Anopheles oswaldoi (Diptera, Culicidae): análise do segundo espaçador interno transcrito (ITS2) do DNA ribossômico e da susceptibilidade à infecção com Plasmodium vivax. / Anopheles oswaldoi (Diptera, Culicidae): analysis of the second internal transcribed spacer (ITS2) of the ribosomal DNA and the susceptibility to infection by Plasmodium vivax.

Mauro Toledo Marrelli

Previous results have suggested that biological differences could exist between specimens of Anopheles oswaldoi captured in the State of Acre and those from the State of Rondônia, Brazil. This species has been appointed as an important malaria vector in localities of Peru and Acre. However, in Rondônia, only a very low percentage of A. oswaldoi fed on malaria patients developed salivary gland infections. In addition, it was suspected that specimens identified as A. oswaldoi captured in open clearings in Costa Marques, Rondônia, were actually A. konderi, and that A. oswaldoi sensu stricto would be restricted to forested areas. These data, together with the difficulties found to identify anophelines of the Oswaldoi group based on morphologic criteria suggest that specimens of A. oswaldoi are members of a complex of cryptic species. The distinction of cryptic species of insects is of critical importance, since different members in a Complex could exhibit differences in ecology, vectorial capacity and response to control measures. DNA sequence analysis and particularly that of the ITS2 region of the rDNA cistron has provided diagnostic characters in some groups of cryptic species becoming a general tool for taxonomic and phylogenetic studies. The first part of the present study was undertaken to determine the extent of differences over the ITS2 sequence of specimens of A. oswaldoi s.l. captured in several localities of South America. The ITS2 of these anophelines were amplified using conserved primers of the 5.8S and 28S regions, cloned and sequenced. The lengths of ITS2 of all mosquitoes captured were about 350 nucleotides, with about 53% GC content. Analysis of the alignment of the sequences, which showed that the similarity varied between 87% and 100%, and analysis of a neighbor-joining tree produced with p-distance using the ITS2 sequences, separated these specimens in four groups. One of them is probably related to A. oswaldoi sensu stricto, and another one can possibly be related to A. konderi. The other two groups may correspond to species the morphologycal identification of which remains to be clarified in the A. oswaldoi complex. These data are evidences that specimens of A. oswaldoi are included in a complex of cryptic species and that the DNA identification could solve some taxonomic questions. A. konderi has been currently considered to be a synonym of A. oswaldoi. Although adults and immature stages of both these anopheline species have identical morphological characters, features of the male genitalia can distinguish these taxa. The second part of this study was conducted in order to compare the susceptibility of A. oswaldoi s.s. and of A. konderi to infection by Plasmodium vivax. The susceptibility was based on the proportion of mosquitoes with oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia and used to obtain F1 progenies. After emergency of adults, male genitalia of mosquitoes of each family were dissected. All families originated from mosquitoes captured in Rondônia corresponded to A. konderi, while about 85.0% of the families from Acre were A. oswaldoi s.s.. F1 progeny of field-captured A. oswaldoi s.s., A. konderi and A. darlingi were fed simultaneously on P. vivax infected blood. Mosquitoes were dissected on day 10-12 after infection and examined for the presence of oocysts and sporozoites. Both A. oswaldoi s.s. and A. konderi developed oocysts in midguts, however, the percentage of oocyst-positives in A. oswaldoi s.s. (13.8%) was higher than in A. konderi (3.3%), and only A. oswaldoi s.s. developed salivary infection with sporozoites (6.9% of positivity). Infection rates in A. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that A. oswaldoi s.s. can transmit P. vivax and suggest that it is more susceptible than A. konderi. Although A. oswaldoi s.s. is an exophilic and zoophilic species, it may be involved in human malaria transmission as it seems to be occuring in the State of Acre.

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