New microsatellite markers for the neotropical malaria vector Anopheles nuneztovari sensu lato

Abstract

ABSTRACT. Anopheles nuneztovari sensu lato consists of cryptic species and genetic lineages, one of which is an important human malaria vector in the northern part of South America. Population structure and evolutionary genetics studies may help in the definition and delimitation of the species and lineages within this species complex, which is relevant information for organizations involved in malaria control efforts. In this study, 10 new microsatellite markers were isolated from 2 repeat-enriched genomic libraries of A. nuneztovari s.l. and were characterized in 37-48 mosquitoes of this species. All loci were highly polymorphic and encompassed 5-25 alleles per locus. The observed (H O ) and expected (H E ) heterozygosities ranged from 0.354 to 0.866 and from 0.613 to 0.932, respectively. Six of the 10 new loci showed significant deviations from the HardyWeinberg equilibrium, and no linkage disequilibrium was detected. The loci described in this study were more polymorphic than the 18 previously characterized loci and appear to be promising markers for use in investigating the fine-scale population genetic structure and the boundaries of the cryptic species and lineages within the A. nuneztovari complex.

Evidence of new species for malaria vector Anopheles nuneztovari sensu lato in the Brazilian Amazon region

“…In the microsatellite analyses, 32 specimens per locality were genotyped for the 12 loci under study (Table 1 ). Of these 12 loci, nine are described in Austin et al [ 39 ]: Anu1, Anu4 , Anu6 , Anu9 , Anu10 , Anu12 , Anu14 , Anu15 and Anu16 ; the other three are described in Cunha-Machado and Scarpassa [ 40 ]: Anu22 , Anu25 and Anu28 .…”

“…The microsatellite loci were amplified by PCR in a final volume of 10 µL reaction solution containing 10–20 ng DNA template, 1.0 μL 10× buffer, 2.1 μL 1 mM dNTPs, 0.3 µL 50 mM MgCl 2 , 0.4 μL 4 mM M13-tailed forward primer [ 44 ], 0.4 μL 4 mM M13-labelled primer (FAM, HEX and TAMRA), 0.8 μL 4 mM reverse primer, 0.2 μL 5 U/µL Platinum Taq DNA Polymerase (Invitrogen Inc., Carlsbad, CA, USA), and 3.8 μL sterile water to complete the total volume. PCR was carried out in 2 steps: the first step consisted of denaturation (68 °C for 2 min, 95 °C for 30 s) followed by 30 cycles of 35 s at 92 °C, 35 s at the primer-specific annealing temperature [ 39 , 40 ], and 35 s at 72 °C.…”

“…The microsatellite markers, developed and characterized previously for A. nuneztovari s.l . [ 39 , 40 ], were analysed for the first time in the present study. These markers are appropriate to estimate intra-population genetic diversity, fine-scale population structure, and also for taxonomic and evolutionary genetic studies of very recently evolved species.…”

Identification and validation of the first EST-SSR markers based on transcriptome of Anopheles darlingi, the primary transmitter of malaria in Brazil

“…nuneztovari (H O = 0.354-0.866 and H E = 0.613-0.932) [9]. Comparing the heterozygosity of A. darlingi in this study with those of other populations of the same such as those from Capanema, in the Pará state (H O = 0.368-0.769 and H E = 0.785-0.987) [10] and Coari, Amazonas state (H O = 0.037-0.833 and H E = 0.177-0.871) [12], higher values are noted.…”

“…Given these indices, research on genetic structures has been carried out in several vector species [4,5] using previously isolated and characterized SSR microsatellite markers. A total of 11 loci have been isolated and characterized in 323 Anopheles marajoara from northern Brazil [6]; Twenty ve loci in 24 to 36 Anopheles albitarsis sensu lato from Puraquequara, Manaus, Amazonas state [7]; Twelve loci in 20 to 25 Anopheles triannulatus s. l. from Manaus [8]; Ten loci in 37 to 48 Anopheles nuneztovari s. l from Manaus and Nova Mazagão, Amapá state [9]; Eight loci in 253 Anopheles darlingi from the states of Amapá, Pará and Mato Grosso [10]; Seven loci in 377 A. darlingi from the states of Acre, Rondônia and Amazonas [11]; Twenty four loci in 21 to 32 A. darlingi from Coari, Amazonas state [12]; ten loci in 440 A. darlingi from Rondônia (found along the Madeira River) [1].…”