New 24 polymorphic DNA microsatellite loci for the major malaria vector Anopheles darlingi and transpecies amplification with another anophelines

Abstract

Anopheles darlingi is a major human malaria vector in the Neotropics. Twenty-four polymorphic microsatellite loci were isolated and characterized in 21-32 individuals collected in Coari (Amazonas, Brazil). The number of alleles per locus ranged from 4 to 11 (average of 7.667). The observed heterozygosity (H O ) varied between 0.037 and 0.833 (average of 0.500), while the expected heterozygosity (H E ) ranged from 0.177 to 0.871 (average of 0.723). Thirteen loci showed a significant deviation from HWE. No linkage disequilibrium was found between the loci.Keywords Anopheles darlingi Á Microsatellites Á Malaria vector Á Amazon Basin Anopheles (Nyssorhynchus) darlingi is the major and the most anthropophilic and endophagic malaria vector in the Brazilian Amazon Basin (Tadei et al. 1998). It is also a significant vector in other countries in South America such as Peru, Colombia and Suriname (Vittor et al. 2006). Although morphology and genetics variation had been described suggesting that A. darlingi is a species complex (Freitas-Sibajev et al. 1995), morphometric and genetic analyses using isozymes, RAPD and ITS2 demonstrated the existence of a single species (Manguin et al. 1999). Studies using mitochondrial and nuclear DNA markers provide support for moderate level of population genetic structure (Mirabello and Conn 2006;Scarpassa and Conn 2007;Mirabello et al. 2008).Eight microsatellite loci were characterized (Conn et al. 2001) but the development of new markers would improve genetic population studies, gene mapping and QTL analysis in this vector. This study reports the isolation and characterization of new 24 microsatellite markers for A. darlingi and cross-amplification in three con-generic species.A genomic library enriched with microsatellite DNA of A. darlingi was constructed (Billotte et al. 1999) from the Genomic DNA extracted (Wilkerson et al. 1995) using a pool of 10 A. darlingi adult specimens newly hatched and unfed collected in Coari, Amazonas, Brazil. The DNA was digested with RsaI restriction enzyme (Invitrogen), and linked to RSA21 and RSA25 adapters. Microsatellite DNA fragments were selected by hybridization with (CT) 8 and (GT) 8 repeats biotin-linked probes and recovered with streptavidin-linked particles (Promega). Selected fragments were linked into a pGEM-T vector (Promega), transformed into Escherichia coli XL1-blue competent cells and inoculated into plates with X-Gal/IPTG/LB agar. After the growth at 37°C the white colonies were transferred onto

Adaptative processes, control measures, genetic background, and resilience of malaria vectors and environmental changes in the Amazon region

“…Considering these controversies and the importance of A. darlingi, Lima et al (2010) constructed a microsatellite DNA library and characterized 24 microsatellite polymorphic loci with a total of 184 alleles for a population in Coari (Amazonas, Brazil). Most repeats for loci were dinucleotides (56.5%), where TG/CA was the most common (42.8%).…”

Isolation and characterization of thirteen polymorphic DNA microsatellite loci for Anopheles triannulatus sensu lato (Diptera: Culicidae) and cross-amplification in four congeneric species

“…Data concerning the isolation and characterization of microsatellite markers in Anopheles species of the Nyssorhynchus subgenus are rare. Only 3 species have been cited: A. marajoara has 11 characterized microsatellite loci (Li et al, 2005), A. darlingi has 8 (Conn et al, 2001) and 24 loci characterized (Lima et al, 2010), and 18 loci have been characterized for A. nuneztovari (Austin et al, 2011). The 13 loci developed for A. triannulatus brought the total to 74 SSR loci for this subgenus.…”

“…The primers were designed in WebSat (Martins et al, 2009) and an M13 tail was added to the 5ꞌ-end of each forward primer (Schuelke, 2000). Thirty-seven primer pairs for microsatellite fragments were amplified (Lima et al, 2010) in a 10-µL reaction volume containing 10-50 ng genomic DNA, forward M13 primer labeled with FAM or HEX (0.4 µM), 0.8 µM reverse primer, 200 µM dNTPs, 1X PCR buffer, 1.5 mM MgCl 2 , and 0.5 U Taq DNA Polymerase (Fermentas, Canada). PCR was performed in 2 steps: denaturation (68°C, 1 min; 94°C, 30 s) followed by 30 cycles of 30 s at 93°C, 35 s at 60°C, 40 s at 68°C.…”

“…Cross-amplification of SSR loci in the genus Anopheles was performed from only 24 microsatellites of A. darlingi tested for A. triannulatus, A. benarrochi, and A. rangeli (Lima et al, 2010). At least 17 loci were amplified in 1 of the 3 species and 8 were detected in all species ranging from 2-5 alleles per locus.…”

Chromosomal localization of actin genes in the malaria mosquito Anopheles darlingi

“…The relevance of An. darlingi as a vector of malaria, its geographic distribution and population structure have been demonstrated in several studies (Tadei et al, 1998;dos Santos et al, 1999;Gilman et al, 2006;Lima et al, 2010). However, crucial genetic and genomic studies on An.…”