SUSCEPTIBILITY OF THE ASIAN HONEY BEE (APIS CERANA) TO AMERICAN FOULBROOD (PAENIBACILLUS LARVAE LARVAE)

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1 SUSCEPTIBILITY OF THE ASIAN HONEY BEE (APIS CERANA) TO AMERICAN FOULBROOD (PAENIBACILLUS LARVAE LARVAE) Kai-Kuang Ho and Yue-Wen Chen* Department of Entomology, National Taiwan University, Taipei, Taiwan 106, R.O.C. Tel & Fax: , kkho@ccms.ntu.edu.tw *Department of Applied Animal Science, National I-Lan Institute of Technology, I-Lan Taiwan 260, R.O.C. ABSTRACT American foulbrood (Paenibacillus larvae larvae) is an important disease of larval European honey bee (Apis mellifera). However, disease signs have never been encountered during hive inspections of Asian honey bee (Apis cerana) colonies in Taiwan. To study the susceptibility of A. cerana larvae to AFB, various doses of P. l. larvae spores were added to larval food and disease development was monitored. Results showed that 1-day-old larvae were most susceptible, next were 2-day-old larvae, while 3-day-old larvae showed no signs of disease even when fed a large dose. At the susceptible age, A. cerana larvae showed higher resistance than A. mellifera larvae when fed the same dose of spores. This resistance by A. cerana larvae apparently was not totally related to their innate immune capability. An important aspect contributing to the resistance of A. cerana was the fact that up to 82.2% of inoculated larvae were removed by adult workers before the capped stage. This adult hygienic behavior effectively decreased the level of spore contamination inside the hive. Keywords: susceptibility, resistance, hygienic behavior, Apis cerana, Apis mellifera, American foulbrood, Paenibacillus larvae larvae INTRODUCTION American foulbrood (AFB) is an important disease of larval European honey bees (Apis mellifera) caused by the bacterium, Paenibacillus larvae supsp. larvae (formerly Bacillus larvae). Only the spore stage of P. l. larvae can initiate the disease, and 1-day-old larvae are highly susceptible, whereas 2-day-old larvae are resistant (Chen et al., 1997). The pathogenesis of P. l. larvae begins when the young larva ingests P. l. larvae spores, which germinate approximately one day later (Bamrick, 1964). After germination the bacteria multiply in the midgut and penetrate into the haemocoele through the gut wall, and the larva ultimately dies from the septicemia. Certain lines of A. mellifera have genetically determined resistance to AFB. This resistance stems from the hygienic removal of infected brood (Thompson, 1964), the filtering role of the proventricular valves of adults (Sturtevant & Revell, 1953), nursing protection of larvae by adults (Thompson & Rothenbuhler, 1957) and bacterial inhibition of larval food (Rose & Briggs, 1969). In Taiwan, Asian honey bees (A. cerana) and A. mellifera are sympatric and share the same resources. Although AFB was first reported in 1967 in A. mellifera colonies (Yen & Chyn, 1971), disease signs in local A. cerana colonies have not been encountered during our hive inspections in Moreover, Asian honey bees show a high resistance in China (Yang, 1982), and no AFB cases have been confirmed in Malaysia (Yusof & Ibrahim, 1995). In contrast, AFB had been Proceedings of the 37 th International Apicultural Congress, 28 October 1 November 2001, Durban, South Africa APIMONDIA 2001 To be referenced as: Proc. 37 th Int. Apic. Congr., 28 Oct 1 Nov 2001, Durban, South Africa ISBN: Produced by: Document Transformation Technologies Organised by: Conference Planners

2 reported in A. cerana colonies in India (Singh, 1961) and Papua New Guinea (Anderson, 1990). The present experiments were designed to determine the susceptibility of Asian honey bees to AFB. MATERIALS AND METHODS Preparation of P. l. larvae cell and spore suspension Vegetative cells of P. l. larvae (ATCC 9545) were grown on Difco brain-heart infusion agar plates supplemented with 0.1 ppm thiamine (BHIT) for three days at 37 C. One colony was placed in a tube which contained three ml BHIT broth and incubated for 24 h at 37 C. The broth was centrifuged at 3,000 g, at 4 C for 30 min and the pellet resuspended with 10 ml of sterilized 0.1M PBS. This suspension contained cells/ml when estimated using a haemocytometer. A spore suspension of P. l. larvae was prepared from the dried remains (scales) of infected European honey bee larvae from the apiary of National Taiwan University. Fifty AFB scales were macerated in 50 ml sterilized distilled water and filtered through a wire netting (150 mesh); the filtrate was centrifuged at 3,000 g, at 4 C for 30 min, the pellet was resuspended with 10 ml sterilized distilled water. The prepared spore suspension which contained spores/ml when estimated using a haemocytometer was kept at 4 C as a stock spore inoculant. The sterilized P. l. larvae spore suspension was prepared by exposure to 2.8% formaldehyde at 37 C for 2 h. These spores were sedimented by centrifugation at 3,000 g for 30 min and washed twice with sterilized distilled water, then incubated at 80 C for 10 min. Its sterility was confirmed by incubating 200 µl of the suspensions on BHIT plates. Oral inoculation in larvae Three A. cerana colonies, each containing six frames of adult workers, were used for this experiment. The queen was confined to a individual frame for 24 h to lay eggs. After oviposition, the experimental comb containing eggs was isolated from the queen in one of two nurse colonies for subsequent development. The egg period was defined to three days. When eggs reached the 1-day-old larval stage, 120 larvae were selected for spore inoculations and the rest were kept for further inoculations on following days. The 1-day-old larvae divided into four experimental groups of 30 larvae according to their location. The distance between groups was at least five rows of cells. One µl of P. l. larvae spore suspension was added to the larval food of each with a micropipette (Gilson, P2). Three doses of spores; 4500, 442 or 21 spores/larva were added and control larvae were added with the same volume of sterilized distilled water. Similarly, on the following days, the 2-day-old larvae were selected and inoculated with 1 µl containing 45,000, 4,500, 450 or 45 spores. Moreover, the 3-day-old larvae were selected and inoculated with 45,000 or 4,500 spores in their food. The position of each larva and the treatment each received was recorded on a transparency film. After the treatments, the test combs were returned to their original nurse colonies until the treated larvae were sealed. The capping rate of larvae was determined and the combs without adult workers were removed and incubated at 34 C, 70% RH until 15 days after egg hatching, after which all inoculated bees were examined for AFB signs. Intrahaemocoeleic inoculation in pupae Combs containing capped cells were randomly selected from A. cerana and A. mellifera colonies without clinical AFB symptoms. The 1-day-old pupae with white eyes were removed from the combs and incubated outside the hive at 34 C for 1 h. The melanizing pupae were discarded. A sterilized syringe (Hamilton, 1710) attached to a sterilized 30-gauge needle (Hamilton, 90030) was inserted.

3 1-2 mm into the segmental membrane between the 3 rd and 4 th terga of the abdomen, and 1 µl suspension of P. l. larvae vegetative cells was injected. Control pupae were injected with the same volume of sterilized 0.1M PBS. The 10-fold dilutions of vegetative cells, ranged from down to 8.3, were injected into pupae. Thirty pupae were injected in each dose. The injected pupae were then placed into the wells of 96-well plates and incubated at 34 C, 70% RH. The pupae were examined for AFB signs at 10 days post-injection. The number of AFB pupae was counted. Dead bees that didn t show the typical AFB symptoms were examined for AFB using the method of Hornizky and Wilson (1989). Hygienic activity toward larval stage Three A. cerana and A. mellifera colonies each containing six and nine frames of adult workers, respectively, were used for this study. Their 1- and 2-day-old larvae, each, were inoculated with 1 µl of viable or sterilized P. l. larvae spores by the oral method, and control larvae were added with sterilized distilled water. After inoculation, the larvae were observed the same way as after the oral inoculation mentioned above. RESULTS Oral inoculation of larvae The dose-mortality relationship of the 1-day-old A. cerana larvae when fed P. l. larvae spores is shown in table 1. The inoculated larvae showed clinical symptoms of AFB, i.e. brood decay to a brownish, viscous mass, even at the lowest dose (21 spores/larva). The AFB symptoms were only observed at the prepupal stage, no pupal case was found in this study. Overall the presence of AFB was low ( %). Many of the P. l. larvae infected larvae ( %) were removed by adult workers before they were capped. At a dose of 4,500 spores, 58.8 % of inoculated larvae were removed before being sealed and only 18.9 % larvae showed AFB symptoms. The total mortality was 74.8%. But in the rest (i.e., 41.2%) of inoculated larvae that reached into the capped stage, only 46.2 % capped larvae showed AFB symptoms and the remainder (53.8%) were healthy when reaching the pre-emergence stage. The similar trend was found at a dose of 442 spores, only 14.1% larvae showed AFB symptoms and there was higher removal of larvae (36.9%) than the controls (7.1%). The mortality increased significantly (P < 0.05) at a dose of 21 spores, but the removal of larvae (14.8%) did not differ from the controls (P > 0.05). The mortality of the 2-day-old larvae increased significantly (P < 0.05) only at heavy doses ( , spores/larva). Even at these heavy doses, only % of total inoculated larvae had AFB. Again, the higher removal ( % vs. 10% in controls) was found in the heavy inoculations. The 3-day-old larvae did not develop AFB symptoms at the doses tested. Intrahaemocoeleic inoculation of pupae Pupae of A. cerana were highly susceptible to P. l. larvae vegetative cells by this inoculation method (table 2). All A. cerana pupae injected with or cells showed AFB symptoms, and 60.0% of those injected with cells. Pupae of A. mellifera also showed similar results. None of the control pupae injected with PBS died and exhibited AFB symptoms, although 6.7% died of other causes. The dose-mortality data significantly fitted the log-probit analysis (χ 2, P < 0.05). The LD 50 of A. cerana and A. mellifera were 41.3 and 94.5 cells per bee, respectively.

4 TABLE 1. Development of American foulbrood in 1-, 2-, and 3-day-old worker larvae of A. cerana, after inoculation with P. l. larvae spores Larval No. spores Removed before AFB Healthy at pre- Corrected age inoculated capped stage (%) (%) emergence (%) mortality 1 (%) 1-day-old c b 22.3 a 74.8 d b 14.1 b 49.0 b 47.1 c a 4.6 a 80.5 c 13.2 b a 0 a 92.9 d 0 a 2-day-old c 3.5 b 68.9 a 23.3 c b 1.1 a 77.2 b 14.1 b a 0 a 85.6 c 4.9 a a 0 a 88.2 c 1.9 a a 0 a 90.0 c 0 a 3-day-old a a a a a a - 1 Corrected mortality = (1 - healthy pupae of treatments / healthy pupae of control) 100% 2 Means in the same column of larval age followed by the same letter are not significantly different ( LSD test, P < 0.05) Hygienic activity toward larval stage There were no significant differences (P > 0.05) in mortality between two species of bees that received the same treatment (table 3). When susceptible larvae were inoculated with viable spores, the number of larvae removed was increased significantly (P < 0.05). However, both mortality and removal of larvae did not differ significantly (P > 0.05) between the control and sterilized-spore groups. Although the percentages of healthy pupae between two species of bees were similar, A. cerana colonies removed more 1-day-old larvae (82.2%) than A. mellifera (32.2%) when inoculated with viable spores and more (37.8%) than A. mellifera (3.0%) when inoculated at the 2- day-old stage. In contrast, A. mellifera showed more (1-day-old, 64.9%; 2-day-old, 25.6%) AFB cases than A. cerana colonies (1-day-old, 10.0%; 2-day-old, 0%).

5 TABLE 2. Development of American foulbrood in 1-day-old worker pupae after injection with P. l. larvae vegetative cells Species No. cells injected Emergence (%) Mortality(%) AFB Other A. cerana M PBS A. mellifera M PBS TABLE 3. Development of American foulbrood in 1- and 2-day-old worker larvae after inoculation with of viable or sterilized P. l. larvae spores Species Larval age Treatment 1 Removed before capped stage (%) AFB (%) Healthy at preemergence (%) Corrected mortality (%) A. cerana 1-day-old I 82.2 d a 7.8 a 91.1 c S 14.4 ab cd 2.5 a W 12.2 ab cd 0 a A. mellifera I 32.2 c 64.9 c 1.1 a 98.7 c S 12.2 ab cd 0 a W 12.2 ab cd 0 a A. cerana 2-day-old I 37.8 c b 27.3 b S 19.1 b c 5.5 a W 14.4 ab cd 0 a A. mellifera I 3.0 a 25.6 b 71.4 b 26.3 b S 3.3 a d 0.2 a W 3.1 a d 0 a 1 I = infectious spores, S = sterilized spores, W = water 2 Means in the same column followed by the same letter are not significantly different ( LSD test, P < 0.05)

6 DISCUSSION This report supports the observations that larval A. cerana are susceptible to P. l. larvae and show typical AFB signs. AFB has not been reported from A. cerana colonies in Taiwan. However, larvae of suitable age inoculated with enough spores have been shown to die of AFB and some also show typical AFB signs at the prepupal stage. Larval age is the key factor that determines their P. l. larvae susceptibility. In A. cerana, 1-day-old larvae were the most susceptible to P. l. larvae and resulted in 74.8% of mortality after spores/larva inoculation. The 2-day-old larvae were less susceptible and resulted in 14.1% of mortality at the same dose of spore inoculation, while 3- day-old larvae were non-susceptible even when inoculated with spores/larva. This general trend was similar to previous studies (the inoculants were the same origin) on the pathogenicity of P. l. larvae to larvae of A. mellifera (Chen et al., 1997). A comparison between these two species of honey beesrevealed that larvae of A. cerana are less susceptible than A. mellifera. Our previous data showed that the LD 50 and LD 95 of P. l. larvae spores to 1-day-old larvae of A. mellifera were 21 and 442 spores, respectively (Chen et al., 1997). However, in this study the same doses resulted in much lower mortality for A. cerana larvae of the same age (table 1). Similar results were found on 2-dayold larvae. Inoculations of spores/larva resulted in 37.2% mortality of A. mellifera larvae (Chen et al., 1997), but only 23.3% of A. cerana larvae. This suggests that one of the critical factors leading to the resistance to AFB in A. cerana is that the larvae are less susceptible to spores of P. l. larvae. In A. mellifera, Rose and Briggs (1969) found brood food from worker cells containing young larvae from AFB resistant lines were more effective in inhibiting spore germination and in reducing the number of viable vegetative cells of P. l. larvae than susceptible lines. Rinderer and Rothenbuhler (1974) found that pollen added to the food of worker larvae of A. mellifera at an age of 6-18 h reduced their susceptibility to AFB. This indicates that food composition can affect the susceptibility of bee larvae to P. l. larvae. Differences have been noted in the composition of larval food between A. mellifera and A. cerana. Takenaka and Takenaka (1996) reported A. cerana royal jelly contained more protein and less carbohydrate than A. mellifera. The larval food of A. cerana may be more effective in reducing the growth of vegetative cells in larval midgut is worth testing in the future because this reduction may contribute to AFB resistance in larvae of A. cerana. Infected larvae quickly die when bacteria enter the haemocoele and result in systemic bacteremia (Davidson, 1973). In young larvae of A. mellifera the time required for vegetative cells to penetrate the gut wall and invade the body cavity varied from days (Bamrick, 1964). In A. cerana, up to 58.8% of inoculated larvae were removed before the capped stage (table 1). The AFB scales contain several volatile acids and other materials that compose the distinct odor (Gochnauer & Shearer, 1981). In this study, inoculant of spore suspension also contained this odor that may entice adult workers to remove. However, table 3 showed the higher removal of inoculated larvae might result from the pathogenicity of spores rather than their distinct odor. This implied that bacteria might have invaded the body cavity of the removed larvae. In A. mellifera 32.2% of inoculated larvae were removed before the capped stage. In contrast, 82.2% of removal happened in precapping A. cerana which may indicate an earlier penetration of gut wall by P. l. larvae in host larvae. Thus the most of infected A. cerana larvae could not reach the capped stage and were effectively removed by adult workers. One individual with the typical AFB signs can produce about 2.5 billions spores (Sturtevant, 1932). In A. mellifera, up to 64.9% of inoculated larvae exhibited AFB signs (table 3). The process of removing this disease individual by the workers would make a heavy contamination toward A. mellifera colonies. However, only 10.0% of larvae showed AFB signs in A. cerana, thus there was a much less chance for contamination.

7 Gilliam and Jeter (1970) reported that A. mellifera adult workers produced agglutinating substances in response to an injection of a vaccine prepared from P. l. larvae. Wilson (1970) reported the LD 50 was 5,000 spores when injecting P. l. larvae spores into the cephalic haemocoele of A. mellifera pupae. These reports showed a considerable immune response occurred in the haemocoele of pupae and adults of A. mellifera. Since normally, it is the vegetative cell of P. l. larvae that invades the body cavity of its host, we injected the vegetative cells into the haemocoele of 1-day-old pupae of both bees. Results showed the LD 50 were 94.5 and 41.3 cells per pupa in A. mellifera and A. cerana respectively. Obviously, A. cerana was more sensitive than A. mellifera when P. l. larvae cells penetrating into the pupal haemocoele. These results suggest that A. cerana pupae may not produce the effective immune response toward the invasion of P. l. larvae cells. In Taiwan, A. mellifera colonies suffer from many serious diseases and enemies, i.e. Varroa mites, wasps, chalkbrood and AFB, while these rarely occurred in A. cerana colonies. Only sacbrood and European foulbrood occasionally occurred in A. cerana colonies during our hive inspection throughout Taiwan. Results of this study identified two factors that may contribute to the resistance to AFB. One factor is the fact that young larvae themselves may be less susceptible to pathogens. The other is, before the capped stage, most of the infected larvae were already removed by adult workers of A. cerana, thus greatly decreased the level of spore contamination inside the hive. REFERENCES ANDERSON, D L (1990) Apis cerana and its associated mites and pathogens in Papua New Guinea. In Ritter, W (eds) Proceedings of the International Symposium on Recent Research on Bee Pathology, 1990, Gent, Belgium; pp BAMRICK, J F (1964) Resistance to American foulbrood in honey bees. V. Comparative pathogenesis in resistant and susceptible larvae. J. Invertebr. Pathol. 6(3): CHEN, Y W; WANG, C H; HO, K K (1997) Pathogenicity of Bacillus larvae to larvae of the honey bee (Apis mellifera). Chinese J. Entomol. 17(1): DAVIDSON, E W (1973) Ultrastructure of American foulbrood disease pathogenesis in larvae of the worker honey bee, Apis mellifera. J. Invertebr. Pathol. 21(1): GILLIAM, M; JETER, W S (1970) Synthesis of agglutinating substances in adult honeybees against Bacillus larvae. J. Invertebr. Pathol. 16(1): GOCHNAUER, T A; SHEARER, D A (1981) Volatile acids from honeybee larvae infected with Bacillus larvae and from a culture of the organism. J. Apicul. Res. 20(2): HORNITZKY, M A Z; WILSON, S C (1989) A system for the diagnosis of the major bacterial brood disease of honeybees. J. Apicul. Res. 28(4): RINDERER, T E; ROTHENBUHLER, W C (1974) The influence of pollen on the susceptibility of honey-bee larvae to Bacillus larvae. J. Invertebr. Pathol. 23(3): ROSE, R I; BRIGGS, J D (1969) Resistance to American foulbrood in honey bees. IX. Effects of honey-bee larval food on the growth and viability of Bacillus larvae. J. Invertebr. Pathol. 13(1): SINGH, S (1961) Appearance of American foulbrood disease in Indian honeybee (Apis indica Fabr.). Indian Bee Journal 23(7/9): STURTEVANT, A P (1932) Relation of commercial honey to the spread of American foulbrood. J. Apicul. Res. 45:

8 STURTEVANT, A P; REVELL I L (1953) Reduction of Bacillus larvae spore in liquid food of honey bees by action of the honey stopper, and its relation to the development of American foulbrood. J. Econ. Entomol. 46(5): TAKENAKA, T; TAKENAKA, Y (1996) Royal jelly from Apis cerana japonica and Apis mellifera. Biosci. Biotech. Biochem. 60(3): THOMPSON, V C (1964) Behaviour genetics of nesting in honeybees. III. Effect of age of bees a resistant line on their response to disease-killed brood. J. Apicul. Res. 3(1): THOMPSON, V C; ROTHENBUHLER, W C (1957) Resistance to American foulbrood in honey bees. II. Differential protection of larvae by adults of different genetic lines. J. Econ. Entomol. 50(6): WILSON, W T (1970) Inoculation of the pupal honeybee with spores of Bacillus larvae. J. Apicul. Res. 9(1): YANG, K F (1982) The characteristics of Apis cerana. In Kansu Apicultural Institute (ed) Report Collections of Apis cerana in China. Kansu Apicultural Institute; Tien-Shui, China; pp YEN, D F; CHYN L C (1971) Studies on a bacterial disease of honeybee in Taiwan. Plant Protection Bulletin13: YUSOF, M R; IBRAHIM, R (1995) Current status of pests and diseases of the honeybee, Apis cerana F., in peninsular Malaysia. In Kevan, P G (ed) The Asiatic hive bee: apiculture, biology, and role in sustainable development in tropical and subtropical Asia. Cambridge, Canada; Enviroquest Ltd; pp

9 SUSCEPTIBILITY OF THE ASIAN HONEY BEE (APIS CERANA) TO AMERICAN FOULBROOD (PAENIBACILLUS LARVAE LARVAE) Kai-Kuang Ho and Yue-Wen Chen* Department of Entomology, National Taiwan University, Taipei, Taiwan 106, R.O.C. Tel & Fax: , kkho@ccms.ntu.edu.tw *Department of Applied Animal Science, National I-Lan Institute of Technology, I-Lan Taiwan 260, R.O.C. A brief introduction of the presenting author Name: Kai-Kuang Ho Affiliation: Dept. of Entomology, National Taiwan Univ., Taipei, Taiwan Position: Professor Education: B. S., National Taiwan Univ., 1958 M. S., Mississippi State Univ., USA, 1969 Ph. D., National Taiwan Univ., 1975 Experience: 1.Professor of the Dept. of Entomology, National Taiwan Univ. Have taught ''Apiculture'' course for 30 years. 2. Consultant of the'' Taiwan Apiculture Association'' for 30 years