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Proper handling of animal tissues from the field to the laboratory supports reliable biomarker endpoints

In the endeavor to assess potential effects to the Gulf of Mexico ecosystem from the Mississippi Canyon 252 incident, referred to as the Deepwater Horizon oil spill, various environmental data have been collected. Whereas initial efforts have included satellite tracking and sediment and water sampling to estimate the geographical scope of oiling, research on biological samples can provide insights into potential physiological responses to oil if it was present in the food web, sediment, or water column. Fish species are ideal model organisms for studying responses to water- and sediment-borne contaminants due to their life history (Jenkins et al. 2014), and several Gulf of Mexico fish species were studied by scientists after this incident. Typical field data collected on fish reflect organism condition and include observations such as fish length, weight, gonad condition, condition factor (weight in relation to length), parasite load, and color of organs (Schmitt and Dethloff 2000). However, if physiological responses occurred due to oil exposure, effects would not be immediately visible using organism-level observations alone. Changes occur first at the organ, tissue, cell, or molecular levels, and these responses can be measured by using biomarker assays (van der Oost et al. 2003).

Book chapter

Endophytic bacteria in grass crop growth promotion and biostimulation

Plants naturally carry microbes on seeds and within seeds that may facilitate development and early survival of seedlings. Some crops have lost seed-vectored microbes in the process of domestication or during seed storage and seed treatment. Biostimulant microbes from wild plants were used by pre-modern cultures to re-acquire beneficial seed microbes. Today some companies have developed or are developing the use of microbes obtained from soils or plant sources to stimulate plant development and growth. Many of these biostimulant microbes are endophytic in plants. Biostimulant products also include humic substances, which appear to function as signal molecules in plants, triggering increased internalization of soil microbes into root cells and tissues. In addition, protein coatings on seeds fuel the growth of seed surface-vectored microbes, increasing microbial activity around and within roots. In this article, we provide evidence of the endophytic nature of many biostimulant microbes, and suggest that many of the beneficial effects of microbial biostimulants stem from their action as endophytes or as participants or stimulants of rhizophagy cycle activity.

Grass Research

Cutaneous mastocytomas in the neotenic caudate amphibians Ambystoma mexicanum (axolotl) and Ambystoma tigrinum (tiger salamander)

Spontaneous mastocytomas studied in 18 axolotls ( Ambystoma mexicanum ) and six tiger salamanders ( Ambystoma tigrinum ) were gray-white, uni- to multilobular cutaneous protrusions from 2 mm to 2 cm in diameter. Tumors were moderately cellular unencapsulated masses that usually infiltrated the dermis and hypodermis with the destruction of intervening tissues. Some tumors were invading superficial bundles of the underlying skeletal muscle. Tumors consisted of mitotically active cells derived from a single lineage but showing a range of differentiation. Immature cells had nearly smooth to lightly cleft or folded basophilic nuclei bordered by a band of cytoplasm with few cytoplasmic processes and containing a few small uniform eccentric granules. Mature cells had basophilic nuclei with deep clefts or folds and abundant eosinophilic cytoplasm with multiple long intertwining cytoplasmic extensions packed with metachromatic granules. The axolotls were old individuals from an inbred laboratory colony. The tiger salamanders were wild animals from a single polluted pond. They could have been old and inbred. Both groups were neotenic. These are the first mastocytomas discovered in cold-blooded animals.

Texas

Reproductive health and endocrine disruption in smallmouth bass (Micropterus dolomieu) from the Lake Erie drainage, Pennsylvania, USA

Smallmouth bass Micropterus dolomieu were sampled from three sites within the Lake Erie drainage (Elk Creek, Twentymile Creek, and Misery Bay, an embayment in Presque Isle Bay). Plasma, tissues for histopathological analyses, and liver and testes preserved in RNALater® were sampled from 30 smallmouth bass (of both sexes) at each site. Liver and testes samples were analyzed for transcript abundance with Nanostring nCounter® technology. Evidence of estrogenic endocrine disruption was assessed by the presence and severity of intersex (testicular oocytes; TO) and concentrations of plasma vitellogenin in male fish. Abundance of 17 liver transcripts associated with reproductive function, endocrine activity, and contaminant detoxification pathways and 40 testes transcripts associated with male and female reproductive function, germ cell development, and steroid biosynthesis were also measured. Males with a high rate of TO (87–100%) and plasma vitellogenin were noted at all sites; however, TO severity was greatest at the site with the highest agricultural land cover. Numerous transcripts were differentially regulated among the sites and patterns of transcript abundance were used to better understand potential risk factors for estrogenic endocrine disruption. The results of this study suggest endocrine disruption is prevalent in this region and further research would benefit to identify the types of contaminants that may be associated with the observed biological effects.

Pennsylvania

Histochemical evidence for nitrogen‐transfer Endosymbiosis in non‐photosynthetic cells of leaves and inflorescence bracts of angiosperms

We used light and confocal microscopy to visualize bacteria in leaf and bract cells of more than 30 species in 18 families of seed plants. Through histochemical analysis, we detected hormones (including ethylene and nitric oxide), superoxide, and nitrogenous chemicals (including nitric oxide and nitrate) around bacteria within plant cells. Bacteria were observed in epidermal cells, various filamentous and glandular trichomes, and other non-photosynthetic cells. Most notably, bacteria showing nitrate formation based on histochemical staining were present in glandular trichomes of some dicots (e.g., Humulus lupulus and Cannabis sativa ). Glandular trichome chemistry is hypothesized to function to scavenge oxygen around bacteria and reduce oxidative damage to intracellular bacterial cells. Experiments to assess the differential absorption of isotopic nitrogen into plants suggest the assimilation of nitrogen into actively growing tissues of plants, where bacteria are most active and carbohydrates are more available. The leaf and bract cell endosymbiosis types outlined in this paper have not been previously reported and may be important in facilitating plant growth, development, oxidative stress resistance, and nutrient absorption into plants. It is unknown whether leaf and bract cell endosymbioses are significant in increasing the nitrogen content of plants. From the experiments that we conducted, it is impossible to know whether plant trichomes evolved specifically as organs for nitrogen fixation or if, instead, trichomes are structures in which bacteria easily colonize and where some casual nitrogen transfer may occur between bacteria and plant cells. It is likely that the endosymbioses seen in leaves and bracts are less efficient than those of root nodules of legumes in similar plants. However, the presence of endosymbioses that yield nitrate in plants could confer a reduced need for soil nitrogen and constitute increased nitrogen-use efficiency, even if the actual amount of nitrogen transferred to plant cells is small. More research is needed to evaluate the importance of nitrogen transfer within leaf and bract cells of plants

Biology

Fungal life-styles and ecosystem dynamics: Biological aspects of plant pathogens, plant endophytes and saprophytes

This chapter discusses various biochemical, genetic, ecological, and evolutionary aspects of fungi that express either symbiotic or saprophytic life-styles. An enormous pool of potential pathogens exists in both agricultural and natural ecosystems, and virtually all plant species are susceptible to one or more fungal pathogens. Fungal pathogens have the potential to impact on the genetic structure of populations of individual plant species, the composition of plant communities and the process of plant succession. Endophytic fungi exist for at least part of their life cycles within the tissues of a plant host. This group of fungi is distinguished from plant pathogens because they do not elicit significant disease symptoms. However, endophytes do maintain the genetic and biochemical mechanisms required for infection and colonization of plant hosts. Fungi that obtain chemical nutrients from dead organic matter are known as saprophytes and are critical to the dynamics and resilience of ecosystems. There are two modes of saprophytic growth: one in which biomolecules that are amenable to transport across cell walls and membranes are directly absorbed, and another in which fungi must actively convert complex biopolymers into subunit forms amenable to transportation into cells. Regardless of life-style, fungi employ similar biochemical mechanisms for the acquisition and conversion of nutrients into complex biomolecules that are necessary for vegetative growth, production and dissemination of progeny, organismal competition, and survival during periods of nutrient deprivation or environmental inclemency.

Advances in Botanical Research

Effects of dietary cadmium on mallard ducklings

Mallard (Anas platyrhynchos) ducklings were fed cadmium in the diet at 0, 5, 10, or 20 ppm from 1 day of age until 12 weeks of age. At 4-week intervals six males and six females from each dietary group were randomly selected, bled by jugular venipuncture, and necropsied. Significant decreases in packed cell volume (PCV) and hemoglobin (Hb) concentration and a significant increase in serum glutamic pyruvic transaminase (GPT) were found at 8 weeks of age in ducklings fed 20 ppm cadmium. Mild to severe kidney lesions were evident in ducklings fed 20 ppm cadmium for 12 weeks. No other blood chemistry measurement, hematological parameter, or tissue histopathological measurement indicated a reaction to cadmium ingestion. Body weight, liver weight, and the ratio of the femur weight to length were not affected by dietary cadmium. Femur cadmium concentration In all ducklings 12 weeks of age declined from the values detected at 4 and 8 weeks of age. Liver cadmium concentrations were significantly higher in relation to the increased dietary levels and in relation to the length of time the ducklings were fed the cadmium diets. At 12 weeks of age the cadmium concentration in liver tissue was twice that in the diet.

Environmental Research

Morphology of certain viruses of Salmonid Fishes. I. in vitro studies of some viruses causing Hematopoietic Necrosis

An electron microscope study was performed on three virus isolates that caused hematopoietic necrosis in salmonid fishes: infectious hematopoietic necrosis (IHN), Oregon Sockeye Disease (OSD), and Sacramento River Chinook Salmon Disease (SRCD). All three isolates were examined by negative staining of fathead minnow (FHM) monolayer tissue culture concentrates and IHN virus was also examined in thin sections of FHM cells. Viruslike particles were observed in infected tissues, but similar structures were not found in uninfected cultures. All three isolates were bullet-shaped, but oval and truncated forms were also observed. Mean measurements of particles from IHN-virus-infected tissue were 158 × 90 mμ. They consisted of an outer coat 15 mμ thick, a core 60 mμ in diameter, subunits about 5 mμ, and an axial pore about 20 mμ in diameter. These particles also were seen budding from the cytoplasmic membrane. Similar particles from SRCD were 159 × 90 mμ and isolates from OSD were 181 × 91 mμ. The three isolates were morphologically indistinguishable from one another and the greater length of OSD was considered insignificant. IHN, SRCD, and OSD viruses were tentatively placed in the rhabdovirus group, but serological studies are needed to determine if they are antigenically identical or should be included as separate members. Biochemical and physical characteristics of these viruses and a comparison with other salmonid viruses is also discussed.

Journal of the Fisheries Research Board of Canada

Coelomic disorders of fishes

Dropsy is a commonly applied term for coelomic distention due to ascites, or the effusion and collection of fluid freely throughout the coelomic cavity. Dropsy, or ascites, is generally a sign of another ongoing disease process, oftentimes one that is multisystemic and impacting coelomic organs and tissues. Dropsy may be caused by a variety of potential etiological agents, both infectious and noninfectious. Stressors such as rapid change in water temperature may predispose fish to bacterial diseases associated with dropsy. Clinically affected fish display coelomic distension of varying degrees of severity. Generally considered a disease syndrome similar to dropsy, affecting specifically Malawi cichlids, a popular group of ornamental fish among aquarium hobbyists. Water belly occurs among salt water pen-reared salmonid fish, including Atlantic salmon, Chinook salmon and rainbow trout. Dietary changes such as reduction of the feeding rate have been reported to improve the condition among affected fish.

Book chapter

Overview of developmental, reproductive, and behavioral/ neurological effects of mercury exposures in wildlife

We review wildlife/mercury literature and our own research findings that demonstrate the relevance of wildlife toxicity data in protecting human health. Methylmercury affects wildlife through reduced adult survival and reproduction, aberrant behavior, immune system effects, and teratogenic effects. Methylmercury can readily cross the blood-brain barrier, is excreted into eggs in birds, and is transferred to young mammals across the placenta and in milk. Its principal effect on wildlife is on neurological functions. Wild mink (Mustela vison) and otter (Lutra canadensis) have died from methylmercury poisoning, with signs of poisoning including anorexia, loss of weight, incoordination, tremors, and convulsions, which are symptoms similar to those experienced by mercury-poisoned humans. Mammals also may experience tonic and clonic convulsions and an increase in fetal anomalies, again paralleling toxic problems in people. Antibody-producing cells can be suppressed by methylmercury. Microscopically, the most notable lesions are in the cerebrum. Extensive vacuolation of hepatocytes in the liver and necrosis and other changes in the appearance of the proximal convoluted tubules of the kidneys are often noted. When harp seals (Pagophilus groenlandicus) were dosed with methylmercury chloride the number of circulating erythrocytes decreased and white blood cell counts greatly increased. The poisoned seals also suffered from uremia, hyperproteinemia, hypercholesterolemia, hyperbilirubinemia, and elevations in lactic dehydrogenase and alkaline phosphatase. In birds, signs of methylmercury poisoning included emaciation and weakness in the extremities, which progressed until the birds died. Mercury poisoning in birds and mammals can be diagnosed from a combination of the signs of poisoning if the animal is still alive, the pathological effects seen in a gross necropsy, the histopathological effects seen with a microscope, and the concentrations of mercury in various tissues. Our studies with mallards (Anas platyrhynchos) suggest that the dietary concentrations of mercury that cause toxicity are lower than those set in fish consumption advisories to protect humans. Because wild mammals and birds are so sensitive to methylmercury poisoning and cannot escape dietary exposure the way humans can, guidelines set to protect wild birds and mammals may very well provide a margin of safety for human health.

Book chapter