Effects of incubation temperature on the upper thermal tolerance of the imperiled longfin smelt (Spirinchus thaleichthys).

Upper thermal limits in many fish species are limited, in part, by the heart's ability to meet increased oxygen demand during high temperatures. Cardiac plasticity induced by developmental temperatures can therefore influence thermal tolerance. Here, we determined how incubation temperatures during the embryonic stage influence cardiac performance across temperatures during the sensitive larval stage of the imperiled longfin smelt. We transposed a cardiac assay for larger fish to newly hatched larvae that were incubated at 9°C, 12°C or 15°C. We measured heart rate over increases in temperature to identify the Arrhenius breakpoint temperature (TAB), a proxy for thermal optimum and two upper thermal limit metrics: temperature when heart rate is maximized (Tpeak) and when cardiac arrhythmia occurs (TArr). Higher incubation temperatures increased TAB, Tpeak and TArr, but high individual variation in all three metrics resulted in great overlap of individuals at TAB, Tpeak and TArr across temperatures. We found that the temperatures at which 10% of individuals reached Tpeak or TArr and temperatures at which number of individuals at TAB relative to Tpeak (ΔN(TAB,Tpeak)) was maximal, correlated more closely with upper thermal limits and thermal optima inferred from previous studies, compared to the mean values of the three cardiac metrics of the present study. Higher incubation temperatures increased the 10% Tpeak and TArr thresholds but maximum ΔN(TAB,Tpeak) largely remained the same, suggesting that incubation temperatures modulate upper thermal limits but not Topt for a group of larvae. Overall, by measuring cardiac performance across temperatures, we defined upper thermal limits (10% thresholds; Tpeak, 14.4-17.5°C; TArr, 16.9-20.2°C) and optima (ΔN(TAB,Tpeak), 12.4-14.4°C) that can guide conservation strategies for longfin smelt and demonstrated the potential of this cardiac assay for informing conservation plans for the early life stages of fish.

Bifenthrin, a Ubiquitous Contaminant, Impairs the Development and Behavior of the Threatened Longfin Smelt during Early Life Stages.

The Longfin Smelt (Spirinchus thaleichthys) population in the San Franscisco Bay/Sacramento-San Joaquin Delta (Bay-Delta) has declined to ∼1% of its pre-1980s abundance and, as a result, is listed as threatened under the California Endangered Species Act. The reasons for this decline are multiple and complex, including the impacts of contaminants. Because the spawning and rearing seasons of Longfin Smelt coincide with the rainy season, during which concentrations of contaminants increase due to runoff, we hypothesized that early life stages may be particularly affected by those contaminants. Bifenthrin, a pyrethroid insecticide commonly used in agricultural and urban sectors, is of concern. Concentrations measured in the Bay-Delta have been shown to disrupt the behavior, development, and endocrine system of other fish species. The objective of the present work was to assess the impact of bifenthrin on the early developmental stages of Longfin Smelt. For this, embryos were exposed to 2, 10, 100, and 500 ng/L bifenthrin from fertilization to hatch, and larvae were exposed to 2, 10, and 100 ng/L bifenthrin from one day before to 3 days post-hatch. We assessed effects on size at hatch, yolk sac volume, locomotory behavior, and upper thermal susceptibility (via cardiac endpoints). Exposure to these environmentally relevant concentrations of bifenthrin did not significantly affect the cardiac function of larval Longfin Smelt; however, exposures altered their behavior and resulted in smaller hatchlings with reduced yolk sac volumes. This study shows that bifenthrin affects the fitness-determinant traits of Longfin Smelt early life stages and could contribute to the observed population decline.

Visual, spectral, and microchemical quantification of crystalline anomalies in otoliths of wild and cultured delta smelt.

Developmental abnormalities in otoliths can impact growth and survival in teleost fishes. Here, we quantified the frequency and severity of developmental anomalies in otoliths of delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish that is endemic to the San Francisco Estuary. Left-right asymmetry and anomalous crystalline polymorphs (i.e., vaterite) were quantified and compared between wild and cultured populations using digital image analysis. Visual estimates of vaterite were validated using X-ray diffraction, Raman spectroscopy, laser ablation ICPMS, and electron probe microanalysis. Results indicated that cultured delta smelt were 80 times more likely to contain a vateritic otolith and 18 times more likely to contain relatively large (≥ 15%) amounts of vaterite. Similarly, cultured fish exhibited 30% greater asymmetry than wild fish. These results indicate that cultured delta smelt exhibit a significantly higher frequency of vestibular abnormalities which are known to reduce fitness and survival. Such hatchery effects on otolith development could have important implications for captive culture practices and the supplementation of wild fish populations with cultured individuals.

Growth, osmoregulation and ionoregulation of longfin smelt (Spirinchus thaleichthys) yolk-sac larvae at different salinities.

Longfin smelt (Spirinchus thaleichthys) is a threatened anadromous fish species that spawns in freshwater to moderately brackish (i.e. 5-10 ppt) reaches of the upper San Francisco Estuary and has declined to ~1% of its pre-1980s abundances. Despite 50+ years of population monitoring, the efficacy of 10+ years of conservation efforts for longfin smelt remain uncertain due to a limited understanding of how the species responds to environmental variation, such as salinity. For example, high mortality during larval stages has prevented culture efforts from closing the life cycle in captivity. Here, we investigated the effects of salinity on longfin smelt yolk-sac larvae. Newly hatched larvae from four single-pair crosses were acutely transferred to and reared at salinities of 0.4, 5, 10, 20 or 32 ppt. We compared whole-body water and sodium ion (Na+) content, notochord length and yolk-sac volume at 12, 24, 48, 72, and 96 hours post-transfer for each salinity treatment. We found that larvae maintained osmotic and ionic balance at 0.4-10 ppt, whereas salinities ˃10 ppt resulted in decreased water and increased whole-body Na+ content. We also found that larvae grew largest and survived the longest when reared at 5 and 10 ppt, respectively, and that yolk resorption stalled at 0.4 ppt. Finally, there were significant but small interclutch variations in responses to different salinities, with clutch accounting for <8% of the variance in our statistical models. Overall, our results indicate that longfin smelt yolk-sac larvae likely perform best at moderately brackish conditions, thus yielding a mechanism that explains their distribution in field surveys and providing key information for future conservation efforts.

Captive Rearing of Longfin Smelt Spirinchus thaleichthys: First Attempt of Weaning Cultured Juveniles to Dry Feed.

The rapid decline of longfin smelt Spirinchus thaleichthys, a threatened euryhaline forage fish in California, is a serious concern for scientists and resource managers. To recover and conserve this species, a captive culture program was initiated, focusing on the collection, captive rearing and breeding of wild broodstock, and the rearing of their offspring. Although progress has been made in the collection of broodstock and the production and culturing of larvae, no studies have evaluated the rearing of juvenile life stages in captivity. The present study examines methodological considerations for culturing F1 juvenile longfin smelt, specifically, the first efforts toward weaning juveniles to a dry commercial pellet feed. Cultured juvenile longfin smelt were fed live Artemia only or co-fed Artemia and dry feed for 62 days, and the effects of feed type on juvenile survival, growth, body condition, and fatty acid profiles were examined. No significant differences were observed between feeding treatments, despite an 80% reduction in Artemia in the co-feeding treatment. Furthermore, examination of fish stomach contents at the end of the trial confirmed the transition to dry feed. This is the first study to indicate successful feeding by longfin smelt on dry commercial pellets, and suggests that juvenile longfin smelt can be fully weaned onto dry feeds. Results of this study are critical for closing the lifecycle of longfin smelt in captivity and developing a successful conservation culture program for this imperiled species.

Polygenic discrimination of migratory phenotypes in an estuarine forage fish.

Migration is a complex phenotypic trait with some species containing migratory and nonmigratory individuals. Such life history variation may be attributed in part to plasticity, epigenetics, or genetics. Although considered semianadromous, recent studies using otolith geochemistry have revealed life history variation within the critically endangered Delta Smelt. Broadly categorizable as migratory or freshwater residents, we examined Restriction site Associated DNA sequencing data to test for a relationship between genetic variation and migratory behaviors. As previously shown, we found no evidence for neutral population genetic structure within Delta Smelt; however, we found significant evidence for associations between genetic variants and life history phenotypes. Furthermore, discriminant analysis of principal components, hierarchical clustering, and machine learning resulted in accurate assignment of fish into the freshwater resident or migratory classes based on their genotypes. These results suggest the presence of adaptive genetic variants relating to life history variation within a panmictic population. Mechanisms that may lead to this observation are genotype dependent habitat choice and spatially variable selection, both of which could operate each generation and are not exclusive. Given that the population of cultured Delta Smelt are being used as a refugial population for conservation, as a supply for wild population supplementation, and currently represent the majority of all living individuals of this species, we recommend that the hatchery management strategy consider the frequencies of life history-associated alleles and how to maintain this important aspect of Delta Smelt biological variation while under captive propagation.

Experimental validation of otolith-based age and growth reconstructions across multiple life stages of a critically endangered estuarine fish.

BACKGROUND: The application of otolith-based tools to inform the management and conservation of fishes first requires taxon- and stage-specific validation. The Delta Smelt (Hypomesus transpacificus), a critically endangered estuarine fish that is endemic to the upper San Francisco Estuary (SFE), California, United States, serves as a key indicator species in the SFE; thus, understanding this species' vital rates and population dynamics is valuable for assessing the overall health of the estuary. Otolith-based tools have been developed and applied across multiple life stages of Delta Smelt to reconstruct age structure, growth, phenology, and migration. However, key methodological assumptions have yet to be validated, thus limiting confidence in otolith-derived metrics that are important for informing major water management decisions in the SFE. METHODS: Using known-age cultured Delta Smelt and multiple independent otolith analysts, we examined otolith formation, otolith-somatic proportionality, aging accuracy and precision, left-right symmetry, and the effects of image magnification for larval, juvenile, and adult Delta Smelt. RESULTS: Overall, otolith size varied linearly with fish size (from 10-60 mm), explaining 99% of the variation in fish length, despite a unique slope for larvae < 10 mm. Otolith-somatic proportionality was similar among wild and cultured specimens. Aging precision among independent analysts was 98% and aging accuracy relative to known ages was 96%, with age estimates exhibiting negligible differences among left and right otoliths. Though error generally increased with age, percent error decreased from 0-30 days-post-hatch, with precision remaining relatively high (≥ 95%) thereafter. Increased magnification (400×) further improved aging accuracy for the oldest, slowest-growing individuals. Together, these results indicate that otolith-based techniques provide reliable age and growth reconstructions for larval, juvenile, and adult Delta Smelt. Such experimental assessments across multiple developmental stages are key steps toward assessing confidence in otolith-derived metrics that are often used to assess the dynamics of wild fish populations.

Complex life histories discovered in a critically endangered fish.

Effective conservation of endangered species requires knowledge of the full range of life-history strategies used to maximize population resilience within a stochastic and ever-changing environment. California's endemic Delta Smelt (Hypomesus transpacificus) is rapidly approaching extinction in the San Francisco Estuary, placing it in the crossfire between human and environmental uses of limited freshwater resources. Though managed as a semi-anadromous species, recent studies have challenged this lifecycle model for Delta Smelt, suggesting the species is an estuarine resident with several localized "hot-spots" of abundance. Using laser-ablation otolith strontium isotope microchemistry, we discovered three distinct life-history phenotypes including freshwater resident (FWR), brackish-water resident (BWR), and semi-anadromous (SA) fish. We further refined life-history phenotypes using an unsupervised algorithm and hierarchical clustering and found that in the last resilient year-class, the FWR (12%) and BWR (7%) comprised a small portion of the population, while the majority of fish were SA (81%). Furthermore, the semi-anadromous fish could be clustered into at least four additional life-history phenotypes that varied by natal origin, dispersal age and adult salinity history. These diverse life-history strategies should be incorporated into future conservation and management efforts aimed at preventing the extinction of Delta Smelt in the wild.

Calibrating temperature reconstructions from fish otolith oxygen isotope analysis for California's critically endangered Delta Smelt.

RATIONALE: Oxygen isotope ratios (δ18 O values) of fish otoliths (ear bones) are valuable geochemical tracers of water conditions and thermal life history. Delta Smelt (Hypomesus transpacificus) are osmerid forage fish endemic to the San Francisco Estuary, California, USA, that are on the verge of extinction. These fish exhibit a complex life history that allows them to survive in a dynamic estuarine environment; however, a rapidly warming climate threatens this thermally sensitive species. Here we quantify the accuracy and precision of using δ18 O values in otoliths to reconstruct the thermal life histories of Delta Smelt. METHODS: Delta Smelt were reared for 360 days using three different water sources with different ambient δ18 Owater values (-8.75‰, -5.28‰, and -4.06‰) and different water temperatures (16.4°C, 16.7°C, 18.7°C, and 20.5°C). Samples were collected after 170 days (n = 28) and 360 days (n = 14) post-hatch. In situ δ18 O values were measured from the core of the otolith to the dorsal edge using secondary ion mass spectrometry (SIMS) to reconstruct temporally resolved thermal life histories. RESULTS: The δ18 Ootolith values for Delta Smelt varied as a linear inverse function of water temperature: 1000 ln α = 18.39 (±0.43, 1SE)(103 TK-1 ) - 34.56 (±1.49, 1SE) and δ18 Ootolith(VPDB) - δ18 Owater (VPDB) = 31.34(±0.09, 1SE) - 0.19(±0.01, 1SE) × T ° C. When the ambient δ18 Owater value is known, this species-specific temperature-dependent oxygen isotope fractionation model facilitated the accurate (0.25°C) and precise (±0.37°C, 2σ) reconstruction of the water temperature experienced by the fish. In contrast, the use of existing general fractionation equations resulted in inaccurate temperature reconstructions. CONCLUSIONS: The species-specific δ18 Ootolith fractionation equation allowed for accurate and precise reconstructions of water temperatures experienced by Delta Smelt. Characterization of ambient δ18 Owater values remains a critical next step for reconstructing thermal life histories of wild Delta Smelt. This tool will provide new insights into habitat utilization, potential thermal refugia, and resilience to future warming for this critically endangered fish.

IsoFishR: An application for reproducible data reduction and analysis of strontium isotope ratios (87Sr/86Sr) obtained via laser-ablation MC-ICP-MS.

The IsoFishR application is a data reduction and analysis tool for laser-ablation strontium isotope data, following common best practices and providing reliable and reproducible results. Strontium isotope ratios (87Sr/86Sr) are a powerful geochemical tracer commonly applied in a wide range of scientific fields and laser-ablation inductively coupled mass spectrometry is considered the method of choice to obtain spatially resolved 87Sr/86Sr isotope ratios from a variety of sample materials. However, data reduction and analyses methods are variable between different research groups and research communities limiting reproducibility between studies. IsoFishR provides a platform to standardize these methods and can be used for both spot and time-resolved line transects. Furthermore, it provides advanced data analysis tools and filters for outlier removal, noise reduction, and visualization of time resolved data. The application can be downloaded from GitHub (https://github.com/MalteWillmes/IsoFishR) and the source code is available, encouraging future development and evolution of this software.

Comparative metabolic ecology of tropical herbivorous echinoids on a coral reef.

BACKGROUND: The metabolic rate of consumers is a key driver of ecosystem dynamics. On coral reefs, herbivorous echinoids consume fleshy algae, facilitating the growth of reef-building calcified organisms; however, little is known about differences among species in their metabolic and functional ecology. Here, we used log-linear (log-log) regression models to examine the allometric scaling of mass and routine metabolic rate for five common herbivorous echinoids on a Hawaiian coral reef: Echinothrix calamaris, E. diadema, Echinometra matthaei, Heterocentrotus mammillatus, and Tripneustes gratilla. Scaling relationships were then contrasted with empirical observations of echinoid ecology and general metabolic theory to broaden our understanding of diversity in the metabolic and functional ecology of tropical herbivorous echinoids. RESULTS: Test diameter and species explained 98% of the variation in mass, and mass and species explained 92.4% and 87.5% of the variation in individual (I) and mass-specific (B) metabolic rates, respectively. Scaling exponents did not differ for mass or metabolism; however, normalizing constants differed significantly among species. Mass varied as the cube of test diameter (b = 2.9), with HM exhibiting a significantly higher normalizing constant than other species, likely due to its heavily-calcified spines and skeleton. Individual metabolic rate varied approximately as the 2/5 power of mass (γ = 0.44); significantly smaller than the 3/4 universal scaling coefficient, but inclusive of 2/3 scaling. E. calamaris and H. mammillatus exhibited the lowest normalizing constants, corresponding with their slow-moving, cryptic, rock-boring life-history. In contrast, E. calamaris, E. diadema, and T. gratilla, exhibited higher metabolic rates, likely reflecting their higher levels of activity and ability to freely browse for preferred algae due to chemical anti-predator defenses. Thus, differences in metabolic scaling appeared to correspond with differences in phylogeny, behavior, and ecological function. Such comparative metabolic assessments are central to informing theory, ecological models, and the effective management of ecosystems.

Top-down control of epifauna by fishes enhances seagrass production.

Predators can influence the structure and function of ecosystems by altering the composition or behavior of herbivore communities. Overexploitation of predators, therefore, may lead to habitat loss by altering important top-down interactions that facilitate habitat-forming species. In seagrass beds, top-down control of algal growth by mesograzers appears to facilitate seagrass production. The indirect consequences of higher-order trophic interactions, however, remain unclear. Although predators may limit the beneficial effects of algal mesograzers, it is also possible that they limit the abundance of invertebrates that consume and foul seagrasses. We used experimental enclosure and exclosure cages to explore the direct and indirect effects of microcarnivorous fishes on epifaunal invertebrates, epiphytic loads, and seagrass growth in a natural eelgrass (Zostera marina) bed in San Diego Bay, California, USA. Contrary to expectations, when fishes were excluded, invertebrate abundance increased by 300-1000%, fouling on eelgrass leaves increased by 600%, and eelgrass production declined by 50%. Despite high densities of predators in enclosures, subsequent effects did not differ from ambient conditions. When predators were excluded, however, abundances of epifauna (including tube-building crustaceans and an eelgrass-grazing limpet) increased dramatically, resulting in reduced seagrass production. Our results are supported by several studies of eelgrass communities in the northeastern Pacific, characterized by coastal upwelling, inverse estuaries, and a voracious seagrass-consuming limpet. These strong, positive, indirect effects of microcarnivores on seagrass production contrast with the beneficial mesograzer paradigm, highlighting the need for hypotheses to be tested across a variety of ecosystems with varying biophysical characteristics.