Urbanization is a significant source of inter- and intra-city environmental variation and is associated with declining population sizes that are increasingly homogeneous. However, whether this shift extends to urban disease ecology and related parasite communities, requires further examination. By comparing the prevalence and diversity of two related parasite genera (host-generalist Plasmodium and host-specialist Haemoproteus) in dark-eyed junco (Junco hyemalis) populations across an urbanization gradient in California, we can determine how broad urban-associated land use changes and localized habitat composition correlate with pathogen communities. Additionally, by examining vector abundance responses, we can begin to assess broader impacts on urban disease transmission and ecology. We report Haemoproteus prevalence decreased in urban habitats, with a larger presence of host-generalist lineages, suggesting urbanization increases homogenization of host-specialist pathogens. Unsurprisingly, the host-generalist pathogen, Plasmodium, showed no correlation with urbanization but prevalence increased with rainfall. Local habitat characteristics had limited effects on Plasmodium infection prevalence, but moderate shrub coverage and low human presence were associated with Plasmodium infections. Lastly, Culex tarsalis, an important vector for Plasmodium and zoonotic diseases, was the only vector to also increase in abundance in response to rainfall. Our results show that broad land use changes associated with urbanization decrease avian parasite biodiversity and highlight localized abiotic and biotic habitat characteristics that may reduce infection prevalence.

Matryoshka RNA viruses (MaRNAV) have been detected using bioinformatics and PCR approaches. They are associated with haemosporidian parasite infections, yet their prevalence and diversity in wild bird populations remain largely unknown. To investigate this, we examined blood samples from wild passerine birds and raptors in the San Francisco Bay Area. Samples were screened for haemosporidian infections followed by RNA sequencing (RNAseq) and reverse transcriptase (RT) PCR to detect MaRNAV. Our analyses identified two novel MaRNAVs (MaRNAV-5 and -6) in various bird species harboring diverse Haemoproteus and Leucocytozoon species. MaRNAV-5, associated with Haemoproteus, exhibited 71.3% amino acid identity to MaRNAV-4 and was found across 15 passerine species. MaRNAV-6, linked to Leucocytozoon, shared 72.9% identity with MaRNAV-3 and was found in 4 raptor species. Prevalence was 44.79% for MaRNAV-5 in infected passerines and 22.22% for MaRNAV-6 in infected raptors. These viruses were not found in uninfected birds and were consistently only in birds infected with haemosporidia. Sanger sequencing revealed high similarity of viral sequences across different bird species. Our findings indicate a high prevalence of MaRNAV among local wild birds, suggesting potential impacts on their health and ecology. We propose a life-cycle model for these viruses where the insect vector is the primary host, and the haemosporidian parasite acts as the virus vector to its next host. Further research is needed to determine the impact of these viruses in avian systems.