Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 14, Number 2, October 2025 | Pages: 1151-1157 | DOI: 10.14421/biomedich.2025.142.1151-1157 ISSN 2540-9328 (online) Study of Sperm Utilization in Female Drosophila melanogaster of bdp and btx Strains Lisa Savitri1*, Kharisul Ihsan2, Elfred Rinaldo Kasimo1, Rochmad Krissanjaya1 1Department of Medical Laboratory Technology, Faculty of Health Sciences, Kadiri University, Jalan Selomangleng No. 1, Kediri, East Java, Indonesia. 2Department of Pharmacy, Faculty of Pharmacy, Public Health, Hospital Administration, Radiology, Universitas Strada Indonesia, Kediri, Indonesia. Corresponding author lisasavitri@unik-kediri.ac.id Manuscript received: 06 August, 2025. Revision accepted: 15 October, 2025. Published: 04 December, 2025. Abstract This study investigates the patterns of sperm utilization in female Drosophila melanogaster of the bdp and btx strains through sequential mating with multiple males. The research was conducted as a descriptive observational study without experimental manipulation. Females of each strain were crossed with four different males in a specific order, with a two-day interval between each mating. F1 offspring phenotypes were recorded daily over a six-day period following the final mating. Data were analyzed descriptively and supported by somatic chromosome reconstruction to identify the male origin of each offspring. The results indicate that sperm utilization in D. melanogaster females occurs in both random and non-random patterns. Non-random utilization was observed when offspring were produced exclusively from the first male, with no contribution from subsequent males. For example, in the ♀btx × ♂N cross, only N♀ and N♂ offspring were produced, indicating that sperm from the first male was preferentially used. In contrast, random sperm utilization was evident in later matings, where offspring from multiple males appeared, suggesting that previously stored sperm remained viable and were used alongside or instead of sperm from later matings. These findings highlight the complexity of post-mating sexual selection in D. melanogaster, particularly the role of female sperm storage and utilization mechanisms. The ability of females to either favor the sperm of a particular male or mix sperm from several males may provide evolutionary advantages in terms of genetic diversity and offspring fitness. This study contributes to a better understanding of reproductive strategies and sperm competition in insects, with potential implications for broader studies in evolutionary biology and genetics. Keywords: Drosophila melanogaster; sperm utilization; sequential mating; sperm competition; chromosome analysis. Abbreviations: black dumpy wings (bdp); black taxi (btx); dumpy wings (dp); First filial generation (F1); Normal (wild-type) strain of Drosophila melanogaster (N); taxi (tx) INTRODUCTION One of the characteristics of living organisms is reproduction, which serves the purpose of preserving the species. Reproduction can occur either sexually or asexually. Sexual reproduction typically involves mating. One example of an animal that reproduces sexually through mating is Drosophila melanogaster. D. melanogaster is a favored model organism in genetic research due to several advantages, including its small size, complex anatomical structure, ease of handling, ability to be easily bred in the laboratory, simple and inexpensive breeding conditions, short life cycle, and the large number of described mutants whose information is readily available. Additionally, it has a small number of chromosomes and features giant chromosomes in the salivary glands of mature larvae (King, 1965). Mating in D. melanogaster begins once the flies reach sexual maturity. In males, sexual maturity is marked by the ability to produce and release sperm, while in females it is indicated by the ability to ovulate ootids. In females, sperm from the male is stored in two seminal receptacles and in the paired spermathecae, the latter being the organ for long-term sperm storage (Clark et al., 1994). Furthermore, Clark et al. (1994) state that female D. melanogaster exhibit efficient sperm utilization. Their study also found that remating increases fertility in females. When a female mates again with another male, there is a phenomenon where the sperm from the second male is preferentially used. This is undoubtedly influenced by factors that regulate when and how sperm is stored. One such factor determining a female's receptivity to remate is a decrease in the amount of stored sperm (Clark et al., 1994). Sperm utilization in fertilization can be random or non-random. Random utilization occurs when offspring from the first male appear in the progeny of subsequent matings with the second, third, or fourth male—in other words, when earlier sperm continue to contribute to later crosses. Non-random utilization occurs when offspring https://doi.org/10.14421/biomedich.2025.142.1151-1157 mailto:lisasavitri@unik-kediri.ac.id 1152 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 1151-1157 from the first male do not appear in subsequent crosses with other males, meaning that each cross produces only the offspring phenotype associated with the male used in that particular mating. MATERIALS AND METHODS Research Design This study is a descriptive observational study, as no specific treatments were applied. The research was conducted by sequentially mating ♀bdp with ♂N, ♂bdp, ♂b, and ♂dp, and ♀btx with ♂N, ♂btx, ♂b, and ♂tx in four different mating schemes, in order to examine sperm utilization in ♀bdp and ♀btx. The data collected were analyzed descriptively using somatic chromosome reconstruction analysis. Population and Sample The population in this study consisted of D. melanogaster obtained from the Genetics Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang. The sample used included D. melanogaster of the following strains: bdp, dp, b, btx, tx, and N. Procedure Medium Preparation The preparation of the medium began with weighing the main ingredients: rajamala banana, cassava tapai, and palm sugar in a 7:2:1 ratio. The banana and tapai were blended with water until smooth. The mixture was then poured into a pot, combined with sliced palm sugar, and cooked over a stove for 45 minutes. Once cooked, the mixture was poured hot into jam jars and immediately sealed with sponge stoppers. The jars were then cooled in a container filled with water. After cooling, the lid was opened to add 5–7 yeast granules and a piece of pupariation paper, then resealed with sponge. Ampule Preparation Pupae that had turned dark were collected from D. melanogaster stock cultures of each strain using a fine brush. These pupae were placed into jars containing the prepared medium, then sealed with a piece of sponge or foam. Mating Procedure In the mating stage, two female genotypes were used: ♀bdp and ♀btx. Each was crossed using four different mating schemes. For ♀bdp, the mating schemes were as follows: ▪ Type I: ♀bdp was first mated with ♂N. After 2 days, she was transferred to a new medium and mated with ♂bdp while ♂N was removed. Two days later, ♂bdp was removed and ♀bdp was crossed with ♂b, followed by ♂dp after ♂b was removed. Two days after the final male was removed, ♀bdp was moved to fresh medium without further mating and released once pupae were observed. F1 phenotypes were recorded and counted from days 0–6. The process was repeated three times. ▪ Type II: ♀bdp was first mated with ♂bdp, then sequentially with ♂b, ♂dp, and ♂N, each with a 2- day interval and replacement of the previous male. After mating with the final male, ♀bdp was transferred to fresh medium without mating and released after pupae appeared. F1 offspring were observed and recorded over six days, and the procedure was repeated three times. ▪ Type III: ♀bdp was mated with ♂b, ♂dp, ♂N, and ♂bdp. The process of transferring to new media and replacing males every 2 days was the same. Phenotypic observations were carried out from day 0 to day 6 and repeated three times. ▪ Type IV: ♀bdp was mated with ♂dp, followed by ♂N, ♂bdp, and finally ♂b. Each male was removed after 2 days, with the female transferred to fresh media. She was released after pupae were observed. F1 phenotypes were recorded daily for six days, and the procedure was repeated three times. For ♀btx, the same procedure was used with different male partners: ♂N, ♂btx, ♂b, and ♂tx. ▪ Type I: ♀btx was sequentially mated with ♂N, ♂btx, ♂b, and ♂tx, each with a 2-day interval and replacement of the previous male. After all matings, ♀btx was moved to fresh medium and released once pupae appeared. F1 phenotypes were observed for six days, and the procedure was repeated three times. ▪ Type II: The mating order was ♂btx, ♂b, ♂tx, and ♂N. The same interval, transfer, and release procedures were followed as in Type I. ▪ Type III: ♀btx was mated in sequence with ♂b, ♂tx, ♂N, and ♂btx. F1 phenotypes were recorded from day 0 to day 6, and the procedure was repeated three times. ▪ Type IV: The mating order was ♂tx, ♂N, ♂btx, and ♂b. Transfers to fresh media occurred every 2 days, with each male removed before the next. Observations and recordings of F1 phenotypes were done for six days and repeated three times. RESULTS AND DISCUSSION Result The characteristics of D. melanogaster strains (Figure 1) used in this study are as follows: a. Normal strain (N) ▪ Bright red eyes ▪ Smooth eye facets ▪ Light brown body color ▪ Wings cover the body and extend beyond body length Savitri et al. – Study of Sperm Utilization in Female Drosophila melanogaster 1153 b. Black strain (b) ▪ Red eyes ▪ Smooth eye facets ▪ Black body color ▪ Wings longer than the body c. Taxi strain (tx) ▪ Red eyes ▪ Smooth eye facets ▪ Light brown body color ▪ Wings longer than the body and lifted upward d. Dumpy wings strain (dp) ▪ Red eyes ▪ Smooth eye facets ▪ Light brown body color ▪ Wings are shortened and notched e. Black taxi strain (btx) ▪ Red eyes ▪ Smooth eye facets ▪ Black body color ▪ Wings longer than the body and lifted upward f. Black dumpy wings strain (bdp) ▪ Red eyes ▪ Smooth eye facets ▪ Dark brownish-black body color ▪ Notched wings Figure 1. The characteristics of D. melanogaster strains: a) Normal strain (N), b) Black strain (b), c) Taxi strain (tx), d) Dumpy wings strain (dp), e) Black taxi strain (btx), and f) Black dumpy wings strain (bdp). Table 1. Phenotypic Count Data. No Type Cross F1 Sex Replicate Ʃ Total Note 1 2 3 1 I ♀bdp><♂N - - - - ♀bdp><♂bdp - - - - ♀bdp><♂b - - - - ♀bdp><♂dp - - - - 2 II ♀bdp><♂bdp bdp ♂ 8 15 - 23 43 Non- Random bdp ♀ 11 9 - 20 ♀bdp><♂b bdp ♂ 11 0 - 11 17 Random bdp ♀ 6 0 - 6 b ♂ 9 0 - 9 17 b ♀ 8 0 - 8 ♀bdp><♂dp bdp ♂ 7 0 - 7 9 Random bdp ♀ 2 0 - 2 dp ♂ 6 0 - 6 12 dp ♀ 6 0 - 6 b ♂ 5 0 - 5 10 b ♀ 5 0 - 5 ♀bdp><♂N bdp ♂ 4 0 - 4 7 Random bdp ♀ 3 0 - 3 dp ♂ 3 0 - 3 11 dp ♀ 8 0 - 8 b ♂ 5 0 - 5 9 b ♀ 4 0 - 4 N ♂ 5 0 - 5 10 N ♀ 5 0 - 5 3 III ♀bdp><♂b - - - - - ♀bdp><♂dp - - - - - ♀bdp><♂N - - - - - ♀bdp><♂bdp - - - - - 4 IV ♀bdp><♂dp - - - - - 1154 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 1151-1157 No Type Cross F1 Sex Replicate Ʃ Total Note 1 2 3 ♀bdp><♂N - - - - - ♀bdp><♂bdp - - - - - ♀bdp><♂b - - - - 5 V ♀btx><♂N N ♂ 8 - - 8 13 Non- Random N ♀ 5 - - 5 ♀btx><♂btx btx ♂ 7 - - 7 10 Random btx ♀ 3 - - 3 N ♂ 6 - - 6 6 ♀btx><♂b b ♂ 2 - - 2 9 Random b ♀ 7 - - 7 N ♂ 1 - - 1 3 N ♀ 2 - - 2 ♀btx><♂tx N ♂ 10 - - 10 12 Random N ♀ 2 - - 2 b ♂ 1 - - 1 7 b ♀ 6 - - 6 6 VI ♀btx><♂btx btx ♂ 11 - - 11 23 Non-Random btx ♀ 12 - - 12 ♀btx><♂b b ♂ 25 - - 25 44 Random b ♀ 12 - - 12 N ♂ 4 - - 4 N ♀ 3 - - 3 ♀btx><♂tx b ♂ 9 - - 9 35 Random b ♀ 19 - - 19 tx ♂ 2 - - 2 tx ♀ 5 - - 5 ♀btx><♂N b ♂ 5 - - 5 14 Random b ♀ 9 - - 9 btx ♂ 4 - - 4 7 btx ♀ 3 - - 3 tx ♂ 5 - - 5 11 tx ♀ 6 - - 6 N ♂ 6 - - 6 10 N ♀ 4 - - 4 7 VII ♀btx><♂b b ♂ 12 - - 12 23 Non-Random b ♀ 11 - - 11 ♀btx><♂tx tx ♂ 9 - - 9 21 Random tx ♀ 12 - - 12 b ♂ 13 - - 13 15 b ♀ 8 - - 8 ♀btx><♂N N ♂ 8 - - 8 15 Random N ♀ 7 - - 7 tx ♂ 10 - - 10 19 tx ♀ 9 - - 9 b ♂ 7 - - 7 12 b ♀ 5 - - 5 ♀btx><♂btx btx ♂ 8 - - 8 15 Random btx ♀ 7 - - 7 tx ♂ 8 - - 8 14 tx ♀ 6 - - 6 b ♂ 6 - - 6 14 b ♀ 8 - - 8 N ♂ 9 - - 9 14 N ♀ 5 - - 5 8 VIII ♀btx><♂tx tx ♂ 7 5 15 27 39 Non-Random tx ♀ 2 3 7 12 ♀btx><♂N tx ♂ 5 20 7 32 69 Random tx ♀ 7 21 9 37 N ♂ 2 5 13 20 37 N ♀ 2 7 8 17 ♀btx><♂btx btx ♂ 2 8 8 18 45 Random btx ♀ 8 12 7 27 Savitri et al. – Study of Sperm Utilization in Female Drosophila melanogaster 1155 No Type Cross F1 Sex Replicate Ʃ Total Note 1 2 3 tx ♂ 1 0 9 10 18 tx ♀ 1 0 7 8 N ♂ 0 0 8 8 16 N ♀ 0 0 8 8 ♀btx><♂b btx ♂ 5 1 6 12 25 Random btx ♀ 3 0 10 13 tx ♂ 2 0 6 8 19 tx ♀ 1 0 10 11 b ♂ 3 5 8 16 26 b ♀ 1 2 7 10 N ♂ 1 0 8 9 18 N ♀ 0 0 9 9 Chromosome Reconstruction Analysis a. Somatic Chromosome Reconstruction ♀bdp 1) ♀bdp X ♂N P bdp bdp X ++ ++ dpb dpb G b dp ++dpb F1 bdp dpb ++ (N) 2) ♀bdp X ♂bdp P bdp bdp X bdp bdp G bdp bdp F1 bdp bdp (bdp) 3) ♀bdp X ♂b P bdp bdp X + + bdp bdp G bdp bdp+ F1 bdp bdp+ (b) 4) ♀bdp X ♂dp P bdp bdp X dpb dpb + + G bdp b+ dp F1 bdp dpb+ (dp) b. Somatic Chromosome Reconstruction ♀ btx 1) ♀btx X ♂ N P btx btx X ++ ++ txb txb G btx b+tx+ F1 btx txb ++ (N) 2) ♀btx X ♂btx P btx btx X btx btx G btx btx F1 btx btx (btx) 3) ♀btx X ♂b P btx btx X + + btx btx G btx btx+ F1 +btx btx (b) 4) ♀btx X ♂tx P btx btx X txb txb + + G btx b+ tx F1 txb btx + (tx) Crosses in bdp Female Individuals Based on the observations, data from cross type I, in which a bdp female was mated with a first male N, showed that the resulting F1 offspring had the N phenotype. Cross type II, between a bdp female and a first male bdp, produced F1 offspring with the bdp phenotype. Cross type III, between a bdp female and a first male b, resulted in F1 offspring with the b phenotype. Cross type IV, with a first male dp, produced F1 offspring with the dp phenotype. These results indicate that all F1 offspring from crosses between bdp females and the first males were non-random, matching the chromosome reconstruction results. For cross type I with a second male bdp (♀bdp × ♂bdp), no data were obtained as the cross had not yet been performed. Cross type II (♀bdp × ♂b) yielded F1 offspring with both bdp 1156 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 1151-1157 and b phenotypes. The number of individuals with b and bdp phenotypes was equal. Cross types III and IV, with second males dp and N (♀bdp × ♂dp and ♀bdp × ♂N), had no data available yet. Cross type I with a third male b (♀bdp × ♂b) also had no available data. Cross type II (♀bdp × ♂dp) produced F1 offspring with bdp, dp, and b phenotypes. Among them, the dp phenotype was more frequent than the bdp and b. Cross types III and IV (♀bdp × ♂N and ♀bdp × ♂bdp) also had no data yet. Cross type I with a fourth male dp (♀bdp × ♂dp) had no data. Cross type II (♀bdp × ♂N) resulted in F1 offspring with bdp, dp, b, and N phenotypes. The dp phenotype appeared most frequently. Cross types III and IV (♀bdp × ♂bdp and ♀bdp × ♂b) had no data as the crosses had not been performed. Overall, observations from crosses between bdp females and second, third, and fourth males showed that the F1 offspring did not only exhibit the phenotypes of the male parent involved in the cross. This contradicts the F1 phenotypes predicted by chromosome reconstruction, suggesting that sperm usage was random. Random sperm usage is evident when offspring resulting from the first male also appear in crosses with second, third, or fourth males. Crosses in btx Female Individuals From the observations, in cross type V (♀btx × ♂N), the F1 offspring had the N phenotype. Cross type VI (♀btx × ♂btx) resulted in btx phenotype. Cross type VII (♀btx × ♂b) produced b phenotype. Cross type VIII (♀btx × ♂tx) yielded tx phenotype. These results indicate that all F1 offspring from btx females crossed with the first males were non-random and matched the reconstructed chromosome outcomes. Cross type V (♀btx × ♂btx) with a second male resulted in F1 offspring with btx and N phenotypes. Cross type VI (♀btx × ♂b) produced b and N phenotypes, with more b individuals than N. Cross type VII (♀btx × ♂tx) resulted in tx and b phenotypes, with equal numbers of each. Cross type VIII (♀btx × ♂N) resulted in tx and N phenotypes, with tx being more frequent. In cross type V with a third male b (♀btx × ♂b), the F1 offspring had b and N phenotypes. Cross type VI (♀btx × ♂tx) resulted in b and tx phenotypes, with more b than tx. Cross type VII (♀btx × ♂N) produced N, tx, and b phenotypes, with tx being the most frequent. Cross type VIII (♀btx × ♂btx) yielded btx, tx, and N phenotypes, with btx being dominant. Cross type V with a fourth male tx (♀btx × ♂tx) yielded b and N phenotypes. Cross type VI (♀btx × ♂N) resulted in b, btx, tx, and N phenotypes, with b being the most frequent. Cross type VII (♀btx × ♂btx) also resulted in btx, tx, b, and N phenotypes, with btx being dominant. Cross type VIII (♀btx × ♂b) produced the same set of phenotypes, with b being the most frequent. All cross data between btx females and the second, third, and fourth males show that F1 offspring did not consist solely of the male parent's phenotype, again contradicting the chromosome reconstruction results. This indicates random sperm usage, as offspring from the first male appeared in crosses with subsequent males. Discussion Sperm Utilization in D. melanogaster btx and bdp Female Individuals Analysis of the data showed that all crosses between ♀bdp and ♀btx females with the first male produced F1 offspring matching the chromosome reconstruction. However, crosses with the second, third, and fourth males yielded F1 phenotypes inconsistent with the expected results, suggesting that sperm utilization in those cases was random. In the first mating, fertilization was non-random because the female had only mated with one male, and thus only one type of sperm was used. In subsequent matings, the appearance of phenotypes associated with the first male's sperm indicates random sperm utilization. This phenomenon is known as sperm displacement or sperm precedence. Sperm displacement occurs when sperm from the first male is physically moved or displaced within the female's sperm storage organs (seminal receptacle and spermatheca) by sperm from a subsequent male. This reduces the likelihood that the first male’s sperm will be used for fertilization. Sperm precedence means that sperm from the second male is more likely to fertilize eggs due to the influence of his seminal fluid. This fluid can suppress the motility and capacitation of the first male's sperm, giving the second male's sperm an advantage. According to Clark et al. (1994), sperm precedence involves two main aspects: (1) physically blocking or displacing earlier sperm, and (2) resisting displacement after copulation. In this study, a single D. melanogaster female was sequentially mated with four different males every two days. This interval aligns with the female's receptivity — her willingness to accept a new mate. After mating, females generally show decreased receptivity due to behavioral and physiological changes, including reduced attractiveness and reluctance to remate (Grillet, 2006; Singh, 2002). These post-mating changes are driven by copulation effects (where male seminal proteins suppress female receptivity to prevent sperm competition) and sperm effects (linked to sperm quantity and usage). The nervous system female receptivity, and stimulation from sperm movement in storage organs influences behavior. Importantly, D. melanogaster females do not wait for stored sperm to be depleted before remating. Thus, sperm from the first male remains present during later matings, enabling sperm competition. Competition arises as new sperm enters storage and competes with earlier sperm. Several factors influence remating frequency and sperm competition, such as sperm count, seminal fluid composition, nutrition, egg laying, testes size, and body size. Specific genes and Savitri et al. – Study of Sperm Utilization in Female Drosophila melanogaster 1157 proteins are known to play roles in sperm competition. In D. melanogaster, sperm from later males is generally more successful. Seminal fluid from subsequent males can suppress capacitation of earlier sperm, without necessarily removing them from storage. Sperm storage organs in females play a key role in prioritizing sperm usage or preventing fertilization by earlier sperm. Male accessory gland proteins are critical in this process (Adrianne et al., 2008). Genes expressed in the spermatheca are known to encode serine proteases, which interact with male proteins and influence fertilization outcomes. These proteases may regulate the internal environment of the spermatheca similarly to human prostate-specific antigen (PSA), which aids semen liquefaction. Other genes, such as trehalase, may help maintain sperm by regulating sugar metabolism. In short, interactions between male seminal proteins and female reproductive proteins determine which sperm succeed. In this study, such interactions likely resulted in sperm from second or later males being favored, which explains why their phenotypes dominated in the F1 generation. CONCLUSIONS Based on the discussion above, a preliminary conclusion can be drawn that sperm utilization in D. melanogaster females of the bdp and btx strains occurs both randomly and non-randomly. Non-random utilization is observed in the first mating; for example, in the cross ♀btx × ♂N (first male), the resulting F1 offspring consisted only of N♀ and N♂ phenotypes. In contrast, random sperm utilization appears in the second, third, and fourth matings — in other words, in crosses involving subsequent males. Acknowledgements: Thank you to the Genetics Laboratory, Faculty of Mathematics and Natural Sciences, State University of Malang, for their support during the completion of this research. Authors’ Contributions: Lisa Savitri designed the study, analyzed the data, and wrote the manuscript. All authors wrote the manuscript and approved the final version of the manuscript. Competing Interests: The authors declare that there are no competing interests. Funding: The authors declare that there are no funding. REFERENCES Adrianne. (2008). An Evolutionary Expressed Sequence Tag Analysis of Drosophila Spermatheca Genes. The International Journal of Organic Evolution 62:11 Anonim. (2008). Siklus Hidup Drosophila. (online) http://zarzen.wordpress.com/2008/09/27/siklus-hidup- drosophila/ diakses tanggal 2 Desember 2011 Borror. (1992). Pengenalan Pelajaran Serangga Edisi Keenam. Gadjah Mada University Press: Yogyakarta Clark. (1994). Variation in Sperm Displacement and Its Association with Accessory Gland Protein Loci in D. melanogaster. (online) http://www.genetics.org/content/139/1/189.full.pdf 1994 diakses tanggal 2 Desember 2011 Corebima, A, D. (2003). Genetika Mendel. Airlangga University Press: Surabaya Grillet. (2006). A Drosophila Male Pheromone Affects Female Sexual Receptivity. (online) http://rspb.royalsocietypublishing.org/content/ 273/1584/315.full.pdf+html diakses tanggal 7 Desember 2011 Jumar. (2000). Entomologi Pertanian. PT Rineka Cipta: Jakarta King, R, C. (1965). Genetics. Oxford University Press: New York Muliati, L. (2000). Pengaruh Strain dan Umur Jantan Terhadap Jumlah Turunan Jantan dan Betina D. melanogaster. Malang: FMIPA Universitas Negeri Malang: Skripsi tidak diterbitkan Pitnick. (1999). Evolution of Multiple Kinds of Female Sperm- Storage Organs in Drosophila. (online) http://online.sfsu.edu/~gs/spicer/pages/spicerpdf/pitnick99.pdf diakses tanggal 7 Desember 2011 Singh. (2006). Female Remating in Drosophila annanassae: Evidence for the Effect of Density on Female Remating Frequency. (online) http://www.springerlink.com/content/n607425vq73n030m/fullt ext.pdf diakses tanggal 7 Desember 2011 Singh. (2002). Female Remating, Sperm Competition and Sexual Selection in Drosophila. (online) http://www.funpecrp.com.br/gmr/year2002/vol3- 1/pdf/gmr0034.pdf diakses tanggal 7 Desember 2011 THIS PAGE INTENTIONALLY LEFT BLANK