Juvenile Hormone Titer and Reproduction of Varroa Jacobsoni In

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Juvenile hormone titer and reproduction of Varroa jacobsoni in capped brood stages of Apis cerana indica in comparison to Apis mellifera ligustica P Rosenkranz, Nc Tewarson, A Rachinsky, A Strambi, C Strambi, W Engels

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P Rosenkranz, Nc Tewarson, A Rachinsky, A Strambi, C Strambi, et al.. Juvenile hormone titer and reproduction of Varroa jacobsoni in capped brood stages of Apis cerana indica in comparison to Apis mellifera ligustica. Apidologie, Springer Verlag, 1993, 24 (4), pp.375-382. hal-00891081

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Juvenile hormone titer and reproduction of Varroa jacobsoni in capped brood stages of Apis cerana indica in comparison to Apis mellifera ligustica

P Rosenkranz NC Tewarson A Rachinsky A Strambi C Strambi W Engels

1 LS Entwicklungsphysiologie, Zoologisches Institut der Universität, Auf der Morgenstelle 28, D-72076 Tübingen, Germany; 2 Department of Zoology, Ewing Christian College, Allahabad-211003, India; 3 Laboratoire de Neurobiologie, CNHS, BP 71, F-13402 Marseille Cedex 9, France

(Received 24 November 1992; accepted 19 February 1993)

Summary — The juvenile hormone (JH) III hemolymph titer of late larval and early pupal stages as determined by radioimmunoassay (RIA) does not differ significantly in Apis cerana and Apis mellifera, particularly in freshly-sealed worker brood. In drone brood, slightly higher JH III hemolymph concentrations were recorded. These results do not agree with the hypothesis that reproduction of the parasitic bee mite Varroa jacobsoni is regulated by host-derived JH. After checking over a thousand capped brood cells containing pupae, it was confirmed that in Apis cerana colonies fertility of female mites is restricted to drone hosts.

Apis cerana indica / Apis mellifera ligustica / juvenile hormone titer / reproduction / Varroa jacobsoni

INTRODUCTION Modern analytical techniques allow the de- termination of hemolymph titer (Rachinsky In honey bees, juvenile hormone (JH) has et al, 1990) and rate of synthesis (Rachin- been studied mostly under aspects of sky and Hartfelder, 1990) even in individu- caste development (Hartfelder, 1990; al bees. Since in arthropods (Downer and Rembold et al, 1992), control of fertility Laufer, 1983) and also in acarids, ticks (Engels et al, 1990), and recently also (Pound and Oliver, 1979; Connat et al, polyethism regulation (Robinson, 1992). 1983) as well as mites (Oliver et al, 1985)

* Present address: Bayerische Landesanstalt für Bienenzucht, Burgbergstr 70, D-91054 Erlangen, Germany exogenous JH was generally found to af- The time of brood cell capping was recorded fect reproduction, it was hypothesized that with the help of transparent sheets. Subsequent- ly larvae were at first taken at 6-h intervals, and this hormone could also regulate oogene- later on less frequently. For each sample 40- sis in the parasitic bee mite Varroa jacob- 120 μl hemolymph from non-parasitized speci- soni, and, in addition, that host-derived JH mens were collected from 3-9 individuals, could be responsible for initiation of repro- pooled, extracted with hexane, coded and duction (Hänel, 1983). Particularly, a high stored at -20°C. After being transported to Eu- JH III hemolymph titer in drone larvae of rope on dry ice, JH III titer determinations were carried out after diol derivatization by radioim- the was consid- early postcapping phase munoassay (Strambi et al, 1981; Rosenkranz et ered to stimulate Varroa vitellogenesis in al, 1990). In the Apis mellifera ligustica colony the original host species, Apis cerana, and only little drone brood was present and was in- likewise in worker larvae of the western sufficient for sampling all required stages, prob- honey bee, Apis mellifera (Hänel and Koe- ably due to high ambient temperatures. niger, 1986). Thus a low JH III titer in late In order to evaluate the actual reproduction of Varroa in the cerana indica 5th instar worker larvae would be a resist- jacobsoni Apis colonies, = 1 100 capped brood cells containing ant factor, mite to restricting reproduction pupae were opened and checked for female drone brood. mites and their offspring. Because of severe However, in the only known case of var- brood infestation with Tropilaelaps clarae, no roatosis tolerance in mellifera, the Af- comparative data could be sampled from the Apis Apis mellifera ligustica colony. ricanized honey bees of Brazil (Engels et al, 1986), fertility of female mites in worker brood is also reduced (Ritter and De Jong, RESULTS 1984) but was not found to be correlated with a low JH III hemolymph titer in worker larvae (Rosenkranz et al, 1990). These Juvenile hormone hemolymph titer data already contradicted the hypothesis in postcapping stages of worker brood of a possible regulation of Varroa reproduction by the host’s JH. Because detailed At the time of brood cell sealing in worker JH III titer measurements were in lacking larvae, the JH III hemolymph titer was cerana larvae, we hemo- Apis analyzed found to be low, ranging < 10 pmol/ml (fig collected from Indian eastern lymph honey 1 a). However, soon after cell capping a bees with mellifera together Apis ligustica steady increase was measured. Peak val- at the same site. The fertili- samples study ues of 70-100 pmol/ml were reached of Varroa females on drone and worker ty ≈ 30-48 h post cell operculation. There hosts of cerana indica was also eval- Apis was not much variation in the different pool uated. data sampled during that period after capping. On average, in 5th instar larvae of Apis cerana indica slightly higher JH MATERIALS AND METHODS concentrations were determined than in Apis mellifera ligustica, but the differences Hemolymph samples were collected during the were statistically insignificant (P > 0.7; spring season, March 1991, from 4 colonies of Friedman test) even around the peak val- cerana indica in Newton hives Apis kept (Tewar- ues. The duration of the JH III maximum son et al, 1992) and 1 colony of Apis mellifera li- was ≈ 24 h, followed by a decrease in titer gustica originating from the US in a Langstroth hive. All experimental colonies had 5 brood reached ≈ 96 h after cell capping, and combs or more and were kept on the campus of again by another increase in the pupal Ewing Christian College, Allahabad, north India. stages (fig 1 a). Juvenile hormone hemolymph titer in postcapping stages of drone brood

Within the first 48 h after brood cell operculation in drone larvae of Apis cerana indica the JH III hemolymph titer varied around 50 pmol/ml (fig 1b) which is clearly more than in worker larvae (fig 1 a). An increase was observed with peak values up to 170 pmol/ml only = 84 h after cell capping. The subsequent decrease before the prepupal/ pupal moult and later reincrease resemble the titer pattern as determined for workers. The few available data for Apis mellifera ligustica drones fall into the range described for Apis cerana indica males. Whether in drone L5 larvae of the western honey bee the JH peak is reached a little earlier has not yet been determined.

Fertility of Varroa jacobsoni in Apis cerana indica brood

Because the first Varroa egg is usually laid = 3 d after brood cell capping, an evalua- tion of the reproductivity of female mites at pupal stages of the host allows a distinct discrimination to be made between fertile and infertile parasite individuals. This was analyzed in several hundred Apis cerana indica worker and drone brood cells = 1 wk after sealing. The results (table I) demon- lymph titer in postcapping stages of drones strate a much lower level of brood infesta- has not been published previously, where- tion in worker than in drone cells. Further- as such data are already known for work- more, no fertile Varroa female was ers and queens (Rachinsky et al, 1990). In detected in the worker brood at all, where- both female castes of Apis mellifera carni- as most of the mites found in drone brood ca the JH III content has also been report- were reproducing. ed recently for the complete period of pre- adult development (Rembold et al, 1992) with similar worker values for all postcap- DISCUSSION ping stages. The regulation of metamorphosis and From the point of view of development, it the involvement in sex- and caste-specific is not surprising that the JH III hemolymph control of JH III production (Hartfelder et titer was found to be almost identical in al, 1993) probably existed long before the of the occurred preimaginal stages of Apis cerana indica splitting genus Apis and A mellifera ligustica. JH III titer data (Ruttner, 1988, 1992). The control of poly- in the duration of the for Apis cerana obtained by radioimmu- morphic patterns in bees noassay (RIA) are presented here for the postcapping period honey appar- first time. Similar results have recently ently allows some variation (Moritz, 1985), been reported for Apis mellifera carnica but this concerns the moment of cell oper- and Africanized bees (Rachinsky et al, culation in particular and not the hormone 1990; Rosenkranz et al, 1990). All these titer peaks correlated with the initiation of and the switch from larval data are in good agreement. metamorphosis to Little is known about JH III in drone de- prepupal-pupal developmental pro- velopment (Hartfelder et al, 1993). Howev- grams. differ- er, the data available on rates of JH syn- Consequently, species-specific thesis in late larval and early pupal drones ences in late 5th instar or prepupal JH III titers are rather as as well as the JH III hemolymph titer val- hemolymph improbable an character related to ues reported here are a little higher but do adaptive parasitiza- not differ much from the worker data. tion by Varroa jacobsoni. In fact, in newly worker larvae and of Therefore, it is improbable that a host- capped prepupae derived JH stimulus enhances Varroa re- Apis cerana indica we found a JH III titer production in drone brood. Particularly the that was even a little higher than in imma- time of the JH peak during the spinning L5 ture Apis mellifera ligustica sampled at the same and also in mellifera drone instar 3-4 d after capping of the ce- study site, Apis carnica et as well as rana brood cells already coincides with the (Rachinsky al, 1990) in Africanized bees from Brazil oviposition of the first Varroa egg and, (Rosen- kranz et All these RIA-based consequently, occurs far too late to stimu- al, 1990). data differ from the Galleria wax test val- late oocyte development in the first gonocycle. Furthermore, there is only 1 peak ues according to which the postcapping JH III titer was described to be within 8 d after capping in the JH III titer as hemolymph in mellifera than in cerana well as in JH III content (Rembold, 1987) higher Apis Apis and not 2, with the second occurring after (Hänel and Koeniger, 1986). There is no for this = 6-7 d, as reported by Hänel and Koeni- explanation discrepancy. ger (1986). An account of a RIA- Therefore, the oft-quoted hypothesis determined course of the JH III hemo- (Ramirez and Otis, 1986; Camazine, 1988; Ruttner, 1992) of a regulation of parasite Résumé — Teneur en hormone juvénile reproduction by host-derived JH in case of et reproduction de Varroa jacobsoni the Varroa-Apis relationship is not sup- dans le couvain operculé d’Apis cerana ported by the hormone titers as measured indica et d’Apis mellifera ligustica. À Al- here with a sensitive and accurate RIA lahabad (Inde du Nord), on a prélevé au (Strambi et al, 1981). Of course this does printemps des échantillons d’hémolymphe not concern the results obtained by JH ap- dans du couvain operculé d’ouvrières et de plication to Varroa females (Hänel, 1983; mâles issu de 4 colonies d’Apis cerana in- Hänel and Koeniger, 1988) which, howev- dica et d’une colonie d’Apis mellifera ligus- er, Milani and Chiesa (1990) were unable tica. On a en outre ouvert plus de 1 000 to reproduce. cellules de couvain renfermant des nym- d’A c indica vérifier la On the other hand, there is no doubt phes pour présence about the dependence of initiation of Var- de Varroa jacobsoni, et on a noté si les femelles de Varroa s’étaient Les roa oogenesis on specific host conditions reproduites. (Rosenkranz, 1990; Steiner, 1991; Rosen- échantillons d’hémolymphe ont été en- kranz and Stürmer, 1993; Steiner et al, voyés en Europe et leur teneur en hormo- 1993). Hemolymph of L5 instars capped ne juvénile III (JH) a été déterminée par less than 24 h earlier was found to stimu- méthode radio-immunologique. late mite egg development. However, in Jusqu’à la mue nymphale, les teneurs host larvae at this stage the JH III hemo- en JH du couvain operculé d’ouvrières dif- lymph titer is very low (Rachinsky et al, fèrent peu chez les 2 races indica et ligus- 1990; Rosenkranz et al, 1990; this paper). tica. Celle des larves de l’abeille indienne In any case, all the available evidence est en moyenne un peu plus élevée que points towards the significance of inhibiting celle de l’abeille européenne (fig 1a). Dans the fertility of female mites reproducing in le couvain de mâles, des teneurs en JH un worker brood as a natural resistance factor peu plus élevées dans l’ensemble que to varroatosis. This phenomenon is well chez les ouvrières (fig 1b) ont été mesu- documented both in the original host spe- rées après operculation. Les valeurs maxi- cies, Apis cerana (Koeniger et al, 1981; males se rencontrent le 2e j après l’opercu- Tewarson, 1987; Tewarson et al, 1992), lation dans le couvain d’ouvrières et le 4e j and has been confirmed by the present dans le couvain de mâles. Dans les 2 cas, study (table I). In the Africanized Apis mel- la présence d’un pic était nette. Le moment lifera scutellata hybrid biotype in Brazil où il se produit, ainsi que les différences (Ritter and De Jong, 1984; Rosenkranz et spécifiques à la race et au sexe, ne ren- al, 1990) and hybrid colonies of Apis mellif- trent pas en ligne de compte pour déclen- era monticola and Apis mellifera ligustica cher la reproduction de Varroa. (Thrybom and Fries, 1991) a similarly low Chez A c indica le parasitisme du cou- rate of Varroa fertility was observed. How- vain par Varroa a été contrôlé 1 semaine ever, the host factors responsible for influ- environ après l’operculation. Les cellules encing reproduction of the Varroa mite still renfermaient déjà des nymphes. Le cou- remain to be detected. vain de mâles était nettement plus parasité que celui d’ouvrières, conformément aux résultats antérieurs. Puisque les femelles ACKNOWLEDGMENT de Varroa pondent leur premier oeuf environ 3 j après l’operculation, il était possible This study was supported by a DAAD overseas au moment du contrôle de différencier de research grant to P Rosenkranz. façon sûre les acariens fertiles des non fertiles. Dans les cellules de mâles, presque 1 a). Bei Drohnenbrut wurden nach dem toutes les femelles d’acariens s’étaient re- Verdeckeln insgesamt leicht höhere JH- produites. En revanche dans les cellules Titer gemessen (Abb 1 b) als bei Arbeiterin- d’ouvrières, pas un seul Varroa n’avait nen. Die Titer-Maxima traten bei Arbeiterin- pondu (tableau I). nenbrut am 2. und bei Drohnenbrut am 4. nach dem Verdeckeln in beiden Ces données, en particulier celles relati- Tag auf, ves à l’évolution de la teneur en JH du Fällen war lediglich ein Peak ausgeprägt. couvain operculé chez l’abeille indienne et Diese Zeitpunkte der JH-Peaks und ebenso die Art- und l’abeille européenne, sont en contradiction geringen Geschlechts- Titerunterschiede kommen avec les résultats précédents, sur lesquels spezifischen als Auslöser für die se fonde l’hypothèse souvent citée de la Milbenfortpflanzung régulation de la reproduction de Varroa nicht in Frage. Bei Apis cerana indica par la JH III présente dans l’hémolymphe wurde der Milbenbefall von Brut etwa eine d’abeilles (Hänel et Koeniger, 1986). Woche nach dem Verdeckelungszeitpunkt kontrolliert. Die Zellen enthielten dann be- Apis cerana indica / Apis mellifera li- reits Puppen. In Übereinstimmung mit frü- gustica / hormone juvénile / reproduc- heren Befunden war die Drohnenbrut we- tion / Varroa jacobsoni sentlich stärker infiziert als die Arbeiterin- nenbrut. Da Varroa-Weibchen das erste Ei ungefähr 3 Tage nach dem Verdeckeln ab- Zusammenfassung — Juvenilhormon- legen, konnte zum gewählten Kontrollzeit- Titer und Fortpflanzung von Varroa ja- punkt sicher zwischen fruchtbaren und un- cobsoni in verdeckelter Brut von Apis fruchtbaren Milben unterschieden werden. cerana indica im Vergleich zu Apis mel- In Drohnenzellen hatten sich fast alle Mil- lifera ligustica. In Allahabad (Nord- benweibchen fortgepflanzt. In Arbeiterin- Indien) wurden im Frühjahr Hämolymph- nenzellen dagegen hatte keine einzige proben von verdeckelter Arbeiterinnen- Varroa-Milbe ein Ei gelegt (Tabelle I). und Drohnenbrut in 4 Völkern von Apis Die hier mitgeteilten Daten, insbesonde- cerana indica und 1 Volk von Apis mellif- re über Verlauf des JH-Titers in verdeckel- era ligustica gesammelt. Außerdem ter Brut sowohl bei der östlichen wie bei wurden über 1000 verdeckelte Brutzellen der westlichen Honigbiene, stehen im Wi- mit Puppen von Apis cerana indica geöff- derspruch zu früheren Befunden, auf net und auf Befall mit Varroa jacobsoni ge- denen eine vielzitierte Hypothese über prüft. Dabei wurde notiert, ob die Milben- Steuerung der Varroa-Fortpflanzung durch weibchen sich fortgepflanzt hatten. Die JH III aus der Bienenhämolymphe basiert Hämolymphproben wurden anschließend (Hänel und Koeniger, 1986). in Europa mittels Radioimmunoassay (RIA) auf ihren Gehalt an Juvenilhormon Apis cerana indica / Apis mellifera ligu- III (JH) analysiert und daraus JH-Titer der stica / Juvenilhormon / Fortpflanzung / Hämolymphe berechnet. Varroa jacobsoni Die so ermittelten JH-Titer von verdeckelter Arbeiterinnenbrut unterscheiden sich bei Apis cerana indica und Apis mellif- REFERENCES era ligustica bis zur Puppenhäutung kaum, die Titer-Werte der Larven indischer Ho- Camazine S (1988) Factors affecting the severi- nigbienen liegen durchschnittlich sogar ty of Varroa jacobsoni infestations on Eur- etwas über denen der europäischen (Abb opean and Africanized honey bees. In: Afri- canized Honey Bees and Bee Mites (Need- Oliver JH Jr, Pound JM, Severino G (1985) Evi- ham GR, Page RE, Delfinado-Baker M, Bow- dence of a juvenile-hormone-like compound man CE, eds) Ellis Horwood Ltd, Chichester, in the reproduction of Dermanyssus gallinae 444-451 (Acari: Dermanyssidae). J Med Entomol 22, Connat JL, Ducommun J, Diehl PA (1983) Juve- 281-286 nile hormone-like substances can induce vi- Pound JM, Oliver JH Jr (1979) Juvenile hor- tellogenesis in the tick Ornithodoros moubata mone: evidence of its role in the reproduction (Acarina: Argasidae). Int J Invertebr Reprod of ticks. Science 206, 355-357 6,285-294 Ramirez BW, Otis GW (1986) Developmental Downer RGH, Laufer H (eds) (1983) Inverte- phases in the life cycle of Varroa jacobsoni, brate Endocrinology. AR Liss, New York an ectoparasitic mite on honeybees. Bee Engels W, Gonçalves LS, Steiner J, Buriolla World 67, 92-97 AM, Cavichio Issa MR (1986) Varroa-Befall Rachinsky A, Strambi C, Strambi A, Hartfelder K von Carnica-Völkern in Tropenklima. Apidolo- (1990) Caste and metamorphosis: hemo- gie 17, 203-216 lymph titers of juvenile hormone and ecdys- Engels W, Kaatz H, Zillikens A, Simoes ZLP, teroids in last instar honeybee larvae. Gen Trube A, Braun R, Dittrich F (1990) Honey Comp Endocrinol 79, 31-38 bee reproduction: vitellogenin and caste- Rachinsky A, Hartfelder K (1990) Corpora allata specific regulation of fertility. Adv Invertebr activity, a prime regulating element for caste- Reprod 5, 495-502 specific juvenile hormone titre in honeybee larvae mellifera J Insect Hänel H (1983) Effect of JH III on the reproduc- (Apis carnica). Phys- tion of Varroa jacobsoni. Apidologie 14, 137- iol 36, 189-194 142 Rembold H (1987) Caste differentiation of the Hänel H, Koeniger N (1986) Possible regulation honeybee - fourteen years of biochemical re- of the reproduction of the honey bee mite search at Martinsried. In: Chemistry and of Social Insects Rembold Varroa jacobsoni (Mesostigmata: Acari) by a Biology (Eder J, host’s hormone: juvenile hormone III. J Insect H, eds) Peperny, Munich, 3-15 Physiol 32, 791-798 Rembold H, Czoppelt C, Grüne M, Lackner B, Hartfelder K (1990) Regulatory steps in caste Pfeffer J, Woker E (1992) Juvenile hormone development of eusocial bees. In: Social In- titers during honey bee embryogenesis and sects, An Evolutionary Approach to Castes metamorphosis. In: Insect Juvenile Hormone and Reproduction (Engels W, ed) Springer, Research (Mauchamp B, Couillaud F, Baehr Heidelberg, 245-264 JC, eds) INRA, Paris, 37-43 Hartfelder K, Tozetto S de O, Rachinsky A Ritter W, De Jong D (1984) Reproduction of Var- (1993) Sex-specific developmental profiles of roa jacobsoni Oud in Europe, the Middle East juvenile hormone synthesis in honey bee lar- and tropical South America. Z Angew Ento- 55-57 vae. Roux’s Arch Dev Biol 202, 176-180 mol 98, Koeniger N, Koeniger G, Wijayagunasekara Robinson GE (1992) Regulation of division of la- NHP (1981) Beobachtungen über die Anpas- bor in insect societies. Annu Rev Entomol 37, 637-665 sung von Varroa jacobsoni an ihren natürlichen Wirt Apis cerana in Sri Lanka. Rosenkranz P (1990) Der Einfluß larvaler Bie- Apidologie 12, 37-40 nenhämolymphe auf die Fertilität von Varroa Milani N, Chiesa F (1990) Some stimuli inducing jacobsoni in europaischen und afrikanisierten oviposition in Varroa jacobsoni Oud. In: Proc- Bienenvölkern. Apidologie 21, 370-372 eedings of the International Symposium on Rosenkranz P, Rachinsky A, Strambi A, Strambi Recent Research on Bee Pathology (Ritter C, Ropstorf P (1990) Juvenile hormone titer W, Van Laere O, Jacobs F, De Wael L, eds) in capped worker brood of Apis mellifera and Apimondia, Ghent, 27-33 reproduction in the bee mite Varroa jacobso- Moritz RFA (1985) Heritability of the postcap- ni. Gen Comp Endocrinol78, 189-193 ping stage in Apis mellifera and its relation to Rosenkranz P, Stürmer M (1993) varroatosis resistance. J Hered 76, 267-270 Ernährungsabhängige Fertilität der Varroa- Weibchen in Arbeiterinnenbrut von Apis mel- Strambi C, Strambi A, de Reggi ML, Hirn MH, lifera carnica und Apis mellifera capensis. In: Delaage MA (1981) Radioimmunoassay of Proc 1st Polish-German Symp Progress in insect juvenile hormones and of their diol de- Varroatosis Research. Stettin, Poland (in rivatives. Eur J Biochem 118, 401-406 press) Tewarson NC (1987) Use of host hemolymph Ruttner F (1988) Biogeography and Taxonomy proteins, seasonal reproduction and a hy- of Honeybees. Springer, Berlin pothesis on nutritional imprinting in the honey bee mite, Varroa jacobsoni, on Apis mellifera Ruttner F (1992) der Naturgeschichte Honigbie- and Apis cerana. In: Chemistry and Biology nen. Ehrenwirth, Munich of Social Insects (Eder J, Rembold H, eds) Steiner J (1991) Oogenese und Embryogenese J Peperny, Munich, 688-689 während des ersten Gonozyklus von Varroa Tewarson NC, Singh A, Engels W (1992) Repro- jacobsoni. Apidologie 22, 460-462 duction of Varroa jacobsoni in colonies of Steiner J, Dittmann F, Rosenkranz P, Engels W Apis cerana indica under natural and experi- (1993) Interrelation between reproduction of mental conditions. Apidologie 23, 161-171 the honey bee (Apis mellifera carnica) para- Thrybom B, Fries I (1991) Development of infes- sitic mite (Varroa jacobsoni) and preimaginal tations by Varroa jacobsoni in hybrid colonies host development during the first gonocycle. of Apis mellifera monticola and Apis mellifera Invertebr Reprod Dev 23, in press ligustica. J Apicult Res 30, 151-155