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Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. ReleasingWolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasingWolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if theWolbachiastrain reduced vector competence. We clearedCulex quinquefasciatusof their naturalWolbachia pipientis wPip infection and transinfected them withWolbachia wAlbB isolated fromAedes albopictus. We show thatwAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected withwPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria,Plasmodium relictum, lineage GRW4, of seven mosquito lines (two withwAlbB; three with naturalwPip infection, and two cleared ofWolbachiainfection) by allowing them to feed on canaries infected with recently collected field isolates of HawaiianP.relictum. We tested 73 groups (N_total= 1176) of mosquitoes forP.relictuminfection in abdomens and thoraxes 6–14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect ofWolbachiaon any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing malewAlbB-infectedC.quinquefasciatusmosquitoes could suppresswPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence forP.relictumifwAlbB became established in local mosquito populations. More broadly, the lack ofWolbachiaeffects on vector competence we observed highlights the variable impacts of both native and transinfectedWolbachiainfections in mosquitoes. 

Plasmodium parasites infect thousands of species and provide an exceptional system for studying host- pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai’i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection pat- terns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores 35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species’ parasitemia levels. Infection prevalence was likely 4–5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus, Hawaii’s most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5–2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens, two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species.