Mercury concentrations in Seaside Sparrows and Marsh Rice Rats differ across the Mississippi River Estuary.

Mercury (Hg) concentrations and their associated toxicological effects in terrestrial ecosystems of the Gulf of Mexico are largely unknown. Compounding this uncertainty, a large input of organic matter from the 2010 Deepwater Horizon oil spill may have altered Hg cycling and bioaccumulation dynamics. To test this idea, we quantified blood concentrations of total mercury (THg) in Seaside Sparrows (Ammospiza maritima) and Marsh Rice Rats (Oryzomys palustris) in marshes west and east of the Mississippi River in 2015 and 2016. We also tested for a difference in THg concentrations between oiled and non-oiled sites. To address the potential confounding effect of diet variation on Hg transfer, we used stable nitrogen (δ15N) and carbon (δ13C) isotope values as proxies of trophic position and the source of primary production, respectively. Our results revealed that five to six years after the spill, THg concentrations were not higher in sites oiled by the spill compared to non-oiled sites. In both species, THg was higher at sites east of the Mississippi River compared to control and oiled sites, located west. In Seaside Sparrows but not in Marsh Rice Rats, THg increased with δ15N values, suggesting Hg trophic biomagnification. Overall, even in sites with the most elevated THg, concentrations were generally low. In Seaside Sparrows, THg concentrations were also lower than previously reported in this and other closely related passerines, with only 7% of tested birds exceeding the lowest observed effect concentration associated with toxic effects across bird species (0.2 µg/g ww). The factors associated with geographic heterogeneity in Hg exposure remain uncertain. Clarification could inform risk assessment and future restoration and management actions in a region facing vast anthropogenic changes.

Immune genotypes, immune responses, and survival in a wild bird population.

Individuals vary in their immune genotype, inbreeding coefficient f, immune responses, survival to adulthood, and adult longevity. However, whether immune genes predict survival or longevity, whether such relationships are mediated through immune responses, and how f affects immune genotype remain unclear. We use a wild song sparrow (Melospiza melodia) population in which survival to adulthood, adult longevity, and f were measured precisely, and in which immune responses have previously been assessed. We investigate four toll-like receptor (TLR) and the major histocompatibility complex (MHC) class IIB exon 2 genes. We test whether immune genes predict fitness (survival to adulthood or adult longevity); whether immune genes predict immune response; whether immune response predicts fitness and whether fitness, immune responses, or immune genotypes are correlated with f. We find that survival to adulthood is not associated with immune gene variation, but adult longevity is decreased by high MHC allele diversity (especially in birds that were relatively outbred), and by the presence of a specific MHC supertype. Immune responses were affected by specific immune genotypes. Survival to adulthood and adult longevity were not predicted by immune response, implying caution in the use of immune response as a predictor for fitness. We also found no relationship between f and immune genotype. This finding indicates that immune gene associations with longevity and immune response are not artefacts of f, and suggests that pathogen-mediated selection at functional loci can slow the loss of genetic variation arising from genetic drift and small population size.

Depauperate major histocompatibility complex variation in the endangered reticulated flatwoods salamander (Ambystoma bishopi).

Reticulated flatwoods salamander (Ambystoma bishopi) populations began decreasing dramatically in the 1900s. Contemporary populations are small, isolated, and may be susceptible to inbreeding and reduced adaptive potential because of low genetic variation. Genetic variation at immune genes is especially important as it influences disease susceptibility and adaptation to emerging infectious pathogens, a central conservation concern for declining amphibians. We collected samples from across the extant range of this salamander to examine genetic variation at major histocompatibility complex (MHC) class Iα and IIß exons as well as the mitochondrial control region. We screened tail or toe tissue for ranavirus, a pathogen associated with amphibian declines worldwide. Overall, we found low MHC variation when compared to other amphibian species and did not detect ranavirus at any site. MHC class Iα sequencing revealed only three alleles with a nucleotide diversity of 0.001, while MHC class IIß had five alleles with a with nucleotide diversity of 0.004. However, unique variation still exists across this species' range with private alleles at three sites. Unlike MHC diversity, mitochondrial variation was comparable to levels estimated for other amphibians with nine haplotypes observed, including one haplotype shared across all sites. We hypothesize that a combination of a historic disease outbreak and a population bottleneck may have contributed to low MHC diversity while maintaining higher levels of mitochondrial DNA variation. Ultimately, MHC data indicated that the reticulated flatwoods salamander may be at an elevated risk from infectious diseases due to low levels of immunogenetic variation necessary to combat novel pathogens.

Identifying genome-wide immune gene variation underlying infectious disease in wildlife populations - a next generation sequencing approach in the gopher tortoise.

BACKGROUND: Infectious disease is the single greatest threat to taxa such as amphibians (chytrid fungus), bats (white nose syndrome), Tasmanian devils (devil facial tumor disease), and black-footed ferrets (canine distemper virus, plague). Although understanding the genetic basis to disease susceptibility is important for the long-term persistence of these groups, most research has been limited to major-histocompatibility and Toll-like receptor genes. To better understand the genetic basis of infectious disease susceptibility in a species of conservation concern, we sequenced all known/predicted immune response genes (i.e., the immunomes) in 16 Florida gopher tortoises, Gopherus polyphemus. All tortoises produced antibodies against Mycoplasma agassizii (an etiologic agent of infectious upper respiratory tract disease; URTD) and, at the time of sampling, either had (n = 10) or lacked (n = 6) clinical signs. RESULTS: We found several variants associated with URTD clinical status in complement and lectin genes, which may play a role in Mycoplasma immunity. Thirty-five genes deviated from neutrality according to Tajima's D. These genes were enriched in functions relating to macromolecule and protein modifications, which are vital to immune system functioning. CONCLUSIONS: These results are suggestive of genetic differences that might contribute to disease severity, a finding that is consistent with other mycoplasmal diseases. This has implications for management because tortoises across their range may possess genetic variation associated with a more severe response to URTD. More generally: 1) this approach demonstrates that a broader consideration of immune genes is better able to identify important variants, and; 2) this data pipeline can be adopted to identify alleles associated with disease susceptibility or resistance in other taxa, and therefore provide information on a population's risk of succumbing to disease, inform translocations to increase genetic variation for disease resistance, and help to identify potential treatments.

Purifying Selection in the Toll-Like Receptors of Song Sparrows Melospiza melodia.

Variation in immune gene sequences is known to influence resistance to infectious diseases and parasites, and hence survival and mate choice, across animal taxa. Toll-like receptors (TLRs) comprise one essential gene family in the vertebrate innate immune system and recognize evolutionarily conserved structures from all major microorganism classes. However, the causes and consequences of TLR variation in passerine birds remain largely unexplored. We examined 7 TLR genes in song sparrows (Melospiza melodia), a species that is studied across North America. We then examined sequences from 4 unduplicated TLRs (TLR1LB, TLR3, TLR4, and TLR15) from birds in 2 parts of the species' range (N = 27, N = 6), tested for evidence of selection, and conducted pilot analyses of the role of TLR heterozygosity in survival. We identified 45 SNPs: 19 caused changes in amino acid sequences and 2 of these were likely deleterious. We found no evidence of codon-level episodic positive selection but detected purifying selection at codons in all TLRs. Contrary to expectations we found no strong correlation between heterozygosity at TLRs and inbreeding coefficient f (estimate ± standard error [SE] = -0.68 ± 0.37, Radj2 = 0.08, F1,25 = 3.38, P = 0.08). In addition, pilot analyses revealed no relationship between TLR heterozygosity and survival (ß ± SE: 0.09 ± 2.00, P = 0.96), possibly due to small sample size. Further analyses of genetic diversity in TLRs are likely to advance understanding of the effects of innate immune gene diversity on the fitness and persistence of wild populations.

Neutral Genetic Processes Influence MHC Evolution in Threatened Gopher Tortoises (Gopherus polyphemus).

Levels of adaptive genetic variation influence how species deal with environmental and ecological change, but these levels are frequently inferred using neutral genetic markers. Major histocompatibility complex (MHC) genes play a key role in the adaptive branch of the immune system and have been used extensively to estimate levels of adaptive genetic variation. Parts of the peptide binding region, sites where MHC molecules directly interact with pathogen and self-proteins, were sequenced from a MHC class I (95/441 tortoises) and class II (245/441 tortoises) gene in threatened and nonthreatened populations of gopher tortoises (Gopherus polyphemus), and adaptive genetic variation at MHC genes was compared to neutral genetic variation derived from 10 microsatellite loci (441 tortoises). Genetic diversity at the MHC class II locus and microsatellites was greater in populations in the nonthreatened portion of the gopher tortoise's range (MHC class II difference in mean A = 8.11, AR = 0.79, HO = 0.51, and HE = 0.16; microsatellite difference in mean A = 1.05 and AR = 0.47). Only MHC class II sequences showed evidence of positive selection (dN/dS > 1, Z = 1.81, P = 0.04). Historical gene flow as estimated with Migrate-N was greater than recent migration estimated with BayesAss, suggesting that populations were better connected in the past when habitat was less fragmented. MHC genetic differentiation was correlated with microsatellite differentiation (Mantel r = 0.431, P = 0.001) suggesting neutral genetic processes are influencing MHC evolution, and advantageous MHC alleles could be lost due to genetic drift.

Mitochondrial DNA Variation in Southeastern Pre-Columbian Canids.

The taxonomic status of the red wolf (Canis rufus) is heavily debated, but could be clarified by examining historic specimens from the southeastern United States. We analyzed mitochondrial DNA (mtDNA) from 3 ancient (350-1900 year olds) putative wolf samples excavated from middens and sinkholes within the historic red wolf range. We detected 3 unique mtDNA haplotypes, which grouped with the coyote mtDNA clade, suggesting that the canids inhabiting southeastern North America prior to human colonization from Europe were either coyotes, which would vastly expand historic coyote distributions, an ancient coyote-wolf hybrid, or a North American evolved red wolf lineage related to coyotes. Should the red wolf prove to be a distinct species, our results support the idea of either an ancient hybrid origin for red wolves or a shared common ancestor between coyotes and red wolves.

Variations in prevalence of viral, bacterial, and rhizocephalan diseases and parasites of the blue crab (Callinectes sapidus).

Prevalence of blue crab diseases and parasites has not been consistently monitored in the Gulf of Mexico. To establish current prevalence levels and to more fully understand population dynamics, commercial landing trends, and effects of future natural and anthropogenic disasters on animal health, we measured the prevalence of white spot syndrome virus (WSSV), Loxothylacus texanus, shell disease, and Vibrio spp. in blue crabs collected from Louisiana in 2013 and the beginning of 2014. We used PCR to detect WSSV and L. texanus infections, visual gross diagnosis for L. texanus externae and shell disease, and standard microbiological culture techniques and biochemical testing for Vibrio spp. We found no crabs infected with WSSV or L. texanus. Absence of L. texanus parasitization was expected based on the sampled salinities and the sampling focus on large crabs. Shell disease was present at a level of 54.8% and was most prevalent in the winter and summer and least prevalent in the spring. Vibrio spp. were found in the hemolymph of 22.3% of the crabs and prevalence varied by site, season, and sex. Additionally, three of 39 crabs tested were infected with reo-like virus.

Prevalence and distribution of three protozoan symbionts in blue crab (Callinectes sapidus) populations across Louisiana, USA.

Louisiana has one of the largest blue crab (Callinectes sapidus) fisheries in the USA, but little is known about blue crab diseases, parasites, and symbionts in this area. In 2013-2014, large juvenile and adult blue crabs were collected at 4 diverse sites to determine the prevalence of the protozoan symbionts associated with black gill disease (Lagenophrys callinectes), buckshot crabs (Urosporidium crescens), and bitter crab disease (Hematodinium perezi). A high aggregate prevalence of L. callinectes (93.2%) was identified across all seasons at all 4 collection sites regardless of salinity. A moderately low aggregate prevalence of U. crescens (22.4%) was identified across all seasons and sites. Prevalence of U. crescens depended on site salinity, with only 10% of infections detected at sites with <6.3 ppt salinity, and no infections detected at the low salinity site. While L. callinectes and U. crescens are commensal parasites of blue crabs, infections can result in unmarketable and unappealing meat. In the Louisiana fishery, H. perezi has been blamed circumstantially for adult mortalities in the low salinity nearshore fishing grounds. Despite this, H. perezi was not detected in any of the large crabs sampled, even from the low salinity sites. The prevalence data reported here for these 3 protozoans are the first to include blue crabs sampled seasonally at multiple locations along the Louisiana coast over the period of a year.

Disease, parasite, and commensal prevalences for blue crab Callinectes sapidus at shedding facilities in Louisiana, USA.

Blue crab diseases, parasites, and commensals are not well studied in the Gulf of Mexico, and their prevalence rates have only been sporadically determined. Commercial soft shell shedding facilities in Louisiana experience high mortality rates of pre-molt crabs, and some of these deaths may be attributable to diseases or parasites. During the active shedding season in 2013, we determined the prevalence of shell disease, Vibrio spp., Lagenophrys callinectes, and Hematodinium perezi at 4 commercial shedding facilities along the Louisiana coast. We also detected Ameson michaelis and reo-like virus infections. Shell disease was moderately prevalent at rates above 50% and varied by shedding facility, collection month, and crab size. Vibrio spp. bacteria were prevalent in the hemolymph of 37% of the pre-molt crabs. Lagenophrys callinectes was highly prevalent in the pre-molt crabs, but because it is a commensal species, it may not cause high mortality rates. Hematodinium perezi was absent in all pre-molt crabs.