Mating affects egg maturation in Anopheles gambiae Giles (Diptera: Culicidae)

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1 June, 24 Journal of Vector Ecology 135 Mating affects egg maturation in Anopheles gambiae Giles (Diptera: Culicidae) Marc J. Klowden and Richard C. Russell Division of Entomology, University of Idaho, Moscow, ID U.S.A. Department of Medical Entomology, University of Sydney and ICPMR, Westmead Hospital, Westmead, NSW 2145, Australia Received 9 October 23; Accepted 8 November 23 ABSTRACT: The effect of mating on egg development and the size of the female accessory glands during the gonotrophic cycle of Anopheles gambiae was investigated. Although females that received a measured meal of blood were more likely to produce an egg batch when they were mated, the increased reproduction was not the result of male accessory gland substances. Changes in the size of the female accessory gland were not observed after mating nor at any time during the gonotrophic cycle, but there was a more rapid increase in its size following emergence in mated females. Journal of Vector Ecology 29 (1): Keyword Index: Mosquito, female accessory gland, male accessory gland substances, reproduction. INTRODUCTION In many insects, substances that are produced by the accessory reproductive glands of the male profoundly affect the physiology and behavior of the female that receives them during mating (Klowden 1999, Gillott 23). One of the most important effects of these substances in mosquitoes is to inhibit the female from mating again for variable periods, sometimes for life (Craig 1967). Although mating behavior in a number of mosquito species is modulated by male accessory gland (MAG) substances alone, these substances do not affect the subsequent mating behavior of the few anophelines that have been studied (Klowden 21). MAG substances have also been implicated in the enhancement of reproductive output, allowing a female that has been marginally nourished to mature eggs that would otherwise not be produced after a blood meal (Klowden and Chambers 1991, Klowden 1993, Uchida et al. 23). Even in autogenous mosquitoes that do not require a blood meal for an initial batch of eggs, MAG substances contribute to the development of the autogenous egg batch (O Meara and Evans 1976, O Meara and Evans 1977, O Meara and Peterson 1985). Thus, the importance of contributions from the male mosquito in affecting the reproductive physiology of the female is well documented. In this study, we examined the mating-induced changes in the reproduction of Anopheles gambiae mosquitoes and the involvement of MAG substances in these changes. We also examined the effects of mating on the growth of the female accessory glands after a blood meal and oviposition, which were shown to change in size during the reproductive cycle of Culex and Aedes mosquitoes (Rosay 1968, Rossignol et al. 1977) and Anopheles maculipennis (Detinova 1962). MATERIALS AND METHODS The mosquito Anopheles gambiae Giles sensu stricto, originating from the Kilimanjaro region of Tanzania, has been reared in our laboratory for the past 7 y. Larvae were maintained at 27ºC and a photoperiod of 14:1 (L:D) on a diet of finely ground Tetramin fish food (Tetra Werke, Germany). Pupae were collected daily and separated by sex in some experiments. Adults were maintained at 27ºC and 8% RH and had constant access to 1% sucrose from cotton wicks. To determine the effect of mating status on egg production, mated or unmated females were given measured amounts of heparinized rat blood by enema (Briegel and Lea 1975) on day 4 post-emergence, and the number of eggs maturing 2 d later in each group was determined by ovarian dissections. Anopheline mosquitoes, including An. gambiae, tend to concentrate the blood meal by excreting quantities of lessconcentrated blood while they feed (Briegel and Rezzonico 1985), and the administration of blood by

2 136 Journal of Vector Ecology June, 24 enema fails to achieve this higher concentration, so the volume administered does not necessarily match the protein content of the same-sized blood meal that would be ingested normally. The enemas did, however, provide a uniform basis upon which to compare experimental groups. Male accessory glands were removed from males at 4 d after emergence and implanted into the hemocele of unmated females through the intersegmental membrane of their abdomens. The wound was allowed to seal by itself. On the following day, the females were given enemas of 1 µl of blood and the percentage that developed eggs was determined 2 d later. Controls consisted of sham-operated females that received the same amount of blood but had the intersegmental membrane torn and nothing implanted. In experiments to correlate morphological changes in the female accessory gland with the reproductive cycle, we dissected mated and unmated females at intervals and measured the two perpendicular dimensions of the elliptically shaped gland with an ocular micrometer. Using the formula for the area of an ellipse, we then determined the areas of the glands visible surfaces. Both mated and unmated females were allowed to blood-feed on an anesthetized laboratory rat at 6 h post-emergence and at 18 h post-emergence prior to dissection. Only mated females were able to oviposit the eggs that matured. RESULTS When measured amounts of blood were administered by enema to An. gambiae females, the percentage that developed eggs increased with the size of the meal (Figure 1). At small blood volumes of 1 µl or less, a significantly greater proportion of mated females matured their eggs compared to those that had not mated. With 1.5 µl of blood, all females developed eggs regardless of mating status. The implantation of a MAG in unmated females had no significant effect on reproduction (Figure 2). The percentage of unmated females that developed eggs from 1 µl of blood did not increase as it did after mating occurred even though a whole MAG was present. The difference in size between the accessory glands of mated and unmated females was significant only at 36 h after adult emergence (Figure 3). The small amount of post-emergence growth that occurred between emergence and 6 h was more rapid in mated females than in unmated females. However, there were no significant changes in size of the gland during the gonotrophic cycle. DISCUSSION Mating is responsible for an increase in egg maturation that is associated with the transfer of male accessory gland substances in Ae. aegypti (Klowden and Chambers 1991), but although mating has a similar effect in female An. gambiae, this effect is not mediated by male accessory gland substances. There was an increase in the percentage of unmated females able to develop eggs from small blood meals as the meal size increased, and with 1.5 µl, all females developed eggs (Figure 1). In contrast, only some mated females were able to mature eggs with only.5 µl of blood and a significantly greater percentage produced eggs with increasing blood meal sizes until all developed eggs with 1.5 µl. Implantation of male accessory glands into unmated females failed to trigger the increased reproduction characteristic of the mated state (Figure 2). Although about 6% of mated females developed eggs, less than 3% of unmated females were able to mature eggs whether or not they had MAG substances present. Uchida et al. (23) observed a similar phenomenon in mated and unmated An. stephensi, and by removing the spermatheca from previously mated females demonstrated that neither the spermatheca nor the sperm stored within it were involved in affecting oogenesis. The stimulus for the effect that mating has on anophelines has yet to be identified. The female accessory gland has a number of functions in other dipterans (Rosetto et al. 1996, Hosken and Ward 1999, Hosken et al. 22), but its specific function in mosquitoes has yet to be determined. Because it is believed to be secretory, one measure of its activity has been a change in the size of the gland that reflects the increased activity of the largely secretory cells that comprise it. Although the increase in its size correlates with certain periods of the gonotrophic cycle in Aedes (Jones and Wheeler 1965, Rossignol et al. 1977) and Culex (Rosay 1968), we were not able to identify any significant increases in size that correlated with the reproductive cycle of An. gambiae and that differed in mated and unmated females, other than a significant difference in its rate of post-emergence growth. Although Detinova (1962) reported that the gland of female An. maculipennis increased in size during ovarian development and decreased during oviposition, she presented no data that could be correlated with other physiological events. The exact function of the gland has yet to be resolved; neither Rosay (1968) nor Giglioli (1963) found any differences in reproduction after the surgical removal of the female accessory gland of mosquitoes. In house flies, the gland was implicated in the ability of sperm to penetrate the egg (Leopold and Degrugillier 1973).

3 June, 24 Journal of Vector Ecology 137 Percentage with eggs (±SE) Unmated females Mated females Blood meal size (µl) Figure 1. The percentage of mated and unmated An. gambiae females able to develop eggs from a measured volume of blood administered through the anus as an enema. Each bar is based on at least 5 insects. The differences in egg development at.75 and 1. µl of blood are significant (P<.5). The control of the switchover to the mated state in An. gambiae is markedly different than in Ae. aegypti, where male accessory gland substances control female physiology to a remarkable extent (Klowden 21). Differences in mating systems will be particularly important in any attempts to control mosquito populations by genetic manipulation. Acknowledgments This research was supported by National Science Foundation grant IBN to MJK. REFERENCES CITED Briegel, H. and A. O. Lea Relationship between protein and proteolytic activity in the midgut of mosquitoes. J. Insect Physiol. 21: Briegel, H. and L. Rezzonico Concentration of host blood protein during feeding by Anopheline mosquitoes (Diptera: Culicidae). J. Med. Entomol. 22: Craig, G. B. Jr Mosquitoes: Female monogamy induced by male accessory gland substance. Science 156: Detinova, T. S Age-grouping methods in Diptera of medical importance. World Health Organization, Geneva. Giglioli, M. E. C The female reproductive system of Anopheles gambiae melas. I. The structure and function of the genital ducts and associated organs. Riv. Marariol. 42: Gillott, C. 23. Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annu. Rev. Entomol. 48: Hosken, D. J., E. Uhia, and I. Ward. 22. The function of female accessory reproductive gland secretion and a cost to polyandry in the yellow dung fly. Physiol. Entomol. 27: Hosken, D. J. and P. I. Ward Female accessory

4 138 Journal of Vector Ecology June, 24 1 Percentage developing eggs MAG Controls Figure 2. The percentage of unmated An. gambiae females that developed eggs with 1 µl of blood, with or without a male accessory gland implanted in the abdomen. Controls had the intersegmental membrane opened and immediately closed. Each group contained at least 25 insects Area of gland (µm 2 ) blood Unmated Mated blood Hours post-emergence Figure 3. The sizes of female accessory glands from unmated and mated female An. gambiae that blood-fed on a laboratory rat at 6 and 18 h after adult emergence. The area was based on two measurements of the face of the gland that were used to determine the area of an ellipse. Each point represents the measurements from 5-13 females, replicated 3 times.

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