A multi-method approach for assessing the distribution of a rare, burrowing North American crayfish species.

Primary burrowing crayfishes face high extinction risk, but are challenging to study, manage, and conserve due to their difficult-to-sample habitat (i.e., terrestrial burrows) and low population densities. We apply here a variety of methods to characterize the distribution, habitat associations, and conservation status of the Boston Mountains Crayfish Cambarus causeyi (Reimer, 1966), an endemic burrowing crayfish found only in the Ozark Mountains of Arkansas, United States. We used species distribution modeling (SDM) on historic occurrence records to characterize the distribution and macro-scale habitat associations of this species. We then ground-truthed SDM predictions with conventional sampling, modeled fine-scale habitat associations with generalized linear models (GLM), and lastly developed and tested an environmental DNA (eDNA) assay for this species in comparison to conventional sampling. This represents, to our knowledge, the first successful eDNA assay for a terrestrial burrowing crayfish. Our MaxEnt-derived SDM found a strong effect of average annual precipitation on the historic distribution of C. causeyi, which occurred most frequently at locations with moderately high average annual precipitation (140-150 cm/yr) within our study region. Cambarus causeyi was difficult to detect by conventional sampling in 2019 and 2020, found at only 9 of 51 sites (17.6%) sampled by searching for and manually excavating crayfish burrows. Surprisingly, habitat suitability predicted from our MaxEnt models was not associated with contemporary C. causeyi occurrences per GLMs. Instead, C. causeyi presence was negatively associated with both sandy soils and the presence of other burrowing crayfish species. Poor SDM performance in this instance was likely caused by the omission of high resolution fine-scale habitat data (e.g., soils) and biotic interactions from MaxEnt models. Finally, our eDNA assay detected C. causeyi from six of 25 sites (24.0%) sampled in 2020, out-performing conventional surveys by burrow excavation for this species. Given the difficulty of studying primary burrowing crayfishes and their high conservation need, we propose that eDNA may become an increasingly important monitoring tool for C. causeyi and similar species.

Increased Temperature Influenced Growth and Development of Lithobates pipiens Tadpoles Exposed to Leachates of the Invasive Plant European Buckthorn (Rhamnus cathartica) and a Triclopyr Herbicide.

Multiple factors including habitat loss, pollutants, invasive species, and disease have contributed to the global decline of amphibians, and further declines can be expected as a result of climate change. Warming temperatures may allow for range expansion of invasive plants, and because herbicides are the primary method to control invasive plants, chemical use may increase. A laboratory experiment was performed to examine the individual and combined effects of leachates from the invasive plant European buckthorn (Rhamnus cathartica, L.) and a triclopyr herbicide (Renovate® 3; 0.21 mg/L), which is commonly used to manage R. cathartica, on northern leopard frog (Lithobates pipiens, Schreber) tadpoles at 2 temperature regimes (20 and 25 °C). We measured tadpole growth weekly and body and intestine morphology at the conclusion of the experiment after 8 wk. In the presence of R. cathartica leachates, tadpole growth increased at 25 °C, but only during the first 3 to 4 wk of the experiment. From week 5 until the end of the experiment, tadpoles were significantly smaller at 25 °C compared with 20 °C, but had more developed limb buds at the end of the experiment (except in the triclopyr treatment). Triclopyr had minimal effects on tadpole growth at the low dose used in this study. These results encourage further examination of potential effects of global climate changes in combination with other environmental factors that may impact amphibian populations. Environ Toxicol Chem 2021;40:2547-2558. © 2021 SETAC.

The influence of nutrient loading on methylmercury availability in Long Island estuaries.

Estuaries provide critical habitat for food webs supporting fish and shellfish consumed by humans, but estuarine ecosystem health has been threatened by increases in nitrogen loading as well as inputs of the neurotoxin, mercury (Hg), which biomagnifies in food webs and poses risk to humans and wildlife. In this study, the effects of nutrient loading on the fate of Hg in shallow coastal estuaries were examined to evaluate if their interaction enhances or reduces Hg bioavailability in sediments, the water column, and concentrations in lower trophic level fish (Fundulus heteroclitus and Menidia menidia). Multiple sites were sampled within two human impacted coastal lagoons, Great South Bay (GSB) and Jamaica Bay (JB), on the southern coast of Long Island, NY, United States of America (U.S.A.). Carbon (C), nitrogen (N), sulfur (S), Hg, and methylmercury (MeHg) were measured in surface sediments and the water column, and total Hg (THg) was measured in two species of forage fish. Minimal differences were found in dissolved and particulate Hg, dissolved organic carbon (DOC), and salinity between the two bays. Across lagoons, concentrations of chlorophyll-a were correlated with total suspended solids (TSS), and water column THg and MeHg was largely associated with the particulate fraction. Methylmercury concentrations in particulates decreased with increasing TSS and chlorophyll-a, evidence of biomass dilution of MeHg with increasing productivity at the base of the food chain. Water column Hg was associated with THg concentrations in Atlantic silversides, while mummichog THg concentrations were related to sediment concentrations, reflecting their different feeding strategies. Finally, higher nutrient loading (lower C:N in sediments) while related to lower particulate concentrations coincided with higher bioaccumulation factors (BAF) for Hg in both fish species. Thus, in shallow coastal lagoons, increased nutrient loading resulted in decreased Hg concentrations at the base of the food web but resulted in greater bioaccumulation of Hg to fish relative to its availability in algal food.

No evidence that crayfish carcasses produce detectable environmental DNA (eDNA) in a stream enclosure experiment.

Environmental DNA (eDNA) is an emerging tool for monitoring invasive and imperiled species, particularly at low densities. However, the factors that control eDNA production, transport, and persistence in aquatic systems remain poorly understood. For example, the extent to which carcasses produce detectable eDNA is unknown. If positive detections are associated with dead organisms, this could confound monitoring for imperiled or invasive species. Here, we present results from one of the first studies to examine carcass eDNA in situ by deploying carcasses of the invasive red swamp crayfish (Procambarus clarkii) in a stream enclosure experiment for 28 days. We predicted that carcasses would initially produce eDNA that would decline over time as carcasses decayed. Unsurprisingly, crayfish carcasses lost biomass over time, but at the conclusion of our experiment much of the carapace and chelae remained. However, no eDNA of P. clarkii was detected in any of our samples at the crayfish density (15 P. clarkii carcasses at ∼615 g of biomass initially), stream flow (520-20,319 L/s), or temperature (∼14-25 °C) at our site. Subsequent analyses demonstrated that these results were not the consequence of PCR inhibition in our field samples, poor performance of the eDNA assay for intraspecific genetic diversity within P. clarkii, or due to the preservation and extraction procedure used. Therefore, our results suggest that when crayfish are relatively rare, such as in cases of new invasive populations or endangered species, carcasses may not produce detectable eDNA. In such scenarios, positive detections from field studies may be more confidently attributed to the presence of live organisms. We recommend that future studies should explore how biomass, flow, and differences in system (lentic vs. lotic) influence the ability to detect eDNA from carcasses.

Effects of temperature, salinity, and sediment organic carbon on methylmercury bioaccumulation in an estuarine amphipod.

Mercury (Hg) is a global contaminant that poses a human health risk in its organic form, methylmercury (MeHg), through consumption of fish and fishery products. Bioaccumulation of Hg in the aquatic environment is controlled by a number of factors expected to be altered by climate change. We examined the individual and combined effects of temperature, sediment organic carbon, and salinity on the bioaccumulation of MeHg in an estuarine amphipod, Leptocheirus plumulosus, when exposed to sediment from two locations in the Gulf of Maine (Kittery and Bass Harbor) that contained different levels of MeHg and organic carbon. Higher temperatures and lower organic carbon levels individually increased uptake of MeHg by L. plumulosus as measured by the biota-sediment accumulation factor (BSAF), while the effect of salinity on BSAF differed by sediment source. Multi-factor statistical modeling using all data revealed a significant interaction between temperature and organic carbon for both sediments, in which increased temperature had a negative effect on BSAF at the lowest carbon levels and a positive effect at higher levels. Our results suggest that increased temperature and carbon loading, of a magnitude expected as a result from climate change, could be associated with a net decrease in amphipod BSAF of 50 to 71%, depending on sediment characteristics. While these are only first-order projections, our results indicate that the future fate of MeHg in marine food webs is likely to depend on a number of factors beyond Hg loading.

Bioaccumulation of methylmercury in wood frogs and spotted salamanders in Vermont vernal pools.

Mercury (Hg) has accumulated in forested landscapes in the Northeastern U.S., and hotspots with enhanced deposition have been identified throughout the region. Due to a variety of favorable landscape characteristics, including relatively high dissolved organic carbon (DOC), fluctuating water levels, and low pH and dissolved oxygen, vernal pools provide ideal conditions for the conversion of Hg to its more toxic and bioavailable form, methylmercury (MeHg). Yet little is known about the concentrations, speciation, and bioavailability of Hg in vernal pools, or its bioaccumulation in vernal pool fauna and potential export into terrestrial systems. We investigated the role of forest cover type on the bioaccumulation of MeHg in wood frog (Lithobates sylvatica) and spotted salamander (Ambystoma maculatum) eggs, larvae, and adults, and investigated relationships among MeHg and water chemistry (pH, DOC). Water samples from pools located in coniferous stands had greater concentrations of THg and MeHg compared to deciduous pool water, and showed significant positive correlation to DOC (r = 0.683, P < 0.001) and correlated negatively with pH (r = -0.613, P < 0.001). Methylmercury levels in amphibian embryos were similar between the two species (L. sylvatica mean = 5.4 ng/g dw; A. maculatum mean = 3.5 ng/g dw). Concentrations of MeHg increased substantially in larvae, and were significantly greater in A. maculatum (mean = 237.6 ng/g ± 18.5 SE) than L. sylvatica larvae (62.5 ng/g ± 5.7 SE). Forest cover type did not explain variation in MeHg concentration among amphibian embryos or larvae. Methylmercury levels in adult tissue samples were significantly greater in A. maculatum (mean = 79.9 ng/g ± 8.9 SE) compared to L. sylvatica (mean = 47.7 ng/g ± 9.7 SE). This research demonstrates that vernal pools are important hotspots where amphibians bioaccumulate MeHg, which may then be transferred to terrestrial ecosystems. The abundance of amphibian larvae suggests they could be important bioindicators for monitoring MeHg loading and bioavailability.

Bioaccumulation of mercury and other metal contaminants in invasive lionfish (Pterois volitans/miles) from Curaçao.

A wide range of ecological and environmental factors influence metal bioaccumulation in fish. Studies of mercury and other metal contaminants in invasive Indo-Pacific lionfish are limited, yet consumption of the invasive predator is increasingly utilized as a management strategy. In this study, we examined the effects of body size, body condition, sex, trophic level, carbon source, diet, depth and capture location on mercury concentrations in lionfish collected from Curaçao. In addition, we examined whether or not a local petroleum refinery is the source of metal contamination in lionfish. Mercury concentrations ranged from 0.008 to 0.106 mg/kg and we found no effect of the petroleum refinery on metal bioaccumulation in lionfish. Low concentrations of metal contaminants indicate lionfish from Curaçao are safe for human consumption.

Influence of sample preparation on estuarine macrofauna stable isotope signatures in the context of contaminant bioaccumulation studies.

The ratios of stable isotopes of carbon and nitrogen provide important information on food sources of aquatic organisms and trophic structure of aquatic food webs. For many studies, trophic position and food source are linked to bioaccumulation and trophic transfer of contaminants from prey to predators. In these cases, it is useful to use measurements on whole organisms to make direct comparisons of contaminant bioaccumulation and food web attributes. There is a great deal of variation in methods used for stable isotope analysis, particularly in the selection of tissue type and sample preparation prior to stable isotope analysis. While there have been aquatic studies that examined methodological differences, few have focused on estuarine organisms. In this study, the effects of depuration and tissue dissection on the stable isotope enrichment of common estuarine invertebrates and fish were examined. Homogenized tissues of non-depurated whole organisms were compared to dissected muscle tissue or depurated whole organisms. A 24 h depuration did not change the mean δ15N and δ13C values for most species examined. Additionally, as expected, significant differences in carbon and nitrogen signatures were found when muscle tissues were compared to whole organisms. However, differences were small enough that food source as inferred by δ13C or trophic level as inferred from δ15N would not be inaccurately represented (differences of <1.9‰ for δ13C and <1.2‰ for δ15N). The results of this study suggest that for these common estuarine fish and macroinvertebrates, stable isotopes ratios of samples can be analyzed without depuration in the same way as samples for contaminant analysis, but differences in tissue types must be taken into account when combining data from different sources.

Effects of chemical management for invasive plants on the performance of Lithobates pipiens tadpoles.

Invasive plants impact amphibians by altering habitat, altering species interactions, and releasing potentially toxic secondary chemicals. Despite being costly and having the potential to affect nontarget wildlife, chemical management is commonly used to control invasive plants. Prior research has indicated that individual effects of invasive plants or herbicides can be harmful to aquatic organisms; however, information is lacking on the combined effect of these factors on amphibians. A laboratory experiment was performed to assess the impact of leachates of the invasive plants Eurasian watermilfoil (Myriophyllum spicatum) and European buckthorn (Rhamnus cathartica), the herbicide Renovate® 3 (triclopyr [3, 5, 6-trichloro-2-pyridinyloxyacetic acid]), and the combined effects of each plant leachate and the herbicide on the growth, morphology, and survival of northern leopard frog (Lithobates pipiens) tadpoles. No effects of treatment on survival were observed. Tadpole exposure to M. spicatum reduced body mass by 17%, exposure to R. cathartica increased body mass by 36%, and exposure to R. cathartica + low herbicide increased body mass by 38% (although only early in the experiment). Exposure to Renovate 3 induced a 16% and 29% decrease in tadpole size in lower (0.22 mg triclopyr active ingredient [a.i.]/L) and higher (0.92 mg triclopyr a.i./L) concentration treatments, respectively. Results from the present study highlight the importance of considering both individual and combined effects of invasive plants and herbicides because they may have different outcomes for tadpole growth and development. Environ Toxicol Chem 2017;36:2958-2964. © 2017 SETAC.

Gene expression profiling of brain samples from patients with Lewy body dementia.

Dementia with Lewy Bodies (DLB) is the second most common neurodegenerative disorder in the elderly. The development and progression of DLB remain unclear. In this study we used next generation sequencing to assess RNA expression profiles and cellular processes associated with DLB in the anterior cingulate cortex, a brain region affected by DLB pathology. The expression measurements were made in autopsy brain tissues from 8 DLB subjects and 10 age-matched controls using AmpliSeq technology with ion torrent sequencing. The analysis of RNA expression profiles revealed 490 differentially expressed genes, among which 367 genes were down-regulated and 123 were up-regulated. Functional enrichment analysis of genes differentially expressed in DLB indicated downregulation of genes associated with myelination, neurogenesis, and regulation of nervous system development. miRNA binding sites enriched in these mRNAs yielded a list of candidate miRNAs participating in DLB pathophysiology. Our study provides a comprehensive picture of gene expression landscape in DLB, identifying key cellular processes associated with DLB pathology.

RNA sequencing of transcriptomes in human brain regions: protein-coding and non-coding RNAs, isoforms and alleles.

BACKGROUND: We used RNA sequencing to analyze transcript profiles of ten autopsy brain regions from ten subjects. RNA sequencing techniques were designed to detect both coding and non-coding RNA, splice isoform composition, and allelic expression. Brain regions were selected from five subjects with a documented history of smoking and five non-smokers. Paired-end RNA sequencing was performed on SOLiD instruments to a depth of >40 million reads, using linearly amplified, ribosomally depleted RNA. Sequencing libraries were prepared with both poly-dT and random hexamer primers to detect all RNA classes, including long non-coding (lncRNA), intronic and intergenic transcripts, and transcripts lacking poly-A tails, providing additional data not previously available. The study was designed to generate a database of the complete transcriptomes in brain region for gene network analyses and discovery of regulatory variants. RESULTS: Of 20,318 protein coding and 18,080 lncRNA genes annotated from GENCODE and lncipedia, 12 thousand protein coding and 2 thousand lncRNA transcripts were detectable at a conservative threshold. Of the aligned reads, 52 % were exonic, 34 % intronic and 14 % intergenic. A majority of protein coding genes (65 %) was expressed in all regions, whereas ncRNAs displayed a more restricted distribution. Profiles of RNA isoforms varied across brain regions and subjects at multiple gene loci, with neurexin 3 (NRXN3) a prominent example. Allelic RNA ratios deviating from unity were identified in > 400 genes, detectable in both protein-coding and non-coding genes, indicating the presence of cis-acting regulatory variants. Mathematical modeling was used to identify RNAs stably expressed in all brain regions (serving as potential markers for normalizing expression levels), linked to basic cellular functions. An initial analysis of differential expression analysis between smokers and nonsmokers implicated a number of genes, several previously associated with nicotine exposure. CONCLUSIONS: RNA sequencing identifies distinct and consistent differences in gene expression between brain regions, with non-coding RNA displaying greater diversity between brain regions than mRNAs. Numerous RNAs exhibit robust allele selective expression, proving a means for discovery of cis-acting regulatory factors with potential clinical relevance.

Transcriptional Regulation of Antimicrobial Host Defense Peptides.

Host defense peptides (HDPs) are of either myeloid or epithelial origin with antimicrobial and immunomodulatory functions. Due to HDP's ability to physically disrupt bacterial cell membranes and profoundly regulate host innate and adaptive immunity, microbial resistance to these peptides is rare. As an important first line of defense, HDPs are mostly present in epithelial cells of the digestive, respiratory or urogenital tracts as well as in the granules of neutrophils, macrophages or intestinal secretory Paneth cells. HDPs are either directly released or inducibly expressed upon exposure to microbes or microbial products, although certain pathogens such as Shigella have evolved an ability to down-regulate HDP synthesis as an immune invasion strategy. Even if a majority of HDPs are induced by infection and inflammation, it is undesirable to augment HDP synthesis and host immunity using pathogen-associated molecular patterns because of an excessive inflammation that is usually accompanied. Recently, several different classes of small-molecule compounds have been identified with the capacity to specifically induce HDP synthesis without triggering extensive inflammatory response. A few HDPinducing compounds even synergize with each other in HDP induction. In this review, we summarized the recent progresses on transcriptional regulation of HDPs by infection and inflammation and by small-molecule compounds. We suggested the potential of dietary regulation of HDPs as a novel antibiotic-alternative strategy to antimicrobial therapy, as oral supplementation of HDP-inducing compounds has shown promise of preventing and controlling infections in humans and several animal species.

Deficits in Metacognitive Capacity Are Related to Subjective Distress and Heightened Levels of Hyperarousal Symptoms in Adults With Posttraumatic Stress Disorder.

Among persons with posttraumatic stress disorder (PTSD), the severity of symptoms and concurrent distress are not fully explained by trauma severity. Interest has consequently arisen in the psychological processes that cause distress and heighten PTSD symptoms. This study accordingly sought to examine whether differences in metacognitive capacity are related to levels of emotional distress, avoidance/numbing, and hyperarousal. Participants were 48 adults with a confirmed diagnosis of PTSD. Comparison groups included 51 adults with HIV and 183 with schizophrenia. Metacognition, emotion recognition, depression, and emotional distress and levels of avoidance/numbing and hyperarousal were assessed concurrently using the Metacognition Assessment Scale-Abbreviated, the Bell Lysaker Emotion Recognition Test, the Beck Depression Inventory, and the Clinician-Administered PTSD Scale. Results revealed that the PTSD group had better ratings of overall metacognitive capacity than the schizophrenia group and specifically poorer levels of metacognitive mastery, or the ability to use metacognitive knowledge to respond to challenges, than the HIV group. Within the PTSD group, poorer metacognitive mastery was linked with greater distress and higher hyperarousal when depression was controlled for statistically. Emotion recognition was not linked with distress or symptom severity. Results are consistent with models in which symptom severity in PTSD is related to the extent to which persons can use knowledge of themselves and others to find ways to respond to distress that match their own unique needs.

Cyclic AMP synergizes with butyrate in promoting ß-defensin 9 expression in chickens.

Host defense peptides (HDP) have both microbicidal and immunomodulatory properties. Specific induction of endogenous HDP synthesis has emerged as a novel approach to antimicrobial therapy. Cyclic adenosine monophosphate (cAMP) and butyrate have been implicated in HDP induction in humans. However, the role of cAMP signaling and the possible interactions between cAMP and butyrate in regulating HDP expression in other species remain unknown. Here we report that activation of cAMP signaling induces HDP gene expression in chickens as exemplified by ß-defensin 9 (AvBD9). We further showed that, albeit being weak inducers, cAMP agonists synergize strongly with butyrate or butyrate analogs in AvBD9 induction in macrophages and primary jejunal explants. Additionally, oral supplementation of forskolin, an adenylyl cyclase agonist in the form of a Coleus forskohlii extract, was found to induce AvBD9 expression in the crop of chickens. Furthermore, feeding with both forskolin and butyrate showed an obvious synergy in triggering AvBD9 expression in the crop and jejunum of chickens. Surprisingly, inhibition of the MEK-ERK mitogen-activated protein kinase (MAPK) pathway augmented the butyrate-FSK synergy, whereas blocking JNK or p38 MAPK pathway significantly diminished AvBD9 induction in chicken macrophages and jejunal explants in response to butyrate and FSK individually or in combination. Collectively, these results suggest the potential for concomitant use of butyrate and cAMP signaling activators in enhancing HDP expression, innate immunity, and disease resistance in both animals and humans.

Polymorphism in glutamate cysteine ligase catalytic subunit (GCLC) is associated with sulfamethoxazole-induced hypersensitivity in HIV/AIDS patients.

BACKGROUND: Sulfamethoxazole (SMX) is a commonly used antibiotic for prevention of infectious diseases associated with HIV/AIDS and immune-compromised states. SMX-induced hypersensitivity is an idiosyncratic cutaneous drug reaction with genetic components. Here, we tested association of candidate genes involved in SMX bioactivation and antioxidant defense with SMX-induced hypersensitivity. RESULTS: Seventy seven single nucleotide polymorphisms (SNPs) from 14 candidate genes were genotyped and assessed for association with SMX-induced hypersensitivity, in a cohort of 171 HIV/AIDS patients. SNP rs761142 T > G, in glutamate cysteine ligase catalytic subunit (GCLC), was significantly associated with SMX-induced hypersensitivity, with an adjusted p value of 0.045. This result was replicated in a second cohort of 249 patients (p = 0.025). In the combined cohort, heterozygous and homozygous carriers of the minor G allele were at increased risk of developing hypersensitivity (GT vs TT, odds ratio = 2.2, 95% CL 1.4-3.7, p = 0.0014; GG vs TT, odds ratio = 3.3, 95% CL 1.6 - 6.8, p = 0.0010). Each minor allele copy increased risk of developing hypersensitivity 1.9 fold (95% CL 1.4 - 2.6, p = 0.00012). Moreover, in 91 human livers and 84 B-lymphocytes samples, SNP rs761142 homozygous G allele carriers expressed significantly less GCLC mRNA than homozygous TT carriers (p < 0.05). CONCLUSIONS: rs761142 in GCLC was found to be associated with reduced GCLC mRNA expression and with SMX-induced hypersensitivity in HIV/AIDS patients. Catalyzing a critical step in glutathione biosynthesis, GCLC may play a broad role in idiosyncratic drug reactions.