Phillip Shults

Haemosporidian parasites of birds are maintained through complex interactions among vertebrate hosts, arthropod vectors, and the parasites themselves, yet their vector–parasite relationships remain poorly resolved under natural conditions in North America. Haemoproteus species are among the most prevalent avian blood parasites and have been implicated in reduced host fitness and malaria-like disease. Here, we combined molecular and histological approaches to investigate Haemoproteus prevalence in wild birds and to identify potential Culicoides biting midge vectors in Kansas. Blood meals from field-collected Culicoides and hunter-collected turkey blood samples were screened using nested PCR targeting the Haemoproteus cytochrome b gene, followed by Sanger sequencing. We additionally dissected and microscopically examined salivary glands as well as turkey blood smears. Nearly all turkey blood samples (95.7%) were positive for Haemoproteus, revealing 2 distinct parasite clades and frequent coinfections. In contrast, Culicoides-based surveillance may have underestimated parasite prevalence in turkeys. Only Culicoides haematopotus consistently tested positive by PCR, and this species was the only one in which Haemoproteus sporozoites were detected in salivary glands. Sequencing of PCR amplicons from unfed C. haematopotus revealed amplification of a Culicoides-specific noncoding genomic region, highlighting limitations of molecular detection alone for vector incrimination. Together, these findings provide field-based evidence that C. haematopotus may serve as a vector of Haemoproteus in wild turkeys in Kansas, emphasizing the importance of integrating molecular and histological data when evaluating vector competence and potential population-level effects.

Culicoides Latreille is a diverse genus of approximately 1,300 species of small biting flies found across the globe, containing numerous species of medical and veterinary importance. Many diseases associated with Culicoides have no specific treatment or vaccine, necessitating prevention through vector control. Current surveillance and control methods are limited by a lack of ecological knowledge about most Culicoides species, including phytophagous (sugar-feeding) behavior. The plants fed on by wild Culicoides are largely unknown, as are the driving forces behind selection of specific plants. To determine which plants midges are sugar-feeding on in nature, Culicoides were collected from five sites within a tallgrass prairie ecosystem in northeastern Kansas. Molecular sugar meal analysis was performed to detect plant DNA within field-collected midges. DNA was extracted from a total of 788 biting midge individuals, with 321 (40.7%) of these successfully amplifying and just 78 (9.9%) producing a sequence with a successful host match. Most matches were only identified to the plant family level with high confidence. Culicoides were found to feed on a diverse assemblage of families, including the Amaranthaceae, Cupressaceae, Solanaceae, and Verbenaceae; however, no significant association was found between Culicoides species and plant family. These results suggest that multiple sugar sources, including plant nectar and fruit, are utilized by biting midges under natural conditions. This information is vital for improving ecologically based control strategies, such as attractive toxic sugar baits, that can reduce disease burden on livestock and wildlife.

Alternative protein sources are needed due to the rising demand and increasing cost of protein ingredients in livestock diets. Mass collection of wild-caught flies from locations with high insect pressure may be an economical and environmentally sustainable approach to supplement livestock feed, but there may be feed safety issues from microbes found in field-caught insects. Therefore, we evaluated a sequencing-based approach to accurately identify potential pathogens in wild-caught flies captured on 2 different livestock farms. In this study, we combined whole-genome shotgun metagenomic sequencing with total RNA-seq to identify a broad range of microbial taxa present in and on wild-caught flies. We describe several databases tailored to the host insect, host animals, and pathogens associated with livestock and humans. Sequences were identified from potentially pathogenic bacteria including Escherichia coli, Gallibacterium anatis, Helicobacterium pullorum, Morganella morganii, Proteus mirabilis, and Providencia alcalifaciens. In addition, sequences from the pathogenic fungi Aspergillus fumigatus and viruses such as the fly pathogen Musca hytrosavirus were found. Despite the limitations of current database curation, a combination of metagenomics and total RNA-seq approaches to taxa identification can provide insight into a broad spectrum of potential pathogens in insects used as supplemental livestock feed.

Vesicular stomatitis (VS) is a viral disease that affects horses, cattle, and swine that is transmitted by direct contact and hematophagous insects. In 2023, a multi-state outbreak of vesicular stomatitis New Jersey virus (VSNJV) occurred in California, Nevada, and Texas, infecting horses, cattle, and rhinoceros. To identify possible insect vectors, we conducted insect surveillance at various locations in San Diego County, CA, including at a wildlife park. CO₂ baited traps set from mid-May to mid-August 2023 collected 2357 Culicoides biting midges and 1215 Simulium black flies, which are insect genera implicated in VSNJV transmission. Insects were pooled by species, location, and date, then tested for viral RNA. Nine RNA-positive pools of Culicoides spp. and sixteen RNA-positive pools of Simulium spp were detected. Infectious virus was detected by cytopathic effect in 96% of the RNA-positive pools. This is the first report of VSNJV in wild-caught C. bergi, C. freeborni, C. occidentalis, S. argus, S. hippovorum, and S. tescorum. The vector competency of these species for VSNJV has yet to be determined but warrants examination. Active vector surveillance and testing during disease outbreaks increases our understanding of the ecology and epidemiology of VS and informs vector control efforts.

We conducted an integrative analysis to elucidate the spatial epidemiological patterns of the Vesicular Stomatitis New Jersey virus (VSNJV) during the 2014–15 epizootic cycle in the United States (US). Using georeferenced VSNJV genomics data, confirmed vesicular stomatitis (VS) disease cases from surveillance, and a suite of environmental factors, our study assessed environmental and phylogenetic similarity to compare VS cases reported in 2014 and 2015. Despite uncertainties from incomplete virus sampling and cross-scale spatial processes, patterns suggested multiple independent re-invasion events concurrent with potential viral overwintering between sequential seasons. Our findings pointed to a geographically defined southern virus pool at the US–Mexico interface as the source of VSNJV invasions and overwintering sites. Phylodynamic analysis demonstrated an increase in virus diversity before a rise in case numbers and a pronounced reduction in virus diversity during the winter season, indicative of a genetic bottleneck and a significant narrowing of virus variation between the summer outbreak seasons. Environment–vector interactions underscored the central role of meta-population dynamics in driving disease spread. These insights emphasize the necessity for location- and time-specific management practices, including rapid response, movement restrictions, vector control, and other targeted interventions.

Insects are a promising source of high-quality protein, and the insect farming industry will lead to higher sustainability when it overcomes scaling up, cost effectiveness, and automation. In contrast to insect farming (raising and breeding insects as livestock), wild insect harvesting (collecting agricultural insect pests), may constitute a simple sustainable animal protein supplementation strategy. For wild harvest to be successful sufficient insect biomass needs to be collected while simultaneously avoiding the collection of nontarget insects. We assessed the performance of the USDA Biomass Harvest Trap (USDA-BHT) device to collect flying insect biomass and as a mosquito surveillance tool. The USDA-BHT device was compared to other suction traps commonly used for mosquito surveillance (Centers for Disease Control and Prevention (CDC) light traps, Encephalitis virus surveillance traps, and Biogents Sentinel traps). The insect biomass harvested in the USDA-BHT was statistically higher than the one harvested in the other traps, however the mosquito collections between traps were not statistically significantly different. The USDA-BHT collected some beneficial insects, although it was observed that their collection was minimized at night. These findings coupled with the fact that sorting time to separate the mosquitoes from the other collected insects was significantly longer for the USDA-BHT, indicate that the use of this device for insect biomass collection conflicts with its use as an efficient mosquito surveillance tool. Nevertheless, the device efficiently collected insect biomass, and thus can be used to generate an alternative protein source for animal feed.

Studies have investigated the potential of using farmed insects in animal feeds; however, little research has been done using wild-caught insects for this purpose. Concerns about inadequate quantities collected, environmental impacts, and the spread of pathogens contribute to the preferred utilization of farmed insects. Nevertheless, by harvesting certain pest species from intensified agricultural operations, producers could provide their animals with affordable and sustainable protein sources while also reducing pest populations. This study explores the possibility of collecting large quantities of pest flies from livestock operations and analyzes the flies’ nutritional content, potential pathogen load, and various disinfection methods. Using a newly designed mass collection-trapping device, we collected 5 kg of biomass over 13 wk, primarily house flies, from a poultry facility. While a substantial number of pests were removed from the environment, there was no reduction in the fly population. Short-read sequencing was used to compare the bacterial communities carried by flies from differing source populations, and the bacterial species present in the fly samples varied based on farm type and collection time. Drying and milling the wild-caught flies as well as applying an additional heat treatment significantly reduced the number of culturable bacteria present in or on the flies, though their pathogenicity remains unknown. Importantly, these disinfection methods did not affect the nutritional value of the processed flies. Further research is necessary to fully assess the safety and viability of integrating wild-caught insects into livestock feed; however, these data show promising results in favor of such a system.

Most population genetic studies concern spatial genetic differentiation, but far fewer aim at analyzing the temporal genetic changes that occur within populations. Vector species, including mosquitoes and biting midges, are often characterized by oscillating adult population densities, which may affect their dispersal, selection, and genetic diversity over time. Here, we used a population of Culicoides sonorensis from a single site in California to investigate short-term (intra-annual) and long-term (inter-annual) temporal variation in genetic diversity over a 3 yr period. This biting midge species is the primary vector of several viruses affecting both wildlife and livestock, thus a better understanding of the population dynamics of this species can help inform epidemiological studies. We found no significant genetic differentiation between months or years, and no correlation between adult populations and the inbreeding coefficient (F_IS). However, we show that repeated periods of low adult abundance during cooler winter months resulted in recurring bottleneck events. Interestingly, we also found a high number of private and rare alleles, which suggests both a large, stable population, as well as a constant influx of migrants from nearby populations. Overall, we showed that the high number of migrants maintains a high level of genetic diversity by introducing new alleles, while this increased diversity is counterbalanced by recurrent bottleneck events potentially purging unfit alleles each year. These results highlight the temporal influences on population structure and genetic diversity in C. sonorensis and provide insight into factors effecting genetic variation that may occur in other vector species with fluctuating populations.