Fundulus Grandis Research Articles

Resilience to changes in salinity and predator diversity in representative Gulf of Mexico estuarine fish predator-prey interactions

Predators strongly influence ecological communities and subsequent ecosystem function by directly consuming prey individuals and indirectly by inciting alterations in prey behavior, termed non-consumptive effects (NCEs). Gulf of Mexico estuaries support complex food webs structured by numerous species interactions maintained in constant environmental flux from variation in salinity. Environmental variation presents a source of physiological stress for estuarine fish and plays a role in their distributions and subsequent interactions. Here, we present the results of a mesocosm experiment examining if acute changes in salinity disrupt NCEs of different predators on estuarine prey. We exposed Gulf killifish (Fundulus grandis) to visual and/or olfactory cues from a common estuarine predator, red drum (Sciaenops ocellatus), and from a freshwater predator that is a transient species in lower portions of estuaries, largemouth bass (Micropterus salmoides), under either decreasing, constant, or increasing salinity. Killifish decreased shoal size and movement in response to olfactory cues or both visual and olfactory cues compared to when they were exposed to no cues or visual cues, demonstrating variable perceived risk dependent on cue type. Behavioral response did not vary by predator species, implying a generalized response to different fish predators. Prey did not change behavior when exposed to decreasing salinity compared to constant salinity. However, prey movement increased with increasing salinity, indicating that salinity changes may provide cues to search for different habitats instead of a stressor. Our experiment indicates that estuarine predator-prey interactions may be resilient to acute salinity changes given the broad tolerances of euryhaline species.

Live‐bait industry as a pathway for movement of nonnative and invasive species: Implications for conservation of native Texas fishes

AbstractObjectiveLive‐bait species are widely used in recreational fishing throughout the United States. However, the use of invasive fish species as live bait has been identified as a common pathway for their introduction into aquatic ecosystems. We assessed the role that the live‐bait industry has in facilitating the introduction of nonnative and invasive species into Texas rivers. We focused on two invasive cyprinodontid species, the Sheepshead Minnow Cyprinodon variegatus and Gulf Killifish Fundulus grandis, which are believed to have been introduced into inland Texas rivers via bait‐bucket releases by anglers.MethodsWe conducted telephone and in‐person surveys of live‐bait shops in Texas on a seasonal basis (summer, fall, winter, spring) for 1 year. Locally owned bait shops and regional aquaculture facilities and hatcheries were included in the surveys.ResultSurveys did not reveal the presence of these invasive cyprinodontids in inland bait shops but did reveal the sale of nonnative Goldfish Carassius auratus and their black color morph, the black salty goldfish. Surveys conducted among inland and coastal live bait shops confirmed the sale of only one nonnative bait fish and six bait items native only to certain regions of Texas. Telephone surveys with aquaculture industry experts revealed that the aquaculture industry was also facilitating the movement of nonnative game and nongame fish species throughout the state.ConclusionGiven the potential for nonnative and invasive species to negatively affect recipient aquatic ecosystems, the continued distribution of such species within the aquaculture industry represents a potential threat in Texas. Our findings highlight the need for close monitoring of the live‐bait industry in the state to prevent further introductions into inland waters and reduce potential ecological risks to native fish populations and overall ecosystem functions and services.

Method dependency of maximum oxygen uptake rate and its repeatability in the Gulf killifish, Fundulus grandis.

The maximum rate at which fish can take up oxygen from their environment to fuel aerobic metabolism is an important feature of their physiology and ecology. Methods to quantify maximum oxygen uptake rate (ṀO2), therefore, should reliably and reproducibly estimate the highest possible ṀO2 by an individual or species under a given set of conditions (peak ṀO2). This study determined peak ṀO2 and its repeatability in Gulf killifish, Fundulus grandis, subjected to three methods to elevate metabolism: swimming at increasing water speeds, during recovery after an exhaustive chase, and after ingestion of a large meal. Estimates of peak ṀO2 during swimming and after an exhaustive chase were repeatable across two trials, whereas peak ṀO2 after feeding was not. Peak ṀO2 determined by the three methods was significantly different from one another, being highest during swimming, lowest after an exhaustive chase, and intermediate after feeding. In addition, peak ṀO2 during recovery from an exhaustive chase depended on the length of time of recovery: in nearly 60% of the trials, values within the first hour of the chase were lower than those measured later. A novel and important finding was that an individual's peak ṀO2 was not repeatable when compared across methods. Therefore, the peak ṀO2 estimated for a group of fish, as well as the ranking of individual ṀO2 within that group, depends on the method used to elevate aerobic metabolism.

Hypoxia-inducible factor 1 alpha protein increases without changes in mRNA during acute hypoxic exposure of the Gulf killifish, Fundulus grandis.

The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses of animals to low oxygen. While the hypoxia-responsiveness of HIF1 is generally attributed to the stabilization of the alpha protein subunit (HIF1α) at low oxygen, several studies on fish report increased tissue levels of HIF1A mRNA during hypoxia, suggesting transcriptional regulation. In the current study, HIF1α protein and HIF1A mRNA were determined in parallel in tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2l-1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1A mRNA levels were unaffected by hypoxia in any tissue. Moreover, HIF1α protein and HIF1A mRNA levels in the same tissues were not correlated with one another during either normoxia or hypoxia. Hence, an increase in HIF1α protein does not depend upon an increase in HIF1A mRNA during acute exposure to low oxygen in this species. The results support the widely accepted mechanism of post-translational protein stabilization, rather than new transcription, during the initial response of fish to hypoxia.

Comparative Phylogeography, Historical Demography, and Population Genetics of Three Common Coastal Fauna in Spartina Marshes of the Northwestern Gulf of Mexico

Coastal wetlands worldwide are experiencing high rates of loss and degradation that may lead to a reduction in diversity in faunal populations. Since salt marsh habitats are subject to a multitude of stressors, evaluations of the genetic diversity, connectivity, and potential resilience of faunal communities within salt marsh habitats are relevant. This study characterizes mitochondrial DNA (mtDNA) diversity for three common faunal residents of salt marshes along the northern Gulf of Mexico. Gulf Killifish (Fundulus grandis) samples were characterized for 1077 bp of the concatenated nucleotide sequence corresponding to the Control Region and Nitrogen Dehydrogenase, Subunits 2 and 5. Daggerblade grass shrimp (Palaemon pugio) samples were characterized using 466 bp of 16sRNA sequence, and phloem-feeding planthoppers (Prokelisia marginata) were characterized using 372 bp of Cytochrome c Oxidase Subunit I (COI) sequence. For F. grandis, our data revealed high levels of haplotypic diversity, evidence of isolation by distance (IBD), and regional population structuring associated with the distribution of two distinct phylogroups and distinct historical demography signatures. P. pugio and P. marginata displayed low levels of haplotypic diversity and evidence of population structure, but both appear to contain only snapshots of the total potential diversity for these species in the Gulf of Mexico. Greater resolution of the patterns of historical demography of Gulf Killifish may be obtained in future studies by including localities from Florida and Mexico. For both P. pugio and planthoppers, future studies would benefit from the characterization of genetic markers with a higher degree of polymorphism. We conclude that despite these three species inhabiting the same habitats along the same stretch of coast, each is subject to a different combination of evolutionary forces, and this study was able to reconstruct differences in how the genetic variation in each of these species emerged, and how it is maintained.

Salinity fluctuations due to urbanization of coastal environments and their potential effect on the genetic divergence of the Gulf killifish (Fundulus grandis)

Salinity fluctuations due to urbanization of coastal environments and their potential effect on the genetic divergence of the Gulf killifish (Fundulus grandis)

Transgenerational effects of parental crude oil exposure on the morphology of adult Fundulus grandis.

The current study involved exposing adult F0 Gulf killifish (Fundulus grandis) to Macondo-252 oil for 36 to 44 days and assessing the effects of this oiling on the swimming performance and morphology in two generations of progeny reared in clean water. Following exposure to oil, the F0 fish were used as broodstock to generate four lineages of F1 fish using a full-matrix mating design derived from the gametes of clean and oil-exposed parents. Later, the four lineages of F1 fish were used as broodstock to create an F2 generation of the same four lineages. We found few differences in embryonic outcome (% dead,% hatched, and% unhatched) in any of the four lineages of F1 and F2 fish. However, as adults, F1 and F2 fish derived from oil-exposed males from the F0 generation had significantly lower critical swimming speeds (Ucrit) than both the control and maternally oil-exposed lineages. Additionally, progeny of oil-exposed fish had altered body shape based on the statistical analysis of two-dimensional landmark-based geometric morphometrics. Fish from oil-exposed lineages showed increased body depth, altered spinal curvature, and changes in the upward angle of projection of the head. Both generations had a significant main effect of maternal and paternal oil exposure on shape; however, F0 paternal oil exposure explained more of the variance in shape across both generations relative to F0 maternal exposure. Our findings demonstrate that parental exposure to oil can impact the shape and aerobic swimming capacity of offspring for at least two generations after the original paternal oiling.

Interindividual variation in maximum aerobic metabolism varies with gill morphology and myocardial bioenergetics in Gulf killifish.

This study asked whether interindividual variation in maximum and standard aerobic metabolic rates of the Gulf killifish, Fundulus grandis, correlates with gill morphology and cardiac mitochondrial bioenergetics, traits reflecting critical steps in the O2 transport cascade from the environment to the tissues. Maximum metabolic rate (MMR) was positively related to body mass, total gill filament length and myocardial oxygen consumption during maximum oxidative phosphorylation (multiple R2=0.836). Standard metabolic rate (SMR) was positively related to body mass, total gill filament length and myocardial oxygen consumption during maximum electron transport system activity (multiple R2=0.717). After controlling for body mass, individuals with longer gill filaments, summed over all gill arches, or greater cardiac respiratory capacity had higher whole-animal metabolic rates. The overall model fit and the explanatory power of individual predictor variables were better for MMR than for SMR, suggesting that gill morphology and myocardial bioenergetics are more important in determining active rather than resting metabolism. After accounting for body mass, heart ventricle mass was not related to variation in MMR or SMR, indicating that the quality of the heart (i.e. the capacity for mitochondrial metabolism) was more influential than heart size. Finally, the myocardial oxygen consumption required to offset the dissipation of the transmembrane proton gradient in the absence of ATP synthesis was not correlated with either MMR or SMR. The results support the idea that interindividual variation in aerobic metabolism, particularly MMR, is associated with variation in specific steps in the O2 transport cascade.

In vitro-in vivo biotransformation and phase I metabolite profiling of benzo[a]pyrene in Gulf killifish (Fundulus grandis) populations with different exposure histories

Chronic exposure to pollution may lead populations to display evolutionary adaptations associated with cellular and physiological mechanisms of defense against xenobiotics. This could result in differences in the way individuals of the same species, but inhabiting different areas, cope with chemical exposure. In the present study, we explore two Gulf killifish (Fundulus grandis) populations with different exposure histories for potential differences in the biotransformation of benzo[a]pyrene (BaP), and conduct a comparative evaluation of in vitro and in vivo approaches to describe the applicability of new approach methodologies (NAMs) for biotransformation assessments. Pollution-adapted and non-adapted F. grandis were subjected to intraperitoneal (IP) injections of BaP in time-course exposures, prior to measurements of CYP biotransformation activity, BaP liver concentrations, and the identification and quantification of phase I metabolites. Additionally, substrate depletion bioassays using liver S9 fractions were employed for measurements of intrinsic hepatic clearance and to evaluate the production of metabolites in vitro. Pollution-adapted F. grandis presented significantly lower CYP1A activity and intrinsic clearance rates that were 3 to 4 times lower than non-adapted fish. The metabolite profiling of BaP showed the presence of 1‑hydroxy-benzo[a]pyrene in both the in vitro and in vivo approaches but with no significant population differences. Contrarily, 9‑hydroxy-benzo[a]pyrene and benzo[a]pyrene-4,5-dihydrodiol, only identified through the in vivo approach, presented higher concentrations in the bile of pollution-adapted fish relative to non-adapted individuals. These observations further the understanding of the evolutionary adaptation of F. grandis inhabiting heavily polluted environments in the Houston Ship Channel, TX, USA, and highlight the need to consider the evolutionary history of populations of interest during the implementation of NAMs.

Prior exposure to weathered oil influences foraging of an ecologically important saltmarsh resident fish.

Estuarine ecosystem balance typically relies on strong food web interconnectedness dependent on a relatively low number of resident taxa, presenting a potential ecological vulnerability to extreme ecosystem disturbances. Following the Deepwater Horizon (DwH) oil spill disaster of the northern Gulf of Mexico (USA), numerous ecotoxicological studies showed severe species-level impacts of oil exposure on estuarine fish and invertebrates, yet post-spill surveys found little evidence for severe impacts to coastal populations, communities, or food webs. The acknowledgement that several confounding factors may have limited researchers’ abilities to detect negative ecosystem-level impacts following the DwH spill drives the need for direct testing of weathered oil exposure effects on estuarine residents with high trophic connectivity. Here, we describe an experiment that examined the influence of previous exposure to four weathered oil concentrations (control: 0.0 L oil m−2; low: 0.1 L oil m−2; moderate: 0.5–1 L oil m−2; high: 3.0 L oil m−2) on foraging rates of the ecologically important Gulf killifish (Fundulus grandis). Following exposure in oiled saltmarsh mesocosms, killifish were allowed to forage on grass shrimp (Palaeomonetes pugio) for up to 21 h. We found that previous exposure to the high oil treatment reduced killifish foraging rate by ~37% on average, compared with no oil control treatment. Previous exposure to the moderate oil treatment showed highly variable foraging rate responses, while low exposure treatment was similar to unexposed responses. Declining foraging rate responses to previous high weathered oil exposure suggests potential oil spill influence on energy transfer between saltmarsh and off-marsh systems. Additionally, foraging rate variability at the moderate level highlights the large degree of intraspecific variability for this sublethal response and indicates this concentration represents a potential threshold of oil exposure influence on killifish foraging. We also found that consumption of gravid vs non-gravid shrimp was not independent of prior oil exposure concentration, as high oil exposure treatment killifish consumed ~3× more gravid shrimp than expected. Our study findings highlight the sublethal effects of prior oil exposure on foraging abilities of ecologically valuable Gulf killifish at realistic oil exposure levels, suggesting that important trophic transfers of energy to off-marsh systems may have been impacted, at least in the short-term, by shoreline oiling at highly localized scales. This study provides support for further experimental testing of oil exposure effects on sublethal behavioral impacts of ecologically important estuarine species, due to the likelihood that some ecological ramifications of DwH on saltmarshes likely went undetected.

Factors affecting DNA barcoding classification accuracy for piscine prey: An experimental assessment with invasive lionfish

The use of DNA barcoding in fish diet studies is becoming more widespread, but the effect of prey digestion on barcoding accuracy has been poorly studied. We conducted a series of controlled feeding experiments, with red lionfish (Pterois volitans) as predators and Gulf killifish (Fundulus grandis) as prey, designed to test the effect of digestion time (12–42 h), prey item (sample) preservation (inside versus outside stomach), and sample preparation (washed versus unwashed) on DNA barcoding classification accuracy. Competitive interactions occurred between lionfish and killifish DNA during PCR amplification when universal COI primers were utilized; thus, primers were designed that matched the same priming site in both killifish and lionfish DNA. The proportion of amplified killifish DNA relative to lionfish DNA per sample was estimated using multiplex quantitative PCR (qPCR) and shown to decrease with longer digestion times, especially when samples were retained within lionfish stomachs for preservation. The effects of digestion and preservation method on amplifiable DNA resulted in significant effects on barcoding accuracy, resulting in erroneous identification of the predator (i.e., self-DNA) for nearly 25% of samples. Removing prey from predator stomachs prior to preservation significantly increased the proportion of amplified killifish DNA from samples, and enhanced barcoding accuracy. Overall, study results have important implications for the probability of false negatives for prey items, as well as false positives for self-DNA, when utilizing barcoding to characterize piscine diet. Results also highlight the need to conduct more method validation research given the increasing prevalence of DNA barcoding as a means to identify specific prey items in fish diet studies.

Reduced biotransformation of polycyclic aromatic hydrocarbons (PAHs) in pollution-adapted Gulf killifish (Fundulus grandis)

Anthropogenic pollution represents a significant source of selection, potentially leading to the emergence of evolutionary adaptations in chronically exposed organisms. A recent example of this scenario corresponds to Gulf killifish (Fundulus grandis) populations inhabiting the Houston Ship Channel (HSC), Texas, USA, which have been documented to have adapted to this heavily contaminated environment. Although not fully elucidated, one particularly important aspect of their adaptation involves the reduced inducibility of the aryl hydrocarbon receptor (AhR) and, potentially, the alteration of major biotransformation pathways. In the present study, we employed a modified Organization for Economic Cooperation and Development (OECD) 319-B test guideline to explore population and sex-related differences in the hepatic biotransformation of six polycyclic aromatic hydrocarbons (PAHs) in F. grandis populations with different exposure histories. Pollution-adapted F. grandis showed significantly lower hepatic clearance of PAHs than non-adapted fish, especially for high molecular weight PAHs (chrysene, benzo[k]fluoranthene, and benzo[a]pyrene), with pollution-adapted females presenting the lowest clearance. The characterization of different phase I biotransformation enzymes revealed that the basal activity of CYP1A, fundamental in the biotransformation of PAHs, was significantly lower in pollution-adapted fish, especially in females, which showed the lowest activity. Contrarily, basal CYP2C9-like activity was significantly higher in pollution-adapted fish. These results demonstrate the importance of exposure and evolutionary histories in shaping organisms' responses to pollution and provide significant evidence of sex-specific biotransformation differences in F. grandis populations.

The influence of hypoxia on the cardiac transcriptomes of two estuarine species - C. variegatus and F. grandis

Increased nutrient loading has led to eutrophication of coastal shelf waters which has resulted in increased prevalence of persistent hypoxic zones – areas in which the dissolved oxygen content of the water drops below 2 mg/L. The northern Gulf of Mexico, fed primarily by the Mississippi River watershed, undergoes annual establishment of one of the largest hypoxic zones in the world. Exposure to hypoxia can induce physiological impacts in fish cardiac systems that include bradycardia, changes in stroke volume, and altered cardiovascular vessel development. While these impacts have been addressed at the functional level, there is little information regarding the molecular basis for these changes. This study used transcriptomic analysis techniques to interrogate the effects of hypoxia exposure on the developing cardiovascular system in newly hatched larvae of two estuarine species that occupy the same ecological niche – the sheepshead minnow (Cyprinodon variegatus) and the Gulf killifish (Fundulus grandis). Results suggest that while differential gene expression is largely distinct between the two species, downstream impacts on pathways and functional responses such as reduced cardiac hypertrophy, modulation of blood pressure, and increased incidence of apoptosis appear to be conserved. Further, differences in the magnitude of these conserved responses may suggest that the length of embryonic development could impart a level of resiliency to hypoxic perturbation in early life stage fish.

Previous oil exposure alters Gulf Killifish Fundulus grandis oil avoidance behavior.

Oil spills threaten the structure and function of ecological communities. The Deepwater Horizon spill was predicted to have catastrophic consequences for nearshore fishes, but field studies indicate resilience in populations and communities. Previous research indicates many marsh fishes exhibit avoidance of oil contaminated areas, representing one potential mechanism for this resilience. Here, we test whether prior oil exposure of Gulf killifish Fundulus grandis alters this avoidance response. Using choice tests between unoiled and oiled sediments at one of three randomized concentrations (low: 0.1 L oil m−2, medium: 0.5 L oil m−2, or high: 3.0 L oil m−2), we found that, even at low prior exposure levels, killifish lose recognition of oiled sediments compared to control, unexposed fish. Preference for unoiled sediments was absent across all oil concentrations after oil exposure, and some evidence for preference of oiled sediments at high exposure was demonstrated. These results highlight the lack of response to toxic environments in exposed individuals, indicating altered behavior despite organism survival. Future research should document additional sublethal consequences that affect ecosystem and food web functioning.

The impact of salinity and dissolved oxygen regimes on transcriptomic immune responses to oil in early life stage Fundulus grandis

Understanding the effects of oil exposure on early life stage fish species is critical to fully assessing the environmental impacts of oil spills. Oil released from the 2010 Deepwater Horizon spill reached habitats where estuarine fish routinely spawn. In addition, estuaries are highly dynamic environments, therefore, fish in these areas are routinely exposed to varying salinity and dissolved oxygen (DO) levels, each of which are known to modulate transcriptional responses. Fish exposed to oil often display altered immune competence, and several studies have shown that Deepwater Horizon oil in particular causes modulation of various immune functions. However, few studies have directly examined how environmental parameters may affect oil-induced immunomodulation, particularly in early life stage fishes when the immune system is still developing. To this end, we examined transcriptional patterns of immune genes and pathways in Fundulus grandis larvae to various oil (0, 15 μg/L), salinity (3, 30 ppt), and DO (2.5, 6 mg/L) regimes in a fully factorial design. Our results suggest that immune pathways are generally activated in all treatment groups with the exception of the Low Salinity/No Oil/Hypoxia treatment where immune pathways are largely suppressed, and the High Salinity/No Oil/Hypoxia treatment where pathways are unchanged. The High Salinity/Oil/Hypoxia treatment had the largest number of enriched immune pathways (44 as defined by IPA and 43 as defined by ConsensusPathDB), indicating that oil under certain environmental conditions has the potential to further modulate immune-related genes, pathways, and responses in fish.

Effects of polycyclic aromatic hydrocarbons and abiotic stressors on Fundulus grandis cardiac transcriptomics

Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Many studies have identified the toxicological effects of PAHs in estuarine and marine fishes, however, only recently has work begun to identify the combinatorial effect of PAHs and abiotic environmental factors such as hypoxia, salinity, and temperature. This study aims to characterize the combined effects of abiotic stressors and PAH exposure on the cardiac transcriptomes of developing Fundulus grandis larvae. In this study, F. grandis larvae were exposed to varying environmental conditions (dissolved oxygen (DO) 2, 6 ppm; temperature 20, 30 °C; and salinity 3, 30 ppt) as well as to a single concentration of high energy water accommodated fraction (HEWAF) (∑PAHs 15 ppb). Whole larvae were sampled for RNA and transcriptional changes were quantified using RNA-Seq followed by qPCR for a set of target genes. Analysis revealed that exposure to oil and abiotic stressors impacts signaling pathways associated with cardiovascular function. Specifically, combined exposures appear to reduce development of the systemic vasculature as well as strongly impact the cardiac musculature through cardiomyocyte proliferation resulting in inhibited cardiac function and modulated blood pressure maintenance. Results of this study provide a holistic view of impacts of PAHs and common environmental stressors on the cardiac system in early life stage estuarine species. To our knowledge, this study is one of the first to simultaneously manipulate oil exposure with abiotic factors (DO, salinity, temperature) and the first to analyze cardiac transcriptional responses under these co-exposures.

Effects of crude oil vapors on the cardiovascular flow of embryonic Gulf killifish

Direct contact with toxicants in crude oil during embryogenesis causes cardiovascular defects, but the effects of exposure to airborne volatile organic compounds released from spilled oil are not well understood. The effects of crude oil-derived airborne toxicants on peripheral blood flow were examined in Gulf killifish (Fundulus grandis) since this model completes embryogenesis in the air. Particle image velocimetry was used to measure in vivo blood flow in intersegmental arteries of control and oil-exposed embryos. Significant effects in oil-exposed embryos included increased pulse rate, reduced mean blood flow speed and volumetric flow rate, and decreased pulsatility, demonstrating that normal-appearing oil-exposed embryos retain underlying cardiovascular defects. Further, hematocrit moderately increased in oil-exposed embryos. This study highlights the potential for fine-scale physiological measurement techniques to better understand the sub-lethal effects of oil exposure and demonstrates the efficacy of Gulf killifish as a unique teleost model for aerial toxicant exposure studies.

Comparison of stocking time and density on Gulf Killifish growth in freshwater ponds

The Gulf Killifish (Fundulus grandis) is a popular baitfish and a candidate for commercial-scale production. For inland production, ponds may need to be stocked at different times and densities to meet market demands. Therefore, in Experiment 1, juveniles were stocked into freshwater ponds at a density of 25,000 fish/ha, with two cohorts (mean total length (TL) = 3.92; 3.52 cm) stocked 1 month apart and evaluated for 12 weeks. Cohort 1 had a lower overall survival (40%) than cohort 2 (79%); with no difference between mean final TL (8.46; 8.00 cm); reaching a market size of 6 cm in 22 days and 32 days respectively. In Experiment 2, juveniles (mean TL = 3.16; 3.23 cm) were stocked into freshwater ponds at two densities (25,000 fish/ha and 50,000 fish/ha). Mean final TL was greater in the lower stocking density (8.68 cm) than the higher stocking density (8.20 cm); with no differences in survival between treatments (75; 72%) respectively. Lower stocking and higher stocking densities reached a market size of 6 cm in 29 and 31 days respectively. These results suggest that multiple cohorts can be stocked and grown to market size in one season at a stocking density of 50,000 fish/ha.

Acute exposure to oil induces age and species-specific transcriptional responses in embryo-larval estuarine fish

Because oil spills frequently occur in coastal regions that serve as spawning habitat, characterizing the effects of oil in estuarine fish carries both economic and environmental importance. There is a breadth of research investigating the effects of crude oil on fish, however few studies have addressed how transcriptional responses to oil change throughout development or how these responses might be conserved across taxa. To investigate these effects, we performed RNA-seq and pathway analysis following oil exposure 1) in a single estuarine species (Cyprinodon variegatus) at three developmental time points (embryos, yolk-sack larvae, free-feeding larvae), and 2) in two ecologically similar species (C. variegatus and Fundulus grandis), immediately post-hatch (yolk-sack stage). Our results indicate that C. variegatus embryos mount a diminished transcriptional response to oil compared to later stages, and that few transcriptional responses are conserved throughout development. Pathway analysis of larval C. variegatus revealed dysregulation of similar biological processes at later larval stages, including alteration of cholesterol biosynthesis pathways, cardiac development processes, and immune functions. Our cross-species comparison showed that F. grandis exhibited a reduced transcriptional response compared to C. variegatus. Pathway analysis revealed that the two species shared similar immune and cardiac responses, however pathways related to cholesterol biosynthesis exhibited a divergent response as they were activated in C. variegatus but inhibited in F. grandis. Our results suggest that examination of larval stages may provide a more sensitive estimate of oil-impacts than examination of embryos, and challenge assumptions that ecologically comparable species respond to oil similarly.

Smells like home: The use of chemically-mediated rheotaxes by Limulus polyphemus larvae

The distribution of horseshoe crab (Limulus polyphemus) larvae and early benthic juveniles is often non-random, with higher abundances typically found in areas of dense submerged vegetation (e.g., seagrass and algae) in coastal and estuarine nursery habitats. To determine if these distribution patterns are mediated by behavioral responses to chemical odors associated with potential settlement sites, we examined whether habitat chemical cues influence the swimming behavior (chemically-mediated rheotaxis) of horseshoe crab larvae at two different periods in their development: immediately after hatching (early-stage) and just prior to metamorphosis (late-stage). The hypothesis that larvae exhibit a positive rheotaxis (upstream movement) in the presence of chemical cues from submerged vegetation and negative rheotaxis (downstream movement) in response to odors from predators was tested in a laboratory T-maze. Larvae were exposed to seawater containing chemical odors from one of four sources: (1) water collected directly from the estuary, and odor water prepared with (2) aquatic vegetation, (3) potential predators, or (4) conspecifics. Behavioral responses of larvae varied with the source of the odor and the developmental stage of the larva. Both larval stages responded to estuarine water and water containing cues from the seagrass Halodule wrightii, a known nursery habitat, but response rates were greater for early-stage larvae. The response of larvae to H. wrightii odor was dose-dependent; upstream swimming response increased with increasing H. wrightii odor concentration. Though juvenile horseshoe crabs are also found within beds of Syringodium filiforme and Acanthophora spicifera, in our experiments early-stage larvae swam downstream away from both cues. In contrast, late-stage larvae exhibited a positive rheotactic response to the odor of A. spicifera. Conspecific odor had no effect on the rheotaxis of either developmental stage tested. Both larval stages unexpectedly responded to predator odors (blue crabs, Callinectes sapidus; killifish, Fundulus grandis) with a positive rheotaxis. When combined with a known positive cue (H. wrightii), killifish predator odor did not alter the rheotactic response of early-stage larvae; however, the addition of the predator odor reversed the upstream swimming behavior of late-stage larvae, likely indicating an alarm response to the presence of the predatory fish. Collectively, our results indicate that the distribution patterns of early benthic horseshoe crabs are a consequence of developmental shifts in oriented swimming behaviors (rheotaxes) that are mediated by chemical odors associated with settlement habitats. These behaviors should aid in the larva's ability to seek out and settle in advantageous habitat, thereby reducing predation and increasing survivorship.