Browsing by Author "Watcher-Weatherwax, William"
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Item A rapid diagnostics test and mobile "Lab-in-a-suitcase" platform for detecting Ceratocystis spp. responsible for rapid `Ōhi`a death(2017-04-23) Atkinson, Carter T.; Watcher-Weatherwax, William; Roy, Kylle; Heller, Wade P.; Keith, Lisa M.We describe a field compatible molecular diagnostic test for two new species of Ceratocystis that infect `ōhi`a (Metrosideros polymorpha) and cause the disease commonly known as Rapid `Ōhi`a Death. The diagnostic is based on amplification of a DNA locus within the internal transcribed spacer region that separates fungal 5.8S ribosomal genes. The assay uses forward and reverse primers, recombinase polymerase, and a fluorescent probe that allows isothermal (40oC) amplification and simultaneous quantification of a 115 base pair product with a battery operated fluorometer. DNA extractions are field compatible and can be done by heating wood drill shavings to 100oC in Instagene® solution containing Chelex® resin to bind potential amplification inhibitors. The initial heat treatment is followed by a short bead beating step with steel ball bearings and zirconium beads to release DNA. DNA is subsequently purified with a magnetic bead based extraction method that does not require silica columns or centrifugation. The assay is designed around a portable “lab-in-a-suitcase” platform that includes a portable fluorometer, miniature centrifuge, and heat block that operate off either 120V AC power sources or a 12 volt battery with a portable inverter, a magnetic rack designed for 1.5 ml tubes and magnetic bead DNA purification, pipettes and consumable reagents and tubes. The entire assay from DNA extraction to results can be performed in less than 90 minutes on up to six independent samples plus a positive and negative control. Sensitivity based on suspensions of Ceratocystis endoconidia (spores) that were added to wood shavings and processed under field conditions by Instagene® magnetic bead DNA extraction was up to 163 spores/mg wood for Species A and 55 spores/mg wood for Species B in 95% of replicates as determined by probit analysis. Sensitivity increased 5–10 fold to 19 spores/mg wood for Species A and 9 spores/mg wood for Species B when extractions were performed with a commercial, silica column based DNA purification kit. The test did not cross react with other common fungi that have been isolated from `ōhi`a.Item Effects of climate and land use on diversity, prevalence, and seasonal transmission of avian hematozoa in American Samoa(2016-01-25) Atkinson, Carter; Utzurrum, Ruth; Seamon, Joshua; Schmaedick, Mark; LaPointe, Dennis; Apelgren, Chloe; Egan, Ariel; Watcher-Weatherwax, WilliamThe indigenous forest birds of American Samoa are increasingly threatened by changing patterns of rainfall and temperature that are associated with climate change as well as environmental stressors associated with agricultural and urban development, invasive species, and new introductions of avian diseases and disease vectors. Long term changes in their distribution, diversity, and population sizes could have significant impacts on the ecological integrity of the islands because of their critical role as pollinators and seed dispersers. We documented diversity of vector borne parasites on Tutuila and Ta‘u Islands over a 10-year period to expand earlier observations of Plasmodium, Trypanosoma, and filarial parasites, to provide better parasite identifications, and to create a better baseline for detecting new parasite introductions. We also identified potential mosquito vectors of avian Plasmodium and Trypanosoma, determined whether land clearing and habitat alterations associated with subsistence farming within the National Park of American Samoa can influence parasite prevalence, and determined whether parasite prevalence is correlated with seasonal changes in rainfall, temperature and wind speed. Three taxonomically distinct lineages of Plasmodium were identified from mosquito vectors and forest birds based on partial sequence data from parasite mitochondrial genes. All three have been described from passerine and galliform birds in Australasia. Two lineages, SCEDEN01 and ORW1, had elongate gametocytes and large schizonts that were consistent with species of Plasmodium in the subgenus Giavannolaia, but were taxonomically distinct from known morphological species of Plasmodium based on a Bayesian phylogenetic analysis of a 478 bp region of the parasite cytochrome b gene. Both are candidates for description as new species. The third lineage (GALLUS02) was detected only in mosquito vectors on Tutuila and was similar in cytochrome b sequence to P. juxtanucleare, a pathogenic species of Plasmodium from chickens and other galliform birds from Australasia, Africa, and South America. Plasmodium relictum, the malarial parasite that has had such a devastating impact on Hawaiian forest birds, was not detected. We observed large, striated trypanosomes in avian hosts from both Tutuila and Ta‘u Islands that fell within the same taxonomic clade as T. corvi and T. culicavium based on 18S ribosomal DNA sequence. We also observed sheathed microfilariae with pointed tails that had some morphological similarities to microfilaria from species of Pelecitus, Struthiofilaria and Eulimdana, but identification will require recovery and examination of adult filarial worms from the connective tissue or body cavities of infected birds. We also observed one or more species of haemococcidians (Isospora, synonym = Atoxoplasma) within circulating lymphocytes from multiple avian host species. Overall prevalence of Plasmodium was higher on Ta‘u (22%, 75/341) than Tutuila (9.2%, 27/294), with most infections occurring in Polynesian starlings, Samoan starlings, Wattled honeyeaters, and Cardinal honeyeaters. Prevalence was relatively constant from year to year and between seasons at individual study sites, but varied among study sites, with highest rates of infection in areas with agricultural activity at Faleasao (37.4%, 73/195, Ta‘u Island) and Amalau Valley (9.7%, 21/216, Tutuila Island). Prevalence in more remote areas of the National Park of American Samoa was lower, ranging from 1.4% (2/146) at Laufuti and Luatele on Ta‘u to 7.7% (6/78) at Olo Ridge on Tutuila. Similar trends were evident for infections with Trypanosoma and filarial worms. Overall prevalence was not influenced significantly by warmer, wet (summer) or cooler, dry (winter) season. We detected Plasmodium infections in Culex sitiens and C. quinquefasciatus through either salivary gland and midgut dissections or PCR amplification of parasite cytochrome b genes in pooled or individual samples of mosquitoes that were collected on Tutuila. Pooled or individual Aedes oceanicus, A. polynesiensis, A. tutuilae, A. upolensis, A. nocturnus, Aedes (Finlaya) (mixed pools of A. samoanus, A. oceanicus, A. tutuilae), Aedes (Stegomyia) (mixed pools of A. aegypti, A. upolensis, A. polynesiensis), and C. annulirostris were negative for Plasmodium, but we detected infections with Trypanosoma through midgut and salivary gland dissections in a single C. sitiens from Amalau Valley, Tutuila and three A. oceanicus from Faleasao, Ta‘u. Two of the A. oceanicus from Faleasao amplified successfully with Trypanosoma primers, but sequences were distinctly different from those obtained from avian hosts. We found a strong association between land use and prevalence of mosquito-transmitted parasites on Ta‘u Island with odds of being infected more than 20 times greater in agricultural plots than more remote native forest. This relationship was evident on Tutuila Island but not statistically significant because of the close proximity of study sites and observed movement of birds between native forest and agricultural land. Our data support previous studies that have suggested that Plasmodium and other vector-borne parasites are part of the indigenous parasite fauna in American Samoa. Transmission dynamics appear to be affected by environmental changes associated with land use practices.Item Genetic diversity of Wolbachia endosymbionts in Culex quinquefasciatus from Hawai`i, Midway Atoll, and Samoa(2016-02) Atkinson, Carter T.; Watcher-Weatherwax, William; LaPointe, Dennis A.Incompatible insect techniques are potential methods for controlling Culex quinquefasciatus and avian disease transmission in Hawai‘i without the use of pesticides or genetically modified organisms. The approach is based on naturally occurring sperm-egg incompatibilities within the Culex pipiens complex that are controlled by different strains of the bacterial endosymbiont Wolbachia pipientis (wPip). Incompatibilities can be unidirectional (crosses between males infected with strain A and females infected with strain B are fertile, while reciprocal crosses are not) or bidirectional (reciprocal crosses between sexes with different wPip strains are infertile). The technique depends on release of sufficient numbers of male mosquitoes infected with an incompatible wPip strain to suppress mosquito populations and reduce transmission of introduced avian malaria (Plasmodium relictum) and Avipoxvirus in native forest bird habitats. Both diseases are difficult to manage using more traditional methods based on removal and treatment of larval habitats and coordination of multiple approaches may be needed to control this vector. We characterized the diversity of Wolbachia strains in C. quinquefasciatus from Hawai‘i, Kaua‘i, Midway Atoll, and American Samoa with a variety of genetic markers to identify compatibility groups and their distribution within and between islands. We confirmed the presence of wPip with multilocus sequence typing, tested for local genetic variability using 16 WO prophage genes, and identified similarities to strains from other parts of the world with a transposable element (tr1). We also tested for genetic differences in ankyrin motifs (ank2 and pk1) which have been used to classify wPip strains into five worldwide groups (wPip1–wPip5) that vary in compatibility with each other based on experimental crosses. We found a mixture of both widely distributed and site specific genotypes based on presence or absence of WO prophage and transposable element markers on Hawai‘i Island (Volcano, Pu‘u Wa‘awa‘a, Laupāhoehoe, Kaumana, Kahuku, Nīnole, and Maulua Gulch), Kaua‘i Island (Kawaikōī, Mōhihi, Kalāheo, Lāwa‘i and Hanapepe) and Midway Atoll. Genotypes from American Samoa were unique and formed their own clade. Based on analysis of ankyrin motifs, wPip strains from Hawai‘i, Kaua‘i, and Midway Atoll were most similar to wPip5 strains of Australasian origin. By contrast, Wolbachia strains from Culex quinquefasciatus collected in American Samoa were most similar to wPip3 strains of American origin. We detected a single Culex mosquito from Pu‘u Wa‘awa‘a on Hawai‘i Island that was infected with a unique wPip3 genotype. This discovery, plus a rarefaction analysis of genotypes from Kaua‘i and Hawai‘i Islands suggests that limited sampling may have underestimated diversity of wPip in our study. Mosquitoes infected with wPip5 and wPip3 are bidirectionally compatible with each other based on prior studies, which would support their ability to coexist within the same population on Hawai‘i Island. Available evidence from prior studies suggests that genotype wPip4 from Africa, the Middle East, Europe, and Asia is bidirectionally incompatible with genotype wPip5 and varies in compatibility with genotype wPip3 depending on geographic origin. Since wPip5 appears to be the most common compatibility group in Hawai‘i based on limited sampling, logical next steps are to 1) expand the current survey to include additional islands and localities, 2) infect a laboratory colony of Hawaiian Culex with wPip4 through tetracycline treatment of Hawaiian mosquitoes and backcross with Culex from Europe, North Africa, and the Middle East that are naturally infected with wPip4, 3) conduct cage trials to confirm bidirectional incompatibilities between Hawaiian Culex infected with wPip4 and wPip5, and 4) conduct field trials to evaluate whether release of incompatible males can be applied at small scales to suppress local populations.