Using DNA to Identify the Source of Invasive Mongooses, \u3ci\u3eHerpestes auropunctatus\u3c/i\u3e (Carnivora: Herpestidae) Captured on Kaua‘i, Hawaiian Islands

Two small Indian mongooses (Herpestes auropunctatus) were live-captured in 2012 at separate locations on the Hawaiian Island of Kaua\u27i, which was previously considered to be free of this invasive species. Genotypes from these two individuals were compared to genotypes of H. auropunctatus from the islands of Hawai\u27i (n =39), O\u27ahu (n =91), Maui (n = 39), and Moloka\u27i (n = 19) to determine the island of origin of the Kaua\u27i individuals. Genotypes were generated from each individual using five microsatellite loci. Genetic clustering was estimated by Bayesian inference of spatial clustering of individuals and clustering of groups of individuals. Both analyses separated the samples into three distinct genetic clusters (K = 3). Kaua\u27i individuals consistently formed a single cluster with individuals from O\u27ahu, whereas Hawai\u27i and Maui formed a second cluster, and Moloka\u27i was the third cluster. Thus, we conclude that the origin of two H. auropunctatus captured on Kaua\u27I was O\u27ahu. All three genetic clusters showed evidence of transportation of mongooses between islands, indicating that sampled islands in the archipelago are capable of acting as both donors and receivers of mongooses

Picky eaters are rare: DNA-based blood meal analysis of \u3ci\u3eCulicoides\u3c/i\u3e (Diptera: Ceratopogonidae) species from the United States

Background: Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) have been implicated in the transmission of a number of parasites and highly pathogenic viruses. In North America, the complete transmission cycles of many of these pathogens need further elucidation. One way to increase our knowledge about the evolution and ecology of Culicoides species and the pathogens they transmit is to document the diversity of vertebrate hosts that Culicoides feed upon. Our objective was to identify the diversity of Culicoides hosts in the United States. Results: We sequenced two vertebrate mitochondrial genes (cytochrome c oxidase subunit 1 and cytochrome b) from blood-engorged Culicoides to identify Culicoides species and their blood meals. We detected the mitochondrial DNA of 12 host species from seven different Culicoides species from three states. The majority of the identified blood meals were from the C. variipennis species complex in California. The hosts included both mammals and birds. We documented new host records for some of the Culicoides species collected. The majority of the mammalian hosts were large ungulate species but we also detected a lagomorph and a carnivore. The bird species that were detected included house finch and emu; the latter is evidence that the species in the C. variipennis species complex are not strictly mammalophilic. Conclusions: These results demonstrate that Culicoides will feed on multiple classes of vertebrates and may be more opportunistic in regards to host choice than previously thought. This knowledge can help with identification of susceptible host species, pathogen reservoirs, and new vector species which, in turn, will improve disease outbreak risk assessments

A molecular analysis to assess codling moth \u3ci\u3eCydia pomonella\u3c/i\u3e L. (Lepidoptera: Tortricidae) predation by orchard birds

The codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is a major economic pest in organic apple orchards. Observational methods, prey removal experiments and correlative experiments with exclosures or nest boxes have demonstrated that birds contribute to the removal of this insect pest. However, the majority of research conducted in the last several decades has taken place outside of the United States and methods for studying biological pest control have advanced dramatically and now include molecular techniques. We conducted a proof-of-concept study to test a DNA-based approach to detect C. pomonella prey in the diets of birds occupying organic apple orchards. We tested published Tortricidae primers, polymerase chain reaction (PCR) and sequencing for detection of C. pomonella in avian feces. We also tested the quality of DNA isolated and purified from fecal samples using two DNA extraction kits. Finally, we field-tested this tool to identify the presence or absence of C. pomonella in the laboratory and from field samples. C. pomonella DNA was amplified in less than 1% of field samples and was successfully sequenced in 0.5%. A single species, the brown-headed cowbird Molothrus ater (Boddaert), had fecal samples positive for C. pomonella DNA. While our results do not provide evidence that birds play a strong role in the control of C. pomonella in western Colorado organic apple orchards, the approach we present is a new tool for understanding bird-mediated ecosystem services, avian feeding ecology, and supporting management decisions for sustainable agricultural practices and farmland biodiversity

Integrating early detection with DNA barcoding: species identification of a nonnative monitor lizard (Squamata: Varanidae) carcass in Mississippi, U.S.A.

Early detection of invasive species is critical to increasing the probability of successful management. At the primary stage of an invasion, invasive species are easier to control as the population is likely represented by just a few individuals. Detection of these first few individuals can be challenging, particularly if they are cryptic or otherwise characterized by low detectability. The engagement of members of the public may be critical to early detection as there are far more citizens on the landscape than trained biologists. However, it can be difficult to assess the credibility of public reporting, especially when a diagnostic digital image or a physical specimen in good condition are lacking. DNA barcoding can be used for verification when morphological identification of a specimen is not possible or uncertain (i.e., degraded or partial specimen). DNA barcoding relies on obtaining a DNA sequence from a relatively small fragment of mitochondrial DNA and comparing it to a database of sequences containing a variety of expertly identified species. Herein we report the successful identification of a degraded specimen of a non-native, potentially invasive reptile species (Varanus niloticus) via DNA barcoding, after discovery and reporting by a member of the public

The 17th Rocky Mountain Virology Association Meeting

Since 2000, scientists and students from the greater Rocky Mountain region, along with invited speakers, both national and international, have gathered at the Mountain Campus of Colorado State University to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of virologists and prionologists through formal presentations and informal discussions concerning science, grantsmanship and network development. This year, approximately 100 people attended the 17th annual Rocky Mountain Virology Association meeting, that began with a keynote presentation, and featured 29 oral and 35 poster presentations covering RNA and DNA viruses, prions, virus-host interactions and guides to successful mentorship. Since the keynote address focused on the structure and function of Zika and related flaviviruses, a special session was held to discuss RNA control. The secluded meeting at the foot of the Colorado Rocky Mountains gave ample time for in-depth discussions amid the peak of fall colors in the aspen groves while the random bear provided excitement. On behalf of the Rocky Mountain Virology Association, this report summarizes the \u3e50 reports

Temporal and Spatial Blood Feeding Patterns of Urban Mosquitoes In the San Juan Metropolitan Area, Puerto Rico

Urban ecosystems are a patchwork of habitats that host a broad diversity of animal species. Insects comprise a large portion of urban biodiversity which includes many pest species, including those that transmit pathogens. Mosquitoes (Diptera: Culicidae) inhabit urban environments and rely on sympatric vertebrate species to complete their life cycles, and in this process transmit pathogens to animals and humans. Given that mosquitoes feed upon vertebrates, they can also act as efficient samplers that facilitate detection of vertebrate species that utilize urban ecosystems. In this study, we analyzed DNA extracted from mosquito blood meals collected temporally in multiple neighborhoods of the San Juan Metropolitan Area, Puerto Rico to evaluate the presence of vertebrate fauna. DNA was collected from 604 individual mosquitoes that represented two common urban species, Culex quinquefasciatus (n = 586) and Aedes aegypti (n = 18). Culex quinquefasciatus fed on 17 avian taxa (81.2% of blood meals), seven mammalian taxa (17.9%), and one reptilian taxon (0.85%). Domestic chickens dominated these blood meals both temporally and spatially, and no statistically significant shift from birds to mammals was detected. Aedes aegypti blood meals were from a less diverse group, with two avian taxa (11.1%) and three mammalian taxa (88.9%) identified. The blood meals we identified provided a snapshot of the vertebrate community in the San Juan Metropolitan Area and have potential implications for vector-borne pathogen transmission

DNA Persistence in Predator Saliva from Multiple Species and Methods for Optimal Recovery from Depredated Carcasses

Molecular forensics is an important component of wildlife research and management. Using DNA from noninvasive samples collected at predation sites, we can identify predator species and obtain individual genotypes, improving our understanding of predator–prey dynamics and impacts of predators on livestock and endangered species. To improve sample collection strategies, we tested two sample collection methods and estimated degradation rates of predator DNA on the carcasses of multiple prey species. We fed carcasses of calves (Bos taurus) and lambs (Ovis aires) to three captive predator species: wolves (Canis lupus), coyotes (C. latrans), and mountain lions (Puma concolor). We swabbed the carcass in the field, as well as removed a piece of hide from the carcasses and then swabbed it in the laboratory. We swabbed all tissue samples through time and attempted to identify the predator involved in the depredation using salivary DNA. We found the most successful approach for yielding viable salivary DNA was removing hide from the prey and swabbing it in the laboratory. As expected, genotyping error increased through time and our ability to obtain complete genotypes decreased over time, the latter falling below 50% after 24 h. We provide guidelines for sampling salivary DNA from tissues of depredated carcasses for maximum probability of detection

Detecting an Elusive Invasive Species: A Diagnostic PCR to Detect Burmese Python in Florida Waters and an Assessment of Persistence of Environmental DNA

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles

Tissue tropisms opt for transmissible reassortants during avian and swine influenza A virus co-infection in swine

Genetic reassortment between influenza A viruses (IAVs) facilitate emergence of pandemic strains, and swine are proposed as a “mixing vessel” for generating reassortants of avian and mammalian IAVs that could be of risk to mammals, including humans. However, how a transmissible reassortant emerges in swine are not well understood. Genomic analyses of 571 isolates recovered from nasal wash samples and respiratory tract tissues of a group of co-housed pigs (influenza-seronegative, avian H1N1 IAV–infected, and swine H3N2 IAV– infected pigs) identified 30 distinct genotypes of reassortants. Viruses recovered from lower respiratory tract tissues had the largest genomic diversity, and those recovered from turbinates and nasal wash fluids had the least. Reassortants from lower respiratory tracts had the largest variations in growth kinetics in respiratory tract epithelial cells, and the cold temperature in swine nasal cells seemed to select the type of reassortant viruses shed by the pigs. One reassortant in nasal wash samples was consistently identified in upper, middle, and lower respiratory tract tissues, and it was confirmed to be transmitted efficiently between pigs. Study findings suggest that, during mixed infections of avian and swine IAVs, genetic reassortments are likely to occur in the lower respiratory track, and tissue tropism is an important factor selecting for a transmissible reassortant

Beneficial and Detrimental Effects of Plasmin(ogen) during Infection and Sepsis in Mice

Plasmin has been proposed to be an important mediator during inflammation/infection. In this study, by using mice lacking genes for plasminogen, tissue-type plasminogen activator (tPA), and urokinase-type PA (uPA), we have investigated the functional roles of active plasmin in infection and sepsis. Two models were used: an infection model by intravenous injection of 1×107 CFU of S. aureus, and a sepsis model by intravenous injection of 1.6×108 CFU of S. aureus. We found that in the infection model, wild-type (WT) mice showed significantly higher survival rates than plasminogen-deficient (plg-/-) mice. However, in the sepsis model, plg-/- or tPA-/-/uPA-/- mice showed the highest survival rate whereas WT and tPA+/-/uPA+/- mice showed the lowest survival rate, and plg+/-, tPA-/-, and uPA-/- mice had an intermediate survival rate. These results indicate that the levels of active plasmin are critical in determining the survival rate in the sepsis, partly through high levels of inflammatory cytokines and enhanced STAT3 activation. We conclude that plasmin is beneficial in infection but promotes the production of inflammatory cytokines in sepsis that may cause tissue destruction, diminished neutrophil function, and an impaired capacity to kill bacteria which eventually causes death of these mice