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Apis iridescent virus and “clustering disease” of Apis cerana
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
Apis Iridescent
31, 368-371
(1978)
Virus and “Clustering L. BAILEY
Rothamstrd
AND BRENDA
Experimentul Received
Station.
Disease” V. BALL
Harprnden,
August
of Apis cerana
Herts.
England
29. 1977
Apis iridescent virus was plentiful in each of several samples of adult individuals of Apis cerana from sick colonies in Kashmir and Northern India. Almost every bee, of those examined individually, was infected with the virus, which caused an easily detectable iridescence in the fat body and most other internal organs. The only other parasites recognized were tracheal mites, but most individual bees and some samples were not infested with these. adults; Apis iridescent virus; Acarapis btvodi; Nosema upis; KEY WORDS: Apis c’rrunu. Malpighantoeha mellijicue; fat bodies: iridovirus.
infested with A. woodi because this is widespread in Britain. Moreover the mite multiplies and spreads more than usual among bees rendered less active than usual for any reason, probably because of increased contact between old infested bees and young susceptible individuals (Bailey, 1958; Bailey and Lee, 1959). Similar inferences can be made about the tracheal mites infesting A. cerunu, because our observations suggest that the disease believed to be caused by them is in fact caused by Apis iridescent virus.
INTRODUCTION Apis iridescent virus, the only example of an iridovirus from Hymenoptera, was first isolated at Rothamsted (Bailey et al., 1976) and has now been found in each of several samples of sick adults of Apis cerana sent to us on different, subsequent occasions. The samples were sent primarily because the bees were known or suspected to be suffering from an infestation with tracheal mites, first reported in India in 1957 and identified on morphological grounds as Acarapis woodi (Michael, 1957; Milne, 1957; Singh, 1957). A sickness of A. cerana has been attributed to such infestations, and the descriptions of this sickness and of annual losses of 25 to 40% of colonies in Northern India due to it (Atwal and Sherma, 1970; Atwal, 1971) are very reminiscent of those of diseases traditionally attributed to infestation of Apis mellifera by A. woodi in Britain during the early part of this century (Bailey, 1958). However, although the lives of individuals are shortened slightly by A. woodi and, as a result, severely infested colonies in Britain are more likely to die in winter than uninfested colonies (Bailey and Lee, 1959; Bailey 1961, 1965), A. woodi has never been shown to make bees visibly sick. When bees are sick, from any cause, they are frequently found to be 0022-201 Copyright All rights
l/78/03
13-0368$01.00/O
0 1978 by Academic Press, Inc. of reproductmn in any form reserved.
MATERIALS
AND METHODS
All samples were of adult bees of Apis colony, and mostly taken at very different times, in Kashmir and Northern India. According to accounts by the consignors of the samples, the colonies were suffering from maladies with very similar signs, although the usual implication that these were signs to be expected from the effects of “Acarapis woodi, ’ ’ together with the indefiniteness of some of them, make it uncertain that only a single disease was involved. However, the most striking and consistent sign was an unusual inactivity, especially in summer, of colonies that frequently formed several small detached clusters of flightless inceranu, each from a different
368
APIS
IRIDESCENT
VIRUS
dividuals, and that often lost many bees crawling on the ground. Large colonies perished within 2 months of becoming visibly affected. Individual bees of the samples were examined microscopically for internal parasites resembling those well-known in Apis mellifera, i.e., Acarapis bz,oodi, Nosema apis. and Malpighatnoeba mellijcae. Iridescent virus was detected either after an extraction process, described below. or by microscopical examination of tissues held beneath water and illuminated from above with an oblique intense beam of white light. Iridescent virus was extracted from bees that were ground up in a 4.1 mixture of 0.01 M potassium phosphate (pH 6.6) + 0.02% of sodium diethyldithiocarbamate (to prevent melanization) and diethyl ether. The mixture was emulsified with one volume of carbon tetrachloride, coarsely filtered, and cleared at 1OOOg for 30 min. and the cleared fluid was centrifuged at 1OOOgfor 30 min or 18,OOOg for 15 min. There was sufficient virus in 10 or more infected bees to produce a bright turquoise iridescent pellet. The whole particles of iridescent virus so obtained were tested serologically in tube precipitation tests using an antiserum prepared against an iridescent virus obtained initially from A. cerana from Kashmir (Bailey et al. 1976). Iridescent virus-infected tissues were seen easily by microscopical examination, best of all in fresh unfixed specimens, but readily enough in bees that had been preserved in formalin or alcohol, although these materials had not always penetrated satisfactorily. Infected tissues appeared bright blue in striking contrast with the surrounding creamy white tissue. Single infected cells could be distinguished in otherwise healthy tissue. RESULTS
Apis iridescent virus was the only recognized parasite that was in all the samples
IN APIS
CERANA
369
and in almost every bee of those examined individually (Table 1). Preparations of the virus, made from all the unpreserved material, were serologically indistinguishable. The fat body was always attacked by iridescent virus (Table 2). but other tissues and organs, including the ovaries, were frequently infected. The midgut seemed least susceptible, at least to the multiplication of sufficient virus to cause iridescence. The bees of colony 5 (Tables 1, 2) were clearly more severely infected, with 75% of all examined tissues infected, than those of colony 4, which had only 29% of examined tissues infected. However, according to the description of these colonies, only colony 4 showed signs of clustering, whereas both were producing crawling bees. The difference between the proportions of bees infected with tracheal mites in the samples from these two colonies is not significant (P > 0.05). DISCUSSION
The obvious conclusion from these observations is that Apis iridescent virus causes the disease that appears as inactivity, clustering, and crawling of adult individuals of A. cerana and that causes the ultimate death of their colony. The only other recognized internal parasites sometimes involved were tracheal mites, but none of the eight available samples had all its bees infested with these, and two of the samples had none. Clearly, if the samples were from colonies all with the same disease, tracheal mites could not have been the cause, nor could they be necessary as vectors of the virus. The proportion of samples that were infested with tracheal mites seems high, but a similar proportion of seemingly normal colonies of A. cerana in the same localities may have been infested. As an example of this possibility, well over 50% of apparently healthy colonies of Apis mellifera have been found infested with A. tz-oodi at one time in Britain (Bailey, 1961). Evidence that Apis iridescent virus is the
370
BAILEY
AND
BALL
TABLE APIS IRIDESCENT
Sample”
VIRUS,
TRACHEAL
MITES
FROM
COLONIES
Region
AND
I
OTHER
WITH
Date
PARASITES
“CLUSTERING
IN SAMPLES
OF ADULT
APB CERANA
DISEASE”
Degree
of infection”
Iridescent
virus
or proportion Tracheal
of bees infected
mites
Other
with:
parasites
I
Kashmir
Nov.
1974
+
911 I
Oil I
2
Kashmir
Aug.
I975
+
0112
O/12
3
Kashmir
Oct.
1976
+
-
0140
4
Kashmir
Dec.
1976
27127
14127
0127
5
Kashmir
Dec.
1976
919
619
019
6
Himachal Pradesh
May
1977
919
019
019
Himachal Pradesh
Jul.
1977
15115
Ill4
011.5
Himachal Pradesh
Jul.
1977
12/14
1114
O/l4
7
8
‘I Samples I, 2. 3. 7, and 8 were unpreserved. samples 4 and 5 were in formalin. and sample 6 was in alcohol. ’ + = an average of about IO” particles of iridescent virus per bee in pooled samples of 20 or more individuals: - = sample too desiccated.
cause of the clustering and crawling sickness in colonies ofA. cerana, though strong, is still only circumstantial. Objective surveys and experimental work, including field tests, with A. cerana in Northern India, are needed to resolve the question. At present, tests have been done only with A. rneflifera in the laboratory (Bailey et al., 1976), and these cannot lead to an under-
standing of the ecology of the virus. However, they have shown that A. mellifera can become infected by it and indicate the possible danger of attempts which are made to establish colonies of A. cerana in regions where it does not occur but Apis rnellifera does. Admittedly, the virus has multiplied quickly and abundantly in A. melliferu only when injected into
TABLE THE
NUMBERS
OF ADULT
OF APB CERANA CONTAINING
INDIVIDUALS
IN SAMPLES
FROM
2
COLONIES
WITH
“CLUSTERING
Iridescent
Sample” 4 5 6
Fat body 27 9 Y
Esophagus s 6
‘I Sample numbers as in Table I. ’ - = Sample inadequately preserved.
Crop 7 8 -
Ventriculus 0 2 3
IRIDESCENT
TISSUES
DISEASE”
tissue”
Rectum 2 Y I
Hypopharyngeal glands I 6 5
Ovaries 0 7 -
APfS
IRIDESCENT
VIRUS
pupae and has produced only slight infections, after about 1 month, when fed to caged adults. It may infect A. ceranu more readily, especially in natural circumstances, although it is characteristic of the iridescent viruses, at least of other insect species, not to cause rapidly fatal diseases. Nevertheless, their eventual effects on populations of insects, such as honey bees, that reproduce slowly, or hardly at all, for relatively long periods, could well be severe. The possibility that A. cerana is an accidental host for an iridovirus that can only complete its infective cycle in other insects seems unlikely. It would mean the almost simultaneous infection by chance of most bees of colonies, enough to cause the cases of severe disease observed. More probably, the virus is effectively transmitted from individual bees in which it first multiplies to others in the colony; so it could be enzootic in A. cerana. The variety of tissues in which it multiplies suggests it could be transmitted via eggs, feces, or gland secretions in food exchanged between adults or supplied by these to larvae. Conceivably one or more species of the external mites of A. cerunu (Crane, 1968) may act as vectors. Alternative hosts cannot be excluded, although, unlike other iridoviruses of the same type, Apis iridescent virus did not multiply when injected into unrelated species. e.g.. Galleriu mrllonellu (Bailey et al, 1976) or species of Noctuidae (Bailey, unpubl.). The two other Apis spp. that occur in India seem most likely as alternative hosts. Successful control ofApis iridescent virus would probably depend on methods that prevent its invasion of colonies and spread within them rather than on the treatment of sick colonies. A knowledge of the ecology of the virus is needed so that such preven-
IN APIS
CERANA
371
tive measures may be devised. Even were these measures not forthcoming, the knowledge gained would probably lead to a better understanding and assessment of damage due to the virus and could well save wasted effort in attempts to control coincident but relatively unimportant parasites. ACKNOWLEDGMENTS We thank Messrs. D. B. Mahindre, Central Bee Research Institute. Srinagar. Kashmir, T. A. Shah, Shah Beekeepers. Srinagar, Kashmir. and Professor G. S. Dogra. Himachal Pradesh University for samples ofA. c’eran(~.
REFERENCES ATWAL, A. S. lY71. Acarine disease problem of the lndian honey bee. Apis indicu F. Amer. Bee J.. 3, 134-135, 186-187. ATWAL, A. S., AND SHARMA, 0. P. 1970. Acarine disease of adult honey bees: Prevention and control. lndian Farming. 20, 39-40. BAILEY, L. 1958. The epidemiology of the infestation of the honey bee. Apis mrllifera L. by the mite, Acurupis n~oo& (Rennie), and the mortality of infested bees. Parasitology. 48, 495-506. BAILEY. L. 1961. The natural incidence of Acurapis ,cwodi (Rennie) and the winter mortality of honey bee colonies. Bee World, 42, 96- 100. BAILEY, L. 1965. The effect of Acarupis ,c,oodi on honey bees from North America. J. Apkult. Rrs.. 4, 10% 108. BAILEY, L.. AND LEE, D. C. 1959. The effect of infestation with Acarapis rr,oodi (Rennie) on the mortality of honey bees. /. Insecf Patho/., 1, 15-24. BAILEY, L.. BALL, B. V.. AND WOODS, R. D. 1976. An iridovirus from bees. J. Gen. Viral.. 31, 459-461. CRANE. E. 1968. Mites infesting honeybees in Asia. Bee World. 49, I l3- 114. MICHAEL. A. S. 1957. Acarine disease found in India. Amer. Bee J.. 97, 107. MILNE, P. S. 1957. Acarine disease in Apis indictr. Bee World. 38, 156. SINGH, S. 1957. Acarine disease in the Indian honey bee (Apis indic,a, F.) Indian Bee J.. 19, 27-28.
OF
INVERTEBRATE
PATHOLOGY
Apis Iridescent
31, 368-371
(1978)
Virus and “Clustering L. BAILEY
Rothamstrd
AND BRENDA
Experimentul Received
Station.
Disease” V. BALL
Harprnden,
August
of Apis cerana
Herts.
England
29. 1977
Apis iridescent virus was plentiful in each of several samples of adult individuals of Apis cerana from sick colonies in Kashmir and Northern India. Almost every bee, of those examined individually, was infected with the virus, which caused an easily detectable iridescence in the fat body and most other internal organs. The only other parasites recognized were tracheal mites, but most individual bees and some samples were not infested with these. adults; Apis iridescent virus; Acarapis btvodi; Nosema upis; KEY WORDS: Apis c’rrunu. Malpighantoeha mellijicue; fat bodies: iridovirus.
infested with A. woodi because this is widespread in Britain. Moreover the mite multiplies and spreads more than usual among bees rendered less active than usual for any reason, probably because of increased contact between old infested bees and young susceptible individuals (Bailey, 1958; Bailey and Lee, 1959). Similar inferences can be made about the tracheal mites infesting A. cerunu, because our observations suggest that the disease believed to be caused by them is in fact caused by Apis iridescent virus.
INTRODUCTION Apis iridescent virus, the only example of an iridovirus from Hymenoptera, was first isolated at Rothamsted (Bailey et al., 1976) and has now been found in each of several samples of sick adults of Apis cerana sent to us on different, subsequent occasions. The samples were sent primarily because the bees were known or suspected to be suffering from an infestation with tracheal mites, first reported in India in 1957 and identified on morphological grounds as Acarapis woodi (Michael, 1957; Milne, 1957; Singh, 1957). A sickness of A. cerana has been attributed to such infestations, and the descriptions of this sickness and of annual losses of 25 to 40% of colonies in Northern India due to it (Atwal and Sherma, 1970; Atwal, 1971) are very reminiscent of those of diseases traditionally attributed to infestation of Apis mellifera by A. woodi in Britain during the early part of this century (Bailey, 1958). However, although the lives of individuals are shortened slightly by A. woodi and, as a result, severely infested colonies in Britain are more likely to die in winter than uninfested colonies (Bailey and Lee, 1959; Bailey 1961, 1965), A. woodi has never been shown to make bees visibly sick. When bees are sick, from any cause, they are frequently found to be 0022-201 Copyright All rights
l/78/03
13-0368$01.00/O
0 1978 by Academic Press, Inc. of reproductmn in any form reserved.
MATERIALS
AND METHODS
All samples were of adult bees of Apis colony, and mostly taken at very different times, in Kashmir and Northern India. According to accounts by the consignors of the samples, the colonies were suffering from maladies with very similar signs, although the usual implication that these were signs to be expected from the effects of “Acarapis woodi, ’ ’ together with the indefiniteness of some of them, make it uncertain that only a single disease was involved. However, the most striking and consistent sign was an unusual inactivity, especially in summer, of colonies that frequently formed several small detached clusters of flightless inceranu, each from a different
368
APIS
IRIDESCENT
VIRUS
dividuals, and that often lost many bees crawling on the ground. Large colonies perished within 2 months of becoming visibly affected. Individual bees of the samples were examined microscopically for internal parasites resembling those well-known in Apis mellifera, i.e., Acarapis bz,oodi, Nosema apis. and Malpighatnoeba mellijcae. Iridescent virus was detected either after an extraction process, described below. or by microscopical examination of tissues held beneath water and illuminated from above with an oblique intense beam of white light. Iridescent virus was extracted from bees that were ground up in a 4.1 mixture of 0.01 M potassium phosphate (pH 6.6) + 0.02% of sodium diethyldithiocarbamate (to prevent melanization) and diethyl ether. The mixture was emulsified with one volume of carbon tetrachloride, coarsely filtered, and cleared at 1OOOg for 30 min. and the cleared fluid was centrifuged at 1OOOgfor 30 min or 18,OOOg for 15 min. There was sufficient virus in 10 or more infected bees to produce a bright turquoise iridescent pellet. The whole particles of iridescent virus so obtained were tested serologically in tube precipitation tests using an antiserum prepared against an iridescent virus obtained initially from A. cerana from Kashmir (Bailey et al. 1976). Iridescent virus-infected tissues were seen easily by microscopical examination, best of all in fresh unfixed specimens, but readily enough in bees that had been preserved in formalin or alcohol, although these materials had not always penetrated satisfactorily. Infected tissues appeared bright blue in striking contrast with the surrounding creamy white tissue. Single infected cells could be distinguished in otherwise healthy tissue. RESULTS
Apis iridescent virus was the only recognized parasite that was in all the samples
IN APIS
CERANA
369
and in almost every bee of those examined individually (Table 1). Preparations of the virus, made from all the unpreserved material, were serologically indistinguishable. The fat body was always attacked by iridescent virus (Table 2). but other tissues and organs, including the ovaries, were frequently infected. The midgut seemed least susceptible, at least to the multiplication of sufficient virus to cause iridescence. The bees of colony 5 (Tables 1, 2) were clearly more severely infected, with 75% of all examined tissues infected, than those of colony 4, which had only 29% of examined tissues infected. However, according to the description of these colonies, only colony 4 showed signs of clustering, whereas both were producing crawling bees. The difference between the proportions of bees infected with tracheal mites in the samples from these two colonies is not significant (P > 0.05). DISCUSSION
The obvious conclusion from these observations is that Apis iridescent virus causes the disease that appears as inactivity, clustering, and crawling of adult individuals of A. cerana and that causes the ultimate death of their colony. The only other recognized internal parasites sometimes involved were tracheal mites, but none of the eight available samples had all its bees infested with these, and two of the samples had none. Clearly, if the samples were from colonies all with the same disease, tracheal mites could not have been the cause, nor could they be necessary as vectors of the virus. The proportion of samples that were infested with tracheal mites seems high, but a similar proportion of seemingly normal colonies of A. cerana in the same localities may have been infested. As an example of this possibility, well over 50% of apparently healthy colonies of Apis mellifera have been found infested with A. tz-oodi at one time in Britain (Bailey, 1961). Evidence that Apis iridescent virus is the
370
BAILEY
AND
BALL
TABLE APIS IRIDESCENT
Sample”
VIRUS,
TRACHEAL
MITES
FROM
COLONIES
Region
AND
I
OTHER
WITH
Date
PARASITES
“CLUSTERING
IN SAMPLES
OF ADULT
APB CERANA
DISEASE”
Degree
of infection”
Iridescent
virus
or proportion Tracheal
of bees infected
mites
Other
with:
parasites
I
Kashmir
Nov.
1974
+
911 I
Oil I
2
Kashmir
Aug.
I975
+
0112
O/12
3
Kashmir
Oct.
1976
+
-
0140
4
Kashmir
Dec.
1976
27127
14127
0127
5
Kashmir
Dec.
1976
919
619
019
6
Himachal Pradesh
May
1977
919
019
019
Himachal Pradesh
Jul.
1977
15115
Ill4
011.5
Himachal Pradesh
Jul.
1977
12/14
1114
O/l4
7
8
‘I Samples I, 2. 3. 7, and 8 were unpreserved. samples 4 and 5 were in formalin. and sample 6 was in alcohol. ’ + = an average of about IO” particles of iridescent virus per bee in pooled samples of 20 or more individuals: - = sample too desiccated.
cause of the clustering and crawling sickness in colonies ofA. cerana, though strong, is still only circumstantial. Objective surveys and experimental work, including field tests, with A. cerana in Northern India, are needed to resolve the question. At present, tests have been done only with A. rneflifera in the laboratory (Bailey et al., 1976), and these cannot lead to an under-
standing of the ecology of the virus. However, they have shown that A. mellifera can become infected by it and indicate the possible danger of attempts which are made to establish colonies of A. cerana in regions where it does not occur but Apis rnellifera does. Admittedly, the virus has multiplied quickly and abundantly in A. melliferu only when injected into
TABLE THE
NUMBERS
OF ADULT
OF APB CERANA CONTAINING
INDIVIDUALS
IN SAMPLES
FROM
2
COLONIES
WITH
“CLUSTERING
Iridescent
Sample” 4 5 6
Fat body 27 9 Y
Esophagus s 6
‘I Sample numbers as in Table I. ’ - = Sample inadequately preserved.
Crop 7 8 -
Ventriculus 0 2 3
IRIDESCENT
TISSUES
DISEASE”
tissue”
Rectum 2 Y I
Hypopharyngeal glands I 6 5
Ovaries 0 7 -
APfS
IRIDESCENT
VIRUS
pupae and has produced only slight infections, after about 1 month, when fed to caged adults. It may infect A. ceranu more readily, especially in natural circumstances, although it is characteristic of the iridescent viruses, at least of other insect species, not to cause rapidly fatal diseases. Nevertheless, their eventual effects on populations of insects, such as honey bees, that reproduce slowly, or hardly at all, for relatively long periods, could well be severe. The possibility that A. cerana is an accidental host for an iridovirus that can only complete its infective cycle in other insects seems unlikely. It would mean the almost simultaneous infection by chance of most bees of colonies, enough to cause the cases of severe disease observed. More probably, the virus is effectively transmitted from individual bees in which it first multiplies to others in the colony; so it could be enzootic in A. cerana. The variety of tissues in which it multiplies suggests it could be transmitted via eggs, feces, or gland secretions in food exchanged between adults or supplied by these to larvae. Conceivably one or more species of the external mites of A. cerunu (Crane, 1968) may act as vectors. Alternative hosts cannot be excluded, although, unlike other iridoviruses of the same type, Apis iridescent virus did not multiply when injected into unrelated species. e.g.. Galleriu mrllonellu (Bailey et al, 1976) or species of Noctuidae (Bailey, unpubl.). The two other Apis spp. that occur in India seem most likely as alternative hosts. Successful control ofApis iridescent virus would probably depend on methods that prevent its invasion of colonies and spread within them rather than on the treatment of sick colonies. A knowledge of the ecology of the virus is needed so that such preven-
IN APIS
CERANA
371
tive measures may be devised. Even were these measures not forthcoming, the knowledge gained would probably lead to a better understanding and assessment of damage due to the virus and could well save wasted effort in attempts to control coincident but relatively unimportant parasites. ACKNOWLEDGMENTS We thank Messrs. D. B. Mahindre, Central Bee Research Institute. Srinagar. Kashmir, T. A. Shah, Shah Beekeepers. Srinagar, Kashmir. and Professor G. S. Dogra. Himachal Pradesh University for samples ofA. c’eran(~.
REFERENCES ATWAL, A. S. lY71. Acarine disease problem of the lndian honey bee. Apis indicu F. Amer. Bee J.. 3, 134-135, 186-187. ATWAL, A. S., AND SHARMA, 0. P. 1970. Acarine disease of adult honey bees: Prevention and control. lndian Farming. 20, 39-40. BAILEY, L. 1958. The epidemiology of the infestation of the honey bee. Apis mrllifera L. by the mite, Acurupis n~oo& (Rennie), and the mortality of infested bees. Parasitology. 48, 495-506. BAILEY. L. 1961. The natural incidence of Acurapis ,cwodi (Rennie) and the winter mortality of honey bee colonies. Bee World, 42, 96- 100. BAILEY, L. 1965. The effect of Acarupis ,c,oodi on honey bees from North America. J. Apkult. Rrs.. 4, 10% 108. BAILEY, L.. AND LEE, D. C. 1959. The effect of infestation with Acarapis rr,oodi (Rennie) on the mortality of honey bees. /. Insecf Patho/., 1, 15-24. BAILEY, L.. BALL, B. V.. AND WOODS, R. D. 1976. An iridovirus from bees. J. Gen. Viral.. 31, 459-461. CRANE. E. 1968. Mites infesting honeybees in Asia. Bee World. 49, I l3- 114. MICHAEL. A. S. 1957. Acarine disease found in India. Amer. Bee J.. 97, 107. MILNE, P. S. 1957. Acarine disease in Apis indictr. Bee World. 38, 156. SINGH, S. 1957. Acarine disease in the Indian honey bee (Apis indic,a, F.) Indian Bee J.. 19, 27-28.