Mating latency, duration of copulation and fertility in four species of the Drosophila bipectinata complex

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1 Indian Journal of Experimental Biology Vol. 52, February 2014, pp Mating latency, in four species of the Drosophila bipectinata complex Akanksha Singh & Bashisth N Singh* Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi , India Received 21 June 2013; revised 1 October 2013 Significant interspecific variations in mean were observed in four species of the Drosophila bipectinata species complex. However, D. bipectinata showed positive correlation between duration of copulation. Similarly, D. malerkotliana showed negative correlation between mating latency and duration of copulation. Likewise, D. pseudoananassae showed positive correlation between mating latency. These results suggest that D. pseudoananassae has distant relatedness from the other three species with respect to mating latency, duration of copulation which supports the previous findings. Keywords: Drosophila bipectinata complex, Interspecific variations, Mating behaviour, Phylogeny Mating behaviour of Drosophila consists of species specific fixed action patterns which are accompanied by orientation movements. Such patterns are referred to as courtship displays and are made up of a number of elements or signals some of which are performed sequentially. The response of the potential mate to the male s pattern results in information being transmitted which enables the two individuals to distinguish conspecifics from non conspecifics, males from females and also the physiological readiness of the females to engage in copulation 1. It is this behaviour that has been extensively studied in fruit flies. Following initiation of the courtship displays, the male may terminate his actions at any point in the sequential performance of the signals or may repeat the full pattern numerous times. If the individual approaches a conspecific virgin female, he usually persists until either copulation occurs or one or both flies terminate the encounter 1. The time spent by the two potential mates till they are ready to go for the copulation is termed as mating latency (time). It is recognised to be an important component of Drosophila mating behaviour and is directly correlated with different components like fecundity, and longevity 2. The female s signals are *Correspondent author Telephone: (O), (R) Mobile: Fax: bashisthsingh2004@rediffmail.com usually more limited in number and diversified. These signals are performed in response to the male s courting overtures and are divisible into two types: rejection responses and acceptance responses. Copulation occurs only if the female responds by performing the acceptance signals. The duration of copulation is species specific but considerable inter individual variation exists among them. It is considered as male determined trait in many Drosophila species and it is the expression of rate of sperm transfer 1. Bateman 3 argued that males should often exhibit a non-discriminating eagerness towards sperm transfer and thus mating, while females should rather exhibit a discriminating passivity. Recent reports suggest that the is not an exclusive part of male mating activity and is determined by both males and females 4. Male mating effort is often measured indirectly using copulation duration 5. This appears under any of two scenarios. First copulation duration can be linked to female remating behaviour where duration correlates positively with remating delay 6. Here males that copulate for longer time reduce competition with rival sperm at a small time investment cost 7. Second prolonged duration of copulation is directly correlated with male sperm investment. It has been demonstrated that the presence of rival males prompts males of D. pseudoobscura to modulate sperm transfer in the form of increase in sperm transfer and alteration in ejaculate transfer 8. In another study it was found that

2 176 INDIAN J EXP BIOL, FEBRUARY 2014 exposure to rival males prior to mating increased duration of mating exposure while it decreased mating duration. Such plastic strategies result in increased reproductive success in competitive environment 9. In D. mojavensis, the variation of copulation duration is influenced by genes in both sexes and also by the size of both sexes 10. It is known that male activity and female receptivity are the main factors responsible for successful mating in Drosophila 11. Different aspects of sexual behaviour of Drosophila such as mating speed, and a correlation between and sexual activity have also been tested in certain species of Drosophila Fulker 14 investigated the relations among mating speed, time of copulation, the number of copulations resulting in fertilization and the number of progeny produced. Similarly, Prakash 15 observed an association between fast mating, repeat mating and the number of offspring produced in D. robusta. The Drosophila bipectinata species complex is a group of four closely related morphologically very similar species: D. bipectinata (Duda 1923), D. parabipectinata (Bock 1971), D. malerkotliana (Parshad and Paika 1964) and D. pseudoananassae (Bock 1971). It is a part of the ananassae subgroup of the large and widely diverged, the melanogaster species group. The females of the four species are indistinguishable morphologically. However, the males can be distinguished on the basis of the pattern of sex comb and pigmentation of abdominal tip 19. They are known to occur in the Oriental-Australian biogeographic zones where all the four species are sympatric over parts of their geographic range 20,21. Evolutionary studies based on sexual isolation, degree of crossability, isozyme variations, polytene chromosome morphology and on the degree of divergence in nuclear and mitochondrial DNA have been done in the members of the complex 19,22. Most of the results point to the fact that D. bipectinata, D. parabipectinata, and D. malerkotliana are closely related to each other, whereas D. pseudoananassae shows distant relatedness from these three. However, behavioural studies have not been done extensively. The only examples of studies done in certain aspects of behaviour are those done on courtship patterns and mating behaviour in the four species 23,24. Recently, Singh and Singh 25 studied remating behaviour in this complex and their findings revealed that D. bipectinata, D. parabipectinata, and D. malerkotliana show similar pattern with respect to remating latency, frequency and in first and second matings while D. pseudoananassae shows entirely different pattern from the rest three members of this complex for these aspects. However, evidence for positive correlation between was reported in D. bipectinata 26. But comparisons related to these aspects were not studied in the four species of this complex before. Therefore, the present study may help us to understand the occurrence of remating in this complex and thereby provide a glimpse into the phylogenetic relationship shared among the members of this complex with respect to mating latency, and. Materials and Methods Drosophila stocks During the course of present study, one strain of each of the four species of the Drosophila bipectinata complex was employed. They are as follows: D. bipectinata-pune (PN 99), D. parabipectinata- Mysore (Mys), D. malerkotliana- Raichur (RC91) and D. pseudoananassae - Brunei (KB284). Procedure Virgin females and males of all four species of the complex were collected and aged for seven days in food vials. With the help of aspirator a single male was placed in a food vial (3 length and 1 diameter) with a single female and allowed to acclimatize to the vial for 30 sec. The pair was observed and courtship time and duration of copulation were recorded for each mated pair. The time elapsed until mating from 30 seconds of confining male and female together is taken as mating latency. was measured from the time of initiation of copulation to termination. Pairs were observed continuously for 1 h and any pair not mating during this period was recorded as unmated. The male was aspirated out from the observation vial after the completion of the copulation. The of all the four species was measured by counting the number of progeny produced by a single mated female. For testing the each mated female was kept in an individual food vial for 3 days and was transferred to a fresh food vial every third day. Three successive changes were made and on the 12 th day the female was discarded from the fourth vial. Total number of flies emerging from each vial (four vials) in twelve days was counted. Data were pooled and mean number of flies per female was calculated. All the experiments were

3 SINGH & SINGH: MATING LATENCY, DURATION OF COPULATION etc. IN DROSOPHILA 177 conducted at 24 C under normal laboratory light conditions during morning hours ( hrs). Statistical analysis One way ANOVA was used to test the variations for mean mating latency, mean in four species of the complex. This was followed by post hoc (Bonferroni t test) test for pair-wise comparisons. Correlation coefficient (r) was calculated in order to test the relationship between mating latency and, between mating latency and and between and. All the tests were performed on the Sigma Stat 2.0 version. Results Mean mating latency, mean were calculated in all four species of the D. bipectinata complex (Table 1). One way ANOVA showed significant variation for mean duration of copulation in all four species of the complex. However, the variation for mean mating latency was found to be non-significant (Table 2A). Post hoc test revealed that D. parabipectinata showed significant difference with D. pseudoananassae and D. malerkotliana for mean. Similarly, D. bipectinata showed significant difference with D. parabipectinata, D. malerkotliana and D. pseudoananassae for. Similarly, correlation coefficient (r) was calculated in order to test the relationship between mating latency and, between mating latency and and between and (Table 2B). There was no correlation between mating latency and in D. bipectinata. Also, there is no correlation between mating latency. However, there was positive correlation between in D. bipectinata. Likewise, there was no correlation between mating latency in D. parabipectinata. In the same way, there were no correlations between mating latency and duration of copulation and between and Table 2 Statistical analysis (A) One way ANOVA for mating latency, duration of copulation in four species of the D. bipectinata species complex Mating latency 1.56 >0.05 NS 5.04 <0.01* Fertility 4.26 <0.01* Note: * Significant, NS - not significant. (B) Correlation coefficient (r) to test the relationship between mating latency and, between mating latency and between and in four species of the D. bipectinata complex Species Comparisons r P F D. bipectinata D. parabipectinata D. malerkotliana D. pseudoananassae Note: *Significant, NS - not significant. P 1.28 >0.05 NS >0.05 NS 6.41 <0.001* >0.05 NS 1.21 >0.05 NS >0.05 NS -5.3 <0.001* >0.05 NS 2.0 >0.05 NS >0.05 NS 6.50 <0.001* >0.05 NS Species Table 1 Mating latency, and number of progeny produced/female in four species of the D. bipectinata complex [Values are mean±se] Number of pairs tested Mating latency in min in min Number of progeny produced/female D. bipectinata ± ± ±20.31 D. parabipectinata ± ± ±42.05 D. malerkotliana ± ± ±34.80 D. pseudoananassae ± ± ±62.97

4 178 INDIAN J EXP BIOL, FEBRUARY However, there was negative correlation between mating latency and in D. malerkotliana. But, there was no correlation between mating latency and between in this species. Similarly, D. pseudoananassae showed positive correlation between mating latency. However, there are no correlations between mating latency and and between. Discussion Successful mating and subsequent reproductive output involve complex interactions between males and females. While males contribute at the level of sperm or accessory protein transfer, females are no less important when it comes to successful mating and subsequent reproduction. The ability to store sperm and/or egg production is very important attribute of females. The process of egg laying which is under the control of female in Drosophila is an outcome of a cascade of events that include progression of eggs through oogenesis (egg production), release of mature oocytes from the ovary (ovulation) and passage of eggs through the oviducts to reach the uterus where they are fertilized and finally the deposition of eggs on the substratum 27. Two Acps SP (Acp 70A) 28 and ovulin 29 have been demonstrated to stimulate the egg laying process in the mated females. These two Acps are not redundant in function but instead act at different stages of the egg laying process. SP acts at the level of oogenesis and perhaps oviposition whereas ovulin is needed to stimulate ovulation. Egg production is a resource intensive process. Since egg production increases dramatically after mating, mated females must allocate more resources towards egg production than virgin females and this egg production may vary among females of the same species as well as among the females of different species. Similarly, storage of sperm by mated females is important for extending the period of after mating. As females are involved in egg production, they also contribute significantly in storage of sperm in storage organ. Sperm are stored in two specialized organs, the single seminal receptacle and the paired spermathecae. The seminal receptacle contains 65-80% of the stored sperm 30. Spermathecae have been suggested to serve as long term storage organs. Both females and males are thought to contribute to the processes involved in sperm storage. Mated female s reproductive tract undergoes a characteristics set of changes in shape that appear to facilitate the movement of the sperm mass towards storage and give sperm access to the sperm storage organs 31. Apart from facilitating sperm entry into storage, Acps also play an essential role in utilization of stored sperm. For example females that receive sperm from males lacking Acps are infertile, even though those females do store a few sperm. However, Miller and Pitnick 32 provided evidence that sperm storage is directly dependent on the length of seminal receptacle as longer the seminal receptacle, more sperm will be stored and thereby more eggs will be fertilized and laid. Therefore, from the above facts it is clear that both females and males are directly or indirectly play role in sperm storage and egg production. In this study our major focus is to discuss interspecific variations for mating latency, in four species of the complex. However, this has been explained in the light of sperm and accessory gland protein transfer which are male characteristics and are involved in the variations among four species of the D. bipectinata complex. Experiments were conducted to investigate the mating latency, in four species of the D. bipectinata complex. Among all the four species studied D. pseudoananassae showed longer and highest as compared to rest three species. This suggests that the transfer of large amount of sperm results in delayed remating by the females of D. pseudoananassae. However, unlike D. bipectinata, D. pseudoananassae does not show positive correlation between duration of copulation. This provides evidence that the males of D. pseudoananassae transfer large amount of sperm at the beginning of copulation. During the rest of the time, perhaps only seminal fluid proteins are transferred which results in decreased sexual receptivity in females that in turn causes increased remating time in this species. This can be the only possible explanation for long duration of copulation and high but at the same time, also a high remating latency 25. Also, it has been suggested that inducing this change in females benefits the males by decreasing the likelihood of sperm competition 33. Lange et al. 34 have demonstrated that in marine hermaphrodite, sperm transfer rate has no correlation with copulation duration. Hence, both stored sperm that are not utilised at a fast rate, as well

5 SINGH & SINGH: MATING LATENCY, DURATION OF COPULATION etc. IN DROSOPHILA 179 as the seminal fluid proteins are involved in decreased receptivity 35 in D. pseudoananassae. Similarly, highly significant positive correlation between mating latency can be explained as follows: the females take much time to become sexually active through different types of displays performed by males. As a result of the high cost incurred by males during courtship the transfer of a large number of sperm into the female tract is selected for, which in turn results in large number of progeny sired by the females. Similarly, D. bipectinata showed positive correlation between and which implies that during copulation more sperm are transferred than the accessory gland proteins. Gilchrist and Partridge 36 suggested that sperm transfer occurs in a block between 6 and 10 min after the start of mating whereas Manier et al 37 have shown that sperm number increased throughout the copulation. This postulate has been supported by Sisodia and Singh 26 who provided evidence for direct relation between in different strains of D. bipectinata. The females lose their receptivity only for short periods of time, after which they are ready to remate 25. This shows that neither sperm nor accessory gland protein play any kind of role in decreasing the receptivity of females. Similar trends were not followed in D. parabipectinata. It shows negative correlation between. This reveals that sperm transfer rate is fast enough and hence a short time is enough for the transfer of large number of sperm in the female tract. Earlier reports provide evidence that D. parabipectinata has been derived from D. bipectinata and bears similarity in nucleotide sequence with D. bipectinata 20. The pattern of sperm utilisation evident in this study does not say anything in contradiction to this. However, D. parabipectinata, similar to D. bipectinata, utilise the sperm that are in store very fast and go for remating 25. As a result the number of progeny produced is higher than D. bipectinata. In the short exhibited by D. parabipectinata males enough accessory gland proteins are not transferred, to check remating much longer. D. malerkotliana which is also one of the wide spread species of this complex shows similar pattern as that of D. parabipectinata and D. pseudoananassae. are negatively correlated in D. malerkotliana. D. malerkotliana females also have a tendency to produce large number of progeny similar to D. parabipectinata and D. pseudoananassae but there is no decrease in receptivity as in the case of D. pseudoananassae females where remating occurs after a long time 25. D. bipectinata species complex belongs to the ananassae species subgroup and therefore its members show certain similarity with D. ananassae, of the ananassae complex from the same subgroup 38. Correlation between these aspects was studied in D. ananassae by Singh and Singh 39. They also found positive correlation between similar to that of D. bipectinata. D. ananassae is the most important species of the ananassae complex, having cosmopolitan distribution endowed with certain unique genetical features that make it a good model for genetical, evolutionary and behavioural studies 40. Similarly, D. bipectinata, is the most important member of the bipectinata complex having wide geographical distribution as D. ananassae in the ananassae complex. Among the four species of the bipectinata complex, the pattern of mating behaviour of D. bipectinata shows similarity to the same of D. ananassae. The present study provides evidence that duration of copulation is not always directly correlated with sperm transfer. Perhaps, longer may result in transfer of accessory gland proteins that are involved in decreased receptivity in females which may affect their ability to remate. Hence, the whole study provides evidence that D. bipectinata, D. parabipectinata and D. malerkotliana exhibit close relatedness with each other and D. pseudoananassae is distantly related to them. Acknowledgement The financial assistance in the form of Meritorious Fellowship to AS and UGC-BSR Faculty Fellowship Award to BNS from the University Grants Commission, New Delhi is gratefully acknowledged. References 1 Spieth H T & Ringo J N, Mating behaviour and sexual isolation in Drosophila in The Genetics and Biology of Drosophila, edited by M Ashburner, H L Carson and J N Thompson, ( Academic Press, London) 3c (1983) Hegde S & Krishna M S, Body size and fitness characters in Drosophila malerkotliana, Curr Sci, 77 (1999) Bateman A J, Intrasexual selection in Drosophila, Heredity, 2 (1948) Hirai Y, Sasaki H & Kimura M T, Copulation duration and its genetic control in Drosophila elegans, Zool Sci, 16 (1999) 211.

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