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Differential expression and localization of four connexins in the ovary of the ayu (Plecoglossus Altivelis)

The post-vitellogenic oocytes of teleost fish are generally unresponsive to maturation-inducing hormone (MIH) until a luteinizing hormone (LH) surge stimulates sensitivity via the acquisition of oocyte-maturational competence (OMC). Heterologous gap junctions (GJs) between granulosa cells and the oocyte have been previously implicated in the regulation of oocyte maturation in various vertebrate species. Although heterologous GJ are present in ovarian follicles of ayu (Plecoglossus altivelis), their role in maturation remains unclear. In the present study, we cloned and characterized complementary DNAs for GJ protein connexin (Cx), and examined the expression pattern of Cx messenger RNAs in the ayu ovary. Four Cx cDNAs with predicted molecular masses of 32.1 (Cx32.1), 34.9 (Cx34.9), 44.1 (Cx44.1), and 44.2 (Cx44.2) kDa, respectively, were cloned. Northern blot analysis revealed that the levels of Cx44.1 and Cx44.2 transcripts were similar during the vitellogenic and ovulatory stages. Cx32.1 transcripts were more abundant during the vitellogenic stage, but their levels declined thereafter. Cx34.9 transcript levels increased during the vitellogenic stage and remained high during the acquisition of OMC. In situ hybridization revealed that Cx44.1 and Cx44.2 signals were restricted to the oocyte, whereas the Cx32.1 and Cx34.9 signals were detected in both cellular fractions. Furthermore, a dye-transfer assay revealed the presence of functional GJs between the oocytes and follicle cells. These results suggest that Cx34.9 contributes to the formation of heterologous GJs between oocytes and granulosa cells. Moreover, GJs formed by Cx34.9 may function during the LH-dependent acquisition of OMC and the MIH-dependent resumption of meiosis in ayu. ?? 2007 Wiley-Liss, Inc.

Molecular Reproduction and Development

Production of haploid gynogens to inform genomic resource development in the paleotetraploid pallid sturgeon (Scaphirhynchus albus)

Order Acipenseriformes (sturgeons and paddlefishes) is an ancient lineage of osteichthyan fishes (>200 million years old) with most extant species at conservation risk. A relatively basal species, the pallid sturgeon, Scaphirhynchus albus, is a federally endangered species native to the Mississippi and Missouri River basins. Hybridization with sympatric shovelnose sturgeon, S. platorynchus, is one of several threats to pallid sturgeon. Current molecular markers cannot reliably distinguish among pure species and multigenerational backcrosses. This information is critical for implementation of management strategies to increase populations through natural reproduction and artificial propagation. Genotypes from a large panel of unlinked single-nucleotide polymorphisms (SNPs) may provide greater resolution of the two species; however, paralogous sequence variants (PSVs) within individuals resulting from an ancient whole genome duplication event confound SNP development. The aim of this study was to produce pallid sturgeon gynogens that contain 100% homozygous DNA contributed by only the maternal parent and have enough DNA for future SNP marker development. When homozygous gynogens are sequenced, heterozygosity at a locus within an individual indicates the presence of incorrectly aligned sequences that contain PSVs; accurate identification of these multi-locus contigs can facilitate their exclusion when developing disomic markers. In this study, we attempted to produce two types of pallid sturgeon gynogens: a) haploid gynogens produced from the activation of pallid sturgeon eggs with ultraviolet-irradiated sperm from the distantly related paddlefish (Polyodon spathula), and b) doubled haploids produced from the activation of pallid sturgeon eggs with irradiated paddlefish milt followed by thermal shock to suppress the first mitotic division. Production of doubled haploids, gynogens with 100% homozygous DNA and double the genome content of haploid gynogens, was pursued because it was originally unknown if haploid gyongens would survive long enough to attain enough genetic material for SNP marker development. We performed flow cytometry and microsatellite genotyping on the specimens in order to confirm haploid and doubled haploid status. Our study was unable to successfully yield doubled haploids; however, we successfully produced haploid gynogens that contained enough nuclear DNA for our future SNP marker development study. Interestingly, this study also produced paddlefish × pallid sturgeon hybrids in the control groups in two separate years; this is the first study to report viable offspring between the paddlefish and a Scaphirhynchus sturgeon species and reflects on the malleability of the genomes of the species in this order.

Louisiana, Arkansas, Missouri, Nebraska, North Dak

Wildlife ecological risk assessment in the 21st century: Promising technologies to assess toxicological effects

Despite advances in toxicity testing and development of new approach methodologies (NAMs) for hazard assessment, the ecological risk assessment (ERA) framework for terrestrial wildlife (i.e., air-breathing amphibians, reptiles, birds, and mammals) has remained unchanged for decades. While survival, growth, and reproductive endpoints derived from whole animal toxicity tests are central to hazard assessment, non-standard measures of biological effects at multiple levels of biological organization (e.g., molecular, cellular, tissue, organ, organism, population, community, ecosystem) have potential to enhance the relevance of prospective and retrospective wildlife ERAs. Other factors (e.g., indirect effects of contaminants on food supplies and infectious disease processes) are influenced by toxicants at individual, population, and community levels, and need to be factored into chemically-based risk assessments to enhance the “eco” component of ERAs. Regulatory and logistical challenges often relegate such non-standard endpoints and indirect effects to post-registration evaluations of pesticides and industrial chemicals, and contaminated site evaluations. While NAMs are being developed, to date their applications in ERAs focused on wildlife have been limited. No single magic tool or model will address all uncertainties in hazard assessment. Modernizing wildlife ERAs will likely entail combinations of laboratory and field-derived data at multiple levels of biological organization, knowledge collection solutions (e.g., systematic review, adverse outcome pathway frameworks), and inferential methods that facilitate integrations and risk estimations focused on species, populations, interspecific extrapolations, and ecosystem services modeling, with less dependence on whole animal data and simple hazard ratios.

Integrated Environmental Assessment and Management

Monitoring of wild fish health at selected sites in the Great Lakes Basin: methods and preliminary results

During fall 2010 and spring 2011, a total of 119 brown bullhead ( Ameiurus nebulosus ), 136 white sucker ( Catostomus commersoni ), 73 smallmouth bass ( Micropterus dolomieu ), and 59 largemouth bass ( M. salmoides ) were collected from seven Great Lakes Basin Areas of Concern and one Reference Site. Comprehensive fish health assessments were conducted in order to document potential adverse affects from exposure to complex chemical mixtures. Fish were necropsied on site, blood samples obtained, pieces of liver, spleen, kidney, gill and any abnormalities placed in fixative for histopathology. Liver samples were saved for gene expression analysis and otoliths were removed for aging. A suite of fish health indicators was developed and implemented for site comparisons and to document seasonal effects and species differences in response to environmental conditions. Organism level (grossly visible lesions, condition factor), tissue level (microscopic pathology, organosomatic indices, micronuclei, and other nuclear abnormalities), plasma factors (reproductive steroid hormones, vitellogenin), and molecular (gene expression) indicators were included. This report describes the methods and preliminary results.

Great Lakes Basin

Introduction to life cycles, taxonomy, distribution and basic research techniques

Avian haemosporidian parasites are a closely related group of apicomplexan parasites with important similarities in their life cycles, development, physiology, and reproduction. Current phylogenies based on mitochondrial and nuclear genes reflect more traditional attempts to classify these organisms based on life history characteristics and morphology, but limited sampling from poorly characterized taxa such as the Garniidae from tropical and subtropical regions continues to limit our understanding of their phylogeny and evolution. Recent advances in molecular diagnostics and the ability to barcode these parasites using mitochondrial cytochrome b sequences have revolutionized the field, but traditional methodology based on microscopy of Giemsa-stained blood smears remains essential for diagnostics and understanding life history characteristics and biodiversity of these organisms. The relative strengths and weaknesses of current methods in wildlife haemosporidian research are discussed. We call for a combination of microscopic, PCR-based, and serological diagnostic methodologies for better estimates of true distribution and other aspects of biology of haemosporidians, particularly in studies on virulence, prevalence, and biodiversity.

Book chapter

A case study: Temporal trends of environmental stressors and reproductive health of smallmouth bass (Micropterus dolomieu) from a site in the Potomac River Watershed, Maryland, USA

Decades of poor reproductive success and young-of-the-year survival, combined with adult mortality events, have led to a decline in the smallmouth bass (SMB; Micropterus dolomieu ) population in sections of the Potomac River. Previous studies have identified numerous biologic and environmental stressors associated with negative effects on SMB health. To better understand the impact of these stressors, this study was conducted at the confluence of Antietam Creek and the Potomac River from 2013 to 2019 to identify temporal changes associated with SMB reproductive health. Surface water samples were collected and analyzed for over 300 organic contaminants, including pesticides, phytoestrogens, pharmaceuticals, hormones and total estrogenicity (E2Eq). Adult SMB were collected and sampled for multiple endpoints, including gene transcripts associated with reproduction (molecular), histopathology (cellular), and organosomatic indices (tissue). In males, biomarkers of estrogenic endocrine disruption, including testicular oocytes (TO) and plasma vitellogenin (Vtg) were assessed. Numerous agriculture-related contaminants or land use patterns were associated with gene transcript abundance in both male and female SMB. Positive associations between pesticides in the immediate catchment with TO severity and E2Eq with plasma Vtg in males were identified. In males, the prevalence of TO and detectable levels of plasma Vtg, liver vitellogenin transcripts ( vtg ) and testis vtg were high throughout the study. Peaks of complex mixtures of numerous contaminants occurred during the spring/early summer when spawning and early development occurs and to a lesser extent in fall/winter during recrudescence. Management practices to reduce exposure during these critical and sensitive periods may enhance reproductive health of these economically important sportfishes.

Maryland

Experimental reintroduction of the federally endangered Santa Cruz Island bush mallow (Malacothamnus fasciculatus var. nestioticus)

Studies of Malacothamnus fasciculatus var. nesioticus were begun in 1995 to understand its distribution, reproductive biology and ecological requirements. After 100+ years of depredation by sheep, two known populations of fewer than 20 plants each survived in 1995. Molecular studies showed that each of the two populations was composed of 1–3 genets. During our study, two additional populations of similar size were discovered. Plants are self-compatible but require insect visitation to augment pollination. Based on seed set, viable embryos, and germination rates, we found no evidence for inbreeding depression. Bush mallow also reproduces vegetatively by rhizomes, the primary means of establishment and persistence in natural populations, and a key feature for maximizing recovery success. Ex situ observations and trial in situ outplantings suggested that supplemental watering was critical to initial survival. We developed a recovery strategy composed of four plots located at varying elevations and aspects. Each plot was enclosed to exclude feral pigs, which posed a continuing threat. Each plot was planted with twelve rooted plants derived from each of three natural populations. Plants were provided supplemental watering for four months. Survivorship after one year ranged from 46% to 91%. Significant differences in survivorship were observed relative to source of plants. After twelve months some plants were flowering and reproducing vegetatively.

California

An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass (Micropterus salmoides floridanus) sampled from the St. Johns River

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass ( Micropterus salmoides floridanus ) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17β-estradiol (E 2 ) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E 2 , VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Florida

Abundance estimation and conservation biology

Abundance is the state variable of interest in most population–level ecological research and in most programs involving management and conservation of animal populations. Abundance is the single parameter of interest in capture–recapture models for closed populations (e.g., Darroch, 1958; Otis et al., 1978; Chao, 2001). The initial capture–recapture models developed for partially (Darroch, 1959) and completely (Jolly, 1965; Seber, 1965) open populations represented efforts to relax the restrictive assumption of population closure for the purpose of estimating abundance. Subsequent emphases in capture–recapture work were on survival rate estimation in the 1970’s and 1980’s (e.g., Burnham et al., 1987; Lebreton et al.,1992), and on movement estimation in the 1990’s (Brownie et al., 1993; Schwarz et al., 1993). However, from the mid–1990’s until the present time, capture–recapture investigators have expressed a renewed interest in abundance and related parameters (Pradel, 1996; Schwarz & Arnason, 1996; Schwarz, 2001). The focus of this session was abundance, and presentations covered topics ranging from estimation of abundance and rate of change in abundance, to inferences about the demographic processes underlying changes in abundance, to occupancy as a surrogate of abundance. The plenary paper by Link & Barker (2004) is provocative and very interesting, and it contains a number of important messages and suggestions. Link & Barker (2004) emphasize that the increasing complexity of capture–recapture models has resulted in large numbers of parameters and that a challenge to ecologists is to extract ecological signals from this complexity. They offer hierarchical models as a natural approach to inference in which traditional parameters are viewed as realizations of stochastic processes. These processes are governed by hyperparameters, and the inferential approach focuses on these hyperparameters. Link & Barker (2004) also suggest that our attention should be focused on relationships between demographic processes such as survival and recruitment, the two quantities responsible for changes in abundance, rather than simply on the magnitudes of these quantities. They describe a type of Jolly–Seber capture–recapture model that permits inference about the underlying relationship between per capita recruitment rates and survival rates (Link & Barker, this volume). Implementation used Bayesian Markov Chain Monte Carlo methods and appeared to work well, yielding inferences about the relationship between recruitment and survival that were robust to selection of prior distribution. We believe that readers will find their arguments compelling, and we expect to see increased use of hierarchical modeling approaches in capture–recapture and related fields. Otto (presentation without paper) also recommended use of hierarchical models in analysis of multiple data sources dealing with population dynamics of North American mallards. He integrated survival inferences from ringing data, abundance information from aerial survey data, and recruitment information based on age ratios from a harvest survey. He used a Leslie matrix population projection model as an integrating framework and obtained estimates of breeding population size using all data.Otto’s approach also permitted inference about biases in estimated quantities. As with the work of Link & Barker (2004), we find Otto’s recommendation to use hierarchical models to integrate data from multiple sources to be very compelling. Alisauskas et al. (2004) report results of an analysis of capture–recapture data for a askatchewan population of white–winged scoters. They used the approach of Pradel (1996) to estimate population growth rate (See the PDF) directly. Estimates for 1975–1985 were quite low, but estimates for the recent period, 2000–2003,increased to values > 1. Parameter estimates for seniority, survival and per capita recruitment (Pradel, 1996) led to the inference that increased recruitment was largely responsible for the improvements in population status and growth. However, various data sources also indicated that this increase in recruitment was likely a result of increased immigration rather than improved reproduction on the area. This latter inference is important from a conservation perspective in indicating the importance of birds in other locations to growth and health of the study population. Lukacs and Burnham presented material to be published elsewhere that dealt with the use of genetic markers in capture–recapture studies. The data sources for such studies are samples of hair or feces, which are then analyzed using molecular genetic techniques in order to determine individual genotypes with respect to a usually small number of loci. Two types of classification error can arise in such analyses. First, if only a small number of loci is examined, then there may be nonnegligible probabilities that multiple individual animals will have the same genotypes. The second type of error arises during the polymerase chain reaction (PCR) process and can result from failure of alleles to amplify (allelic dropout) or from PCR inhibitors in hair and feces that produce the appearance of false alleles or misprinting (Creel et al., 2003). Lukacs and Burnham developed models that formally incorporate possible misclassification of samples resulting from these errors. These models permit estimation of parameters such as abundance and survival in a manner that properly incorporates this uncertainty of individual identity. We anticipate that noninvasive sampling based on molecular genetic analyses of hair or feces will become extremely important for some species, and that the models of Lukacs and Burnham will become very popular for such analyses. MacKenzie & Nichols (2004) discuss the use of occupancy (proportion of patches or habitat area that is occupied) as a surrogate for abundance. In cases of territorial species and where birds occur at low densities, the number of occupied patches may provide a reasonable estimate of abundance. In other cases, occupancy can be viewed as providing information about one tail of the abundance distribution, P (N = 0). The motivation for considering occupancy as a surrogate for abundance is that occupancy is based on so–called presence–absence surveys that are frequently less expensive of time and effort than methods that estimate abundance directly. We describe one set of models that can be used to estimate occupancy for a single season and another that can be used to estimate parameters such as local probabilities of extinction and colonization that are associated with occupancy dynamics. We outline a possible hybrid approach that combines occupancy data with data on marked individuals in order to betterexplore the mechanisms underlying occupancy dynamics. These five presentations made for an interesting session containing useful information and recommendations for future work. A number of themes connecting these presentations could be emphasized. For example, two of the presentations considered alternatives to standard capture–recapture sampling that can be used to draw inferences about abundance, or a portion of the abundance distribution, with field methods that should be less expensive than usual capture–recapture approaches of handling animals. We believe that the most important theme of the session was the emphasis on the processes responsible for changes in abundance. In particular, we are excited by the potential for using hierarchical models as a means of investigating relationships among vital rates and as a means of combining multiple sources of data relevant to system dynamics. Indeed, we expect the importance of this session theme to be reflected in the content and presentations of the next EURING meeting.

Animal Biodiversity and Conservation

Interaction of rearing environment and reproductive tactic on gene expression profiles in Atlantic salmon

Organisms that share the same genotype can develop into divergent phenotypes, depending on environmental conditions. In Atlantic salmon, young males of the same age can be found either as sneakers or immature males that are future anadromous fish. Just as the organism-level phenotype varies between divergent male developmental trajectories, brain gene expression is expected to vary as well. We hypothesized that rearing environment can also have an important effect on gene expression in the brain and possibly interact with the reproductive tactic adopted. We tested this hypothesis by comparing brain gene expression profiles of the two male tactics in fish from the same population that were reared in either a natural stream or under laboratory conditions. We found that expression of certain genes was affected by rearing environment only, while others varied between male reproductive tactics independent of rearing environment. Finally, more than half of all genes that showed variable expression varied between the two male tactics only in one environment. Thus, in these fish, very different molecular pathways can give rise to similar macro-phenotypes depending on rearing environment. This result gives important insights into the molecular underpinnings of developmental plasticity in relationship to the environment. ?? 2005 The American Genetic Association.

Conference Paper

A body composition model with multiple storage compartments for polar bears (Ursus maritimus)

Climate warming is rapidly altering Arctic ecosystems. Polar bears ( Ursus maritimus ) need sea ice as a platform from which to hunt seals, but increased sea-ice loss is lengthening periods when bears are without access to primary hunting habitat. During periods of food scarcity, survival depends on the energy that a bear has stored in body reserves, termed storage energy, making this a key metric in predictive models assessing climate change impacts on polar bears. Here, we developed a body composition model for polar bears that estimates storage energy while accounting for changes in storage tissue composition. We used data of dissected polar bears ( n = 31) to link routinely collected field measures of total body mass and straight-line body length to the body composition of individual bears, described in terms of structural mass and two storage compartments, adipose and muscle. We then estimated the masses of metabolizable proteins and lipids within these storage compartments, giving total storage energy. We tested this multi-storage model by using it to predict changes in the lipid stores from an independent dataset of wild polar bears ( n = 36) that were recaptured 8–200 days later. Using length and mass measurements, our model successfully predicted direct measurements of lipid changes via isotopic dilutions (root mean squared error of 14.5 kg). Separating storage into two compartments, and allowing the molecular composition of storage to vary, provides new avenues for quantifying energy stores of individuals across their life cycle. The multi-storage body composition model thus provides a basis for further exploring energetic costs of physiological processes that contribute to individual survival and reproductive success. Given bioenergetic models are increasingly used as a tool to predict individual fitness and population dynamics, our approach for estimating individual energy stores could be applicable to a wide range of species.

Article

Bisphenol A and 17 alpha-ethinylestradiol-induced transgenerational differences in expression of osmoregulatory genes in the gill of medaka (Oryzias latipes)

Embryonic bisphenol A (BPA) and 17α-ethinylestradiol (EE2) exposure can have far reaching health effects in fish, including adult onset transgenerational reproductive abnormalities, anxiety, and cardiac disorders. It is unknown whether these two environmental estrogens can induce transgenerational abnormalities in the gill. The present study examined transgenerational effects of BPA or EE2 exposure on genes that are critical for osmoregulation in fish. Medaka ( Oryzias latipes ) embryos were exposed to either BPA (100 μg/L) or EE2 (0.05 μg/L) for the first 7 days of embryonic development and never thereafter for the remainder of that generation (F0) and in subsequent generations of this study (F1, F2, and F3). Expression of osmoregulatory genes ( NKAα1a , NKAα1b , NKAα1c , NKAα3a , NKAα3b , NKCC1a, and CFTR ) were examined in gills of the first-generation (F0) adults which were directly exposed as embryo and in the fourth-generation adults (F3), which were never exposed to either of these environmental estrogens. Significant alterations in expression of osmoregulatory genes were observed in both F0 and F3 generations. Within the F0 generation, a sex-specific expression pattern was observed with a downregulation of osmoregulatory genes in males and an upregulation of osmoregulatory genes in females. At the F3 generation, this pattern reversed with the majority of the osmoregulatory genes upregulated in males and downregulated in females, suggesting that exposure to BPA and EE2 during embryonic development induced transgenerational impairment in molecular events associated with osmoregulatory functions in subsequent generations. These adverse outcomes may have impacts on physiological functions related to osmoregulation of fish inhabiting contaminated aquatic environments.

Aquatic Toxicology

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

Abundance of host fish and frequency of glochidial parasitism in fish assessed in field and laboratory settings and frequency of juvenile mussels or glochidia recovered from hatchery-held fish, central and southeastern Texas, 2012-13

In 2012–13, the U.S. Geological Survey (USGS), in cooperation with the U.S. Fish and Wildlife Service (USFWS), completed the first phase of a two-phase study of mussel host-fish relations for five endemic mussel species in central and southeastern Texas that were State-listed as threatened on January 17, 2010: (1) Texas fatmucket ( Lampsilis bracteata ), (2) golden orb ( Quadrula aurea ), (3) smooth pimpleback ( Quadrula houstonensis ), (4) Texas pimpleback ( Quadrula petrina ), and (5) Texas fawnsfoot ( Truncilla macrodon ). On October 6, 2011, the USFWS announced the completion of a status review and determined that the five mussel species warranted listing under the Endangered Species Act; however, listing of these species at that time was precluded by higher priority listing actions, and currently (December 2014), they remained unlisted. Freshwater mussels are long-lived, sedentary organisms that spend their larval stage as obligate parasites on the gills or fins of fishes, and many of these larvae, which are referred to as “glochidia,” can survive only on a narrow range of host-fish species. Results from both study phases are likely to provide information useful for propagation of rare mussels, reintroduction of host fish, population and reproduction monitoring, habitat restoration and enhancement, and adaptive management. The abundance of host fish, frequency of parasitism in fish, and frequency of juvenile mussels or glochidia recovered from hatchery-held fish was assessed by collecting fish and mussels at 14 sites distributed among seven streams in central and southeastern Texas (juvenile mussels and glochidia were not differentiated in hatchery-held fish). All fish collected and assessed in this study were wild-caught. Qualitative surveys of the resident mussel communities were made, focusing on the five candidate species. A subsample (3 percent in 2012 and 19 percent in 2013) of the fish collected during aquatic biota surveys was submitted to the USFWS San Marcos National Fish Hatchery and Technology Center to collect juvenile mussels and glochidia recovered from the host fish, which were held for 28 days in holding tanks to allow time for most of the attached glochidia to release from the gills of the fish after transforming into juvenile mussels. All fish not sent to the hatchery were assessed for glochidia in the field or in the USGS Texas Water Science Center laboratory in Austin, Tex. Juvenile mussels and glochidia that were recovered from fish at the hatchery were submitted for use in the second phase of this study, the development of deoxyribonucleic acid (DNA) identification keys to determine mussel and host-fish relationships through DNA-based molecular identification (DNA typing of the juvenile mussels and glochidia). Reporting on the results of DNA-based molecular identification research is beyond the scope of this report. In 2012, the majority of the fish that were collected, in terms of total number and species types, belonged to the sunfish family Centrarchidae (centrarchids; 1,277 individuals and at least 10 species). Redbreast sunfish ( Lepomis auritus ) was the most common species collected in 2012 (603 individuals), but the largemouth bass ( Micropterus salmoides ) species was caught at all 10 sites. The largest number of species (19) was collected at the San Saba Menard site (San Saba River near Menard, Tex.) on May 22, 2012. In 2013, most of the fish that were collected, in terms of total number and species types, were centrarchids (763 individuals) and cyprinids (10 species), respectively. Blacktail shiner ( Cyprinella venusta ) was the most common species collected in 2013 (287 individuals), but bluegill ( Lepomis macrochirus ) was the only species that was caught at all nine sites. The largest number of individuals (382) and species (19) was collected from the Colorado Columbus site (Colorado River near Columbus, Tex.) on June 11, 2013. A minimum of two fish (any species) parasitized with glochidia was collected from each of the 10 sites sampled during 2012. The highest percentage of parasitized fish (19.1 percent) was measured at the Guadalupe Victoria site (Guadalupe River near Victoria, Tex.). The catfish family Ictaluridae (ictalurids) exhibited the highest proportion of parasitized fish (12.1 percent). Of the nine sites sampled in 2013, the Pedernales Fredericksburg site (Pedernales River near Fredericksburg, Tex.) had the highest proportion of parasitized fish at 22.7 percent. Ictalurids again exhibited the highest frequency of parasitism (26.5 percent). Of the fish that were not sent to the hatchery but assessed for glochidia in the field or in the laboratory in 2012, at least 13 species were parasitized, and longear sunfish ( Lepomis megalotis ) was the species with the highest percentage of parasitized individuals (17.3 percent). Of the fish that were not sent to the hatchery but assessed for glochidia in the field or in the laboratory in 2013, only eight species were parasitized, and flathead catfish ( Pylodictis olivaris ) was the species with the highest percentage of parasitized individuals (42.9 percent). With the exception of the San Antonio Charco site, fish were submitted to the hatchery from all sampling sites in 2013. During the first sampling period in 2013 (April 1–5), slightly more than half (16 out of 29) of the fish species (on a per site basis) that were submitted to the hatchery released juvenile mussels and glochidia. Compared to the other sampling periods in 2013, substantially fewer glochidia per fish were present on fish submitted to the hatchery during the second sampling period in 2013 (April 29–May 2). Although only two sites were sampled during the third sampling period in 2013 (June 10–11), more juvenile mussels and glochidia were recovered at the hatchery during this sampling period (107) than were recovered during the first two sampling periods in 2013 combined (102). An average of 17 juvenile mussels or glochidia was recovered per largemouth bass submitted to the hatchery from the Guadalupe Victoria site during the third sampling period. A total of 19 fish species collected at nine sites was submitted to the hatchery in 2013, and 14 of these species had juvenile mussels or glochidia that were recovered at the hatchery. The three most productive species, in terms of the average number of juvenile mussels or glochidia recovered, were longear sunfish, spotted bass, and largemouth bass, each of which averaged more than two juvenile mussels or glochidia recovered per individual.

Texas

Yosemite toad (Anaxyrus canorus) transcriptome reveals interplay between speciation genes and adaptive introgression

Genomes are heterogeneous during the early stages of speciation, with small ‘islands’ of DNA appearing to reflect strong adaptive differences, surrounded by vast seas of relative homogeneity. As species diverge, secondary contact zones between them can act as an interface and selectively filter through advantageous alleles of hybrid origin. Such introgression is another important adaptive process, one that allows beneficial mosaics of recombinant DNA (‘rivers’) to flow from one species into another. Although genomic islands of divergence appear to be associated with reproductive isolation, and genomic rivers form by adaptive introgression, it is unknown whether islands and rivers tend to be the same or different loci. We examined three replicate secondary contact zones for the Yosemite toad ( Anaxyrus canorus ) using two genomic data sets and a morphometric data set to answer the questions: (1) How predictably different are islands and rivers, both in terms of genomic location and gene function? (2) Are the adaptive genetic trait loci underlying tadpole growth and development reliably islands, rivers or neither? We found that island and river loci have significant overlap within a contact zone, suggesting that some loci are first islands, and later are predictably converted into rivers. However, gene ontology enrichment analysis showed strong overlap in gene function unique to all island loci, suggesting predictability in overall gene pathways for islands. Genome-wide association study outliers for tadpole development included LPIN3, a lipid metabolism gene potentially involved in climate change adaptation, that is island-like for all three contact zones, but also appears to be introgressing (as a river) across one zone. Taken together, our results suggest that adaptive divergence and introgression may be more complementary forces than currently appreciated.

Molecular Ecology