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Background  Malaria infection in mosquitoes is traditionally detected by microscopic examination for  Plasmodium  oocysts and sporozoites. Although PCR is now widely used, the presence of parasite DNA in a mosquito does not prove that sporogony is achieved. Thus, detection of sporozoites by microscopy is still required to definitively identify vector mosquitoes. The aim of this study was to confirm sporogony of avian  Plasmodium  spp. in  Culex pipiens pallens  and  C. inatomii  caught from the wild.    Findings  Mosquitoes collected at two sites in Japan were dissected and examined by microscopy for  Plasmodium  oocysts and sporozoites. DNA was extracted from the midgut and salivary gland of infected mosquitoes, and the infecting  Plasmodium  species was identified by sequencing 478 bp of cytochrome  b . Oocysts, or both oocysts and sporozoites, were found in 3.94 and 0.46 % of  C. p. pallens  and  C. inatomii , respectively. Four (CXPIP09, GRW4, GRW11 and SGS1) and three cytochrome  b  lineages (CXINA01, CXINA02 and CXQUI01) were confirmed to achieve sporogony in  C. p. pallens  and  C. inatomii , respectively. One mosquito each of  C. p. pallens  and  C. inatomii  was co-infected with two different  Plasmodium  lineages.    Conclusions  These findings demonstrate that  C. p. pallens  and  C. inatomii  are natural vectors of four and three lineages of avian  Plasmodium  spp., respectively. The data indicate that a systematic procedure combining microscopy and PCR is a feasible and reliable approach to identify natural vectors of wildlife malaria.

Sporogony and sporozoite rates of avian malaria parasites in wild Culex pipiens pallens and C. inatomii in Japan