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Possible regulation of the reproduction of the honey bee mite Varroa jacobsoni (Mesostigmata: Acari) by a host's hormone: Juvenile hormone III
J. Insect Physiol.
Vol. 32,
No. 9, pp. 791-798,
0022-1910/86$3.00t 0.00 Perpmon Journals Ltd
1986
Printed in Great Britain
POSSIBLE REGULATION OF THE REPRODUCTION OF THE HONEY BEE MITE J’ARROA JACOBSON1 (MESOSTIGMATA: ACARI) BY A HOST’S HORMONE: JUVENILE HORMONE III HEINZ HKNEL* and NIKOLAUS KOENIGER Institut fiir Bienenkunde (Polytechnische Gesellschaft), Fachbereich Biologie, der J.W.-Goethe-Universitiit, Frankfurt a.M. Karl-von-Frisch-Weg, D-6370 Oberursel, F.R.G.
Ah&net-With its first haemolymph meal from honey bee larvae the parasitic mite Vurroa jacobsoni obtains different amounts of juvenile hormone III depending on the species and sex of the larva. Drone larvae of both Apis meNifera and A. cerana contain more than 5 ng/ml juvenile hormone in the haemolymph during the first 60 h after brood cell capping. Worker brood of A. mef~ifera contains 3-7 ng/ml whereas A. cerana does not reach 1 ng/ml during the first day after sealing. Juvenile hormone concentrations lower than 4 ng/ml seem to be one factor to prevent reproduction of the mites. Varroa, having parasitized on winter bees (low juvenile hormone titre) show 8% reproduction after beeing transfered to suitable brood cells. Mites from old summer bees (high juvenile hormone titre) show 76% reproduction. Varroa from winter bees transfered to larvae which were treated with juvenile hormone show 18%, and mites parasitizing winter bees sprayed with juvenile hormone showed 39% reproduction. Juvenile hormone effectscould be demonstrated down to the cellular level in obgenesis. Our results suggest a model for a 2-fold influence of host juvenile hormone on Varroa: The first influence on the adult bee which might be necessary for the cell-entering behaviour and preparation of the ovary. The second one on the bee larva obviously triggers yolk incorporation into the eggs of the mite and induces oviposition. Key Word Index:
Juvenile hormone (III), Apis, Vurroa, host influence, parasite, reproduction
MATERIALS
INTRODUCI’ION
Vurroa jacobsoni is a natural parasite of the Eastern honeybee (&is cerana). Recently this mite has been found parasiting the Western honey bee (Apis mellifera), where it causes severe damage to the infested colonies (Smirnov, 1979). The life cycle of Varroa is restricted to bees and their colonies. Adult, mated female mites stay on workers or drones where they feed on haemolymph. For reproduction the mite leaves the adult bee and enters into a brood cell on a 4th stage larva. Shortly afterwards, the bees seal the brood cell and with the metamorphosis of the bee larva the reproduction of Vartoa is carried out. The duration of brood cell capping is about 11 days and after this the young adult bee emerges and the mite, together with the offspring, is set free to switch over on adult bees. So, an elaborated synchronisation between the reproduction of the mite and the life cycle of the honey-bee is expected. This is the subject of our research. Juvenile hormone which plays a major role in the development, individual, and social life of the honey bee and in the reproductive process of numerous arthropods in general, attracted our interest. In earlier experiments we described an enhancing effect of juvenile hormone on the reproduction of Vurrou if applied to the parasitized early-spinning of larva of A. rneliiferu worker brood (HIinel, 1983). *Present address: Hoechst AG, Abteilung fiir Chemotherapie, Postfach 800320, 6230 Frankfurt/M. 80, F.R.G. 79
AND METHODS
Galleria-rest To measure the juvenile hormone concentration in winter bees of known age, A. mellifera drone larvae and pupae, and A. ceranu larvae and pupae, and A. cerana larvae and pupae, the Galleriu-test was applied (de Wilde et al., 1968). We purificated the extracts with TLC (Schooley, 1977). In our experiments one Galleria-unit (GU) corresponded to 13.5 pg juvenile hormone III (Sigma Corp.). Each value for A. ceruna was determined by analysing some 120 Gulleriupupae. Parallel measurements of A. melriferu haemolymph confirmed the comparability of our Gulleriu bioassay and the HPLC-method (Hagenguth, 1979). Insects Emerging A. mellijkru worker bees were collected daily, marked with shellac, and kept in queen-right colonies to obtain dated bees. Larvae and pupae were dated according to the scheme of Rembold ef al. (1980). 250 ~1 pooled haemolymph was collected for each single measurement. We were unable to obtain drone pupae of A. ceranu older than 250 h after the moult because of the season and a limited stay in Malaysia. Maintaining the mites Vurroa were collected from worker bees in queenright colonies at various seasons. With forceps, we carefully transferred them to groups of worker bees (n = 2&30) in small cages. The bees were supplied
HEINZ Hjim~
192
and NIKOLAUSKOENIGER
‘. ._
. . .. .L
‘5. ‘rn
..
400 Start
of
1.vorroo feedlng
r*g
After
hotchlng
Fig. 1. Juvenile hormone concentration in larvae and pupae of A. cerana and A. mellijeraworkers. Values for A. me~riferra after Rembold and Hagenguth (1981) (star). Abscissa indicates the age of the bee larvae after emerging from the egg. S = sealing of the brood cell. with sugar-water and in experiments extending 1 week a piece of pollen comb was added. The cages were kept in an incubator under suitable conditions (70% r.h., 34°C). Treatment with juvenile hormone (Sigma Corp.)
It was dissolved in acetone (Merck p-a.) and diluted 1:9 with distilled water. In the spraying experiments, each group of bees with the parasitic mites was sprayed with 3 ml (375 pg juvenile hormone) solution using a Power-Pack@ spraying device. Control groups received the solvent. For the treatment of mites and larvae a Hamilton@ syringe was used to apply volumes down to 1 ~1. Transfer of mites into cells
For the reproduction experiments cells of A. mellifera worker or drone brood were selected which were sealed 18 h before. There was virtually no spinning at this stage. The lid was opened partially with a blade and the mite was carefully placed in it. Slight pressure on the lid was sufficient for resealing. The combs were kept without bees in an incubator at 34°C and 70% humidity. Cells were opened shortly before the emergence of the bees to examine the mite offspring. Histology of the mites
Effects of juvenile hormone on the reproductive physiology of the mites was investigated by histological methods as well. Mites were kept on winter bees (see before) and sprayed with different concentrations of juvenile hormone. One set of groups received 3 mg once only (dissolved as described before) for each 30 bees with mites. The other groups received 200 pg for each 200 bees with mites 4 times at daily intervals. In each set, control groups of the same size were sprayed with the solvent. From the first groups (3 mg juvenile hormone) female mites were fixed in alcoholic Bouin fluid (Romeis, 1968) and embedded in methylmetacrylate (Hoechst AG, unpubl. method). The 3qm sections were stained according to Ladewig (Roulet, 1948). The stain was suitable to demonstrate the different stages of yolk incorporation into the eggs. In the other set (200 pg) every day after the first spraying 15 mites were fixed in 5% formaline (Merck
p.a.). The genital system was removed and embedded in an 1% agar (Merck p.a.) cube. The cube was then transferred to Paraplast@ (Lancer Corp.) and sectioned (4-5 pm). The serial sections were stained according to Mayer (Romeis, 1968). Size determination and documentation of the egg appearance was carried out with a Zeiss inverted microscope containing a measuring device. RESULTS
Juvenile hormone titre of Apis larvae
The results of the titre determinations of worker and drone brood are shown in Figs 1 and 2. Worker broods of both species A. cerana and A. melrifera show one significant peak in the juvenile hormone concentration after the sealing of the cell when spinning behaviour occurs. The rise in A. mellifera is earlier and more delayed than in A. cerana, but it reaches over 15 ng/ml of juvenile hormone. After the distinct decline when 20-hydroxyecdysone reaches a high concentration (Hagenguth and Rembold, 1978), both species remain at low levels till there is a second peak during the pigmented pupal phase (Fig. 1). Drone broods of both species show two distinct peaks after the sealing of the cell. The concentration of juvenile hormone in male larvae never falls below 5 ng/ml in the first 60 h after capping. The first peak in A. cerana is detectable at the first moment feeding in Varroa. In A. mellifera it is some 18 h later. In both species the first egg of Varroa may appear before there is a second peak in juvenile hormone concentration (Fig. 2). After the decline during pupation there is no second peak like in worker pupae. In Table 1 the juvenile hormone titre at the critical time for Varroa reproduction is summarized for the two sexes and species. High juvenile hormone titres always cause a high percentage of reproduction. Varroa parasitizing winter bees Worker bees during the winter live for several months until the next spring. They have juvenile hormone titres below 20 ng/ml throughout their life (Fluri et al., 1982) except for the period of nectar collection in spring. In Table 2, we show the results
Regulation of the reproduction of the honey bee mite JH
793
I
a.......
A.mrlllfero
.-.
A.cerotw
kr
‘m.. . . .. ...... . . . . . . . . ... ...pm.._..a.*+ (hl 300
350
400 450 Afterhatching
Startof fesdlng
Fig. 2. Juvenile hormone concentrations in larvae and pupae of A. mellifera and A. ceruna drones. For legend see Fig. 1. Table 1. Juvenile hormone concentrations in larvae of Apis me/lifers and A. cerana drones and workers &16 h after the sealing of the cell. Observed reproduction of Varroa on these trues of brood JHIII (ngiml)
Reproduction of Varroa
Species
Caste
A. mellifera A. cerana
Drone
8-15
Drone
15-30
A. m&fern
Worker
A. eerana
Worker
2.5-l (Rembold and Hagenguth, 1981) 0.043
Frequent
Always Depending on several factors None
JHIII = juvenile hormone III.
of 8 experiments with winter bees of known age and Varroa to compare the juvenile hormone titre of the bee and the offspring which is produced by these mites. The titre in all groups was well below 13 ng/ml and there was only little reproduction in three of the groups. There Seems to be no obvious periodicity in juvenile hormone titre during the first 25 days of the winter bees. In a second set of experiments, nine groups of undated winter bees with parasitic Vurroa were kept in the incubator for various times. Thereafter the mites were transfered into suitable brood cells. As Table 3 shows there is a slight effect of the duration of parasitation in the incubator. Winter bees which were taken from the field at air temperatures below 0 (“C) seem to have only little effect on the induction of reproduction of the parasitic mites. Incubation times of 7-21 days resulted in increased offspring production of the mites. The percentage of mites with Table 2. Effect of the age of winter bees on which Varroa was parasitic before the mite was transferred to a suitable brood cell Age of bee (days)
JHIII in the bee (ngiml)
Varroa
(n)
Mites with offspring (Oh)
24 21 19 19 17 14 13 5
4.9 8.2 11.5 11.9 6.2 9.1 10.6 4.6
38 21 21 29 28 29 29 30
0 5 0 0 13 I 0 0
JHIII = juvenile hormone III. There is no significant increase (P > 0.01) of mite reproduction with .. “.. . me age o* me we.
offspring never exceeded 35% in any of these experiments. Varroa parasitizing summer bees As it is known that the juvenile hormone content of summer bees rises during ageing (Rutz et al., 1976), we investigated the effect of summer bees (older than 12 days) on the reproduction of mites parasitic on them. In the last column of Fig. 3 the results of 4 sets of experiments are given: 76 + 22% of the mites developed offspring in the cells. After this set of experiments, we investigated to see if the addition of juvenile hormone to mites on winter bees could mimic the reproduction-enhancing effect of old summer bees. In 15 groups of bees, we took mites from winter bees and added juvenile hormone to the larva in the brood cell. From a total of 475 mites, 81 developed offspring (18 + 17%). If we sprayed the bees with the parasitic mites with juvenile hormone and kept them for three additional days on the bees, 170 from 708 developed offspring in the cells (39 f 28%, 8 groups). Table 3. Effect of the duration on which mites were parasitic on bees in an incubator on the offspring rate of the mites Time in the incubator
(days)
(n)
With offspring (“/) (2 f SD)
0
135 132 71 9 245 12 23 21 15
5*7 7&l 18 0 5_+8 25 35 29 0
1 3 5 6 7 10 21 31
Varroa
Significance (P z 0.01)
as.
sig. n.s. “S.
sig. sig. sig. n.s.
HEINZHKNELand NIKOLAUS KOENIGER
194
iB . ..*.... . . type . . . . . .of . . . bee . . . .. lone type of JHlarva bee treatment . .. 29 mites (n) 663 475 706 Fig. 3. Influence of external juvenile hormone on reproduction of Varroa.WB = mites kept on winter bees; SB = mites kept on summer bees (age 12 days). Juvenile hormone treatment either on larvae (2nd column) or on adult bees (3rd column). Standard deviation and number of mites in each test indicated. WB ... . none
.
WB . . .. . . .dri. WB . . . . . . “6ii..
If the results of Fig. 3 are compared, it is obvious that juvenile hormone increases the capacity of offspring production in those mites which were kept on winter bees. Effect of unphysioiogically high amounts of juvenile hormone on the ovary of Varroa
Serial sections of 12 mites sprayed with 3 mg juvenile hormone per group of bees fixed from 24 to 238 h after the treatment showed remarkable irregularities in the yolk incorporation. Vacuoles of different size appeared in the largest eggs, or the egg was 2-3 times larger than the biggest one in the control group. In the mites fixed 100-200 h after spraying, the nucleus was separated from the apparently coagulated yolk material. Generally the appearance of large yolk bodies was different from the control mites which showed perfectly fine granulated material in their biggest eggs irrespective of the time tixed. In two serial sections of mites which we took
from worker brood cells of A. cerana (no reproduction), the same type of fine yolk material was visible as in our control groups. In two Figures (4 and 5) we show the effect of juvenile hormone on the yolk material. Figure 4 shows part of the ovary of a control mite. In the biggest egg only fine yolk material is visible. In all the juvenile hormone-treated mites we detected changes as in Fig. 5. Here only coarse yolk material is present and the egg increases remarkably in size, although the mite stayed on an adult bee. Such an appearance was never observed in a control mite. Effect of physiological amounts of juvenile hormone on the ovary of Varroa
The results of the investigation on the genital system of mites sprayed with physiological amounts of juvenile hormone on bees are summarized in Table 4. It shows clearly there is no effect of physiological amounts of juvenile hormone on the size of the
Table 4. Size and yolk state of the biggest egg and appearance of the organum lyriforme (OL) in Varroa after juvenile hormone treatment Treatment Solvent control
200 pg JHIII Time after 1 treatment (h)
Enn
OL
Size @m) 8*s
Ena
OL
Size (pm) R*s
30 54 18 102 126 174 218
b c c c,d c,d d c,d
i+ + + 0 0 0 0
2.9 f 1.O 3.2 f 0.3 3.4 f 0.4 3.2 f 0.3 3.6 f 0.4 3.6*0.5 3.5 f 0.7
a a a a a c
0 0 0 0 0 0
3.2 f 3.5 f 3.3 f 2.8 f 3.1 f 3.9 f
0.4 0.5 OS 0.4. 0.7 0.3
*Only 6 badly fixed ovaries analysed. Abbreviations: -: not investigated. + +: heavy proliferation of the OL. f: slight proliferation. 0: no proliferation. a: fine yolk material. b: partly coarse yolk at the edges of the egg. c: half of the biggest eggs coarse. d: disturbances in yolk incorporation. JHIII: juvenile hormone III.
Fig. 4. Histological section of an ovary of a control mite. The biggest egg and some smaller eggs are visible. At the edges of the egg some basophihc material is deposited.
Fig. 5. Histological section of an ovary of a juvenile hormone-treated mite. The biggest egg with c:oarse yolk bodies and the nucleus is visible. Note the size of the egg.
795
Regulation of the reproduction of the honey bee mite biggest egg in Varroa. The organum lyriforme, which is thought to be the yolk-forming organ, appears proliferated shortly after the first treatment. This effect decreases steadily. There is no visible proliferation of this organ in the control groups. As early as 30 h after the start of the experiment, some coarse yolk material is visible in some of the biggest eggs of the juvenile hormone-treated groups. These disturbances are apparent throughout the whole period. In the control group, only the last set of ovaries showed some bigger yolk particles after 2 18 h on bees in the incubator. DISCUSSION
Juvenile hormone influence on Varroa in the brood cell
The general observation made by everyone who investigates Vurroa is, that reproduction on the drone brood is better than that on the worker brood. In a previous paper, we reported that there is a considerable enhancement of reproduction if juvenile hormone is added to the early spinning larvae and that this effect is limited to a short period of time (Hanel, 1983). This phenomenon for a sensitive phase for juvenile hormone seems to be widely distributed among insects: Locusta migratoria is only susceptible during a short period of the 5th instar (Dhadialla and Wyatt, 1981), and the Rhodniw prolixus during a period of 24 days in the 5th stage (Nijhout, 1983). If juvenile hormone is missing at that time it cannot be replaced by a later treatment (Davey, 1981). That is what we found in Vurroa as well. Therefore it was necessary to measure the titre in the developing brood of A. cerana and A. mell$era, because Varroa begins to feed shortly after sealing of the cell, when the larvae begin to spin. There are also possibilities for host-parasite interactions other than hormones. In the brood cell, kairomones or tactile stimuli by the bee larvae might induce a blockade of the parasite’s reproduction. As Figs 1 and 2 show, there is a remarkable difference in juvenile hormone contents of both species. The juvenile hormone concentration of A. ceruna worker brood seems to rise too late and too little to induce the yolk incorporation into the mite eggs. Perhaps this is one of the reasons for the lack of offspring in A. cerana worker brood (Koeniger et al., 1981), and thus for the comparatively little interference, the mite causes in its natural host. Figure 3 indicates that an enhancement of juvenile hormone on the larvae alone is not as effective as an enhancement of juvenile hormone concentration on the adult bee which Vurroa parasitizes before entering the cell. Juvenile hormone influence of the ad& bee
The parasitization of the mites on adult bees where they encounter different titres of juvenile hormone seems to be of some importance for the numerous processes in connection with the reproduction of the mite. Titre values showed a significant increase in juvenile hormone in old summer bees. Winter bees do not reach these concentrations until they start to collect nectar in spring (Fluri et al., 1982). Accordingly, there was little to no reproduction of the mites which parasitized winter bees of varying age (Table 2).
797
Mites which parasitized old summer bees showed (Fig. 3) relatively high percentages of reproduction. These bees contain up to nearly 55 ng/ml juvenile hormone in the haemolymph (Rutz et al., 1976). Preliminary experiments showed that Varroa parasitizing winter bees contain little juvenile hormone III, corresponding to the low level in their hosts (Hlnel, unpubl.). By using radioactive precocene II, autoradiography of mite cryo-sections might show whether there is a corpus allatum homologue organ in Varroa. Thus it remained for us to clarify, if spraying juvenile hormone on parasitized winter bees could mimic the physiological conditions of summer bees as far as Vurroa reproduction is concerned. Figure 3 shows that there is a significant intIuence of juvenile hormone on the percentage of reproduction in mites, although the 76 + 22% from summer bees is not achieved. Applying the t-test for the comparison of means (P > O.OOl), juvenile hormone treatment of both kinds leads to a reproductive success, significantly different from the controls. Additionally the treatment of the mites on adult bees is different from that of juvenile hormone treatment on larvae. We assume, that the juvenile hormone concentration of the adult bee has an influence on the behaviour of the mite: to enter a brood cell. Preliminary experiments (Hiinel, unpubl.) show that mites from old summer bees enter brood cells more frequently than mites parasitic on young bees. Barabanova (1983) and Smimow (1979) found only little invasive behaviour of mites in the late autumn. During this time of low juvenile hormone titre in the bees, there are still a fair number of brood cells available. Preliminary counts in early spring (Hiinel, unpubl.) showed that cell entering with successful reproduction is linked to the first collecting flights on sunny days. At that time bees with the first high titre occur. The influence of juvenile hormone on the process of yolk incorporation calls for comment. As we showed, the physiological amounts of juvenile hormone sprayed on winter bees with mites influenced only the organum lyriforme and the neurosecretory system (Hlnel, 1986). No enhanced incorporation of yolk into the egg occurred and it would not be likely because the adult bee does not contain the amounts of protein which are available in larvae. Only because of the precise timing, the mite is able to complete its reproductive cycle within the cell. If the developmental period of a worker bee is only l-2 days shorter than average, as in Apis melltfera capensis Esch., Varroa is unable to produce normal numbers of offspring (Moritz and Hiinel, 1983). A model of a 2-fold push in reproduction in Varroa develops, We assume that the period on old summer bees causes the differentiation of the cells of the yolk-forming organum lyriforme in the mites for the coming reproductive cycle. This was also shown by the histological observations. Additionally, the mite enters the brood cell to receive a second pulse of juvenile hormone from the larvae. This seems to participate in the initiation of the incorporation of yolk material into the biggest egg of the mite. This 2-step process resembles the condition in Rhodnius (Abu Hakima and Davey, 1975 and 1977). There is some evidence for a function of juvenile hormone or
HEINZ HINEL and NIKOLAUSKOENIGER
798
hormone-like substances ‘in ticks and mites (Oliver et al., 1984; Pound and Oliver, 1979; Oliver et al., 1985). Our results do not finally prove the dependence of* Vurroa on the host’s hormone but the reproduction experience shown here, together with the histological sections and earlier work on the neurosecretory system (Hiinel, 1986) indical/e, that juvenile hormone influences the mite’s reproduction. The results of the hormone treatments might lead to a new approach in the control of Vurroa. It is known from several other insects that high concentrations of juvenile hormone have a lethal effect on developing stages (Buschor et al., 1984). Generally additional juvenile hormone seems to have no disturbing influence upon the bee colony (Rutz et al., 1976) thus it could be a candidate for a hormonal
juvenile
Varroa
control.
Acknowledgements-The
authors like to thank the Deutsche Forschungsgemeinschaft for the grant Ko 400/l-2. Mr Makhdzir Mardan, Jabatan Perlindungan Tunbuhan, Fakulti pertanian Malaysia, Serdan, Selangor, Malaysia provided a colony of A. ceruna. Professor Maschwitz (Frankfurt) enabled the senior author to carry out experiments in Malaysia.
REFERENCES Abu-Hakima R. and Davey K. G. (1975) Two actions of juvenile hormone on the follicle cells of Rhodniusprolixus. Can. J. Zool. 58, 1187-1188. Abu-Hakima R. and Davey K. G. (1977) The action of juvenile hormone on the follicle cells of Rhodnius prolixus : The importance of volume changes. J. exp. Biol. 69, 3344. Barabanova V. V. (1983) Pischtchewaritjelej fermentej Varroa jacobsoni. Vest. Zool. 1983 81-83. Buschor J., Beyeler P. and Lanzrein B. (1984) Factors responsible for the initiation of a second oocyte maturation cycle in the ovoviviparous cockroach Nuuphoeta cinerea. J. Insect Physiol. 30, 241-249.
Davey K. G. (1981) Regulation of cell volume in follicle cells of Rhodnius by JH. In Juvenile Hormone Biochemistrv (Ed. by Pratt -Ci. E. and Brooks G. T.), pp. 233-240. Elsevier North-Holland Biomedical Press, Amsterdam. Dhadialla T. S. and Wyatt G. R. (1981) Competence for juvenile hormone stimulated vitellogenin synthesis in the fat body of female and male of Locusta migratoria. In Juvenile Hormone Biochemistry (Ed. by Pratt G. E. and Brooks G. T.), pp. 257-262. Elsevier North-Holland Biomedical P&s,-Amsterdam. Fluri P.. Liischer M.. Wille H. and Gerin L. (1982) Chanaes in weight of the pdaryngeal gland andsome haemolyiph titres of juvenile hormone, protein and vitellogenin in worker honey bees. J. Insect Physiol. 28, 61-68. H&e1 H. (1983) Effect of JH,,, on the reproduction of Varroa jicobsoni. Apidologie ‘i’4, 137-142.-
Hiinel H. (1986) Effect of juvenile hormone (III) from the host Apis melrifera (Insecta: Hymenoptera) on the neu-
rosecretion of the parasitic mite Varrou jacobsoni (Atari: Mesostigmata). Exp. uppl. Acarol. In press. Hagenguth H. (1979) Mikroanalytische Bestimmung von Juvenil-hormontitern der Honigbiene. Dissertation, University of Munich. Hagenguth H. and Rembold H. (1978) Kastenspezifische Modulation des Ecdysteroidtiters bei der Honigbiene. Mitt. dt. Ges. angew. Ent. 1, 296298.
Koeniger N., Koeniger G. and Wijayagunasekara N. H. P. (1981) Beobachtungen iiber die Anpassung von Vurroa jacobsoni an ihren natiirlichen Wirt Apis cerana in Sri Lanka. Apidologie 12, 37-40. Moritz R. F. A. and Hiinel H. (1984) Restricted development of the parasitic mite Vurroa jacobsoni Oud. In the Cape honeybee Apis met&era capensis Esch. Z. angew. Ent. 97, 91-95. Nijhout H. F. (1983) Definition of a juvenile hormone sensitive period in Rhodnius prolixus. J. Insect Physiol. 29, 667-677.
Oliver J. H. Jr, Pound J. M. and Severino G. (1984) Effects of the antiallatotropin precocene 2 on the reproduction the chicken mite Dermanyssus gallinae. In kdvances in Invertebrate Reproduction Vol. 3 (Ed. bv Enaels W.). pp. 621. Elseiier North-Holland Biomedical Press; Amsterdam. Oliver J. H. Jr, Pound J. M. and Severino G. (1985) Evidence of a juvenile hormone-like compound in the reproduction of Dermanyssus gallinae (Atari: Dermanyssidae). J. med. Ent. 22, 281-286. Pound J. M. and Oliver J. H. Jr (1979) Juvenile hormone: evidence of its role in the reproduction of ticks. Science 206, 355-357.
Rembold H. and Hagenguth H. (1981) Modulation of hormone pools during post embyonic development of the female honey bee castes. In Int. Conf. Regulation oflnsect Development and Behaviour, Part I (Ed. by Kloza M.), pp. 427-440. Wroclaw Techn. Univ. Press, Karpacz, Poland. Rembold H., Kremer J. P. and Ulrich G. M. (1980) Characterization of postembryonic developmental stages of the female castes of the honey bee, Apis mellfera L. Apidologie 11, 29-38. Romeis B. (1968) Mikroskopische Technik, 16th edn. R. Oldenbourg, Miinchen. Roulet F. (1948) Methoden der Pathologischen Histologie, 1st edn. Springer, Wien. Rutz W., Gerig L., Wille H. and Liischer M. (1976) The function of juvenile hormone in adult worker honey bees, Apis mellifera. J. Insect Physiol. 22, 14851491.
Schooley D. A. (1977) Analysis of the naturally occuring juvenile hormones-their isolation, identification, and titer determination at physiological levels. In Annlyfical Biochemistry of Insects (Ed. by Turner R. B.), pp. 241-287. Elsevier North-Holland Biomedical Press, Amsterdam. Smirnow A. M. (1979) Aktuelle Ergebnisse der Sowietischen Wissenschaft iiber Atiologie, Pathogenie, Epizootologie, Diagnose und Bekiimpfung der Varroatose. In Bekiimpjiing und Vorbeugung der Varroatose (Ed. by Harnaj V. and Rousseau M.), pp. 60-74. Apimondia, Bucharest. deWilde J., Staal G. B., deKort C. A. D., deLoof A. and Baard G. (1968) Juvenile hormone titer in the haemolymph as a function of photoperiodic treatment in the adult Colorado potato beetle (Leptinotarsa decemlieata) (Say). Proc. K. ned. Akad. Wet. Sect. C 71, 321-326.
Vol. 32,
No. 9, pp. 791-798,
0022-1910/86$3.00t 0.00 Perpmon Journals Ltd
1986
Printed in Great Britain
POSSIBLE REGULATION OF THE REPRODUCTION OF THE HONEY BEE MITE J’ARROA JACOBSON1 (MESOSTIGMATA: ACARI) BY A HOST’S HORMONE: JUVENILE HORMONE III HEINZ HKNEL* and NIKOLAUS KOENIGER Institut fiir Bienenkunde (Polytechnische Gesellschaft), Fachbereich Biologie, der J.W.-Goethe-Universitiit, Frankfurt a.M. Karl-von-Frisch-Weg, D-6370 Oberursel, F.R.G.
Ah&net-With its first haemolymph meal from honey bee larvae the parasitic mite Vurroa jacobsoni obtains different amounts of juvenile hormone III depending on the species and sex of the larva. Drone larvae of both Apis meNifera and A. cerana contain more than 5 ng/ml juvenile hormone in the haemolymph during the first 60 h after brood cell capping. Worker brood of A. mef~ifera contains 3-7 ng/ml whereas A. cerana does not reach 1 ng/ml during the first day after sealing. Juvenile hormone concentrations lower than 4 ng/ml seem to be one factor to prevent reproduction of the mites. Varroa, having parasitized on winter bees (low juvenile hormone titre) show 8% reproduction after beeing transfered to suitable brood cells. Mites from old summer bees (high juvenile hormone titre) show 76% reproduction. Varroa from winter bees transfered to larvae which were treated with juvenile hormone show 18%, and mites parasitizing winter bees sprayed with juvenile hormone showed 39% reproduction. Juvenile hormone effectscould be demonstrated down to the cellular level in obgenesis. Our results suggest a model for a 2-fold influence of host juvenile hormone on Varroa: The first influence on the adult bee which might be necessary for the cell-entering behaviour and preparation of the ovary. The second one on the bee larva obviously triggers yolk incorporation into the eggs of the mite and induces oviposition. Key Word Index:
Juvenile hormone (III), Apis, Vurroa, host influence, parasite, reproduction
MATERIALS
INTRODUCI’ION
Vurroa jacobsoni is a natural parasite of the Eastern honeybee (&is cerana). Recently this mite has been found parasiting the Western honey bee (Apis mellifera), where it causes severe damage to the infested colonies (Smirnov, 1979). The life cycle of Varroa is restricted to bees and their colonies. Adult, mated female mites stay on workers or drones where they feed on haemolymph. For reproduction the mite leaves the adult bee and enters into a brood cell on a 4th stage larva. Shortly afterwards, the bees seal the brood cell and with the metamorphosis of the bee larva the reproduction of Vartoa is carried out. The duration of brood cell capping is about 11 days and after this the young adult bee emerges and the mite, together with the offspring, is set free to switch over on adult bees. So, an elaborated synchronisation between the reproduction of the mite and the life cycle of the honey-bee is expected. This is the subject of our research. Juvenile hormone which plays a major role in the development, individual, and social life of the honey bee and in the reproductive process of numerous arthropods in general, attracted our interest. In earlier experiments we described an enhancing effect of juvenile hormone on the reproduction of Vurrou if applied to the parasitized early-spinning of larva of A. rneliiferu worker brood (HIinel, 1983). *Present address: Hoechst AG, Abteilung fiir Chemotherapie, Postfach 800320, 6230 Frankfurt/M. 80, F.R.G. 79
AND METHODS
Galleria-rest To measure the juvenile hormone concentration in winter bees of known age, A. mellifera drone larvae and pupae, and A. ceranu larvae and pupae, and A. cerana larvae and pupae, the Galleriu-test was applied (de Wilde et al., 1968). We purificated the extracts with TLC (Schooley, 1977). In our experiments one Galleria-unit (GU) corresponded to 13.5 pg juvenile hormone III (Sigma Corp.). Each value for A. ceruna was determined by analysing some 120 Gulleriupupae. Parallel measurements of A. melriferu haemolymph confirmed the comparability of our Gulleriu bioassay and the HPLC-method (Hagenguth, 1979). Insects Emerging A. mellijkru worker bees were collected daily, marked with shellac, and kept in queen-right colonies to obtain dated bees. Larvae and pupae were dated according to the scheme of Rembold ef al. (1980). 250 ~1 pooled haemolymph was collected for each single measurement. We were unable to obtain drone pupae of A. ceranu older than 250 h after the moult because of the season and a limited stay in Malaysia. Maintaining the mites Vurroa were collected from worker bees in queenright colonies at various seasons. With forceps, we carefully transferred them to groups of worker bees (n = 2&30) in small cages. The bees were supplied
HEINZ Hjim~
192
and NIKOLAUSKOENIGER
‘. ._
. . .. .L
‘5. ‘rn
..
400 Start
of
1.vorroo feedlng
r*g
After
hotchlng
Fig. 1. Juvenile hormone concentration in larvae and pupae of A. cerana and A. mellijeraworkers. Values for A. me~riferra after Rembold and Hagenguth (1981) (star). Abscissa indicates the age of the bee larvae after emerging from the egg. S = sealing of the brood cell. with sugar-water and in experiments extending 1 week a piece of pollen comb was added. The cages were kept in an incubator under suitable conditions (70% r.h., 34°C). Treatment with juvenile hormone (Sigma Corp.)
It was dissolved in acetone (Merck p-a.) and diluted 1:9 with distilled water. In the spraying experiments, each group of bees with the parasitic mites was sprayed with 3 ml (375 pg juvenile hormone) solution using a Power-Pack@ spraying device. Control groups received the solvent. For the treatment of mites and larvae a Hamilton@ syringe was used to apply volumes down to 1 ~1. Transfer of mites into cells
For the reproduction experiments cells of A. mellifera worker or drone brood were selected which were sealed 18 h before. There was virtually no spinning at this stage. The lid was opened partially with a blade and the mite was carefully placed in it. Slight pressure on the lid was sufficient for resealing. The combs were kept without bees in an incubator at 34°C and 70% humidity. Cells were opened shortly before the emergence of the bees to examine the mite offspring. Histology of the mites
Effects of juvenile hormone on the reproductive physiology of the mites was investigated by histological methods as well. Mites were kept on winter bees (see before) and sprayed with different concentrations of juvenile hormone. One set of groups received 3 mg once only (dissolved as described before) for each 30 bees with mites. The other groups received 200 pg for each 200 bees with mites 4 times at daily intervals. In each set, control groups of the same size were sprayed with the solvent. From the first groups (3 mg juvenile hormone) female mites were fixed in alcoholic Bouin fluid (Romeis, 1968) and embedded in methylmetacrylate (Hoechst AG, unpubl. method). The 3qm sections were stained according to Ladewig (Roulet, 1948). The stain was suitable to demonstrate the different stages of yolk incorporation into the eggs. In the other set (200 pg) every day after the first spraying 15 mites were fixed in 5% formaline (Merck
p.a.). The genital system was removed and embedded in an 1% agar (Merck p.a.) cube. The cube was then transferred to Paraplast@ (Lancer Corp.) and sectioned (4-5 pm). The serial sections were stained according to Mayer (Romeis, 1968). Size determination and documentation of the egg appearance was carried out with a Zeiss inverted microscope containing a measuring device. RESULTS
Juvenile hormone titre of Apis larvae
The results of the titre determinations of worker and drone brood are shown in Figs 1 and 2. Worker broods of both species A. cerana and A. melrifera show one significant peak in the juvenile hormone concentration after the sealing of the cell when spinning behaviour occurs. The rise in A. mellifera is earlier and more delayed than in A. cerana, but it reaches over 15 ng/ml of juvenile hormone. After the distinct decline when 20-hydroxyecdysone reaches a high concentration (Hagenguth and Rembold, 1978), both species remain at low levels till there is a second peak during the pigmented pupal phase (Fig. 1). Drone broods of both species show two distinct peaks after the sealing of the cell. The concentration of juvenile hormone in male larvae never falls below 5 ng/ml in the first 60 h after capping. The first peak in A. cerana is detectable at the first moment feeding in Varroa. In A. mellifera it is some 18 h later. In both species the first egg of Varroa may appear before there is a second peak in juvenile hormone concentration (Fig. 2). After the decline during pupation there is no second peak like in worker pupae. In Table 1 the juvenile hormone titre at the critical time for Varroa reproduction is summarized for the two sexes and species. High juvenile hormone titres always cause a high percentage of reproduction. Varroa parasitizing winter bees Worker bees during the winter live for several months until the next spring. They have juvenile hormone titres below 20 ng/ml throughout their life (Fluri et al., 1982) except for the period of nectar collection in spring. In Table 2, we show the results
Regulation of the reproduction of the honey bee mite JH
793
I
a.......
A.mrlllfero
.-.
A.cerotw
kr
‘m.. . . .. ...... . . . . . . . . ... ...pm.._..a.*+ (hl 300
350
400 450 Afterhatching
Startof fesdlng
Fig. 2. Juvenile hormone concentrations in larvae and pupae of A. mellifera and A. ceruna drones. For legend see Fig. 1. Table 1. Juvenile hormone concentrations in larvae of Apis me/lifers and A. cerana drones and workers &16 h after the sealing of the cell. Observed reproduction of Varroa on these trues of brood JHIII (ngiml)
Reproduction of Varroa
Species
Caste
A. mellifera A. cerana
Drone
8-15
Drone
15-30
A. m&fern
Worker
A. eerana
Worker
2.5-l (Rembold and Hagenguth, 1981) 0.043
Frequent
Always Depending on several factors None
JHIII = juvenile hormone III.
of 8 experiments with winter bees of known age and Varroa to compare the juvenile hormone titre of the bee and the offspring which is produced by these mites. The titre in all groups was well below 13 ng/ml and there was only little reproduction in three of the groups. There Seems to be no obvious periodicity in juvenile hormone titre during the first 25 days of the winter bees. In a second set of experiments, nine groups of undated winter bees with parasitic Vurroa were kept in the incubator for various times. Thereafter the mites were transfered into suitable brood cells. As Table 3 shows there is a slight effect of the duration of parasitation in the incubator. Winter bees which were taken from the field at air temperatures below 0 (“C) seem to have only little effect on the induction of reproduction of the parasitic mites. Incubation times of 7-21 days resulted in increased offspring production of the mites. The percentage of mites with Table 2. Effect of the age of winter bees on which Varroa was parasitic before the mite was transferred to a suitable brood cell Age of bee (days)
JHIII in the bee (ngiml)
Varroa
(n)
Mites with offspring (Oh)
24 21 19 19 17 14 13 5
4.9 8.2 11.5 11.9 6.2 9.1 10.6 4.6
38 21 21 29 28 29 29 30
0 5 0 0 13 I 0 0
JHIII = juvenile hormone III. There is no significant increase (P > 0.01) of mite reproduction with .. “.. . me age o* me we.
offspring never exceeded 35% in any of these experiments. Varroa parasitizing summer bees As it is known that the juvenile hormone content of summer bees rises during ageing (Rutz et al., 1976), we investigated the effect of summer bees (older than 12 days) on the reproduction of mites parasitic on them. In the last column of Fig. 3 the results of 4 sets of experiments are given: 76 + 22% of the mites developed offspring in the cells. After this set of experiments, we investigated to see if the addition of juvenile hormone to mites on winter bees could mimic the reproduction-enhancing effect of old summer bees. In 15 groups of bees, we took mites from winter bees and added juvenile hormone to the larva in the brood cell. From a total of 475 mites, 81 developed offspring (18 + 17%). If we sprayed the bees with the parasitic mites with juvenile hormone and kept them for three additional days on the bees, 170 from 708 developed offspring in the cells (39 f 28%, 8 groups). Table 3. Effect of the duration on which mites were parasitic on bees in an incubator on the offspring rate of the mites Time in the incubator
(days)
(n)
With offspring (“/) (2 f SD)
0
135 132 71 9 245 12 23 21 15
5*7 7&l 18 0 5_+8 25 35 29 0
1 3 5 6 7 10 21 31
Varroa
Significance (P z 0.01)
as.
sig. n.s. “S.
sig. sig. sig. n.s.
HEINZHKNELand NIKOLAUS KOENIGER
194
iB . ..*.... . . type . . . . . .of . . . bee . . . .. lone type of JHlarva bee treatment . .. 29 mites (n) 663 475 706 Fig. 3. Influence of external juvenile hormone on reproduction of Varroa.WB = mites kept on winter bees; SB = mites kept on summer bees (age 12 days). Juvenile hormone treatment either on larvae (2nd column) or on adult bees (3rd column). Standard deviation and number of mites in each test indicated. WB ... . none
.
WB . . .. . . .dri. WB . . . . . . “6ii..
If the results of Fig. 3 are compared, it is obvious that juvenile hormone increases the capacity of offspring production in those mites which were kept on winter bees. Effect of unphysioiogically high amounts of juvenile hormone on the ovary of Varroa
Serial sections of 12 mites sprayed with 3 mg juvenile hormone per group of bees fixed from 24 to 238 h after the treatment showed remarkable irregularities in the yolk incorporation. Vacuoles of different size appeared in the largest eggs, or the egg was 2-3 times larger than the biggest one in the control group. In the mites fixed 100-200 h after spraying, the nucleus was separated from the apparently coagulated yolk material. Generally the appearance of large yolk bodies was different from the control mites which showed perfectly fine granulated material in their biggest eggs irrespective of the time tixed. In two serial sections of mites which we took
from worker brood cells of A. cerana (no reproduction), the same type of fine yolk material was visible as in our control groups. In two Figures (4 and 5) we show the effect of juvenile hormone on the yolk material. Figure 4 shows part of the ovary of a control mite. In the biggest egg only fine yolk material is visible. In all the juvenile hormone-treated mites we detected changes as in Fig. 5. Here only coarse yolk material is present and the egg increases remarkably in size, although the mite stayed on an adult bee. Such an appearance was never observed in a control mite. Effect of physiological amounts of juvenile hormone on the ovary of Varroa
The results of the investigation on the genital system of mites sprayed with physiological amounts of juvenile hormone on bees are summarized in Table 4. It shows clearly there is no effect of physiological amounts of juvenile hormone on the size of the
Table 4. Size and yolk state of the biggest egg and appearance of the organum lyriforme (OL) in Varroa after juvenile hormone treatment Treatment Solvent control
200 pg JHIII Time after 1 treatment (h)
Enn
OL
Size @m) 8*s
Ena
OL
Size (pm) R*s
30 54 18 102 126 174 218
b c c c,d c,d d c,d
i+ + + 0 0 0 0
2.9 f 1.O 3.2 f 0.3 3.4 f 0.4 3.2 f 0.3 3.6 f 0.4 3.6*0.5 3.5 f 0.7
a a a a a c
0 0 0 0 0 0
3.2 f 3.5 f 3.3 f 2.8 f 3.1 f 3.9 f
0.4 0.5 OS 0.4. 0.7 0.3
*Only 6 badly fixed ovaries analysed. Abbreviations: -: not investigated. + +: heavy proliferation of the OL. f: slight proliferation. 0: no proliferation. a: fine yolk material. b: partly coarse yolk at the edges of the egg. c: half of the biggest eggs coarse. d: disturbances in yolk incorporation. JHIII: juvenile hormone III.
Fig. 4. Histological section of an ovary of a control mite. The biggest egg and some smaller eggs are visible. At the edges of the egg some basophihc material is deposited.
Fig. 5. Histological section of an ovary of a juvenile hormone-treated mite. The biggest egg with c:oarse yolk bodies and the nucleus is visible. Note the size of the egg.
795
Regulation of the reproduction of the honey bee mite biggest egg in Varroa. The organum lyriforme, which is thought to be the yolk-forming organ, appears proliferated shortly after the first treatment. This effect decreases steadily. There is no visible proliferation of this organ in the control groups. As early as 30 h after the start of the experiment, some coarse yolk material is visible in some of the biggest eggs of the juvenile hormone-treated groups. These disturbances are apparent throughout the whole period. In the control group, only the last set of ovaries showed some bigger yolk particles after 2 18 h on bees in the incubator. DISCUSSION
Juvenile hormone influence on Varroa in the brood cell
The general observation made by everyone who investigates Vurroa is, that reproduction on the drone brood is better than that on the worker brood. In a previous paper, we reported that there is a considerable enhancement of reproduction if juvenile hormone is added to the early spinning larvae and that this effect is limited to a short period of time (Hanel, 1983). This phenomenon for a sensitive phase for juvenile hormone seems to be widely distributed among insects: Locusta migratoria is only susceptible during a short period of the 5th instar (Dhadialla and Wyatt, 1981), and the Rhodniw prolixus during a period of 24 days in the 5th stage (Nijhout, 1983). If juvenile hormone is missing at that time it cannot be replaced by a later treatment (Davey, 1981). That is what we found in Vurroa as well. Therefore it was necessary to measure the titre in the developing brood of A. cerana and A. mell$era, because Varroa begins to feed shortly after sealing of the cell, when the larvae begin to spin. There are also possibilities for host-parasite interactions other than hormones. In the brood cell, kairomones or tactile stimuli by the bee larvae might induce a blockade of the parasite’s reproduction. As Figs 1 and 2 show, there is a remarkable difference in juvenile hormone contents of both species. The juvenile hormone concentration of A. ceruna worker brood seems to rise too late and too little to induce the yolk incorporation into the mite eggs. Perhaps this is one of the reasons for the lack of offspring in A. cerana worker brood (Koeniger et al., 1981), and thus for the comparatively little interference, the mite causes in its natural host. Figure 3 indicates that an enhancement of juvenile hormone on the larvae alone is not as effective as an enhancement of juvenile hormone concentration on the adult bee which Vurroa parasitizes before entering the cell. Juvenile hormone influence of the ad& bee
The parasitization of the mites on adult bees where they encounter different titres of juvenile hormone seems to be of some importance for the numerous processes in connection with the reproduction of the mite. Titre values showed a significant increase in juvenile hormone in old summer bees. Winter bees do not reach these concentrations until they start to collect nectar in spring (Fluri et al., 1982). Accordingly, there was little to no reproduction of the mites which parasitized winter bees of varying age (Table 2).
797
Mites which parasitized old summer bees showed (Fig. 3) relatively high percentages of reproduction. These bees contain up to nearly 55 ng/ml juvenile hormone in the haemolymph (Rutz et al., 1976). Preliminary experiments showed that Varroa parasitizing winter bees contain little juvenile hormone III, corresponding to the low level in their hosts (Hlnel, unpubl.). By using radioactive precocene II, autoradiography of mite cryo-sections might show whether there is a corpus allatum homologue organ in Varroa. Thus it remained for us to clarify, if spraying juvenile hormone on parasitized winter bees could mimic the physiological conditions of summer bees as far as Vurroa reproduction is concerned. Figure 3 shows that there is a significant intIuence of juvenile hormone on the percentage of reproduction in mites, although the 76 + 22% from summer bees is not achieved. Applying the t-test for the comparison of means (P > O.OOl), juvenile hormone treatment of both kinds leads to a reproductive success, significantly different from the controls. Additionally the treatment of the mites on adult bees is different from that of juvenile hormone treatment on larvae. We assume, that the juvenile hormone concentration of the adult bee has an influence on the behaviour of the mite: to enter a brood cell. Preliminary experiments (Hiinel, unpubl.) show that mites from old summer bees enter brood cells more frequently than mites parasitic on young bees. Barabanova (1983) and Smimow (1979) found only little invasive behaviour of mites in the late autumn. During this time of low juvenile hormone titre in the bees, there are still a fair number of brood cells available. Preliminary counts in early spring (Hiinel, unpubl.) showed that cell entering with successful reproduction is linked to the first collecting flights on sunny days. At that time bees with the first high titre occur. The influence of juvenile hormone on the process of yolk incorporation calls for comment. As we showed, the physiological amounts of juvenile hormone sprayed on winter bees with mites influenced only the organum lyriforme and the neurosecretory system (Hlnel, 1986). No enhanced incorporation of yolk into the egg occurred and it would not be likely because the adult bee does not contain the amounts of protein which are available in larvae. Only because of the precise timing, the mite is able to complete its reproductive cycle within the cell. If the developmental period of a worker bee is only l-2 days shorter than average, as in Apis melltfera capensis Esch., Varroa is unable to produce normal numbers of offspring (Moritz and Hiinel, 1983). A model of a 2-fold push in reproduction in Varroa develops, We assume that the period on old summer bees causes the differentiation of the cells of the yolk-forming organum lyriforme in the mites for the coming reproductive cycle. This was also shown by the histological observations. Additionally, the mite enters the brood cell to receive a second pulse of juvenile hormone from the larvae. This seems to participate in the initiation of the incorporation of yolk material into the biggest egg of the mite. This 2-step process resembles the condition in Rhodnius (Abu Hakima and Davey, 1975 and 1977). There is some evidence for a function of juvenile hormone or
HEINZ HINEL and NIKOLAUSKOENIGER
798
hormone-like substances ‘in ticks and mites (Oliver et al., 1984; Pound and Oliver, 1979; Oliver et al., 1985). Our results do not finally prove the dependence of* Vurroa on the host’s hormone but the reproduction experience shown here, together with the histological sections and earlier work on the neurosecretory system (Hiinel, 1986) indical/e, that juvenile hormone influences the mite’s reproduction. The results of the hormone treatments might lead to a new approach in the control of Vurroa. It is known from several other insects that high concentrations of juvenile hormone have a lethal effect on developing stages (Buschor et al., 1984). Generally additional juvenile hormone seems to have no disturbing influence upon the bee colony (Rutz et al., 1976) thus it could be a candidate for a hormonal
juvenile
Varroa
control.
Acknowledgements-The
authors like to thank the Deutsche Forschungsgemeinschaft for the grant Ko 400/l-2. Mr Makhdzir Mardan, Jabatan Perlindungan Tunbuhan, Fakulti pertanian Malaysia, Serdan, Selangor, Malaysia provided a colony of A. ceruna. Professor Maschwitz (Frankfurt) enabled the senior author to carry out experiments in Malaysia.
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Davey K. G. (1981) Regulation of cell volume in follicle cells of Rhodnius by JH. In Juvenile Hormone Biochemistrv (Ed. by Pratt -Ci. E. and Brooks G. T.), pp. 233-240. Elsevier North-Holland Biomedical Press, Amsterdam. Dhadialla T. S. and Wyatt G. R. (1981) Competence for juvenile hormone stimulated vitellogenin synthesis in the fat body of female and male of Locusta migratoria. In Juvenile Hormone Biochemistry (Ed. by Pratt G. E. and Brooks G. T.), pp. 257-262. Elsevier North-Holland Biomedical P&s,-Amsterdam. Fluri P.. Liischer M.. Wille H. and Gerin L. (1982) Chanaes in weight of the pdaryngeal gland andsome haemolyiph titres of juvenile hormone, protein and vitellogenin in worker honey bees. J. Insect Physiol. 28, 61-68. H&e1 H. (1983) Effect of JH,,, on the reproduction of Varroa jicobsoni. Apidologie ‘i’4, 137-142.-
Hiinel H. (1986) Effect of juvenile hormone (III) from the host Apis melrifera (Insecta: Hymenoptera) on the neu-
rosecretion of the parasitic mite Varrou jacobsoni (Atari: Mesostigmata). Exp. uppl. Acarol. In press. Hagenguth H. (1979) Mikroanalytische Bestimmung von Juvenil-hormontitern der Honigbiene. Dissertation, University of Munich. Hagenguth H. and Rembold H. (1978) Kastenspezifische Modulation des Ecdysteroidtiters bei der Honigbiene. Mitt. dt. Ges. angew. Ent. 1, 296298.
Koeniger N., Koeniger G. and Wijayagunasekara N. H. P. (1981) Beobachtungen iiber die Anpassung von Vurroa jacobsoni an ihren natiirlichen Wirt Apis cerana in Sri Lanka. Apidologie 12, 37-40. Moritz R. F. A. and Hiinel H. (1984) Restricted development of the parasitic mite Vurroa jacobsoni Oud. In the Cape honeybee Apis met&era capensis Esch. Z. angew. Ent. 97, 91-95. Nijhout H. F. (1983) Definition of a juvenile hormone sensitive period in Rhodnius prolixus. J. Insect Physiol. 29, 667-677.
Oliver J. H. Jr, Pound J. M. and Severino G. (1984) Effects of the antiallatotropin precocene 2 on the reproduction the chicken mite Dermanyssus gallinae. In kdvances in Invertebrate Reproduction Vol. 3 (Ed. bv Enaels W.). pp. 621. Elseiier North-Holland Biomedical Press; Amsterdam. Oliver J. H. Jr, Pound J. M. and Severino G. (1985) Evidence of a juvenile hormone-like compound in the reproduction of Dermanyssus gallinae (Atari: Dermanyssidae). J. med. Ent. 22, 281-286. Pound J. M. and Oliver J. H. Jr (1979) Juvenile hormone: evidence of its role in the reproduction of ticks. Science 206, 355-357.
Rembold H. and Hagenguth H. (1981) Modulation of hormone pools during post embyonic development of the female honey bee castes. In Int. Conf. Regulation oflnsect Development and Behaviour, Part I (Ed. by Kloza M.), pp. 427-440. Wroclaw Techn. Univ. Press, Karpacz, Poland. Rembold H., Kremer J. P. and Ulrich G. M. (1980) Characterization of postembryonic developmental stages of the female castes of the honey bee, Apis mellfera L. Apidologie 11, 29-38. Romeis B. (1968) Mikroskopische Technik, 16th edn. R. Oldenbourg, Miinchen. Roulet F. (1948) Methoden der Pathologischen Histologie, 1st edn. Springer, Wien. Rutz W., Gerig L., Wille H. and Liischer M. (1976) The function of juvenile hormone in adult worker honey bees, Apis mellifera. J. Insect Physiol. 22, 14851491.
Schooley D. A. (1977) Analysis of the naturally occuring juvenile hormones-their isolation, identification, and titer determination at physiological levels. In Annlyfical Biochemistry of Insects (Ed. by Turner R. B.), pp. 241-287. Elsevier North-Holland Biomedical Press, Amsterdam. Smirnow A. M. (1979) Aktuelle Ergebnisse der Sowietischen Wissenschaft iiber Atiologie, Pathogenie, Epizootologie, Diagnose und Bekiimpfung der Varroatose. In Bekiimpjiing und Vorbeugung der Varroatose (Ed. by Harnaj V. and Rousseau M.), pp. 60-74. Apimondia, Bucharest. deWilde J., Staal G. B., deKort C. A. D., deLoof A. and Baard G. (1968) Juvenile hormone titer in the haemolymph as a function of photoperiodic treatment in the adult Colorado potato beetle (Leptinotarsa decemlieata) (Say). Proc. K. ned. Akad. Wet. Sect. C 71, 321-326.