Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

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.

A multidimensional density dependent matrix population model for assessing risk of stressors to fish populations.

Modeling exposure and recovery of fish and wildlife populations after stressor mitigation serves as a basis for evaluating remediation success. Herein, we develop a novel multidimensional density dependent matrix population model that analyzes both size-structure and age class-structure simultaneously. This modeling approach emphasizes application in conjunction with field monitoring efforts (e.g., through effects-based monitoring programs) and/or laboratory analysis to link effects due to stressors to outcomes in populations. We applied the model to investigate Atlantic killifish (Fundulus heteroclitus) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin with effects on fertility and survival rates. The Atlantic killifish is an important and well-studied model organism for understanding the effects of pollutants and other stressors in estuarine and marine ecosystems. For each exposure concentration, the corresponding plots of total population size, population size structure, and age structure over time were generated. The present study serves as an example of how a multidimensional matrix population model can integrate effects across the life cycle, provide a linkage between endpoints observed in the individual and ecological risk to the population as a whole, and project outcomes for multiple generations.

The effect of population density on growth and survival of a Neotropical annual killifish.

We used a field experiment to test the effects of population density on the growth rate and survival of Austrolebias bellottii, a Neotropical annual killifish. Effects differed between the sexes: males at high densities achieved a smaller final size and experienced higher mortality while no such effects were observed in females. This sex-specific effect could be an indirect consequence of mate competition.

Life-time exposure to waterborne copper IV: Sperm quality parameters are negatively affected in the killifish Poecilia vivipara.

In previous studies, we have shown that copper (Cu) is significantly accumulated in various tissues of killifish Poecilia vivipara following chronic exposure. Also, we showed that chronic metal exposure disrupted energy production and growth in this species. In the present study, we aimed to evaluate if chronic exposure to this metal could also affect reproductive parameters of P. vivipara males (sperm quality). In order to test that, newborn (<24 h-old) fish were exposed to two concentrations of waterborne Cu (5 and 9 µg/L) for 345 days. After exposure, fish were euthanized and the testes were collected for sperm analysis. We could observe that exposed animals had reduced sperm motility and period of motility. Also, the sperm of exposed fish had reduced plasma membrane integrity, mitochondrial functionality and DNA integrity when compared to sperm of control animals. It is suggested that the well-known association of Cu with elevated oxidative damage, endocrine disruption and energetic disturbance are involved with the observed outcomes. The results obtained in the present study show that chronic exposure to environmentally relevant concentrations of waterborne Cu caused reductions in all parameters used to evaluate sperm quality. Therefore, it is concluded that life-time exposure to this metal may disrupt fish reproduction and negatively affect the maintenance of its populations.

Combined effects of salinity, temperature, hypoxia, and Deepwater Horizon oil on Fundulus grandis larvae.

Oil spills have polluted the marine environment for decades and continue to be a major source of polycyclic aromatic hydrocarbons (PAHs) to marine ecosystems around the globe, for example during the 2010 Deepwater Horizon spill. Although the toxicity of PAHs to fish has been well studied, their effects combined with abiotic stressors are poorly understood. The goal of this study was to describe the combined impacts of crude oil and environmental stressors on fish larvae, a sensitive life stage. Gulf killifish (Fundulus grandis) larvae (<24 h post-hatch) were exposed for 48 h to high energy water accommodated fractions (HEWAF; total PAHs 0-125 ppb) of Macondo oil from the Deepwater Horizon spill under different combinations of environmental conditions (dissolved oxygen 2, 6 ppm; temperature 20, 25, 30 °C; salinity 3, 10, 30 ppt). Even under optimal environmental conditions (25 °C, 10 ppt, 6 ppm) larval survival and development were negatively affected by PAHs, starting with the lowest concentration tested (∼15 ppb). Hypoxia and high temperature each increased the adverse effects of HEWAF on development and mortality. In contrast, salinity had little effect on any of the endpoints measured. Importantly, expression of the detoxifying gene cyp1a was highly induced in PAH-exposed larvae under normoxic conditions, but not under hypoxic conditions, potentially explaining the enhanced toxicity observed under hypoxia. This work highlights the importance of considering how suboptimal environmental conditions can exacerbate the effects of pollution on fish early life stages.

Rapid growth and large body size in annual fish populations are compromised by density-dependent regulation.

We tested the effect of population density on maximum body size in three sympatric species of annual killifishes Nothobranchius spp. from African ephemeral pools. We found a clear negative effect of population density on body size, limiting their capacity for extremely fast development and rapid growth. This suggests that density-dependent population regulation and the ephemeral character of their habitat impose contrasting selective pressures on the life history of annual killifishes.

Effects of passive integrated transponder tagging on cortisol release, aerobic metabolism and growth of the Gulf killifish Fundulus grandis.

The effects of passive integrated transponder (PIT) tagging on cortisol release, standard metabolic rate (SMR) and daily specific growth rate (GS ) were evaluated in the Gulf killifish, Fundulus grandis, a small estuarine fish native to the Gulf of Mexico. Cortisol release by individual fish was measured non-invasively prior to PIT tagging, immediately after tagging and once per week for 1 month following tagging. Within the first 2 h of tagging, cortisol release rates were significantly elevated compared with values measured prior to tagging and significantly higher than that of fish handled identically except not implanted with PIT tags. By 1 week after PIT tagging, cortisol release rates returned to control levels. SMR, determined by intermittent-flow respirometry and GS , defined as per cent change in body mass per day, were measured prior to PIT tagging and weekly for 1 month after tagging. Neither SMR nor GS was significantly different in tagged v. untagged fish for the duration of the study. One month after tagging, haematocrit, plasma cortisol, blood glucose and blood lactate did not differ between tagged and untagged individuals. Therefore, after a transient stress response that subsides within 1 week, PIT tagging had no significant effects on these physiological variables in F. grandis, validating its use as a method of marking this and other small fishes.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species.

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ±â€¯36 mg) were about 12 and 630 µg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 µg/L and a no-observed-effect concentration of 2.1 µg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 µg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Dispersion/reaggregation in early development of annual killifishes: Phylogenetic distribution and evolutionary significance of a unique feature.

Annual killifishes are members of the Aplocheiloidea and live in ephemeral habitats that desiccate regularly during the dry season and refill during the rainy season. Populations of these fishes survive the dry season by producing drought-resistant diapausing eggs that are buried in the substrate. When the pool refills during the rainy season the juveniles hatch, grow rapidly and reproduce until the pool desiccates again during the next dry season. The association with such unpredictable habitats has led to the evolution to a variety of developmental adaptations such as a dispersed/reaggregation phase of the deep blastomeres, three possible diapause stages, extreme tolerance to high salinity and anoxia, an efficient DNA repair system and an extremely short life span. Here, we review the course of the dispersed/reaggregation phase, its evolution and phylogenetic distribution and diversity within the Aplocheiloidea. The phenomenon of blastomere dispersion/reaggregation in these fishes was first described in the 1960s and 70s. Blastomeres of most teleost fishes segregate into three groups that give rise to the enveloping cell layer, the yolk syncytial layer and the deep blastomeres that will form the embryo itself. When epiboly commences, the deep blastomeres form a more or less coherent cell sheet with a so called embryonic shield at it marginal zone marking the area where gastrulation takes place. In annual killifishes, the deep blastomeres segregate when epiboly starts and disperse when epiboly commences. After epiboly has been completed, the deep blastomeres are randomly distributed and migrate all over the enveloping cell layer. After several days they start to reaggregate and form the actual embryo that starts gastrulation. The evolutionary origin and mechanism behind this peculiar developmental pathway have puzzled developmental biologists for almost 50 years. However, several of these annual killifishes (Nothobranchius furzeri, Austrofundulus limnaeus, Austrolebias charrua and Austrolebias bellottii) have become model organisms in studies on developmental physiology, aging and stress tolerance. This has led to the establishment of modern genetic techniques such as transgenesis and cell fate mapping that are now used to tackle questions about the origin and mechanisms behind the dispersal/reaggregation phase.

Combined effects of cadmium exposure and temperature on the annual killifish (Nothobranchius furzeri).

Freshwater organisms are increasingly exposed to combinations of stressors. However, because it is time-consuming and costly, research on the interaction of stressors, such as compound toxicity and global warming on vertebrates, is scarce. Studies on multigenerational effects of these combined stressors are almost nonexistent. In the present study, we tested the combined effects of 4 °C warming and cadmium (Cd) exposure on life-history traits, biomarkers, bioaccumulation, and multigenerational tolerance in the turquoise killifish, Nothobranchius furzeri. The extremely short life cycle of this vertebrate model allows for assessment of sublethal and multigenerational effects within 4 mo. The applied Cd concentrations had only limited effects on the measured endpoints, which suggests that N. furzeri is more resistant to Cd than fathead minnow and rainbow trout. In contrast, the temperature increase of 4 °C was stressful: it delayed female maturation and lowered adult mass and fecundity. Finally, indications of synergistic effects were found on peak fecundity and embryonic survival. Overall, these results indicate the importance of studying chronic and multigenerational effects of combined stressors. Environ Toxicol Chem 2018;37:2361-2371. © 2018 SETAC.

Transcriptomic alterations during ageing reflect the shift from cancer to degenerative diseases in the elderly.

Disease epidemiology during ageing shows a transition from cancer to degenerative chronic disorders as dominant contributors to mortality in the old. Nevertheless, it has remained unclear to what extent molecular signatures of ageing reflect this phenomenon. Here we report on the identification of a conserved transcriptomic signature of ageing based on gene expression data from four vertebrate species across four tissues. We find that ageing-associated transcriptomic changes follow trajectories similar to the transcriptional alterations observed in degenerative ageing diseases but are in opposite direction to the transcriptomic alterations observed in cancer. We confirm the existence of a similar antagonism on the genomic level, where a majority of shared risk alleles which increase the risk of cancer decrease the risk of chronic degenerative disorders and vice versa. These results reveal a fundamental trade-off between cancer and degenerative ageing diseases that sheds light on the pronounced shift in their epidemiology during ageing.

Assessment of gonadal and thyroid histology in Gulf killifish (Fundulus grandis) from Barataria Bay Louisiana one year after the Deepwater Horizon oil spill.

We examined gonads and thyroid glands of Gulf killifish (Fundulus grandis) 1yr after the Deepwater Horizon oil spill. F. grandis were trapped from two impacted sites in Barataria Bay (Bayou St. Denis, Bay Jimmy) and an un-impacted site in East Texas (Sabine Pass). The greatest number of F. grandis were collected at Sabine Pass. F. grandis collected at Bayou St. Denis were smaller and had smaller Fulton condition factor scores than fish collected at Sabine Pass. Sex ratios were biased roughly 2:1 in favor of females at Sabine Pass and Bayou St. Denis. Gonad-somatic index (GSI) in males from Sabine Pass was double that of fish from Bay Jimmy while germinal epithelium thickness of the testes was 2.7 fold smaller in males from the impacted site. GSI and oocyte diameters in females from Bayou St. Denis were significantly smaller than females from Bay Jimmy or the reference site. There were no differences in thyroid follicle cell height. While total polyaromatic hydrocarbons at the impacted sites were no different from the reference site, the impacted sites did have greater concentrations of benzo[a]pyrene in sediment pore water. The finding of smaller GSI and testicular germinal epithelium in males from an impacted site suggest that exposure to a combination of oil and dispersants may adversely impact testicular function.

The P-type ATPase inhibiting potential of polyoxotungstates.

Polyoxometalates (POMs) are transition metal complexes that exhibit a broad diversity of structures and properties rendering them promising for biological purposes. POMs are able to inhibit a series of biologically important enzymes, including phosphatases, and thus are able to affect many biochemical processes. In the present study, we analyzed and compared the inhibitory effects of nine different polyoxotungstates (POTs) on two P-type ATPases, Ca2+-ATPase from skeletal muscle and Na+/K+-ATPase from basal membrane of skin epithelia. For Ca2+-ATPase inhibition, an in vitro study was performed and the strongest inhibitors were determined to be the large heteropolytungstate K9(C2H8N)5[H10Se2W29O103] (Se2W29) and the Dawson-type POT K6[α-P2W18O62] (P2W18) exhibiting IC50 values of 0.3 and 0.6 µM, respectively. Promising results were also shown for the Keggin-based POTs K6H2[CoW11TiO40] (CoW11Ti, IC50 = 4 µM) and Na10[α-SiW9O34] (SiW9, IC50 = 16 µM), K14[As2W19O67(H2O)] (As2W19, IC50 = 28 µM) and the lacunary Dawson K12[α-H2P2W12O48] (P2W12, IC50 = 11 µM), whereas low inhibitory potencies were observed for the isopolytungstate Na12[H4W22O74] (W22, IC50 = 68 µM) and the Anderson-type Na6[TeW6O24] (TeW6, IC50 = 200 µM). Regarding the inhibition of Na+/K+-ATPase activity, for the first time an ex vivo study was conducted using the opercular epithelium of killifish in order to investigate the effects of POTs on the epithelial chloride secretion. Interestingly, 1 µM of the most potent Ca2+-ATPase inhibitor, Se2W29, showed only a minor inhibitory effect (14% inhibition) on Na+/K+-ATPase activity, whereas almost total inhibition (99% inhibition) was achieved using P2W18. The remaining POTs exhibited similar inhibition rates on both ATPases. These results reveal the high potential of some POTs to act as P-type ATPase inhibitors, with Se2W29 showing high selectivity towards Ca2+-ATPase.

Antifungal and Ichthyotoxic Sesquiterpenoids from Santalum album Heartwood.

In our continuing study on a survey of biologically active natural products from heartwood of Santalum album (Southwest Indian origin), we newly found potent fish toxic activity of an n-hexane soluble extract upon primary screening using killifish (medaka) and characterized α-santalol and ß-santalol as the active components. The toxicity (median tolerance limit (TLm) after 24 h at 1.9 ppm) of α-santalol was comparable with that of a positive control, inulavosin (TLm after 24 h at 1.3 ppm). These fish toxic compounds including inulavosin were also found to show a significant antifungal effect against a dermatophytic fungus, Trichophyton rubrum. Based on a similarity of the morphological change of the immobilized Trichophyton hyphae in scanning electron micrographs between treatments with α-santalol and griseofulvin (used as the positive control), inhibitory effect of α-santalol on mitosis (the antifungal mechanism proposed for griseofulvin) was assessed using sea urchin embryos. As a result, α-santalol was revealed to be a potent antimitotic agent induced by interference with microtubule assembly. These data suggested that α-santalol or sandalwood oil would be promising to further practically investigate as therapeutic agent for cancers as well as fungal skin infections.

Time-limited environments affect the evolution of egg-body size allometry.

Initial offspring size is a fundamental component of absolute growth rate, where large offspring will reach a given adult body size faster than smaller offspring. Yet, our knowledge regarding the coevolution between offspring and adult size is limited. In time-constrained environments, organisms need to reproduce at a high rate and reach a reproductive size quickly. To rapidly attain a large adult body size, we hypothesize that, in seasonal habitats, large species are bound to having a large initial size, and consequently, the evolution of egg size will be tightly matched to that of body size, compared to less time-limited systems. We tested this hypothesis in killifishes, and found a significantly steeper allometric relationship between egg and body sizes in annual, compared to nonannual species. We also found higher rates of evolution of egg and body size in annual compared to nonannual species. Our results suggest that time-constrained environments impose strong selection on rapidly reaching a species-specific body size, and reproduce at a high rate, which in turn imposes constraints on the evolution of egg sizes. In combination, these distinct selection pressures result in different relationships between egg and body size among species in time-constrained versus permanent habitats.

Embryonic development of the annual killifish Austrofundulus limnaeus: An emerging model for ecological and evolutionary developmental biology research and instruction.

BACKGROUND: Austrofundulus limnaeus is an annual killifish from the Maracaibo basin of Venezuela. Annual killifishes are unique among vertebrates in their ability to enter into a state of dormancy at up to three distinct developmental stages termed diapause I, II, and III. These embryos are tolerant of a wide variety of environmental stresses and develop relatively slowly compared with nonannual fishes. RESULTS: These traits make them an excellent model for research on interactions between the genome and the environment during development, and an excellent choice for developmental biology laboratories. Furthermore, A. limnaeus is relatively easy to maintain in a laboratory setting and has a high fecundity, making it an excellent candidate as an emerging model for studies of development, and for defining the limits of developmental buffering in vertebrates. CONCLUSIONS: This study reports for the first time on the detailed development of A. limnaeus and provides a photographic and illustrated atlas of embryos on the two developmental trajectories possible in this species. Developmental Dynamics 246:779-801, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Phototoxic potential of undispersed and dispersed fresh and weathered Macondo crude oils to Gulf of Mexico Marine Organisms.

Crude oils contain a mixture of hydrocarbons, including phototoxic polycyclic aromatic hydrocarbons (PAHs) that have the ability to absorb ultraviolet (UV) light. Absorption of UV light by PAHs can substantially increase their toxicity to marine organisms. The objective of the present study was to examine the potential for phototoxicity of fresh and naturally weathered Macondo crude oils alone and in combination with the dispersant Corexit 9500 to mysid shrimp (Americamysis bahia), inland silverside (Menidia beryllina), sheepshead minnow (Cyprinodon variegatus), and Gulf killifish (Fundulus grandis). Acute toxicity tests were conducted using combinations of natural or artificial sunlight and low-energy water-accommodated fractions (WAFs) of fresh and weathered Macondo crude oils collected from the Gulf of Mexico. Studies were also conducted to compare the phototoxicity resulting from natural and artificial sunlight. Fresh Macondo crude oil was more phototoxic than weathered crude oils, both in the presence and in the absence of UV light. Differences in toxicity between fresh and weathered crude oils were likely attributed to lighter-ringed PAHs in fresh crude oils. Phototoxic PAHs were relatively resistant to weathering compared with lighter-ringed PAHs. The addition of Corexit 9500 to crude oil increased toxicity compared with tests with crude oil alone, by increasing phototoxic PAH concentrations in WAFs. Macondo crude oils had the potential to be phototoxic to Gulf of Mexico marine organisms if specific light conditions and PAH concentrations were present during the Deepwater Horizon oil spill. Environ Toxicol Chem 2017;36:2640-2650. © 2017 SETAC.

Embryonic-only arsenic exposure in killifish (Fundulus heteroclitus) reduces growth and alters muscle IGF levels one year later.

Arsenic is a contaminant of drinking water and crops in many parts of the world. Epidemiological studies have shown that arsenic exposure is linked to decreased birth weight, weight gain, and proper skeletal muscle function. The goal of this study was to use killifish (Fundulus heteroclitus) as a model to determine the long-term effects of embryonic-only arsenic exposure on muscle growth and the insulin-like growth factor (IGF) pathway. Killifish embryos were exposed to 0, 50, 200 or 800ppb AsIII from fertilization until hatching. Juvenile fish were reared in clean water and muscle samples were collected at 16, 28, 40 and 52 weeks of age. There were significant reductions in condition factors, ranging from 12 to 17%, in the fish exposed to arsenic at 16, 28 and 40 weeks of age. However, by 52 weeks, no significant changes in condition factors were seen. Alterations in IGF-1R and IGF-1 levels were assessed as a potential mechanism by which growth was reduced. While there no changes in hepatic IGF-1 transcripts, skeletal muscle cells can also produce their own IGF-1 and/or alter IGF-1 receptor levels to help enhance growth. After a 200 and 800ppb embryonic exposure, fish grown in clean water for 16 weeks had IGF-1R transcripts that were 2.8-fold and 2-fold greater, respectively, than unexposed fish. Through 40 weeks of age, IGF1-R remained elevated in the 200ppb and 800ppb embryonic exposure groups by 1.8-3.9-fold, while at 52 weeks of age, IGF-1R levels were still significantly increased in the 800ppb exposure group. Skeletal muscle IGF-1 transcripts were also significantly increased by 1.9-5.1 fold through the 52 weeks of grow-out in clean by water in the 800ppb embryonic exposure group. Based on these results, embryonic arsenic exposure has long-term effects in that it reduces growth and increases both IGF-1 and IGF-1R levels in skeletal muscle even 1year after the exposure has ended.

Cross-resistance in Gulf killifish (Fundulus grandis) populations resistant to dioxin-like compounds.

The Houston Ship Channel (HSC) in Houston, Texas is an aquatic environment with a long history of contamination, including polychlorinated dibenzodioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals. Populations of Gulf killifish (Fundulus grandis) from the HSC have adapted to resist developmental cardiac deformities caused by dioxin-like compounds (DLCs). Contaminants in the HSC have acted as a strong selective pressure on resident Gulf killifish populations. Rapid adaptation can lead to fitness costs, some as a direct result of the mechanisms involved in the adaptive process, whereas other adaptations may be more general. To explore potential fitness costs, we evaluated two Gulf killifish populations with documented resistance to DLC-induced cardiac teratogenesis (Patrick Bayou and Vince Bayou), and one previously characterized reference population (Gangs Bayou). We also characterized a previously unstudied population from Galveston Bay as an additional reference population (Smith Point). We tested the sensitivity of F1 larvae from these four populations to two classes of pesticides (pyrethroid (permethrin) and carbamate (carbaryl)) and two model pro-oxidants (tert-butyl hydroquinone (tBHQ) and tert-butyl hydroperoxide (tBOOH)). In addition, we explored their responses to hypoxia and measured resting metabolic rates (M.O2). Both adapted populations were cross-resistant to the toxicity of carbaryl and both pro-oxidants tested. There were no population differences in sensitivity to permethrin. On the other hand, one reference population (Gangs Bayou) was less sensitive to hypoxia, and maintained a lower M.O2 . However, there were no differences in hypoxia tolerance or resting metabolic rate between the second reference and the two adapted populations. This investigation emphasizes the importance of including multiple reference populations to clearly link fitness costs or cross-resistance to pollution adaptation, rather than to unrelated environmental or ecological differences. When compared to previous literature on adapted populations of Fundulus heteroclitus, we see a mixture of similarities and differences, suggesting that F. grandis adapted phenotypes likely involve multiple mechanisms, which may not be completely consistent among adapted populations.