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Biological, Environmental and Agricultural Sciences 2(2017)49-58 Available online at www.academicpress.org

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Comparison of Phylogenetic Relationships Between Populations of Chrysopidae Family (Insecta: Neuroptera) in Tehran and Kermanshah Provinces Alinaghi Mirmoayedi *1, Marzieh Marami1, Danial Kahrizi2, Kheirollah Yari3 1Department

of Plant Protection, Razi University, Kermanshah, Iran. Department of Agronomy and Plant Breeding, Razi University, Kermanshah, Iran. 3Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran. 2

ARTICLE INFO

ABSTRACT

Article history: Received 20 May 2017 Revised 3 July 2017 Accepted 17 July 2017 Available online 15 August 2017

473 samples of Chrysopidae were collected from different locations in Kermanshah and Tehran provinces. Among them nine species identified as follows, Chrysoperla kolthoffi, Ch. sillemi, Ch. lucasina, Ch. carnea, Dichochrysa prasina, Italochrysa vartianorum, Suarius nanus, Chrysopa viridana, Ch. pallens. Twenty-four specimens totally were used. Three specimens from each of four identical species from Kermanshah and Tehran were chosen to make the molecular analysis. Each of them was ground separately using a micropestle in a 1.5 ml microtube with 50 μl of DNA extraction buffer (5 mM Tris-HCl and 0.5 mM EDTA pH 8.0). Then genomic DNA was extracted. In order to electrophoresis, 1.5 % agarose gel was used and the bands stained by using 0.5 μg/ml ethidium bromide and UV rays were used to make the bands visible and to make photography. Nineteen RAPD primers were used for PCR, 13 of them showed polymorphisms. 133 electrophoresis bands were produced, 126 of them showed polymorphism and totally shaped 94.7% of polymorphism. Between the Ch. lucasina and Ch. kolthoffi of Kermanshah there was a maximum of similarity, i.e. 0.4382, but Ch. carnea of Tehran and Ch. carnea of Kermanshah had a 0.3333 of genetic similarity. ©2017Published by AcademicPress.org

Keyword: Chrysoperla spp. Genetic diversity Kermanshah RAPD Tehran

1. Introduction Brooks (1997) undoubtedly is one of the big names in studying the genera of Chrysopidae family on a world scale. However, concerning the Middle East and especially the fauna of Chrysopidae of Iran keys of identification by Aspöck et al. (1980, 2001) and Hölzel (1966, 1967) are of utmost importance. Fauna of Chrysopidae of Iran was vastly investigated by different authors, although some of them studied only different predatory, ecological or biological control roles of Chrysoperla carnea as an important generalist predator of aphids, larvae of Lepidoptera, thrips, scale insects treehoppers nymphs, etc. Hölzel (1966, 1967) an Austrian entomologist was a pioneer to study the fauna of Chrysopidae of Iran, followed by *

Corresponding author. E-mail address: [email protected]

Farahbakhsh (1961), Heydari (1965, 1995) and (Mirmoayedi (1995, 1999a, 1999b, 2001, 2002), Yassayiee and Mirmoayedi, 1998; Mirmoayedi and Thierry, 2002). There were also Iranians authors who studied the fauna of Chrysopidae in a limited region of Iran such as, Shakarami (1997) who has studied the Chrysopidae fauna of Lorestan and Farahi et al. (2009) who studied the fauna of Chrysopidae of North-East and East of Iran, Ghahari et al. (2010) have studied the Chrysopidae fauna of some rice fields of Iran. The populations of complex species of Ch. carnea was the subject of vast studies in the world, especially in Europe, Japan and USA. Different authors used different methods to reveal distinction and identification of these hidden species

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existing in carnea complex. For example, Thierry et al. (1992, 1998) used the measurement of claws and biometrical methods to differentiate species of carnea complex from each other, we in our study of carnea complex have used the claw measurement and also molecular study of RAPD markers to study the hidden species of carnea complex in Tehran and Kermanshah. Henry et al. (1996, 2002, 2003, 2006, 2010, 2013) and Wells and Henry (1992) have used special digital acoustic instruments to register the voice as well as the dances of different species in carnea complex and thus by compare of the graphs obtained to differentiate different species in carnea complex. In the past thirty years, insect molecular systematics has undergone remarkable progress. Advances in analysis of nuclear and mitochondrial material lead to data generation and accumulation of large amounts of DNA sequences. The cytochrome oxidase I, rRNA16S, 18S, and elongation factor-1 alpha genes have been vastly used among the insects, their use resulted in a reveal of phylogenetic relations and diversity between different groups (Caterino et al., 2000). Although the RAPD marker was extensively used for the reveal of molecular phylogeny, populations similarity and diversity, populations studies in forensic science, and detection of insecticides during the past thirty years. However, there were always pro and cons for its use in insect systematics. Gozlan et al. (1997) used the RAPD-PCR method to distinguish between different species of Orius (Homoptera, Anthocoridae) they were successful to differentiate between species but differentiation of strains from distinct geographical origins was impossible due to the high level of polymorphism. RAPD-PCR was used to evaluate the existence of diversity between two populations of cockroaches species Periplanata americana and Blatella germanica. Ten random primers were used for PCR, after electrophoresis many bands were obtained which could easily differentiate between two species (Neekhra et al., 2012). Jain et al. (2010) expressed that RAPD markers are well suited for genetic mapping, plant and animal breeding applications and DNA fingerprinting. With particular utility on the study of population genetics, RAPD markers can provide an efficient assay for polymorphism, which allows rapid identification and isolation of chromosome specific fragments, and in

other studies DNA fingerprints studies based on RAPD, was very different from one species or from one primer to the other. On the other hand, two independently isolated cell lines from the lepidopteran Phthorimaea operculella produced nearly identical profiles with only minor differences (Léry et al., 2003). Genomic DNA sequences differing in only one base may not be amplified in RAPD protocol or may result in a complete change in the number and size of amplified segments of DNA (Hoy, 1994). Intraspecific variation between specimens of one species of insects was studied by different authors in the past, we mention some of them. Tauber and Tauber (1986) found that eco-physiological traits play a pivotal role in the genetic diversity of north American populations of Ch. carnea, they are two interpopulation and intrapopulation factors, although prey and photoperiod can vary independently, the separate traits tend to covary to form coadaptive sets. Sosa-Gomez (2004) has studied five populations of the velvet bean caterpillar Anticarsia gemmatalis Hübner, (Insecta: Lepidoptera: Noctuidae) from Londina (Brazil), Florida (USA), La Virginia (Argentina), Passo Fundo (Brazil), and has found that the RAPD analysis was able to discriminate individuals from the same geographical population, he concluded that the overall genetic diversity represented by the Gst value was 0.1402, suggesting that variation between the five A. gemmatalis populations was low (14%) compared to the variation within each population (86%). Grapputo et al. (2005) used mtDNA of COII genes and AFLP technique to reveal inter and intraspecific variation of Colorado beetles, Leptinotarsa decemlineata in the United States and Europe. They have made their studies on the specimens collected from five locations in the US (Colorado, Idaho, Kentucky, Minnesota and New Brunswick) and eight locations in Europe (Spain, France, North and South Italy, Poland, Estonia, Russia and Finland), and sequencing of the amplified mtDNA from 109 beetles from 13 populations resulted in 20 different haplotypes, the percentage of variable sites was 3.81%. Black et al. (1992, 2001) have demonstrated that the use of RAPD-PCR technique was successful in revealing genetic variation in four aphid species, the green wheat aphid (Schizaphis graminum (Rondani), the Russian wheat aphid (Diuraphis noxia (Mordvilko), the pea aphid (Acyrth osiphon pisum (Harris), and the brown ambrosia aphid

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(Uroleucon ambrosiae (Thomas). In contrast with allozyme surveys, RAPD-PCR revealed large amounts of genetic variations among individuals in each of these species. Vaughn and Antolin (1998) have studied the populations of parasitoid wasp Diaeretiella rapae (Hymenoptera: Braconidae) in an environment with their host aphids, using RAPD-PCR, they found 11 codominant and 34 dominant RAPD polymorphisms that conformed to Mendelian segregation patterns. A nested analysis of variance indicated extensive genetic differentiation among six populations of D. rapae. Zhou et al. (2000) have sampled Heliothis armigera in six locations (five in Israel, and one in Turkey) and their genetic relationship was analyzed using RAPD-PCR. Three 10-oligonucleotide primers revealed 84 presumptive polymorphic loci that were used to estimate population structure. Results showed a low level of genetic distances among Israeli and Turkish populations. Dvorak et al. (2011) studying Intraspecific variability of natural populations of Phlebotomus sergenti have used seven RAPD primers to analyze eleven field samples from localities in Turkey, Israel, Syria, and Uzbekistan, specimens from each country formed a unique clade, but specimens from Uzbekistan, originating from one locality, formed the most homogeneous clade with almost identical band patterns. Tiple et al. (2009) have used RAPD-PCR for differentiating between different species of Lycaenidae butterflies, they have used 7 decamer primers totally 114 bands produced, among them110 were polymorphic with 96.49% of similarity. Dendrograms based on similarity coefficient grouped 5 species into two distinct clusters. Lushai et al. (2002) by use of RAPD, molecular markers were successful to analysis of populations of winged aphids Sitobion avenae, collected in one year on experimental plants and they could differentiate the presence of several novel genotypes on the different hosts, they used the data obtained to draw a dendrogram in which three main branches existed, one clustering aphids caught on agricultural crops (wheat and barley), and the other two separating the insects colonizing the native grasses cocksfoot and Yorkshire fog. Rejish et al. (2012) used 40 RAPD primers to find diversities between Trichogramma chilonis populations of different regions of India and have found that 8 of the primers constituted 19% polymorphic markers, they

have plotted a dendrogram based on data obtained and observed that the populations of wasps from SH1 (Uttar Pradesh) and LK3 (Uttar Pradesh) formed a closed sub-cluster with minimum genetic distance, but wasp populations from Maharashtra (PN1) and Punjab (PJ1) clustered to the extremes of dendrogram, which was plausible as these two populations inhabited the ecosystems significantly variable concerning temperature and humidity. Kim and Sappington (2004) using RAPD-PCR, have studied boll weevil (Anthonomus grandis Boheman) populations from eighteen locations across eight US states and NorthEast Mexico. to find genetic variations within and between weevil populations, sixty-seven reproducible bands from six random primers was obtained. Among all population, genetic and geographical distances were positively correlated. Gene flow between southcentral, western and eastern regions were limited but migration between locations within regions was relatively frequent up to distances of ∼300–400 km. Although they reported that estimate of effective migration by use of RAPD was much lower than those estimated data obtained already from mtDNA-RFLP method. Skoda et al. (2012) have collected samples of Cochliomyia hominivorax from different locations in Brazil, Nicaragua, Panama, Jamaica, Mexico, and have used RAPD-PCR to determine primers for distinguishing of species and also to differentiate geographical origin of samples. Numerical analyses of RAPD-PCR products from the 3 primers (OPG-10, OPE-12, and OPJ-8) between 40 used primers, resulted in clear-cut population grouping of each fly sample according to its geographical origin and also distinguishing this species from 7 other species including Ch. macellaria. Mirmoayedi et al. (2012) have used RAPD to find individual and sex genetic diversity among each genus and between two genera of Chrysopa and Chrysoperla species and have found that there were maximum of genetic diversity and minimum of genetic similarity between Chrysopa male (Chrysopa-M) and Chrysoperla female (Chrysoperla-F) populations, in contrast there was maximum of genetic similarity and minimum of genetic diversity between Chrysoperla-M and Chrysoperla-F in one hand and Chrysopa-M and Chrysopa-F in other hand. There were also more genetic similarities among males and females of Chrysopa or Chrysoperla, than between male of

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Chrysopa with female of Chrysoperla or vice versa. Mirmoayedi et al. (2013) have used RAPD for distinguishing relationship between different Myrmeleontidae species, seventeen primers were used to study 36 specimens of Myrmeleontidae, which produced patterns that clearly distinguished the 12 species analyzed. The analysis of data by use of principal components analysis was done by use of coefficient of similarity of Jackard. Palpares libelloides and Creoleon griseus grouped in one clade, having 0.21 genetic similarity in their DNA structure, although the former is morphologically classified in subfamily Palparinae, and the latter in subfamily Myrmeleoninae and both species have a close relationship to Cueta parvula. Mirmoayedi et al. (2014) have used RAPD for distinguishing the genetic diversity between nine species of Chrysopidae family of Kermanshah province, they found that the maximum of genetic similarity based on Jacquard index was observed between Ch. pallens and Ch. viridana which was % 78.5 (= 0.785) and they have clustered in one clade. The aim of the present study was to find the genetic diversity between different populations of any single species of Chrysopidae in two different locations of Tehran and Kermanshah, and to find if the geographical barrier could play a role in establishing of intra-specific genetic diversity amongst the Chrysopidae species which was collected by us from Tehran and Kermanshah. 2. Materials and methods 2.1. Collect of Specimens 123 specimens of Chrysopidae were collected from different locations in Kermanshah and Tehran provinces. Among them nine species were identified. Their species names are as follows; Ch. kolthoffi, Ch. sillemi, Ch. lucasina, Ch. carnea, D. prasina, I. vartianorum, S. nanus, Ch. viridana, Ch. pallens. 350 specimens were collected from different locations in Tehran province, 8 species were identified, their species names are as follows; Ch. kolthoffi, Ch. carnea, Ch. lucasina, Ch. sillemi, Anisochrysa amseli, Ch. dubitants, Ch. pallens, S. nanus. 2.2. Extraction of Genomic DNA Twenty-four specimens totally were used; three specimens from each species were chosen to make the mo-

lecular analysis. The determination of species was done following morphological characters. For molecular studies, each of them was ground separately using a micropestle, then they have been centrifuged using a 1.5 ml. micro-tube with 50 μl of extraction buffer (5 mM Tris-HCl and 0.5 mM EDTA pH 8.0), thereafter, washed with a 550 μL of buffer. Each tube was capped and warmed at 65º C for 50 min. The solution was centrifuged at1300 rpm, for seven minutes. The supernatant was transferred to a new tube, 550 μl of chloroform was added and the tube was vortexed for a few seconds. Then the new solution was centrifuged at 13000 rpm for 15 min, the supernatant was transferred to a new 1.5 ml tube and 750 μl of cold isopropanol alcohol was added. The tube was laid on in a freezer, to stay for an overnight, after this lapse of time the tube was centrifuged again at 13000 rpm for 15 min, the supernatant was extracted and 200 μl of 70% cold ethanol alcohol was added for washing, and again, centrifuged at 7000 rpm for 5mn, the obtained DNA was dried at ambient laboratory temperature and was mixed with a 50 μl TE buffer and preserved in 20º C in a freezer for further analysis. 2.3. RAPD primers Nineteen RAPD primers were used, 13 of them showed multiple polymorphisms. 133 electrophoresis bands were produced, 126 of them showed polymorphism and totally they have shaped 94.7% of polymorphism. The sizes of bands were between 3003500 bp. The best primers with maximum (100%) of polymorphism were OPA1, OPA7, OPA10, PKB b, PKB c, PKB d, OPH1, OPH5, OPH12, OPH20, POP Fe, N7, N13 and those primers which formed the minimum (82.86%) of polymorphism were OPA2, OPA3, OPA11, OPA18, POA20, OPB a. Polymorphism was calculated by dividing the Number of the polymorphic bands to the sum of total bands. 2.4. RAPD-PCR We used the method of Williams et al. (1990). The commercially prepared PCR mixture kit by Arian Gene Gostar Company, Tehran was used for PCR (Table 1). All the tubes were sterilized, and 10 ng of DNA of each specimen was mixed with 20 μl of PCR mixture and the final solution of PCR was brought to 25 μl. For each specimen, each of 19 primers provided by Arian Gene Gostar Company. Tehran was used

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with three replications. The primers have been kept frozen in -20º C, each primer was diluted to 10 μl prior to being mixed with PCR mixture. PCR was done in a Mastercycler machine made by Corbett Company. DNA polymerase was added and PCR was done as

follows; initial denaturation, one cycle, 1 min at 94° C, 44 cycles of (denaturation, 1 min each at 94° C, annealing, 1 min 36° C, primers extension, 1 min at 72° C), and final primers extension one cycle, at 72° C, for 5 min.

Table 1. The component of a PCR mixture plus DNA from each specimen. Components PCR Buffer (10 x) dNTP (10 mM ) MgCl2 (50 mM) Taq Polymerase ddH2O Primer (10 µM) DNA (10 ng) Total volume

Concentration (µl) 2.5 0.55 0.75 0.3 13-15 2.5 5.0 25

2.5. Electrophoresis To do electrophoresis, 1.5 % agarose gel was used, and the bands stained by using 0.5 μg/ml ethidium bromide, UV Rays by (Bio-Rad Gel Doc 2000) was used to make the bands visible, and to make photography. Adobe-photoshop CS5.was used for the graduation of the bands.

were calculated for each RAPD primer according to the formula: PIC = 1 – R (Pij) 2, in which, Pij is the frequency of its pattern revealed by the jth primer summed across all patterns revealed by the primers (Botstein et al., 1980). Index (h) (Nei, 1973) and Shannon’s Information index (I) (Lewontin, 1974) were calculated for gene diversity estimation.

2.6. Data analysis The POPGEN 1.32 (University of Alberta and Center for International Forestry Research) and GENALEX 6.2 and NTSYS-Pc (Applied Biostatistics, Inc, NY.) were used for analysis of data and for parsimony calculations, the method of Swofford (1993), was used. PIC (Polymorphic information content) values

3. Results when we speak of "Tehran" here, we mean different suburban regions of the province Tehran and equally when we are speaking about Kermanshah we mean species which were collected in different counties of Kermanshah province. As in Table 2.

Table 2. Matrix of data of genetic similarity based on Jackard's coefficient comparing Chrysoperla spp. of carnea complex of Tehran province (T) with Kermanshah province(K). 1- Ch. lucasina T 2- Ch. sillemi T 3- Ch. kolthoffiT 4- Ch. carnea T 5- Ch. lucasina K 6- Ch. sillemiK 7- Ch. kolthoffiK 8- Ch. carnea K.

1 2 3 4 5 6 7 8

1

2

3

4

5

6

7

8

1 0.6162 0.4269 0.4875 0.4851 0.5053 0.3617 0.3370

1 0.5064 0.4594 0.4329 0.3870 0.3 0.2705

1 0.5238 0.3510 0.3448 0.2962 0.2972

1 0.3222 0.3132 0.3108 0.3333

1 0.6279 0.4382 0.4875

1 0.5128 0.4533

1 0.5156

1

The minimum of genetic similarity was observed between Ch. sillemi of Tehran and Ch. carnea of Kermanshah, and the maximum of genetic similarity was seen between Ch. lucasina and C. sillemi both of Kermanshah. Dendrogram of similarity based on

coefficient of similarity (Fig. 1) shows that Ch. lucasina and Ch. sillemi of Tehran have composed a cluster while Ch. sillemi of Kermanshah has formed another cluster with Ch. kolthoffi of Kermanshah, on the other hand, the Ch. kolthoffi and Ch. carnea both

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from Tehran have formed a cluster but Ch. carnea of Kermanshah has formed another cluster with Ch. lucasina of Kermanshah. When we look at Table 2, we see that, between the Ch. lucasina and Ch. sillemi of Kermanshah there is a maximum of similarity, i.e. 0.6279, but Ch. carnea of Tehran and Ch. carnea of

Kermanshah were positioned distantly apart from each other so there is a 0.33 of genetic similarity between them. A graphic presentation based on principal component analysis of the genetic proximity or distance of Chrysoperla spp. in Tehran and Kermanshah is shown in Fig. 2.

Fig.1. Dendrogram based on coefficient of similarity of species of Carnea complex in Tehran and Kermanshah. Definition of abbreviations are as follows; LucaT: Ch. lucasina Tehran, SillT: Ch. sillemi Tehran, KoltT: Ch. kolthoffi Tehran, CarT: C. carnea Tehran, SillK: Ch. sillemi Kermanhah, KolK: Ch. kolthoffi Kermanshah, CarK: Ch. carnea Kermanshah, LucaK: Ch. lucasina Kermanshah.

Fig. 2. Three-dimensional graph of principal component analysis (PCA) indicating genetic proximity and distance between different populations of Ch. carnea, Ch. sillemi, Ch. kolthoffi and Ch. lucasina in Tehran and Kermanshah.

3. Discussions RAPD primers use, produced examples of successes and failures in reveal of genetic diversity between insect populations in the past, we give examples of some of them here, Wilkerson et al. (1993) have studied two morphologically indistinguishable taxa

within the Anopheles gambiae complex, A. gambiae Giles and A. arabiensis Patton., they used fifty-seven RAPD primers and Twenty-one of the primers (37%) between A. Gambiae and A. Arabiensis and almost all other primers gave bands which could potentially serve as markers for these species and twenty-one pri-

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mers satisfied the criteria they searched for and a subset of thirteen of these primers were applied to DNA samples from thirty individuals of each species. Seven of those primers functioned in a completely diagnostic manner for the colony populations, giving species-specific banding patterns for each individual tested. The other primers failed as markers because the bands which appeared diagnostic on the pooled DNA samples proved to be present in both species when applied to larger population samples. Lin et al. (2009) expressed that the advantages of RAPD were its simplicity, low cost, being rapid, use of arbitrary primers, no need of initial genetic or genomic information and the requirement of only tiny quantities of target DNA, they have used a total of 160 primers between different populations of sweet potatoes and eight primers showed consistent amplified band patterns among the plants with variations within and between varieties. Our results proved and were in accordance with the expression made by Williams et al. (1990) that RAPD primers could reveal polymorphisms between a wide variety of species. Concerning our study compare between different populations of the same species of neuropteran of Chrysopidae family, as we showed in Table 2, we saw that a 0.2962 (%29.62) of genetic similarity existed between Ch. kolthoffi specimens collected from Tehran and those collected in Kermanshah. And when we compared the genetic similarity between Ch. lucasina collected in Tehran and Kermanshah the genetic similarity is 0.4851equivalent to % 48.51, and the figures for genetic similarities for Ch. sillemi and Ch. carnea both found in Kermanshah and both found in Tehran were successively 0.4533 (%45.33) and 0.4594 (%45.94), there is also a 0.2705(27.05%) similarity between Ch. sillemi found in Tehran and Ch. carnea found in Kermanshah and 0.3132(31.32%) similarity between Ch. sillemi found in Kermanshah and Ch. carnea found in Tehran, between Kermanshah and Tehran there is a distance of 520 kilometers, so it is possible that the distance between them could play a role as geographical barrier causing a genetic diversification between populations of the same species of Tehran and Kermanshah from each other. Bamehr et al. (2014) studied diversity among populations of ladybird Cryptolaemus montrouzieri in different regions in Mazanderan using UPGMA and NTSYS-pc

softwares, the populations were clustered into two distinct clades and genetic similarity of the populations ranged from 0.48 (between Tonekabon and Freydunkenar populations) to 0.78 (Chalus and Behshahr insectarium, the genetic diversity between different populations of ladybird Ch. montrouzieri in various regions of Mazendaran was (0.48-0.78) which was more than the genetic diversity that we have found among the populations of Ch. kolthoffi in Tehran and Kermanshah (0.2969) and between the populations of Ch. lucasina in Tehran and Kermanshah (0.4851), so we saw lesser similarity and more diversity between different populations of the same species in Tehran and Kermanshah. Yellow stem borer Scirpophaga incertulas Walker, is considered as the most dangerous pest of rice in south east Asia. Suman (2015) used RAPD markers, ten arbitrary 10-mer oligonucleotide primers were used to amplify genomes of yellow stem borer populations. A total of 104 bands were amplified, of which 99 (95.1%) were polymorphic. He has found thirty-three unique bands usable for diagnostics. Genetic similarity among YSB populations varied from 0.24 to 0.651, with an average of 0.415 indicating that wide genetic variation existed between YSB populations at a molecular level. Farahpour- Haghani et al. (2014) using RAPD-PCR studied the genetic similarity of different populations of Chilo suppressalis in Guian and Mazanderan provinces in Iran and found that there were significant genetic diversity among populations in central Guilan rice plantations due to vast rice plantation in that region, there were also high genetic diversity among populations of two provinces of Guilan and Mazanderan, although more genetic similarity was observed between eastern Guilan specimens with west Mazendaran populations because these regions are located near to each other with little distance between them. RAPD was used in cockroaches, P. americana and B. germanica, the number and size of amplified products varied depending upon the sequence of random primers and genotypes used, a total of 75 discrete amplified products were obtained out of which 34 products exhibited diagnostic as well as the species-specific pattern. On an average 12.5 bands per primer were scored. RAPD-PCR was used for assessing genetic diversity in Indian lac insects and by use of fifty polymorphic markers to establish precise genetic distances, 26 RAPD primers have produced

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169 polymorphic bands sufficient for distinguishing species to complement pertinent taxonomic studies. Some of the primers were useful to reveal intraspecific level differences (Ranjan et al., 2011). To assess the polymorphism between populations of Plutella xylostella resistant to two insecticides acephate and spinosad and strains attacking Cry2Ab-Bt cottons, RAPD primers was used, and the results showed that the overall genetic similarity between the three populations treated with spinosad was 27 to 56%, acephate resistant strains maximum similarity of 68% was observed between F1 generation and F2 generation of Delhi strain and minimum similarity 27% was observed between F1 and F2 of Karnataka strains (Sunitha et al., 2015). A study to find polymorphism between different populations of blackfly Simulium gravelyi Populations living in different altitudes by use of RAPD-PCR, showed that the adults of S. gravelyi population at site 5 (1590 m) had significantly higher heterogeneity (P < 0.01, t-test) than of the other sites. On the other hand, site 1 (290 m) had significantly lower heterogeneity (Anbalagan et al., 2012). References Anbalagan, S., Bharathiraja, C., Pandiarajan, J., Dinakaran, S., Krishnan, M., 2012. Use of random amplified polymorphic DNA (RAPD) to study genetic diversity within a population of blackfly, Simulium gravelyi from palni hills, peninsular India. Biologia, 67 (6): 11951203. Aspöck, H., Aspöck, U., Hölzel, H., 1980. Die Neuropteren Europas. Goecke et Evers, Krefeld Vol 1 and 2. Aspöck, H., Hölzel, H., Aspöck, U., 2001. Kommentierter Katalog der Neuropterida (Insecta, Rhaphidioptera, Megaloptera, Neuroptera) der Westpaläarktis. Denisia, 02. Oberösterreiches Landes Museum. Bamehr, S., Mohammadi-Sharif, M., Hadizade, A., Karimi, J., 2014. Genetic diversity of mealybug ladybird, Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) populations in Mazandaran province by RAPD marker. Plant Pests Research, 4 (3): 15-24. Black, W. C., Duteau, N. M., Puterka, G. J., Nechols, J. R., Petorini, J. M., 1992. Use of the random amplified polymorphic DNA polymerase chain reaction (RAPDPCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae). Bulletin of Entomological Research, 82 (2): 151-159. Black, W. C., Baer, C. F., Antolin, M. F., Duteau, N. M., 2001. Population genomics, genome wide sampling of

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