Flagellated protozoa (Trypanosomidae) in the honey bee, Apis mellifera, in Australia

124

NOTES

Flagellated

Protozoa

(Trypanosomidae)

Apis mellif

era,

This note records, to the best of our knowledge, for the first time in Australia, the presence of a flagellated protozoon, of the family Trypanosomidae in the honey bee, Apis mellifera. The flagellate probably belongs to the genus Crithidia. It is suspected that there is possibly a causative relationship between the flagellate and an abnormal mortality of adult worker honey bees. For many years, the Apicultural Research Unit of the Victorian Department of Agriculture has maintained a diagnosis service to the industry in this State. Every year a large number of samples of adult honey bees showing abnormal mortality, is received at the Unit. In the majority of cases the cause of mortality can be attributed to one of the common diseases of bees. There are, however, a significant number of samples received, on which no diagnosis, in terms of known diseases of adult bees, can be given. In recent years, sufficient evidence has been accumulated to permit a diagnosis of approximately half of the samples, previously reported as “cause of mortality unknown,” as “suspected virus infection.” During the latter part of 1964 and early 1965, a close examination of those remaining samples, to which no cause of mortality could be attributed, showed the presence of a very actively motile organism. Examination with ordinary and phase-contrast microscopy led us to suspect that this organism was a flagellated protozoon. To obtain a more positive identification, fixed material was submitted to an authority for further examination. Results of this examination showed the organism to belong to the family Trypanosomidae and the genus Cvithidia (R. B. McGhee, 1965, University of Georgia, Athens, Georgia; private communication) . The generic classification is

in

in

the

Honey

Bee,

Australia

subject to review on further examination of cultured material. The material examined in the present instance was taken from the hindgut of adult bees found disabled and crawling on the ground in front of colonies in the experimental apiary of the Apicultural Research Unit at Ferntree Gully, Victoria. Similar organisms have been found in specimen material submitted from many parts of Victoria. At the present time there is no concrete evidence that the organism under review is pathogenic to bees. However, this flagellate has been found in disabled bees from colonies showing abnormal mortality, and in which no other known disease of adult bees could be

FIG. 1. Protozoa bee. Giemsa stain.

in rectal fluid of worker Approx. X 1000.

honey

125

NOTES

worker signs.

found. This would therefore tend to indicate a possible causative relationship between the flagellate and the abnormal mortality of honey bees. It has been found from examination of a large number of adult bees that the flagellates are most likely to be found in adult worker bees in which the rectum is distended with an almost clear fluid, containing clearly visible bodies in suspension, consisting of coagulated pollen grains. Figure 1 shows a photomicrograph of a smear of the rectal contents of an adult

A Modified

Azan

The author wishes to thank Dr. G. Ettershank, Dr. G. Davis, and Mr. R. Gladwyn of Monash University for their advice, guidance, and assistante with the microscopic preparations.

D. F.

the

Georgia

Coastal

Accepted November

Body

Plain

LANGRIDGE

Department Of AgracuZture, Apicultural Research Unit, Ferntree Gully, Victoria, Australia

A. Huger (J. Insect Pathol., 3, 338-341, 1961) briefly reviewed the use of acid or alkali pretreatment for staining polyhedra with different stains and presented two methods for staining capsular virus inclusion bodies (granules) in which he used different acid pretreatments and different lengths of time in the iron alum mordant and in Heidenhain’s iron hematoxylin. J. N. Hubschman (Stain Technol., 37, 379380, 1962) reviewed some modifications of the mallory triple connective-tissue stain in which azocarmine is substituted for acid fuchsin and then presented a simplified process using this stain for crustacean tissue. When this simplified azan technique was applied to acid-treated sections of lepidopterous larvae infected with a nuclear-polyhedrosis virus, the polyhedral inclusion bodies stained bright red. In order to increase the contrast between infected and noninfected cells, fast green FCF was added to the counter-staining solution. All specimens were fixed in Bouin Duboscq Brasil, and paraffin sections were cut at 4-6 p in thickness. with

the above-mentioned

ACKNOWLEDGMENTS

Staining

Inclusion

1 In cooperation Experiment Station.

bee, showing

Technique

1.5, 1965

for

Viruses’ STAINS

Solution I. Dissolve 0.1 g of azocarmine G in 100 ml of distilled water and boil the SO~Ution for 5 minutes. Allow to cool and add 2 ml of glacial acetic acid. Filter before use. Solution ZZ. Dissolve in 100 ml of distilled water: 1.0 g phosphotungstic acid, 0.1 g aniline blue (water soluble), 0.5 g orange G, 0.2 g fast green FCF. STAINING

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

PROCEDURE

Toluene via alcohols to water 50 percent acetic acid, 5 minutes Distilled water rinse, 2 minutes Azocarmine (Sol. I), 15 minutes Distilled water rinse, 5 seconds Aniline, 1 percent in 95 percent alcohol, 30 seconds Distilled water rinse, 5 seconds (should be changed often) Counterstain (Sol. II), 15 minutes 50 percent alcohol, 10 seconds Absolute alcohol, 2 changes, 30 seconds each Toluene, 2 changes Mount in neutral, synthetic mounting medium