To understand local transmission of vector-borne diseases, it is important to identify potential vectors, characterize their host feeding patterns, and determine if vector-borne pathogens are circulating within the region. This study simultaneously investigated these aspects of disease transmission by collecting engorged mosquitoes within two rural study sites in the central Red River Valley of North Dakota. Mosquitoes were identified, midguts were excised, and the blood was expelled from the midguts. DNA was extracted from blood meals and subjected to PCR and direct sequencing to identify the vertebrate origin of the blood. Using different primer sets, PCR was used to screen for two types of vector-borne pathogens, filarioid nematodes and hemosporidian parasites. White-tailed deer were the primary source of blood meals for the eight aedine mosquito species collected. None of the 288 deer-derived blood meals contained filarioid or hemosporidian DNA. In contrast, 18 of 32 Culex tarsalis and three of three Cx. pipiens blood meals contained avian blood, representing eight different species of birds. Of 24 avian-derived blood meals examined, 12 contained Plasmodium DNA, three of which also contained Leucocytozoon DNA (i.e., dual infection). Potential confounding effects resulting from parasite acquisition and development from previous blood meals (e.g., oocysts) were eliminated because host blood had been removed from the midguts prior to DNA extraction. Thus, specific parasite lineages/species could be unequivocally linked to specific vertebrate species. By combining mosquito identification with molecular techniques for identifying blood meal source and pathogens, a relatively small sample of engorged mosquitoes yielded important new information about mosquito feeding patterns and hemosporidia infections in birds. Thorough analyses of wild-caught engorged mosquitoes and other arthropods represent a powerful tool in understanding the local transmission of vector-borne and zoonotic diseases.

Vertebrate reservoirs of arboviruses are often infected with microfilariae (MF). Laboratory studies have shown that MF can enhance the infectivity of arboviruses to mosquitoes. Soon after being ingested, MF penetrate the mosquito midgut. If the host blood also contains virus (i.e., vertebrate is dually infected), penetrating MF may introduce virus into the hemocoel. This can transform otherwise virus-incompetent mosquito species into virus-competent species and simultaneously accelerate viral development, allowing mosquitoes to transmit virus sooner than normal. This phenomenon is termed microfilarial enhancement of arboviral transmission. The prevalence of MF is very high in many passerine populations in North America. Therefore, we investigated if microfilarial enhancement could have facilitated the establishment and rapid spread of West Nile virus (WNV) across the mid-western United States. Our investigations revealed that mosquitoes, WNV, and passerine MF do interact in nature because; 1) 17% of 54 common grackles (Quiscalus quiscula L.), 8% of 26 American robins (Turdus migratorius L.), and 33% of three eastern kingbirds (Tyrannus tyrannus L.) were concurrently microfilaremic and seropositive to WNV; 2) feeding activities of mosquitoes overlapped temporally with the appearance of MF in the blood of common grackles; 3) mosquitoes fed on common grackles and American robins in nature; and 4) mosquito ingestion of two taxonomically distant species of passerine MF (i.e., Chandlerella quiscali and Eufilaria spp.) resulted in penetration of mosquito midguts. To estimate the theoretical effect that MF enhancement could have on WNV transmission in areas of high MF prevalence, vectorial capacity values were calculated for Culex mosquitoes feeding on common grackles, whereby MF enhancement was either invoked or ignored. For Cx. pipiens, vectorial capacity increased over three-fold when potential effects of MF were included in the calculations. For Cx. tarsalis, the effect was less (i.e., 1.4-fold increase). Closer attention should be paid to the potential of MF to enhance mosquito transmission of arboviruses.

Chandlerella quiscali is a filarial nematode parasite of the common grackle (Quiscalus quiscula), a widespread bird species found throughout most of North America. Worms collected from wild-caught birds were morphologically identified as C. quiscali and tested for the presence of Wolbachia, an alphaproteobacterial endosymbiont required for reproduction and maturation by many filarial species. Although various methods, including PCR, in situ hybridization and immunohistology, were employed, we were unable to detect evidence of colonization with Wolbachia. Due to the widespread distribution of the grackle host, localization within the host, and high-infection rates, C. quiscali may be among the most easily obtainable of Wolbachia-free filarial species. Further studies of C. quiscali and other Wolbachia-free filarial species may help to shed light on the reason(s) that some filarial species require Wolbachia while others do not.

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