Browsing by Author "Atkinson, Carter T."
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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 Changes in prevalence of avian malaria on the Alaka`i Plateau, Kaua`i, 1997-2007.(2010-06) Atkinson, Carter T.; Utzurrum, Ruth B.We determined prevalence of malarial infections in samples of native and non-native forest birds that were sampled at three locations on the Alaka`i Plateau between 1994-1997 and again between 2007-2009. The three sites spanned the elevational range of the plateau and were located at Kawaikōī Stream (1100 m), the upper drainage of Mōhihi Stream (1250 m) and the vicinity of Halepa`akai Stream near Sincock’s Bog (1350 m). We detected a dramatic and significant increase in prevalence of avian malaria both at the lower (Kawaikōī) and upper (Halepa`akai) ends of the Alaka`i Plateau during the past decade. Overall prevalence of infection increased threefold from 11% to 30% at Kawaikōī Stream and tenfold from 2% to 20% at Halepa`akai Stream. Much of this increase is likely a result of local transmission, since two sedentary native species, `Elepaio and Kaua`i `Amakihi, have experienced some of the largest increases in prevalence. Curiously, prevalence has not changed significantly at Mōhihi Stream and remains at approximately 10%. We also detected avian trypanosomes (Trypanosoma sp.) in both recent and historic blood samples from Nutmeg Mannikins (Lonchura punctulata) that were captured on the plateau. This is the first report of this mosquito-transmitted blood protozoan from the Hawaiian Islands and evidence indicates that it has been a previously undetected blood parasite in the islands for at least 15 years and likely longer. We found no evidence to indicate that the parasite has spread to native Hawaiian forest birds, but our sample sizes are limited. While our study was not designed to detect the specific factors responsible for the changes in prevalence of malaria at lower and upper portions of the plateau, the results are consistent with predicted increases in prevalence that might be expected in a warming climate and clearly show that environmental conditions necessary to support transmission of malaria now exist throughout major portions of the Alaka`i Plateau. Additional field work to identify larval habitat for mosquitoes, adult mosquito distribution and density, and the relative role of human activity, feral ungulates, and interactions between changing climatic conditions and the deeply dissected topography of the plateau may help to identify why some areas have experienced significant increases in malarial prevalence.Item Changes in the prevalence of avian disease and mosquito vectors at Hakalau Forest National Wildlife Refuge: a 14-year perspective and assessment of future risk(2016-03-10) LaPointe, Dennis A.; Gaudioso-Levita, Jacqueline M.; Atkinson, Carter T.; Egan, Ariel; Hayes, KathleenThroughout the main Hawaiian Islands, introduced mosquito-borne disease has had, and continues to have, a profound impact on the distributions and abundance of native Hawaiian forest birds. Populations of remaining native forest birds are largely restricted to high elevation forests where mean temperatures are marginal for vector and parasite development and limited availability of larval mosquito habitat constrains mosquito populations and disease transmission. Hakalau Forest National Wildlife Refuge (HFNWR) was established for the preservation of endemic avifauna in 1985. Since its creation, native bird communities there have remained intact and most species populations are stable or increasing. However, avian malaria had been detected at HFNWR in the past and, in light of documented climate change, new concerns have been raised regarding the long-term fate of the refuge’s forest birds. To examine the possible changes in avian malaria transmission at HFNWR we sampled forest birds for blood parasites, trapped adult mosquitoes and surveyed larval mosquito habitat at three sites during 2012 and compared our results with similar data collected between 1998 and 1999. We tested blood samples by polymerase chain reaction (PCR), immunoblotting, and microscopy to determine prevalence of acute and chronic infection and used attractive gravid traps to sample the vector mosquito Culex quinquefasciatus. Our study documented spatial trends and temporal changes in the prevalence of avian malaria, mosquito presence, larval mosquito habitat and feral pig activity at HFNWR. We found evidence of local transmission in high elevation forests, a general pattern of increasing prevalence at lower elevations and along a South to North gradient and a two-fold decrease in the prevalence of avian malaria in the intervening 14 years. Despite considerable effort, we were unable to detect larval C. quinquefasciatus and captured only one adult indicating that the vector of avian malaria has a very limited presence at HFNWR. We did, however, document the establishment of another invasive mosquito, Aedes japonicus japonicus, and its occurrence in tree fern cavities and rock pools as larval habitat in the lower forests of HFNWR. We suggest that interspecific competition by A. j. japonicus and predation by a suite of native predators may provide biotic resistance to the establishment of permanent C. quinquefasciatus populations. While current predictions of climate change in the Hawaiian Islands include a gradual warming and enhanced transmission by mid-century, the current cooling trend recorded at high elevation HFNWR illustrates the importance of monitoring to document fine scale temporal and site specific changes in prevalence. Long term changes in precipitation may have a more profound effect on local transmission of malaria than temperature and we may have already seen some potential impacts of an extended drought at HFNWR with a decrease in feral pig activity and pig-associated larval mosquito habitat and increases in stream-associated larval mosquito habitat.Item Economical environmental sampler designs for detecting airborne spread of fungi responsible for Rapid `Ōhi`a Death(2019-02-10) Atkinson, Carter T.; Roy, Kylle; Granthon, CarolinaWe designed two new samplers for monitoring airborne particulates that rely on either natural wind currents (Passive Environmental Sampler) or a battery-operated fan (Active Environmental Sampler). Both samplers are significantly less expensive than commercial devices such as Rotorod® and Burkard Samplers that are used in the agricultural and health science industries. They are economical enough to be deployed in large numbers across broad landscapes. We evaluated their use for detecting airborne spread of ambrosia beetle frass that may contain infective spores of the fungi (Ceratocystis lukuohia and C. huliohia) that are responsible for Rapid `Ōhi`a Death (ROD), a newly documented pathosystem on Hawai`i Island. We compared performance of the new samplers to Rotorod® Model 20 Samplers by releasing synthetic polyethylene spheres (12–160 µm in diameter) and also Xyleborus spp. frass known to contain C. lukuohia and C. huliohia propagules under controlled laboratory and field conditions. Overall, the Active Environmental Sampler proved to be 3–4 times more effective in capturing polyethylene spheres and 2–3 times more effective in capturing frass than either the Passive or Rotorod® Samplers. Significant differences between the Passive and Rotorod® Samplers were not detected. For the frass release experiment, C. lukuohia DNA was detected once by qPCR in an Active Environmental Sampler and C. huliohia DNA was detected during two different trials, once with an Active Environmental Sampler and once with a Passive Environmental Sampler. No detections were made with Rotorod® Samplers. Both Active and Passive Samplers were used in the field for detection of airborne dispersal of C. lukuohia and C. huliohia at Orchidlands Estates in the Puna District of Hawai`i Island. We found that airborne dispersal of potentially infective beetle frass was uncommon over short distances with qPCR detections in up to 10% of weekly sampler collections.Item Effectiveness of rapid 'ōhi'a death management strategies at a focal disease outbreak on Hawai'i Island(2021-02-03) Roy, Kylle; Granthon, Carolina; Peck, Robert W.; Atkinson, Carter T.The ongoing spread of rapid ‘ōhi‘a death (ROD) in the Hawaiian Islands threatens the long-term sustainability of ‘ōhi‘a lehua (Metrosideros polymorpha) forests throughout the state. First identified in the Puna district of Hawai‘i Island in 2014, the disease caused by the novel fungi Ceratocystis lukuohia and Ceratocystis huliohia has now spread island-wide and was recently detected on Kaua‘i, O‘ahu, and Maui. The leading hypothesis for the spread of ROD is through airborne ambrosia beetle frass particles that contain viable Ceratocystis propagules, thus management efforts focus on containing this frass. At the time of this study (2017–2018), the Waipunalei site was the northernmost outbreak of ROD on Hawai‘i Island. The focal nature of the outbreak and accessibility of the location provided the opportunity to monitor the effectiveness of two types of proposed management methods to reduce the airborne spread of potentially infective ambrosia beetle frass: tree felling and insecticide treatments. We placed 23 passive environmental samplers (PES), which monitored for airborne frass and wood particles containing C. lukuohia and C. huliohia in a grid that spanned the outbreak area over 22 weeks. Cross-vane panel traps with 1:1 methanol:ethanol lures were attached to nine of the PES to document wood-boring ambrosia and cerambycid beetle populations during the latter three months of the study. Monitoring with PES began three weeks before management and continued for one month after the last infected trees were felled. Glass microscope slides from the 23 PES were examined for airborne ambrosia beetle frass and wood particles by microscopy. DNA was extracted from the slides and tested by qPCR (quantitative polymerase chain reaction) for C. lukuohia and C. huliohia. We also investigated the correlation of beetle gallery counts with tree height and tested the efficacy of Bifen I/T insecticide (active ingredient: bifenthrin 7.9%) for preventing beetle attacks on the cut surface of ‘ōhi‘a bolts (tree stem sections). Beetle trapping data revealed that the area supports a diverse community of wood-boring beetles, some of which likely attack ‘ōhi‘a and may facilitate the spread of ROD. The number of beetle galleries on felled ‘ōhi‘a trees decreased linearly as tree height increased. We also observed significantly fewer beetle attacks on Bifen I/T treated ‘ōhi‘a bolts than non-treated bolts, but gallery formation nearly ceased in both treated and control bolts by week three. Ceratocystis lukuohia DNA was detected twenty-six times and C. huliohia was detected five times in the PES throughout this study. DNA detections were correlated to frass and wood counts, and the number of felled trees were correlated to wood particle counts but not frass counts. Both the timing and distribution of detections across the sampling grid indicate that tree felling may have reduced airborne detections of Ceratocystis DNA soon after tree felling was completed. A subsequent increase in detections after tree felling ceased may indicate that incomplete removal of infected trees and the appearance of new infections in previously asymptomatic trees could have allowed airborne detections of potentially infectious fungal propagules to once again increase.Item Efficacy of a commercial cararypox vaccine for protecting Hawai`i `Amakihi from field isolates of avipoxvirus.(2010-09) Atkinson, Carter T.; Wiegand, Kimberly C.; Triglia, Dennis A.; Jarvi, Susan I.At least three variants of avian pox virus are present in Hawai’i - Fowlpox from domestic poultry and a group of genetically distinct viruses that cluster within two clades (Pox Variant 1 and Pox Variant 2) that are most similar to Canarypox based on DNA sequence of the virus 4b core protein gene. We tested whether Hawai’i ‘Amakihi can be protected from wild virus isolates with an attenuated live Canarypox vaccine that is closely related to isolates that cluster within clade 1 (Pox Variant 1) based on sequence of the attenuated Canarypox virus 4b core protein. Thirty-one (31) Hawai`i ‘Amakihi (Hemignathus virens) with no prior physical evidence of pox infection were collected on Mauna Kea from xeric, high elevation habitats with low pox prevalence and randomly divided into two groups. One group of 16 was vaccinated with Poximmune C® while the other group received a sham vaccination with virus diluent. Four of 15 (27%) vaccinated birds developed potentially life-threatening disseminated lesions or lesions of unusually long duration, while one bird never developed a vaccine-associated lesion or “take”. After vaccine-associated lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox, a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 1 (Pox Variant 1) or a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 2 (Pox Variant 2). Similarly, three random groups of five unvaccinated ‘Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated ‘Amakihi challenged with Fowlpox had transient infections with no clinical signs of infection. Mortality in vaccinated ‘Amakihi that were challenged with Pox Variant 1 and Pox Variant 2 ranged from 0% (0/5) for Pox Variant 1 to 60% (3/5) for Pox Variant 2. Mortality in unvaccinated ‘Amakihi ranged from 40% (2/5) for Pox Variant 1 to 100% (5/5) for Pox Variant 2. While the vaccine provided some protection against Pox Variant 1, serious side effects and low efficacy against Pox Variant 2 make it risky to use in captive or wild honeycreepers.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.Item Population genetic structure of rare and endangered plants using molecular markers(2013-02) Raji, Jennifer; Atkinson, Carter T.This study was initiated to assess the levels of genetic diversity and differentiation in the remaining populations of Phyllostegia stachyoides and Melicope zahlbruckneri in Hawai`i Volcanoes National Park and determine the extent of gene flow to identify genetically distinct individuals or groups for conservation purposes. Thirty-six Amplified Fragment Length Polymorphic (AFLP) primer combinations generated a total of 3,242 polymorphic deoxyribonucleic acid (DNA) fragments in the P. stachyoides population with a percentage of polymorphic bands (PPB) ranging from 39.3 to 65.7% and 2,780 for the M. zahlbruckneri population with a PPB of 18.8 to 64.6%. Population differentiation (Fst) of AFLP loci between subpopulations of P. stachyoides was low (0.043) across populations. Analysis of molecular variance of P. stachyoides showed that 4% of the observed genetic differentiation occurred between populations in different kīpuka and 96% when individuals were pooled from all kīpuka. Moderate genetic diversity was detected within the M. zahlbruckneri population. Bayesian and multivariate analyses both classified the P. stachyoides and M. zahlbruckneri populations into genetic groups with considerable sub-structuring detected in the P. stachyoides population. The proportion of genetic differentiation among populations explained by geographical distance was estimated by Mantel tests. No spatial correlation was found between genetic and geographic distances in both populations. Finally, a moderate but significant gene flow that could be attributed to insect or bird-mediated dispersal of pollen across the different kīpuka was observed. The results of this study highlight the utility of a multi-allelic DNA-based marker in screening a large number of polymorphic loci in small and closely related endangered populations and revealed the presence of genetically unique groups of individuals in both M. zahlbruckneri and P. stachyoides populations. Based on these findings, approaches that can assist conservation efforts of these species are proposed.Item Use of whole blood samples preserved in DNA lysis buffer for serological detection of avian malaria in Hawaiian forest birds(2020-06-08) Atkinson, Carter T.Two buffers that are commonly used to preserve whole blood for polymerase chain reaction (PCR) diagnostics, tris-ethylenediaminetetraacetic acid (TEN) and tris-sodium dodecyl sulfate-ethylenediaminetetraacetic acid (SDS-EDTA), were evaluated to determine whether they can also be used to preserve blood for serological studies to detect antibodies to avian malaria. TEN buffer had no effect on antibody binding as measured by enzyme-linked immunosorbent assay (ELISA) or Western blotting. By contrast, the SDS-EDTA buffer completely abolished all antibody binding. Efforts to restore binding by dialysis and concentration of the samples were not successful. Addition of sodium dodecyl sulfate (SDS) and Proteinase K to samples preserved in TEN buffer was also evaluated, because this treatment is sometimes used to render samples non-infectious prior to shipping. This treatment abolished all antibody binding by both ELISA and Western blotting. TEN buffer appears to be good for preserving whole blood samples for both PCR and serological studies, making it possible to simultaneously preserve blood samples for both PCR and serological diagnostic tests in a single tube.