Ribosomal DNA second internal transcribed spacer sequence studies of Culicid vectors from an endemic area of Dirofilaria immitis in Spain

Abstract

Studies were performed in an endemic area of Dirofilaria immitis in Spain to genetically characterize the potential mosquito vector species present by means of DNA sequencing and elucidate which of these species may be involved in the transmission. The rDNA ITS-2 sequences of two Culex pipiens haplotypes, H1 and H2, Aedes (Aedimorphus) vexans, Fredwardsius vittatus, Ochlerotatus (Ochlerotatus) caspius, Anopheles (Anopheles) atroparvus, and Anopheles (Anopheles) plumbeus were obtained. F. vittatus and An. plumbeus were detected for the first time. Results on abundance, presence, and activity suggest that the month of August is the period of higher transmission risk, with C. pipiens, Ae. vexans, and An. atroparvus present simultaneously. Population studies indicate that C. pipiens may be considered the most important potential vector, while Ae. vexans, An. atroparvus, and O. caspius being involved in transmission only sporadically. The absence of larval dirofilarial infection agrees with the very low prevalences known in endemic areas.

Culex pipiens as a potential vector for transmission of Dirofilaria immitis and other unclassified Filarioidea in Southwest Spain

“…pipiens higher abundances and parasite development in the vector ( Roiz et al, 2014 andSassnau et al, 2014). Regardless of the observed monophasic distribution, mosquitoes were nevertheless captured throughout the year, as previously ( Bargues et al, 2006 andRoiz et al, 2015). This probably reflects an outstanding environmental adaptation capacity of Cx.…”

“…According to Bargues et al (2006) and Morchón et al (2007), the analysis of nuclear rDNA ITS-2 sequences allows validation of the classification and haplotype characterization of mosquitoes. These studies suggested that haplotype H1 may be genetically ascribed to Cx.…”

“…pipiens ITS-2 polymorphisms was assessed on the dirofilarial positive mosquitoes, by amplifying rDNA with primers 5.8S (5′-TGTGAACTGCAGGACACATG-3′) and 28S (5′-ATGCTTAAATTTAGGGGGTA-3′) ( Bargues et al, 2006). The thermal cycler was set at 94 °C for 5 min, followed by 30 cycles with a denaturation step for 30 s at 94 °C, annealing for 30 s at 50 °C, and extension for 1 min at 72 °C, and a final extension for 7 min at 72 °C.…”

“…pipiens ITS-2 sequences were edited with BioEdit ( Hall, 1999) aligned with known Culicidae sequences available in the GenBank-EMBL using CLUSTAL-W version 2.0 (Thompson et al, 1994). This served as the starting point for assignment to a known genetic haplotype, using an approach previously followed by Bargues et al (2006) and Morchón et al (2007), based on the construction of multiple sequence alignment between all most similar ITS-2 sequences of mosquitoes available from GenBank-EMBL (Supplementary data 1).…”

Molecular Characterization of Culex Theileri from Canary Islands, Spain, a Potential Vector of Dirofilaria Immitis

“…The molecular markers used for the characterisation of Cx. theileri were selected according to previous results having shown their usefulness for mosquitoes in general or Culicids in particular: small subunit or 18S rRNA gene [18][19][20][21] and the second internal transcribed spacer ITS-2 [22][23][24][25] within the nuclear rDNA, and the cytochrome c oxidase subunit I cox1 [9, within the mtDNA. This finding represents a new record for vector species not only for the Canary Island fauna but also for the whole Spanish country where the only mosquito species involved in D.…”