Insights into the introduction history and population genetic dynamics of the Argentine black-and-white tegu (Salvator merianae) in Florida.

Invasive species are a major threat to global biodiversity. The US state of Florida is especially susceptible to the spread of exotic reptiles due to its subtropical climate, disturbed habitats, and robust pet trade. The Argentine black-and-white tegu (Salvator merianae) is a large, omnivorous lizard currently established in two different regions of Southern Florida. These two populations pose potential threats to sensitive ground nesting species such as gopher tortoises, American crocodiles, and migratory birds. At present, the introduction histories of these populations and the degree to which they are connected by gene flow are largely unknown. To address these issues, we genotyped S. merianae from Hillsborough and Miami-Dade Counties at ten microsatellite loci to assess intrapopulation genetic diversity, the degree of gene flow between populations, and compare the plausibilities of several potential introduction scenarios. Our results indicate that both populations have low genetic diversity [mean number of effective alleles across loci in both populations = 2.09 and are highly differentiated from each other (GST = 0.170; G″ST = 0.545)]. In addition, our results suggest that these populations underwent a bottleneck event prior to their divergence. We discuss what our results suggest about the histories of Florida's invasive tegu populations, as well as how they inform ongoing management strategies.

Insights into the Introduction History and Population Genetic Dynamics of the Nile Monitor (Varanus niloticus) in Florida.

Invasive species are widely recognized as important drivers of the ongoing biodiversity crisis. The US state of Florida is especially susceptible to the proliferation of invasive reptiles, and nonnative lizards currently outnumber native lizard species. At present, there are 3 documented breeding populations of the Nile monitor (Varanus niloticus) in different regions of Southern Florida, and these populations are considered potential dangers to threatened, fossorial endemics, such as burrowing owls, American crocodiles, and gopher tortoises. Nevertheless, at present, both the introduction histories of these populations and the degree to which they are connected by gene flow are not known. To address these issues, we genotyped V. niloticus from Cape Coral, Homestead Air Reserve Base, and West Palm Beach at 17 microsatellite loci and conducted a variety of analyses to assess both intrapopulation genetic diversity, the degree of gene flow between populations, and the most likely introduction scenario. The results of our analyses demonstrate that all 3 populations have limited genetic diversity (mean number of effective alleles across loci in all 3 populations ~ 2.00) and are highly differentiated from one another (G ST = 0.268; G″ST = 0.628). Our results also suggest that these populations resulted from independent introduction events that occurred within the past few decades. Consequently, we advise that wildlife managers focus management efforts on containment of existing populations and intensification of monitoring efforts on potential migration corridors.

Soy glyceollins regulate transcript abundance in the female mouse brain.

Glyceollins (Glys), produced by soy plants in response to stress, have anti-estrogenic activity in breast and ovarian cancer cell lines in vitro and in vivo. In addition to known anti-estrogenic effects, Gly exhibits mechanisms of action not involving estrogen receptor (ER) signaling. To date, effects of Gly on gene expression in the brain are unknown. For this study, we implanted 17-ß estradiol (E2) or placebo slow-release pellets into ovariectomized CFW mice followed by 11 days of exposure to Gly or vehicle i.p. injections. We then performed a microarray on total RNA extracted from whole-brain hemispheres and identified differentially expressed genes (DEGs) by a 2 × 2 factorial ANOVA with an false discovery rate (FDR) = 0.20. In total, we identified 33 DEGs with a significant E2 main effect, 5 DEGs with a significant Gly main effect, 74 DEGs with significant Gly and E2 main effects (but no significant interaction term), and 167 DEGs with significant interaction terms. Clustering across all DEGs revealed that transcript abundances were similar between the E2 + Gly and E2-only treatments. However, gene expression after Gly-only treatment was distinct from both of these treatments and was generally characterized by higher transcript abundance. Collectively, our results suggest that whether Gly acts in the brain through ER-dependent or ER-independent mechanisms depends on the target gene.

Use of RT-PCR in conjunction with a respiratory pathogen assay to concurrently determine the prevalence of bacteria and SARS-CoV-2 from the nasopharynx of outpatients.

Introduction: COVID-19 has emerged as a highly contagious and debilitating disease caused by the SARS-CoV-2 virus and has claimed the lives of over 7.7 million people worldwide. Bacterial co-infections are one of many co-morbidities that have been suggested to impact the outcome of COVID-19 in patients. The goals of this study are to elucidate the presence of bacteria in the nasopharynx of SARS-CoV-2 positive and negative patients and to describe demographic categories that may be associated with the detection of these organisms during one of the initial waves of the COVID-19 pandemic. Methods: To this end, we investigated SARS-CoV-2 and bacterial co-detection from outpatient RT-PCR testing in Texas. Results: The results indicate that Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae were the most frequently detected bacteria in both SARS-CoV-2 positive and SARS-CoV-2 negative patients and that these bacteria were present in these two patient populations at similar proportions. We also detected Staphylococcus aureus in a significantly larger proportion of males relative to females and people under 65 years of age relative to those 65 and over. Finally, we observed that SARS-CoV-2 was more commonly detected in Hispanics compared to non-Hispanics; however, low disclosure rates make volunteer bias a concern when interpreting the effects of demographic variables. Discussion: This study describes the bacteria present in the nasopharynx of SARS-CoV-2 positive and negative patients, highlights associations between patient demographics and SARS-CoV-2 as well as bacterial co-detection. In addition, this study highlights RT-PCR based molecular testing as a tool to detect bacteria simultaneously when SARS-CoV-2 tests are performed.

Modelling the Caenorhabditis elegans gonad over developmental time using the Distal Tip Cell marker lag-2p::gfp.

Development is a process that occurs over time, but defects are often scored at the end point of the process being studied. We are interested in understanding the molecular basis of gonad development in Caenorhabditis elegans and have used the Distal Tip Cell marker lag-2p::gfp to develop a larval size model of gonad growth. We found that gonad length demonstrates two distinct phases relative to larval length, with a breakpoint in mid-L3 stage. We hope that this model will help determine at what point in gonad development our genes of interest act.

Linear methods for analysis and quality control of relative expression ratios from quantitative real-time polymerase chain reaction experiments.

Relative expression quantitative real-time polymerase chain reaction (RT-qPCR) experiments are a common means of estimating transcript abundances across biological groups and experimental treatments. One of the most frequently used expression measures that results from such experiments is the relative expression ratio (RE), which describes expression in experimental samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to one or more experimental or nonbaseline condition) in terms of fold change relative to calibrator samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to a control or baseline condition). Over the past decade, several models of RE have been proposed, and it is now clear that endogenous reference gene stability and amplification efficiency must be assessed in order to ensure that estimates of RE are valid. In this review, we summarize key issues associated with estimating RE from cycle threshold data. In addition, we describe several methods based on linear modeling that enable researchers to estimate model parameters and conduct quality control procedures that assess whether model assumptions have been violated.

Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development.

BACKGROUND: The Mexican axolotl (Ambystoma mexicanum) is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic) form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum) that typically undergo a metamorphosis. RESULTS: Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph) and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs) were identified as unique to the axolotl (n = 76) and tiger salamander (n = 292) than were identified as shared (n = 108). All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. CONCLUSIONS: Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome-wide reduction in mRNA abundance across loci, including genes that regulate hypothalamic-pituitary activities. This suggests that an axolotls failure to undergo anatomical metamorphosis late in the larval period is indirectly associated with a mechanism(s) that acts earlier in development to broadly program transcription. The axolotl hopeful monster provides a model to identify mechanisms of early brain development that proximally and ultimately affect the expression of adult phenotypes.

Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration.

BACKGROUND: Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. RESULTS: Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences. CONCLUSION: Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

Genetic variation in Plethodon cinereus and Plethodon hubrichti from in and around a contact zone.

Climate change poses several challenges to biological communities including changes in the frequency of encounters between closely related congeners as a result of range shifts. When climate change leads to increased hybridization, hybrid dysfunction or genetic swamping may increase extinction risk-particularly in range-restricted species with low vagility. The Peaks of Otter Salamander, Plethodon hubrichti, is a fully terrestrial woodland salamander that is restricted to ~18 km of ridgeline in the mountains of southwestern Virginia, and its range is surrounded by the abundant and widespread Eastern Red-backed Salamander, Plethodon cinereus. In order to determine whether these two species are hybridizing and how their range limits may be shifting, we assessed variation at eight microsatellite loci and a 1,008 bp region of Cytochrome B in both species at allopatric reference sites and within a contact zone. Our results show that hybridization between P. hubrichti and P. cinereus either does not occur or is very rare. However, we find that diversity and differentiation are substantially higher in the mountaintop endemic P. hubrichti than in the widespread P. cinereus, despite similar movement ability for the two species as assessed by a homing experiment. Furthermore, estimation of divergence times between reference and contact zone populations via approximate Bayesian computation is consistent with the idea that P. cinereus has expanded into the range of P. hubrichti. Given the apparent recent colonization of the contact zone by P. cinereus, future monitoring of P. cinereus range limits should be a priority for the management of P. hubrichti populations.

A model of transcriptional and morphological changes during thyroid hormone-induced metamorphosis of the axolotl.

Anuran (frog) metamorphosis has long-served as a model of how thyroid hormones regulate post-embryonic development in vertebrates. However, comparatively little is known about urodele (salamander) metamorphosis. We conducted a detailed time-course study of induced metamorphosis in the Mexican axolotl (Ambystoma mexicanum) that probed metamorphic changes in morphology and gene expression in the skin. Using morphometrics, quantitative PCR, histology, and in situ hybridization we demonstrate that the development of transcriptional markers is fundamental to the resolution of early metamorphic events in axolotls. We then use linear and piecewise linear models to identify a sequence of morphological and transcriptional changes that define larval to adult remodeling events throughout metamorphosis. In addition, we show that transcriptional biomarkers are expressed in specific larval and adult cell populations of the skin and that temporal changes in these biomarkers correlate with tissue remodeling. We compare our results with other studies of natural and induced metamorphosis in urodeles and highlight what appear to be conserved features between urodele and anuran metamorphosis.

Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma).

BACKGROUND: Thyroid hormones (TH) induce gene expression programs that orchestrate amphibian metamorphosis. In contrast to anurans, many salamanders do not undergo metamorphosis in nature. However, they can be induced to undergo metamorphosis via exposure to thyroxine (T4). We induced metamorphosis in juvenile Mexican axolotls (Ambystoma mexicanum) using 5 and 50 nM T4, collected epidermal tissue from the head at four time points (Days 0, 2, 12, 28), and used microarray analysis to quantify mRNA abundances. RESULTS: Individuals reared in the higher T4 concentration initiated morphological and transcriptional changes earlier and completed metamorphosis by Day 28. In contrast, initiation of metamorphosis was delayed in the lower T4 concentration and none of the individuals completed metamorphosis by Day 28. We identified 402 genes that were statistically differentially expressed by > or = two-fold between T4 treatments at one or more non-Day 0 sampling times. To complement this analysis, we used linear and quadratic regression to identify 542 and 709 genes that were differentially expressed by > or = two-fold in the 5 and 50 nM T4 treatments, respectively. CONCLUSION: We found that T4 concentration affected the timing of gene expression and the shape of temporal gene expression profiles. However, essentially all of the identified genes were similarly affected by 5 and 50 nM T4. We discuss genes and biological processes that appear to be common to salamander and anuran metamorphosis, and also highlight clear transcriptional differences. Our results show that gene expression in axolotls is diverse and precise, and that axolotls provide new insights about amphibian metamorphosis.

Transcriptional response of Mexican axolotls to Ambystoma tigrinum virus (ATV) infection.

BACKGROUND: Very little is known about the immunological responses of amphibians to pathogens that are causing global population declines. We used a custom microarray gene chip to characterize gene expression responses of axolotls (Ambystoma mexicanum) to an emerging viral pathogen, Ambystoma tigrinum virus (ATV). RESULT: At 0, 24, 72, and 144 hours post-infection, spleen and lung samples were removed for estimation of host mRNA abundance and viral load. A total of 158 up-regulated and 105 down-regulated genes were identified across all time points using statistical and fold level criteria. The presumptive functions of these genes suggest a robust innate immune and antiviral gene expression response is initiated by A. mexicanum as early as 24 hours after ATV infection. At 24 hours, we observed transcript abundance changes for genes that are associated with phagocytosis and cytokine signaling, complement, and other general immune and defense responses. By 144 hours, we observed gene expression changes indicating host-mediated cell death, inflammation, and cytotoxicity. CONCLUSION: Although A. mexicanum appears to mount a robust innate immune response, we did not observe gene expression changes indicative of lymphocyte proliferation in the spleen, which is associated with clearance of Frog 3 iridovirus in adult Xenopus. We speculate that ATV may be especially lethal to A. mexicanum and related tiger salamanders because they lack proliferative lymphocyte responses that are needed to clear highly virulent iridoviruses. Genes identified from this study provide important new resources to investigate ATV disease pathology and host-pathogen dynamics in natural populations.

Assessment of intra and interregional genetic variation in the Eastern Red-backed Salamander, Plethodon cinereus, via analysis of novel microsatellite markers.

The red-backed salamander (Plethodon cinereus) has long-served as a model system in ecology, evolution, and behavior, and studies surveying molecular variation in this species have become increasingly common over the past decade. However, difficulties are commonly encountered when extending microsatellite markers to populations that are unstudied from a genetic perspective due to high levels of genetic differentiation across this species' range. To ameliorate this issue, we used 454 pyrosequencing to identify hundreds of microsatellite loci. We then screened 40 of our top candidate loci in populations in Virginia, Pennsylvania, and Ohio-including an isolated island population ~ 4.5 km off the shore of Lake Erie (South Bass Island). We identified 25 loci that are polymorphic in a well-studied region of Virginia and 11 of these loci were polymorphic in populations located in the genetically unstudied regions of Ohio and Pennsylvania. Use of these loci to examine patterns of variation within populations revealed that South Bass Island has low diversity in comparison to other sites. However, neither South Bass Island nor isolated populations around Cleveland are inbred. Assessment of variation between populations revealed three well defined genetic clusters corresponding to Virginia, mainland Ohio/Pennsylvania, and South Bass Island. Comparisons of our results to those of others working in various parts of the range are consistent with the idea that differentiation is lower in regions that were once glaciated. However, these comparisons also suggest that well differentiated isolated populations in the formerly glaciated portion of the range are not uncommon. This work provides novel genetic resources that will facilitate population genetic studies in a part of the red-backed salamander's range that has not previously been studied in this manner. Moreover, this work refines our understanding of how neutral variation is distributed in this ecologically important organism.

Head injury and pulmonary embolism: a retrospective report based on the Pennsylvania Trauma Outcomes study.

OBJECTIVE: We retrospectively examined the database of the Pennsylvania Trauma Systems Foundation to determine the risk of pulmonary embolism in adult patients sustaining isolated head trauma or multiple injuries, including head trauma, to answer two questions: What is the incidence of symptomatic pulmonary embolism during hospitalization in a trauma center in patients who have sustained a head injury? Are patients with head injuries more at risk for pulmonary embolism than trauma patients without head injuries? METHODS: We determined the total number of adult submissions per year to the Pennsylvania Trauma Outcomes Study from 1992 to 1996. Age, sex, Glasgow Coma Scale score, Abbreviated Injury Score for head injury, Injury Severity Score, intensive care unit days, hospital days, and the presence or absence of head injury, spinal injury, pelvic fractures, and/or femur fractures were recorded. Statistical techniques to evaluate their correlation with the incidence of pulmonary embolism included chi(2) testing, linear regression analysis, Kendall analysis, and logistic regression analysis. RESULTS: The average incidence of symptomatic pulmonary embolism in head-injured patients occurring during their acute hospital stay was 0.38%. This rate was not significantly greater than the 0.27% incidence of pulmonary embolism in patients without head injury. Factors that significantly increased this incidence were age greater than 45 years, Injury Severity Score greater than 15, male sex, and the presence of pelvic or femur fractures or of spinal cord injury. CONCLUSION: We found no evidence that head injury is a significant independent risk factor for development of symptomatic pulmonary embolism during the acute hospitalization of the trauma patient.

Genomics of a metamorphic timing QTL: met1 maps to a unique genomic position and regulates morph and species-specific patterns of brain transcription.

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect quantitative trait locus (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele-specific chromosome rearrangement on linkage group 2 that uniquely brought functionally associated genes into linkage. Furthermore, we found that more than 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation.

Microarray-based identification of transcription factor target genes.

Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF activity, the experimental setup, the statistical analysis of the microarray data, and the validation of target genes.

Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum.

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.

Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum.

Ambystomatid salamanders offer several advantages for endocrine disruption research, including genomic and bioinformatics resources, an accessible laboratory model (Ambystoma mexicanum), and natural lineages that are broadly distributed among North American habitats. We used microarray analysis to measure the relative abundance of transcripts isolated from A. mexicanum epidermis (skin) after exogenous application of thyroid hormone (TH). Only one gene had a >2-fold change in transcript abundance after 2 days of TH treatment. However, hundreds of genes showed significantly different transcript levels at days 12 and 28 in comparison to day 0. A list of 123 TH-responsive genes was identified using statistical, BLAST, and fold level criteria. Cluster analysis identified two groups of genes with similar transcription patterns: up-regulated versus down-regulated. Most notably, several keratins exhibited dramatic (1000 fold) increases or decreases in transcript abundance. Keratin gene expression changes coincided with morphological remodeling of epithelial tissues. This suggests that keratin loci can be developed as sensitive biomarkers to assay temporal disruptions of larval-to-adult gene expression programs. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander, thus setting the stage for future investigations of TH disruption in the Mexican axolotl and other salamanders of the genus Ambystoma.