Drosophila Strains

Kumar, Sanjay & Singh, Arvind Kumar, 2017, Neocaridina ikiensis Mendivil & Cardoso 2022, n. sp., Zoological Studies 56 (1), pp. 1-10 : 2-3

Drosophila Strains

Drosophila ananassae flies were collected from fifteen different eco-geographical localities of India ( Fig. 1) by net sweeping method from fruits and vegetable markets. Place of collection, their abbreviation, latitude and time of collection are given in table 1. After bringing the flies to laboratory, naturally impregnated females were cultured in separate food vials to establish isofemale lines. Individual fly from isofemale lines was used for allozyme analysis. It was tried to utilize maximum number of flies for genetic analysis, emerging in the first generation itself but in some cases, flies derived from second or third generations of isofemale lines were also used. This practice was followed only because it is not possible to analyze all the populations together. Similar methods have also been adopted by earlier workers ( Ayala et al. 1974). The isofemale lines were maintained on simple yeast-agar culture medium at 24 ± 1°C with 12 hour cycle of lightdark period.

Native Gel Eletrophoresis

For this purpose, a single fly was homogenized in 50 μl 20 mM Tris buffer (pH 7.4) and the homogenate was centrifuged at 12000 rpm at 4°C for 10 minutes (Kumar and Singh 2013). Supernatant was separated into two aliquots and subjected to 8 percent native polyacrylamide gel electrophoresis in 25mM Tris and 250 mM Glycine electrode buffer (pH 8.2) at 200 V for 4 hour at 4°C. In-gel staining for enzymes was done according to Shaw and Prasad (1970) and Ayala et al. (1972). The locus and allele designations were done following the standardized genetic nomenclature for enzyme coding loci ( Lakovaara and Saura 1971). Genetic variability of fifteen natural populations was assessed by analyzing seven enzyme systems ACPH ( EC. 3.1.3.2 Acid phosphatase), XDH ( EC. 1.1.1.204 Xanthine dehydrgenase), APH ( EC. 3.1.3.1 Alkaline phosphatase), AO ( EC. 1.2.3.1 Aldehyde oxidase), EST ( EC. 3.1.1.1 Esterase), MDH ( EC. 1.1.1.37 Malate dehydrogenase) and ME ( EC. 1.1.1.40 Malic enzyme) corresponding to 12 loci (Acph1, Acph2, Xdh, Aph2, Aph3, Ao1, Ao2, Est2, Est3, Est4, Mdh and Me).

Statistical Analysis

Quantitative data obtained for allozyme frequencies of all fifteen Indian natural populations of D. ananassae were utilized to derive genetic variability estimates, F-statistics and Nei’s genetic identity ( Nei 1972). Since, variation in the allelic or genotypic frequencies in different populations lead to population sub-structuring or subdivision, testing various parameters of F-statistics become imperative to study genetic differentiation among these populations. Genotype and allele frequencies were estimated using GENEPOP, version 4.2 ( Rousset 2008; http://kimura.univ-montp2. fr/~rousset/Genepop.htm). Genetic variability was recorded as mean observed (HO) and expected ( HE) heterozygosity by using software GenAlEx 6.5 ( Peakall and Smouse 2012). Population inbreeding coefficient ( FIS) was calculated to deduce the level of inbreeding due to population sub-structuring and also the departure of HO from Hardy-Weinberg Equilibrium ( Hedrick 2005). Population structure analysis was done using traditional F-statistics following Wright (1951) by using GenAlEx 6.5 ( Peakall and Smouse 2012). Genetic identity (I) approach was also utilized to determine the pattern of geographic variation among Indian natural populations of D. ananassae . It was computed by using GenAlEx 6.5 ( Peakall and Smouse 2012) according Nei (1972). To test ‘ isolation by distance’ effect, the values of genetic distance and geographic distance were correlated. Based on these results, an un-rooted dendrogram has been constructed by UPGMA using genetic similarity index, DendroUPGMA ( Garcia-Vallve et al. 1999; http://genomes.urv.cat/ UPGMA /).