A Comparison of Non-Destructive Visceral Swab and Tissue Biopsy Sampling Methods for Genotyping-by-Sequencing in the Freshwater Mussel Fusconaia askewi.

Limiting harm to organisms caused by genetic sampling is an important consideration for rare species, and a number of non-destructive sampling techniques have been developed to address this issue in freshwater mussels. Two methods, visceral swabbing and tissue biopsies, have proven to be effective for DNA sampling, though it is unclear as to which method is preferable for genotyping-by-sequencing (GBS). Tissue biopsies may cause undue stress and damage to organisms, while visceral swabbing potentially reduces the chance of such harm. Our study compared the efficacy of these two DNA sampling methods for generating GBS data for the unionid freshwater mussel, the Texas pigtoe (Fusconaia askewi). Our results find both methods generate quality sequence data, though some considerations are in order. Tissue biopsies produced significantly higher DNA concentrations and larger numbers of reads when compared with swabs, though there was no significant association between starting DNA concentration and number of reads generated. Swabbing produced greater sequence depth (more reads per sequence), while tissue biopsies revealed greater coverage across the genome (at lower sequence depth). Patterns of genomic variation as characterized in principal component analyses were similar regardless of the sampling method, suggesting that the less invasive swabbing is a viable option for producing quality GBS data in these organisms.

Long-term patterns in inland fish kills associated with cold-shock and winter stress: a regional case study from Texas.

Colder water temperatures are generally regarded as a stressful period for fishes (i.e., winter stress syndrome), which can be exacerbated by cold-shock stress associated with major arctic freezes. Although cold-shock stress and mass mortalities are well documented for coastal marine fishes, few studies report the effects of winter stress or cold-shock stress on inland fishes. The purposes of this study were to describe patterns in inland fish mortalities associated with winter stress syndrome and with cold-shock stress in Texas as a regional example of inland fish mortalities associated with colder water temperatures. Using fish mortality reports (1969-2021) recorded by state agency biologists, colder water temperature mortalities occurred in 66% (N = 35) of the years, with greatest percentages of the reports occurring during three major arctic freezes in 1981, 1983 and 2021. The majority of reports were from urbanized counties (79%) and from lentic habitats (56%). Seventeen taxa and 1,021,217 individuals were estimated to be killed during the 53s years. Numbers of inland fish mortalities were greater during major arctic freeze years than non-major arctic freeze years, attributed primarily to mortalities of non-native fishes (e.g., blue tilapia Oreochromis aureus, suckermouth catfish Hypostomus plecostomus). Numbers of native fish mortalities, primarily clupeids and catostomids, were not different between major arctic freeze years and non-major arctic freezes. The 43,000 inland fish mortalities reported during major arctic freeze years are in stark contrast to the 35 million coastal marine fish mortalities. Proposed mechanisms to explain cold-shock mortalities in coastal waters (e.g., species within the northern extent of their range, lack of access to deeper water) are similar in inland waters, yet inland waters do not have the same level of mortalities. Consequently, the disparities between mortalities in coastal and inland waters are not readily discernable at this time.

Coping With Urban Habitats Via Glucocorticoid Regulation: Physiology, Behavior, and Life History in Stream Fishes.

As environments become urbanized, tolerant species become more prevalent. The physiological, behavioral and life-history mechanisms associated with the success of such species in urbanized habitats are not well understood, especially in freshwater ecosystems. Here, we examined the glucocorticoid (GC) profiles, life-history traits, and behavior of two species of fish across a gradient of urbanization to understand coping capacity and associated trade-offs. We studied the tolerant live-bearing Western Mosquitofish (Gambusia affinis) for two years and the slightly less tolerant, egg-laying, Blacktail Shiner (Cyprinella venusta) for one year. We used a water-borne hormone method to examine baseline, stress-induced, and recovery cortisol release rates across six streams with differing degrees of urbanization. We also measured life-history traits related to reproduction, and for G. affinis, we measured shoaling behavior and individual activity in a novel arena. Both species showed a trend for reduced stress responsiveness in more urbanized streams, accompanied by higher reproductive output. Although not all populations fit this trend, these results suggest that GC suppression may be adaptive for coping with urban habitats. In G. affinis, GC recovery increased with urbanization, and individuals with the lowest stress response and highest recovery had the greatest reproductive allotment, suggesting that rapid return to baseline GC levels is also an important coping mechanism. In G. affinis, urban populations showed altered life-history trade-offs whereas behavioral traits did not vary systematically with urbanization. Thus, these tolerant species of fish may cope with anthropogenically modified streams by altering their GC profiles and life-history trade-offs. These results contribute to understanding the mechanisms driving species-specific adaptations and thereby community structure in freshwater systems associated with land-use converted areas.

Asymmetric introgression between fishes in the Red River basin of Texas is associated with variation in water quality.

When ecologically divergent taxa encounter one another, hybrid zones can form when reproductive isolation is incomplete. The location of such hybrid zones can be influenced by environmental variables, and an ecological context can provide unique insights into the mechanisms by which species diverge and are maintained. Two ecologically differentiated species of small benthic fishes, the endemic and imperiled prairie chub, Macrhybopsis australis, and the shoal chub, Macrhybopsis hyostoma, are locally sympatric within the upper Red River Basin of Texas. We integrated population genomic data and environmental data to investigate species divergence and the maintenance of species boundaries in these two species. We found evidence of advanced-generation asymmetric hybridization and introgression, with shoal chub alleles introgressing more frequently into prairie chubs than the reciprocal. Using a Bayesian Genomic Cline framework, patterns of genomic introgression were revealed to be quite heterogeneous, yet shoal chub alleles were found to have likely selectively introgressed across species boundaries significantly more often than prairie chub alleles, potentially explaining some of the observed asymmetry in hybridization. These patterns were remarkably consistent across two sampled geographic regions of hybridization. Several environmental variables were found to significantly predict individual admixture, suggesting ecological isolation might maintain species boundaries.

Effect of preservation on fish morphology over time: Implications for morphological studies.

It has long been recognized that the process of preserving biological specimens results in alterations of body shape, though detailed studies examining the degree to which morphological changes occur throughout the preservation process are lacking. We utilize geometric morphometric analyses, an increasingly common tool for examining shape variation in a wide variety of biological disciplines, to examine the effects of formalin and ethanol preservation on the body shape of 10 freshwater fish species over time: from fresh specimens to eight weeks after preservation. We found significant changes in body shape among fresh and formalin fixed specimens. Furthermore, changes in body shape continue to occur after subsequent ethanol preservation. Two fish species collected at multiple localities show significant morphological differences for a limited number of morphometric characters. However, the significance, or lack thereof, often changed inconsistently from one stage of preservation to another. We conclude that morphometric analyses would ideally be performed on fresh specimens. However, recognizing that this is not always feasible, it is important to be aware of the morphometric changes that can occur during preservation.

Temperature-mediated feeding between spring-associated and riverine-associated congeners, with implications for community segregation.

Freshwater fish communities segregate along water temperature gradients attributed in part to temperature-mediated physiological processes that affect species fitness. In spring complexes of southwest USA, spring complexes with narrow range of water temperatures are dominated by a community of fishes (i.e., spring-associated fishes), whereas riverine habitats with wide-range of water temperatures are dominated by a different community of fishes (i.e., riverine-associated fishes). The purpose of this study was to test a prediction of the concept that temperature-mediated species performance is a mechanism in maintaining community segregation. We predicted that a spring-associated fish (Largespring Gambusia Gambusia geiseri) would feed first and more often in a pairing with a riverine-associated fish (Western Mosquitofish G. affinis) at an average spring temperature (23 °C) and that the riverine-associated fish would feed first and more often in a pairing with the spring-associated fish at a warm riverine temperature (30 °C). Among four trails consisting of 30 pairings, at the spring complex temperature (23 °C), Largespring Gambusia had a greater number of first feeds (mean ± 1 SD, 5.0 ± 0.82) than Western Mosquitofish (2.5 ± 1.73) and had greater mean number of total feeds (1.9 ± 0.31) than Western Mosquitofish (0.81 ± 0.70). At the riverine environment temperature (30 °C), Western Mosquitofish had a greater number of first feeds (5.25 ± 1.71) than Largespring Gambusia (2.5 ± 1.73) and had greater mean number of total feeds (2.78 ± 1.05) than Largespring Gambusia (0.94 ± 0.68). Our findings suggest that temperature-mediated species performance could be maintaining segregation between the two fish communities. This study benefits our understanding of distributional patterns and improves threat assessments of stenothermal aquatic organisms.

Occurrence and amount of microplastic ingested by fishes in watersheds of the Gulf of Mexico.

Ingestion of microplastics by fishes could be an emerging environmental crisis because of the proliferation of plastic pollution in aquatic environments. Microplastics in marine ecosystems are well documented, however only one study has reported percent occurrence of microplastics in freshwater fishes. The purpose of this study was to quantify the occurrences and types of microplastics ingested by fishes within several freshwater drainages of the Gulf of Mexico and an estuary of the Gulf of Mexico. Among 535 fishes examined in this study, 8% of the freshwater fishes and 10% of the marine fishes had microplastics in their gut tract. Percentage occurrence of microplastics ingested by fishes in non-urbanized streams (5%) was less than that of one of the urbanized streams (Neches River; 29%). Percent occurrence of microplastics by habitat (i.e., benthic, pelagic) and trophic guilds (herbivore/omnivore, invertivore, carnivore) were similar. Low but widespread occurrences among drainages, habitat guilds, and trophic guilds indicate proliferation of plastic pollution within watersheds of the Gulf of Mexico, but consequences to fish health are unknown at this time.

Can Species Distribution Models Aid Bioassessment when Reference Sites are Lacking? Tests Based on Freshwater Fishes.

Recent literature reviews of bioassessment methods raise questions about use of least-impacted reference sites to characterize natural conditions that no longer exist within contemporary landscapes. We explore an alternate approach for bioassessment that uses species site occupancy data from museum archives as input for species distribution models (SDMs) stacked to predict species assemblages of freshwater fishes in Texas. When data for estimating reference conditions are lacking, deviation between richness of contemporary versus modeled species assemblages could provide a means to infer relative biological integrity at appropriate spatial scales. We constructed SDMs for 100 freshwater fish species to compare predicted species assemblages to data on contemporary assemblages acquired by four independent surveys that sampled 269 sites. We then compared site-specific observed/predicted ratios of the number of species at sites to scores from a multimetric index of biotic integrity (IBI). Predicted numbers of species were moderately to strongly correlated with the numbers observed by the four surveys. We found significant, though weak, relationships between observed/predicted ratios and IBI scores. SDM-based assessments identified patterns of local assemblage change that were congruent with IBI inferences; however, modeling artifacts that likely contributed to over-prediction of species presence may restrict the stand-alone use of SDM-derived patterns for bioassessment and therefore warrant examination. Our results suggest that when extensive standardized survey data that include reference sites are lacking, as is commonly the case, SDMs derived from generally much more readily available species site occupancy data could be used to provide a complementary tool for bioassessment.

Salmonellae in fish feces analyzed by in situ hybridization and quantitative polymerase chain reaction.

The potential of fish to transfer salmonellae from heterogeneous aquatic biofilms into feces was assessed in controlled aquarium studies with Suckermouth Catfish Hypostomus plecostomus and with biofilms inoculated with salmonellae. Neither the presence of catfish nor inoculation with salmonellae had detectable effects on the abundance of the microbial community. Densities of the microbial community were about 10(5) cells/mL in the water during a 1-week period, whereas densities of the microbial community increased 10-fold (10(6) to 10(7) cells/mg) in catfish feces during the same period. Salmonellae were detected by both quantitative polymerase chain reaction (qPCR) and situ hybridization in water samples immediately after inoculation, in numbers of about 10(4) cells/mL, representing up to 20% of the cells of the microbial community. Numbers decreased by three orders of magnitude within the first 3 d of the study, which represented only 0.01% of the community, and became undetectable after day 5. In catfish feces, numbers of Salmonella initially increased to up to 6% of the cells of the community but then declined. These results suggest that Salmonella are not biomagnified during gut passage, and thus, fish only provide a means for the translocation of this pathogen.

Metacercarial distribution of Centrocestus formosanus among fish hosts in the Guadalupe River drainage of Texas.

We examined the gills of wild fish collected from central Texas for Centrocestus formosanus metacercariae to determine whether this temperature-restricted parasite had invaded the thermally dynamic Guadalupe River via an introduced population in its thermally stable tributary, the Comal River. We collected fish from three sites in the Guadalupe River near its confluence with the Comal River (upstream, at, and downstream) and one site in the Comal River. Centrocestus formosanus infected 14 of the 25 species examined (56.0%) and 171 of the individual fish (27.1%). Several of the infected fish represent new host records for the parasite, and two are listed as species of special concern by the state of Texas. Mean metacercarial intensities varied from 8 to 616 among species, and the highest recorded intensity was greater than 800 in two Guadalupe roundnose minnow Dionda nigrotaeniata. Among the 24 species examined from the Guadalupe River, 11 (45.8%) were infected with C. formosanus. Thorough surveys at the study sites yielded no living specimens of the first obligate intermediate snail host (red-rim melania Melanoides tuberculatus), which must be present to perpetuate the parasite. Thus, the infections were probably due to drifting cercariae that had been shed into the water column upstream of the study area in the Comal River. We therefore investigated spatial patterns in cercarial acquisition using caged fish to determine whether drifting cercariae were present in the water column at the study sites. Of 57 uninfected blacktail shiners Cyprinella venusta exposed to Guadalupe River water downstream from and at the confluence, 52 (91.2%) became infected with C. Formosanus metacercariae at a mean rate of 4 metacercariae/d. This finding extends the known geographic range of this invasive exotic parasite and is the first report of the life cycle being advanced in the fish assemblage of a thermally variable temperate stream in the USA.

Mercury contamination of the fish community of a semi-arid and arid river system: spatial variation and the influence of environmental gradients.

Mercury (Hg) contamination of aquatic ecosystems is a global environmental problem. Data are abundant on Hg contamination and factors that affect its bioaccumulation in lake communities, but comparatively little information on riverine ecosystems exists. The present study examines fish Hg concentrations of the Lower Rio Grande/Rio Bravo del Norte drainage, Texas, USA and several of its major tributaries in order to assess whether spatial variation occurs in fish Hg concentrations in the drainage and if patterns of Hg contamination of fish are related to gradients in environmental factors thought to affect Hg concentrations in fish communities. Fish, invertebrates, sediments, and water quality parameters were sampled at 12 sites along the lower Rio Grande/Rio Bravo del Norte drainage multiple times over a one-year period. Spatial variation was significant in fish Hg concentrations when fish were grouped by literature-defined trophic guilds or as stable isotope-defined trophic levels, with highest concentrations found in the Big Bend region of the drainage. Mercury in fish in most trophic guilds and trophic levels were positively related to environmental factors thought to affect Hg in fish, including water column dissolved organic carbon (DOC) and sediment Hg concentrations. It is likely that fish Hg concentrations in the Big Bend region are relatively high because this section of the river has abundant geologic Hg sources and environmental conditions which may make it sensitive to Hg inputs (i.e., high DOC, variable water levels). Results from the present study indicate that Hg contamination of the Rio Grande/Rio Bravo del Norte has substantial implications for management and protection of native small-bodied obligate riverine fish, many of which are imperiled.

Spatial and temporal distribution of the Asian fish tapeworm Bothriocephalus acheilognathi (Cestoda: Bothriocephalidea) in the Rio Grande (Río Bravo del Norte).

Recent collections of the Asian fish tapeworm Bothriocephalus acheilognathi in the Rio Grande have raised concern about the potential impacts on Rio Grande endemic and imperiled fishes. The objectives of this study were to determine distribution and definitive hosts of the Asian fish tapeworm within the Rio Grande drainage and to quantify occurrences and abundances. In total, 1,992 fish spanning 11 families were collected and examined for Asian fish tapeworms in the Rio Grande and the Pecos and Devils rivers. The parasite was collected from red shiners Cyprinella lutrensis, Tamaulipas shiners Notropis braytoni, sand shiners N. stramineus, river carpsuckers Carpiodes carpio, plains killifish Fundulus zebrinus, western mosquitofish Gambusia affinis, blue suckers Cycleptus elongatus, blacktail shiners Cyprinella venusta, proserpine shiners Cyprinella proserpina, and Manantial roundnose minnow Dionda argentosa, with the latter four species being new host records. Monthly collections of red shiners from Big Bend National Park exhibited prevalence levels above 15% in January-March and December and below 10% during April-June and October. With over 50% of the Rio Grande icthyofauna in Texas considered imperiled, the occurrence and pathological effects of the Asian fish tapeworm in combination with reduced water quantity and quality and increased habitat fragmentation are of concern for these taxa.

Detection of salmonellae from fish in a natural river system.

Sediment, water, and fish gut samples taken at three sites near the headwaters of the San Marcos River, Texas, were analyzed for salmonellae Salmonella spp. by culture and molecular techniques. While enrichment cultures from sediment and water samples from the two uppermost sites were negative for salmonellae in polymerase chain reaction analyses, both sediment and water samples were positive at the downstream site. At all sites, salmonellae were present in the guts of different fishes (e.g., largemouth bass Micropterus salmoides, channel catfish Ictalurus punctatus, common carp Cyprinus carpio, and suckermouth catfish Hypostomus plecostomus). The highest percentage of detection (33% of analyzed fish) occurred at the downstream site, whereas detection percentages at the upper two sites were 18% and 17%. Detection of salmonellae was usually limited to one segment of the gut (i.e., upper or lower part). Serovars were highly variable among individuals and differed between the upper and lower gut in the only individual (a common carp) that had salmonellae in both gut segments. In situ hybridization demonstrated that salmonellae were normally associated with particulate material in the gut and occurred in highly variable numbers ranging from an occasional organism to a majority of the gut microbe population. These results demonstrate the presence of different serovars of potentially human pathogenic salmonellae among four ecologically distinct fishes within natural environments. They also suggest that salmonellae are not components of the indigenous microbial community in fish intestines but rather are ingested with particulate material.

First record of Bothriocephalus acheilognathi in the Rio Grande with comparative analysis of ITS2 and V4-18S rRNA gene sequences.

Bothriocephalus acheilognathi is an introduced tapeworm in North America often reported as a serious ecological threat to native fishes. In this paper, we report the first record of B. acheilognathi in the Big Bend region of the Rio Grande in Texas (known as the Río Bravo del Norte in Mexico). Identification of B. acheilognathi was confirmed by morphologic and genetic techniques (sequences of ITS2 and V4-18S rRNA genes). Its prevalence was 27% and its intensity ranged from 1 to 5 individuals in a January 2006 collection of 115 red shiners Cyprinella lutrensis. In addition, it was found in the Tamaulipas shiner Notropis braytoni, a Rio Grande endemic and a new host record. The occurrence of B. acheilognathi might have negative ecological impacts on endemic fishes in the Rio Grande. Several of the fishes that could serve as definitive hosts are of conservation concern. Its occurrence also might affect the success of reintroducing the Rio Grande silvery minnow Hybognathus amarus, which is federally listed as endangered, in this portion of the Rio Grande.