PHYLOGENETIC RELATIONSHIP OF AN INVASIVE DROSOPHILID, indianus AND CLOSELY RELATED SPECIES OF (DIPTERA) BASED ON ESTERASE PATTERNS

M. S. MEGEED, OLA A. GALAL AND M. M. ABDEL-RAZEK

Genetics Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh, Egypt

aprionus indianus (Gupta, 1970) is a ular markers have indicated a close rela- Z drosophilid belongs to the genus and tionship between Zaprionus species and subgenus Zaprionus (Drosophilidae). It is the immigrans-Hirtodrosophila radiation a new potential pest for numerous fruit within . These markers, to- crops that exhibits a wide geographical gether with alloenzymes and quantitative distribution throughout the tropics and traits, have been used to describe the temperate regions. Based on the various probable scenario for the expansion of Z. locations where this organism has been indianus from its center of dispersal (Afri- found, it is believed that Z. indianus lives ca) to regions of Asia (ancient dispersal) on 80 host plants, making this species the and the Americas (recent dispersal) most ecologically diverse drosophilid (Commar et al., 2012). Due to its evolu- (Yassin and David, 2010). tionary history, ecological and morpholog- ical diversity, the Zaprionus genus seems Drosophilids are saprophagic spe- to be a good model for comparative stud- cies that develop in decomposing plant ies with the melanogaster subgroup. The material including fruits, leaves and flow- similarities between species of the genus ers as well as fungi. Species of the group Zaprionus and species of the subgroup melanogaster tend to use decomposing melanogaster in terms of their evo- fruits, flowers and other plant parts as lutionary characteristics and their ecologi- substrates for feeding and mating. Species cal diversity have been highlighted in evo- of the genus Zaprionus also mate on flow- lutionary studies (De Setta et al., 2009; ers and fruits (Schmitz et al., 2007; 2011). Although the phylogenetic rela- Markow and O’Grady, 2006 & 2008). In tionships within the Zaprionus genus had addition, Z. indianus feeds on the bacteria been recently proposed (Yassin et al., and yeast found in decomposing fruits, 2008), its taxonomic positioning in the principally on the yeast Candida tropicalis Drosophilidae family remains a matter for (Gomes et al., 2003). There have been discussion. proposals to reclassify the genus Zaprionus as a subgenus or group of the Isozyme patterns showed a pro- genus Drosophila because various molec- nounced differentiation in many organ- ______Egypt. J. Genet. Cytol., 44:281-290, July, 2015 Web Site (www.esg.net.eg) 282 M. S. MEGEED et al. isms including . They are still MATERIALS AND METHODS amongst the quickest and cheapest marker systems to develop, and remain an excel- Insects lent choice to identify low levels of genet- Two natural populations of Z. ic variation (Ferguson et al., 1995). Ester- indianus as well as wild type of D. ase isozyme is one of the lipid- melanogaster and D. simulans were col- hydrolyzing enzymes which have a great lected from Kafr El-Sheikh Governorate, significance in the field of genetics and Egypt. Samples of Z. indianus adults were toxicology (Callaghan et al., 1994). In collected from two different cities in Kafr insects, esterase genes have shown high El-Sheikh localities; KafrEl-Sheikh (A) rates of intraspecific and interspecific var- and Sidi Salem (B). A stock colony was iation. The level of esterase may established from the natural collected flies found to be highly variable depending on and maintained at 25±2C on the standard the life stage, sex, tissue, hormones, strain, Drosophila medium (cornmeal, agar, mo- food, environmental conditions and nu- lasses, yeast and anti-fungal agent; propi- onic acid). (Or- merous other factors (Devorshak and Roe, egon-K) stock population was used as a 1999; Villatte and Bachmann, 2002; Li et standard laboratory strain for comparison. al., 2005; Baffi et al., 2007). All these studies have suggested that the esterase Polyacrylamide gel electrophoresis isozymes exhibit high level of polymor- (PAGE) phism in Drosophila and other organisms, and this polymorphism offers adaptive Electrophoretic patterns of esterase flexibilities to these species. isozymes were studied for both wild type natural populations and standard laborato- This study aimed to focus on the ry strain. Samples were prepared from phylogenetic relationship among D. mela- whole body of adults by homogenizing nogaster, its sibling species D. simulans 250 mg flies in 500 µl of 20% sucrose and Z. indianus, a divergent species be- according to El-Fadly et al. (1990). Ho- mogenates were centrifuged at 12000 rpm longing to Drosophilidae. Two natural for 15 min at 4C. populations of Z. indianus and natural populations of D. melanogaster and D. Esterase isozyme patterns were an- simulans; belonging to the melano- alyzed using 7.5% polyacrylamide gel gaster species group, were used. A poly- electrophoresis according to the method of acrylamide gel electrophoresis was ap- Davis (1964). An equal volume (30 μl) of plied to study the esterase isozyme band- supernatant was carefully loaded to each ing patterns in the four populations com- well. Esterase bands were detected on the pared to D. melanogaster (Oregon-K gel as described by Vallejos (1983) using strain) as a standard laboratory strain. α-naphthyl acetate as substrate and subse- RELATIONSHIP OF AN INVASIVE DROSOPHILID, Zaprionus indianus 282 AND CLOSELY RELATED SPECIES OF DROSOPHILIDAE quent color development with fast blue tions; A and B are having eight and ten RR salts. After the appearance of bands, polymorphic bands, respectively. the gels were photographed. On the other hand, the electropho- Phylogenetic analysis retic bands showed wide variation in their intensities ranging from faint to dark, re- The data presented in Table (1) flecting different activities in the tissues of generated from esterase isozyme banding these populations. patterns were introduced to SPSS package program according to binary values of (1) The observed isozyme patterns can and (0) for the presence and absence of be explained in terms of allelic differ- bands, respectively. The genetic distances ences. Isozymes are all functionally simi- among the genotypes were assessed based lar forms of enzyme including all poly- on Jaccard's similarity coefficient mers of subunits produced by different (Jaccard, 1901) using the Unweighted Pair gene loci or by different alleles at the Group Method with Arithmetic mean same locus. Their electrophoretic (UPGMA) analysis (Nei, 1973). mobilities are the result of different size and shapes of enzyme molecules and their RESULTS AND DISCUSSION variation is a good indicator of genetic diversity (Shannon, 1968). So, electropho- Esterase polymorphism resis separation of isozymes has been widely used both in taxonomic and genet- The electrophoresis results of es- terase isozyme patterns presented in Fig. ic studies in Drosophila. In this regard, (1) give an evidence of esterase polymor- Galego et al. (2006) described six loci phisms of the three analyzed species under coding for esterases in Z. indianus, four of investigation. Figure (1) shows that natu- which encode α-esterases and two encode ral populations of D. melanogaster and D. β-esterases. Two of these loci, Est-3 (four simulans (from the melanogaster sub- alleles) and Est-2 (two alleles), were pol- group), Z. indianus (from Zaprionus ge- ymorphic. Alloenzyme studies indicate nus) and the standard D. melanogaster that the distribution of genetic variability laboratory strain (Oregon-K) show a high at the α-esterase 3 locus in Z. indianus is degree of polymorphism. A total of 18 influenced by natural selection, including bands were detected (Table 1), two bands selection by insecticides and selection were monomorphic and the other 16 bands stemming from climatic variation (Galego were polymorphic, with 88.89% polymor- and Carareto, 2007 & 2010). Plasticity in phism. As it appears in Fig. (1), D. mela- the distribution of allele frequencies for nogaster (Oregon-K) is having seven pol- the Est-3 locus may also have contributed ymorphic bands, D. melanogaster and D. to the successful spread of this organism simulans are having each four polymor- given that esterases perform multiple es- phic bands, and at last Z. indianus popula- sential functions in insects. 282 M. S. MEGEED et al.

Phylogenetic relationship phological and cyto-taxonomical analysis that D. simulans show close similarity It has been noticed that the number with D. melanogaster (Capy and Gibert, of species specific bands ranged from 6 2004; Nolte and Schlötterer, 2008). This is bands (for D. melanogaster and D. expected since these species are closely simulans) to 12 bands (for Z. indianus; related. Rakshit and Chatterjee (2012) also population B). Each esterase band was noted that the evolutionary distance of Z. considered as a separate character and indianus is far away from melanogaster scored 1 (present) or 0 (absent) to obtain a species group of Drosophila. rectangular binary data matrix (Table 1). Esterase patterns are important tool A similarity matrix of the species for genetic differentiation analysis and studied was obtained using Jaccard’s coef- evolutionary relationship of Drosophila ficient. As it appears from the data in Ta- species (Nascimento and De Campos ble (2), the maximum similarity coeffi- Bicudo, 2002). Commar et al. (2012) cient of 0.692 was found between the two mentioned that various molecular markers Z. indianus populations; A and B, indicat- with alloenzymes and quantitative traits ing a high degree of genetic similarity have indicated a close relationship be- between them. tween Zaprionus species and the The relatedness between the spe- immigrans-Hirtodrosophila radiation cies studied for this investigation was cal- within Drosophila. culated by Jaccard’s similarity coefficient Species of the subgenus Zaprionus method using the above data mentioned in and subgroup melanogaster would share a Table (2). Phylogenetic analysis based on last common ancestor at least as old as the esterase isozymes profile was constructed divergence of the Sophophora and Dro- using the UPGMA procedure (Fig. 2). sophila subgenera (Russo et al., 1995; Figure (2) shows that D. melano- Tamura et al., 2004). In addition to their gaster (the laboratory strain; Oregon-K shared ecological characteristics, the his- and the natural population) and D. toric and contemporary geographic coex- simulans form one cluster unit of the mel- istence between species of the subgroup anogaster species group whereas the other melanogaster and the subgenus Zaprionus cluster unit consists of the two Z. indianus suggest that these two groups of species populations; A and B. The phylogenetic passed through a period that allowed the tree reveals that there is a high degree of transfer of transposable elements during diversity existing between Zaprionus and their diversification. The invasive poten- melanogaster species. tial of various species of both genus, such as D. melanogaster (David and Capy, These results support the previous 1988), D. simulans (Hamblin and Veuille, taxonomical data obtained from mor- 1999) and Z. indianus (Gupta, 1970) may RELATIONSHIP OF AN INVASIVE DROSOPHILID, Zaprionus indianus 282 AND CLOSELY RELATED SPECIES OF DROSOPHILIDAE have promoted horizontal transfer events Callaghan, A., V. Boiroux, M. Raymofld (Carareto, 2011). and N. Pasteur (1994). Prevention of changes in electrophoretic mo- Drosophila melanogaster and Dro- bility of overproduced esterase sophila simulansas were considered the from organophosphate-resistant dominant species before the invasion of Z. mosquitoes of the Culexpipiens indianus (Valente et al., 1989; Santos and complex. Med. Veterin. Entomol., Valente, 1990; Valiati and Valente, 1996). 8: 391-394.

SUMMARY Capy, P. and P. Gibert (2004). Drosophila melanogaster, Drosophila Two natural populations of simulans: so similar yet so differ- Zaprionus indianus; collected from two ent. Genetica, 120: 5-16. different cities in Kafr El-Sheikh gover- norate, Egypt, were analyzed for esterase Carareto, C. M. (2011). Tropical Africa as variability in comparison with natural a cradle for horizontal transfers of population of both of D. melanogaster and transposable elements between D. simulans. Drosophila melanogaster species of the genera Drosophila (Oregon-K) was also used as a standard and Zaprionus. Mol. Genet. Ele- laboratory strain. The electrophoresis re- ments, 1: 179-186. sults gave an evidence of esterase poly- Commar, L. S., L. G. Galego, C. R. Ceron morphisms in the studied populations with and C. M. Carareto (2012). Taxo- 88.89% polymorphism. Phylogenetic nomic and evolutionary analysis of analysis based on Jaccard’s similarity co- Zaprionus indianus and its coloni- efficient of esterase patterns showed that zation of Palearctic and D. melanogaster and D. simulans popula- Neotropical regions. Genet. Mol. tions formed one cluster unit of the mela- Biol., 35: 395-406. nogaster species group whereas other cluster unit consisted of the two Z. David, J. R. and P. Capy (1988). Genetic indianus populations. variation of Drosophila melano- gaster natural populations. Trends Genet., 4: 106-111. REFERENCES Davis, R. J. (1964). Disc electrophoresis. Baffi, M. A., C. D. Pereira, G. R. L. de II. Method of application to human Souza, C. R. Ceron and A. M. sperum proteins. Ann. N. Y. Acad. Bonetti (2007). Esterase profile in Sci., 121: 404-427. the postembryonic development of Rhipicephalus microplus. Pesq. DeSetta, N., M. A. Van Sluys, P. Capy Agropec. Bras., 42: 1183-1188. and C. M. Carareto (2009). Multi- 282 M. S. MEGEED et al.

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RELATIONSHIP OF AN INVASIVE DROSOPHILID, Zaprionus indianus 282 AND CLOSELY RELATED SPECIES OF DROSOPHILIDAE

Table (1): Presence and absence of esterase bands of studied Drosophilidae species.

Drosophila Zaprionus Zaprionus Drosophila Drosophila melanogaster indianus indianus melanogaster simulans (Oregon-K) (A) (B) 1 1 1 1 1 1 2 0 0 0 1 1 3 0 0 0 0 1 4 0 0 0 1 1 5 1 0 0 1 1 6 1 1 0 1 0 7 0 0 1 1 1 8 1 1 1 0 0 9 1 0 0 1 1 10 1 0 0 0 1 11 0 0 1 0 0 12 0 1 0 1 1 13 1 0 1 0 0 14 0 1 0 0 0 15 1 1 1 1 1 16 0 0 0 0 1 17 1 0 0 0 0 18 0 0 0 1 1 Total 9 6 6 10 12

Table (2): Jaccard similarity coefficient of studied Drosophilidae species calculated from esterase banding patterns.

Drosophila mel- Zaprionus Drosophila mel- Drosophila anogaster (Ore- indianus anogaster simulans gon-K) (A) D. melanogaster 0.364 D. simulans 0.364 0.333 Z. indianus (A) 0.357 0.333 0.231 Z. indianus (B) 0.312 0.200 0.200 0.692

222 M. S. MEGEED et al.

Fig. (1): Isozymes profile of α-esterase of different Drosophilidae species. Lane 1: D. melanogaster (Oregon-K); Lane 2: D. melanogaster; Lane 3: D. simulans; Lane 4: Z. indianus (A); Lane 5: Z. indianus (B).

Fig. (2): UPGMA phylogenetic tree resembles the phy- logenetic relationship between the different Drosophilidae species based on Jaccard simi- larity indices.