Assessment of the Effects of Double-Stranded RNAs Corresponding to Multiple Vitellogenesis-Inhibiting Hormone Subtype I Peptides in Subadult Female Whiteleg Shrimp, Litopenaeus vannamei.

Vitellogenesis-inhibiting hormone (VIH) negatively regulates reproduction in shrimp and other decapod crustaceans. In order to assess the effects of transcriptional silencing by multiple VIH subtype I sinus gland peptides (SGPs) on ovarian maturation in female whiteleg shrimp, Litopenaeus vannamei, we synthesized five dsRNAs targeting Liv-SGP-A, -B, -C, -F, and -G and injected them into subadults. The following treatments were employed: sgpG-dsRNA (targeting Liv-SGP-G), sgpC-dsRNA (targeting Liv-SGP-C), and mixed-dsRNA (targeting Liv-SGP-A, -B, and -F). The expression of Liv-SGP-G in eyestalks was significantly decreased at 10, 20, and 30 days after the injection of sgpG-dsRNA In addition, it was significantly decreased at 10 and 30 days after the injection of mixed-dsRNA. The expression of vitellogenin (Vg) gene expression in the ovaries, and concentrations of Vg protein in the hemolymph, were not changed by the administration of any dsRNA treatment (the ovaries remained immature in all treated individuals and contained mostly oogonia and previtellogenic oocytes). Although the administration of dsRNAs corresponding to multiple VIHs did not promote ovarian maturation, this is the first report of the co-transcriptional repression of Liv-SGP-G by the injection of dsRNA for homologous genes (Liv-SGP-A, -B, and -F). These results indicate that subadults can respond to the techniques of transcriptional silencing.

Vitellogenin offsets oxidative costs of reproduction in female painted dragon lizards.

Vitellogenesis ('yolking' of follicles) is a bioenergetically costly stage of reproduction requiring enlargement of the liver to produce vitellogenin (VTG) yolk precursor proteins, which are transported and deposited at the ovary. VTG may, however, serve non-nutritive anti-oxidant functions, a hypothesis supported by empirical work on aging and other life-history transitions in several taxa. We test this hypothesis in female painted dragon lizards (Ctenophorus pictus) by examining covariation in VTG with the ovarian cycle, and relative to reactive oxygen species (ROS) including baseline superoxide (bSO). Plasma VTG decreased prior to ovulation, when VTG is deposited into follicles. VTG, however, remained elevated post-ovulation when no longer necessary for yolk provisioning and was unrelated to reproductive investment. Instead, VTG was strongly and positively predicted by prior bSO. ROS, in turn, was negatively predicted by prior VTG, while simultaneously sampled VTG was a positive predictor. These findings are consistent with the hypothesis that VTG functions as an anti-oxidant to counteract oxidative stress associated with vitellogenesis. The relationship between bSO and VTG was strongest in post-ovulatory females, indicating that its function may be largely anti-oxidant at this time. In conclusion, VTG may be under selection to offset oxidative costs of reproduction in egg-producing species.

Energetic investment associated with vitellogenesis induces an oxidative cost of reproduction.

Oxidative stress is a potential cost of reproduction, but conclusive evidence for this relationship is lacking. The goal of this study was to serially assess across a seasonal gradient the relationship between reproduction, circulating plasma energy metabolites and oxidative state. Here, we examine a study animal ideally suited to test for the oxidative costs of reproduction: the Allen Cays Rock Iguana. Female rock iguanas reproduce at varying frequencies, often skipping years, allowing for a comparison between reproductive and non-reproductive females during the same narrow, annual breeding season. This feature of iguana life history enabled us to address not just sex and seasonal differences in physiology, but also potential oxidative costs of reproduction in females. Male and female iguanas were sampled during the early (vitellogenic), late (gravid) and post-reproductive seasons. Ultrasound examinations were performed on females to quantify reproductive investment, and blood samples were collected for physiology assays, which included reactive oxygen metabolites (d-ROMs), antioxidants, triglycerides, free glycerol and glucose. The early reproductive season was characterized by significant increases in reproductive female's triglycerides, free glycerol and oxidative stress compared to late and post-reproductive periods and non-reproductive females and males during all sampling periods. Antioxidants were significantly elevated during the early reproductive season for reproductive females, non-reproductive females and males when compared to late and post-season. Follicle number in early reproductive females was positively related to d-ROMs, triglycerides and free glycerol, negatively related to antioxidants and showed no relationship with glucose. Measures of oxidative stress, d-ROMs and oxidative index were positively correlated with circulating levels of the lipid metabolite free glycerol during the early reproductive period, but this relationship weakened in the late season and disappeared in the post-season. Broadly, this study supports the hypothesis that the relationship between reproduction and oxidative stress is driven by energy investment, being greatest during early reproduction when vitellogenesis is occurring.

Secondary vitellogenesis persists despite disrupted fecundity in amphipods maintained on metal-contaminated sediment: X-ray fluorescence assessment of oocyte metal content.

Melita plumulosa is an epibenthic, detritivorous amphipod found in estuaries along the eastern coast of Australia. It has been utilized as a test organism in rapid ten to thirteen days reproduction toxicity tests for sediment quality assessment. The fecundity of females in the toxicity test has been found to be inhibited by exposure of the amphipods to contaminated sediments enriched with zinc and other metals. This study investigated the proposal that interference in vitellogenesis is the cause of reproductive toxicity of metals in crustaceans. Inspection of the ovaries from amphipods on day 6 of the test either from control or Zn/Pb/Cd/Cu-spiked sediment, that were nearing completion of vitellogenesis, showed that the females in all treatments were producing similar numbers of oocytes undergoing secondary vitellogenesis. The distribution of the Zn, Cu and Pb in the oocytes and ventral caeca of females was examined by X-ray fluorescence microscopy. Elemental mapping revealed a dense accumulation of Zn in primary oocytes and a uniform distribution of Zn and Cu in the secondary oocytes in all treatments. Zn and Cu were also observed to be uniformly distributed in the ventral caeca. Pb was not detected in either of these tissues. The apparent normal morphology and the typical number of oocytes undergoing secondary vitellogenesis suggest that vitellogenesis was not being disrupted by Pb displacing Zn in the metal-binding domain of vitellogenin in amphipods exposed to the contaminated sediment during the test. Alternative mechanisms for the reproductive toxicity of amphipods exposed for six days to metal-contaminated sediment are discussed.

Structural and performance costs of reproduction in a pure capital breeder, the Children's python Antaresia childreni.

Females often manage the high energy demands associated with reproduction by accumulating and storing energy in the form of fat before initiating their reproductive effort. However, fat stores cannot satisfy all reproductive resource demands, which include considerable investment of amino acids (e.g., for the production of yolk proteins or gluconeogenesis). Because capital breeders generally do not eat during reproduction, these amino acids must come from internal resources, typically muscle proteins. Although the energetic costs of reproduction have been fairly well studied, there are limited data on structural and performance costs associated with the muscle degradation required to meet amino acid demands. Thus, we examined structural changes (epaxial muscle width) and performance costs (constriction and strength) over the course of reproduction in a pure capital breeder, the children's python (Antaresia childreni). We found that both egg production (i.e., direct resource allocation) and maternal care (egg brooding) induce muscle catabolism and affect performance of the female. Although epaxial muscle loss was minimal in nonreproductive females, it reached up to 22% (in females after oviposition) and 34% (in females after brooding) of initial muscle width. Interestingly, we found that individuals with higher initial muscular condition allocated more of their muscle into reproduction. The amount of muscle loss was significantly linked to clutch mass, underscoring the role of structural protein in egg production. Egg brooding significantly increased proteolysis and epaxial loss despite no direct allocation to the offspring. Muscle loss was linked to a significant reduction in performance in postreproductive females. Overall, these results demonstrate that capital-breeding females experience dramatic costs that consume structural resources and jeopardize performance.

Bioenergetic components of reproductive effort in viviparous snakes: costs of vitellogenesis exceed costs of pregnancy.

Reproductive effort has been defined as the proportion of an organism's energy budget that is allocated to reproduction over a biologically meaningful time period. Historically, studies of reproductive bioenergetics considered energy content of gametes, but not costs of gamete production. Although metabolic costs of vitellogenesis (MCV) fundamentally reflect the primary bioenergetic cost of reproductive allocation in female reptiles, the few investigations that have considered costs of reproductive allocation have focused on metabolic costs of pregnancy (MCP) in viviparous species. We define MCP as energetic costs incurred by pregnant females, including all costs of maintaining gestation conditions necessary for embryogenesis. MCP by our definition do not include fetal costs of embryogenesis. We measured metabolic rates in five species of viviparous snakes (Agkistrodon contortrix, Boa constrictor, Eryx colubrinus, Nerodia sipedon, and Thamnophis sirtalis) during vitellogenesis and pregnancy in order to estimate MCV and MCP. Across all species, MCV were responsible for 30% increases in maternal metabolism. Phylogenetically-independent contrasts showed that MCV were significantly greater in B. constrictor than in other species, likely because B. constrictor yolk energy content was greater than that of other species. Estimates of MCP were not significantly different from zero in any species. In viviparous snakes, MCV appear to represent significant bioenergetic expenditures, while MCP do not. We suggest that MCV, together with yolk energy content, represent the most significant component of reptilian reproductive effort, and therefore deserve greater attention than MCP in studies of reptilian reproductive bioenergetics.

Adverse outcome pathways and ecological risk assessment: bridging to population-level effects.

Maintaining the viability of populations of plants and animals is a key focus for environmental regulation. Population-level responses integrate the cumulative effects of chemical stressors on individuals as those individuals interact with and are affected by their conspecifics, competitors, predators, prey, habitat, and other biotic and abiotic factors. Models of population-level effects of contaminants can integrate information from lower levels of biological organization and feed that information into higher-level community and ecosystem models. As individual-level endpoints are used to predict population responses, this requires that biological responses at lower levels of organization be translated into a form that is usable by the population modeler. In the current study, we describe how mechanistic data, as captured in adverse outcome pathways (AOPs), can be translated into modeling focused on population-level risk assessments. First, we describe the regulatory context surrounding population modeling, risk assessment and the emerging role of AOPs. Then we present a succinct overview of different approaches to population modeling and discuss the types of data needed for these models. We describe how different key biological processes measured at the level of the individual serve as the linkage, or bridge, between AOPs and predictions of population status, including consideration of community-level interactions and genetic adaptation. Several case examples illustrate the potential for use of AOPs in population modeling and predictive ecotoxicology. Finally, we make recommendations for focusing toxicity studies to produce the quantitative data needed to define AOPs and to facilitate their incorporation into population modeling.

Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Swimming physiology of European silver eels (Anguilla anguilla L.): energetic costs and effects on sexual maturation and reproduction.

The European eel migrates 5,000-6,000 km to the Sargasso Sea to reproduce. Because they venture into the ocean in a pre-pubertal state and reproduce after swimming for months, a strong interaction between swimming and sexual maturation is expected. Many swimming trials have been performed in 22 swim tunnels to elucidate their performance and the impact on maturation. European eels are able to swim long distances at a cost of 10-12 mg fat/km which is 4-6 times more efficient than salmonids. The total energy costs of reproduction correspond to 67% of the fat stores. During long distance swimming, the body composition stays the same showing that energy consumption calculations cannot be based on fat alone but need to be compensated for protein oxidation. The optimal swimming speed is 0.61-0.67 m s(-1), which is approximately 60% higher than the generally assumed cruise speed of 0.4 m s(-1) and implies that female eels may reach the Sargasso Sea within 3.5 months instead of the assumed 6 months. Swimming trials showed lipid deposition and oocyte growth, which are the first steps of sexual maturation. To investigate effects of oceanic migration on maturation, we simulated group-wise migration in a large swim-gutter with seawater. These trials showed suppressed gonadotropin expression and vitellogenesis in females, while in contrast continued sexual maturation was observed in silver males. The induction of lipid deposition in the oocytes and the inhibition of vitellogenesis by swimming in females suggest a natural sequence of events quite different from artificial maturation protocols.

Toxication or detoxication? In vivo toxicity assessment of ozonation as advanced wastewater treatment with the rainbow trout.

Ozonation as advanced wastewater treatment method is an effective technique for micropollutant removal. However, the application of this method carries the inherent danger to produce toxic oxidation byproducts. For an ecotoxicological assessment conventionally treated wastewater, wastewater after ozonation and ozonated wastewater after sand filtration were evaluated in parallel at an operating treatment plant via the fish early life stage toxicity test (FELST) using rainbow trout (Oncorhynchus mykiss). The FELST revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW and to the ozonated water after sand filtration. Hence sand filtration obviously prevents from adverse ecotoxicological effects of ozonation. An additional test with yolk-sac larvae resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treated wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. Adverse ozonation effects may have been a result of the conversion of chemicals into more toxic metabolites. However, sand filtration reduced toxication effects indicating that these oxidation byproducts are readily degradable or adsorbable. The results indicate that in any case ozonation should not be applied without subsequent post treatment appropriate for oxidation byproducts removal (e.g. sand filtration).

The metabolic cost of avian egg formation: possible impact of yolk precursor production?

Little is known about the energy costs of egg production in birds. We showed in previous papers that, during egg production, European starlings (Sturnus vulgaris) undergo a 22% increase in resting metabolic rate (RMR) and that the maintenance and activity costs of the oviduct are responsible for 18% of the variation in elevated laying RMR. Therefore, other energy-consuming physiological mechanisms must be responsible for the remaining unexplained variation in elevated laying RMR. Yolk precursor [vitellogenin (VTG) and very-low-density lipoprotein (VLDL)] production is likely to be costly because it signifies a marked increase in the biosynthetic activity of the liver. We documented the pattern of yolk precursor production in response to daily injections of 17beta-estradiol (E2) in zebra finches (Taeniopygia guttata). Based on this pattern we carried out an experiment in order to evaluate the metabolic costs of producing VTG and VLDL. Our E2 treatment resulted in a significant increase in plasma VTG and VLDL levels within the natural breeding range for the species. Although RMR was measured during the period of active hepatic yolk precursor production, it did not differ significantly within individuals in response to the treatment or when comparing E2-treated birds with sham-injected birds. This could mean that yolk precursor production represents low energy investment. However, we discuss these results in light of possible adjustments between organs that could result in energy compensation.