Long-term climatic stability drives accumulation and maintenance of divergent freshwater fish lineages in a temperate biodiversity hotspot.

Anthropogenic climate change is forecast to drive regional climate disruption and instability across the globe. These impacts are likely to be exacerbated within biodiversity hotspots, both due to the greater potential for species loss but also to the possibility that endemic lineages might not have experienced significant climatic variation in the past, limiting their evolutionary potential to respond to rapid climate change. We assessed the role of climatic stability on the accumulation and persistence of lineages in an obligate freshwater fish group endemic to the southwest Western Australia (SWWA) biodiversity hotspot. Using 19,426 genomic (ddRAD-seq) markers and species distribution modelling, we explored the phylogeographic history of western (Nannoperca vittata) and little (Nannoperca pygmaea) pygmy perches, assessing population divergence and phylogenetic relationships, delimiting species and estimating changes in species distributions from the Pliocene to 2100. We identified two deep phylogroups comprising three divergent clusters, which showed no historical connectivity since the Pliocene. We conservatively suggest these represent three isolated species with additional intraspecific structure within one widespread species. All lineages showed long-term patterns of isolation and persistence owing to climatic stability but with significant range contractions likely under future climate change. Our results highlighted the role of climatic stability in allowing the persistence of isolated lineages in the SWWA. This biodiversity hotspot is under compounding threat from ongoing climate change and habitat modification, which may further threaten previously undetected cryptic diversity across the region.

Genomic structural variation in Barramundi Perch Lates calcarifer and potential roles in speciation and adaptation.

Advancements in genome sequencing and assembly techniques have increased the documentation of structural variants in wild organisms. Of these variants, chromosomal inversions are especially prominent due to their large size and active recombination suppression between alternative homokaryotypes. This suppression enables the 2 forms of the inversion to be maintained and allows the preservation of locally adapted alleles. The Barramundi Perch (BP; Lates calcarifer) is a widespread species complex with 3 main genetic lineages located in the biogeographic regions of Australia and New Guinea (AUS + NG), Southeast Asia (SEA), and the Indian Subcontinent (IND). BP are typically considered to be a protandrous sequential hermaphrodite species that exhibits catadromy. Freshwater occupancy and intraspecific variation in life history (e.g. partially migratory populations) exist and provide opportunities for strongly divergent selection associated with, for example, salinity tolerance, swimming ability, and marine dispersal. Herein, we utilize genomic data generated from all 3 genetic lineages to identify and describe 3 polymorphic candidate chromosomal inversions. These candidate chromosomal inversions appear to be fixed for ancestral variants in the IND lineage and for inverted versions in the AUS + NG lineage and exhibit variation in all 3 inversions in the SEA lineage. BP have a diverse portfolio of life history options that includes migratory strategy as well as sexual system (i.e. hermaphroditism and gonochorism). We propose that the some of the life history variabilities observed in BP may be linked to inversions and, in doing so, we present genetic data that might be useful in enhancing aquaculture production and population management.

Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae.

BACKGROUND: The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. RESULTS: We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary. CONCLUSIONS: Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

Pikeperch muscle tissues: a comparative study of structure, enzymes, genes, and proteins in wild and farmed fish.

Pikeperch (Sander lucioperca) is a freshwater species and an internationally highly demanded fish in aquaculture. Despite intensive research efforts on this species, fundamental knowledge of skeletal muscle biology and structural characteristics is missing. Therefore, we conducted a comprehensive analysis of skeletal muscle parameters in adult pikeperch from two different origins, wild-caught specimens from a lake and those reared in a recirculating aquaculture system. The analyses comprised the biochemical characteristics (nucleic acid, protein content), enzyme activities (creatine kinase, lactate dehydrogenase, NADP-dependent isocitrate dehydrogenase), muscle-specific gene and protein expression (related to myofibre formation, regeneration and permanent growth, muscle structure), and muscle fibre structure. The findings reveal distinct differences between the skeletal muscle of wild and farmed pikeperch. Specifically, nucleic acid content, enzyme activity, and protein expression varied significantly. The higher enzyme activity observed in wild pikeperch suggests greater metabolically activity in their muscles. Conversely, farmed pikeperch indicated a potential for pronounced muscle growth. As the data on pikeperch skeletal muscle characteristics is sparse, the purpose of our study is to gain fundamental insights into the characteristics of adult pikeperch muscle. The presented data serve as a foundation for further research on percids' muscle biology and have the potential to contribute to advancements and adaptations in aquaculture practices.

Transcriptome analyses in juvenile yellow perch (Perca flavescens) exposed in vivo to clothianidin and chlorantraniliprole: Possible sampling bias.

The St. Lawrence River is an important North American waterway that is subject to anthropogenic pressures including intensive urbanization, and agricultural development. Pesticides are widely used for agricultural activities in fields surrounding the yellow perch (Perca flavescens) habitat in Lake St. Pierre (Quebec, Canada), a fluvial lake of the river where the perch population has collapsed. Clothianidin and chlorantraniliprole were two of the most detected insecticides in surface waters near perch spawning areas. The objectives of the present study were to evaluate the transcriptional and biochemical effects of these two pesticides on juvenile yellow perch exposed for 28d to environmental doses of each compound alone and in a mixture under laboratory/aquaria conditions. Hepatic mRNA-sequencing revealed an effect of chlorantraniliprole alone (37 genes) and combined with clothianidin (251 genes), but no effects of clothianidin alone were observed in perch. Dysregulated genes were mostly related to circadian rhythms and to Ca2+ signaling, the latter effect has been previously associated with chlorantraniliprole mode of action in insects. Moreover, chronic exposure to clothianidin increased the activity of acetylcholinesterase in the brain of exposed fish, suggesting a potential non-target effect of this insecticide. Further analyses of three clock genes by qRT-PCR suggested that part of the observed effects of chlorantraniliprole on the circadian gene regulation of juvenile perch could be the result of time-of-day of sacrifice. These results provide insight into biological effects of insecticides in juvenile perch and highlight the importance of considering the circadian rhythm in experimental design and results analyses.

Paternal-effect-genes revealed through sperm cryopreservation in Perca fluviatilis.

Knowledge about paternal-effect-genes (PEGs) (genes whose expression in the progeny is influenced by paternal factors present in the sperm) in fish is very limited. To explore this issue, we used milt cryopreservation as a specific challenge test for sperm cells, thus enabling selection amidst cryo-sensitivity. We created two groups of Eurasian perch (Perca fluviatilis) as a model - eggs fertilized either with fresh (Fresh group) or cryopreserved (Cryo group) milt from the same male followed by phenotypic-transcriptomic examination of consequences of cryopreservation in obtained progeny (at larval stages). Most of the phenotypical observations were similar in both groups, except the final weight which was higher in the Cryo group. Milt cryopreservation appeared to act as a "positive selection" factor, upregulating most PEGs in the Cryo group. Transcriptomic profile of freshly hatched larvae sourced genes involved in the development of visual perception and we identified them as PEGs. Consequently, larvae from the Cryo group exhibited enhanced eyesight, potentially contributing to more efficient foraging and weight gain compared to the Fresh group. This study unveils, for the first time, the significant influence of the paternal genome on the development of the visual system in fish, highlighting pde6g, opn1lw1, and rbp4l as novel PEGs.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Riverscape genetics of the orangethroat darter complex.

Freshwater darters belonging to the orangethroat darter species complex, or Ceasia, are widely distributed in the Central and Southern United States, with ranges that span both glaciated and unglaciated regions. Up to 15 species have been recognized in the complex, with one, Etheostoma spectabile, having a widespread northern distribution and another, Etheostoma pulchellum, having a sizeable southern distribution. The other species in the complex have much more restricted distributions in unglaciated regions of the Central Highlands. We sampled 384 darters from 52 sites covering much of the range of Ceasia and evaluated patterns of genetic diversity, genetic structure, and pre- and post-glacial patterns of range contraction and expansion. We anticipated finding much stronger signals of genetic differentiation and diversification in unglaciated regions, given the higher species diversity and levels of endemism reported there. Surprisingly, microsatellite genotyping revealed two well-differentiated genetic clusters of E. spectabile in samples from glaciated regions, one confined to the Illinois River basin and another found in the Wabash drainage and Great Lakes tributaries. This suggests that there was expansion from two isolated glacial refugia, with little subsequent post-glacial gene flow. Fish collected from throughout the unglaciated region were less genetically differentiated. Fish assigned to Etheostoma burri and Etheostoma uniporum based on collection sites and morphological characters were not genetically differentiated from E. spectabile samples from the region. Hybridization and introgression occurring in the Central Highlands may confound genetic delineation of species in this region of high endemism and diversity.

DNA Methylation Profiling of Ovarian Tissue of Climbing Perch (Anabas testudienus) in Response to Monocrotophos Exposure.

Epigenetic modifications like DNA methylation can alter an organism's phenotype without changing its DNA sequence. Exposure to environmental toxicants has the potential to change the resilience of aquatic species. However, little information is available on the dynamics of DNA methylation in fish gonadal tissues in response to organophosphates. In the present work, reduced-representation bisulfite sequencing was performed to identify DNA methylation patterns in the ovarian tissues of Anabas testudienus exposed to organophosphates, specifically monocrotophos (MCP). Through sequencing, an average of 41,087 methylated cytosine sites were identified and distributed in different parts of genes, i.e., in transcription start sites (TSS), promoters, exons, etc. A total of 1058 and 1329 differentially methylated regions (DMRs) were detected as hyper-methylated and hypo-methylated in ovarian tissues, respectively. Utilizing whole-genome data of the climbing perch, the DMRs, and their associated overlapping genes revealed a total of 22 genes within exons, 45 genes at transcription start sites (TSS), and 218 genes in intergenic regions. Through gene ontology analysis, a total of 16 GO terms particularly involved in ovarian follicular development, response to oxidative stress, oocyte maturation, and multicellular organismal response to stress associated with reproductive biology were identified. After functional enrichment analysis, relevant DMGs such as steroid hormone biosynthesis (Cyp19a, 11-beta-HSD, 17-beta-HSD), hormone receptors (ar, esrrga), steroid metabolism (StAR), progesterone-mediated oocyte maturation (igf1ar, pgr), associated with ovarian development in climbing perch showed significant differential methylation patterns. The differentially methylated genes (DMGs) were subjected to analysis using real-time PCR, which demonstrated altered gene expression levels. This study revealed a molecular-level alteration in genes associated with ovarian development in response to chemical exposure. This work provides evidence for understanding the relationship between DNA methylation and gene regulation in response to chemicals that affect the reproductive fitness of aquatic animals.

Effects of flow velocity on the growth performance, antioxidant activity, immunity and intestinal health of Chinese Perch (Siniperca chuatsi) in recirculating aquaculture systems.

The cultivation of Chinese Perch (Siniperca chuatsi) in recirculating aquaculture systems (RASs) has become a common trend. To explore the effect of flow velocity on the growth performance, antioxidant activity, immunity and intestinal health of Chinese Perch in RAS, 240 Chinese Perch with an initial weight of 70.66 ± 0.34 g were selected and randomly divided into 4 groups: control group [CK, 0 body length per second (bl/s)], low flow velocity (LF, 0.4 bl/s), middle flow velocity (MF, 0.8 bl/s) and high flow velocity (HF, 1.2 bl/s) for a 56-days experiment. The results showed that the flow velocity significantly increased the weight gain rate and feed intake in Chinese Perch. At 1.2 bl/s, the flow velocity increased the intestinal trypsin content and intestinal villus length. Furthermore, the relative expression of appetite-related genes showed a tendency to increase, and the relative expression of appetite-inhibiting genes had a significant decrease in HF. Regarding immune-related indicators, the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST) were significantly higher in MF and HF. However, the activities of lysozyme (LZM) significantly decreased. Moreover, the activities of total superoxide dismutase (T-SOD) and catalase (CAT) were significantly higher in the CK group than in the other groups. Excessive flow velocity also caused the mRNA level of most immune-relevant genes to markedly decrease. With regard to intestinal health, the intestinal content sequencing results showed that MF could increase the intestinal diversity index of Chinese Perch. In addition, with increasing flow velocity, the relative abundance of Proteobacteria gradually increased, while the proportion of Firmicutes decreased. In conclusion, although the high flow velocity could promote growth, feeding, and digestion, inhibit fat deposition and increase the intestinal microbial abundance, the flow velocity caused stress, which leads to a decline in immunity and increases the death rate and the risk of intestinal disease in Chinese Perch. These findings provide theoretical support for the development of RASs for Chinese Perch.

Optimal sample type and number vary in small shallow lakes when targeting non-native fish environmental DNA.

Non-native fish have been shown to have deleterious impacts on freshwater ecosystems in New Zealand. Early detection is critical for their effective management. Traditional capture-based techniques may not detect newly introduced fish, especially if they are present in low abundance. Molecular techniques that target environmental DNA (eDNA) have been shown, in many instances, to be more sensitive, cost-effective and require lower sampling effort. However, appropriate sampling strategies are needed to ensure robust and interpretable data are obtained. In this study we used droplet digital PCR assays to investigate the presence of two non-native fish in New Zealand, the European perch (Perca fluviatilis) and rudd (Scardinius erythrophthalmus) in three small lakes. Samples were collected from water and surface sediment at near-shore and mid-lake sites. Probabilistic modelling was used to assess the occupancy of fish eDNA and develop guidance on sampling strategies. Based on the detection probability measures from the present study, at least six sites and five replicates per site are needed to reliably detect fish eDNA in sediment samples, and twelve sites with eight replicates per site for water samples. The results highlight the potential of developing monitoring and surveillance programs adapted to lakes, that include the use of assays targeting eDNA. This study focused on small shallow lakes, and it is likely that these recommendations may vary in larger, deeper, and more geomorphologically complex lakes, and this requires further research.

Comparative genomics revealed drastic gene difference in two small Chinese perches, Siniperca undulata and S. obscura.

Siniperca undulata and S. obscura (Centrarchiformes: Sinipercidae) are small Chinese perches, living in creeks and streams in southern China. While they have sympatric distribution and occupy similar macrohabitat, their body sizes and ecological niches have many differences. Determining the genome sequences of S. undulata and S. obscura would provide us an essential data set for better understanding their genetic makeup and differences that may play important roles in their adaptation to different niches. We determined the genome sequences of both S. undulata and S. obscura using 10× genomics technology and the next-generation sequencing. The assembled genomes of S. undulata and S. obscura were 744 and 733 Mb, respectively. Gene family analysis revealed that there were no overlap between S. undulata and S. obscura in terms of rapid expanding and rapid contracting genes families, which were related to growth, immunity, and mobility. Positive selection analyses also cooperated that the function of selected genes involve growth, athletic ability, and immunity, which may explain the preference of different niches by S. undulata and S. obscura. Pairwise sequentially Markovian coalescent analyses for the two species suggested that populations of both S. undulata and S. obscura showed a rising trend between 90 and 70 Ka probably due to the mild environment during the last interglacial period. A stage of population shrinking occurred from 70 to 20 Ka, which was in with the Tali glacial period in eastern China (57-16 Ka).

Corticosteroid plasma kinetics and gonadal receptor gene expression during the reproductive cycle in female Eurasian Perch: Investigation of the roles of corticosteroids in vitellogenesis.

To improve the quality of reproduction in Eurasian perch, Perca fluviatilis L., which is a promising candidate for Eurasian freshwater aquaculture that is currently cultivated in recirculating aquaculture systems (RAS), investigating the hormones that mediate and affect reproduction in this species is indispensable. The literature defines a group of four major corticosteroids (11-deoxycorticosterone, 11-deoxycortisol, corticosterone and cortisol) that might mediate critical stages of reproduction in female perch. Unfortunately, neither the basic roles nor the kinetics of these four corticosteroids throughout the reproductive cycle of female perch have been well defined to date. In this study, we therefore elucidated the plasma kinetics of these four corticosteroids during the reproductive cycle of domesticated female perch while monitoring the expression of the different receptors and enzymes that mediate their production and possible functions. Additionally, we performed an in vitro experiment during late vitellogenesis to investigate the possible direct roles of these steroids during that stage. Our results revealed that these four corticosteroids were detectable throughout the reproductive cycle, and the levels of most of them (11-deoxycorticosterone, 11-deoxycortisol, and cortisol) fluctuated significantly depending on the stage of reproduction. 11-Deoxycorticosterone and 11-deoxycortisol exhibited their highest levels, 1.8 ng/ml and 58 ng/ml, respectively, at the beginning of the reproductive cycle. By the end of the reproductive cycle, 11-deoxycortisol and cortisol plasma levels exhibited a surge, reaching 58 ng/ml and 150 ng/ml, respectively. During the perch reproductive cycle, the corticosteroid receptor complex is not regulated only at the hormone level, as the expression levels of all corticosteroid receptor genes showed a progressive and similar decline. In vitro exposure of vitellogenic oocytes to some of these corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) induced an increase in yolk globule diameter and a decrease in the density of yolk globules, which indicates the involvement of both of these hormones in yolk globule coalescence. Taken together, these results implicate corticosteroids in the reproductive cycle, although the related cellular mechanisms remain to be investigated.

Multiple lines and levels of evidence for avian zoochory promoting fish colonization of artificial lakes.

Understanding how obligate freshwater organisms colonize seemingly isolated ecosystems has long fascinated ecologists. While recent investigations reveal that fish eggs can survive the digestive tract of birds and successfully hatch once deposited, evidence for avian zoochory in natura is still lacking. Here, we used a 'multiple lines and levels of evidence' approach to demonstrate possible bird-mediated colonization of lakes by the European perch (Perca fluviatilis). We studied a set of newly-formed and isolated artificial lakes that the public is either prohibited to access because of gravel extraction or allowed to access (mainly for angling). The motivating observation is that a large proportion of prohibited-access lakes (greater than 80%) were colonized by European perch even though stocking by anglers and managers never occurred. Three supplementary lines of evidence supported avian zoochory. First, European perch spawning occurs when waterfowl abundance is very high. Second, European perch lays sticky eggs at shallow depths where they can be eaten by waterfowls or attached to their bodies. Third, genetic analyses suggested that European perch actually migrate among lakes, and that distances moved match with daily flight range of foraging waterfowl. Together, multiple lines of evidence point to avian zoochory as a probable pathway for fish colonizing remote or newly-formed freshwater ecosystems.

Phylogeography, hybridization, and species discovery in the Etheostoma nigrum complex (Percidae: Etheostoma: Boleosoma).

The history of riverine fish diversification is largely a product of geographic isolation. Physical barriers that reduce or eliminate gene flow between populations facilitate divergence via genetic drift and natural selection, eventually leading to speciation. For freshwater organisms, diversification is often the product of drainage basin rearrangements. In young clades where the history of isolation is the most recent, evolutionary relationships can resemble a tangled web. One especially recalcitrant group of freshwater fishes is the Johnny Darter (Etheostoma nigrum) species complex, where traditional taxonomy and molecular phylogenetics indicate a history of gene flow and conflicting inferences of species diversity. Here we assemble a genomic dataset using double digest restriction site associated DNA (ddRAD) sequencing and use phylogenomic and population genetic approaches to investigate the evolutionary history of the complex of species that includes E. nigrum, E. olmstedi, E. perlongum, and E. susanae. We reveal and validate several evolutionary lineages that we delimit as species, highlighting the need for additional work to formally describe the diversity of the Etheostoma nigrum complex. Our analyses also identify gene flow among recently diverged lineages, including one instance involving E. susanae, a localized and endangered species. Phylogeographic structure within the Etheostoma nigrum species complex coincides with major geologic events, such as parallel divergence in river basins during Pliocene inundation of the Atlantic coastal plain and multiple northward post-glacial colonization routes tracking river basin rearrangements. Our study serves as a nuanced example of how low dispersal rates coupled with geographic isolation among disconnected river systems in eastern North America has produced one of the world's freshwater biodiversity hotspots.

Detection of the threatened snail darter Percina tanasi in the Tennessee River system using environmental DNA.

The distribution of many fishes that occupy large rivers is poorly known, in part due to the difficulties of sampling for them. This is especially true for small-bodied or rare species, such as the snail darter Percina tanasi, 44, 469-488; 1976). This federally listed (threatened) species has a limited distribution in the Tennessee River system in Alabama and Tennessee, where it is known from a few large tributaries or small rivers. In Alabama, P. tanasi was previously known from only one locality, but has recently been found in two additional, widely separated systems. These new records raise questions regarding the accuracy of our current understanding of the range for this species. Particularly, is P. tanasi present throughout the main stem Tennessee River, and is this species dispersing into new areas from source populations in the river? To clarify the distribution of P. tanasi in Alabama, 83 unique sites were surveyed using environmental DNA analysis. This cost-effective detection tool reduces the difficulty associated with empirically sampling large rivers for small fishes. Approximately 42% of sites sampled were positive for P. tanasi DNA. This study confirmed the known localities of P. tanasi in the Bear Creek, Elk River and Paint Rock River. Several new localities were also discovered throughout the main stem Tennessee River and in Shoal Creek, near Florence, Alabama. These findings can inform biologists about where to prioritize conservation efforts and further could lead to studies assessing movement and relatedness between populations in this system.

Molecular Analysis and Sex-specific Response of the Hepcidin Gene in Yellow Perch (Perca Flavescens) Following Lipopolysaccharide Challenge.

Hepcidin antimicrobial peptide (hamp) is active in teleosts against invading pathogens and plays important roles in the stress and immune responses of finfish. The response of hamp gene was studied in yellow perch (yp) (Perca flavescens) challenged with lipopolysaccharides to understand if this immunity response is sex-specifically different. The cloned hamp gene consists of an open-reading frame of 273 bp and encodes a deduced protein of 90 amino acids (a.a.), which includes a signal peptide of 24 a.a., a pro-domain of 40 a.a. and a mature peptide of 26 a.a. Yp hamp involves 8 cysteine residues with 4 disulfide bonds, and a protein with an internal alpha helix flanked with C- and N-terminal random coils was modeling predicted. RT-qPCR was used to analyze the relative abundances (RAs) of hamp mRNA in the livers of juvenile female and male yellow perch challenged with lipopolysaccharide. The expression levels of hamp were significantly elevated by 3 h (RA = 7.3) and then peaked by 6 h (RA = 29.4) post-treatment in females but the peak was delayed to 12 h (RA = 65.4) post-treatment in males. The peak mRNA level of challenged males was shown 7.6-fold higher than females. The post-treatment responses in both genders decreased to their lowest levels by 24 h and 48 h. Overall, female perch had an earlier but less-sensitive response to the lipopolysaccharide challenge than male.

Incipient speciation in allopatric Etheostoma rupestre (Percidae: Etheostomatinae) lineages, with the description of three new subspecies.

In recent years, new species descriptions for the North American darters have proliferated. Most species concepts accepted by contemporary ichthyologists require that a valid species be both monophyletic and diagnoseable, yet many lineages exhibit modal or range differences in morphological characteristics without individuals being diagnosable. Such scenarios present difficulties with regards to proper taxonomic recognition of divergent lineages and often prohibit appropriate conservation action. Following the example of recent authors, we provide meristic, geometric morphometric, and pigmentation data to support the recognition of three subspecies of Etheostoma rupestre, a species endemic to the Mobile Basin. These morphological data cohere with previous genetic work for E. rupestre. The nominate subspecies Etheostoma rupetsre rupestre (Tsais Rock Darter) is endemic to the Tombigbee River and Black Warrior River watersheds in Alabama and Mississippi and is characterized by having lower numbers of lateral blotches, lower range and mean of lateral line scales, lower modal number of scales above the lateral line, and lower degrees of nape squamation than other subspecies. Etheostoma rupestre piersoni (Shamrock Darter), ssp. nov., is endemic to the Cahaba and Alabama River Watersheds in Alabama and is characterized by intermediate counts of lateral blotches and higher scale counts and nape squamation than E. r. rupestre. Etheostoma rupestre uphapeense (Jade Darter), ssp. nov., is restricted to several small, disjunct populations in the Coosa and Tallapoosa watersheds in Alabama, Georgia, and Tennessee. Etheostoma r. uphapeense is characterized by having a higher mean number of lateral blotches than both other subspecies and higher scale counts than E. r. rupestre. While E. r. rupestre and E. r. piersoni are widespread and abundant within their respective ranges, E. r. uphapeense has a disjunct range and is often uncommon where it occurs. Etheostoma r. uphapeense should be monitored where it occurs to discern population trends.

Recolonization origin and reproductive locations, but not isolation from the sea, lead to genetic structure in migratory lagoonal fishes.

The assessment of connectivity in marine ecosystems is a requirement to adequate fisheries management. In this study we have selected two commercially exploited migratory species, European perch (Perca fluviatilis) and European smelt (Osmerus eperlanus), to evaluate the connectivity between the Curonian Lagoon and the coastal Baltic Sea. Our results indicate that isolation between the coastal lagoon and the adjacent sea area does not lead to the formation of genetic structure in migratory fish species. However, both species do register subpopulations coexisting in the area without interbreeding. This indicates that the fisheries management for migratory fishes in coastal lagoons affects a wider area than just the coastal lagoon. European perch, being a postglacial recolonizer from various refugees, has four different subpopulations, while the mechanism that maintains this division remains unexplored. The feeding migrations of European perch to the coastal zone suggest that the reproduction might occur elsewhere and that the factors for genetic structure suggested at the Baltic Sea scale might operate during these migrations. For European smelt, we discuss the existence of two different ecotypes, one lagoonal and one diadromous, and the different registered spawning locations as explicative causes for the maintenance of two genetically divergent clusters. The lagoonal ecotype reproduces and spawns inside the Curonian Lagoon while the diadromous one lives in the open Baltic Sea, performing spawning migrations to the lagoon and the mouth of Nemunas river, thus, maintaining the genetic divergence among them. However, our results indicate that there are no differences in size between both clusters, while the lagoonal population is expected to be smaller, forbidding the determination of two genetically different ecotypes. We conclude that there are no geographically and genetically separated populations of these two species in the lagoon-sea- terrestrial inlets continuum, and unified stock management for the coastal Baltic Sea and the Curonian lagoon is required.