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Comparison of microscopy and metabarcoding to identify pollen used by the critically endangered rusty patched bumble bee, Bombus affinis

Taxonomic analysis of pollen collected by bees can provide insights into their host plant use, providing information about the plant species selected for targeted conservation strategies. The two main identification approaches used are morphological analysis of pollen samples affixed to microscope slides (i.e., microscopic palynology) and molecular analysis of samples. Both methods are widely used for freshly collected materials and have been compared in multiple studies, yet their application to archived samples remains to be explored. Archived samples may be particularly useful for the study of rare or protected species, particularly when historical foraging patterns are uncertain. We used both methods to analyse pollen collected by the endangered rusty patched bumble bee species, Bombus affinis Cresson, applied to museum-archived specimens. Pollen samples were removed from the corbiculae of bumble bees originally collected in Michigan between 1914 and 1974. Samples included 24 rusty patched bumble bees each with large pollen loads on both corbiculae, allowing for pollen from the same bee to be analysed using both methods. DNA metabarcoding detected more plant taxa than light microscopy, and DNA barcoding also had higher taxonomic resolution when compared to taxa determined using light microscopy. In many instances, pollen could only be confidently identified to tribe or family with light microscopy. Discrepancy between methods decreased when taxa identified via DNA metabarcoding were binned into ecologically relevant groups corresponding to those identified using light microscopy. Although binning demonstrated smaller within-method variance, there was still minimal correspondence between the two methods. Results indicate there are benefits and biases unique to each method and highlight the utility of binning taxonomic results to morphological or ecological groupings.

Insect Conservation and Diversity

After-hatch and hatch year Buff-breasted Sandpipers (Calidris subruficollis) can be sexed accurately using morphometric measures

Determining the sex of birds quickly in the field can help in studies of behavior and distribution, and when selecting particular sexes for deploying tracking devices or collecting samples. However, discerning males from females is difficult in species that are plumage monomorphic and have overlapping sexual-size dimorphism, as in Buff-breasted Sandpipers Calidris subruficollis . We developed three discriminant functions to sex Buff-breasted Sandpipers based on measurements of live birds captured in Brazil whose sex was confirmed with molecular techniques. We validated these discriminant functions using morphometric measures from other independent samples of known-sex live birds from wintering (Brazil), migration (Texas), and breeding (Alaska) sites. Discriminant functions derived from birds captured in Brazil accurately sexed ≥88% of the validation sample from Brazil, Texas, and Alaska. Errors in classification occurred among males on the wintering (0–5%) and breeding (8–12%) grounds, and females during migration (0–11%). Discriminant functions worked well because of the substantial sexual size dimorphism present in the species, with male traits being in general 5.2–10.4% larger than female traits. The size of morphological traits did not vary by age (after controlling for sex) for birds sampled on the wintering grounds and during migration. Our results indicate that discriminant functions can be used to sex after-hatch year (AHY) Buff-breasted Sandpipers throughout their range, and for hatch year (HY) birds during their first southbound migration and winter. Being able to accurately sex both AHY and HY birds using only morphological measurements will improve studies of the ecology and population structure of this species and enhance the application of conservation measures.

Wader Study

Chapel Hill bisphenol A expert panel consensus statement: Integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure

This document is a summary statement of the outcome from the meeting: “ Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health ” sponsored by both the NIEHS and NIDCR at NIH/DHHS, as well as the US-EPA and Commonweal on the estrogenic environmental chemical bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane; CAS# 80-05-7). The meeting was held in Chapel Hill, NC, 28–30 November 2006 due to concerns about the potential for a relationship between BPA and negative trends in human health that have occurred in recent decades. Examples include increases in abnormal penile/urethra development in males, early sexual maturation in females, an increase in neurobehavioral problems such as attention deficit hyperactivity disorder (ADHD) and autism, an increase in childhood and adult obesity and type 2 diabetes, a regional decrease in sperm count, and an increase in hormonally mediated cancers, such as prostate and breast cancers. Concern has been elevated by published studies reporting a relationship between treatment with “low doses” of BPA and many of theses negative health outcomes in experimental studies in laboratory animals as well as in vitro studies identifying plausible molecular mechanisms that could mediate such effects. Importantly, much evidence suggests that these adverse effects are occurring in animals within the range of exposure to BPA of the typical human living in a developed country, where virtually everyone has measurable blood, tissue and urine levels of BPA that exceed the levels produced by doses used in the “low dose” animal experiments.

Reproductive Toxicology

Complex immune responses and molecular reactions to pathogens and disease in a desert reptile (Gopherus agassizii)

Immune function plays an important role in an animal's defense against infectious disease. In reptiles, immune responses may be complex and counterintuitive, and diagnostic tools used to identify infection, such as induced antibody responses are limited. Recent studies using gene transcription profiling in tortoises have proven useful in identifying immune responses to various intrinsic and extrinsic stressors. As part of a larger experiment with Mojave desert tortoises ( Gopherus agassizii ), we facilitated the transmission of the pathogenic bacteria, Mycoplasma agassizii (Myag), to naïve adults and measured innate and induced immune reactions over time. Specifically, we evaluated clinical condition, presence of Myag in the nasal/oral cavity, induced antibody responses specific to Myag, and measured molecular reactions (gene transcript profiles) in 15 captive tortoises classified as naïve, exposed, or infected and 14 wild tortoises for comparison. Myag was confirmed inside the nasal/oral cavity in exposed tortoises within 30–60 days of introduction to infected animals, yet we did not detect Myag specific induced antibody responses in these individuals until 420–595 days post exposure. Surprisingly, we found no overall differences in the gene transcript profiles between our experimental treatment groups throughout this study. This work highlights the complexities in assessing immune function and diagnosing pathogen related infections in tortoises and other reptiles.

Ecology and Evolution

Molecular characterization of Bathymodiolus mussels and gill symbionts associated with chemosynthetic habitats from the U.S. Atlantic margin

Mussels of the genus Bathymodiolus are among the most widespread colonizers of hydrothermal vent and cold seep environments, sustained by endosymbiosis with chemosynthetic bacteria. Presumed species of Bathymodiolus are abundant at newly discovered cold seeps on the Mid-Atlantic continental slope, however morphological taxonomy is challenging, and their phylogenetic affinities remain unestablished. Here we used mitochondrial sequence to classify species found at three seep sites (Baltimore Canyon seep (BCS; ~400m); Norfolk Canyon seep (NCS; ~1520m); and Chincoteague Island seep (CTS; ~1000m)). Mitochondrial COI (N = 162) and ND4 (N = 39) data suggest that Bathymodiolus childressi predominates at these sites, although single B. mauritanicus and B. heckerae individuals were detected. As previous work had suggested that methanotrophic and thiotrophic interactions can both occur at a site, and within an individual mussel, we investigated the symbiont communities in gill tissues of a subset of mussels from BCS and NCS. We constructed metabarcode libraries with four different primer sets spanning the 16S gene. A methanotrophic phylotype dominated all gill microbial samples from BCS, but sulfur-oxidizing Campylobacterota were represented by a notable minority of sequences from NCS. The methanotroph phylotype shared a clade with globally distributed Bathymodiolus spp. symbionts from methane seeps and hydrothermal vents. Two distinct Campylobacterota phylotypes were prevalent in NCS samples, one of which shares a clade with Campylobacterota associated with B. childressi from the Gulf of Mexico and the other with Campylobacterota associated with other deep-sea fauna. Variation in chemosynthetic symbiont communities among sites and individuals has important ecological and geochemical implications and suggests shifting reliance on methanotrophy. Continued characterization of symbionts from cold seeps will provide a greater understanding of the ecology of these unique environments as well and their geochemical footprint in elemental cycling and energy flux.

Delaware, Georgia, Maryland, New Jersey, North Car

A review of the key genetic tools to assist imperiled species conservation: analyzing West Indian manatee populations

Managers faced with decisions on threatened and endangered wildlife populations often are lacking detailed information about the species of concern. Integration of genetic applications will provide management teams with a better ability to assess and monitor recovery efforts on imperiled species. The field of molecular biology continues to progress rapidly and many tools are currently available. Presently, little guidance is available to assist researchers and managers with the appropriate selection of genetic tools to study the status of wild manatee populations. We discuss several genetic tools currently employed in the application of conservation genetics, and address the utility of using these tools to determine population status to aid in conservation efforts. As an example, special emphasis is focused on the endangered West Indian manatee (Order Sirenia). All four extant species of sirenians are imperiled throughout their range, predominately due to anthropogenic sources; therefore, the need for genetic information on their population status is direly needed.

Journal of Marine Animals and Their Ecology

A new tool for studying waterfowl immune and metabolic responses: Molecular level analysis using kinome profiling

Here, we describe the design of an Anas‐ specific kinome peptide array that can be used to study the immunometabolic responses of mallard and American black duck to pathogens, contaminants, and environmental stress. The peptide arrays contain 2,642 unique phosphorylate‐able peptide sequences representing 1,900 proteins. These proteins cover a wide array of metabolic and immunological processes, and 758 Gene Ontology Biological processes are statistically significantly represented on the duck peptide array of those 164 contain the term “metabolic” and 25 “immune.” In addition, we conducted a comparison of mallard to American black duck at a genetic and proteomic level. Our results show a significant genomic and proteomic overlap between these two duck species, so that we have designed a cross‐reactive peptide array capable of studying both species. This is the first reported development of a wildlife species‐specific kinome peptide array.

Ecology and Evolution

Investigations of novel unsaturated bile salts of male sea lamprey as potential chemical cues

Sulfated bile salts function as chemical cues that coordinate reproduction in sea lamprey, Petromyzon marinus . 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3kPZS) is the most abundant known bile salt released by sexually mature male sea lampreys and attracts ovulated females. However, previous studies showed that the male-produced pheromone consists of unidentified components in addition to 3kPZS. Here, analysis of water conditioned with mature male sea lampreys indicated the presence of 4 oxidized, unsaturated compounds with molecular weights of 466 Da, 468 Da, and 2 of 470 Da. These compounds were not detectable in water conditioned with immature male sea lampreys. By using mass spectrometry, 4 A-ring unsaturated sulfated bile salts were tentatively identified from male washings as 2 4-ene, a 1-ene, and a 1,4-diene analogs. These were synthesized to determine if they attracted ovulated female sea lampreys to spawning nests in natural streams. One of the novel synthetic bile salts, 3 keto-1-ene PZS, attracted ovulated females to the point of application at a concentration of 10 -12 M. This study reveals the structural diversity of bile salts in sea lamprey, some of which have been demonstrated to be pheromonal cues.

Journal of Chemical Ecology

Methods for measuring denitrification: Diverse approaches to a difficult problem

Denitrification, the reduction of the nitrogen (N) oxides, nitrate (NO 3 − ) and nitrite (NO 2 − ), to the gases nitric oxide (NO), nitrous oxide (N 2 O), and dinitrogen (N 2 ), is important to primary production, water quality, and the chemistry and physics of the atmosphere at ecosystem, landscape, regional, and global scales. Unfortunately, this process is very difficult to measure, and existing methods are problematic for different reasons in different places at different times. In this paper, we review the major approaches that have been taken to measure denitrification in terrestrial and aquatic environments and discuss the strengths, weaknesses, and future prospects for the different methods. Methodological approaches covered include (1) acetylene-based methods, (2) 15 N tracers, (3) direct N 2 quantification, (4) N 2 :Ar ratio quantification, (5) mass balance approaches, (6) stoichiometric approaches, (7) methods based on stable isotopes, (8) in situ gradients with atmospheric environmental tracers, and (9) molecular approaches. Our review makes it clear that the prospects for improved quantification of denitrification vary greatly in different environments and at different scales. While current methodology allows for the production of accurate estimates of denitrification at scales relevant to water and air quality and ecosystem fertility questions in some systems (e.g., aquatic sediments, well-defined aquifers), methodology for other systems, especially upland terrestrial areas, still needs development. Comparison of mass balance and stoichiometric approaches that constrain estimates of denitrification at large scales with point measurements (made using multiple methods), in multiple systems, is likely to propel more improvement in denitrification methods over the next few years.

Ecological Applications

Anticoagulant rodenticide toxicity to non-target wildlife under controlled exposure conditions

Much of our understanding of anticoagulant rodenticide toxicity to non-target wildlife has been derived from molecular through whole animal research and registration studies in domesticated birds and mammals, and to a lesser degree from trials with captive wildlife. Using these data, an adverse outcome pathway identifying molecular initiating and anchoring events (inhibition of vitamin K epoxide reductase, failure to activate clotting factors), and established and plausible linkages (coagulopathy, hemorrhage, anemia, reduced fitness) associated with toxicity, is presented. Controlled exposure studies have demonstrated that second-generation anticoagulant rodenticides (e.g., brodifacoum) are more toxic than first- and intermediate-generation compounds (e.g., warfarin, diphacinone), however the difference in potency is diminished when first- and intermediate-generation compounds are administered on multiple days. Differences in species sensitivity are inconsistent among compounds. Numerous studies have compared mortality rate of predators fed prey or tissue containing anticoagulant rodenticides. In secondary exposure studies in birds, brodifacoum appears to pose the greatest risk, with bromadiolone, difenacoum, flocoumafen and difethialone being less hazardous than brodifacoum, and warfarin, coumatetralyl, coumafuryl, chlorophacinone and diphacinone being even less hazardous. In contrast, substantial mortality was noted in secondary exposure studies in mammals ingesting prey or tissue diets containing either second- or intermediate-generation compounds. Sublethal responses (e.g., prolonged clotting time, reduced hematocrit and anemia) have been used to study the sequelae of anticoagulant intoxication, and to some degree in the establishment of toxicity thresholds or toxicity reference values. Surprisingly few studies have undertaken histopathological evaluations to identify cellular lesions and hemorrhage associated with anticoagulant rodenticide exposure in non-target wildlife. Ecological risk assessments of anticoagulant rodenticides would be improved with additional data on (i) interspecific differences in sensitivity, particularly for understudied taxa, (ii) sublethal effects unrelated to coagulopathy, (iii) responses to mixtures and sequential exposures, and (iv) the role of vitamin K status on toxicity, and significance of inclusion of supplemental vitamin K or menadione (provitamin) in the diet of test organisms. A more complete understanding of the toxicity of anticoagulant rodenticides in non-target wildlife would enable regulators and natural resource managers to better predict and even mitigate risk.

Book chapter

The rising tide of ocean diseases: Unsolved problems and research priorities

New studies have detected a rising number of reports of diseases in marine organisms such as corals, molluscs, turtles, mammals, and echinoderms over the past three decades. Despite the increasing disease load, microbiological, molecular, and theoretical tools for managing disease in the world's oceans are under-developed. Review of the new developments in the study of these diseases identifies five major unsolved problems and priorities for future research: (1) detecting origins and reservoirs for marine diseases and tracing the flow of some new pathogens from land to sea; (2) documenting the longevity and host range of infectious stages; (3) evaluating the effect of greater taxonomic diversity of marine relative to terrestrial hosts and pathogens; (4) pinpointing the facilitating role of anthropogenic agents as incubators and conveyors of marine pathogens; (5) adapting epidemiological models to analysis of marine disease.

Frontiers in Ecology and the Environment

Pneumonia in bighorn sheep: Risk and resilience

Infectious disease was an important driver of historic declines and extirpations of bighorn sheep (Ovis canadensis) in North America and continues to impede population restoration and management. Domestic sheep have long been linked to pneumonia outbreaks in bighorn sheep and this association has now been confirmed in 13 captive commingling experiments. However, ecological and etiological complexities still hinder our understanding and control of the disease. We provide an overview of the current state of knowledge about the biology and management of respiratory disease in bighorn sheep and propose strategies for moving forward. Epizootic pneumonia in bighorn sheep is polymicrobial. Mycoplasma ovipneumoniae, a bacterium host-specific to Caprinae and commonly carried by healthy domestic sheep and goats appears to be a necessary primary agent. All-age epizootics following introduction of M. ovipneumoniae along with other pathogens into bighorn sheep populations are usually severe (median mortality 47%) but fatality rates vary widely, from 15 – 100%. Disease severity may be influenced by the strain of M. ovipneumoniae, by secondary bacterial and viral pathogens, and by factors affecting transmission and host immunity. Once introduced, M. ovipneumoniae can persist in bighorn sheep populations for decades. Carrier dams transmit the pathogen to their susceptible lambs, triggering fatal pneumonia outbreaks in nursery groups, which limits recruitment and slows or prevents population recovery. The result is that demographic costs of pathogen persistence often outweigh the impacts of the initial invasion and die-off. There is currently no effective vaccine or antibiotic for domestic or wild sheep and to date, no management actions have been successful in reducing morbidity, mortality, or disease spread once pathogen invasion has occurred. Molecular-based strain typing suggests that spillover of M. ovipneumoniae into bighorn sheep populations from domestic small ruminants is ongoing, and that consequences of pathogen invasion are amplified by movements of infected bighorn sheep. Therefore, current disease management strategies focus on reducing risk of spillover from reservoir populations of domestic small ruminants and on limiting transmission among bighorn sheep. A broad array of approaches has been tried and more are needed to prevent pathogen introduction, induce disease fadeout in persistently infected populations, and promote population resilience across the diverse landscapes bighorn sheep inhabit. A comprehensive examination of disease dynamics across populations could help elucidate how disease fades out naturally and if population resilience can be increased in the face of infection. Cross-jurisdictional adaptive management experiments and transdisciplinary collaboration, including partnerships with members of the domestic sheep and goat community, are needed to facilitate innovation and speed progress towards sustainable solutions for managing pneumonia to protect and restore bighorn sheep populations.

California, Idaho, Nevada, Oregon, Utah, Washingto

Influence of offshore oil and gas structures on seascape ecological connectivity

Offshore platforms, subsea pipelines, wells and related fixed structures supporting the oil and gas (O&G) industry are prevalent in oceans across the globe, with many approaching the end of their operational life and requiring decommissioning. Although structures can possess high ecological diversity and productivity, information on how they interact with broader ecological processes remains unclear. Here, we review the current state of knowledge on the role of O&G infrastructure in maintaining, altering or enhancing ecological connectivity with natural marine habitats. There is a paucity of studies on the subject with only 33 papers specifically targeting connectivity and O&G structures, although other studies provide important related information. Evidence for O&G structures facilitating vertical and horizontal seascape connectivity exists for larvae and mobile adult invertebrates, fish and megafauna; including threatened and commercially important species. The degree to which these structures represent a beneficial or detrimental net impact remains unclear, is complex and ultimately needs more research to determine the extent to which natural connectivity networks are conserved, enhanced or disrupted. We discuss the potential impacts of different decommissioning approaches on seascape connectivity and identify, through expert elicitation, critical knowledge gaps that, if addressed, may further inform decision making for the life cycle of O&G infrastructure, with relevance for other industries (e.g. renewables). The most highly ranked critical knowledge gap was a need to understand how O&G structures modify and influence the movement patterns of mobile species and dispersal stages of sessile marine species. Understanding how different decommissioning options affect species survival and movement was also highly ranked, as was understanding the extent to which O&G structures contribute to extending species distributions by providing rest stops, foraging habitat, and stepping stones. These questions could be addressed with further dedicated studies of animal movement in relation to structures using telemetry, molecular techniques and movement models. Our review and these priority questions provide a roadmap for advancing research needed to support evidence-based decision making for decommissioning O&G infrastructure.

Global Change Biology

Predator-specific mortality of sage-grouse nests based on predator DNA on eggshells

Greater sage-grouse (hereafter sage-grouse; Centrocercus urophasianus ) populations have declined across their range. Increased nest predation as a result of anthropogenic land use is one mechanism proposed to explain these declines. However, sage-grouse contend with a diverse suite of nest predators that vary in functional traits (e.g., search tactics or hunting mode) and abundance. Consequently, generalizing about factors influencing nest fate is challenging. Identifying the explicit predator species responsible for nest predation events is, therefore, critical to understanding causal mechanisms linking land use to patterns of sage-grouse nest success. Cattle grazing is often assumed to adversely affect sage-grouse recruitment by reducing grass height (and hence cover), thereby facilitating nest detection by predators. However, recent evidence found little support for the hypothesized effect of grazing on nest fate at the pasture scale. Rather, nest success appears to be similar on pastures grazed at varying intensities. One possible explanation for the lack of observed effect involves a localized response by one or more nest predators. The presence of cattle may cause a temporary reduction in predator density and/or use within a pasture (the cattle avoidance hypothesis). The cattle avoidance hypothesis predicts a decreased probability of at least one sage-grouse nest predator predating sage-grouse nests in pastures with livestock relative to pastures without livestock present during the nesting season. To test the cattle avoidance hypothesis, we collected predator DNA from eggshells from predated nests and used genetic methods to identify the sage-grouse nest predator(s) responsible for the predation event. We evaluated the influence of habitat and grazing on predator-specific nest predation. We evaluated the efficacy of our genetic method by deploying artificial nests with trail cameras and compared the results of our genetic method to the species captured via trail camera. Our molecular methods identified at least one nest predator captured predating artificial nests via trail camera for 33 of 35 (94%) artificial nests. We detected nest predators via our molecular analysis at 76 of 114 (67%) predated sage-grouse nests. The primary predators detected at sage-grouse nests were coyotes ( Canis latrans ) and corvids ( Corvidea ). Grazing did not influence the probability of nest predation by either coyotes or corvids. Sagebrush canopy cover was negatively associated with the probability a coyote predated a nest, distance to water was positively associated with the probability a corvid predated a nest, and average minimum temperature was negatively associated with the probability that either a coyote or a corvid predated a nest. Our study provides a framework for implementing an effective, non-invasive method for identifying sage-grouse nest predators that can be used to better understand how management actions at local and regional scales may impact an important component of sage-grouse recruitment.

Idaho

A space‐for‐time substitution reveals the long‐term decline in genotypic diversity of a widespread salt marsh plant, Spartina alterniflora, over a span of 1500 years

Clonal populations face a trade‐off between sexual recruitment and vegetative growth and, once established, may undergo continuous declines in genotypic diversity if their sexual recruits make poor competitors. The geological history of delta formation in the Lower Mississippi River Valley was used to age eight S. alterniflora marshes for use in a space‐for‐time substitution ranging over 1500 years, in order to determine the long‐term effects of clonal growth on genotypic diversity in natural populations. 2 We also predicted that highly heterozygous clones are competitively superior, leading to an increase in the overall level of genetic diversity as a marsh ages and/or to an increasingly positive relationship between clone size and individual heterozygosity, and that the clumping of ramets within clones will occur over increasingly large distances as populations age, while the clumping of genetically related clones will become less pronounced as intraclonal competition begins to obscure the initial effects of localized seedling recruitment. Using molecular markers to differentiate clones, we documented a decline in clonal richness at the rate of approximately 1% 100 years −1 that was accompanied for the first 300–500 years by an increase in the distance over which clumping of ramets within genets occurred. Older populations, in the 500–1500‐year range, showed evidence of clone fragmentation. The spatial clustering of kin was observed for only two marshes, and exhibited no clear relationship with marsh age. Whereas the overall level of genetic diversity was consistent among marshes and showed no clear relationship with marsh age, the relationship between heterozygosity and individual clone size became increasingly pronounced within older marshes. Our results suggest that under natural conditions S. alterniflora marshes will rarely reach ages sufficient for the loss of all clonal diversity, or for the effects of inbreeding and drift to pose a significant threat to population viability.

Journal of Ecology

Rapid diagnosis of avian influenza virus in wild birds: Use of a portable rRT-PCR and freeze-dried reagents in the field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal. Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene. The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

Journal of Visualized Experiments

Smoke-induced seed germination in California chaparral

The California chaparral community has a rich flora of species with different mechanisms for cuing germination to postfire conditions. Heat shock triggers germination of certain species but has no stimulatory effect on a great many other postfire species that are chemically stimulated by combustion products. Previous reports have shown that charred wood will induce germination, and here we report that smoke also induces germination in these same species. Smoke is highly effective, often inducing 100% germination in deeply dormant seed populations with 0% control germination. Smoke induces germination both directly and indirectly by aqueous or gaseous transfer from soil to seeds. Neither nitrate nor ammonium ions were effective in stimulating germination of smoke-stimulated species, nor were most of the quantitatively important gases generated by biomass smoke. Nitrogen dioxide, however, was very effective at inducing germination in Caulanthus heterophyllus (Brassicaceae), Emmenanthe penduliflora (Hydrophyllaceae), Phacelia grandiflora (Hydrophyllaceae), and Silene multinervia (Caryophyllaceae). Three species, Dendromecon rigida (Papaveraceae), Dicentra chrysantha, and Trichostema lanatum (Lamiaceae), failed to germinate unless smoke treatment was coupled with prior treatment of 1 yr soil storage. Smoke-stimulated germination was found in 25 chaparral species, representing 11 families, none of which were families known for heat-shock-stimulated germination. Seeds of smoke-stimulated species have many analogous characteristics that separate them from most heat-shock-stimulated seeds, including: (1) outer seed coats that are highly textured, (2) a poorly developed outer cuticle, (3) absence of a dense palisade tissue in the seed coat, and (4) a subdermal membrane that is semipermeable, allowing water passage but blocking entry of large (molecular mass > 500) solutes. Tentative evidence suggests that permeability characteristics of this subdermal layer are altered by smoke. While the mechanism behind smoke-induced germination is not known, it appears that smoke may be involved in overcoming different blocks to germination in different species. For example, in Emmenanthe penduliflora, NO 2 in smoke was sufficient to induce germination, and most forms of physical or chemical scarification also induced germination. For Romneya coulteri, NO 2 alone failed to induce germination, and scarified seeds required addition of gibberellic acid. In Dicentra chrysantha, none of these treatments, nor smoke alone, induced germination, but germination was triggered by a combination of soil burial followed by smoke treatment. Smoke-stimulated species differed substantially in the duration of smoke exposure required to induce germination, and this was inversely correlated with tolerance to smoke exposure. We suggest that such differences in response may affect postfire community structure.

California

Characterization of the putatively introduced red alga Acrochaetium secundatum (Acrochaetiales, Rhodophyta) growing epizoically on the pelage of southern sea otters ( Enhydra lutris nereis )

Ecological associations between epibionts (organisms that live on the surface of another living organism) and vertebrates have been documented in both marine and terrestrial environments, and may be opportunistic, commensal, or symbiotic (Lewin et al. 1981, Holmes 1985, Allen et al. 1993, Bledsoe et al. 2006, Pfaller et al. 2008, Suutari et al. 2010). Although epibiont proliferation is frequently reported on slow-moving, sparsely haired organisms such as manatees and sloths, reports from densely furred, highly mobile mammals are much less common. There are reports of epizoic algae for several species of pinnipeds (Kenyon and Rice 1959, Scheffer 1962, Baldridge 1977, Allen et al. 1993), which rely to varying degrees on both pelage and blubber for thermoregulation, but the phenomenon has not been widely described. Scheffer (1962) noted that red algae was fairly common on the pelage of northern fur seals (Callorhinus ursinus), pinnipeds for which fur likely makes a comparatively high contribution to thermoregulation (Donohue et al. 2000). For species with pelage that plays a critical role of thermal insulation, it seems implausible that an epibiont would persist on healthy individuals that devote significant energy resources toward grooming and actively maintaining their coat. Biological characteristics of epibiont settlement and attachment, and physiological requirements of epizoic species play key roles in their successful colonization and potential host impacts. To investigate this relationship, we explore a novel discovery of an epizoic alga from southern sea otters, including describing algal development on sea otter hair and molecular identification of the algae.

Marine Mammal Science