PUBLICATIONS of MARTHA GILLIAM Bee Jour. 107:256. 1967. Mellifera L.) from Various Elevations. Experi-Entia 26:1006-1007. 1970

B00K #1

PUBLICATIONS OF MARTHA GILLIAM

1. Blocker, Martha Gilliam. Studies on honey bee (Apis mellifera L.) hemolymph. M.S. Thesis. University of Wyoming, 50 pages. 1966.

2. Gilliam, M. and Shimanuki, H. Total hernocyte counts in hemolymph of immature honey bees. Amer. Bee Jour. 106:316. 1966.

3. Gilliam, M. and Shimanuki, H. Progress report: studies on honey bee blood. Amer. Bee Jour. 107:256. 1967.

4. Gilliam, M. and Shimanuki, H. In vitro phagocytosis of Nosema apis spores by honey-bee hemocytes. Jour. Invertebr. Path. 9:387-389. 1967.

5. Gilliam, M. and Shimanuki, H. Coagulation of the hernolymph of the larval honeybee. The XXIst Internationa] Apicultural Congress, p. 469, University of Marylan d, lE67 . Apimondia Publishing House, Bucharest, Romania. 1969.

Gilliam, M. and Jeter, W. S. Synthesis of agglutinating substances in adult honeybees against Bacillus larvae. Jour. Invertebr. Path.- 16:69-76. 1910.

7. Gilliam, M. and Shimanuki, H. Coagulation of hemolyrnph of the larval honey bee (Apis mellifera L.). Experientia 26:908-909 . 1970.

8. Gilliam, M. and Shimanuki, H. Total hemocyte counts of honey bee larvae (Apis mellifera L.) from various elevations. Experi-entia 26:1006-1007. 1970.

9. Gilliam, M. Microbial sterility of the intestinal content of the immature honey bee, Apis mellifera. Ann. Entomol. Soc. Amer.64:315-316. 1971.

10. Qil!!1q,_M. and Shimanuki, H. Blood cells of the worker honeybee. Jour. Apic. Res. 10:79-85. 1971.

11. Gillia_m, M. and Jackson, K. K. Proteins of developing worker honey bees, Apis mellifera. Ann. Entomol. Soc. Amer. 65:516-517. 1972.

12. Gilliam, M. and McCaughey, W. p. Total amino acids in developing worker honey bees (Apis mellifera L.). Experientia 28:142-143. 1972.

13. Gilliam, M. Gel filtration of the hemolyrnph of the honey bee (Apis mellifera L.). Experientia 28:341. 1972.

14. Gilliam, M. and Jackson, K. K. Enzymes in honey bee (Apis rnellifera L.) hemolymph. Comparative Biochem. Physiol. 428:423-42i. 1972.

15. Gilliam, M. and Prest,D.B. Fungi isolated frorn the intestinal contents of foraging worker honey bees, Apis mellifera. Jour. Invertebr. Path. 20:101-103. 1972.

16. Gilliam, M. Lack of effect of deficient diets on hemolymph proteins of adult worker honeybees. In Insect and Mite Nutrition, ed. J.G. Rodriguez, p.281-286, North- Holland Publishing Co., Amsterdam. 1972. (By invitation) 2

17. Gilliam, M. Age-dependent variation of differential haemocyte counts of developing worker honeybees. Jour. Apic. Res. 12:61-64. 1973.

18. Gilliam, M. Immunological studies on the honey bee (Apis mellifera L.). Ph.D. Dissertation, University of Arizona, 38 pages. Also Dissertation Abstracts Internation al 34:1 I 668- 1 1678. 1973 .

19. Gilliam, M. and Valentine, D. K. Unusual electrophoretic behavior of proteins from honey bee hemolymph. Comparative Biochem. Physiol. 458:463-466. 1973.

20. Gilliam, M. and Valentine, D. K. Enterobacteriaceae isolated from foraging worker honey bees, Apis mellifera. The XXIVth International Apicultural Congress of Apimondia, Buenos Aires, Argentina, p. 404-407. Apimondia Publishing House, Bucharest, Romania. 1973.

2t. Gilliam, M. and Taber, S., III. Microorganisrns and diseases encountered in continuous bee production. Amer. Bee JoLrr. 113:222-223. 1973.

22. Gilliam, M. Are yeasts present in adult worker honey bees as a consequence of stress? Ann. Entomol. Soc. Amer. 66:1176. 1973.

23. Gilliam, M. Total amino acids in pollen fumigated with ethylene oxide. Environmental Entomol. 2:881-882. 1913.

24. Gilliam, M. and Valentine, D. K. Enterobacteriaceae isolated from foraging worker honey bees, Apis mellifera. Jour. Invertebr. Path. 23:38-4I. 1974.

25. Gilliam, M. and Morton, H. L. Enterobacteriaceae isolated from honey bees, Apis mellifera, treated with2,4-D and antibiotics. Jour. invertebr. Path. 23:42-45. [gl+.

26. Argauer, R. J. and Gilliam, M. A fluorometric method for determining oxytetracycline in treated colonies of the honey bee, Apis mellifera. Jour. Invertebr. Path. 23:51-54. 1974.

27. Gillia-m, M., Prest, D. B. and Morton, H. L. Fungi isolated from honey bees, Apis mellifera, fed2,4-D and antibiotics. Jour. Invertebr. Path. 24:213-217.- 1974.

28. Gilliam, M., Wickerham, L.J., Morton, H. L. and Martin, R. D. Yeasts isolated from honey bees, Apis mellifera, treated with 2,4-D and antibiotics. Manuscripts VII Ann. Mtg. Soc. Invertebr. Path. , p.29, Tempe, Arizona. 1974.

29. Prest, D. B., Gilliam, M., Taber, S., III and Mills, J. P. Fungi associated with discolored honey bee, Apis mellifera, Iarvae and pupae. Jour. Invertebr. Path. 24:253- 255. t974.

30. Gilliam, M., Wickerham, L. J., Morton, H. L. and Martin, R. D. Yeasts isolated from honey bees, Apis mellifera, fed2,4-D and antibiotics. Jour. Invertebr. Path. 24:349-356. 1974.

3r. Gilliam, M. and Argauer, R. J. How long is Terramycin stable in diets fed to honey bee colonies for disease control? Amer. Bee Jour. 115 230 and 234. 1975.

32. Gilliam, M. The absence of yeasts in nectars of selected Arizona plants attractive to honey bees, Apis mellifera. Ann. Entomol. Soc. Amer. 68:705-706. 1975. 33. Gilliam, \4. and Argauer, R. J. Stability of oxytetracycline in diets fed to honeybee colonies for disease control. Jour. Inveitebr. P-ath. 26:383-386. 1975.

34. Gilliam, M. and Valentine, D. K. Bacteria isolated from the intestinal contents of F^tqg_tlg wgrker honey bees, Apis mellifera: the genus Bacillus. Jour. Invertebr. Path. 28:275-276. 1976.

35. Gilliam, M. Yeasts and honey bees. Jour . Aiz. Acad. Sci. 1 l:46-47. 1976.

36. Gilliam, M. and Prest, D. B. The mycoflora of selected organs of queen honey bees, Apis mellifera. Jour. Invertebr. Path.29:235-237. 1977.

37. Gilliam, M.,- Morton, H. L., Prest, D. 8., Martin, R. D. and Wickerham, L. J. The mycoflora of adult worker honey bees, 4pis mellifera: effects of 2,4,5-T and caging of beecolonies. Jour. Invertebi. Path.30:50-54. 197i.

38. Gilliam, M., Taber, s., III and Argauer, R. J. Degradation of oxytetracycline in medicated sucrose and honey stored by caged honey bees, Apis m6llifera. Jour. Invertebr. Path. 31 : 128-130. 1978.

39. Gilr{3ln, M., Taber, s., III and Rose, J. B. chalkbrood disease of honey bees, Apis mellifera L.: a progress report. Apidologie 9:75-89. 1978.

40. Gilliam, M. Bacteria belongilg to the genus Bacillus isolated from selected organs of queen honey bees, Apis mellifera. Jour. Invertebr. path. 31:389-391 . 1978.

41. chalkbrood - status Qiltiaryr M. today- and hopes for control. Amer. Bee Jour. 118:468-471. 1978. (By invitation)

42. Gilliam,_M,, Jaber, S., III and Rose, J. B. Research on chalkbrood disease of honey bees. XXVIth International Congress of Apiculture of Apirnondia, Adelaide, Australia, p. a51-458, Apimondia Publishing House, Buiharest, Romania. igla. 43. Gilliam, M., Taber.l._, II! 1nd Rose, J. B. Research on chalkbrood disease of honey bees. Apiacta 13:67-75. 1978. (By invitation)

44. Gilliam, M. and Dunham, D. R. Recent isolations of Bacillus pu-!-yifaqtens from powdery scales of honey bee, Apis mellifera, larvae. Jour. Inv-ertebr. Path. 32:222- 223. 1978.

45. Gi[gm, M. Fungi. In Honey Bee Pests. Predators. and Diseases, ed. R. A. Morse, p. 78-101, cornell university Press, Ithaca, New York. 1978. (By invitation)

46. Gilliam, M. and Morton, H. L. Bacteria belonging to the genus Bacillus isolated from honey bees, Apis mellifera, fed2,4-D and antifioti=cs. np{aorcgie92n-zzt. l9ig.

47. Gil_liam, M. Microbiology of pollen and bee bread: the yeasts. Apidologie 10:43-53. 1979.

48. Gilliam, M., Taber,^S., IIIan_d Argauer, R. J. Degradation of Terramycin in honey. Amer. Bee Jour. ll9:720, 722, 723. 1979. 4

49. Gilliam, M. Microbiology of pollen and bee bread: the genus Bacillus. Apidologie 10:269-274. 1979.

50. Gilliam, M. Le couvain platre. Sante de I'Abeille 54:119-183. 1979. (By invitation)

51. Gilliam, M., Taber, S.,III and Argauer, R. J. Degradation of oxytetracyclinein sugar syrup and honey stored by honeybee colonies. Jour. Apic. Res. 18:208-211. 1979.

52. Loper, G. M., Standifer, L.N., Thompsotr, M.J. and Gilliam, M. Biochemistryand microbiology of bee-collected almond (Prunus dulcis) pollen and bee bread. I. Fatty acids, sterols, vitamins and minerals. Apidologie 11:63-73. 1980.

53. McCaughey, W. F., Gilliam, M. and Standifer, L. N. Amino acids and protein adequacy for honey bees of pollens from desert plants and other floral sources. Apidologie I 1:75-86. 1980.

54. Gilliam, M. Control of chalkbrood disease of honey bees, Apis mellifera L.: difficulties and possibilities. XVI Int. Congr. Entomol. Abst., p.426, Kyoto, Japan. 1980. (By invitation)

55. Gilliam, M., McCaughey, W. F. and Wintermute, B. Amino acids in pollens and nectars of citrus cultivars and in stored pollen and honey from honeybee colonies in citrus groves. Jour. Apic. Res. 19:64-12. 1980.

56. Standifer, L. N., McCaugh€y, W. F., Dixon, S.8., Gilliam, M. andLoper, G. M. Biochemistry and microbiology of pollen collected by honey bees (Apis mellifera L.) {rom qlmgnd (irynus dulcis). II. Protein, amino acids and enzymes. Apidologie lI:163-171. 1980.

57. Gilliam, M. Control of chalkbrood disease of honey bees, Apis mellifera L.: difficulties and possibilities. (In Japanese) Honeybee Science l:159-162. 1980. (By invitation)

58. Gilliam, M. and Argauer, R. J. Oxytetracycline residues in surplus honey, brood nest hougy, and larvae after medication of colonies of honey bees, Apis mellifbra, with antibiotic extender patties, sugar dusts, and syrup sprays. Envirbn. Entomol. 10:479- 482. 1981.

59. Gilliam, M., McCaughey, W. F. and Moffett, J. O. Amino acids in the floral nectar of cotton. Apidologie 12:125-132. 1981.

60. Gilliam, M. Nutrition of honey bee queen, drone, and worker brood and adults. Beekeeping Short Course Syllabus, p.23, Coop. Extension, Coll. of Agric., Wash. St. Univ. 1981. (Byinvitation)

61. Gilliam, M. Control of chalkbrood disease of honey bees: difficulties and possibilities. Amer. Beekeeping Fed. 37th Ann. Meeting Proc. Inform. Sess., p.5. 1981. (By invitation)

62. Gilliam, M. Amino acids in the floral nectar of cotton (Gossypium spp.). Jour. Ariz.- Nev. Acad. Sci. 16:17. 1981. 5

63. G-illiam, M. and Argauer,_ R. Terrarnycin J. residues in surplus and brood nest honey- after medication of honeybee-1981. colonies by three different melhods. Glean. Bee Cult. 109:545-546, 550-551.

64. G_illiam, M. qd Algqqer-,-R. J._ Terrarnycin in diets, bees, and stores. (In Japanese) Honeybee Science 3:55-62. 1982. (By invitation)

65. Gilliam, M. and Argauer,-R. J. Oxytetracycline residues in honey from colonies of 4zu m,etli_lef medicated for diseasecontrol. Invertebrate Pathology and Microbial Control, III Int. Colloq. Invertebr. Path. and XV Ann. Mtg. Soc. Invertebr. Path. Abst., p. I27, Brighton, England. 1982.

66. Gilliam, M., Taber, s., III and Richardson, G. v. chalkbrood disease and hygienic behavior of honey bees. Glean. Bee Cult. I 1 1 :258, 264-265. 1983.

67. Gilliam, M., Taber, S., III and Richardson, G. v. Hygienic behavior of honey bees in relation to chalkbrood disease. Apidologie 14:29-39. 't-g8l.

68. Gilliam, M. and Lore^n_2, B. J. Gram-positive cocci from apiarian sources. Jour. Invertebr. Path. 42:187-195. 1983.

69. ch4-s.tensen, M. and Gilliam, M. Notes on lhe Ascosphaera species inciting chalkbrood in honey bees. Apidologie t4:29t-29j.-T98f .

70. Gilliam, M., Moffett, J. o. and Kauffeld, N. M. Examination of floral nectar of citrus, cotton, and Arizona desert plants for rnicrobes. Apidologie 14:299-302. 1983.

71. Gilliam, M. Recent research on chalkbrood disease of honey bees. The XXIXth Irternatiora!Qqlgr.ess Apiculture _o_f of Apimonclia, Budapeit, Hungary, p.224-225, Apimondia Publishing House, Bucharest, Rornania. 1983.

72. Gilliam, M., Taber, S., III and Richardson, G. v. selective breeding of honey bees for.hygienic behavior to control chalkbrooc't disease. XVII Ann. Mtgl Soc. Invertebr. Path. Program & Abst., p. 38, Davis, California. 1984.

73. Gilliam, M., Buchmann, S. L.,Lorenz, B.J. and Roubik, D. w. Thevulturebeeand path. program Eagillus.spp._XYII Ann. Mtg. soc. Inverrebr. & Abst., p. 61, Davis, California. 1984.

74. Gilliam, M., Buchmann, S. L. and Lorenz, B. J. Microbial floraof thelarval glovt|lo^n.s of the solitary bees, Centris pallicla and Anthophora sp. Apidologie 15:1- 10. 1984.

75. Gilliam, M. Letter to F.C.A.A.A.-New Zealand chalk brood. Australian Bee J. 65:7. 1984. (By invitation)

76. Gilliam, M..,. Buchm?nn, !. L.,Lorenz,.B. J. and Roubik, D. w. Microbiology of the F|vul provisions g{ thg.slilgless bee, Trigona hypogea, an obligate necrophage. Biotropica l7:28-31. 1985,

77. Gilliam, M. Microbes from apiarian sources: Bacillus spp. in frass of the greater wax moth. Jour. Invertebr. Path. 45:218-224. 1985. u-?' B ,,1t #79 rflHH BLOOD, or hemolymph, of the I honey bee (Apis mellifera L.) is a pale yellorvish liquid containing blood cells variously referred to as hemocytes, Total Hemocyte Counts In blood corpuscles, or leucocytes. Blood vessels are not present in insects. The Hemolymph of Immature hernolyrnph fills the spaces in the body cavity and bathes the sulfaces of the tissues. Honey Bees Little research on the total henocyte count (THC) of honey bees has been by MARTHA GILLIAM2 ond HACHIRO SHIMANUKI3 reported. Szyszkin (1958) ancl Kostecki Entomology Reseorch Division, ( 1965) fotrnd that the nutttber of hemo' Agr. Res. Serv., USDA, Loromie, Wyo.a cytes per rnnr3 of hernolynrph tends to decrease rvith age in the adult honey bee. The purpose of our investigation determine the THC's of im- lvas to if but decreased at day 6, From the 6th Table Il.-Comparison of Total Hemo- bees decreased u'ith age. mature honey to the 15th day, the number of cells cyte Counts in llemolymph of Drone for THC, To obtain hemolymph a gradually increased (Table I and Fig- and Worker Larvae and Pupae. insect rvith a sterile rve punctured the ure I). The THC's of drone, rvorker hemolymph hypoderrnic needle. The Iarvae, and pupae never differed by Age (days) and the rvound was rvhich exuded flom more than a few cells (Table II). Our sex of bees Average THC* 0.5 mark Thoma drarvn to the of a results showed that the THC's of im- pipette and diluted Laruae rvhite-cell-dih-rting mature honey bees did not decrease mark rvith Toisson's fluid (1.0 5, u'orker 10,0+0 to the 1l rvith age. g sodiurn chloride, 8.0 g sodium sulfate, 5, drone 10,720 30 ml glycerin, 15 rng crystal violet, 160 Table I.-Total Hemocyte Counts in 6, rvorker 9,1 20 ml distilled rvater). After thoroughly Hemolymph of Immature Honey Bees. 6, drone 8,200 nrixing the fluid in the pipette for 2 Pupae minutes, rve discarded the first 3 drops Age (days) Average THC* 12, worker 9,020 and trsed the fourth dlop fol the count, 12, drone 8,960 Laruae enrploying a Spencer bright-line hemo' 5 10,040 *Average individual samples cytometer with improved Neubauer rul- of 5 ing. \Ye included the cells in the 4 6 8,120 8,660 References corners and central square in the count, Kostecki, R. 1965. Investigation'--on Uo 9,520 and rnultiplied the sum by 40 to give the haemocytes and haemolymph of the number of cells pel mrn3. If the Pupae t2 9,020 honeybees. Jour. Apic. Res. 4:49-54. cells 'rvere unevenly distributed, rve dis- Szyszkin, B. 1958. Fornriennyie 15 l l,920 A. carded the sample. The average THC's elementy gemolimfy miedonosnoj pczoly. rvere based on 5 indiviclual samples' *Average of 5 individual samples Uch. Zap. B. G. P. L 15:14l-162. o T'HC's rvere taken for larvae rvhen the bees were 5, 6, 7, and 8 daYs old and for pupae rvhen they rvere 12 and 15 days old. We began the counts at 5 days because the younger larvae rvere a too srnall to use for the counts. We rvere a) z unabie to drarv sufficient quantities of hernolyrnph before the 5th day of age. a) \Ve did not tttilize nrature bees since Kostecki (1965) published a rvell-docu- F mented report on the TIIC's of adult nz bees of various ages. Horvever, rve did t coIlrpare THC's of drones rvith those of rvorker larvae of the same age. a pupae hacl more he- F Fifteen-day-old z nlocytes per ttrnrS of hernolymph than l 6 o othcr irnrrtatul'e stages. In the irnntature o 5 stages, the TIIC's rvere high at day 5 trl F o 4 I The data contained herein constitute o nart of c thcsis subnlitted by the scnior u i"uthor in -forpartial fulfillment of the re- r ouirements the degree of llaster of Scioncc, Unirersity of Wyonring. J :Present address: Department of }Iicro- F Arizona, Tucson. o biolclgy, University of F 3Plesent adtlress: Bee Disease Labora- torl'. Asricullural Rescarch CFnter, F-\T Buildini A, Bcltsvillc, Ilar]'lan(1. r Published with approval of the Direc- 56?89lOll1213 tor, tVt'or,'ing Agricttllulal F:-\l','tilrlarrt AGE OF BEES IN DAYS St:rtiorr. as Journal Artielc 304. Reprinted from October, 1966, American Bee Journal

/ t t -t -tfl eased bees might contain the causativc organism of the infection. progrett Honey bee hemolymph w'as tested for Knport, SfuJrno the presence of various clotcing factors which exist in human blood. The blood of this insect did not clot because of on JJorny Rnn R/o"J' the absence of a factor knorvn as proconvertin and because it contained a circulating heparin - like anticoagulant. by MARTHA G|[UAM2 qnd HACHIRO SHIMANUKIs However, bee blood and human blood Entomology Reseorch Division, Agr. Res. Serv., USDA do contain seven other common coagulation factors. From the standpoint of Loromie, WYominga evolution, this information was quite interesting. mHE blood, or hemolymPh, of the blood to determine the number of blood Literature Cited hon.y bee (APis mellilera L.) is cells per mm3 of hemolyrnph. As the I Bishop, G. H. 1923. Body fluid of yellorvish liquid containing blood elevation increased, the number of blood a pale the honey bee larva. I. Osmotic pres- cells variously referred to as hemocytes, cells increased. A definite correlation sure, specific gravity, pH, O, capacity, blood corpuscles, or leucocytes. Accord- existed bet'rveen the log'o number of CO: capacity, and buffer value, and ing to Bishop (1923), the blood of the hemocytes per mm3 and the log'o eleva' their changes with larval activity and larvae is a clear fluid rvhich comprises tion from lvhich the lan'ae were ob' metamorphosis. Biol. Chem. 58:543- 25-30% of the whole body weight. tained. This suggested that honey bee J. 565. Insect blood is not carried in blood hemocytes might contain a respiratory vessels. It fills the spaces in the body pigment similar to hemoglobin or hemo- Jones, J. C. 1962. Current concepts cavity and bathes the surfaces of the cyanin rvhich is a special carrier of concerning insect hemocytes. Am. Zool. organs. Wigglesrvorth (1961) states that respiratory gases. Further studies to 2:209-246. most hemocytes lie on organ surfaces, confirm this finding are anticipated in Nelson, J. A. 1915. The emblyology but some do circulate in the blood' the future. of the honeybee. Princeton University lVhen blood cells are associated rvith While performing total hemocYte Press. Princeton, New Jersey. 282 p. tissues, they become elongated. Freely counts (THC) upon bees infected rvith Wigglesrvorth, V. B. 1961. The prin- circulating hemocytes are round to oval. Nosema apis, it was noted that the ciples of insect physiology. Methuen Most of the blood cells appear to orig- THC was greatly reduced. To deter- and Cornpany Ltd., London. 516 p. inate by mitotic or amitotic multipli- mine what rvas happening to the blood cation of existing blood cells. Nelson cells of the diseased bees, a suspension t. (1915) states that the leucocytes are of Nosema spores (108/ml) rvas added produced in the mesoderm of the em' to bee blood in a depression slide. The bryo. slide was observed at 430X at room The four best knorvn functions of temperature. lVhen the N. apis spore insect blood cells are phagocytosis, for- suspension rvas added to the hemo- mation of connective tissue around for- lymph, the hemocytes remained rela- eign material, secretion of enzymes, and tively stationary. The spores appeared coagulation of the hemolymph (Jones, to be attracted to the blood cells by 1962). According to Wigglesrvorth some kind of chemotactic action. The (1961), the natural immunity of insects spores entered through the cell t'alis, to bacteria is chiefly a phagocytic im- and no pseudopods rvere observed. As munity. The primary factor in acquired the blood cells became gradually filled immunity seems to be an increase in with spores, a characteristic invagina' phagocytic rate. tion of the cell occurred. The blood quantity We found that honey bee blood con- cells then ruptured due to the tained seven different types of blood of spores ingested, and the cell contents cells in addition to trvo types of stem spilled out. This entire process took cells. All hemocytes rvere ultimately approximately 45 minutes. All types derived from the stem cells. The dom- of blood cells rvere affected. Nosema inant type of blood cell varied rvith the spores in sufficient quantities thus age but not with the sex of the insect. tended to destroy hemocytes. The de- could eventually Five - day - old larvae were obtained struction of blood cells from various elevations ranging from rveaken the insect, and possibly lead to 159 feet belorv sea level to 7200 feet its death. This process might explain honey bees above sea level. Total hemocyte counts some of the symptoms of (THC) were made upon the larval infected 'iith Nosema apis. The total hemocyte count was a good diagnostic ! The data contained herein constitute test for Nosema disease since the THC oart of a thesis submitted bv the senior significantly lorver iuthor in -foroartial fulfillment of the re- of infected bees rvas ouirements the degree of Master of than that of healthy bees. The spores Science. Universitv of Wyoming. rPredent address: Department of luicro- could also be observed in the hemo- biolosv. Universitv of Arizona, Tucson. rPrAsent address: Bee Disease Labora- lymph. tory, Agricultural Research Center, Belts- The blood from healthy bees rvas ville. Maryland. -a Fublished rvith approval of the Direc- found to be relatively bacteria and fungi tor. lvyoming AgriCultural Experiment Horvever, the hemolymph of dis- Station,-as Joumai Article 318. free. Reprinted from July, 1967, American Bee Journal Vol. 107 (7): 256 I LIri

l I l l .J { I

{

i InYitro PHAGOCYTOSIS OF Nosenra apis SPORES BY i !' ,t IIONEY-BEE HEMOCYTES

Manrrre Gr"r.reu e:vn Hecrrmo Snr.velruxr

Reprinted frour JounNar, or INwntsBRAtr PrrHor.ocv, Vol' 9, No' 3, September 1967 Copyright @ 1967 by Academic Press Inc. Prhfieil dn U.S.A. l { \

l Reprintecl frorn JOUR\.\L Of- I\\'EIi'I'EBR.\TE I'ATHOLOGf, \'ol. 9, No. 3, September 1967 Cor:r-right aO 1967 by' Acaclenric Press Inc. Priried in U.S.A.

JOURNAL OF INVERTEIJRATE PATIIOLOGY 9, 387_389 (1967)

In Yitro Phagocytosis of Nosentu rrlris Spores by Honey-Bee Hemocytesr'2

I\'[.qnfi{e Crr-lrerts AND HACHIno SHrltaNuxra

Entomology Research Diaision, Agricultural Researclr Seraice U.S. Department of Agriculture, Laramie, Wgoming

Acceptetl Septernber 12, 1966

Phagocl'tozed Nosema apis spores caused hemocl'tes of the honey bee, Apis nrcIlifera, to mpture. This phenornenon may be responsible for the weakened state and the ensuing death of the insect.

IxrxopugrroN ejected rvith feces to contaminate the food or water of other bees, thus trans- Nosema disease of the honey bee, mitting the protozoan. Apis melliferd, is caused by a microscopic A graclual weakening of infected insects single-celled protozoan> Nosenw a'Stis, be- is quite characteristic; diseased bees are longing the order N{icrosporidia. The to unable to lly rvithout alighting frequently. life cycle of this organism has not yet been Also the stomach is swollen, ancl becomes elucidated. Flou'ever, in one stage of its rvhite in color because of the spores. Thc developnrent, Nosenta apis forms highly disease is usually cliagnosed by removing resistant spores s'hich refract light so that, the abdomen rvith forceps and pulling out rvithcxrt staining, they can be seen ever.r the digestive tract; the gut contents are clearly urrclcr the microscopc. then smeared upon a slide and observed The trar.rsmitted through pathogen is under the rnicroscope for the presence of food, rl,rrter, equipment contaminated or spores. Nose?r?n spores. First the young para- w'ith Professor Zander (f909), rvho cliscov- sites gro$' ancl rnultiply rvithin the stom- ered the causative organism of nosema ach; then they enter the epithelial cells disease, stated thr,rt the midgut of the bee lining the midgut (Grout, l9{9). When is the main site of this parasite. Flon'ever, epithelial cells break, the the *'rrlls of tlie Steche (1960) found Nosemo api.s in the spores are liberated. They carl no\v be ovaries and mandibular glands. Also Soko- ' The tlirta contained herein cotrstitrttecl a ptrrt lov ancl Grobov (1963) found lJosema of tr thesis sul;rnitted by the senior author iu par- spores in the hemolymph, pharvngeal tial fullillnent of the reqttircttients fcir the clegree bees of llaster of Science, Universitl'of \\'1'onring. gland, and thorercic gland of infected 'Publisliecl *'ith approval of the Director, and notecl that Nosenlo spores introducetl \\'yorning Agrictrltural Erperinrent Station, as l'ith hen-rolvrnph into healthy bees caused Journal Article 302. typical clevelopment of the disease; there- " I're'sent aclrlress: Depirrtruent of \licrobiology, fore they postulatecl that the organism University of Arizortrr, Trtcson. causing noserra disease can probably go " Present acltlress: Bee l)ise'ase Lal;orttorl', i\gri- culttrral Ilt'search Center, Beltsvillc, \larl'lirncl. from thc hemolymph into the gut as s'ell

387 38E GILLIA}T AND SHIMANUKI

as from the gut into the hemolymrrh. Ou hemglymplr in a clepression the other hand, Miiller (1925) slicle, and 0.02 obr"i"d no cc of a l0t/ml suspension differences betlveen of No,senm (lrtis the bloocl spores rvas of healthy "orp.,r"l", added. The .slicle ruas observe

l',i

f.i J K L t .:l l#ffiffiffi Frc. 1. Phagocytosis of Noserna apis spores by honey-bee hemocl'tes.

This destmction of blood cells g'ould even- arrcl mctamorphosis. J. BioI. Clrcnt., 58, 543- JbD. insect, and cleath rvoulcl tually t'eaken the Gnour, R. A. 1949. "The Hive and the Honey resuli. The process may therefore possibly Bee," D.rdant and Sons, 652 pp' Ilamilton, explain the slmptoms of honey bees in- Ilhnois. fectcd $'ith Noselw apis. Nli;rr-rn, K. f925. Uber die korpuskultiren Ele- bei der errvachsenen The TFIC proved a goocl cliagnostic test mente der Bhrtfliissigkeit Honigbiene (Apis mellifca L.). Erlanger that of irlfectecl bees rvzrs for l,losento since Jahrb. Bienenkuncle, 3, 5-27. Iou'er than that of herrlthl'bees' The spores Soxor-or', V. P., -lnrr Gnotov, O. F. 1963. Nosctna coukl also be obsen'ed in the hemoll'mph. spores in the hemoly-mph of the bee. Pclrclo- xodstao, T, 39. REnEnaxces Srrcrrr, \Y. 1960. Atiologie uncl Therapie cler Noserrratose rler llonigbierrc, Z. Bienenforsch., .49-52. Brsuor, C. H. 1923. Ilodl' fltricl of tlie honel' bee 5, larvi'te. I. Oslnotic pr..essttre, speciffc gr:lvit)', Z.rxmn, E. 1909. Tierische Parasiten als Krank- pH, O" caprrcitl', COr cilpilcitl', and buffer lreitst-rreger Bei der Bicne . Leipziger Bie- vnltre, ancl their changes *'ith lar-val activitl' nen:t+., 24, I1i-I30, 1M-l66. 5

rl COAGULATION OF HBMOLYMPH OF THE LARVAL HONEYBEE MARTHA GILLIAM ANd HACHIRO SHIMANUKT

mellitero' was' Poolecl larval hemolymph of blood of the honev-exist bee. 'in APis coagulants that human blood. The tested for the presence of-the various in i"rii- inJi"i ted' that bee blood contained the following coagulants found human blootl : 1. Partial tltromboPlastins 3. Antihemophilic globulin 3, I)lasma tliromboplastin antecedent .{. Proaccelerin 5. Plasma thromboplastin component 6. Hageman factor ?. Stuart factor blood, but the The concentrations in the hemolymph were less than in human p[esent' coagulants were, nevertheless, addition, it con- Hemolymph did ;;i-;ibit"*ur" it lacked proconvertin. In tained t circulating heparin-like anti-coagulant'

Irom Proc.-Apirnond,ia XXI Int. !.pic. Congr., pr h69. University of Maryiand', 145i.- Publishing llouse.- Bucharest, Romania. Published in l_969. Paper presenied al ihe xJrst, rnte.naii o::al- r-pi-cur-turar- co::gress - lla:.^y1ai:c, rg5? Coagulation oi Heinolyroph oi lhe Larval Honey p,"t

Marrha ClLLLer9/ ani. I{achiro Shinanukt2/ Entonolog-y Resea;'ch Division, Agricultu-ral Research serwice,

U. S. Deparrr,nent of Agz.icuJ-ture, Lare:nie, Wyoning

Previous invesiigaiions j-n ihe fiel-d of invertebrate brood. coagul-aiion have consisied prilar.il-y of nozphological description ra-"her i;han id.en'i:ificaiion of tr.e cherdcal_ subsiances involved. in the clotiing process. ilor+eve::, Gregoire (t91r, ],]g|.7, lg5il siudied pa't telns of blood coaguJ-a't:-on in ve.z'i-ous insects by using phase-

coni;:'ast nicroscopy. A1so, j-n crr:siaceans, insecis, md spid.e:'s, the biood may coniain cel"] s waich libe:'aie substances that initiate or pz'onoie coagulaticn (i'facfa-i.l-ane and Robb--Snith, l96L). The puzpose of ihis invesiigai,ion was to d.eiernine whether the henolynph of the honey bee, Apis q:l-iifera, contains clotting fac-r,ors

:d'enilcal- io those found in huiaan bl ood. and. why tbe henol;mph does no.u

a^^*.'1 vuGSu:a -+^uc.

J/ Part of a thesis subniti;ed by i;b.e senior au'thor in partial fulfrllnent of ihe requirenents for ihe degree of Master of Science, Uni-versiiy of Wyoning. 2/ P=esent adc:ess: Depa-r'inent of l,(icrobiology, universiiy of Arizona, :}r;csoli, .!,yizona.

2/ P=eseri acdress: Bee Disease Labc:aioz'y, Agricur-turar B.esearch

Cente:', Beltsviil-e, Mraryland., I\LA,TERIAIS AI{D }MfIiODS

Henolyurph was obtained. by gentry puncturing larvae of various ages with a steri-le hypoderrnic need.le and d.rawing the fluid which emd.ed. fron the wound into a capilrary pipette. The henol;nnph was pooied in sheIl vials and s'i;ored. ai -20oc. (gtood of pupae and. adu'lts was not used. because of -r,he difficulty of obtaining sufficient quantities - ) This poolec l-anral- honey bee henolyoph was then .Lested. for the presence of the blood coagulants i;hat exist in hr:nan blood (rie. r).

Normal plasma and serun obiained fron hunan volunteers were used. as stand.ards. rnseci henoLya.ph was treaied, in exactly ihe same nanner as human b100c. The foll0wing d.ei;e:minations for "o^r- ulants were raaCe in tri_plicate on each sample: 1. One-stage prothronbj.n tine 2. Pari;ial thronboplastin tjne 3. ThronbopS-astin generation tesi /r. Thronbin clot time 5. Antihenophilic globulin (Facior VIII) 6. Plasma thronboplastin anieced.ent (Factor XI) 3

plcritla 1

BL00D C0-4.$ILI!TICU S3}il1.i3 Ci I.IA_!T

?lssue extract Plale] a'is UC. . Ca++ Proacceleiin Proaccelez'i-n Proconveriin -F;:'biheinophi li c g 3-obu3in Stueri factor PLasna thrornbopiasiin co:nponen'u Stua:'t facbor Plasraa ihronboplasii.n anbecedenb llageraan facbor

' Proi,h#rabinase + Prothro:abin + gfoYa thronboolas*r,in

?roacceLerin Pr"oconveriin

seYonbin fiibrinogen ---)labrt.n 4 7 - Recar cificaticn iine and. nonspecifrc nixing test for anticoag'*1an'r,s

8. Proaccele:'in (iactor V) 9. Proconve:.tin (Facto: VIi)

l-0. Piasc.a thronboplasiin conoonent (Factor IX) 1l-. Hagenan factor (Fac.i;o: XfI)

12. S'r,ua-z.t facicr (Faci;or X)

ESINTS The results of the coaguiation tesis are given in Tabres 1 and 2 and !ig. 2- The croiiing ti-r:.es of honey bee h.eraolpnph in each test i+ere a'r, leasi t',,+o io three ii-nes as long as tha'r, of birnan pJ-asna, probably because of the louer conceni;ration of coagulants Pi'esen-c in bee b'l ood - The absence of proconvertin from the. her.ollaph pleven-bed the fomraiion of a ciot in tne tesi of one-stage p:.o-r,hronbin ti.ne. Tabie 2 sholrs ihai; the la:,vai heno-r )ruch ieng.bhened the lecarcrficaiion time of hr:ren plasna. Thus, a cireulating anti_ coagulani and ihe absence of p-z'oconver.,in probabJ-y a.ccouni for ihe negaiive thronbin cloi; tj:le of bee btood The thronboplasiin gene:'a-tion c*rvbs of la-rval hernol-ynph and hr:nan (Fic- blood 2) were sinilaz'. Therefore, the henoly.nph was caoabl-e of generating in-r,rinsic thronboplastin i,riih .i;he aid. of hr,nan sera and. p1atelet,s. 5

?:ttt5 L

FJS]L?S 0F COic:L{?rO}t TESTS

Coagulati o t:.u=! r!/

l-asna l.arvql- henol:pph Oae-s'lage p:robhronbin ii-ne ]-E"B ) 2 hours Pariia'! thronrboplasii_n tine 2) "1 365"8 Thror,:oln clob tine 10"8 ) 2 hours Aniiheno;:hil-i QQr c giobulin Vvr) -.1 r,n L)J. )^ Plasna bh:-onboplasLin anLeceient 9?.5 2i.7.a Proaccelerirr 6l+"0 203-o Proconverlin 2L"2 ) 2 hou:s PLasna thro:rbcplastin cor,:ponen.b oJ") ^O/4U) rU^ iiagenae iect,or tll a JU()e< 355.3 Siuar-i facuor 20.2 37z.lt

3/ Average of three deter:r.inations !/ fn seccnds u:rless oihe=..iise noieC 6

tt F;;. 2

nESiJt?S 0F RfC:tJCIFiC:iTfCt.I ?Ii"E .*i.;D !,ICNSPECIFfC

LiI{i}iG :ES? tC3 .U:?fCOACinStitS

Subs:rate Cloiting tine-x-

O"5 nI hr::nan plasna 3 raj.nuies O"5 nl larval henoly:,ph ) 2 hours O"25 ni-htn:n plasna, O"25 nL laz'vaL henol;aipir t. ai_^..r^^

-x-Average of Lhree detendnat:-ons !:i.J!i 2

?J J'il!'S Cf '-.'.-:i.C_.-. O?LiS':'Iti

\\ \\ \ '\/'v DISCUSSJ_ON

The rests ind.i-cate thai; bee henolynph coniains the following coagulants found in hunan blood: 1. Pa:iia1 -biironboplestins

2. An-uihenophil-ie g3-obuJ-in

3 - Plasna tirronbcplasiin a-irteceCent /r. Proaccelez'in

5. Plasma thronboplas.i;in semFonent

6. Hagenan fac-bo-:, Stuari 7. factor I l The conceairaiions in the henolynlch were less than in huran blood, but the coagulanils Lrere, never-bhel_ess, present. .i The one-s'Lage prothronbin -bine -reathened. 1 r+as in i,he bee heno_ lynph by the absence of'p:oco:rvert:n. The conceniratlon of ii_ssue thronbop-lasii.n was neg'ligi-bie, but, tae pai,-tial tirronboplastin concen'r,3aiion u'as deiectabl_e. Siace ihe bee bIood gave a nornal -birronboo-l as-,,in genela'uion ilme cuf.ve, henolyrrph r,las obviously capable of generating ini;:.insic thronboplasiin when proconveriin was supplied by hr:nan serun. The :'eealcifi.cation and nonspecif.r c mixing iesis for antl- coagulan"Ls indicated thai a circur aiirrg anlicoagulant was p=esert in the h.eloJ-iti-oh since coagulaiion cf h-.:an piasr'.,a nas ienihened. by adiilion 'he of bee b-rooc.. This c::'culating aniicoagulant cou'ic also -che erclair scc.e of i:he failu:'e oi henoJ_y:rph io clot in the 'ch:"c:bir efot i;r.e det,errainat_cn 9 The clol;iing systen for hone;r bee heinol-ynrph was similar io that of nan, except that honey bee henoryoph had. no proconvertin and. contained. lesspr anounts of othe:- coagulants; as noted., a circu-

Iai:i.ng anticoagulant probably was presenp in henolpph. Hor,rever, the sys-"en must be ad.equate for the bee since it was selected. by evolution 10

REFENN,iCES

Gregoi:'e, C. l-95;... Bl-ood coagulati.on in arthropods. If. Phase- con't33st nicroseopic obselvations on hemoiyr:rph coagulat'i.on in sixty-one species ol insects. Bl-ooC 6, L..l73-!!g8- Gregoi:'e, C. Lcl57. Stud.ies by phase-cont'1'ast rnicroseopy on d.isiri- bution paiterns of henolyn;oh coagulation in insects. Sniihsonian Insi. Misc. Collectlons 8/1(6), l-35 Gregoi:'e, C. 1959. F\:.r-r,her obse:sraiions on dj-st:'ibution patte-rns

of he::ioiynph in neot'ropical insee'cs. Srni.'i;hsonrgn fnsi. Misc. Ccl-lections. V9O) , l-23. *"r"*., md Robb-Snith, A. H. T. ,196'.. t'trhnciions of

tbe Bl-ood..tt Academic Press fnc., New Yoz'k. rl (p

Ilopriutecl fr,rnr Jounx-rr, or l-rvalrrur'rrc P'\l IIoLocY \tolrrnre 16, Nrrnrber I, Ju'ly 1970 Copyright @ f97O by Acadernic Press Inc. Printecl irr U. S. A,

.IoURNAL OF r\\'ERTEDR.{TE pAfHOLoCy 16, 69-70 (1970)

Synthesis of Agglutinating Substances in Adtrlt Honeybees Again.st Bacillus lan;ae1

N'Ienrrt.L Grlu'ur: AND WAYtsuR\ S. JEIER Department ol l\ticrobiolog,y and tr[eclical Techrtology, IJniaersity of Arizona, Tucsort, Ari=ona 8572I

Receiaed Nouentber 10, 1969

Adult rvorker honel'bees, Apis nrcIlifera, produced agglutinating substances in response to an injection of a vaccine prepared from Bacillrr laroae, tlte causative organism of Amelican foulbroocl (AFB).

Ixrnolugrrox trIaranrer-s AND IUETTIoDS

Much controversy has existecl conceming Vaccine prepat'atiotr. Cultures of B.laroae the possibility of antibody production by were gro\\.n in brain-heart infusion broth insects. For example, Glaser (1918) re- ( Difco ) sr.rpplemented rvith 0.0lVa thiamine ported an acquired agglutinin formed by hydrochloricle for 72 hr at 37oC. Au equal grasshoppers in response to an injection of volrrrre of 0.67o formalinizecl saline rvas Bacillus poncei, but Briggs (1958) was Lur- added to tlre culture rvhich rvas allorved to successful in his attempts to ffnd agglutinins stand at roonr temperature for 3 clays. After for various particulate atltigens in the conffrming the bacterial sterility of the cul- hemolymph of lepidoptelons larvae. Yet, ture, the organisms lvere seclimented by Gary et al. (f9a8) reported that Bacillus centrifugation at 15009 for 30 min. Subse- lctraae cells were agglutinated by the he- quently', the supematant fluicl was reno\ted, molymph of bees from a colony infectecl and tlie bacteria \\,ere lesuspended in 100 rvith American foulbroocl (AFB). ml of 03Va forn-ralinized saline. Ptior to use, The purpose of this investigation rvas to the vaccine rvas c'lilutecl rvith 0.857o saline detennine rvherthcr aggltltitlins could be b1' comparison rvith a lrePhelonletric stiur- producecl cxperimenttrlly in honey bees in dard to contain 12 X lOs orgariisms (both resporlse to arrtigenic stimulation b;r B. sporcs ancl vegetative cells) per ml. laraae vaccine. Bee injection ancl bleetling. Aclult rvorker honey bees rvere anesthetized rvith carbon clioxide arid injected indivicluallv s'ith 5 pl I 'fhe data contained herein constitute part of of the vaccine. Injections wcre nrade into a clissertation subnritted by the senior author in partial fulfillment of the rerluirenrents for the the thorax using a microlitel spinge ancl a degree of Doctor of Philosophy, University of 27-gauge needle. Control bces receivecl 5 pl Arizona. This investigrttion rvas supportecl by Co- of 0.85Va saline, carbon clioxidc onll', a stab operative Agreement Crant No' 12-1-1-100-9062 in the thoras rvith a 27-gnuge needle, or no (33) from the Apicultur:e Reseirrch Brrrnch of the treatment. Etrch group containecl approxi- USD.,\. 150 bees. hours later 9 Present adch'e,ss: LISDI\ Bee Researc'h Labo- matel)' Trventy-fs1r ratorv, Tucson, Arizona 85719. the bees u'ere again anesthetizecl s'ith car- 69 70 crl-I,rAlr AND JEIIR bon dioxicle. Nlost of the insects u'ere de- REsur-rs err'n Drscussrox capitatecl rvith a scalpel, and the drop of An agglutination titer of 1280 rvas ob- hemolymph that exucled from the thorax served ri'ith anti-B' lara{Le hemolymph. All ruot *'ith a capillarl' pipettc' All controls rvere negirtive. The experiment rvas "oll""t"cl hemoll,rlph s'trs centlifugecl at 25009 for repeatecl fivc tinrcs, ancl the.sar.rre titer rvas 20 rnin to tetttot'e il1s hemoc)'-tes. Thc: olitained in circh case. The strme results hemoly'mph from each group of bees rvas u'ere obtained regardless of the injection poolei in acid-s'rrshecl vials ancl storecl at site or tlie body regiou from rvhich thc -70'C prior to ttse' bloocl u'as drtruer. These ffndings indicate The site of injection and body region that agglutinating substances are produce_d from rvhich the blood rvas clrarvn rverc ancl disieminated rapiclly u'ithin the bocly varied in some experiments. In these, bees of the honey bee. No agglutinatiorr rvirs ob- rvere either injectecl betrveen the third aricl served lvhen S. tlnmpson, an trntigenically fourth abdominal segments ancl then blecl unrelated organism, rvas enploved as the frorn the thorax, or thel' rvere injected irr test substance. On the other hand, an ag- the thoras ancl blecl from the abclornen' glutination titer of 20 s'as observed rvhen Agglutinatiort Tests. Beginning with a b. subtili.s, a relatecl species, rvas usecl. The appearcd to be 1:20 dilution of hemolvr-nph, 2'fold dilutions agglutinating materials thus to 1:2560 were preParecl in 0.85% NaCl' relatively specific for B. Iarooe. One nrilliliter of the antigen, B. laraue vtc- Acxxo$'Lrocltli'r.rs cine, rvits added to I ml of etrch hemoll'n-rph clilutiou in a test hrbe. Salinc replacccl We thanh Dr'. \t. D. Levin of the USDA for his for-her--tech- eithel the antigen or antiserunr for controls' cooperation ancl N'lrs. Karen Jackson nicil a-ssistence. Dr. H' Shinanuhi of the USDA inctrbatecl at 37'C iu a u'ater All tLrbes lvere Bee I)isease Laboratorl in Bcltsville, trIarylnnd, bath for 18 hr trncl ollsen'ecl for agglutinn- supplieil the Bncillrrs laruae cultures. tion. To test fol nonspecific cross-rcactivitl' of Rlrnnuscrs the hemol1'mph u'ith antigenically untelatetl Bnrccs, J. D. f958. Ilurnoral inrmnnity in lepi- organisms, Salnronella tlrcmpsort' vacciuc clopterous larvae. J. Exp. Zool., f38, 15il188' C' I., ar-o lftnno, A' rvis employ'ed in agglutination tests. To G.,tnr, N. D., Nrl,sox, J' evidence of resistance of u'ith a relatecl bacterium, 19.18. Serological shorv specificitv larvae ancl l'orliers to Bocillus lan:ae. I- Econ' a Bacillus subtilis cellular suspension tvirs Entontol., 41, 661-663. used. Comparable nurnbers of bces u'erc Gr-esrn, R. W. 1918' On the esistence of ir-u- used in all cleterminations. mrrnity plinciples in insects. Psyclrc,25' 39-46' 7

PURCHASED 8Y THE UNIIED STAITS Ogt'nnrmrnT oF AcRrcuLruRE FoR Separatum EXPERIENTI.\ 2d, 90S (1970) OFFIBIAI USE Birkhiuser Verlag, Llasel (Schtreiz)

Coagulation of Hemolymph of the Larval Honey Bee (Apis mellifera L.\ Previous investigations in the fielcl o{ invertebrate follos-ing determinations for coagulants rvere made in bloocl, or henroll'nrph, coagulation have consisted pri- triplicate on each sanrple using the nrethods of Ercrler-- marily of nrorphological description rather than identifica- EERGUR T: tion of the chcmical substances in'i'olved in the clotting l. One-stage prothrornbin time. process. For exanrple, GtecorRnl-3 stuclied patterns of 2. Partial thromboplastin tinte. Llood coagulation in various insects bv using phase- 3. Thromboplastin gencration test. contrast microscopr'. The ptrrpose of this investigation 4. Thrombin clot tinre. rvas to deternrine rvhethcr the henroh-nrph of the honey 5. Antihemophilic globulin (Factor YIII). bee, Apis mellit'era L., contains clotting factors similar to 6. Plasma thromboplastin antecederlt (Factor \I). those found in hunran blood antl rrhl' the hemolvntph 7. Recalcification tinre and nonspecific nrixing test ckres not coagulate. for anticoagulants. Flemolyruph n'as obtained b1' genth- Iruncturing lan'ae s. Proaccelcrin (I;actor \') hy'potlernric neccllc and of variotrs ages *-itlr a sterilc 9. Proconr-ertin lttactor r-i t;. a dra*'irtg the fltricl rrhich exucletl frorlr the s'ouncl irtto 10. Plasnra thronrboplastin cotuponcnt (Factor I\). 'fhc shell capillary pipctte. hcmoll'nrph rvas pooled in 11. Hagenran factor (I:actur \lI). and adtllts vials and storetl at - 2lloC. (Rlood of ptrpae 12. Strrart factor (Iractor X). s.as not used becerttse of the tlifficulty of obtaining strf- This poolccl larr-al honel' bee hertro- 'l'hc' results of the coagulati()ll tcsts arr' given in Tablt's I ficicnt quantitics.) 'l'hc l1'nrph ilas tl'ren testt'tl for the prescnce of the bloocl ancl II ancl in tht: Irigure. clotting tinrr-s of hotrt'v bce hcrnoh-mph in each test \-!'rc at lt'ast 2-3 tinrcs as long coagnlirnts that exilt irt httnlatr bloocl. 'l'hr Scrrrnal pllrsnta antl scrtttrl tlbtainecl fronr htrtnan vol- as that of hunran plasnra. absence of proconvt'rtin rrntrcrs s'cre ttsctl :ts stanclzrrtls' Inscct hctuolr-nrph rvas fnlnr the hcnrolvnrph prcr-entt'tl tlrt- fornration of a clot treatccl in exactlv thc sarne nlallner as httmatr bloocl. Thc in the tt-st of onc-stagc prtttlrrotnlrirr titnt'. 15. 8. 1970 Specialia 909

Table I. Results of coagulation tests Table II shorvs that the larval hemolymph lengthened the recalcification tinre of human plasma. Thus, a circu- Coagulation tirne D Coagulation test " lating anticoagulant and the absence o{ proconvertin may Hunran Larval plasma hemolymph possibly account for the negative thrornbin clot time of bee blood. One-stage prothronrbin time f 8.8 >2h The thromboplastin generation time curves of larval Partial thronrboplastin time 53.2 365.8 hemolymph and human blood (Figure) s'ere similar. Thronrbin clot time 1C.8 >2h Therefore, the hemolyrnph rvas capatrle of generating Antihenrophilic globulin 88.3 I 50.3 intrinsic thromboplastin rvith the aid of human sera and Plasmathromboplastinantecedent 92,5 217.O platelets. The concentration of tissue thromboplastin rvas 64,0 203.0 Proaccelerin negligible, partial throarboplastin concentration Proconvertin 2+.2 >2h but the Plasmathronrboplastincomponent 83.5 285.0 \vas detectable. Hageman factor 186.2 355.3 The clotting system for honey bee hemolymph rvas Stuart factor 20.2 372.1 similar to that of man, except that honey bee hemolymph had no proconvertin and contained lesser amounts of . D Average of 3 determinations, In seconds unl.ess othenlise noted. other coagulants; as noted, a circulating anticoagulant probably rvas present in hemolymph. The coagulants in Table II. Results of recalcification time and nonspecific rnising test bee hemolymph might have had a different chemical for anticoagulants nature and thus rvere less suitable substrates, enzymes, or co-factors in the system. Substrate Clotting timer

0,5 ml human plasma 3 min 0.5 ml larval hemoll'mph >2h Zusamnent'a.ss?rzg. Ilaemolymphe der Honigbiene Apis 0.25 ml human plasma, 0.25 ml larval hemolymph 4 min mellilica *'urde auf das Vorhandensein von verschiedenen Koagulantien untersucht, die im menschlichen Blut vor- r Average of 3 determinations. kommeo. Die Haemolymphe gerann nicht, rveil sie kein Proconvertin enthielt. Zudem konnte ein zirkulierendes Anticoagulans nachgelviesen $'erden. Menrne. Grllrarr5 and llecnrRo Ssluawuxr6'? . }llJll.lail plasl]ia o Bee leln0lylilgh Bee Di,sease Laboratory Entontology Research Diuisi,on, A gri culkval Re s e ar ch S eru i ce, US Depariment ol Agriailhne, Laramie {lVyonting USAI,19 Febntary 1970.

ees t C. Gnecorn.e, Blood 6, 1173 (f951). .eU t C, Gnrcotne, Srnithson. misc. Collns 131t, 1(19-r7r. I C, Gnrcornr, Smithson. misc. Collns t.tg, I (1959). a J. W. ErcnrlBERGER, Labonlory Llelhods in Blooil Coaguloliott (Harper and Row, NewYork 1965). 5 Present address: U.S,D.A. Bee Research Laboratory,2000 E. Allen Road, Tucson (Arizona 857f9, US.A,). -0 1 2 3 I' . Present address: U.S.D.A. Bee Disease Investigations, Agricultural Research Center, Beltsville (Ilaryland 20705, USA). lncuwlion tims ? Published with approval of Director, Wyoming Agricultural Results of thromboplastin generation test. Experiment Station, as Journal Article 310. Separatum EXPERIENTI^ 26, 1006 (1970) Birkhiuser Verlag, Basel (Schrveiz)

Total Hemocyte Counts of Honey Bee Larvae (Apis melliJeraL.) from Various Elevations The hemolymph, or blood, of the honey bee, .{pi,s The ar-erage of total hernocyte connts (TllC) of the ntellifera L., is a palc 1'ellorvish fluicl containing blood 5-dar--old larval honev bees arc shos.n in llablc I. cells referred to 'r'ariouslv as hemocytes, blood cor- --\nalr-sis shorvecl a rcgression cocflicicnt of 0.10351 'rvhich J puscles, or lcucocl-tes. The blood of larvae contprises rvas significant at thc 1f 'o level of confidence (Table II). 25-31o/o of the total boclv rveightl. Insect hemolr-nrph is Therefole, a deiinite relationship es'istecl lrctrveen the not carriccl jn blood vessels; it fills the spaces of the boch- THC ancl the clevation {ronr rvhich thc. inscct u'as obtained cavity and bathes the surfaces of tissues. In vertebrate (Irigurc). llore circulating blood cells s-ere present in blood, there is a definite correlation bets'ccn the total lan-ae from higher elevations. nurnber of crythrocl'tes and elevation. The present paper describes a study of the circulating hen'rocytes of honer' bee larvac obtained from various elevations- -Iotal Combs containing larval honev bees s'ere shipped air Tablt'I. hernocl'tc counts of -i-day-old honev bee larvae from mail from localities at various elevations ranging from various locations 159 feet belorv sea level to 7200 fect above sea level. I)ates of the shipnrents u'ere such that lan ae Nere about days old ri-hen thev arrived. Indiviclual 5-da1'-old Sanrple Location fronr Elcvation .{tg. 5 nurnber rvhich larvae (feet) Tl{C/rnms larvae rvere then punctured rvith a sterile hypodermic s'ere obtained henroll'mph needle, and the hemoll'urph l'hich exucled from the s'ound'was drau-n to thc 0.5 mark of a Thoma rvhitc-ceil cliluting pipette and dilutecl to thc 11 marli rvith Toisson's 1 \\'r.stnlorland, Calif . -- 159 2'c,+7 j,570 fluid (1.0 g sodium chloride, 8.0 g sodiunr sr.rlfate, 30 ml 2 Baton Rouge, La. 35 3 Dalis, Calif. (1) 60 -5,6S0 glvcerin, 15 mg crystal violet, 160 ml distilled u'ater). + Beltsvillt', 1ld. 61 +,r76 rvas thoroughil' r.nixed in the pipette for .'\fter the fluid 5 Ottarra, Canada 2io 6,093 2 nrin, the first three drops s'e're discarcled, and the count 6 I)avis, Calif. (2) 300 5,266 s-as macle of thc fourth. -'\ Spencer bright-linc hemoc\-to- 7 Columbus, Ohio 760 7,1+0 nreter with inrproved Neubauer ruling rvas used to count 8 \ladison, \\'is. 858 7,992 the cells in the 4 corners and the central square. Thcn 9 11h16x, \€'1s\'6rrk (2) 950 +,053 rvas b1' 40 to give thc nulnber of l0 Ithaca, \eivYork (1) 1,300 5,8+0 the sum nrultiplied 'Iucson, cells/mm3. If thc cells werL' unevcnll' distributcd, tire lt ^{riz. 2,.5+3 i,()67 sample rvas cliscarded. average total counts reported 12 Logan, t'tah +,7.i3 9,1 10 -\ll 13 Laranric, \\'vo. 7,?00 r0,000 s,ere the results of cortnts fronr 5 saltlples, all taken fronl 5-daf'-old larvae. Tlie effect of elevation ott the' cotlnts rvas determinetl bv regression corrc'lation studics in rvhich logro totzrl hemocr.te counts ancl logro elevation s'erc J used since thcr usc of logarithnrs appeared to givc the best corrcl:rtion. I G. fl. Ilrsuor, ,f . biol, Ciheru. i.'i, .t'l.l (19-2.1). q

2- of the Intestinal Content of The gut of each bee rvas plated in quadruplicate on the lllicrobial Sterility yeast nutrient agar, Honey Bee, -Cpis mellilerar'2'g follorving media: Lindegren agar, the Irnmature potato dextrose agar, brain heart infusion agar, EI\IB igar, and NlacConkey agar. Trvo plates of each medium TIARTHA GILLIA}'I {rom each bee lvere incubated at 37'C and trvo at room Entomologl' Research Division, Agr-.-Rgs. Serv', USDA, temperature. All plates rvere incubated aerobically for Tucson, Arizona 85719 14 days. In addition, 2 tubes of fluid thioglvcollate medium White ( 1921) stated that the intestinal content of (Difco) rvhich supports the grorvth o{ aerobic as rvell as freshly emerged honey bees, lpis ntellifera L., rvas micro- anaerobic microorganisms u'ere inoculated rvith a loopful bially sterile. Horvever, he gave no data to support this oI homogenate from each bee. These tubes rvere incubated conclusion. Kluge (1963) attributed this sterilitv to the at 37'C and examined for 10 daYs. antibiotic properties of larval food. IV{oreover, rvhen she As a control, the 8 distilled rvater rvashings through liberated young bees from their cells shortly before which the excised guts had been passed rvere examined emergence and placed them in a sterile environment, in the same manner. they rvere microbially sterile for the remainder of their lives. Haisig and Kamburov (1966) found yeasts and RESULTS molds in a sma'll percentage of larvae rvhich lvere at No microorganisms rvere isolated from the guts of any least 3 days olcl. Horvever, Lotmar (1946) stated that larvae, pupae, or nervly emerged honey bees. Only 7 prior to emergence the entire intestinal tract of bees rvas aerobic colonies o{ bacteria rvere isolated from the first iterile, but her observations were based solely on histo- 3 rvashings of distilled rvater through rvhich the guts logical examination oI the alimentary tract. were passed. No organisms rvere found in the last 5 The present paper reports the results of microbial washings. examination of the intestinal contents o{ immature rvorker honey bees (larvae, pupae, and nervly emerged adults) ISCUSSION to determine rvhether sterility exists until after emerg- Under the conditions employed in this experiment, the ence of the adults. Since these earlier rvorks failed to guts of larvae, pupae, and nervly emerged rvorker honey the give experimental data to support the conclusions of bees are sterile. The isolation of the 7 aerobic bacterial investigators, I examined the intestines oi immature colonies {rom the rvashings of the outside of the gut honey bees, making use of the best techniques available may have been air contaminants since no microorganisms today. This information is needed for studies of the rvere found in any homogenates. The normal microbial effect of specific microorganisms on honey bee nutrition flora of the gut must develop sometime after emergence |*. and disease. of the adult. Food consumption after emergence probably plays a large role in the intestinal microbial complement' IIATERI.\LS AND \IETHODS The microbial flora of the honey bee appears to be the Eighty larvae, pupae, and nervly emerged rvorker honey result of chance, depending on the food source of the bees rvere obtained from colonies at the Tucson Bee Re- colony. Preliminary experiments in my laboratory indi- search Laboratory. After the insects lvere removed from cate that yeasts, fungi, and bacteria are found in the their cells, each rvas individually passed through 8 intestines of adult rvorker bees a ferv days after emerg- separate rinses of sterile distilled ll'ater to remove ex- ence. This confirmation and documentation of earlier ternal microorganisms. Then the entire alimentary tract claims of the sterility of the gut of the immature honey (esophagus to rectum) was removed quickly to avoid bee should greatly simplify the establishment of gnotobio- air contamination, and immediately passed through 8 tic bee colonies for studies on the role of microorganisms separate 'rvashings of sterile distilled water to remove in disease and nutritional processes. extraneous matter and microorganisms. These guts vere individually transferred to separate sterile tissue grinders REFERENCES CITED containing 2.5 ml of 0.85% sterile NaCl. They u'ere Haisig, M., and G. Kamburov. l%6. Yeasts from the homogenized by hand, usually lor 2-3 min; horvever, lirvae'of healthy honey bee colonies. Vet. Arh. rvith a ferv guts it was necessary to homogenize for as 36: 6649. [In Serbo-Croatian, English surnmary.] long as 10 min. A loopful of the mixture from each Kluge. R 1963. Untersuchungen iiber die Darmflora bee rvas streaked on solid Difco@' media in petri dishes. ?er Honigbiene, Apis nellifca. Z. Bienenforsch. 6: 14149. r Hvmenootera: Apidae. Lotmar, R. 1946. Uber Flagellaten und Bakterien in 2Tliis uoik uas done in cooperation uith tbe l-niversity of Honigbiene (Af is nrcllifica). Agricultural Experiment-Station, Tucson. Received for Diinndarm der Arizona Schrveiz. Bienen-Ztg. 2: 49-76. oublication' Tulv 2J. 1970. 3 Endorsecl- and communicated by L. N. Standife r. tDfhite, P. B. 1921. The normal bacterial flora of the I llention of a proprietary nr6duct or comPany name does not imply endorsemenl by the USDA. honey bee. J. Pathol. Bacteriol. 21:6178.

Rebrinted' lront tlte Axxels oF TrrE ENTo\roLoGrcAL Socrrrv or AlrentcA Volume 6,1. \umber l, pp. 315-316, Januarv 1971 PURCHASID BY THt UNIITD SIAIiS DEPARTMENT OF AGRIOUI.TI.]RI FEfi OFFICIAL UST

Jotrrnal of ,4picultural Researclt 10(2) : 79-85 (1971)

BLOOD CELLS OF THE WORKER HONEYBEEI,2

Ma,nrH,q, Grr-lra,u3 AND HACHIRo Surua.Nurr4 Entontology Research Diyision, Agricultural Research Serrice, U.S. Departntent o;/ Agriculture, Laramie, l4/yoming 82070, U.S.A.

Manuscript received for publication 29th October 1970 Summary Worker honeybee haemolymph, or blood, was found to contain 7 types of haemocytes and 2 kinds of sten-r cells. However, not all 7 types are present at any one life stage of the insect.

I ntroduction The literature concerning the haematology of the honeybee (Apis mellifera) is con- fusing. Nelson (1924) believed that only one type of blood cell existed in feeding larvae prior to capping; a second type, the lymphocyte, appearing later. Fyg09a2) reported that a single colourless nucleated blood cell was found in the haemolymph of the honeybee. Metalnikoffand Toumanoff(1930) described two kinds of haemocytes from larvae: proleucocytes comprising 85){ of these cells, the rest being larger mature cells with lighter-staining cytoplasm. Wigglesworth's book (1961) divides honeybee larval blood cells into two groups on the basis of the ratio of nucleus to cytoplasm. Miiller (1925), Toumanoff (1930, 1951), Morgenthaler (1953), and Shishkin (1957/58) observed three types of blood cells, but the different classification systems used com- plicates comparison of their results. Kostecki (1964) recognized two groups ol blood corpuscles-young haemocytes or prohaemocytes and adr-rlt haemocytes or leucocytes in addition to several transitory forms. He divided these ceils further, into 7 types, which appeared as differentiation proceeded. The prohaemocytes gradually disappeared as the insect aged (see also Kostecki, 1965);they multiplied by mitosis whereas the older cells multiplied by amitosis. Wille and Vecchi (1966) described 8 elentents in the haemolymph olthe adr-rlt summer honey bee and a ninth referred to as "concentration of haemocytes". In addition, the oenocytes, which may not be trne blood cells, are also found in the haemolymph (Morgenthaler, 1953). Mr-rttowski (1921) described them as excretory in function, and Wigglesworth (1961) thought they removed waste products from blood or ger-rerally regulated its physical and chemical composition. Snodgrass (1925) stated that the pericardial fat-ceils formed a layer across the dorsal part of the pericardial sinus above the heart and next to the hypodermis of the body wall, but according to Wigglesworth (1961), the function of the pericardial cells is the segregation and storage of waste products. These cells have the ability to absorb colloidal particles from the blood.

I The data contained herein constituted part of a thesis submitted by the senior author in partial lulfilment of the requirements for the degree of Master of Science, University of Wyoming, Laramie. u Published with approval of the Director, Wyoming Agricultural Experiment Station, as Journal Article 430. 3 Present address: Bee Research Laboratory, Tucson, Arizona 85719, U.S.A. 1 Present address: Bee Disease lnvestigations, Beltsville, Md 20705, U.S.A. 80

Photomicrograph (700x) and drawing ol oenocyte (Fig. l), pericardial cell (Fig. 2), and proleucocyte (Fig. 3). 81

Photomicrograph (700 x ) and drawing of ner-rtropl.ril (trig. 4), eosinophil (Fig. 5), and basophil (Fig. 6). 82

The prescrtt invcstigation was urade to examine blood cells in haemolyr.nph fiom worker honeybees at various developmental stages and ages and to develop a stan- dardizecl repeatable uretl-rod of exarnination and a unilorm nomenclature. Materials and Methods A laying queen was con{rr.red by a queen-excluder cage to an area olbrood conrb in a colony. All samples of bees were obtained fiom this colony, so the age of the bees was known. The first eggs were found after 24 hours, and the ages of all subsequer.rt samples were determined by assigning these {irst eggs the age of I day. Differential haemocyte counts rvere made upon haemolymph from worker eggs, larvae, pllpael and adults of different ages. Individual eggs were ground on a slide, smeared, and air-dried. Haemolymph was obtained from larvae by gently pr-rncturing the ir,sects with a sterile hypodermic rreedle. The flLrid which exuded from the wound was drawn into a capillary tube, Haemolymph from pupae and aduits was best obtained by severing the head and drawing the blood which exuded from the thorax into a capillary tr-rbe. (Haemolymph drawu from the adult abdomen was sometimes contaminated with micro-organisms, since it was difficult to avoid puncturing the gut.) Each drop of haemolymph was placed on a slide, smeared, and air-dried, and all smears were stained with Wrigl-rt's blood stain for I minr-rte ar.rd destained for 2 minutes with Wright's buffer (3.315 g mollobasic potassium phosphate, 1'28 g diabasic sodium phosphate, distilled water to 500 ml). The slides were washed with tap water, air-dried, and examined with the oil- immersion objective of a light microscope. The haemolymph from approximately 700 bees was examined in the colrrse of this str"rdy.

Results Honeybee haerrrolyrnph contains 7 distinct types of blood cells plLrs 2 types ol stcrn cells as lbllor,vs: l. The oenocyte (I'rig. 1) is acell ol'ectoclerural origin which is l'ormcd in the hypo, dcrrris (Morgenthaler, 1953). Oenocytes have a dian-reter oi9'0 l4'0 pr. ln or-rr opi- nion, they are stem cells lrom which haemocytes are lormed. Mitotically dividing oenocytes have been observed, and these give rise to prolencocytes. Oenocytes have a basophilic to r.reutrophilic cytoplasm, with a lighter-stnining nncleus which is usually ncu t roplr ilic. 2. Thc pericardial cell (Fig. 2) is a neutrophilically staining cell (10'0 20.0 p irr diarneter) in which the nucleus is seldonr visible. This type ol'cell is also a stent cell fbr leLrcocyle formatiorr. The mitotic division ol a pericardial cell results in the 1omration ol two ner"rtrophilic haemocytes (neutrophils). 3. The proleucocyte (Fig. 3) contains cytoplasm which stains neutrophilically, as does tl.re nucleus. The nucleus, which occr,rpies most of the cell, stains more lightly thar"r the cytoplasm. These haemocytes have a diameter oi3'5 6'0 ir; they are lound only in eggs and larvae (Table l). 4. The neutrophil (Fig. 4) is a neutrophilically staining haemocyte in which the granular nucleus is so large that the cytoplasm is seldom visible. These cells (diameter 3'0 7'0 p) are lound in every developmental stage of the worker honeybee (Table l). 5. The eosinophil (Fig. 5) is a haemocyte with a large red-staining granular nucleus. The hyaline cytoplasm is seldom visible. These cells (diameter 3'0-6.0 p) are observed in every developmental stage of the worker honeybee (Table l). 83

6. The basophil (Fig. 6) has a diarrrcter of 2"0-4'5 pr. The glanr"rlar nuclcus is tleep purple and occupies most of the cell. This cell is absent in the egg bLrt is foLrnd in ail other stages (Table l). However, the haemolyrnph of old adult worker bees may or rnay not contain a few basophils. 7 . Tl.re normal leucocyte (Fig. 7) has a diameter of 3.0-7.0 p and possesses a slightly eosirrophilic rruclelts which is granular, with a neutrophilic to basophilic cytoplasni. It is present in the lTaemolymph of all stages of bees, but is sometir.nes abselt in the egg (Table l). 8. Tlre pycnonucleocyte (Fig. 8) is 12.0-18.0 pL long andi.5-12.0 pr wicle (Morgen- thaler, 1953). Tl.re contpact, slightty neutrophilic nucleus is small in relatiol to the abundarlt cytoplasm. The lightly neutrophilic to eosinophilic cytoplasm is gsually ragged or torn on one or more sides. This cell is absent in eggs and pupae but is always present in yottrrg adult bees. I{aen.rolyrnph of larvae and old adLrlf bees nray or nlay not contain pycnonucleocyles (Table l). 9. The hyalinocyte (Fig.9) is 7.0-11.0 p long and 3.5 7.0 p wide. The nucleus is eosinophilic to neutrophilic depending upon the age ofthe insect; in older adult bees. it is more eosinophilic. The cytoplasm is slightly neutrophilic in younger bees and hyaline in the older insects. The hyalinocyte is the last type of blood .ell to app.at ar.rd is seldom for-rnd before the adult stage (Table l).

Trnu L Tvpes ol haelr]ocvte in developn.ren:1J:?i* o1'the worker honeybee (+ : prcsenr;

Lile stage o.f Normal bee Proleucotyte Neutrophil Eo.sinophil Basophil !eucot.yte Pycnonucleor:yte Hyalinocyte

+ + ++ larvae + + +++ l: pupa + +++ young adull + +++ + + old adult + + ].+ +

Discussion Worker honeybee haentolyntph thus contains 7 distinct types of haenrocytes in ad{i- tion to 2 kinds of stem cells, but not all 7 types are folrnd at any one life stage. The terms below are in general use in insect as well as mammalian haematology, with the exception of "hyalinocyte". Proleucocytes are observed only in eggs and larvae; hyalinocytes appear or-rly in adr-rlt insects. Eosinophils and neutrophils are present in the haemolymph of all developmental stages and ages of the honeybee. Normal leucocytes are generally found in all ages of the insect but are sometimes absent in the egg. The basophrl is absent in the egg and is sometimes absent in the older adult bee. The pycnonircleo- cyte appears consistently only in the young adult. The possibility exists that some of the haemocytes observed are transitory forms. Also, a particular type ol haemocyte may transform into another kind of blood cell. The proleucocyte is the youngest blood celi, and the hyalinocyte the most advanced, in terms of the life stages in which they are found. Haemolymph from drones and queens contains the same 7 types of haemocytes. 84

t, 8

Pl"rotomicrograph (700x) and drawing of normal leucocyte (Fig. 7), pycnonucleocyte (Fig. 8), and hyalinocyte (Fig. 9). 85

However, the relative proportions ol the various kinds of blood cells vary from caste to caste. Wright's blood stain allows excellent differentiation ol honeybee haemocytes. Other blood stains were r-rsed, br-rt they were not as satisfactory.

Acknowledgement We thank the Journal o/ Apicullural Research lor publishing the photographs in colour. The cost of this reproduction was paid for by the U.S. Department of Agricr-rlture.

References Fvc, W. (1942) Das Bienenblr-rt. Sclty,eiz. Bienenztg 65(3) : 120 122 Kosrecxt, R. (1964) Elements anatomiqr-res de I'h6molynrphe des abeilles (Apis mellifica L.). Bull. apk:. Doc. sci. rech. Inf.7(2) : l5l 182 (1965) Investigations on the haemocytes and haetnolymph of honeybees. J. apic. Re.i. - 4(1) : 49-54 Mt.raLxtxonR, S. & TourllNorn, C. (1930) l-es cellr"rles sangr,rines et la phagocytose chez les larves d'abeilles. C.r. Sdanc. Soc. Biol. 103 : 965 961 MonceNrHar-nn, P. W. (1953) Blutuntersuchr.rngen bei Bienen. Mitt..schn,eiz. ent. Ge.s.26(4) :245- 257 Mut-t-lr<, K. (1925) Uberdiekorpr.rskr-rlziren Elementerler BJiitfllissigl

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