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USGS2021

Genetic diversity is considered important but interpreted narrowly in country reports to the Convention on Biological Diversity: Current actions and indicators are insufficient

International agreements such as the Convention on Biological Diversity (CBD) have committed to conserve, and sustainably and equitably use, biodiversity. The CBD is a vital instrument for global conservation because it guides 195 countries and the European Union in setting priorities and allocating resources, and requires regular reporting on progress. However, the CBD and similar policy agreements have often neglected genetic diversity. This is a critical gap because genetic diversity underlies adaptation to environmental change and ecosystem resilience. Here we aim to inform future policy, monitoring, and reporting efforts focused on limiting biodiversity loss by conducting the largest yet evaluation of how Parties to the CBD report on genetic diversity. A large, globally representative sample of 114 CBD National Reports was examined to assess reported actions, progress, values and indicators related to genetic diversity. Although the importance of genetic diversity is recognized by most Parties to the CBD, genetic diversity targets mainly addressed variation within crops and livestock (a small fraction of all species). Reported actions to conserve genetic diversity primarily concerned ex situ facilities and legislation, rather than monitoring and in situ intervention. The most commonly reported status indicators are not well correlated to maintaining genetic diversity. Lastly, few reports mentioned genetic monitoring using DNA data, indigenous use and knowledge of genetic diversity, or development of strategies to conserve genetic diversity. We make several recommendations for the post-2020 CBD Biodiversity Framework, and similar efforts such as IPBES, to improve awareness, assessment, and monitoring of genetic diversity, and facilitate consistent and complete reporting in the future.

Biological Conservation↗

USGS2019

Toxicokinetics of imidacloprid-coated wheat seeds in Japanese quail (Coturnix japonica) and an evaluation of hazard

Birds are potentially exposed to neonicotinoid insecticides by ingestion of coated seeds during crop planting. Adult male Japanese quail were orally dosed with wheat seeds coated with an imidacloprid (IMI) formulation at either 0.9 mg/kg body weight (BW) or 2.7 mg/kg BW (~3 and 9% of IMI LD50 for Japanese quail, respectively) for 1 or 10 days. Quail were euthanized between 1 and 24 h post-exposure to assess toxicokinetics. Analysis revealed rapid absorption (1 h) into blood, and distribution to brain, muscle, kidney and liver. Clearance to below detection limits occurred at both dose levels and exposure durations in all tissues within 24 h. Metabolism was extensive, with 5-OH-IMI and IMI-olefin detected at greater concentrations than IMI in tissues and fecal samples. There was no lethality or overt signs of toxicity at either dose level. Furthermore, no evidence of enhanced expression of mRNA genes associated with hepatic xenobiotic metabolism, oxidative DNA damage or alterations in concentrations of corticosterone and thyroid hormones was observed. Application of the toxicokinetic data was used to predict IMI residue levels in liver with reasonable results for some field exposure and avian mortality events. It would appear that some affected species are either consuming larger quantities of seeds or exhibit differences in ADME or sensitivity than predicted by read-across from these data.

Environmental Science & Technology↗

USGS2015

Microbial infections are associated with embryo mortality in Arctic-nesting geese.

To address the role of bacterial infection in hatching failure of wild geese, we monitored embryo development in a breeding population of Greater white-fronted geese ( Anser albifrons ) on the Arctic Coastal Plain of Alaska. During 2013, we observed mortality of normally developing embryos and collected 36 addled eggs for analysis. We also collected 17 infertile eggs for comparison. Using standard culture methods and gene sequencing to identify bacteria within collected eggs, we identified a potentially novel species of Neisseria in 33 eggs, Macrococcus caseolyticus in 6 eggs, and Streptococcus uberis and Rothia nasimurium in 4 eggs each. We detected seven other bacterial species at lower frequencies. Sequences of the 16S rRNA genes from the Neisseria isolates most closely matched sequences from N. animaloris and N. canis (96 to 97% identity), but phylogenetic analysis suggested substantial genetic differentiation between egg isolates and known Neisseria species. Although definitive sources of the bacteria remain unknown, we detected Neisseria DNA from swabs of eggshells, nest contents, and cloacae of nesting females. To assess the pathogenicity of bacteria identified in contents of addled eggs, we inoculated isolates of Neisseria , Macrococcus , Streptococcus , and Rothia at various concentrations into developing chicken eggs. Seven-day mortality rates varied from 70 to 100%, depending on the bacterial species and inoculation dose. Our results suggest that bacterial infections are a source of embryo mortality in wild geese in the Arctic.

Alaska↗

USGS2013

Worldwide phylogenetic relationship of avian poxviruses

Poxvirus infections have been found in 230 species of wild and domestic birds worldwide in both terrestrial and marine environments. This ubiquity raises the question of how infection has been transmitted and globally dispersed. We present a comprehensive global phylogeny of 111 novel poxvirus isolates in addition to all available sequences from GenBank. Phylogenetic analysis of the Avipoxvirus genus has traditionally relied on one gene region (4b core protein). In this study we expanded the analyses to include a second locus (DNA polymerase gene), allowing for a more robust phylogenetic framework, finer genetic resolution within specific groups, and the detection of potential recombination. Our phylogenetic results reveal several major features of avipoxvirus evolution and ecology and propose an updated avipoxvirus taxonomy, including three novel subclades. The characterization of poxviruses from 57 species of birds in this study extends the current knowledge of their host range and provides the first evidence of the phylogenetic effect of genetic recombination of avipoxviruses. The repeated occurrence of avian family or order-specific grouping within certain clades (e.g., starling poxvirus, falcon poxvirus, raptor poxvirus, etc.) indicates a marked role of host adaptation, while the sharing of poxvirus species within prey-predator systems emphasizes the capacity for cross-species infection and limited host adaptation. Our study provides a broad and comprehensive phylogenetic analysis of the Avipoxvirus genus, an ecologically and environmentally important viral group, to formulate a genome sequencing strategy that will clarify avipoxvirus taxonomy.

Journal of Virology↗

USGS2017

Population genetic structure and gene flow of Adélie penguins (Pygoscelis adeliae) breeding throughout the western Antarctic Peninsula

Adélie penguins ( Pygoscelis adeliae ) are responding to ocean–climate variability throughout the marine ecosystem of the western Antarctic Peninsula (WAP) where some breeding colonies have declined by 80%. Nuclear and mitochondrial DNA (mtDNA) markers were used to understand historical population genetic structure and gene flow given relatively recent and continuing reductions in sea ice habitats and changes in numbers of breeding adults at colonies throughout the WAP. Genetic diversity, spatial genetic structure, genetic signatures of fluctuations in population demography and gene flow were assessed in four regional Adélie penguin colonies. The analyses indicated little genetic structure overall based on bi-parentally inherited microsatellite markers ( F ST =-0.006–0.004). No significant variance was observed in overall haplotype frequency (mtDNA Φ ST =0.017; P =0.112). Some comparisons with Charcot Island were significant, suggestive of female-biased philopatry. Estimates of gene flow based on a two-population coalescent model were asymmetrical from the species’ regional core to its northern range. Breeding Adélie penguins of the WAP are a panmictic population and hold adequate genetic diversity and dispersal capacity to be resilient to environmental change.

Antarctic Science↗

USGS2017

A comparison of honey bee-collected pollen from working agricultural lands using light microscopy and ITS metabarcoding

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010–2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts.

Environmental Entomology↗

USGS2010

Human enteric viruses in groundwater indicate offshore transport of human sewage to coral reefs of the Upper Florida Keys

To address the issue of human sewage reaching corals along the main reef of the Florida Keys, samples were collected from surface water, groundwater and coral [surface mucopolysaccharide layers (SML)] along a 10 km transect near Key Largo, FL. Samples were collected semi-annually between July 2003 and September 2005 and processed for faecal indicator bacteria (faecal coliform bacteria, enterococci and Clostridium perfringens) and human-specific enteric viruses (enterovirus RNA and adenovirus DNA) by (RT)-nested polymerase chain reaction. Faecal indicator bacteria concentrations were generally higher nearshore and in the coral SML. Enteric viruses were evenly distributed across the transect stations. Adenoviruses were detected in 37 of 75 samples collected (49.3%) whereas enteroviruses were only found in 8 of 75 samples (10.7%). Both viruses were detected twice as frequently in coral compared with surface water or groundwater. Offshore, viruses were most likely to be found in groundwater, especially during the wet summer season. These data suggest that polluted groundwater may be moving to the outer reef environment in the Florida Keys.

Florida↗

USGS2021

The presence of denitrifiers in bacterial communities of urban stormwater best management practices (BMPs)

Stormwater best management practices (BMPs) are engineered structures that attempt to mitigate the impacts of stormwater, which can include nitrogen inputs from the surrounding drainage area. The goal of this study was to assess bacterial community composition in different types of stormwater BMP soils to establish whether a particular BMP type harbors more denitrification potential. Soil sampling took place over the summer of 2015 following precipitation events. Soils were sampled from four bioretention facilities, four dry ponds, four surface sand filters, and one dry swale. 16S rRNA gene analysis of extracted DNA and RNA amplicons indicated high bacterial diversity in the soils of all BMP types sampled. An abundance of denitrifiers was also indicated in the extracted DNA using presence/absence of nirS, nirK , and nosZ denitrification genes. BMP soil bacterial communities were impacted by the surrounding soil physiochemistry. Based on the identification of a metabolically-active community of denitrifiers, this study has indicated that denitrification could potentially occur under appropriate conditions in all types of BMP sampled, including surface sand filters that are often viewed as providing low potential for denitrification. The carbon content of incoming stormwater could be providing bacterial communities with denitrification conditions. The findings of this study are especially relevant for land managers in watersheds with legacy nitrogen from former agricultural land use.

Maryland↗

USGS2025

Late Quaternary environmental change in eastern Beringia

Eastern Beringia (Alaska and western Yukon) is an extensive, high-latitude region of North America that remained largely unglaciated throughout the Quaternary. Consequently, its sedimentary deposits preserve long-term environmental records that have intrigued scientists for nearly a century. Recent advances in palaeoecological proxies and dating methods have proved critical in addressing long-standing questions about regional late Quaternary environmental change. At the same time, they have led to new and sometimes controversial hypotheses. This review covers recent discoveries and unresolved questions focused on the period 57,000–10,000 calendar years before C.E. 1950 (cal yr BP). The middle Wisconsin interstadial (57,000–30,000 cal yr BP) was a period of relative warmth in eastern Beringia, compared with the late Wisconsin (30,000–14,000 cal yr BP). Early in the interstadial occasional Picea woodland was present amongst widespread shrub tundra. Palaeoecological, sedimentary and isotopic data indicate that climate was cooler and drier than the Holocene, with high rates of aeolian activity. Megafauna typically associated with the ‘mammoth steppe’ ecosystem (woolly mammoth [ Mammuthus primigenius ], horse [ Equus ] and steppe-bison [ Bison priscus ]) were present in some abundance. The transition towards late Wisconsin cold-stage conditions (35,000–30,000 cal yr BP) coincided with the establishment of the Bering Land Bridge and featured expansion of spatially varied, herbaceous vegetation, sometimes associated with deep active layers. Sedimentary DNA ( seda DNA) and macrofossil evidence show vegetation was not a prairie-like grassland, and the term “steppe-tundra” is a better descriptor. Permafrost pore-ice isotopic (δ 18 O) records suggest a step change in one or more climate drivers ca. 30,000 cal yr BP, by which time steppe-tundra was established across eastern Beringia. It remains uncertain whether Picea survived cold-stage conditions within isolated refugia, or whether it recolonized from south of the Laurentide-Cordilleran ice sheets. Genetic data suggest that Picea probably survived in situ ; however, there is no definitive fossil evidence to support this. The end-Pleistocene transition from steppe-tundra to shrub tundra began ca. 15,000 cal yr BP and took place within decades at local scales. The expansion of woody taxa coincided with rising sea levels, reduced sea-ice extent and an abrupt shift in atmospheric circulation that enhanced precipitation. During this time, Earth's orbital configuration caused high early-summer temperatures and strong seasonality, creating growing conditions very different from today. The vegetation consisted of Salix and Betula shrub tundra with open areas of herbs and graminoids. During the deglacial warming trend, the Younger Dryas oscillation (12,800–11,700 cal yr BP) was variably expressed. It is generally evident in records affected by adjacent oceans but can be absent at sites in continental areas. These past conditions and paleoenvironmental changes have implications for contemporary issues: hypotheses about Pleistocene mammalian extinction; sensitivity of eastern Beringia to major oceanic reorganizations and high-frequency climate variability; the nature of woody plant expansion with climate warming; grazing, hydroclimate and fire as controls over ecosystems; the efficacy of “Pleistocene rewilding” for carbon capture.

Beringia↗

USGS2004

A comparison of genetic ariation between an anadromous steelhead, Oncorhynchus mykiss , population and seven derived populations sequestered in freshwater for 70 years

In 1926 cannery workers from the Wakefield Fisheries Plant at Little Port Walter in Southeast Alaska captured small trout, Oncorhynchus mykiss , from a portion of Sashin Creek populated with a wild steelhead (anadromous O. mykiss ) run. They planted them into Sashin Lake which had been fishless to that time and separated from the lower stream by two large waterfalls that prevented upstream migration of any fish. In 1996 we sampled adult steelhead from the lower creek and juvenile O. mykiss from an intermediate portion of the creek, Sashin Lake, and five lakes that had been stocked with fish from Sashin Lake in 1938. Tissue samples from these eight populations were compared for variation in: microsatellite DNA at 10 loci; D-loop sequences in mitochondrial DNA; and allozymes at 73 loci known to be variable in steelhead. Genetic variability was consistently less in the Sashin Lake population and all derived populations than in the source anadromous population. The cause of this reduction is unknown but it is likely that very few fish survived to reproduce from the initial transplant in 1926. Stockings of 50–85 fish into five other fishless lakes in 1938 from Sashin Lake did not result in a similar dramatic reduction in variability. We discuss potential explanations for the observed patterns of genetic diversity in relation to the maintenance of endangered anadromous O. mykiss populations in freshwater refugia.

Alaska↗

USGS2017

Potential concerns with analytical Methods Used for the detection of Batrachochytrium salamandrivorans from archived DNA of amphibian swab samples, Oregon, USA

Oregon↗

USGS1988

Chemical characterization and mutagenic properties of polycyclic aromatic compounds in sediment from tributaries of the Great Lakes

Sediments from four inshore industrial sites and a reference site in the Great Lakes were extracted with solvents and characterized chemically for polycyclic aromatic compounds (PACs). An aqueous phase and a crude organic extract were obtained. The crude organic extract was further resolved into fractions A-2 (polycyclic aromatic hydrocarbons) and A-3 (nitrogen-containing polycyclic aromatic compounds), which were analyzed for PACs by gas chromatography and gas chromatography-mass spectrometry. The extracts and fractions were tested for mutagenicity in three assays: Ames, rat hepatocyte unscheduled DNA synthesis, and Chinese hamster ovary hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT). Sediments from the industrial sites contained 27 to 363 μg/g total PACs; the reference site, less than 1 μg/g. Qualitative differences in the residue profiles among the sites were attributable to the probable sources of the PACs (petroleum versus combustion). Only one industrial site yielded measurable (0.1 μg/g or more) concentrations of individual nitrogen-containing PACs. In the Ames assay, only the highest doses of the A-2 fractions from two sites approached positive results. Conversely, the crude organic extract and A-2 and A-3 fractions from all sites induced unscheduled DNA synthesis. Crude organic extracts and the A-2 and A-3 fractions from all industrial sites gave well-defined dose-response relations in the CHO/HGPRT assay. We established the presence of chemical mutagens in sediment that could be correlated with neoplasms in fish from many of the sites; however, the mutagenicity of the sediment extracts was not completely related to the degree of contamination by PACs. We also discuss the utility of mutagenicity assays in the evaluation of complex chemical mixtures and recommend the use of a CHO/HGPRT-type assay in which cells are not required to proliferate in the presence of potential interfering chemicals.

Environmental Toxicology and Chemistry↗

USGS2002

Transport of microspheres and indigenous bacteria through a sandy aquifer: Results of natural- and forced-gradient tracer experiments

Transport of indigenous bacteria through sandy aquifer sediments was investigated in forced- and natural-gradient tracer teste. A diverse population of bacteria was collected and concentrated from groundwater at the site, stained with a DNA-specific fluorochrome, and injected back into the aquifer. Included with the injectate were a conservative tracer (Br- or Cl-) and bacteria-sized (0.2-1.3-??m) microspheres having carboxylated, carbonyl, or neutral surfaces. Transport of stained bacteria and all types and size classes of microspheres was evident. In the natural-gradient test, both surface characteristics and size of microspheres affected attenuation. Surface characteristics had the greatest effect upon retardation. Peak break-through of DAPI-stained bacteria (forced-gradient experiment) occurred well in advance of bromide at the more distal sampler. Transport behavior of bacteria was substantially different from that of carboxylated microspheres of comparable size.

Environmental Science & Technology↗

USGS2012

Shifts in identity and activity of methanotrophs in arctic lake sediments in response to temperature changes

Methane (CH 4 ) flux to the atmosphere is mitigated via microbial CH 4 oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH 4 emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH 4 oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH 4 oxidation potential of 37.5 μmol g −1 day −1 in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR of pmoA and of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in 13 C-labeled DNA obtained by SIP demonstrated that the type I methanotrophs Methylobacter , Methylomonas , and Methylosoma dominated carbon acquisition from CH 4 in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH 4 , especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship ( R = 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.

Alaska↗

USGS2016

Effects of Climate and land use on diversity, prevalence, and seasonal transmission of avian hematozoa in American Samoa

The 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.

HI↗

USGS2025

Cell penetrating peptide-mediated delivery of gene-silencing nucleic acids to the invasive common reed Phragmites australis via foliar application

As a popular tool for gene function characterization and gene therapy, RNA interference (RNAi)-based gene silencing has been increasingly explored for potential applications to control invasive species. At least two major hurdles exist when applying this approach to invasive plants: (1) the design and screening of species- and gene-specific biomacromolecules (i.e., gene-silencing agents or GSAs) made of DNA, RNA, or peptides that can suppress the expression of target genes efficiently, and (2) the delivery vehicle needed to penetrate plant cell walls and other physical barriers (e.g., leaf cuticles). In this study, we investigated the cell-penetrating peptide (CPP)-mediated delivery of multiple types of GSAs (e.g., double-stranded RNA (dsRNA), artificial microRNA (amiRNA), and antisense oligonucleotide (ASO)) to knock down a putative phytoene desaturase ( PDS ) gene in the invasive common reed ( Phragmites australis spp. australis ). Both microscopic and quantitative gene expression evidence demonstrated the CPP-mediated internalization of GSA cargos and transient suppression of PDS expression in both treated and systemic leaves up to 7 days post foliar application. Although various GSA combinations and application rates and frequencies were tested, we observed limitations, including low gene-silencing efficiency and a lack of physiological trait alteration, likely owing to low CPP payload capacity and the incomplete characterization of the PDS-coding genes (e.g., the recent discovery of two PDS paralogs) in P. australis . Our work lays a foundation to support further research toward the development of convenient, cost-effective, field-deployable, and environmentally benign gene-silencing technologies for invasive P. australis management.

Mississippi, Ohio↗

USGS2014

Impact of stressors on transmission potential of Renibacterium salmoninarum in Chinook salmon

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD) affecting several species of Pacific salmon. The severity of BKD can range from a chronic infection to overt disease with high mortality as in the case of large losses of adult Chinook salmon ( Oncorhynchus tshawytscha ) in the Great Lakes during late 1980s. The goal of this study was to empirically evaluate how environmental stressors relevant to the Great Lakes impact R. salmoninarum disease progression and bacterial shedding, the latter parameter being a proxy of horizontal transmission. In the first study (Aim 1), we focused on how endogenous host thiamine levels and dietary fatty acids impacted resistance of Chinook salmon to R. salmoninarum . Juvenile fish were fed one of four experimental diets, including a (1) thiamine replete diet formulated with fish oil, (2) thiamine deplete diet formulated with fish oil, (3) thiamine replete diet formulated with soybean oil, and (4) thiamine deplete diet formulated with soybean oil, before being challenged with buffer or R. salmoninarum . We observed significantly higher mortality in the R. salmoninarum infected groups relative to the corresponding mock controls in only the thiamine replete diet groups. We also observed a significant effect of time and diet on kidney bacterial load and bacterial shedding, with a significant trend towards higher shedding and bacterial load in the fish oil – thiamine replete diet group. However, during the course of the study, unexpected mortality occurred in all groups attributed to the myxozoan parasite Ceratomyxa shasta . Since the fish were dually-infected with C. shasta , we evaluated parasite DNA levels (parasitic load) in the kidney of sampled fish. We found that parasite load varied across time points but there was no significant effect of diet. However, parasite load did differ significantly between the mock and R. salmoninarum challenge groups with a trend towards longer persistence of C. shasta DNA in fish dually-infected with R. salmoninarum . Overall, results in Aim 1 indicated: 1) that the experimental diets impacted bacterial but not parasitic infection patterns, 2) that low thiamine levels may reduce the severity of R. salmoninarum infection, and 3) that fish infected with R. salmoninarum may be less able to clear a secondary infection with a parasite. The second study (Aim 2) focused on the role that temperature plays in the progression of BKD from the asymptomatic infected state to a diseased state. Lake Michigan Chinook salmon were infected with R. salmoninarum at a common intermediate water temperature and, at 2 weeks post-infection, were split into three temperature groups (cool, intermediate and warm). Fish held at the cool temperature (8°C) had significantly greater mortality following challenge, significantly higher levels of bacteria in the kidney, and shed significantly greater amounts of bacteria into the water relative to fish held at the intermediate (12°C) and warm (15°C) temperatures. Thus, our results support the hypothesis that, for BKD, warm temperature stress does not contribute to greater disease progression and increased bacterial shedding. Our laboratory results are consistent with field epidemiological observations that BKD mortality in the Great Lakes is commonly associated with declining water temperatures in the fall or when water temperatures begin to increase but are still cool after over-wintering.

Report↗

USGS2003

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida ( C. albicans , C. tropicalis , C. krusei , C. parapsilosis , C. glabrata and C. lusitaniae ) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Applied and Environmental Microbiology↗