that there lYere t1\ro morPhologically 

different types of Perinlstis apls. Each lvas heterothallic 

and capable of causing chalkbrood disease. The trvo varieties \\,ere 

not capable of beir-rg crossed with olle another' One varicty, the 

usual one in primary cases of chalkbrood (cases of actual outbreaks 

of the clisease in the bee colony), had small cysts. Thc other, more 

commonly found in secondary cases (rvhere the fungtls developed 

on coml.)s th.-rt had been kept outside the hive), had much larger 

c1.sts arrd rvas stalked. This large-fruitecl variety preferred lorv 

tcnrpett-rttrres, especialll' during c\'si fornlation' The optimum tenrpurnt,-,r" 

for cysi formation in the large'-fruitc'd varicty \vas 20" 

-elsius, and irr the small-fruitecl r'ariet;', 30'Celsirrs' 

Prirkschl (1953) clesigrrated the snrall-frr,ritercl form, originally 

narrred Prrioptis apis, as Pericy-slis c;ris variety 'tlrtor (Maasst-n) 

lLt 

!:-.;,,&g*r ri'}-u 

T' 

Proks 

(1-e34 

Zobl. 

and I 

lisher 

since 

namt 

ety u 

rial. 

varie 

SK, 

ceae 

phae 

For 1 

gro\ / 

sepal 

for tl 

for tt 

bee, 

mold 

phae 

In 

ber c 

bees 

Di; 

Ch 

ish Ir 

Bartl 

Giar; 


1.94!, 

1957. 

Bl 

opis 

cia ti: 

the r 


forni 

in h, 

othc 

Dakt

Prcikschl etzobl and the large-fruited form described by Maurizio 

(\g34, 1935) as Pericystis npi- variety maior (Maurizio) Prcikschl et 

iobl. in the United States, Spiltoir (1955) studied the life cycle, 

and spiltoir and olive (1955) reclassified the'fungus and establisheci 

a new genus and family, Ascosphaern and Ascosphaeraceae' 

since the naie Pericysfis had been previously used as -a -generic 

name in the red algae. spiltoir and olive (1955) valiclated the variety 

under Ascosphnern npis vaiety major without seeing any materiat. 

rney also establishecl the type I'ariety as Ascosphaern apis 

variety apis. 

Skou (fgZZ) compared cultures of members of the Ascosphaeraceae 

and designated Ascosphaeraceae as the only family in Ascosphaerales 

.,.de. the series Plectomycetes in the class Ascomycetes. 

For the first time, all members of the Ascosphaeraceae were 

grown side by side. He raised Ascosphaera npis variety mnior .to a 

Ieparate ,p".i*, as Ascosytlnera rnaior; Ascosphnera npis was retained 

foi the small-fruited form; and Ascosplnera prolilterda was erected 

for the new /scosphaera species found associated with the solitary 

bee, Megaclile cetituncttlctiis t. Bettsia alaei, the saprophytic pollen 

mold, is-the sole member of the only other genus in the Ascosphaeraceae. 

ln'1.974, Stejskal described Arrhenosphaers crsnei as a new member 

of the Asiosphaeraceae. This organism causes chalkbrood in 

bees in Vcnczucla. 

Distribution 

Chalkbrood has been reported from Europe, including the British 

Isles, for many years (Anderson 1934, 1938, Bailey 1963,1968' 

Barthcl 1971, Betts 1919, 1932, 1951, Deans 7940, Dreher 1938' 

Giatrffret and Taliercio 1'967, Lunder 1972, Maassen 1913, Matus 

ancl sarbak, 1974, Maurizio 1934, Mclellan 1.964, N{orgenthaler 

1944, Roussy 1'962, Taberly and Monteita 1967, Zander 1919)' In 

1957, Seal reported chalkbrood in Nerv Zealancl' 

Ilaker ancl Torchio (1968) reported the first record of Ascosphaera 

a;ris from the United states. Their isolates from utah rvere associatecl 

r,r,ith the leafcutter bee, Megncltile inertttis Provancher, and 

tlre soil-nesting bee, Anthophora Ttacifica 

Cresson. Later Thomas 

and Luce (1g7r) reported chalkbroocl from honey bees in California, 

ancl Hitchcock and Christensen (1972) fotind the disease 

in honey bee larYae in Nebraska and \Vyoming and noted that 

' other occrtrrences of the disease in California, Minnes0ta, North 

Dakota, ancl Montana had recently corne to their attention' Giliiam

i:' 

':, 

82 PESTS, PREDATORS, AND DISEASES 

and Taber (7973) reported chalkbrood in Ariz'na, and Co.nor 

(1974a) found chalkbroocl in ohio. By 1974, the disease had been 

found in at least 35 states (De jong 1976). 

.Gochnauer, Hugh6s, and Conner (1972) reported the first record 

of chalkbrood in Ca.ada in honey bees from British Columbia 

and Saskatchewan. Nelson, Barker, Blancl, Corner, Soehngen, and 

vilieneuve (1976) surveyed 5,374 coronies in five prorlinces of 

Canada and found that 32 per cent of these colonies hid mummies 

in the frames. Ilorvever, is per cent of the infected colonies had 

less than ten cells rvith charkbrood. Ii is possible that the disease 

rvas Llnreported for some years in Canacla ancl the u'ited states 

(Gochna'er, Furgala, ancr shimanuki1975), since cornmercial beekeepers 

indicated that trre discase had been prese.t for decacies 

but rvas considered insignificant in comparison to American foul_ 

brood (Connor 1974b). 

Etiology' 

According to Bailey honey bee larvae 

!!9-67), 

are most susceptibie 

to chalkbrood disease if they i.gesi spores of Ascosprtnern aTtis i"h"r-, 

they are threc to four clal'5 ord anci then are crriileci briefly trvo 

clays latcr imrnediatell, after they are sealeci in their cells to pirpatc. 

Because of the chilling factor, the affectecr rarvae are usualri iJr'a 

on.the peripherl. of the broocl area. For this reasorr, it rr.as once 

believed that only drone ra^'ac *'ere affectecr since tire' are frequerrtly 

on the periphery of the brood nest. 

spores 

. the^ th" germin ate in t,iro in trre gui of the larvae n hich 

is alm'st anaerobic. Horvever, the mycelitim is aerobic and, there, 

fore, survi'es in three to four-day,-olcr larvae better thai, in 

younger brood because of the shortei time it is deFrrived of oxygen 

(lt"y 1967). The spores germi'ate p;rrticurarry ln the hi.d encl 

of the gut. When the larva becomes sealecl in its ce.ll to 

the mycelium 

f,rpate, 

breaks out of the hincl end of the bor1r. (Bailey t'0621. 

since Ascosphaera apis is a heterothalric orga'ism, the iarva is 

transformed into a rvhite chalklike munlnlr,, rvilen the rtrvcelium of 

only o.e strain (+ or --) infects a rarva. hihen the myceiia of both 

strains grorv in a larva, fruiting bocries are for'retl ,r., th" outsicre 

of the dead larva, and tl-re r,r**1, becomes gr.a1, 1o black (Figure 

5.2). 

At first the clead larvae are covered u,ith a flulfi, rvhite gro.rvth of 

myceli.r and are srvollen to thc size of the ccll. Latcr, the clead 

larvae dry irrto harcl, shrunken, chalklike lrrinps, rvhich nav be- 

Figu 

Snri 

con 

of t. 

mul 

of li 

in tl 

mie 

boa' 

Bi 

infe, 

u'itl 

ancl 

took 

ck-s; 

tire.: 

post 

mair 

tion-.

or 

?n 

rd 

ria 

1d 

of 

3S 

ad 

SC 

:ES 

ies 

tl- 

ble 

en 

["o 

+5 

;; 

[." 

l"- 

i.hreln 

;en 

:nd 

rte, 

t7\. 

rls 

iof 

Ith 

ide 

i's- 

I 

!of 

lad 

rDe - 

. 

: 

i 

i 

t 

i 

:k' 

h-. *"'t-t 

1 :f' ^: 

;.i: ; 

L:;", ':' ''i' 

FiFnfi 

vd 

Figne 5.2. Chalkbrood; close-up of mummies in worker comb' (Photo by M' V' 

Smith) 

come gray to black if fruiting bodies are formed (Figure 5'3)' Many 

of the*celis in heavily infected colonies may remain sealed, and the 

mummies will rattle if the comb is shaken. However, the remains 

of larvae can be found in sealed or unsealed cells. Most larvae die 

in the upright stage. Also, adult bees generally remove the mummies, 

whici can be found at the colony entrance and on the bottom 

board. 

Barthel (1971) and Matus and sarbak (1974) stated that natural 

infection could occur in two ways, either by ingestion of spores 

rvith food or via the body surface from spores adhering to combs 

ancl cell walls. Howevei, N{aurizio (1934) thought that infection 

took place through the mouth and not through the cuticle or spiracles; 

whereas Ro-ussy (1962) found that the sPores germinated on 

the surface of larvae. Gochnauer, Furgala, and Shimanuki (1975) 

postulated that once a colony rvas infected, the sPores could remain 

viable on the combs and eventually germinate when conditions 

became favorable, and the disease could then reappear. They 

!i* 

i.::.. t. . . la''- i: i'::;.;." 

.:., . .- , ::rtl:,,-.r.i.:.'ii::,.;:,1..:,,i:. -:.,,,:.1"r;j",1.,::t.-,.

'#i:,'. 

f{d:, 

-L=*: 

". i 

Figure 5,3. Chalkbrood mummie_s.. Initiaily thedead rarvae remain white; they are 

entirely permeated by the vegetative hyphae of Ascosphneya apii- s."u"i'roorurition 

occurs later and causes the larvae to take a characteristic dari or black colcr. (photo 

by D. M. Menapace) 

also suggested that Ascasphoera npis might survive in soir, find 

its way into the food chain of honey bees, and be transrnitted to 

larvae via contaminated brood food. 

,A'lthough the ability of infection to spread is probably low (Bailey 

1963), the spores are quite resistantlnd remain inflctive for at 

least 15 years (Toumanoff 1951). Maurizio (1994) and Betts (1951) 

thought that the disease was probably carriecl by honey, and therefore, 

one should avoid feeding honey from infected colonies. 

Maurizio (1934) found the fungus in the intestines of bees from an 

infected colony and stated that the organism winterecl in bees and 

in honey. De Jong and Morse (7926) found Ascosplwern apis in the 

honey sac contents of adult worker bees from infected colonies 

and shorl'ed that spores were passed from bee to bee in food 

exchange. They also repoited that queen bees could transmit the 

disease. The results of Mehr, Menapace, Wilson, and Sackett 

(1976) suggested that the disease mighi be carried by pollen. How_ 

.e' 

P 

r( 

sl 

fe 

br 

'be 

in 

br 

(1 

tir 

tt' 

P': 

br, 

c(l 

be 

dir 

tir 

Ce 

1 

on 

pl; 

exl 

bo: 

th; 

mc 

bee 

car 

tict 

T 

dur 

pla 

apI 

(1e. 

por 

hiv, 

virc 

cha 

A 

hacl

y ar( 

ii.r li,:'n 

, ['hr;ttt 

iind 

.'d to 

iailey 

itlr at 

: r e51) 

:hcre- 

..rnies. 

')lll an 

s aird 

in tlie 

li.rnics 

iood 

rit thc 

r,:kett 

llon,- 

i.i 

I 

( 

I 

F l 

I 

FUNGI 

ever, infections rvere obtainecl by feeding colonies spores with 

pollen only during a pollen dearth (De Jong 1976). Stejskal (1974) 

reported that honey bee larvae could be infected with Arrhenosphaera 

cranei Stejskal rvhen fed nectar containing spores or when 

fed by worker bees that had cleaned infected cornbs. 

Further spread of the disease occurs mostly through drifting of 

bees from infected colonies. However, transfer of spores by the 

beekeeper also occurs when contaminated tools and combs are 

interchanged in infected and healthy colonies (Barthel 1971). 

Several factors seem to contribute to the development of chalkbrood 

disease in bee colonies. As mentioned previorisly, Bailey 

(7967) found that honey bee larvae are most susceptible to infection 

when they are three to four days old and are chilled briefly 

tn'o dayg later immediately after they are sealed in their cells to 

pupate. Thcrcfore, hc notcd that the brood secnrs morc likely to 

be-chillecl in spring rvhen chalkbrood is rnost evident because 

colonies are then gron'ing rapiclly and the ratio of brood to adult 

bees is the largest. He postulatecl that chilling enables oxygen to 

diffuse into the gut and reactivate the myceliurn, which then continues 

to grorv when the brood temperature is restored to 35o 

Celsirrs. 

Seal (1957) found that a colony whicl-r is badly ventilated and 

only partly occupicd during 'ivinter provided an excellent restirrg 

place. for spores of Ascosphaerc apis. Then rvhen tl're brood nest 

expanded in spring or early sulnmer and covered the combs harboring 

the spores, the larvae coulci becorne infected. FIe suggested 

that races of bees r'vith a tendcncy to excessive srvarndr-rg are the 

mbst strsceptible to chalkbrood since the departure of srvarming 

bees leavc-s too large a brood charnber for the remaining bees to 

care for properly. Also, inbred lines of honey bees seem to be partictrlarly 

susceptible to infection (Moeller and Williams 1976). 

Dallrnann (1966) found that chalkbrood occurs particularly 

during rainy summers in apiaries that are located in moist cool 

placcs. Gochr-rauer (1963) stated that fungus infections of bees 

appear in colonies lvith excessive hive moisture. Also, Lunder 

(L972) attribr.rted the occurrence of chalkbrood to rvet rveather, 

Lroor for.rging conditions for long pr:riods, rveak bee colollies, open 

hivcs, and genetic factors. He questioned rt'hethe-r the acid environment 

resultirrg from grovving air pollution might promote 

clr alkbrclocl developrnent. 

A possible connection betn'een chalkbrood and heather locations 

h.rcl been suggestecl, but Deans (19-10) stated that this did not 

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PESTS, PREDATORS, AND DISEASES 

appear to be tl're'case in Scotlarrd. Hou'el'er, Dreher (1938) thouS;ht 

the frequent and persistent occurrence of chalkbroocl in l'reathc'r 

regiorrs in Germany lvas due priniarily to the predclminarrt prr.'rctice 

of skep beekeeping errrd the'darnp occarLic climate. 

Barthel (1.971) thought tlrat cl"raikbrood had becomc partially 

epidenric in the German Der.rrocratic Republic becansc' of the high 

relative humiditl. and high temperatures. Cury (1951) believed that 

chalkbrood occurrecl rnairrll, in hot months only in poorly aired 

hives. I{iglily hydratecl horrel' rvas found to create dampness and 

permit dcvekrpment of chalkbrood, and the administration of antibiotics 

in svrup seenls to favor the grou'th of fnngi (Taltarly 1962). 

Chalktrroc,ci also seems to occur in colonics that are first rveakened 

by another disease. Deans (19.10) in Scotlar-rcl .found that ir-r 

aln'rost every serioLls casc of chalkbroocl, there u'ere '"'er)' felv 

foraging bees because of an attack of acarinc disease. Once weakened 

b1' otl-rer causes, the beres did not seem to clean or-rt tl"rc' cells 

to kcep the disc,rse irr cht'ck or to eradicatc it. He strggcsted that 

in scvcre cases of chalkbrood, sorne other circumstance had first 

rveakcned the stock. 

Mattrs and Sarbak (1974) four-rd tl-rat chalkbrood occurrecl in apiaries 

rvhere secollLlar)' faclors such as unsaLisfactorl' beekeeping 

or another disease had occurrc'c1. \{aurizio (1934) found seconclarv 

cases of chalkbrood on coml,s affeciecl u'ith European forrlbrood. 

\{illc (1954) noted that chalkbrr.rod seemecl to occur jointll' rvith 

noscrna disease, bactcrial sr.pticen.rias, and rickettsioses rather than 

alone; ancl a possiblc relatior-rship betu'een sacbrood disease ancl 

chalkblood has bcen srrggested (N4chr, Menapace, \\rilson, arrd 

Sacke'tt 1976; Moellcr and lVilliarns 1976). Also, I)reher (1938) 

observed that the furrgus seerned to grou' first in injurcd brood. 

Ciarrffret and Taliercia (7967) attribr-rtec'l the' sprc.ad of fungal 

discas': of bees to thc use of antibiotics that upset the equilitrrium 

of the intestinal flora of bees aird therefore allorve'd the fungi to 

flourisli. Flumiclity, lack of verrtilatjon, lvatery fcxld, and genetic 

factors \vere suggested as other aids in tl're spread of infection. 

Gilli;rm and Taber (7973) reported chalkbrood in colonies that 

had been fed excessive anrounts of pollern ancl extensivcly n-ranipulated. 

Sorr-re of the.se color-ries also hacl other cliscases such as 

Anrericau foulbrood. Also, Ilitchcock arrtl Christenserr (1972) 

found clrrll

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T 

FUNGI 

rvilcl bees, play a part in spreacling chalkbrbod disease (Bailey 

1963). In England, Ascosphoern n;ris was found in a dead pupa of a 

leafcutter bee (Melville and Dade 1944) and in the cell of a masor-t 

bee. (Clout 1956). In the United States, Baker and Torchio (1968) 

reported that Ascosphaera altis rvas associated rvith the feces of both 

a ieafcutter bee, L4egnchile inernis, and a soil-nesting bee, Antln- 

7tlrcrn pacifica, and also with the cell lining adjacent to the feces of 

Antlrciphoira pttcit'icn. The cells of bcrth bee species contained living 

noninfected prepupae. The fungus rvars also isolated from the feces 

of another soil-ncsting bee, Anthophorn peritomnc Cockerell, in Utah 

(Tcrrclricr 1971) a^d fiom Megachile rottndnta (: Megachile pacit'ica) 

Fabricius (Thomas and Poinar 1973). 

Batra, Baha, and Bohart (1973) also found Ascosplnera apls associated. 

with Atttttophora pncifica, sPecies of Megachile, and the alkali 

bee Noiilia melcnderi Cockerell. They noted the importance of the 

leservoir of inoculum in the nests of lvild bees. some individuals 

of most bee species in an area forage on the same croPs and thus 

permit ut-t ""Cha.tge of inoculum via the flolr'ers. Moreover, they 

iecovered the same fungi from the honey stomachs of several 

species of bees. 

In Denmark, Skou (7972) reported Ascosphaera proliperdn .Skou 

causing chalkbrood in Megachile centunculLris L. Ascosplraera nnitr 

(I'roksihl et Zobl) Skou n'as also found to cause chalkbrood in 

thcse bees, although it seemed to be only a facultative parasite 

(Norgaarcl F{olm and Skou 1972). Thev concluded that the ascospot"s 

of the fungus stick to the bodies of the bees as they pass 

through one cell after another when emergirrg. Therefore, this is 

or-rc mlthod of transfer of the organism from one generation to the 

next. 

Treatn'rent 

No chernotherapeutic agent is registered for usc against chalkbrood 

clisease in the United States (Cochnauer, Furgala, and 

shimanuki 1975). Usually the losses caused by the disease have not 

been consiclered serious enorrgh to justify research on treatment 

(Bailey 1963). Since the adult bees l5enerally carry the dead brood 

out oi the hive, the clisease often disappears rn"'ithout intervention 

from the beekeeper. Dreher (1938) emphasized the imprortancc of 

br-rilring ,r'r.r,-t-tr-t i*s and not allolving them to accumulate' Also, 

Dcarrs (1940) stated that the disease $'as not a probiem in strong 

colonies. 

i)estruction of affectecl combs or sect;.ons of combs in severe 

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PESTS, PREDATOIIS, AND DISEASES 

cases has been recomnrended (Betts 1951, Gochnauer, Furgala, 

and Shimanuki 7975, Kenn'ard i932). Kenrvard (1932) suggested 

rendering good-combs safe, after removal of shrunken larvae, by 

exposurL' to sun rays or by' fumigating rvith sulfur fumes or 40 per 

cent formalir-r. Giauffret, Tostain-Caucat, arrd Taliercio (1969) and 

Thornas and Luce (7972) found that fumigation *"ith ethy.lene oxide 

killed Asc-osTshnertt npis in infected combs. 

Moisture accumulalion and poor ventilation in hit'es shor.rld be 

preventcd since cold and damp rt'eather encourages the development 

of chalkbrood. Zander (7919) em1-rhasized the importance of 

rrvrapping coloiries during ninter and kecping them dry. Seal 

(7957) stated that serious spread of the disease could be prevented 

by' closirrg the l-rives for rvirrti:r ancl keeping the hives clear of long 

grass to prevent darnpness and to alion'ac-lecluate verrtilation. One 

can also enlarge the entrance to a colony to aid vcrrtilatiorr (Gochnauer, 

Furgala, and Shimanuki 1975). 

Seal (1957) recomment-led strengthening b.rdly diseased colonies 

by atlclirrg young adult bees and hatchir-rg broocl and feeding sugar 

synrp. He also stated that chalkbrood could be pre.r'ented by not 

allorving the- bees to rvinter irr too large a brood c}-ranrbcr. 

Lunder (7972) suggestecl the use of resistant qlleens, and Mraz 

(7973) recourmeuded requeening u'itir nonsuscei)iible strains. Recerrth' 

Nelson (1975) nrade crosses hetween Nerv Zealand and 

Califclr-r-ria slocks of bees and for.rncl that the resuiting stock tvas 

lcss affcctcd by chalkbrood than the Califomia stock. 

Tabarly (7962) reportcd that chalkbrood disease dtcreases in 

affecLecl colclnies lvl-rerr l-roney rvith a rvater corrtr-nt above 19 per 

ccnt is replaced u'ith a more concentrated horrer containing less 

tharr 17 per cent r.t'ater. 

Several authors havet condtrcted experiments on tire use of 

chemicals to control chalklrrood. Elbe and \4/eide (19(.1) founcl that 

0.7 per cent thl,rnol prr.vented thc gron'th of Asrrrspllacra apis in 

culture. When the th1'mol solrrtiorr rvas sprayed on infected combs, 

the' disearsc disappearcd. lf c>rvever, it n.as necessctrv to spray evcry 

courb ancl thc inrrer n'alls of the brotld chambcr. The bc.es n.or-rld 

not accept 0.7 per ce-nt thymol in s1'rup. Thet'also reported tl-rat 

cl-rloridcs of potassiunr, calcirrm, and soditrnr and iumts of various 

chcnrical substances shon'ecl no inhikriting effect orr lhe grorvth of 

the fungus. 

Barthel (7971) folrntl that thvniol in a 2 per cent soltrtion l.racl 

a fungi:,t;:tic cffect irr 2tl minutes c.r.r,{scojir/rn,'i',i rtirii in i'itro. IJe 

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L*' '-1.;.r 

FUNGI 89 

stated that stimulation of the cleaning instinct of bees is the principal 

control rneasure. 

Dallmann (7966) tested the disinfectant "Fesia-Form" (formaldehycle 

base) on bee colonies infected rvith chalkbrood. He 

spriyed the solution on brood combs, the inside Parts of the hive, 

aird ihe liight board. Within a week after spraying, the adult bees 

from severely infected colonies had removed the mummies, and 

no reinfection rvas observecl throughout the year. He found that a 

4 per cent solution of "Fesia-Form" killed the fungus. Barthel 

(1971) performed in aitro tests on the activity of fungicides against 

Ascosphae rn npis and reported that 4 Per cent "Fesia-Forn'r" killed 

the spores after 30 minutes. 

Antimycotics and antiseptics have been tested for possible use in 

the treatment of chalkbrood. Giauffret and Taliercio (1967) reported 

that amphotericin B lvas the most effective agent of those 

they tested against AscosTthnera apis. However, it was not stable. 

Acticlione exhibited a high toxicity for bees, and griseofulvin did 

not inhibit the grorvth of Ascosphnern apis. The antiseptics tested 

rvere quite stable but were more toxic for bees than the antimycotics. 

For example, cetyl-trirnethyi amnronium rvas toxic at levels 

of about 0.5 grams per hive. 

Thomas and Luce (1'972) reported that sorbic acid and methyl 

parahydroxybenzoate inhibited Ascos1thnera apis in ctrlture' Later 

T'aber, Sackett, and Mills (7975) fed sorbic acid and sodium propionate 

in pollen-sugar patties to badly infected bee colonies. 

Chalkbroocl disappeared seven days after the treatment started. 

There were no symptorns of toxicity in the beles, and no reduction 

in sealed brood occurred. N4oeller ancl lVilliams ('1976) found that 

feeding infected colorries 250 parts per million benomyl in sugar 

sy'rup reduced infection. 

Author's Opinions 

The origin of chalkbrood disease in the United States is a mystery. 

At least trvo possibilities exist. one view is that chalkbrood 

diseasc has been in this country for many years but lvas considered 

lrnimportant. Arrother vierv is that the fung,us n'as only recentll' 

introducecl . Some of the peoplg rvho believe that the disease has 

bee.n present for years also thirrk that it recently has becorne more 

rvideipreacl and has increased in severity. If this is true, tire fungus 

nrav have mutate'd or strains of bees may have become nlore susi:eptible. 

Tl-re. first possibility seL.ms rnore reason.rble since n'Iir:ro- 

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90 PESTS, PREDATOI{S, AND DISEASES 

organisms mutate readily and multiply quickly. The possibility 

exists that strains of Ascosphtrerrt tlllis ir-r the United states cliffc'r 

from those found in othcr parts of the rvorld. 

The fungus may have been present in solitary or wild bees in this 

conntry for manv years. There is no rvay of knorving this since 

little n'ork has been done on the microflora of these bees. These 

bees ma1' have sen'ed as a reservoir of inoculun for honey bees. 

The fungus may have entered on imported polletr. Also, the 

lvide-spread use of antibiotics to control bee diseases may have 

increase.d the incidence of chalkbrood clisease by upsetting the 

normal intestinal microflota of bees. 

Chalkbrood may be a st.css-related disease of ho.ey bees since 

abnormal, starved, a^d co.fi.et1 bees see.r nlore susceptible. 

Also, some bee colonies are more adept at re'roving the diieasecl 

lar'ae (sec Nelso', Barker, Bl.rnd, Corner, Soeh.g;en, ancl Ville_ 

ncuve 1976, Gilliam, T;rber, a.d Itose unpublished observations). 

These corrjectures den'ronstrate that thcrr- ale rnany interesting 

avenues of research on ch.rlkbror.d clisease that neecl [o be investigated. 

Particularly, the taxo'omy and infe.ctivity of straii.rs of 

Ascos7tl711gv11 apis and the susceptibilitr, of strains of honey' bees 

under various management proceclures ncecl tc be examineci. 

Bettsia aluei, the Pollen Mold 

Bettsin nii'ri (tsetis) skou, formerll' Ascosplncra slut:i and, periaptis 

sh,ci, is a fungus that is saprophvtic on pollen stored by honey 

bees i. cells of the conrb. 'fherefore, it is commonly referied to as 

pollen mold. lt does not attack brc-roc-l (Betts 1932). 

Pollcn that is mold1, x'ith l)eftsirr ali,ei mat, be n'ristaken for clralkbrclclcl. 

Ho\r'eVer, it breaks up easilt, into fragments representing 

the origirral pollen loads (Betts 1951). 

Although Ascosphst:ro cPls and Bettsiit oluei are sinrilar, the latter 

has a lorr,' opti'ru'r ter.perature for spore germination and gro*'th, 

and therefore usualll' occurs in earlv spri.g after a *'ei'rvinter 

(Bailey 1963). Chaiktrrood gerrerall' occurs later. Also, the fruiting 

bodies of Bcttsitt aluei are small compared ri.ith those of Ascosythae ra 

npis, and the. spores rvithin are single. 

Ilistorr. 

Be'tts (1912.r) first de.scribed the Frollen mold anel namecl iL perialstis 

ali'ci. Shc obsen'ed that sonte combs on n'hich the fungus 

\vas present contained celis that r\-ere no longer rvhite but had be- 

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FUNGI 91 

collle gra)'l due to the formation of cysts' She also gave a detailed 

,llo.ptiotogical description of the organism 

In 1919, Betts coniiarecl Bcflsin n/t'r'i n'ith the chalkbroocl fungus 

ancl noted that the two organisms, although similar' were.distinct' 

Bettsia ali,ei clid not gro!v ft brood nest temPelature' and the. cysts 

contained simple spherical transparent spores' In contrast" the 

chalkbroocl fungus gretv u'ell at brood. rrest temPeratule' and the 

.iiut, .o,-rtui.eaipnJrical bodies each of rvhich cont'rined a number 

(1919) and Ctaussen (1921) confirmed 

.i";;';;","r.'Z".acr 

Betts's observations. 

Burnside (1927) clescribed a pollen fungus and named it Oaulnria 

lni,rnrrutu' Betts callecl his attention to the similarity between this 

'it^g.,, and Belfsin a/uel' Subsequently' Burnside (11:a; compared 

.r.rf,".r.", received frorrr Matlrizio in dn'itzerland and Betts in En- 

;;;:i 

with isolates frot.u the Unitecl States and agreed that Ouularis 

Tnritraecoln was indeed Rettsia nlt'ei' Therefore' he withdrew the 

'r''on," hc g,avc to thc fungus in1927' 

- Mur.irio (7929) pointJd out that as early as .1919 Morgenthaler 

inSrr,itzerlandhaclsuspectedthatBcff.sinnlt-,eiwasheterotlrallic, 

and she confirmecl that the cysts arise only when- the two opposite 

mating t1'pes corne together' In 1934' she found that both Ascospiirrruiriii 

and Betfsia aluei exist in two forms based on the dif- 

ference ^- in the size of the cvsts' 

Spiftoi, (1955) established that Ascrlsplnert npis and Ascos1thaera 

t-luei were ascolnycetes' Spiltoir and Olive (1955) reclassificd the 

genus Pericqstis onJgut'" beriqlstis aluei .the name Ascoslthaern alaei 

without examining f"ertile culiures of the organism' Then Skou 

(tgZZ) scparated Arcorpl"c'o alaei .from the genus Ascosplnera 

and founde.l u ,,"* j"'ius Bcrfsia n'ith the genus typeBet.tsia aluei' 

H" utro reported thal Betrsia aluei grel poorly or not at all on ordinary 

media but grerv rvell and' produced spore cysts on a m.edium 

containing honey, y'east extract' and pollen' The organism-did not 

start growt}t ot rJonl tenrperature but grevn' fairly well at 18" 

Ceisius. 

Distribution 

Bcttsitt n/uri has been found on pollen in hives of honey bees 

in Great Britain (Betts 1912a, 1g7ib, 1979' 1'932' 1951)' switzerlancl 

(Mauti zJo7()29), the Unitecl States-(I3urnride1'977 '19,3^4;Batra' 

Batra, and Rohart tdril, France (Giauffret arrcl Taliercio 1967), and 

!)elrmark (Skou 7972)' I)ettsin rtluei was also identified as the cause 

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of a m1'cosis in a pup.r of au unitlentified 

Iand (l'honras and poinar 1923). 

Etiology 

ir.ild bee from Nerv Zea_ 

Bcttsin alttci is colntnon in hives during \\,in{cr ilhcl earlrr 

and sro'vs on polten stored i" .r*tr'i;?,,r'ibir'ii,'',;#1rtffI: 

renro\red from the hive (Skou 7972). Th,e pollen ,;;;*; ;; rvhich 

it develops are rende'ed us.iess (iiuun.et o,-,a ruri"'..io isozl. 

The organism does not gro*'in ce,s that are fi'ect rvith pollen 

arrd finished rvith a lay,er of hor,*y on top before they are sealed 

r'ith n'ax' Flowerrer, it gro*'s rver'in ."r;'";;;;;"0 ,rlt'in"i""^", .. 

where tire honey on top l.ras been remor,r.d (Skou 1922). 

since the spores clo-not die duri.g the heat of ,.,r_r.,rr.,"r, the1, 

are taken to the .e*'hive b;' sr'a.mi.,! bees and g";i.,u," * i,ni., 

one to five days at 1.5"r. 1b" celsius i2u,la", rgi'gr. e.."iAi,lg to 

Giauffret and Taiierci o (1967), B et tsi a,riaci aevelc,p. ;,; ;;i;; *.r" 

readill'at a te'peratr-rre belo*, 30'Celsius ancl is f.uncl particr,rlarll, 

in tlre coki season in rvr,akenea trives. 

The cor.rtents of the cells att;icked b1. the fungus dr1, into hartl 

plugs that often split i.t. layers. rr,"r" p;.€, :;; p"i_""i". oy 

myceliurn. Combs,on rvhich the fungus î, i."r".i,..,o1,-l;n,o,,_, 

cells in which the fungus is gray o*if; to the formatiori of c.sts. 

Treatn-rcnt 

Bettsitr cruei is undoubtcdry a norm.-rr inhabitant .f trre hearthy 

bee hive (Betts 1912b). ,Evidentiy p;ir;; morcr has n,t rreen con- 

sidcred a scrir)us problem, n"i ,n"r"i,rre tir control 

tlcerrrcd 

measLlrcs 

* n'''".*.r., are 

11'. Hor'e'er, n"tir s;r ) rectrur'rcnrrec., soa k- 

ini; molcly co.rrbs in l'atc.r for 24 ho,r* ancr the'proppirrg the,r. 

ui) and s;'ringing tht,rn to rr..rsh otrt the nrold; I)ollcn. Tfie crllnLrs 

shourcl trren bc alrorvc.r to clrl' ona, ir.desirccl, s1'rirrgctr agairr. 

Aftenvards the cells containing ver;, 1lu.0 Iunrpls of ;rlrllL_11 m'st be 

ilil."."t leaving the micirib in which the Lrees .1,.' rr,.riJ-.,.,or" 

Za.der (7919) reported that *.ith trre arrir..rr o{ r,u,arnrer lvc;rt}rer, 

thc spoilcd nrnrr", "f 1-ffl.'.,.- "frr:..,:., rcnrt,r.etl fr.onr t6c hi'r,. 

I I(r\\'0\,('r., this clc,rr' 

r, a,. a p r u g r i t ; "i;: ;l is.,',:.lii ; i nl;:: ::in:,; ll:.,ft :ffi :,j., l; 

the poilen. Therc.fore,.the,b_ecs gnorr,A; ccll> io renrove thc pollcn 

masses from the c()urbs. He r.,'c,'rnrnre.cJt l k,,"pi;;'.",,r,..r.1r' 0.', 

a.d. r.r'raplrir.rg tirem cluri.c *,intt_r to aioicl the gror'th ,rf B.l;.;; 

rili't'i or.r l'rolierr. 

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Stonebrood 

FUNGI 

Stonebrood disease of honey bees is rare and is considered to be 

of minor importance to beekeepers. Both brood and adult bees are 

susceptible io attack by several species of fungi belonging to the 

g".,.ri As:rtergilltLs. As'tergillus flnurs Link causes stonebrood most 

irecluently, ittnougtr Asltergilltt: funignt us Fresenius is occasionally 

encounterecl. 

Infection of larvae is more often recognized than infection in 

aclult bees, and infecied lan'ae and pupae are transforrned into 

hard stonelike mummies after death. The abdomens of infected 

adult bees also become n-rumrnified. 

History 

Stonel'rood lvas first elescrilred by Maassen in 1906 in Germany' 

He isolatecl and identifi ed Aspergillus flnous from the dead bees and 

noted that affected larvae and pupae had been transformed into 

hard leatl'rery mummies. 

Bahr (1916) in Denmark reported finding stonebrood on rare 

occasions. Betts (1919) speculated that the disease might have been 

imported to Britain from continental Europe. Zander (1919) described 

stonebrood as appearing suddenly and disappearing again' 

\n 7923, Vincerrs reported stonebrood in France. N{orgenthaler 

(7927) clcscribed stonelrrood caused by Aspergilltrs tttgcr Van 

Tieghem. 

Tournanoff (1928) cor-icluded that mortality in some cases resultecl 

from toxic products elaborated by the fungus in the bee's 

ir-rtestine and liberited bv the digestive juices of the gut. Burnside 

(i930) founcl that the actitrn of the pathogenic funS;i on bees is both 

phi,sical ancl chcmical in t-rature" Tissues are pe'netrated by the 

inrlcelium and are digested by enzymes produced by the fungus' 

Burrrside clemonstrated that a transient toxic substance in a strain 

of Aspergillus flauus n'as the cause of fatal poisoning ir-r bee-s. In 

aclclition, he found that Aspe13illus fltn,us, Asryrgillus orqzae (fthlt''trrg) 

Cohn , Asytcrgilltts eflustts Tiraboschi (: Asl'ergillus otyzne 

IAhiburgl Cohn viriet,t effusus [Tiraboschi] Ohara), As1tergillus 

;,ariisiiicr,s S1'reare, Aspcrgilltrs (: flnwLs-ttryztte AspcrSiIIus oryznc?), 

rls;rg1'qi/111-e fi ntigt tus, AsTte rgilltrs nitltilttr ts (Eidan) Winter, Aspe rgil- 

Irts ocltrnceus Wilhelm, atrcl tnemL.'ers of the grotrpr Aspergilltrs 

il/rnri.ls Lirrk killecl bees n-hen inoculatcd experimentalll'. Aspcrgil' 

'ltts 

flnttus attacks bt-es more frequentll' than other Aspergilli, but 

rlslrcr.qil/rrq ftnrigntus u'as highly pathogcnic. Also, lspcr;gi/lrrs 

. ,ri I :': 

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rtidulcuts, Aspcrgilrus rtigtr, members of the Asytcrgiilus grnucus 

group, and ,4spcrgillus oclirscctrs attack bees in natr_rrc_.' 

Dreher (1953) obse^'ecr several spontaneous outbreaks of stonebroocl 

in.G-ermany...He, stated that a spontaneous rL.covery \.\,as 

impossible because the bc-es c.urcl not remove tl,re mummies fron.r 

the cells since thcy rvere connectecl to the cell rvall by nrvcelia. 

He obsen'ed no toxic effect of AsTtergilllle flnt,us and.lo.r.i,rJJ,n tnut 

an epidemic clutbreak clf sto.ebrood probabr), ()ccurs rvhcn a 

highli'virulcnt strain of the fungus att.rcks coronies that have a 

lon'rt'sist;nce. 

Bailey (196s) stated there is no experirnental e'iclence tu shor' 

that Aspergillus flnuu5 and Aslteryilltts t'unignttLs a." p.irmry p"rf-,n_ 

gens even though trrese organisrr,s occasionally multiply'ii be,es. 

Mcl-eila. (1964) fotrnd an outbreak of st''ebruoJ '1ir1o ,gittu, 

flauus) i. scotlanr-l that 'r,r'as the first rcportccr occ''.ence of the 

disease in thc Ur-ritecl Kingdorn in 13 1.ears. 

- Mitroiu, Popa, Serba., ancl To*a jt966) a.d Giauffret ancl Ta_ 

liercir (1967) tested ar-rti.rycotic crren-ricars agai.st the causative 

agents of stoncbrood. Hoivc'er, these chc'riials ru"." .,o, iested 

against the fungal cliseascs itt zriuo. 

Distribution 

Stoncbrood has been re.portecl fronr Europe (N{aassen 1906, 

Bahr 1916, Zancler 1919, virric'rts'J.923, Touman.ff 792g, Dadc 1949, 

Bailey 1963, 

P:-"1:"11953, Mcleltan 7964, Giauffret a.ct Taliercio 

1967), North America (Bur'sicre 1930, steirrhaus 19-19, Bailel. igall, 

and Verrczuela (Stejskal 1959). 

Etiol()E), 

Aspcrgillus 

flartus anrl occasionallrt Asll{rtilllts t'urrtigntus and otl.rer 

of AspergitltL-s i^fect 

,speci11. ana kllr both larval u,-,,r u.l,rlt ho.rev 

Lrees. 'l'hcse fungi are ubiqr-rit.us a.d are commoni,r, fon.cl ir", .# 

They are. pathogenic for other insect_s ancl can cause resp.,i-irr1, 

dise.ases in man and other ar.rimals. 

Lar'ae in sealed arrd unsealcd celrs may.be affcciecl as,*.elr as 

pupae. - Capped pupae are less strsceptitle (Curv 1951). Most 

infected lar'ae die iri the searecl stage L.efore puptrti'', a.cr adult 

sunlmcr bee's secnr pa.ticularly susceptiblc-, altiroirgir they ,'n1,-,1;g 

at any age' (Drche r 1953). 

Ilecs are attacked 'vherr ilrc. fu'g;rr sp(rres are ir.,gestecr (Bur.side 

1930). After the i;pclr.es ger.niinate r"ithin the alintc.',nta.r, .,n,,oi,-rtl* 

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,L*' r:-1r 

FUNGI 

resulting m1'celia may attack all the softer tissttes' Spores can' 

ho*'eve-r, glrminate on the cuticle, and the mycelia rvill then 

penetrate the tissues. 

As the fungus invacles the tissues, the body of the larva and the 

abclorrrenoftheaclultbeebecomeharclened'Ininfectedlarvae,tlre 

fungus clevelops rapidly, passing,through the cuticle and fornring 

a cira.acteristic wltitish-1'ellorv tittg ot: collar behind the head' 

Withirr one to three days, it envelopes the rt'hcle larva as a false 

,t ir't. fn" fungus then produc"s 'pot"s on the external part of the 

cleucl larvn, ind the color changes to green' This grorvth is 

f "* a"ty. AsTt e r gilltts fl nu t r s has u-y"lloot- gtee n color' antl .Asp.e rgil - 

iu,s ftttri.gatur'1, g.uy-g,een' The tiisease, tittt"t a murnmification of 

brood, arrcl the *u*"*i". are solid and hard' At times' the fungal 

;p;;"; are formecl in such large numbers that they completclv fill 

the comb celis that coutain n'rummified larvae' 

Adtrltbeesusuallyallowanybroodkilledbystonebroodtorenrain 

itr combs fo. some tirne, or they only partially renlove it since 

destruction of the cell rvalls is often necessarll for complete removal 

(Br.rrnside 1930). Dreher (1953) found that the mumtnies rverc not 

ioor" in the cells but *,ere atiached to the cell *'all by aerial mycelia' 

Thc first noticeable s)'mPtoms of stonebrood in adult becs are 

abnormal restlessness, i""bi"t'"tt, ancl paralysis' The abdomen is 

generally dilated. Spores form-earlies.t and most abundantly near 

in* n"ui. Ihe abdomen of dead adult bees frequently shows mumrnification 

similar to the entire body of the larvae' It does not 

deca1., and the intcrior often becomei hard as a result of the fungal 

g.c,r.ith. I'he fungus forrns spores on clead adult bees' especially 

i,n the transition parts of the thorax and abdornen' 

Usually the coiony is not seriousll' affected since only a small 

percentage of adult t"., ot larvac is infected' Hon'ever' death of 

i',atrrrullyinfected colorries has bcetr observed (Dreher 1953)' Cases 

are knorvn u'here scarcely any brood clisease occurred' yet adult 

becs clied in large numbers (Betts 1919)' 

Bailey (1963) iuggested that because of the raritv of the disease 

und tt-r" fact that tir"e causatir.e fungi are comnlon, the sporcs ma1' 

c.ruse ciiseast' only lvherr they are prescnt in subnormal larvae or 

prcpupae. If muih of the bioocl dies, then the coiony may die 

bcc:rust' it rvill be rveakenecl and the remirining brood and agirrg 

aclults may Lre susceptible to aitack by the fungus' 

l.he methocl of naiural spread of the disease is not knolvn. Betts 

(1919) stated that stoncbroocl *as undoubtedly disserninated by 

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PESTS, PREDATORS, AND DISEASES 

beekeepe'rs bv the interchange of combs fro'r diseased to healthy 

colonies. she als. believed that hone;' fr.m i.fectecl conrbs fed to 

bees u'ould cause the disease. 

ciauffret and raliercio (7967) stated trrat the spread of fungar 

diseases was probably related to the use of antibioiics rvhich upset 

the equilibrium of the .rormal intestinal flora of bees. They also 

listecl l-rumidity, lack of ve.tilation, food or supplies containing 

too much rvater, and genetic factors as possibre iiat i., the development 

of fungal diseases. Also, Maassen (1906) suggcsted that 

it rvas probable that conrb cells fillect ivith polren ari"the places 

n'here the' fungus first de'elops; he noted that this had noi been 

proven. cur1. (1951) believed that lan'ae ancl adult bees are attacked 

more freque-ntly *'hen the humiditf is high and that stonebro'd 

is transmitted by.pollen- or hone1, iontairiing fungal spores 

lflf:f are ingested.with the food. Grigortsovskaia u,.,i go.odui 

(7972) fed spores of Aspergiiltrs funtigatis and Aspcrginus ttiger in 

sugar syrup to bees of 'arious ages. Aftcr three io flur clayi, the 

bees became hairless a.cl unable to fl1,. The yourrge, beJs died 

fi rst. 

Attempts to infect colonies artificiailv have been unsuccessful 

(Bailey 1963). Therefore, stressecl or abnor.ral bees may Lre rnore 

susceptible, and tc'rxins (Toumarroff 192g, Burnsider 1930j, perhaps 

a-flatoxins, produced by the fungi might be responsiblc'for the 

disease. B,'rside (1930) co'clucrecl that the pathogerricity oi the 

fungi that attack bees,thr'up;h the gut ivall ippeais t' be cletcrmi'ed 

by' the ability of the spores and m'celia to resist the actio' 

of the intestinal fluids. 

Trt'atrne nt 

fhcrc is 'o krror'r'n treatnrcnt ftrr sttrêhrood (Bailcy. r963, ciauffret 

and Taliercio 1967). Hol'ever, in se'ere cur"r, Betts (1919, 

1951) recornmcnded burning trre bees, combs, a.cl alr contents of 

the hi','e and then disinfecting the hi'e. To sa'u,e a mircily diseasecr 

c'lony, she suggested shaki.g the. bees onto ne*, equipnre't, clis_ 

infecti'g tlie old hi'e, ancl burning all cor-.bs. white^ doirrg this 

rvork, one slrould protect the c1'es, m.trth, and nose- *'ith a ,ii"ta 

and a v'et ha.dkerchief to re'tluce thc risk of i.fecti.n. Betts enrphasized 

tl'r.at the honev from c.lonies infectecl rvith sto.ebrood is 

rrot s.rfe for htrnran consunrption since As,tt ygi!!1,, /rri,rr-s has been 

knorvn to gr

healthl' 

rs fed to 

f fungal 

:h upset 

rey also 

:rtaining 

,: devell:ed 

that 

.: places 

rot been 

r are atrt 

stone- 

.i spores 

Borodai 

rrrger in 

.r1's, the 

:es died 

: ccc'ssf ul 

're-. more 

ucrhaprs 

for the 

of the 

rl'eter- 

' .-rction 

j, Giarifi:. 

(1919, 

', ,.'nts of 

, .eased 

. rt, clis- 

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r-'* 

FUNGI 

only the brood was affected rvere treated by brushing the bees 

into artificial swarm boxes and feeding the bees in these boxes for 

two days in a cool darkened room. Hives and accessories were 

sterilized and the frames provided with foundation. The bees rvere 

then returned to the old hives and regularly fed urrtil comb was 

built. He noted no relapse. 

Giauffret, Tostain-Caucat, and Taliercio (7969) found that fumigating 

combs with ethylene oxide for 1,5 hours at 22o Celsius killed 

Aspergillus flaaus. Also, Giauffret and Taliercio (1967) studied the 

possibility of treating fungal diseases with antimycotics and antiseptics. 

They determined the in aitro sensitivity of trvo strains of 

Aspergillus flaaus to antimycotics and compared the toxicity of these 

products for adult bees and brood. They found that nystatin ancl 

thiabendazole rvere most effective against Aspergillus flauus. Actidione 

was the most toxic test substance to adult bees and larvae. 

Horvever, they noted that studies on the activity of these antimycotics 

against fungal diseases should be done in aiao.lr{itroiu, 

Popa, Serban, and Toma (1966) found that stamycin, a mycostatic 

agerrt, inhibited development of cultures of Aspergillus niger and 

Aspergillus funigatus. 

Gochnauer, Furgala, and Shimanuki (1975) stated that no treatment 

for stonebrood is required since bees remove diseased brood, 

ancl the colony appears to recover spontaneously. Ho'wever, 

Zander (1919) found that rvhen bees attempted to remove larvae 

affected by stonebrood, they gnawed au'ay the celi walls and 

reached the mummies only nith difficulty. Therefore, they lvere 

onll' partly successful in their attempts. Those larvae that could 

not be removed were covered with propolis so that only the heads 

remained visible. 

Maassen (1906) observed that bees were not very suscePtibie to 

the disease. The susceptibility increased if the hive temperature 

rvas high, if the hive was poorly ventilated, or if the bees rvere 

corrfined for a long time. 

Other Diseases 

Mel.rnosis 

F,vg (193a) discovered in sterile queell bees a disease that affected 

tl-re reproductive sy'stem. The disease n'as designated H-mel;rnosis 

(frorn the German "Hefe" mearriug yeast) to distinguish it from 

another melanosis (B-rnelanosis) caused by a bacteriurn. The s1'str'matic 

position of the yeast-like microorganism c.rusinS; H-melarrosis 

is still undetermined.

PISTS, PREDATORS, AND DISEASES 

According to Fyg (1961\, the organism probabll' errters the re.productive 

organs through the sting chamber and vaginal orifice. It 

produces in the oviducts and ovaries a melanosis or black coloration. 

J'he poison sac ancl poison.gland of the queen are also affected 

and contain large black srvellings. These sn'ellings exert 

pressure on the oviduct, and egg laving ceases. Since infected 

queens become sterile, Ctrry. (1951) recomnrended that tl-rey be 

replaced, althougl'r such queens are superseded by a nerv queen 

(Bailcy'1963). 

Healthy r-luc.ens can tre infected bv vagirral inoculations in the 

laboratory (Fyg,796q, and adult rvorkers and drones have been 

infected n,ith cultures injected into the thorax (Fy'g 1936). However, 

direct feeding of the pathogen to be.es has uot produce.d 

infection. Unfortunatelv, nothing is kno'rvn about ihe incidence of 

the disease (Bailey 7968). 

The pathogen Frroduces lvhite., smooth, sl'riny colc,rnies on beerlvort 

agar at 30' Celsius. These colonies never darken even in old 

cultures (F1'g 196a). Fvg (i964) contends tl-rat this organism is not 

idcntical rrith the org;.rnism Mclunscllrt nrors npis described by 

Orosi-P6l (1936, 1,939). 

Orcjsi-Pal (1936) found a livir-rg matecl queen that had ceascrl to 

Iav eggs. The genital orifice was plugged rvith a brown substance 

in n'hich the sting n'as embeddecl. This "anal rvacl" rvas lodged 

soliclll'betn'een the last tr.vo abdominal segments aird the stirrger. 

He cultivated spores from the 'rvad and or.aries of this queerr a'cl 

also fourrd tl-re organisnr in hind guts of infected worker bee.s. T'he 

n'rost strikir-rg sign of the disease in rvorker bees was an evagination 

of the hiridgut. He gave the name of Melnnosella nrttrs npis to the 

pathogenic agent and n'as able to cause mortality in worker bees 

within 24 hours after feeding them pr-rre culturts of the organism. 

Corrtarski (7937138) found a fungiform organism betrveen the 

ovarioles of a queen suffering from ovarian atrophy. I{orvever, Fyg 

(1964) examined 224 cases of ovarian atroplrl' of queens ancl clid 

not encounter this condition. 

Polte' and Neshatave'a (1969) stated that the agent Meranoseiln 

urLlrs opis corresponds morpl-rologicallv and culturally to AurettltnsidiLtrtt 

pullultris (Del3aly) Arnaud \: Pullulnrin Ttullularrs [DeBary] 

Llerkl-rout). They' injccted 'r'orker bees in the thorax and abclome.n 

rr'ith Arrrcirbttsitihrm Ttullulans fronr cultule collectiorrs and from 

qucens nitir nrelanosis. Infection began in the regions injected; 

and no pathological differences \\'ere observed i. strains from 

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rubstauce 

; lodged 

r stinget'. 

,r-en and 

.,tes. The 

,rgirration 

rris to thc 

rker bees 

rrr;.ruism. 

u'ceu the 

errcr, Fyg 

s ;rnd did 

.l tlnnttsclln 

Atrrtobttsi- 

IDeBary] 

lLrdomen 

arrd fronr 

,, injected, 

,rirrs from 

i4 

i: 

a 

Ix1 3 

t j 

t 

:, 

t! 

! 

b 

a; 

FUNGI 

queens and culture collections. They postulated that the melanosis 

organism is introduced into the hive lvith honeyder,r' honey. 

Gontarski (1950) found n-relanized hypopharyngeal glands in 

about 15 per cent of the pollen-collecting bees'from a colony. He 

thought that queens became infected when they rvere fed the 

hypopharyngeal gland secretion by diseased rvorkers. Nectar-collecting 

bees from the same colony rvere not infected. Therefore, 

pollen \vas suggested as the source of infection. 

Other Molds 

Fungi are commonly found as saprophytes on and inside bees 

ancl on brood combs. Most of the fungi collected by the rvidespread 

foraging of bees are probably unable to become established 

within the bee or the beehive (Burnside 1927). Horever, after the 

death of the bee, sorne of the fungi germinate and mummify the 

softer tissues inside the exoskeleton. Othcrs do not germinate 

n'ithin the bee either before or after death. 

Betts (1912b), Rurnside (7927), and Maurizio (1931) described 

fungi associated with hives. Combs that have been used for broocl 

rearing and for the storage of honey and pollen are susceptible 

to overgrowtl'r by fungi if sufficient moisture and proper temperatures 

for the growth of the fungi are present (Burnside 7927). 

Damage is generally limited to poorly ventilated hives, rveak colonies, 

and cornbs in unoccupied hives. Through the proper regulation 

of humidity within the hive, the bees are able to protect 

thc-mse-lves and their stores from attack by fungi. 

Br.rrnside (7927) noted that the Penicillia are the most common 

group within the beehive. He frequently fourrd entire combs, and 

at times after the death of the bees, all the combs of a hive overgrorvn 

with these organisms. Badly infected combs are not readily 

accepted by the bees. He added that actual parasitism of bees has 

rrrrt been established for any species of Penicillitnrr. T'he Aspergilli 

occur far less abundantly in the hive, and species of Mucor do 

lroi grotr, u'ell on brood combs. 

Ficlitz (1925) reported that Triclndernn lignortrrr (Tocle) FIarz arrd 

,\lrrror ntucctict Brefelcl found on mummified bees $'ere pathogcnic 

to brood ancl adult bees n'hen intrclduced into healthv colonies on 

L,rtrtrrl combs. Nicholls (1,934) and Chorvdhury (1953) also found 

tlr.rt liiclrodtntta lignorurn causecl disease in bees. In Tasn-rania, 

ilrt' furrgrrs appeared tcl be associated lvith No,serrrr apis Zander 

(\iclrolls i934). Becs fed Trichod(rml lignonlnr spores in sug;rr

lOO PESTS, PREDATORS, AND DISEASES 

syrup died (Grigortsovskaya and Boroclai 7972). Myceliunr was 

found in the epithelium of the miclgut and in muscles, and the 

gut contained many fungal spores. 

Mucor hiennlis Wehmer rvas reported to be virulent for young 

adrrlt bees rvhen tl'rey \vere exposed to a temperature of 20' Celsius 

(Burnside 1935). Hon'ever, the normal temperature of the brood 

nest is abor.'e the tolerance of lvhrcor liennlis. Moreove'r, when bees 

are old enough to leave the hive, they are no longer susceptible. 

Therefore, this disease is of little importance. 

Queen larl,ae in sealed cells have beerr reported to be attacked 

by AsTtergillus niger (Burnside 1939, Prest, Gilliam, Taber, and 

Mills 1974). In the latter paper, the fungus r,r'as also isolated from 

discolored rvorker and drone larvae. 

In f)enrnark, a case of a disease similar to stonebrood rvas described 

and attributed to a fungus of the €ienus Clauiceps (Cor,r,an 

188i/82). Drone t.'rood n'as attacked first, then rvorker brood, and 

finalll'adult bees. 

Cury (1951) described a disease of iarvae arrd adult bees caused 

by Rhizopus equinus Costatin ar-rd Lucet rvhich was obtained via 

the pollen of florvers contaminated rvith horse manure. In addition, 

he stated that in cold countries Sco|rttlcrioltsis brqticnulis (Saccardo) 

Bainier caused a disease called yellorv brood and black 

brood. The affectcd larvae died and turned yellow or black accorclirrg 

to the age of the fungus and strongly adhered to the walls 

of the ce.il. The disease does not affect protected broocl and generalll' 

disappears rvhen the temperature riscs. 

Yeasts 

Yeasts have been fourrd in nectar (tsetts 1920, Lochhead and 

Heron 1.929, Batra, Batra, and Bohart 7973), honey (Falrian and 

Quinet 1928, l.ochhead and Farrell 1931, l.ochheacl and lvlclr{aster 

1931, Tvsset and Rautiin De l-a Ror, 1974), bee bread or stored 

pollerr (lVilson and Man'irr 1929, Pain ancl Maugerrct7966, Egorova 

arrd Bab'eva 7967, Egnrova 1971), hives (Betts 7928a,1928b,1928c, 

1929), apiarv soils (Lochhe.rd ancl Farrell1930), and bees (see Batra, 

Batra, and Bohart 1973 arrcl Gilliam, \{ickerham, Morton, and 

N4artin 1974). 

Yeasts are not considercd to be pathogL'l1s of honey trees, although 

soltlc osmophilic species ciiuse fermentation of honey. 

How'ever, Giordani (1952) isol.-rted a veast belonging to thc genus 

Torulopsis from the digestir,e tract of bce.s suffe'ri.g from a tiisease 

of unkrrorvrr nature. \{hen this yeast n'.rs fe'd to healthl.bee colo- 

..*i*-. L\F@.! 

t- 

ni 

dr 

(B 

m 

19 

ad 

ln 

Pa 

8r( 

cla 

ber 

COI 

val 

bet 

res 

larr 

ASS 

tior 

\ 

col< 

m)/l 

Ton 

197, 


Cc'l S( 


(Gil 

bee: 

be ir 

A, 

It 

L-ibr, 

ihc r

:lium n'as 

i, and the 

ior t'oung 

0" Celsius 

the brood 

then bees 

r:;ci-'ptible' 

c attacked 

aLrer, and 

lated from 

tl rvas ders 

(Con'alr 

,rood, and 

ees causcd 

'tained via 

:. In addi- 

.nrr/is (Sacr 

ricl black 

r nCCordihe 

u'alls 

'rud gener- 

irhead and 

:.rL.rian and 

lr{c}vlaster 

i or stored 

,6, Lgorova 

2,Sb, 1928c, 

; (see Batra, 

itr tr-rn, and 

v bees, aloi 

honey. 

r the gerrus 

l .r disease 

iv bee colo- 

iJi$slu-.- 

FUNGI 

nies, it developed in the digestive tract of the bees and caused 

death. Injection of some species of yeasts into bees causes death 

(Burnside 1930, Batra, Batra, and Bohart 1923). Also, some yeasts 

rnay ferme.rt the provisions of honey bees (Betts 1920, Moreaux 

1949, vecchi 1959) and cause sickness or death of the larvae or 

adult bees that ingest these provisions. 

. In fact, yeasts may be beneficial to honey bees by playing a role 

in the production and preservatio. bee bread (Ego'roia 7977, 

Pain and Maugenet 1966) and by supplying 'f vitamins and other 

gron'th factors (Egorova 1977,Batra, Batra, and Bohart 1923). Gior_ 

dani (1957) fed caged bees s,ccharorrrr/ccs species, Torula species, or 

bee-collected pollen in sugar syrup. From these experiments, she 

concluded that the yeasts had approximately the same nutritional 

value for young bees as pollen. Moreover, the Torula species gave 

better results than pollen. Hajsig (19s9) cites the r"r,-,ir, of oiher 

researchers to point out that yeasts have a favorable effect on the 

larval development of bees. Much nlore research is needed to 

assess the effect of yeasts (and other microorganisnrs) on the nutrition 

and physiology of honey bees. 

Yeasts are more readily isolated from adult honey bees from 

colonies that have been (1) treated with antibiotics such as terram1'cin 

(Toma5ec 1957, Hajsig 1959, Mitroiu, popa, Serban, ancl 

Toma 1966, Gilliarn 1973, Gilliam, Wickerham, Morton, ancl Marti. 

7974), (2) treated rvith herbicides (Gillian-r, Wickerham, Morton, 

and Martin 1974), (3) caged (Gilliam 1973), (4) suffering from dis_ 

cases not caused.by yeasts (Betts 1928c, Lavie 1951, Kamburov 

and l{ajsig 1963, Gilliam 7973), or (5) receiving cleficient diets 

(Giliiam 7973). since ferv yeasts are founcl in the intestines of aclult 

b.es from healthy free-flyi.g colonies, tl-reir presence in bees n-ray 

tre indicative of stress conditions (Gilliam lgfu). 

Acknon.lcdgmcnt 

I thturk Alfred D. straughan of the usDA National A5;ricultural 

Librarl', Beltsville, Mar14and, for translatinp; into Engtish man1, of 

the citcd references. 

i.lt 

,d 

i "rl 

'_.4:1.1,: + 

!L: ,: 

n."!-ri 

.l'.1'.; 

iq'rt 

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.:,,i e 

r:a .t 

:. ': :; 

::,-:. a ':t 

,, rt l* 

::f 

..-*,* 

i *,; 

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.* I 

.,:j, .l 

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'--:t?.q 

.;j:. 

a" 

#.

Apidologie, 1978, 9 (3),213-222. 

;rJlfi [i /, 

iili Ti,Jti?,,11,,T' 

BACTERIA BELONGING TG THE GENUS BACILZUS ISOLATED 

FROM HONEY BEES, APIS MELLII7ERA, FED 2,4-D AND 

ANTTBTOTTCS (l) 

Martha GILLIAM 

U. S. Department of Agriculture, Science and Education Administration, Bee Research Laboratory 

2000 E. Allen Road, Tucson, Arizona 85719 


Howard L. MORTON 

U. S. Department of Agriculture, Science and Education Administration, 

Rangelands Weed and Brush Control 

2000 E. A llen Road, Tucson, Arizona 8 57 I9 

SUMMARY 

The guts of388 adult worker honey bees.,4pls melli,fera, lrom caged control colonies. from colonies 

fed (2.4 dichlorophenoxy) acetic acid (2,4 D), and from colonies fed a combination oloxytetracvcline and 

fumagillin were examined over a period of l3 months lor organisms olthe genus Bacillus. One hundred 

and ten organisms belonging to 13 species were identified. Bacillus megaterium, B. subti!is, and B. pumilus 

were the most frequently isolated organisms and were found in bees in all three treatment 

groups. The antibiotics and 2.4-D reduced the number of bee guts containing Eacll11s. No Bacll1ts organisms 

were isolated during the hot summer months of June Septembcr. Thus. weather may also influence 

the composition of the gut microflora. 

INTRODUCTION 

For several years, we have been examining the intestinal microflora ol honey 

bees, Apis melli,fera, with the ultimate goal ol determining the role of microorganisms 

, in the nutrition and physiology of these insects. One aspect ol this investigation has 

concerned the effects of antibiotics used to ccntrol bee diseases and of pesticides on 

the gut microflora of bees. Thus, we isolated and identified enteric bacteria 

i (Gtt-uau and MonroN, 1974), molCs (GlllrAM et al., 1974 a), and yeasts (Grr-r-rAM et 

(l) Mentionofaproprietaryproductorcompan)'namedoesnotconstituteanendorsementbvtheU.S.Depart 

ment of Agriculture.

214 M. GILLIAM AND H. L. MORTON 

al,,1974 b) from the guts ofadult worker bees from control colonies, from colonies fed 

a combination of the antibiotics oxytetracycline (TM-25) and fumagillin (Fumidil B), 

and from colonies fed the herbicide (2,4-dichlorophenoxy) acetic acid (2,4-D). From 

this work, we concluded that the antibiotics depressed the growth of 

Enterobacteriaceae, molds, and yeasts and that 2,4-D caused a proliferation of 

intestinal yeasts but had little effect on the molds or Enterobacteriaceae. 

Recently we reviewed the literature concerning bacteria of the genus Bacillus that 

have been isolated from honey bees and reported the isolation of 14 species from 

the guts of foraging worker bees (Gllrtau and VaI-ENuNr, 1976). Since information 

about the incidence of these sporeforming rods in honey bees is scant and since we 

wished to assess the effects of 2,4-D and the antibiotics on these organisms, we report 

here the results of examination of the guts of adult worker bees from control colonies, 

from colonies fed 2,4-D, and from colonies fed a combination of oxytetracycline and 

fumagillin. 

MATERIAL AND METHODS 

The procedures for establishing, caging, maintaining, and feeding bee colonies were described in 

detail by Grlrrenr and MonroN (1974). Two tests were conducted to obtain data throughout the 

year. In the first test, each of the three hives receiving a different treatment (control, herbidide-fed, and 

antibiotic-fed) was placed in a separate 12x l2x 9-ft Saran mesh cage. In the second test, nine hives 

were used, and the three colonies receiving the same treatment were placed in the same cage. Thus, in 

the two tests, bees were examined from a total of 12 colonies : four control colonies. four colonies fed 2.4 

D, and four colonies fed the antibiotics. 

Briefly, the procedure was to feed all colonies l-lb pollen patties (maintenance diet), which were replenished 

weekly. Colonies receiving the herbicide were fed the dimethylamine salt of 2,4-D at a concentration 

of I 000 ppm active ingredient by weight in 60 o/o sucrose-water solution from a jar placed directly 

above the frames containing brood (MonroN and Morprrr, 1972). The mixture of herbicide 

and syrup was replenished twice a week. Colonies fed antibiotics were given the maintenance diet plus 

0.5 g TM-25 and 0.5 g Fumidil B in addition @ 60o/o sucrose-water solution. Control colonies received 

the maintenance diet and 60 o/o sucrose-water solution. Fresh water was available to all colonies. 

In the hrst test, three adult worker bees lrom each colony were examined weekly. The intestinal 

tracts (esophagus to rectum) were aseptically removed and individually homogenized in 2.5 ml of sterile 

0.85 o/o NaCl as previously described (Gtr-ru.u and PnEsr, 1972). A loopful of the homogenate 

from each bee was streaked in duplicate on trypticase soy agar (BBL) and nutrient agar (Difco) in petri 

dishes. All plates were incubated under aerobic conditions at 37 oC for 14 days. 

In the second test, every three weeks the intestines oftwo bees from each ofthe nine colonies were individually 

homogenized in 2.5 ml of sterile thioglycollate l3 5 C medium (BBL). Each homogenate was 

streaked in duplicate on trypticase soy agar. nutrient agar. and eugonagar (BBL). One plate was incubated 

at 25 oC and one at 37 oC under aerobic conditions lor 14 days. 

All resulting bacterial colonies were stained by the Gram method and, il necessary. were restreaked 

on plates of the same medium used for initial isolation to obtain pure cultures. Gram-stained slides of 

the cultures were examined for spores. The size. shape. and location of the spores within the sporangia 

and the morphology of the vegetative cells were noted. Bacteria belonging to the genus Bacillus were 

maintained on slants of nutrient agar. They were then tested and identified according to GonooN et 

al. (1973).

BACILLUS IN BEES 2t5 

In the first test, bees were sampled from July l97l to January 1972. In the second test, they were 

sampled from September 1971 to August 1972 though all the herbicide-fed colonies had died by April 

19'12, a control colony died inMay 1972, and an antibiotic-fed colony died in June 1972. We attribute 

the death of the herbicide-fed colonies to the ovicidal and larvicidal effects of 2,4-D (MonroN and 

Morrrrr, 1972). 

RESULTS AND DISCUSSION 

One hundred and twelve Bqcillus organisms were isolated, and 110 belonging to 

l3 species were identified from the 388 bees that we examined (Table l). We were 

unable to identify two isolates, one resembling B. alvei and the other resembling B. 

sphaericus. Since we were interested in determining the number of bee guts containing 

Bacillus organisms but not the total number of Bacillus cells per bee gut, more 

isolates were identified than are shown in the tables to estimate adequately the number 

of species present. B acillus megaterium, B . subtilis, and B . pumihls were found in the 

bee guts most frequently. These organisms were isolated from bees in all three treatment 

groups. 

Organism 

Bacillus megaterium 

B. subtilis 

B. pumilus 

B, licheniformis 

B. circulans 

B. alt,ei 

B. coagulans 

B. brevis 

B. cereus 

B. sphaericus 

B. Jirmus 

B. laterosporus 

B. polymyxa 

Unidentified 

u388 bee guts examined 

TasL. l. - Bacillus Isolated From Control Bees, Bees Fed 2,4-D, 

and Bees Fed Oxytetracycline and Fumagillin" 

Number of bee guts 

containing the organism 

Table 2 gives the results of isolations in the first test. Bacillus licheniformis and 

B , subtilis were found most frequently, in 10 and eight bee guts, respectively. Filteen 

of the isolates (in l4 bees) were found in control bees, nine were found in bees fed2,4- 

D and only three were found in bees fed antibiotics. Thus, in this test, the antibiotics 

greatly reduced the number of bee guts containing Bacillus. ln fact, the three guts 

z1 

2l 

21 

l0 

l0 

8 

3 

3 

) 

2 

1 

I 

I 

2

216 M. GILLIAM AND H. L. MORTON 

from bees fed antibiotics that contained the organisms were collected the same 

day. No guts of bees fed antibi

Organism 

Bacillus subtilis 

B. subtilis 

B. coagulans 

B. coagulans 

B. pumilus 

B. coagulans 

B. subtilis 

B. subtilis 

B. subtilis 

B. megaterium 

Unidentified 

B. Jirmus 

B. megaterium 

B. megaterium 

B. rnegaterium 

B. pumilus 


B. brevis 

B. circulans 

B. megaterium 

B. sphaericus 

B. subtilis 

B. circulans 

B. subtilis 

B. tnegaterium 


B. pumilus 

B. subtilis 

B. tneg\terium 

B. tnegalerium 


B. pumilus 

B. pumilus 

B. subtilis 

B. circulans 

B. ah,ei 

B. circulans 

B. circulans 

B. brevis 

B. circulans 

B. subtilis 

B. alvei 

BACILLUS IN BEES 

TasL. 3. - Bacillus Isolated From Honey Bees - Second Test 

) rrom 

) same 

I u.. 

1 from 

\ same 

I u.. 

1 from 

) same 

) u.. 

) lrom 

) same 

\ u.. 

I i'u.n 

/ same 

) u.. 

) r,o' 

) same 

) u.. 

) to. 

) same 

) u.. 

A 

H 

H 

H 

H 

A 

c 

C 

A 

A 

c 

C 

H 

H 

H 

H 

H 

A 

A 

A 

c 

C 

c 

c 

C 

C 

H 

A 

C 

C 

C 

H 

tOl19/'11 

t0/19/7 | 

t0/t9 7 | 

tt/ 9/7t 

tt/ 9/1\ 

t1/ 9/'7 | 

t1/ 9/11 

111 9/7 | 

ltl 9/7t 

tt/ e/71 

tt/ e/71 

1t/30/7 1 

tt/3017 t 

tt/3017 t 

t2/2t/7 t 

t2/2t/7 t 

tl t t/72 

1l tt/72 

1/ tt/72 

t/ tt/72 

2/ t/72 

2/ t/72 

2/ t172 

2/ t172 

2/ t/72 

2/ t/72 

2l t/72 

2/ t/72 

2/ tl72 

2/ r/72 

2123/72 

) /)1/1'' 

2/23172 

2/23172 

) /)7/1) 

)/)2,/1) 

2l23l72 

1/)1/1') 

3l t4/72 

3lt4172 

3l t4/72 

3l t4/72 



I 

2 

I 

I 

2 

I 

I 

I 

I 

I 

I 

I 

I 

I 

2 

I 

I 

I 

2 

I 

I 

I 

I 

2 

I 

I 

I 

I 

I 

-l 

2t'7

2t8 M. GILLIAM AND H. L. MORTON 

Organism 

B. laterosporus 

Unidentified 


B. subtilis 

B. putnilus 

B. trtegateriwn 

B. pwnilus 

B. circulans 

B, circulans 

B. subtilis 

B, cereus 

B. pwnilus 

B. subtilis 

B. ntegaterium 

B. pumilus 

B. pumilus 

B. pwnilus 

B. alyei 

B. alvei 

B. megaterium 

B. ntegateriurn 

B. purnilus 

B. purnilus 


I t'u' 

/ same 

) u.. 

) 

'i 

I 

from 

same 

bee 

from 

same 

bee 

from 

same 

bee 

lrom 

same 

bee 

from 

same 

bee 

aC - control; H : 2,4-DtA : antibiotics 

I 

Treatmenta 

Date 

isolated 

3/ 14/72 

3l 14172 

3l 14172 

3l 14172 

3l t4,12 

3l t4112 

4l 4112 

4l 4172 

4l 4112 

4l 4112 

4t 4112 

4l 4112 

4t 4172 

41 4112 

4l2sl12 

4125t12 

5l t6112 

sl t6172 

5l t6112 

5l t6l12 

sl t6t12 

5l t6112 

5l t6112 

5l t6llZ 

sl t6112 



second test but were not isolated in the hrst test. All isolations of B. alvei and all but 

two of B. pumilus were made after the first test was terminated. Perhaps, if the first 

test had extended beyond January 1972, these organisms would have been isolated. 

However, Bacillus licheniformis was isolated lrom October l97l through December 

1971. Therefore, it is surprising that it was not found in bees in the second 

test. 

Of special interest is the fact that no Bacillus organisms were isolated lrom bee 

guts during the hot summer months of June, July, August, and September. Tysssr 

and DURAND (1968) examined the midgut and hindgut of adult worker bees in 

France from October to March and reported that 29 o/o of the microorganisms isolated 

I 

I 

2 

6 

2 

I

BACILLUS IN BEES 2r9 

were Gram positive. Similarly, Froonove and GusevA, (1964) concluded that 

intestines of adult worker bees always contain spore formers in winter. In contrast, 

EL-LsnHy and Er-SIsnu (1912) examined microorganisms from the surfaces, crops, 

and intestines of adult worker bees in Egypt during the flowering of citrus (March to 

May) and of cotton (June to August) and concluded that aerobic sporeforming bacilli 

were the most frequently encountered organisms representing 61-83 7o of the 

flora. We also examined foraging worker bees from free-flying colonies in December 

(GIrrIeu and VaLENIINE, 1916) and found Bacillus organisms in all bee 

guts. Therefore, climate, weather, and vegetation-may influence the composition of 

the gut microflora. 

Tysssr and DunaNo (1968) reported that the low population of spore formers 

in the intestinal contents of bees indicated that these bacilli are there in transit 

since sporogenous bacteria are present in soils and are found only accidentally and in 

limited numbers on the floral organs ol plants. Thus they postulated that bees do not 

have much chance of being heavily contaminated. Moreover, they thought that the 

high osmotic pressure and relatively low pH of the intestinal content of bees would 

also limit the population of bacilli. El-LErrHv and Er--Srnlct (1972), in contrast, 

postulated that the predominance of spore-forming bacilli on the surface of bees as 

well as in the crop and gut may indicate that bacteria normally present on flowers continuously 

enter the alimentary canal. They reported that conditions in the gut were 

favorable for these bacteria and that the source of food influences the composition of 

the microflora of the gut. 

The bees that we used in our tests were obtained from caged colonies and thus 

were not foraging on flowers. Therefore, only soil, wind, and food, and water could 

have served as sources of inocula. In fact, as shown in Table-4, more bee guts contained 

organisms belonging to the gents Bacillas than contained Enterobacteriaceae 

(Grr-rnvr and MonroN 1974) or molds (Grlrrl.u et al., 1974 a). Only the yeasts that 

appear to be indicative of stressed bees (Grr-rtAv' et al., 1974 b) were found in more bee 

guts from colonies treated with 2,4-D and untreated colonies. 

T,q.nL. 4. - Number of Bee Guts Containing Bacteria, Yeasts, and Molds 

Microorganism 

Bacillus spp. 

Enterobacteriaceae 

Yeasts 

Molds 

aC : control; H : 2,4-Dt A = antibiotics 

Treatment groupu 

c H A 

44 

l3 

20 

25 

l5 

95 

15 

)5 

t2 

t2 

8

224 M, GILLIAM AND H. L. MORTON 

Almost allthe Bacillzs organisms isolated in the present study have been previously 

isolated from honey bees (see Grluelr and Va.r-rNrrNr, 1976). The exception was 

B. sphaericus which is a new record of an organism associated with honey 

bees. Bacillus megaterium was found most frequently in bee guts in the present study 

but was not isolated from foragers (GIllIalt and Ver.eNrrNn, 1976); Bacillus polymyxa 

was isolated most frequently from foraging bees. 

Thus, differences exist in the intestinal microflora of honey bees from caged colonies 

and from free-flying colonies. Also, feeding 2,4-D or a combination ol oxytetra, 

cycline and fumagillin to bees causes shifts in the microflora. 

ACKNOWLEDGEMENTS 

Received in March 1978. 

We thank Mr. Richard MARTIN. Mr. Randall JETER. and Mrs. Dinorah DUNHAM lor their ex 

cellent technical assistance. 

ZUSAMMENFASSUNG 

ZUM GENUS BACILLUS GEHIiRENDE BAKTERIEN. DIE AUS MIT 2.4 T) 

uND ANTIBlorlra cErUrttnrEN BIENEN. Aprs vELLtFER.4. ISoLtERT wuRDEN 

Der Darminhalt von 388 erwachsenen Honigbienenarbeiterinnen wurde auf sporenbildende Bakterien 

des Genus Bocl1lls untersucht. Die Bienen wurden vier Kontrollvolkern entnommen, die Pollenteig 

und Zuckerlijsung erhielten, von vier weiteren Volkern, die Pollenteig und in Zuckerlosung I 000 ppm des 

Herbizids (2,4-Dichlorphenoxy) - essigsbure (- 2,4-D), und noch von vier weiteren Volkern, die TM-25 

(Oxytetrazyklin) enthaltenden Pollenteig und Fumidil B (Fumagillin) in Zuckerlosung erhielten. Alle Volker 

wurden in Kifigen gehalten. Fiir die Untersuchungen wurden das ganze Jahr hindurch Bienen entnommen. 

Der Darmtrakt (Speisercihre bis Enddarm) wurde homogenisiert und auf geeigneten mikrobiologischen 

Medien ausgebreitet, tm Bacillus zu isolieren. Es wurden 110 zum Genus Bociilrls gehrirende Bakterien 

aus den untersuchten Bienen identifiziert. Bacillus megaterium, B. subtilis wd B. pumilus wurden 

am hduhgsten bei allen drei Versuchsgruppen isoliert. Das Herbizid und die Antibiotika verminderten die 

Anzahl der Bienendhrme. die Bacillus enthielten. und wdhrend der heissen Sommermonate von Juni- 

September wurden keine Bacillus-Organismen isoliert. Ein Vergleich dieser Ergebnisse mit unserer lriiher 

veroffentlichten Arbeit iiber die Mikroflora des Darms bei Sammelbienen zeigt. dass Unterschiede bestehen 

in der Darmmikroflora von Honigbienen aus gekdfigten und aus frei fliegenden Vtjlkern. Eine Fiitterung 

der Bienenvrilker mit 2,4-D oder Kombination von Oxytetrazyklin und Fumagillin verursacht Verdnderungen 

in der Darmmikroflora der erwachsenen Arbeiterinnen. Zusetzlich mcigen Klima, Wetter 

und Vegetation die Zusammensetzung der Mikroflora im Darm beeinflussen. 

RESUM€ 

BACTERIES DU GENRE BACILLUS ISOLEES A PARTIR D'ABEILLES, 

APIS MELLIFER,4, APRES ADMINISTRATION DE 2,4 D ET D'ANTItsIOTIQUES 

On a examin6 le contenu intestinal de 388 ouvriires adultes du point de vue des bacteries appartenant 

au genre Bacillus. Les abeilles provenaient de 4 colonies t6moins qui recevaient de la pite de pollen et du

BACILLUS IN BEES 

sirop; de 4 autres colonies qui recevaient de la pdte de pollen et du sirop renfermant 1 000 ppm de l'herbicide 

acide 2,4-dichlorophenoxyac6tique Q,a-D); et de 4 autres colonies qui recevaient de Ia pAte de pollen 

renfermant du TM-25 (oxyt6tracycline) et du sirop additionn6 de Fumidil B (fumagilline). Toutes les colonies 

etaient maintenues en cages. 

Les abeilles 6taient pr6levees pour analyses tout au long de l'annee. Le tractus intestinal (de l'asophage 

au rectum) 6tait homog6n6is6 et 6tal6 sur un milieu microbiologique appropri6 pour isoler Bacillus. 

A partir des abeilles examin6es on a pu identifier 1 I I bact€ries du genre Bacillus. Dans Ies 3 groupes 8ocillus 

megaterium, B, subtilis et B. pumilus furent les plus frequents. L'herbicide et les antibiotiques ont reduit 

le nombre d'intestins d'abeilles renfermant Bacillus et aucun Bacillus n'a pu €tre isol6 durant les mois 

chauds de juin a septembre. Si I'on compare ces r6sultats avec notre travail prec6demment publi6 sur ia 

microflore intestinale des abeilles butineuses. on s'apergoit que des differences existent entre la microflore 

intestinale des abeilles encag6es et celle des abeilles volant librement. L'administration de 2.4-D ou d'une 

association d'oxyt6tracycline et de lumagilline provoque des changements dans la microflore intestinale 

des ouvridres adultes. En outre le climat, ie temps et la v6g6tation peuvent influencer sa composition. 

REFERENCES 

Er--Lrrrny. M. A., Er-Srg.q.Br, K. B., 1972. - External and internal microflora of the honey bees (Apis 

mellifera L.). EUpt. J. Microbiol., 7, 79-87. 

Froonova, G. N., Gusrvl, N. V., 1964. - [Spore-forming microorganisms of the intestines of adult 

honey bees.i. Leningrad. Vet. (nst.,26,94 100. 

Grrrreu, M., MonroN, H. L.. 1974. - Enterobacteriaceae isolated from honey bees, ,4pls mellifera, 

treated with 2,4-D and antibiotics. J. Invertebr. Pathol., 23, 42-45. 

Grruru, M., Pnesr, D.8., 1972. - Fungi isolated from the intestinal contents of foraging worker honey 

bees. ,4pls mellifera. J. Invertebr. Pathol.,20, 101-103. 

Grllrllr, M., VerrNrrNr D. K., 1976. - Bacteria isolated from the intestinal contents of foraging worker 

honey bees, Apis mellifera.'the genus Bacillus. J. Invertebr. Pathol.,28,275-216. 

Grrrreu, M., Pnssr, D. B., MonroN , H. L, 1974 a. - Fungi isolated lrom honey bees, Apis mellifera, fed 

2.4 D and antibiotics. J. Invertebr. Pathol., 24, 213-217. 

Gnulu, M.. WrcrBnHelr, L. J., Monror.r. H. L., MenrrN, R. D., 1974 b. - Yeasts isolated from honey 

bees, ,4pis mellifera, fed 2,4-D and antibiotics. J. Invertebr. Pathol.,24,349-356. 

GonooN, R. E., HevNrs, W. C.. Hon-Nlv PnNc, C.. 19i3. - The genus Bacillus. USDA Hand-book, 

427, t-283. 

MonroN, H. L., Morrnrr, J. O.. 1972. - Ovicidal and larvicidal effects of certain herbicides on honey 

bees. Environ. Entomol., l. 611-614. 

TBssrr, C., Dun.qNn, C., 1968. - Contribution a l'6tude du microbisme intestinal des abeilles butineuses 

saines (1pis mellifica L.) : d6nombrement et 6tude des groupements constitutifs (premier m6moire). 

Bull. Apicole, I l, 107- 118. 

22r

Apidolo gie, 197 9, l0 (l), 43-53. 

MICROBIOLOGY OF POLLEN AND BEE BREAD : 

THE YEASTS 


U.S. Departmettt of Agriculture 

Science and Education Adminislratiott 

Bee Researeh Laboratory 

2000 East Allen Road 

Tucson, Arizona 85719 

SUMMARY 

PI.IRCHASED BY ME UITITPD STNI S 

DEPIRTTETTT OF TGRICULTURE FOR 

0frrclAr us€ 

One-hundred and thirteen yeasts belonging to seven genera were isolated from almond (Prunus 

communis) pollen from the flower, from pollen pellets from traps placed onbee (Apis mellifera) hives in 

the orchard, and from pollen stored in comb cells of the hive (bee bread) for one, three, and six weeks. 

Torulopsis magnoliae was the most common isolate and was found in all pollen samples except pollen 

from the flower. Thus, the bees may have added it to the pollen. The number of isolates and species 

decreased with time and storage. In general, most of the yeast species from pollen from the flower and 

the trap were not found in bee bread. AIso, yeast isolates from pollen from the flower and the trap 

fermented more sugars and assimilated more carbon compounds than those from bee bread. 

INTRODUCTION 

The nutritional requirements of honey bees, Apis mellifera, are met by the 

collection of pollen, nectar, and water. Nectar is the primary source of 

carbohydrates; pollen provides proteins, lipids, vitamins, and minerals. Foraging 

worker bees collect pollen that is then packed into cells of the brood comb by other, 

generally younger, bees, and a small cover of honey is deposited on the pollen to 

prevent spoilage. This store of pollen, which has undergone chemical changes, is 

called bee bread. Bee bread is consumed by adult bees and is fed to larvae. 

Pollen and bee bread,differ biochemically. For example, bee bread contains 

more reducing sugars than pollen from the same plant species (Cesrerl, 19 l2). Also, 

bee bread contains vitamin K (Hevolx and VIvINo, 1950) and a milk-digesting

44 

M. GILLIAM 

enzyme (HIrcHcocr, 1956); pollen collected from the legs of foraging bees does 

not. AvprrsLAN (1935) found that bee bread made from birch pollen contained six 

times as much lactic acid as hand-collected birch pollen. 

The conversion of pollen to bee bread and the accompanying biochemical changes 

have often been postulated to be the result of microbial action, principally a lactic acid 

fermentation caused by bacteria and yeasts (Foorn, 195i; Heyoar, 

1958)' CnEvrcnrn (1950) conducted microbiological analyses of fresh pollen and 

pollen stored in comb cells and reported four phases ol microbial development in 

fermenting pollen that occurred during seven days from the time of the appearance of 

lactic acid bacteria, yeasts, indole-producing bacteria (Escherichia), and sporulating 

aerobic bacteria. The first phase lasted 12 hours and was characterized by the 

development of a heterogenous group of microorganisms including yeasts. In the 

second phase, anaerobic lactic acid bacteria (Streptococcus) utilized growth factors 

produced by the yeasts and putrefactive bacteria and lowered the pH ol the 

pollen. The third phase was characterized by the disappearance of Streptococcus and, 

the development of Lactobacillus that produce more acid than Streptococcus. The 

fourth phase, which began at the end of the sevenin aay, was characterized by the 

disappearance of the lactic acid bacteria and certain yeasts due to the large quantity of 

lactic acid produced. The pollen became microbially sterile, and the pH was 

approximately 4. During the course of this study, CnevrcHrr isolated from lresh and 

fermented pollen 77 groups ol yeasts that he considered to be sources of proteins, 

lipids, and necessary growth factors. 

Pen and MAUGENET (1966) reported that three microbial genera (Lactobacillus, 

Pseudomonas, and Saccharomyces) were important in the modification of pollen during 

storage. Lactobacillus caused a lactic acid fermentation that stabilized the pollen by 

increasing the acidity, but the roles of Pseudomonas and, Saccharom).ces were not well 

understood. They thought that pseudomonads probably contributed to the 

anaerobiosis required by Lactobacillus and to the degradation of the walls ol pollen 

grains since they developed rapidly as soon as the pollen was packed by bees into comb 

cells but disappeared almost totally after two to three days. Then lactic acid was 

produced by Lactobacillus. This lactic acid fermentation was completed by the end ol 

about l5 days though the responsible organisms maintained a stationary population lor 

several months. The yeasts, which were present in small numbers initially, increased 

after fermentation and subsisted in stored pollen longer than other organisms. These 

researchers seeded pollen sterilized by gamma irradiation with Lactobacillus and, 

concluded that a pure lactic acid fermentation produced an unappetizing product of 

poor nutritive value for bees. Thus, they thought that the yeasts played the most 

important role from the nutritional standpoint. It has also been rcported that the 

enzyme systems involved in the lermentation of pollen are similar to yeast enzyme 

systems (Oruuuru, 1943).

YEASTS IN POLLEN AND BEE BREAD 

Econova (1971) isolated a bacterium that she named Lactobacteriurn pollirtis 

from bee bread samples collected in the spring and summer. This organism occurred 

either in pure culture or in mixed cultures with yeasts. In a mixed culture with the 

yeasts the bacterium survived for up to six months without subculturing. However 

pure culture Lactobacterium pollinis had to be subcultured every 30 days. Thus, it 

would seem that the yeasts were either using the lactic acid produced by the bacterium 

or were providing necessary growth factors. 

Table I summarizes the available literature on yeasts isolated from pollen and bee 

bread. Yeasts belonging to l2 genera have been identified. Other researchers noted 

yeasts in pollen and bee bread, but the organisms were not identified (BErrs, 1928; 

Lruos and MecH.loo, 1975; WnsoN and MARvIN, 1929). 

Since relatively little is known about the microbiology of pollen and bee bread and 

since the yeasts seem to be important in the elaboration of bce brcad. I examined pollen 

and bee bread for these organisms. In this paper, bec brcad is defined as poilen 

packed by bees in comb cells of the hive. 

MATERIALS AND METHODS 

Fifteen colonies of bees were placed in an almond, Prunus communis, orchard in February 1976 in 

the vicinity ol Davis, California; almond pollen was collected hourly from pollen traps placed on these 

hives. The pollen was frozen in liquid nitrogen within 15-30 minutes after collection and was stored in a 

freezer until used. On the basis of uniform color and size of the pellets, the pollen collected was at least 

99.8 o,/o almond pollen. 

Also. four colonies of honey bees were established in September 1976 on new equipment and were 

maintained in a polyethylene green house. Each hive contained nine frames, one with sealed and unsealed 

brood and the others containing only newly drawn combs. At lour hour intervals two almond pollen pel 

lets were placed into each cell on both sides of three newly drawn combs from each hive. During the 

four-hour intervals, the bees packed the pollen into the cells. After two three days. most ofthe comb cells 

were at least 112-314 filled with packed pollen. The bees had access only to water and the almond 

pollen. Then three combs from each colony containing the packed pollen were placed in a clean room 

maintained al34 oC and 55-60 % R.H. to s\imulate storage in the hive and to avoid possible overgrowth of 

the pollen by molds. 

The following samples of almond pollen were collected for analyses : fresh pollen collected by hand 

from the flower in March 1977; pollen pellets removed from the bees' legs by traps placed on hives in the 

almond orchard; and bee bread stored in comb cells for one, three. and six weeks. Each bee bread sample 

was a composite collection from all the combs that contained pollen pellets. 

Each pollen or bee bread sample was divided into four sub-samples of approximately 0.75 g 

each. Then each of the lour sub-samples was homogenized by hand in 2.5 ml of sterile 0.85 o/o NaCl in a 

glass tissue grinder. The homogenates were plated (0.1 ml) in duplicate on acidified yeast extract-malt 

extract agar containing I o/o glucose, pH 3.7-3.8 (YMA) (MInBn et al., 1976). One plate from each subsample 

was incubated at 26oC and one al 37oC under aerobic conditions. When yeast colonies 

appeared, they were checked for purity and transferred to slants ol YMA and yeast extract-malt extract 

agar with l7o glucose (YM-l) (WrcrnnHerr,r, 195 1). Morphological and physiological tests were 

conducted according to WIcKERHAM (1951), and isolates were identified according to BARNETT and 

PANKHURST (1974) and Looorn (1970). 

45

46 

M. GILLIAM 

Tenl. 1. - Yeasts previously isolated from pollen and bee bread 

Yeast 

Candida paraosilosis 

Candida reukaufii 

(: M e t s c h ni ko w i a r eukaufi) 

Candida tenuis 

Cry ptoc o ccu s neofor mans 

Debaryomyces disporus 

(-- Pichia dispora) 

Endomycopsis bultonii 

Hansenula anomala 

Met s c hni ko wia reukaufi 

Rhodotorulaflava 

(: Cryptococcus flavus) 

Rhodotorula glutinis 

Rhodotorula glutinis var. glutinis 

R h o d o t or ul a muc i la gi no s a 

(: Rhodotorula rubra) 

Sac c haro my ce s cerevisiae 

S ac charo my ce s hetero genicus 

Saccharomyces mellis 

(: Saccharomyces bisporus var. mellis 

Saccharomvces rosei 

Saccharomyces rouxii 

Sac c haro my co de s ludwigii 

Sp oro bo Io my c e s albo-rubscens 

Torula chartaruzr (identity not known) 

Torulopsis candida 

Torulopsis etchellsii 

Torulopsis famata 

(: Torulopsis cdndida) 

Torulopsis globosa 

(: Citeromyces matritensis) 

Torulop si s inc onspicua 

Torulopsis magnoliae 

Torulopsis sake 

(: Candida sake) 

Torulopsis stellata 

Zy gosac c haro my c e s b ailii 

(- Saccharomyces bailii var. bailii) 

Bee bread 

) 

) 

) 

D 

Pollen carried by 

wild bees 

Bee bread 

Provisions 

Indian corn pollen 

) 

Provisions 


Provisions 

Bee bread 

Stored pollen 

) 

) 

) 

Provisions 


Bee bread 


Bee bread 


Bee bread 

RESULTS 

D 

) 

) 

D 

Reference 

Econovn and Bag'pvn, 1967 

)) 

) 

D 

Knrcan-vnr Rrr, 1970 

Econove and Ben'pv.q, 1967 

Bnrnr er al.,1973 

AnrzeN et al., 1967 

t) 

Bnrna et al.,1973 

AnrzeN et al., 1967 

Brrne et a1.,1973 

Econova and Bls'sv.{, 1967 

PerN and MlucrNrr. 1966 

)t 

Barnr el al.,1973 

D 

AruzaN et al., 1967 

Econov,q, 1971 

AnrznN et al., 1967 

Econovn and Brs'evn. 1967 

) 

AnrznN e/ al.. 1967 

Econovn and Ban'nvl, 1967 

One-hundred and thirteen yeasts were isolated by the sampling procedure used, 

and 110 were identified (Tabl. 2). The three yeasts that I was unable to identify were 

different species. The unidentified isolate from bee bread stored in the comb for three 

weeks was a pink mucoid yeast that did not grow in the medium used for assimilation 

)) 

D 

D 

)) 

D


Tenl.2. - Yeasts isolated from almond pollen and bee bread. 

C/yptococcus albidus var. albidus 

Kloeckera apiculata 

Cryptococcus albidus var. dffiuens 

C andida guilliermondii var. guilliermondii 

Rhodotorula pilimanae 

Torulopsis norvegica 

Candida parapsilosis 

C ry p tococ cus laure ntii v ar. laurentii 

Hansenula anomala vat. anomala 

R ho do t orula gluti ni s v ar. glutini s 

Rhodotorula rubra 

Unidentified 

C an dida gui lli e r mo ndii v ar. gui llier mon dii 


M e t s c hni ko w i a p ulc herri ma 

R ho dot orula glutini s v ar. glutini s 

Rhodotorula pallida 


Cryptococcus flavus 

Cry p lo co ccus laurentii var. magnus 

R ho do torula glutini s v ar. glutini s 


Cryptococcus albidus var. albidus 

Unidentified 


Cryptococcus dlbidus var. dffiuens 

Cryptococcus albidus var. albidus 

Yeasts 

Isolates from pollen from the flower 

Isolates from pollen from the bees' Iegs 

Isolates from bee bread stored in the comb for 1 week 

Isolates from bee bread stored in the comb for 3 weeks 


tests; it produced acid without gas in the fermentation tubes containing glucose, 

galactose, maltose, sucrose, melibiose, and raffinose. Neither of the two unidentified 

isolates from pollen from the flower fermented any of the sugars that were tested, but 

both assimilated a large number of carbon compounds. 

8 

5 

4 

4 

2 

2 

l9 

t0 

I 

I 

I 

2l I 

I 

1 

9 

) 

I 

9 

3 

1 

47

48 M. GILLIAM 

Torulopsis magnoliae was the most common isolate and was found in all pollen 

samples except pollen from the flower. Candida guilliermondii var. guilliermondii 

was the second most common isolate but was found only in pollen from the flower and 

pollen from the trap. Cryptococcus albidus was isolated a total of 18 times from 

pollen from the flower and from bee bread stored for 3 weeks and 6 weeks. Most of 

the other isolates were obtained from pollen from the flower. 

The number of isolates and species decreased with time and storage 

(Tabl. 3). Pollen from the flower contained the most species though this sample and 

pollen from the trap yielded an equal number of isolates. Thus, almond pollen from 

the flower contained a diverse yeast flora that diminished upon collection by bees and 

storage in the hive. Torulopsis magnoliae was the predominant isolate from bee 

bread. 

TasL' 3. - Numbers of yeast species and isolates from almond pollen and bee bread. 

Flower 

Pollen trap 

Comb cells alter 1 week 

Comb cells after 3 weeks 

Comb cells after 6 weeks 

Source of pollen Total species isolated 

Total isolates 

DISCUSSION 

Yeasts belonging to seven genera were isolated from almond pollen and bee 

bread. organisms belonging to Torulopsis, candida, and cryptococcys comprised 

85 o/o of the isolates. Torulopsis magnoliae was the most common isolate representing 

43 o/o of the yeasts. This organism was also the most common yeast isolate from the 

guts of adult worker honey bees in Arizona (Grllrllr et al., 1974 Gu-rtav et al., 

19'17). Since z. magnoliae was not found in pollen from the flower but first appeared 

in trapped pollen and was then found in all bee bread samples, the bees may have added 

it to the pollen. Also, biochemical changes that occurred in trapped pollen and bee 

bread obviously allowed the organism to flourish. 

New records of yeasts from pollen and bee bread are Candida guilliermondii var. 

guilliermondii, Cryptococcus albidus var. albidus, cr. albidus var. dffiuens, cr. 

laurentii var. laurentii, Cr. laurentii var. magnus, Kloeckera apiculata, Metschnikowia 

pulcherrima, Rhodotorula pallida, Rh. pilimanae, and Torulopsis norvegica. of these, 

c. guilliermondii, cr. albidus, Kl. apiculata, and M. pulcherrima have been isolated 

lrom honey bees (see Grr-r-ranr et at., 1974). 

t2 

5 

4 

3 

1 

32 

32 

24 

12 

l3


All the isolates but two (Hansenula anomala var. anomala and M. pulcherrima) 

were asporogenous. Sporulating yeasts are not often found in bees (Econova and 

BAB'EVA, 1967;Glr,rtnl,retal., l974;Gnul.Metal.,L977)or beebread(Econovaand 

BAB'EVA, 1967). Candida guilliermondii contains strains that are incapable of 

mating; mating strains of C. guilliermondii are classified in the heterothallic species 

Pichia guilliermondii. Thus, P. guilliermondii is the perfect form of C. guilliermondii 

var. guilliermondii. Microscopic observations of cultured mixtures of my strains of C. 

guilliermondii var. guilliermondii did not reveal any indication of conjugation or 

sporulation. This is not surprising since most strains of the organism have been found 

to be sexually unreactive (WtcrEnnan and BunroN, 1954). 

Candida guilliermondii var. guilliermondii was the second most common isolate 

but was found only in pollen from the flower and from the pollen trap. Thus, bee 

bread was not a suitable environment for this organism. In general, most of the yeasts 

from pollen from the flower and trap were not found in bee bread, although Cr. albidus 

and Rh. glutinis var. glutinis were found in both. Thus, the physical and chemical 

properties of pollen and bee bread may influence the composition of the yeast flora. 

CnsvrcHlr (1950) observed that most of the yeasts isolated from fresh and 

fermented pollen did not ferment any sugars. However, Econova and BAB'EVA 

(1967) found that the majority of species they isolated fermented those sugars found in 

the bee colony or nectar, specifically glucose, sucrose, and maltose. Some of the 

isolates fermented galactose, but the enzyme splitting lactose was absent in almost all 

the yeasts isolated. They concluded that the yeast flora of the honey bee colony 

consists of species adapted to live in the medium and that the epiphytic yeast species of 

insect-pollinated plants enter the yeast flora of bee intestines and are also found in bee 

bread. Similarly, Wtr-soN and MnRvrN (1929) had also reported that none of the 

yeasts they isolated from pollen and bee bread fermented lactose, although glucose and 

sucrose were fermented. A few of the isolates fermented raffinose. maltose and 

galactose. 

My results on the fermentative properties of yeasts from pollen and bee bread are 

in agreement with those previously reported. None of the isolates fermented 

lactose. Only one yeast, 11. anomala var. anomala, from pollen from the flower, 

fermented maltose. Isolates from pollen from the flower and the trap fermented more 

sugars that those from bee bread. Over half of these isolates fermented glucose and 

sucrose. Some also fermented galactose, melibiose, and raffinose. However, if gas is 

produced in the tube containing raffinose, the yeast may be fermenting the raffinose 

molecule 113,2/3, or completely (vlN oln Wer-r, 1970). Eighty percent of the 

isolates from bee fermented glucose and sucrose; no other sugars tested were 

fermented. Of the total isolates from all sources, 72o/o fermented glucose, and 65 o/o 

fermented sucrose. 

Eighty-three percent of the isolates grew in the osmotic pressure medium. Only 

two isolates, both from the flower, grew at 5 oC, but 66 o/o of the isolates grew at 

49

50 

M. crLLrAM 

37 oC. However, the ability to grow at this higher temperature was most evident in 

yeasts isolated from pollen from the trap and bee bread stored for one week. 

Carbon assimilation tests used in yeast taxonomy are more sensitive than 

fermentation tests for detecting the presence of yeast enzyme systems (wrcrnnnl,lr, 

195 l). These tests employ a chemically defined medium to which a single organic 

compound is added as the sole source of carbon. A standardized suspension of the 

yeast to be tested is then added to the medium. Assimilation reactions test the ability 

or inability of a yeast to use a specific carbon compound oxidatively. I routinely use 

the 38 carbon compounds listed by WrcrsRrreNa (1951). 

Although fewer yeasts were isolated from bee bread (49 minus one that did not 

grow in the assimilation medium) than pollen (64), 50 o/o more of the isolates from 

pollen than from bee bread assimilated maltose, cellibiose, trehalose, melezitose, Dxylose, 

D-arabinose, D-ribose, L-rhamnose, ethyl alcohol, and adonitol. Forty 

percent more of the pollen isolates than bee bread isolates assimilated melibiose, L- 

arabinose, D-glucosamine hydrochloride, alpha-methyl-D-glucoside, pyruvic acid, and 

DLlactic acid. In fact, none of the isolates from bee bread assimilated lactic acid, 

although 42o/o of those from pollen did. Inulin,.soluble starch, dulcitol, calcium 2keto-D-gluconate, 

and citric acid were also assimilated by a larger percentage of 

isolates from pollen than bee bread. Percentages of the other compounds assimilated 

by isolates from pollen and from bee bread differed by less than 25 7o, and most 

differed by lO o/o or less. 

Thus, a larger percentage of yeasts from pollen from the trap and from the flower 

assimilated more carbon compounds and fermented more sugars than did those from 

bee bread. More of these compounds may be available in pollen than bee bread. The 

number of yeast species also decreased in bee bread. Therefore, the biochemical 

changes that have occurred in bee bread may limit the species of yeasts that are able to 

survive. If the yeasts are responsible for biochemical changes in pollen as it converted 

to bee bread, then these changes may start to occur'as soon as the bees touch the pollen 

to pack it in the pollen baskets on the legs. FoorE (1957) speculated that stored 

pollen could be inoculated with microorganisms by the bees as it is mixed with nectar 

or regurgitated honey both while the foraging bee moistened the grains to make a 

suitable mass to carry to the hive and during packing of the pollen into a comb cell by 

other bees in the hive. 

EcoRov.q, (1971) noted that yeasts form vitamins, mainly those of the B group, in 

bee bread and were therefore necessary for honey bees. She also thought that lactic 

acid bacteria obtain vitamins and amino acids from yeasts. Cnlvrcurr (1950) 

examined eight groups of yeasts from pollen and bee bread and found that seven grew 

without vitamins, and one did not. The vitamin deficiency test revealed that 13 % of 

my isolates grew in the absence of vitamins. Thus, they were capable of synthesizing 

all the vitamins required for growth. Most of these yeasts were isolated from pollen 

from the flower and the trap.


Starch was synthesized by l7 o/o of the isolates, although no yeasts from pollen 

from the trap or from bee bread stored for one week possessed this ability. Gelatin 

was liquified by 15 o/o of the isolates indicating that proteases were produced by some 

isolates from all samples. 

Because the pH of the pollen and bee bread samples was 4.2-4.5, a favorable 

environment for the growth of fungi existed. Lavte (1960) reported that pollen 

collected by bees contains an active antibiotic factor that is derived both from the plant 

and from the bee. However, pollens do not contain any anti-fungal substances. Therefore, 

the years and molds are not affected. 

When reporting the results of microbiological analyses of pollen, one should 

always give the pollen species and source. For example, PRIN and MauceNnr (1966) 

emphasized that their results on microflora of pollen and bee bread were probably valid 

only for their region or for a region of similar climate. Thus, my results revealed a 

more diverse yeast flora than reported by other workers. However, the role of yeasts 

in biochemical modifications of pollen and in bee nutrition are not defined (Pan and 

MaucrNnr, 1966). Hopefully the present paper will encourage other researchers 

from other regions to examine the yeast flora of bees, pollen, and bee bread. Only 

after thorough microbiological analyses have been completed can work begin on the 

contribution of specific groups of microorganisms to bee nutrition. 

ACKNOWLEDGMENTS 

Received for publication in August 1978. 

I thank Mrs. Dinorah DL.NHAM for her excellent technical assistance; Dr. L. N. SrlNorrrn, 

Mr. Harold DoN, and Mr. John MIlt-s lor the pollen samples; Dr. L. J. WtcrsnHeN4 for microscopic 

examination of the yeasts; and Mrs. Rachael AtcazB for translating into English the paper by PltN and 

MAUGENET. 


DIE MIKROBIOLOGIE VON POLLEN UND BIENENBROT : DIE HEFEN 

Die folgenden Proben von Mandelpollen (Prunus communis) wurden auf das Vorkommen von Hefen 

untersucht : Handgesammelter Pollen aus den Bhiten; Pollenhdschen, mittels Pollenfallen an den 

Bienenkdsten in der lr{andelkultur unmittelbar von den Beinen der Bienen abgestreilt; und Bienenbrot, in 

den Wabenzellen liir eine, drei und sechs Wochen gelagert. Homogenate von jeder Probe wurden auf 

angesduerten Hefeextrakt-Malzextrak-Agar mil I o/o Glukose ausgebracht. 

Es wurden I 13 verschiedene Hefen isoliert, und I 10 von ihnen wurden identifiziert. Mandelpollen aus 

den Bhiten enthielt eine vielfiiltige Hefenflora, die sich nach der Einsammlung durch die Bienen und 

Einlagerung im Volk verringerte. Organismen aus den Genera Torulopsis, Candida lund Cryptococcus 

stellten 85 o/o der Isolate. Am hdufigsten war Torulopsis magnoliae mit 43 o/o sdmtlicher Hefen. Da diese 

Art im Pollen aus den Bhiten nicht gefunden wurde, sondern erstmals im Pollen aus der Pollenfalle und 

dann in sdmtlichen Proben von Bienenbrot, kdnnte es sein, dass sie von den Bienen in den Pollen gebracht 

worden war. 

51

52 M. GILLIAI\4 

Candida guilliermondii var. guilliermondii war das zweithdLufigste Isolat, aber es wurde nur im Pollen 

aus der Bhite und aus der Falle gefunden. Bienenbrot war also kein geeignetes Milieu ftir diesen 

Organismus. Ganz allgemein kann gesagt werden, dass die meisten Helen aus dem aus Bh.iten oder aus der 

Falle stammenden Pollen im Bienenbrot nicht gefunden wurden. 

Aus Bhiten oder aus der Pollenlalle stammende Pollenhefen fermentierten eine grcissere Anzahl von 

Zuckern und assimilierten mehr Kohlenstoffverbindungen als Hefen aus Bienenbrot. Keine der Hefen 

fermentierte Laktose und nur ein einziges Isolat fermentierte Maltose. Die biochemischen Verdnderungen, 

die im Bienenbrot erfolgen, scheinen also die Zahl der Hefearten, die imstande sind zu iiberleben, zu 

beschriinken. 

RESUME 

MICROBIOLOGIE DU POLLEN ET DU PAIN D'ABEILLES : LES LEVURES 

On a recherch6 les levures dans les 6chantillons suivants de pollen d'amandier (prunus communis): 

pollen r6colt6 i la main sur les fleurs, pelotes de pollen pr6lev6es sur les pattes des abeilles par des trappes 

placees sur les ruches dans le verger d'amandiers, pain d'abeilles stock6 dans les cellules des rayons pendant 

une semaine, trois semaines ou six semaines. Des homog6nats de chacun de ces 6chantillon, oni 6t6 

d6pos6s sur agar d l'extrait de levures acidifi6 et d I'extrait de malt, renfermant 1 % de glucose. 

Cent trente levures ont 6t6 isol6es et cent dix d6termin6es. Le pollen d'amandier pr6leve sur les fleurs 

renfermait une flore diversifi6e de levures, qui diminuait suite ir la r6colte par les abeilles et au stockage 

dans la ruche. l,es organismes appartenant aux genres Torulopsis, Candida et Cryptococcus constituaient 

85 % des isolat's. Torulopsis magnoliae, I'isolat le plus commun, repr6sentait 43 7o des levures. puisqu'on 

ne l'a pas trouv6 dans le pollen pr6lev6 sur les fleurs, mais qu'il est apparu pour la premidre fois dans le pollen 

r6colte par trappes et a 6t6 retrouve ensuite dans tous les 6chantillons de pain d'abeilles, ce sont les 

abeilles qui ont dri I'ajouter au pollen. 

Candida guilliermondii var. guilliermondii vienten seconde position, mais n'a 6t6 trouv6 que dans le pol 

len pr6lev6 sur les fleurs et dans celui r6colt6 par trappes. Le pain d'abeilles est donc un milieu qui ne convient 

pas d cet organisme. En g6n6ral la plupart des ievures pi6r.nt., dans le pollen de fleurs et le pollen de 

trappes n'ont pas 6t6 retrouv6es dans le pain d'abeilles. 

['es levures provenant du pollen de fleurs et du pollen de trappes ont fait fermenter plus de sucres et ont 

assimil6 plus de compos6s carbon6s que celles provenant du paind'abeilles. Aucune des levures n,a fait fermenter 

le lactose et un seul isolat a fait fermenter le maltose. Il se peut donc que les changements biochimiques 

qui surviennent dans le pain d'abeilles limitent les espdces de levures capables de survivre. 

REFERENCES 

AnrzaN D., Poe.l A. Mnnoru p., Toua C., SnnnlN M., CnrseN I., Donne V., 1967. _ Conservation du 

pollen par des radiations ionisantes. Buil. Apicote,10, 43_50. 

AvrlstrN G. A.' 1935' - Recent work on the chemical composition of pollen. Bee World, 16, 92. 

BnnNnrr J. A., PlNrHunsr R. J., 1974. - ( A New Key to the Yeasts r, North Holland. Amsterdam. 

BerRA L. R., Barn.l s. w. T., BoHART G. E., 1973. - The mycoflora of domesticated and wild bees 

(Apoidea). Mycopathol. Mycol. appt., 49, 13-44. 

Brrrs A. D., 1928. - Hive yeasts. - IIt. Bee World,9, 154-155. 

CnsrBu-D.B., 1912.-Thebehaviorofthehoneybeeinpollencollecting. U.S.Dept.Agr.Bull., l2I.


CnsvrcH6 V., 1950. - Mikrobiologie pylov6ho kva(eni. Publ. Fac. Sci. Univ. Masaryk,323, 103-130. 

Econove A. I., 1971. - [Preservative microflora in stored pollen]. Veterinariya, S' 40-41. 

EcoRovA A. I.. Bnn'rvr I. P., 196?. - [ Yeast flora of the honey bee (Apis mellifcaL.)]. Akad' Nauk 

,4rnr. SS,!R (Ser. Biol. - Med. Nauk),2, 127-132. 

Foorr H. L., lg57 . - Possible use of microorganisms in synthetic bee bread production. Amer. Bee J. ,97, 

4'.76-4'.t8. 

Grr-rr.cN4 M., WrcrEnnlM L. J., MoRroN H. L., ManrIN R. D., 1974. - Yeasts isolated from honey bees, 

Apis mellfera, fed 2, 4-D and antibiotics. J. Invertebr. Pathol., 24, 349-356' 

Grrrrnrrl M.. MoRroN H. L., Pnrsr D. B., MlnrtN R. D., Wtcrrnuelr.r L. J., 1977. - The mycoflgra of 

adult worker honeybees, Apis mellfera.'effects of 2,4, 5-T and caging of bee colonies' J. Invertebr. 

Pathol.,30' 50-54. 

KRE6ER,VAN RrJ N.J. W., 1970. - Genus 5. Endomycopsis Dekker. In < The Yeasts: A Taxonomic 

Study, r ed. J. Lodder, 166-208, North-Holland, Amsterdam. 

Hevoer M. H., 1958. - Pollen - pollen substitutes - beebread. Amer. Bee J., 98' 145-146. 

Hayo.qr M. H., Vrvnqo A.8., 1950. - The changes in the thiamine, riboflavin, niacin and pantothenic 

acid content in the food of female honeybees during growth with a note on the vitamin K activity of 

royal jetly and beebread. Ann. Entomol. Soc. Amer', 43' 361-367. 

HrrcHcocK J. D., 1956. - A milk-digesting enzyme in pollen stored by honey bees. Amer. Bee J.,96,487- 

489. 

LAvrE P., 1960. - Les substances antibact6riennes dans la colonie d'abeilles (Apis mellifca L.). Ann. 

Abeille, 3, 103-183, 201-305. 

Lsr,.ros M.V.F., Mncneoo J. O., 1975. - Caracterizacio da microflora bacteriana normal de gel6ia real, 

larvas, pr6-pupas, imagos, mel e p6len da adelha Apis melliftra adansonii. Anais Congr. Brasil. Apicult.,3,191-198. 

Loooen J.(ed.), 1970. - rThe Yeasts: A Taxonomic study,t North-Holland, Amsterdam. 

Mrr-rBn M. W., PH.lrr H. J., MrneNol M., HEro W. B., Srlnr*.rEn W. T., 1976. - Torulopsis sonorensis, a 

new species of the genus Torulopsis. Int. J. Syst' Bacteriol., 26' 88-91' 

OruNuxr K., 1943. - Uber den Gaswechsel der Pollen. Y. Acta Phytochim. Japan, 13,93-98. 

pArN J., MeucrNer J., 1966. - Recherches biochimiques et physiologiques sur le pollen emmagasin6 par 

les abeilles. Ann. Abeille, 9' 209-236. 

vAN DER War-r, J. F., 1970. - Criteria and methods used in classification. In < The Years : A Taxonomic 

Study, r ed. J. Lodder, 34-113, North-Holland, Amsterdam. 

WtcrBnnrll, L.J., 195 1. - Taxonomy of yeasts. U.S. Dept. Agric. Tech. Bull.' 1029. 

Wrcrrnnau, L. J., BunroN, K. A., 1954. - A clarification of the relationship of Candida guilliermondii to 

other yeasts by a study of their mating types. "/. Bacteriol.,68, 594'597. 

WrLsoN, H. F., MenvrN, G. 8., 1929. - On the occurence of the yeasts which may cause the spoilage of 

honey. J. Econ. Entomol.' 22' 513-517. 

53

Degradation of 

Terramycin in Honey' 

by MARTHA GILLIAM, STEPHEN TABER, lll 

Agriculturol Reseorch, Science qnd Educqtion Adminislrolion, U. S. Deportmenl of Agriculture 

Corl Hoyden Bee Reseqrch Cenler, 20OO Eqst Allen Rood, Tucson, Arizono 85719 

qnd ROBERT J. ARGAUER 

Agriculturol Reseorch, Science qnd Educotion Administrotion, U. S. Deportment of Agriculture 

Agricuhurol Environmenlq! Quqlity lnslilule, Beltsville, Morylond 2O7Os 

NIMAL soluble powder Terramy- 

1 

fl cin (oxyterracycline) is used by 

beekeepers to control American foulbrood 

and European foulbrood diseases 

of honey bees, Apis mellifera. For treatment, 

the antibiotic is incorporated into 

various diet formulations including sugar 

dusts, sugar syrup feedings, sugar 

syrup sprays, antibiotic extender patties 

(Wilson et al., 1971 ), pollen patties 

(Gilliam and Argauer, 1975), and 

honey. 

Honey for human consumption must 

not contain Terramycin. Thus, we have 

conducted experiments to determine 

the time required for degradation of 

'Ierramycin in medicated sugar syrup 

and medicated honey stored by caged 

bees and by bee colonies. Details of 

these experiments are reported in the 

recent papers by Gilliam et al. (1978, 

1979). The present paper is a briefer 

sunmary of this work. 

In the experiments with caged bees, 

we wished to determine whether Terranrycin 

in the medicated syrup and 

honey fed to the bees became rnore 

concentrated after it was stored by 

thern in comb cells. Eckert ( 1953 ) 

found that this happened with sulfathiazole. 

In addition, we wished to 

follou' the rate of degradation of Terramycin 

in both the worker bees and 

in the cells packed with medicated syrup 

and honey after short-term (one 

rveek ) and long-term ( two weeks ) 

rnedication. 

First, we obtained bees from seven 

colonies that had not been treated with 

Terramycin and had no apparent disease. 

Then groups of these bees were 

placed in wire-screen cages with a side 

that could be opened to remove bees 

for analyses. In one test, each cage 

contained 600-700 bees, and in the 

1 Mention of a proprietary product or 

company name does not constitute an endorsement 

of this product by the U. S. 

Departrnent of A8riculture. 

720 

other test, 1 100-1400 bees were placed 

in each cage. Each cage contained a 

top bar with a strip of foundation on 

u'hich the bees built comb, and a caged 

mated queen was suspended from the 

side of this bar. Distilled water and 

test honey or syrup were available to 

the bees from capped bottles with holes 

drilled through the lid. The bottles 

were inverted over wire-screen holes 

on the top of the cage. The cages 

were maintained at 29oC and 50-60% 

RH without lights except when solutions 

were changed or samples collected. 

For each test, there were 18 cages, 

three each given one of the following 

six treatments: (1 ) nonmedicated sy- 

rup (control ) ; (2 ) medicated syrup, 

one-week treatment; (3) medicated syrup, 

two-week treatment; (4) nonmedicated 

honey (control); (5) medicated 

honey, one-week treatment; (6) medicated 

honey, two-week treatment. 

The syrup was a 50/6 (w/v) sucrose 

solution. The medicated syrup contained 

0.3 g of animal-soluble powder 

Terramycin (contains 25 g of oxytetracycline 

hydrochloride/lb of formulation; 

Pfizer) per 100 g of sucrose. Both 

the nonmedicated and medicated syrups 

were prepared fresh daily. 

The honey was collected from colonies 

that had not received Terramycin. 

Nonmedicated honey was prepared 

by adding enough distilled water to the 

honey to produce .the same refractive 

index as the 5O/6 syrup; this was a 

48-49% (v/v) solution. The nonmedicated 

honey was prepared once a week 

and was stored in the refrigerator. The 

rnedicated honey was prepared each 

day before use by adding an amount 

of Terramycin to give approximately 

the same amount present in the medicated 

syrup. 

Food coloring was added to the diets 

so we could determine the type of sugar 

or honey present (treated or un- 

1/= //) 

treated) and the approximate time that 

it was packed into the cells. Seventyfive 

milliliters of the honey or sucrose 

solution were provided to each cage of 

bees each day. Each bee consumed 

approximately 3 pg of. Terramycin/day. 

'Ihe distilled water was changed as 

needed. After the one- and two-week 

feedings of medicated honey or syrup, 

nonmedicated honey or syrup was supplied 

for the remainder of the test. 

Samples (2 ml) of stored honey or 

syrup for analyses were collected by 

placing a small capillary tube attached 

to a vacuum apparatus through the 

rvire screen of the cage into the comb 

cells. The cells from which the honey 

or syrup were removed were indicated 

by a n-rark on the wire screen in front 

of them so subsequent samples would 

not be collected from the same cells. 

Samples of bees taken for analyses consisted 

of 10 live bees collected from 

each treatment group of three cages. 

Syrup, honey, and extracts of homogenized 

bees were analyzed for Terramycin 

by a fluorometric method (Argauer 

and Gilliam, 1974). 

In both tests, the highest levels of 

.ferramycin in bees and stores were 

recorded at the end of the period during 

which the medication was fed. 

Subsequently, the Terramycin degraded 

at a rapid rate. We were not able to 

find any appreciable increase in concentration 

in the stores except at the 

end of the medication period when the 

amount of Terramycin was sometimes 

about double that supplied in the food. 

In stored syrup, the Terramycin had 

completely degraded by three to five 

weeks after the start of the one-week 

feeding, and in stored honey, it had 

completely degraded by five to six 

weeks after the start of both the oneand 

two-week feedings. The Terramycin 

concentration in stored honey was 

higher than in stored syrup, although 

degradation of Terramycin in both 

American Bee Journal

kinds of stores occurred at approxirnatcly 

the same rate. 

The Terramycin in the bees also degladed 

in five to six t'ceks after treatrnent 

\\'as begun. In a little more than 

half the cages, rnorc Terramycin found 

its r'r'ay into the storcs than into the 

bees. 'I'he bees leceiving syrup lived 

Iongcr than those receiving honey, although 

periodic examinations of bees 

for nosema disease and septicemia \rere 

negative. 

In experiments n'ith bee colonies, lve 

follolr'ed the deeradation of tlvo concentrations 

of Terramycin in stored 

rnedicated syrup and honey. Fourteen 

bee colonies u'ere maintained and fed 

pollen in a grecnhouse. These colonies 

s'ere divided into groups of three colonies 

each and gir,en the follou'ing 

treatments: nredicated honev (1X conccntration 

of Ter|arnycin): medicatcd 

honel l2X concentration of Terrarnycin): 

rncdicated syrup (1X concentration 

of Terrarnl'cin ) ; rnedicated syrup 

2X concentration of Terramycin). 

'l'*o colonies sclvcd as controls. 

Thr' 1X solutiorrs of rrredicated honey 

and s1'rup \\'erc prepared as described 

for the experirnents *'ith caged bees. 

The 2X solutions contained tivice that 

arnount of Telrarnycin (0.6 g per 100 

rnl of honey or syrup). Each colony 

rcceived 200 nrl of the appropriate 

solution each da,v. Gleen food coloring 

n'as added to all rnedicated diets to 

rnark the stores. The medicated diets 

\\'ere fed until suf ficient comb was 

built and adequate stores rvere available 

for sarnpling. This required nvo 

to ser.'cn n eeks. Thereafter, nonrnedicated 

solutions l'ithout dye rvere given 

to thc colonies, and the frames containing 

the dyed stores t'ere placed in the 

box above the brood ncst. 

Sarnples of stored honey or syrup 

gele collected one u'eek after the experinrent 

lr'as begun and then each 

week thereafter for 1B n'eeks. All sarnples 

n'ere analyzed for Terramycin by 

the f luororrrctric rnethod. 

In thc tcst \r'ith bee colonies, the 

concontrations of Terrarnycin increased 

only in one kind of storc, honey containing 

a lX concentration of Terrarnycin, 

and that occurred only after 

the first 'n'eek of feeding. The rate of 

degradation u'as similar in honcy and 

syrup. In general, a r-apid loss of Terlanrycin 

nas cvident for eight rvceks 

after the beeinning of the medication. 

'Ihereafter, degradation proceeded slorvly. 

In rnost cases, the Tcrrarr.rycin degraded 

by six to nine rvecks after the 

rnedication ended. A srnall residual 

fluorcscence was found in stored rnedi 

cated honey and syrup for as long as 

1B rvceks after the start of the treatrnent. 

This was probably not due to 

Terramycin since the drug is known 

to form nonactive degradation products 

rvhich are fluorescent (Day et al., 

l978). This small residual fluorescencc 

in honey u.hich has contained Terramycin 

nright be used as an indicator of 

previous medication with Terramycin. 

Although Terramycin in medicated 

solutions stored by caged worker bees 

completely degradcd by three to five 

\\'eeks after the start of treatment, a 

loiv level of fluorescence \r'as still detectable 

in rriedicatcd stores in bee col. 

onies for as long as 16 rreeks after 

medication had ceased. The differenccs 

in caeed adult bees and in bee 

colorries t'ould be both nutritional and 

physiological since the bec colonies 

*'ele fed pollcn, u'ere rearing brood, 

and thc bccs rvcre f lying. Horvever, 

rhe reasons for the differenccs in fluorescence 

are not clear. It is possible 

that Terlamvcin in stores in bee colonies 

binds to sor)rethins supplied by 

the bces during collection and inversion 

that is not added by caged adult 

bees. The acid pH, hypertonicity, viscosity, 

and buffering pou'er of the organic 

acids protect Terrarnycin in honev 

(Ciharnbonnaud, 1968). As mentioned, 

the low level of fluorescence 

probably rcprescnts a degradation product 

with no antibacterial activity. 

In colonies not confined to a greenhouse, 

thc Terramycin content of stores 

would bc diminished by dilution rvith 

nectar. Also, pasteurization of honey 

for consur.nption u ould further reduce 

the activity of the antibiotic. Since in 

our greenhouse colonies the combs containing 

the medicated stores werc 

rnoved to the box above thc brood nest, 

the stores g'ere ntaintained at a lower 

ternperature that would tend not to 

cause the Terramycin to degrade as 

quickly. 

Wilson (1974) emphasizcd that alnrost 

alu'ays u'hen residues of antibiotics 

are detected in honey, the arnount 

of treated sylup fed .rvas large enough 

so that the s'orliel bees stored the liquid 

in combs in the brood nest as honey. 

Surplus honey stored by the bees 

above the brood nest was alu'ays free 

of Terrarnycin regardless of the method 

of application iantibiotic extender patties, 

dusting), duration of treatment, 

ul season rr'hen applied. 

Thus, rnost of the Terramycin fed 

to bees degrade s w'ithin six weeks or 

so, although small amounts of the antibiotic 

may be detected in stores f or 

longer peliods depending on factors 

such as temperature, availability of. 

nectar, and abundance of stores in the 

hivc whcn medication is begun. At 

least six w,eeks should be allorved betwecn 

the last fceding of Terramycin 

in syrup or honey and extraction of 

honey. It is also rvise never to extract 

brood nest honey if Terramycin has 

been fed, especially if it has been fed 

in dusts or antibiotic extender patties. 

In future work, n.e hope to analyze 

brood nest honey and surplus honey 

fronr apiary colonies that have been 

fed Terrarnycin in dusts, syrup feedings, 

syrup sprays, antibiot.ic extender 

prttios, and pollen palties. 

BEFEBENCES 

Argauer, B. J, aJlat U. Glrrlar[. 1974. A 

fluorometric method for determining 

oxytetracycline in treated colonies of 

the honey bee, Apls mellifen. J, hvert€br. 

Pathol., ?,3.5L-54. 

Chambouaud. J. P. 1968. Contribution 

ra la rpcherchc des antibiotiquos dans le 

miel. Bu]l Aplc. Inf. Docm. Sclent. 

Tech., 11:133-198. 

Day, S, T., ltr/. G. Crouthamel, I,. C, uartlnelli, 

aral J. t. E. Ma. 1978. Mechanism 

of fluorometric analysis of tetracycline 

involving metal complexation. 

J. Pbarmaceut. Scl., 67;1518.1523. 

Ecksrt, J. E, 1953. The use of sulfathiazole 

in relation to American foulbrood 

disease of honey bees. J, Ecorr, E!tomol., 

46 :382-383 

eIUIarn, M. artl B. J. Arg'auer. 1975. How 

long is Terraml-cin stable in diets fed 

to honey bee coLonies for disease control? 

Amer, Beo J. U5:230 and 234. 

Gilliam, u,, S. taber IfI, anal 4,, J. &gauer. 

1978, Degradation of ox]'tetrac,'cline 

in medicated sucrose and hone]' 

stored by caged honey bees, Apls melUfera. 

J. Ilvertebr. Pathol., 31:123-130. 

Gllllm, U,, S. Taber IEI, ancl B. J, Atgauer. 

1979, Degradation of oxytetrac)'cline 

in syrup and honey stored by 

honeybee colonies. J. Aplc, Bs., in 

press, 

Wilsor, .w. f. 197{. Residues of oxytetrac]-cline 

in hotrel' stored by Apls 

mellijera, Envlron. EutotDol., 3:674-6?6. 

Wllson, fP, T,, J. B. EUlott, anat J. D. 

Ilitchcock. 19?1. Antibiotic extender 

patties for control of American foul 

brood. J, Aplc. Bes., 10:143-147. 

Reprinted from October, 1979, American Bee Journal 

October 1979 Vol. 119 (10): 720, 722, 723 

723

A p idolo gie, 197 9, lO (3), 269 -27 4' 

MICROBIOLOGY OF POLLEN AND BEE BREAD : 

THE GENUS BACILLUS 


U,S. Department of Agriculture, Science and Education Administalion 

Carl Hayden Bee Research Center 

2000 East Allen Road, Tucson, Arizona 857 19 

SUMMARY 

PROPERTY OF USDA 

FOR OFFICIAL USE ONLT 

Forty-one bacteria belonging to the genus Bacillus were isolated from almond, Prunus dulcis (- P. 

amygdalus - P. communis), pollen from the flower; from pollen pellets from traps placed on hives of 

honeybees, Apismellifera,inthealmondorchard;andfrompollenstoredinthecombcellsof thehive(bee 

bread) for one, three, and six weeks. Thirty-three ofthe 4t isolates were B. subtilis, the only species 

associated with all pollen and bee bread samples. Bacillus megaterium, B. licheni,fnrmis, B. pumilus, and 

B. circulans were also isolated. Since the greatest number of Bacillus isolates and species were found in 

pollen from the trap, the foraging bees may have added the organisms to the pollen. 

INTRODUCTION 

Recently, as the first effort in an attempt to define the microflora olpollen and bee 

bread, I isolated and identified ,l l3 yeasts from almond, Prunus communis, pollen ffom 

the flower; from pollen pellets removed from the bees' legs by traps placed on hives of 

honey bees, Apis mellifera, in the orchard; and from pollen stored in comb cells of the 

hive (bee bread) for one, three, and six weeks (Grllralt, 1979). Torulopsis magnoliae 

was the most common isolate and was apparently added to pollen by the bees since it 

was found in all samples except from the flower. The number of isolates and species 

decreased with time and storage, and most of the yeast species lrom pollen from the 

flower and the trap were not found in bee bread. In addition, yeast isolates lrom 

pollen from the flower and the trap fermented more sugars and assimilated more carbon 

compounds than those from bee bread. 

I / -t

214 

N{. GILLIA]t,I 

The second effort was ccncerned with Bacilltts organisms associated rvith pollen 

and bee bread. Members of the genus Bncil/rs are rod-shaped bacteria that are 

capable of forming endospores aerobically. These organisms are commonly 

associated rvith worker honey bees (El-Lrnuv and Er Srsenr. 1972: Grllrana and 

v^q.r-rNrrNE. 1976; Gnual,a and MonroN, l97g). Also, Econova (1971) lound that 

species of Bacillus accounted for 17 oto of the microorganisms that she isolated lionr 

bee bread" However, though these isoiates had proteolytic enzymes. they did not 

participate in the lactic fermentation that has been reported to be responsible lor the 

conversion of pollen to bee bread (CnrvlcHrK, 1950: parx and MaucrNi,r. 

1966). Otherwise. little work has been conducted on members of the genus Baci/lus 

found in pollen and bee bread. WHIrr (1906) lound that pollen stored in the cells of 

combs containing European foulbrood contains manv B.alt,ei 

organisms' CHsvrcrrlx (1950) reported that sporulating aerobic bacteria. lactic acid 

bacteria, ,'easts. and indole-producing bacreria were present in pollen at the time the 

fermentation began. Also, Macnloo (1971) reported an interesting association 

between the pollen of Melipona quaclrifasciata and a Bacillus organism. The Bacillus 

appeared to predigest the pollen. and elimination of the organisrn caused destruction oi 

the comb cells by the workers and the eventual death of the colon1,. 

Reported here are the results of the examination of pollen and bee bread fur 

organisms belonging to the genus Bacillus, These organisms are known for their 

ability to produce antibiotics (Karz and Dnuarn , l97]'): terminally methyl_branchecl 

fatty acids (KeNnna, 1977); and numerous enzymes (Barrrs.r et at., l97g) including 

pectinases (Luuur and Duurxorn. l9T6), cellulases (sEnzrorr.ro and rrur. l9T4)" 

and commerciaily significant efizymes such as amylases. proteases" j-glucanases, and 

isomerases (Focenrv et al., 1974 a. 1914 b). Because of the wide range ol metaLrolic 

activities of these organisms, they may be important in the production and preseryation 

of bee bread. 

M.{TERIALS AND METHODS 

f)etails ofmethods used to coilect anti preserve aimond pollen and to establish anci maintain the bee 

c.ololies that receired tht packed almond peliets are given by Grr-r_rlrrr (1979). Samples were pollen lrom 

the flcwer and tiom the bees'Iegs and bee bread stored in comb celis tbr i.lne. rhree. ancl six rveeks. Each 

polien anci bee bread sample rvas divided rnro four sul;-samples of approximarely 0.75 g each- 'rheri each 

ol the four srrb'sampies was homogenize

tsACILLUS IN POLL.EN 271 

All colonies rvere stained by the Gram method and examtneci for spores' The size' shape' and 

Iocation of the spores within the sporangia were noted, and the morphology of ttre Vegetati!'e cells was 

determined. Although most cultures contained sporangia and free spores after incubation ltrr 48 hours. a 

few had to be incubaied lor as long as 12

272 M. GILLIAM 

TABL. 1. - Bacillus isolated from almond pollen and bee bread. 

Organism Number of 

Bacillus subtilis 

B. subtilis 

B. subtilis (atypical) 

B, megaterium 

B.lichenifurmis 

B, circulans 

B. subtilis 

B. subtilis 

B. pumilus 

B. subtilis 

S.lichenifurmis 

Isolates from pollen from the flower 

lsolates from pollen from the bees' legs (pollen trap) 

Isolates from bee bread stored in the comb for I week 


lsolates from bee bread stored in the comb for 6 weeks 

o From a total of 80 plates in the experiment. 

isolateso 

antibiotic factor that is derived both from the bee and the plant, and this factor was 

active against B. alvei and 8. larvaebut had no anti-fungal properties. Thus, it may 

limit the numbers and species of Bacillus without affecting the yeasts. In any case, it 

appears that a specific microflora does occur in pollen and bee bread. and it is 

particularly noticeable in regard to Bacillus spp. Moreover, 66 different peptide 

antibiotics are produced by strains of B. subtilis, and most of these are active against 

gram-positive bacteria though some are active against yeasts and molds (Brnov, 

1974). Thus, the picture becomes quite complicated. 

If lactic acid preserves stored pollen by the same process that occurs in green food 

materials stored in silos (HavneK, 1958), some analogies may be drawn from what is 

known of the role of Bacillus in silage. For example, bacteria of the genus Bacillus, 

yeasts, and molds affect the stability of silage on its removal from the silo for leeding 

(woor-rono, 1978). Also, Bacillr.rs organisms apparently have the ability to 

contribute to the lactic acid and acetic acid content of silage. However, they do not 

contribute to the same extent as the lactic acid bacteria, and they are less efficient than 

lactic acid bacteria in the production of lactic acid from water-soluble carbohvdrates 

2 

ll 

I 

I 

7 

3 

I 

I 

8 

4 

2

BACILLUS IN POLLEN 

(woorrono , lg'17). However, at this time the role of these organisms in pollen and 

bee bread is unknown. Only additional experimental work will elucidate the 

contributions of micro-organisms to the nutrition of honey bees' 

ACKNOWLEDGEMENT 

I thank Dr. L. N. SreNorrsn for the pollen samples' 


2'.13 

Received.[or publication in April 1979' 

MIKROBIOLOGIE VON POLLEN UND BIENENBROT: 

DAS GENUS.B,4CILLUS 

Die lolgenden Proben von Mandelpollen (Prunus dulcis - P. amygdalus : P. communis) wurden auf 

das vorkommen von aerobischen, sporenbildenden Bakterien aus dem Genus Bacllfus untersucht: 

- handgesammelter Pollen aus den Bliiten; 

- Pollen, der mittels Pollenfallen an vtilkern in Mandelpflanzungen von den Beinen der Bienen 

abgestreift worden war; 

- Pollen in Wabenzellen (Bienenbrot), der dort eine' drei oder sechs Wochen lang gelagert war' 

Homogenate von jeder Probe wurden auf Platten mit saurem Niihragar (pH 5) und Eugon-Agar 

(pH 7) ausgebracht. 

41 Bakterien aus dem Gents Bacillus wurden isoliert und bestimmt' 33 von den Isolaten waren 

B.subtilis, die einzige Art, die in allen Proben von Pollen und Bienenbrot vorkam' Ausserdem wurde 

Bacillus megaterium, B. lichenifurmis, B. pumilus und B. circulans isoliert. Da die grcisste Zahl von 

Bacillus-lsolaten und -Arten in pollen aus der Falle gefunden wurde, krinnte es sein, dass diese Organismen 

durch die Sammelbienen in den Pollen gelangten' 

Obwohl Bacillus-Organismen im Pollen wesentlich weniger hdufig waren als Hefen, ktinnen sie doch 

wegen der Breite ihrer Stoffwechselaktiviteten - z.B. in der Produktion von Antibiotika und Enzymen - 

von Bedeutung sein. 

RESUMF 

MICROBIOLOGIE DU POLLEN ET DU PAIN D'ABEILLES : 

LEGENRE BACILLUS 

On a recherch6 les bact6ries a6robies sporulantes appartenant au genre Bacillus dans les 6chantillons 

suivants de pollen d'amandier, Prunus dulcis (: P' amygdalus: P' communisl ; pollen r6colt6 ir la main 

sur les fleurs; pollen pr6lev6 sur les pattes d'abeilles, Apis mellifera; des trappes plac6es sur les ruches 

situ6es dans le verger d'amandiers; et pollen stock6 dans les cellules des rayons de la ruche (pain d'abeilles) 

pendant I semaine, 3 ou 6 semaines. Des homog6nats de chacun des 6chantillons ont 6t6 d6poses sur agar 

nutritif acidifi6 (pH 5) et sur agar eugon (pH 7)' 

euarante et une bact6ries du genre Bacillus ont 6t6 isol6es et identifi6es. Trente-trois d'entre elles 

€ta\eni Bacillus subtilis, seule espdce associ6e ir tous les 6chantillons de pollen et de pain d'abeilles' On a

274 

M. GILLIAM 

6galement isol6 B. megaterium, B.licheniformis, B. pumilis et B. circulans. puisque le plus grand nombre 

de bact6ries isol6es et d'espdces a 6t6 trouv6 dans le pollen provenant de trappes. on peut supposer que ce 

sont les abeilles butineuses qui ont ajout6 ces organismes au pollen. 

Bien que les organismes de type Bacittus soient bien moins nombreux dans le pollen que les levures, 

ils peuvent avoir un r6le important d cause du large domaine de leurs actilitd's mdtaboliques. telles que la 

production d'antibiotiques et d'enzymes. 

REFERENCES 

BAPrIsr J. N.. Mervoel- M.. GtIrp-Na R. L.. 1978. - Comparative zone eieetrophoresis of enzvmes in the 

genus Bacilias. Int. J. St,st. Bacteriol.,28.229 ?44. 

Bnnnv J.. 1914. - Recent developments of antibiotic research and classification of antibiotics according 

to chemical structure. Adv. Apttl. lVicrohicl., I8. .109,406. 

CnrvrcHtr v" 1950. - Mikrobiologie pylov6ho kvaieni. Pubt. Fctc..!r:i. L'rrr., trIasar1 k,323, 103,130. 

Econova A. L. 1971. - lPreservative microflora in stored pollen.l tr;prsrj11qri1'a,8, 40 41. 

Er--LrtrHv M. A.. Er--Sreael K. B". 1972. - External anci inlernal microflora of the hone1, bees {,,lpls 

melli,fera L.). Eg1,pt. J. tr[icrobiol.,7.79 8'7. 

Focenrv W. M., GntrrrN P. J.. Jovcn A. I\4.. 19?4. - Enzymes of Bacillus species-part l" process 

Biochem.,9, 11-18. 24. 

Focrnrv W. M.. GntrrrN P. J.. JclYCr A. M., 1974. - Enzymes of Bacitius species,part 2. process 

Biochem., 9, 2,1.29. 31. 33. 35" 

Gtu-rav lvr.. 1979. I\licrobiologv of pollen and bee bread : the i'easts. 4pitlotugie,l0,43 5_1. 

Gtirtnlr M., Monrox H. L.. l9i8. - Bacteria belonging to the genus Bacillus isolated from honev bees, 

Apis mellifera, fed 2.4-D and antibiotics. Apidologie, 9, 213-221. 

Grtrtav M.. V.{nN'rtNe n- K., 1976. - Bacteria isnlated from the intestinal contents olforaging worker 

honey bees. Apis melli"fera I the genus Bacillus. J. Intertebt. pathol.,2g, Z,'t5 276. 

GonocrN R. 8.. HavNns w. c.. Hon NAy PANG c.. 19?3. - The genus Bacillus. usDA Hantlbook, 

42:. 

Havoar: N4. I{.. 1958. - Pollen pollen substitutes bee breacl. Amer. Bee J., gg, 145 116. 

K'rNe nn T., 1977. - Fatty acids of the genus Bacillus.' an e-xample of branchecl chain 

preference. Bacteriol. Reu., 41, 391-418. 

Kerz E', DEMAIN A.L", 197'1. - The peptide antibiotics of Bacillus.'chernistry. biogenesis. and possible 

functions. Bacteriol. Rev., 41, 449-414. 

La;uuE J., DultaNoln V. C.. 1976. - Recherche de l'activit6 pectinolytique chez le genre Bociltus" ,4nn. 

Micrabiol. Inst. Pasteur, 127 A,423-427. 

Lavrr P.. 1960. - Les s,bstances antibact6riennes dans Ia colonie d'abeilles (Apis nrelli-fert L.). .4nn. 

Abeille.3. 103 183. 201-305. 

I\'[.qcHaoo J' O', 1971. - Simbiose entre as abelhas sociais brasileiras (Meliponinae. Apidae) e uma 

especie de bacteria. Ciencia e Cultura (Sao paulo),23,625-633. 

PAIN J., Mrucplnr J., 1966. - Recherches biochimiques et phvsioiogiques sur le pollen emmagasin6 par 

les abeilles. Ann. Abeilte" 9, 209-2-76. 

SpnznorlLo A., Taur S. M., 1974. - Cellulase de bacterias isoladas de " ninhos D de Ata laet,igata, 

Smith. Ciencia e Cultura (Sao Paulo), 26, 957-960. 

Wstrr G. F., 1906. - The bacteria of the apiary. L:SDA Tech..!e,ies, 14, I 50. 

WoorpoRo M. K.. 1977. - Studies on the significance of three Bacillus species to thc ensiling prLrcess. 

J. Appl. Bacreriol., 4t, 447-452. 

Woorrono M. K., 1978' - Antimicrobial effects of mineral acids. organic acids. salts and sterilizing 

agents in relation to their potential as silage additives. J. Brir. Grasslantl ioc.,33, l-ll 136.

La santJ ae ltabeiJ-le, #54, L979 

179-183 

. une colonie spdciale- 

,es sont sorties avant 

rses et, dans les ruches 

irarque aucune atteinte 

'. rvain- 

Photo Regard. 

,nt 0tre fort prdjudicia- 

'rcole lorsqu'elles s6visr, 

effet, beaucoup de lar- 

, pure perte puisqu'une 

sans donner les ouvrie- 

.lu'ayant exige soins et 

,ic nonrbreus:s bi,'.r- 

,a rdcolte s ajoutant aux 

: pour obtenrr des cada- 

', jt')rD€nt les possibilitds 

'-, sur le profit escornptd 

ians certains cas, consi- 

,:olitique d'6limination 

's et le repeuplement 

;sid6rdes comme rdsis- 

)rtement nettoyeuses 

.i mycoses et ceci ne 

:1u'd partir de lignees 

d'une race pure adap- 

''rmatologiques du lieu 

Andr€ FEGARD. 

)) 

lui donnerez 

complete. 

N. 54 - 1979 

J 

lE COUVAIN PLATn€ 

Etat actuel de nos connaissances 

et de nos recherches en vue d'une maitrise de cette maladie 

(American Bee Journal, Traduction lsabclle VAlI-LANT] 

Martha Gi l-I.i"am 

INTROI}UCTIOFJ ; 

Le ccuvain platre est une maladie qui 

touche le cotrvain d'abeilles et qui est 

caus6e par le e:lrampignon : AscosFiraeta 

Apis. Les larves malades se nlomiiienl 

fig, l), et les momies deviennent blarr 

ches b cause du mycelium dr,r champignon 

{Fig. 2). Pourtent, si des mycelia de 

Sexes opposds (+ et --) se regror.rpent. il 

se forme des spores i I'intdrieur des corps 

f iuctil:r',::; :: 'L;. lJl cl les tlonlil:s du-viennr:nt 

iortcee.', glises ou itoires (i:ii;. 'i1. g,t 

t:-ouve ces nrotnies a I'enlrtie ries ruches, 

sur le plancher et dans les cellules de colo' 

nies infest6es. 

Maasson (1913J, en Allemagne, publia les 

premi6res observaticns concernailt Ie couvain 

piit16. En 1916, il d6crivit le chanrpi' 

qnon ct I'appela Pericystis apis. ['!rrs tarcl. 

aux Etats-Un;s, Spiltar (1955) a itudi6 le 

cycle de vie et Spiltar et Olive (1955) ont 

rdclassifi6 le champignon et ont 6tabli une 

nouvelle espdce, Ascophaera, puisque le 

nom Pericystis avait d6ja 6t6 employ6 pour 

!'algue rouge. 

HEP,tRTITION GEOGRAPHIOUE : 

L6 couvain pl?rtrd a 6t6 signal6 depuis 

plusieurs anndes en Europe, les lles Britan' 

'niques 

incluses. En 1957. Seal signala le 

'couvain pl6tr6 en Nouvelle 26lande. 

Baker et Torchio (1968) publierent leur 

premier rapport sur A. apis aux Etats-Unis. 

Les cas isol6s qu'ils signalbrent iurent associ6s 

b l'abeille coupeuse de feuilles, 

tulergachile inermis, et ir I'abeille qui nidifie 

dans le sol, Antophora pacificia Plus tard, 

Thomas et Luce (1972) signalOi'ent la maladie 

chez des abeilles productrices de miel 

en Californie ; Hitchcock et Christenserr 

(1972) trouvdrent la maladie clrez des lar' 

ves d'abeilles au Nebraska et en Wyonring 

et notbrent que d'autres cas de maladie 

avaient atti16 leur attention en Californie, 

Minnesota, Dakota du Nord et Montana. 

Gilliam et Taber (1973) signaldrent la mala. 

die en Arizona et Conner (1974) en Ohio. 

Nelson (1976) visita 5.374 colonies dans 

cinq provinces du Canada en 1975 et d6couvrit 

que 32 orb de ces colonies avaient 

N" 54 - 1979 

,d'.:s nrorriics sur ieurs cadres. Celrendent, 

ii oo des coloriics touchdes avaient moins 

d': ifi ccllules atteintes. Une visite sinti' 

laire a dti cffc.;tudr: en 1976 dans; I'Ouost 

Canadien, qrri rr';vilait que 33 u,o des p;rquets 

d'abeilles et 20 o,a des colonies sddentaires 

avaicnt le couvain pl5trd. 

La maladic est maintenant largernent ddvetoppie 

aux Etats-Unis et au Canada. Err 

1976, on avait d6tectd le couvain pl5tr6 dans 

33 des 48 i.lets dt: cotttineni {avec l-la''vatl) 

c'; iJarrs C des [t provinr.iT'1 falt,..iiclrt.rt,:" iiJl' 

n:ipJCe ct Vvilscrr. 1!7C). 

L'ETIOLOGIE : 

Malheureusement, on connait peu de choses 

sur I'dpicldmiologie et la pathog6ndse 

dc la nralaciie. Selon Bai!ey (1967). lcs lrrves 

r.J abeilles sont plus sensiblers i Iir ;rralac.lic. 

Si elles ingereni des spores de A. aprs 

quand elles ont 3 ou 4 jours, elles meurent 

alors 2 jor-rrs aprbs leur operculation. Ensuite, 

les spores germent dans l'intestin 

des larves. D'abord, les larves mortes sont 

recouvertes d'un duvet blanc de mycelia et 

sont gonflees ,1 la taille de la celluie. Plus 

lard, elles se dessdchent et deviennent des 

morceaux durs et ratatinds, comme de la 

craie, qui peuvent devenir grls oLr noirs si 

Ies corps {rrrciifiies se fornrent. On pu'trt 

troLvcr lcs restes des larves dans dcs.ccilules 

opercul6es ou pas. La plupart des 

larves nreurent au stade d6roul6. l-es abeilles 

arJultes retircrrt g;6n6ralement les nromies 

que l'on peut retrouver i I'entrde des 

ruches ou sur le plancher. 

ll y a eu des contreverses en ce qui 

concerne I'origine de I'iniection de A. Apis, 

) savoir si elle provient C une ingestion 

de spores par la nourriture, ou par le corps 

lui-nrcrne .1 parlir de s;rores se l.rout,ant sur 

les cadres ou sur Ies parois des cellules. 

Gochnauer (1975) affirmait qu'une fois que 

la colonie dtait infest6e, les spores pou- 

'/aient rester actifs, et ainsi la nraladie 

pouvait riapparaitre. ll suggrdra aussi que 

I'A. Apis pouvait survivre dans le sol, s'introduire 

dans la chaine alimentaire ce I'a- 

[:eillr: et ainsi contaminer les larves par 

la nourriture. 

175 

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Bren que I'on considdre comrne peu importante 

la propacation de I'infection (Bailey, 

1963) les spores sont trAs 16sistantes 

et restent infectieuses pendant 15 ans (Toumanoff 

, 1951 ). 

l',laurizio (1934) et Betts [1951) pensaient 

que la maladie 6tait probablement transportde 

par le miel et. par consdqr-rent, l'on 

devait iviter de nourrir avec du miel provenant 

de colcnies irifect6cs fr,{aurizio (1934) 

trouva le ciranrpignon daris les intestins 

d'abeilles et d6clara quc I'organisnre hibernait 

dans les abeilles et dans le miel 

De Jong et Morse (1976) trouvdrent I'A. 

Apis dans le contenu des jabots des abeilles 

adultes et montrdrent que les spores 

passaient d'abeille en abeille par l'6change 

de nourriture. 

Une arrtre extension arrive surtout par 

les C6p!acemenis ii'abeille: i,;i.ovenant de 

cr,!,rnies rc;cs: irs. C::p.:idi::;. l,r ce ll.;tr;tinaticn 

peut arrivcr aLrssi a ceuse cle l'apiculteur, 

quand celui-ci interchange des cadres 

et des outils de colonies infest6es 

avec des colonies saines (Barthet, 1971J. 

Parmi les autres sources possibles de 

contamination, il y a le pollen (fulehr, 1976), 

le vent, I'eau, les reines (De Jong et Morse, 

1976 - l\4oeller et Williams, 1976) et 

les paquets d'abeilles (Nelson, 1977). 

On a 6tabli que beaucoup de facteurs 

contribuaient au d6veloppement de cette 

maladie (voir Gillian, 1978). lls comprennent 

un refroidissement des larves, une 

faible ventilation des ruches, un temps frais 

et humide, des tempdratures et une humiditd 

6lev6es, des colonies faibles pour I'hi" 

vernage, une humidjt6 trop forte dans la 

ruche, la pollution de l'air, l'utilisation d'antibiotiques 

favorisant le ddveloppement de 

champignons, des colonies souffrant d'autres 

maladies, du cou,rain endomma96, des facteurs 

g6netiques, une nourriture aqueuse, 

ei d;s nranipi.ilatir:rrs trop fresqr-ientes des 

colonies. 

ll a 6td suggijrci que d'autres insectes, 

principalement les abeilles sauvages ou 

solitaires, contribuaient A la propagation 

de ia nraladie. Ascosphaera apis a 6t6 associ6e 

ou a 6t6 trouvde chez les abeilles 

coupeuses de feuilles, les abeilles . magoos 

r, celles qui nidifient dans le sol, et 

les abeilles alkali (Baker et Torchio, 1968; 

Batra (1973; Chout, 156; Stephen et Undurraga, 

1965). Batra (1973) remarqua I'importance 

de la capacit6 de contamination 

par les colonies d'abeilles sauvages. Certaines 

butineuses de la plupart des espdces 

d'abeilles d'une m6me zone, se retrouvent 

sur les m6mes cultures et ainsi 

180 

i,rlrnlettent une contar.nirrati(,n par les fleurs. 

l)e plus, elles rdcupcrent les mdmes cham. 

pignons provenant des jabots d'abeilles 

d'ar.rtres espdces. 

Traitement : 

ll n'existe aucun agent chirniothdrapeutique 

pour lutter contre le couvain platrd aux 

Etrrts-Unis. Auparavant, lcs pertes occasionnies 

par la inaladie n'dlaient pas assez 

s6rieuses pour jLrstifier une rccherche sur 

Lin traitenrent. Pt.risqtre les alreilles adr.rltes 

gydtilr-alenrent entportent le r;orrvairr rnort 

hors de la ruclre. la rrralcdie disparait souvent 

sans I'intervention de l'apicuiteur. Dans 

des cas qraves, la deslrrrction de cadres 

touchds, ou de scctions de cadres, a 6td 

recommandde IBetts, 195l : Gochnauer et 

al, 1S751. Giauffret et al f1969) et Thontas 

et i-ir,.rc (l!112) lrouvuitlt qll.i llr lun-rigati,tn 

i I'o;.)rde ci'ithyl..;rc lrr.rii l'A. ltpis. 

ll far.rdrait probablerrerrl eviter I'accurlulation 

d'hunridit6 et une verrtilation suifisante, 

puisqu'il parait que le tentps froid et humide 

encourage le ddveloppernent de la maladie. 

Zander (1919) insista sur I'importance 

d'envelopper les colonies pendant I'hiver 

et de les tenir au sec. Seal (1957) disait 

qL'on pouvait 6viter Une extension s6rieuse 

de la maladie en. fernrant les ruches pour 

l'hiver et en nettoyant I'herbe autour des 

ruches pour 6viter I'humiditd et permettre 

une bonne ventilation. On peut aussi 6larqir 

I'entr6e de la ruche pour favoriser la 

ventilation (Goclrrrauer et al. t9751. 

Seal (1957) reccmrnancla de renf orcer les 

ruches fortenrent nralades en ajoutant de 

;eunes abeilles adultes et du couvain opercr,rld 

et en les nourrissant de sirop de 

sucre. ll dit aussi que la nraladie poirvait 

etre 6vitde en ne laissant pas les abeilles 

hiverner dans un corps de ruche trop grarrd. 

Lunder (19'/2) sug96ra I'enrploi de reines 

r6sistantes, et Mraz (1973) recolnmanda de 

remplacer les reines par des souches noll 

sensibles aux maladies. R6cernrnent, Nelson 

{'19751 fit des croisemerlts entre des 

souches de Nouvelle-Zelande et des sou. 

ches de Californie et trouva que la souche 

obtenue 6tait moins atteirrte de couvain 

pl6t16 que la souche de Californie. 

Plr,rsieurs auteurs ont nrene des expdriences 

au sujet de I'utilisation de mtidicaments 

pour contr0ler la maladie. D'une 

nranidre gtin6rale, ces tentativcs n'ont pas 

renrport6 de succds, ) cause de la toxicit6 

des mddicaments pour les abeilles, du fait 

que beaucoup de medicaments n'dtarent pas 

acceptables pour les abeilles, et b cause 

du terrips et du travail exig6s pour ddsin- 

N" 54 - 1979 

lectcl iorrt. 

W'ide {1${' 

mol empi:' 

Apis en ctrl 

6tait vapor 

maladie di' 

saire de , 

parois intt. 

abeilles n', 

mol dans 

que le tl' 

avait un u 

dans les 2' 

ll affirnra . 

nettoyaqe , 

vention. 

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*d*ifffi3* 

ff1i$t *r*ilffi,**+lt#*r#ffi# 

.Ct'*jr:-:j*lk;i*,.,r;*'j.' j;;- r.;rr*", ,l-' ',j,.,r.,.,i;,;.*,î-n;*i'. 'n:'..;'''Ji.i$*;;. - .- ''*i'''

trininaticr par lcs lleurs 

i,ire:rt les ii.,ren'"i .nnr. 

dcs . jabr ts d'abeilles 

i:gent chimiothcrapeuti. 

:a- le couvain platrd aux 

r|it, les peries occasion. 

l;e n'6taient pas assez 

.fier une recherche sur 

,, te les abeilles adultes 

,i ient le couvain mort 

maladie disparait soucn 

de l'apiculteur. Dans 

destruction de cadres 

.iions de cadres, a 6td 

. 1951 ; Gochnauer et 

ri al. (1969) et Thomas 

.rent que la fumigation 

r .r,l lA r\n:; 

r1leiri dvitar i lr,;c;irn.]..il;t. 

,,r ventilatit,;, suif isanie, 

l'' temps froid et humi- 

,.Sppernent de la nrala- 

,:,ista sur i'rnrportance 

,ries pendant I'hiver 

;. Seal [1957j cjisait 

,,re eXtens,^rr StirieUSe 

, rrrant les rr.tches pour 

.I'herbe autour des 

, humidit6 et permettre 

,i On peut aussi 6lar- 

,'he pour favoriser la 

'r et al, '1975). 

'' anda de renforcer les 

,:;lades en ajor,itant de 

.r;s et du couvain oper- 

'rrrissant de sirop de 

.rr: la maladie pouvait 

,:,sant pas les abeilies 

irs de ruche trop grand. 

',',rr I'enrploi de reines 

:3) ieco,;t;i,anda dc 

par des souches non 

', .s Bticemment, Neii 

.)isements entre des 

,-Z6.lande et des sorl- 

: trouva que la souche 

, atteinte de couvain 

. te Californie 

ont mene des exp6- 

:'uiilisation de rn6dica- 

,;r la maladie. D'une 

3 tentatives n'ont pas 

i cause de la toxicitd 

,- les abeilles, du fait 

rrran rents n'6taient pas 

. abeilles, et a cause 

,,1 exig6s pour ddsin- 

N-, 54 - 1979 

tectcr toutc la ruche. filrr a*s1r_r,lc, Elbe et 

Weide {'liit;i) trouvircnt QLre Cr ' 'ti de thy' 

nrol enrpechait le devclopperrrJrrt cie l'A. 

Apis en culture. Ouand la solution de thymol 

dtait vaporis6e sur les cadres infestdes, la 

nr:;lirdie disparaissait. Pourtani, il fut neccs" 

saire de vaporiser chaqLre cacjre et lcs 

parois intdrieures du corps clc ruche. Les 

abeilles n'accepteraient pas il.'i lo de thy. 

nrol rlairs du sirop, Barthei ii971), trouva 

c;ile le thymol ciair: une srl.jt;on A 2 o,'o 

avait un e{fet destructeur du charnpignorr 

dans lcs 20 nrinutes (sur I'A. apis in vitro), 

ll affirnra qtre la stirnulation de Iirrstinct dt: 

nettoyage Stait ia prlnciprlle nresur"e de pro' 

ven tion. 

Dalhman (1936) a test6 le desinfectant 

. Fesia-Forrn " (base formol dehydel sur 

des colonjes rrrlest6rs de couvain platri. 

ll a vaporis6 la solutiorr sur les cadres de 

couvuirr, st-rr les parois rnicrnes clc ii ru- 

:ir.: 3t Sr.;; llr pltnche {j r::;:li. Lil.: :.1-rrr',r,|,, 

:,p;-Js la v.til,-)iis0tion, ir:s abeilies aOiiitl:; 

de colorrics sirieusenrent inlesl6es. ava;en', 

retird les nronries et on ne pouvaii revoir 

d'infection cette ann6e-la. ll trouva qu'une 

solution:l 4 qo de. Fcsia Form " tuait 

le champignon. Barthel (1971) a f ait des 

expiriences in vitro sur l';:clion ties fun(li. 

cides Il) contre I'A. apis et rapporta qUc 

4 o, Ce " 

Fesia Form, tuair ies spores 

aprds 30 minutes. 

Des antimycosiques et antiseptiques ont 

dt6 exp6riment6s en vue d'un usage possible 

dans le traitement du couvain plitri. 

Ciauff ret et Taliercio (t 967) rapportent rlLrc 

!':rnphetericine B dtaii la plus efficacc. 

Poii.riani, elle n'titait pas stable. l-es arrtiseptiques 

test6s 6taient plr.rs stables, nlais 

plus toxiques pour les abeilles que les ant 

jmycosiqrres. Moeller €.t Williai-;r (f !r76) 

trou','erent que 25 P ppr-n de berromyl rlans 

du sirop de sucre semblaient avoir un eftet 

positif pour rddirire, mais norr pas alinriner 

ci'ii,platai;:ent l'infci:i;on c.lu couva;;i pi;itre. 

Thomas et Luce ('1972) rapporteient que 

I'acide sorbique et le nrdthyl parahydroxybenzoate 

inhibaient I'A. apis en ctrlture. Plus 

tarcl, Taber et al. {1975) ont nris d.: i'acidc 

sorbique et du sodiurl prepionate dans des 

petites boulettes de pollen et de sucre, 

dorrrrdes b des colonies s6rieusenreni atteintes. 

La maladie disparut 7 jours aprds 

le debLrt du traiter;rent. ll n'y eut aucun 

synrpt0me de toxicit6 chez les abeilles et 

aLrcrJr)e rdduction cle c:otrvain opercult!. 

L'utilisation de la turnigation d'oxyde d'e- 

[1) fungi : clranrpignons 

N. s4 - 1979 

thylirre et la silection d'abcitles resistanics 

arr ccitvairr plltrc, parilisserrt lr:s moyens 

lcs plus pronrettcurs cle controlcr la mirladie, 

trien qu'une recherche soit a faire pour 

tester l'efficacite de l'oxyde d'cthylcne 

corrtrc I'A. epis, ot Jrorrr sclcctionncr e1 

6lever des abcillt:s risislantes. 

Fig. 1 : larves d'abeilles nronrifi6es par rlne 

infcctirir cl'A. lrli: ;tc:'.j..(,t)'.ilt,;ic. 

I a;s it,trinres soni itl.rlrciii.,;. 

Fig. 2 : Espbce norr accoiipl6e d'Ascosl;hae 

ra apis. fJycelia seir!enr€:nl. 

F'; j 

Fig. 3 : Espdce d'A. apis accoupl6e, montrant 

dus spores dans les corps 

{rr.rct if eres. 

-.1. 

j'- -" '. r-, ! 

!i 

';. 

1 _,j 

.,1::..'j1 f, -:i 

t i...,' 

j 

A.tr:' -.-1" ^.\ 

i;.j'lr 

181 

: 

h;, 

tr, 

F.,i

Hypolhescs : 

L'oriqine cJe la inalaclic atrx'Itats-Uttil r:s; 

un mystore. ll exisic au nroins delrx possibilitds- 

Certains pensent que cette rnalrdie 

dtaient prisente rians le prys depLris lorr5t" 

teillps, mai:i .rir: i):i !r ) arccorriait t)3:t d i;ii 

portance. Fr,rsilr;g bcaucotip de nricrc'biologistes 

compdtents ont exanrine les rnaladies 

d'aheilles depuis Ionr.;tcnrps, il sr:rnble intprobable 

qu'ils n'aient pas exar-ninti le cor.rvain 

pliird s'il exist;rit. Je strspeci.t: les 

apiculteurs d'avoir crir a une autre inaladie 

de champignon, le couvain p6trifi6. qtri elle; 

aussi donne des ntomies. Ceux qui pL'irsent 

que la nialadie existait deprris des atrirules 

pensent aussi nle, rticeintrtettt, e!le s'es't 

propa96e et 1;'r-'st aggravee. Si cela es: 

vrai, c'est quc ie chanrpicuron s'esi trans 

{or:rtti oti bien igire les aociiic,s sc}tli iirl\/enues 

plus sensibles. En effet. les espcces 

6lr:.'r. ;1;1i5 clrt.; Itats'lJrriS D:1ilvcllt 0lrr: difttrr,.c:;li 

ii ili: cCli*s 11|e I'i,:'t irllve :|,t,:t.[-s 

ciarr.g le trrlrt,ie. 

Lc chanrpignon peut a,,'oir'exist6 chez des 

abeilles solitaires ou sarivages dans le pay's 

depuis longiemps. Ceci ne peut 6tre v6rifi6, 

puisque peu de choses ont 6t6 faites sur 

la microflore de ces abeilles, laquelle a pr,r 

servir de foyer de contamination pour les 

abeilles productives de miei. 

D'autres pensent que le champignon ne 

fut introduit que 16cemment, peut-itre sur 

du pollen import6. Puis, l'usage de plirs en 

plus grand d'antibiotiques dans le traitement 

des nraladies d'aceilles peut avoir aug' 

nrent6 l'!nciderrce de la rnaiadie, en botrle' 

versant la nricroflore intest!nale norntale; 

des abeilles.' 

Le couvain platrd pert 6tre une maladie 

due b un sr.ress puisqrie des abeilles artornrales, 

affam6es et elfernrees, sernblent 

plus sensibles. ll y a aussi des colonies 

nroins aptes que d'auires ?r retirer les iarrres 

malades. 

Ces conjectlrres nrontrent qu'i! y a Ce 

nombreuses pistes ini6ressantes de recherche 

sur la maladie, clLri devraient 6tre examindes. 

Plus partict:iierenrertl, il faLrdrl:it 

6tudier la classification et la virLtlence dcs 

espdces de A. apis, ainsi que la sensibrlrt6 

des souches d'ahellles. 

Rapport de recherche : 

L'un des obstacles majeurs dans la conduite 

d'une recherche sur Ie couvain pl6tre 

a 6t6 la difficult6 de provoquer des in[ections 

chez les abeilles ir 6tudier. Ces infections 

nous infornreraient sur les,. contment 

D, " quand " et " pourquoi " les 

182 

- -n . 

"i 

i-; l,';.' 

,.i ' ',r.,..1 

": 

. :l 

. . .,- i *+nt.1:-ir{d:}!. 

alreillcs r;Lrccclrrrbcnt ir ccttt: nrala,lrc ct ellcs 

t)ornrr:llrilicrlt dclalcrrrcrrt arrx cherclrirurs de 

iairc dc:; cxpi:ricnces :;rrr les nrctlroclr:s dc 

traitcnrent. Donc, rrou:; avons nrcnei des 

experietrces polrr infcctel-du couvain de 

colorrics d'rrrr rur;hcr ct cirr couvairt rlaintcnll 

cn irrr:ul.ratcrrr'. Lcs :pcrierrce 

sont dans li: r api)ort 16ccrrt dc 

Gillianr et al {1978). 

Llcaucoul.r cl'essiri:;, la 1;lupart ayant etd 

Sirrrs sr.rcCas ou norr rc;:rociuctifs, ont 6tc 

j:.;tir pour infecter lc coirvairr dans des 

colorries cle rucher avec de I'A. A.pis dans 

des conditions varices. affectarrt ?r la fois 

l'agcrrt ;ratlroqorre et lcs r;olonies. Ccs es, 

sais inclucnt: 

ir,nrploi cl'inocrrlats priparr)s a partir' 

cle cultrrre artificielles d'lt. apis, orr i 

I'inversr-', des supcnsions a partir de 

larves naturellenrent infest6es, 

- l'introcluction dc l'inocrrlat en v;rDori, 

SiiliOil dt:rirOIt r;t.iIit, en (jaillltrX Ljr:j 

1lollcr,, 

-- I'r.,iit1tloi rle rnoilir-'s avec d jverlopyrenrent. 

soit v6g6tatif, soit sporLrl€ de 

- I'altciration du rapport abeilles adLrl- 

- et l'introduction de nlomies moulues 

l'agent pathogdne, 

tes et couvain, par enldvement d'abeilles 

adultes orr de cacires oe couvain 

avant I'intro(iuction dc l'inoculat. 

dans du pollen. 

. !ji. ' : 

..: r'/. ,.. 

..- '1:. 

,... 

, - t:l 

L': 

' \ ,iT.i 

. . ,l 1.. .. 'i l? . .i,.^ 

krig ;j i. ':i 

r,J '. ';t l'n' 

r:'lr' i i' ,?rl t ,lr 

: . :{:r: .,1 

.,. i : 

' ",.,' 

' ' l':l 

t,':t'...l 

i.-..ii 

!:. i.r' ' : 

...: ,'r 

Fiq.4 : l-arves nrornifices p;rr urre irrfection 

d'A. apis accoupi6e. Les rnomies 

sont gris fonci orr noires. 

Lt,:, inicciiorrs les plt;s stivcreg frrrr,,1 

plovorlLr6es rlans des colonies qi,i avaicn,r 

dte nourries Ce pollen non contamine, afin 

de faire de l'dlevagre rJe couvain. De celles-ci 

on avait retiri rrn ccrlain nonrbre 

d'abeilles rclultcs avant d'avoir vapoiisti le 

couvain d'une suspension de trois rlomies 

noires (sporul6es) dans 9C nrl de 5 o,a cje 

sucrose darrs de I'eau. Ce traitelnent fut 

appliqui trois fois par jour, le 1", le 3'et 

le 5' .jour de I'experience et il provoqua de 

lourdes infections (10 9o du courrainJ :,u 

6' jour. Pourlant, ntOr.nc ce traitentent fut 

N, 54 - 1g7g 

filo I i 

furcri 

Ainsr 

colo:' 

int 1;L;; 

la d iLr 

/-\ ' 

i nfec l 

nfJt[; 

relirr 

rilisat 

tenir 

in oct,' 

D", 

st l;. 

plStr-r 

:a it 

d'a Lrt r 

Pou rr ' 

mific. 

aill:l:,i 

rl irf , 

/et;' 

atia(i. 

par I 

savo i 

sont 

De 

ren ( 

r)'ar' 

leu r..; 

jours 

Lan 

f ro, i: 

Des 

l6s 

la 

Sur 

t,j 

api s 

I'A. a 

la rv e. 

pous: 

cies i, 

Dc 

tion' 

sur l, 

ri i, i rr. 

avoil - 

la bc, 

des l. 

les.; 

POUS S 

tion: 

E nsr. 

e ntit 

Les 

pas, 

ves c 

N":

'lt A ceite nraladie et elles 

.'lemei,rt aux chercheurs de 

rnces sur les nrdthodes de 

'. nous avon: men6 des 

; infe'eter du couvain de 

.;lrer et dtr couvain nrain- 

'ir. Les d6tails de I'exp6- 

,.i le rapport rdcent de 

i$.1. 

::tis, la plupart ayant dt6 

,rrn reproductifs, ont 6td 

ier le couvain dans des 

)r avec de I'A. Apis dans 

rriees. affectant i la fois 

' et les colc'nies. Ces es- 

,,,:ulats prepards b partir 

,.tificielles d'A. apis, ou i 

:; supensions ir partir de 

'. f ement infest6es, 

Je linoculat crr '.,eporir:rp 

et7/otr, en gate;u:< de 

. ronries avec d6veloppeegdtatif, 

soit sporul6 de 

.rleO€. 

. r rapport abeilles adul- 

,n, par enlevement d'abeil- 

,'tr de caores de couvain 

..::ion de I'inoculat, 

,ron de momies moulues 

;rn- 

rrtrifiies pai une infectiln 

.i(.coupl6e. Les morrries 

lC)rrCd oU noires. 

ir:s plus sdvdres furent 

rr:s colonies qui avaient 

. llen non contamin6, afin 

.rge de couvain. De cel- 

.!iird un certain nornbre 

avant d'avoir vaporis6 le 

nension de trois momies 

dans 90 ml de 5 ortr de 

i'eau. Ce t.aitement f ut 

par jour, le 1", le 3' et 

..i-ier ce et il provoqua de 

it 0 o/o du couvain) au 

rn6me ce traitement fut 

N. 54 - 1979 

nloins e{ficace dans d'autre:. iolonies qui 

furent traitdes a des p6riodes ,-,lus tardives. 

Ainsi, les diff drences de sensibilitd des 

colonies d'abeilles peuvent etre un facteur 

important dans la nranifestation de la rnaladie. 

A cause de la difficultd i introduire des 

infections dans des colonies dc rucher, 

nous avons in,/ent6 des techniqUes pour 

retirer lc cor-rvain des cadr"es, pour la ste. 

rilisalion en surface du couvain, pour ntain 

tenir le couvain dans un incubair:lrr., et porrr 

inoculer du couvain avec l'A. aprs. 

D'abord, nous avons cherche ir diterrnincr 

si I'A. apis causail la nralaclie du couvairr 

plafrd, ou bien si simplelrent, elle envahil: 

sait lr:s larves dr:;i tuics orr blessdes pc 

d'autrcs choses. {;r:t1s dgrl';arc hypofhes, 

pourrait 6tre appuyde par le fait de la momifieation 

des lan'sl ntortes. ItJols espirorr:, 

; irsr.:i tiiitrr,rinlr .li l,r n:r;r]l;lrcaiiitt viijitl 

cj'rnfeclions par C.lvelcp;re;irr:lrl i lr Irri,; 

vcq6tatif ct sporule, et si l'A. a;)is peui 

attaquer dcs larves par Ia cuticLtle ctfort 

par la bouche. De plus, nous espdrons 

savoir si les jeunes et les vieilles larves 

sont 6galenrent sensibles ) l'infection. 

Des larves de bellules desoperculies {urent 

16unies et divis6es en deux groupes 

C i;r:e, lcs petites et lcs Erondcs, -d'aprds 

leurs tailles. Les petites larves avaieni 3.4 

jours, et les grandes avaient 4,5 b 5,5 jours. 

La moitid des larves furent tu6es par rcfroidissement 

avec de la neige carbonique. 

Des qroupes rle larves furent cnsuitr inocu. 

lds ave

Chalkbrood - Status Today and Hopes for Control' - Golden Rule Honey

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