Intake and growth histories modulate bone morphology, microarchitecture, and mineralization in juvenile green turtles (Chelonia mydas).

Compensatory growth (CG) is accelerated growth that occurs when food availability increases after food restriction. This rapid growth may be associated with sublethal consequences. In this study, we investigated the effects of food restriction and subsequent realimentation and CG on bone structure in juvenile green turtles (Chelonia mydas). Turtles were fed ad libitum food for 12 weeks (AL), restricted food for 12 weeks (R), or restricted food for 5 weeks followed by ad libitum food for 7 weeks (R-AL). R-AL turtles demonstrated partial CG via enhanced food conversion efficiency (FCE) upon realimentation. After the 12th week, gross morphology (GM), microarchitecture, and mineralization of the right humerus of each turtle were analyzed. Many GM measurements (including proximal and maximal bone lengths, bone widths, and shaft thickness), most measurements of bone microarchitecture (excluding cortical and trabecular thickness and trabecular separation), and all mineralization measurements were labile in response to intake. We examined the possibility that changes in nutrient allocation to bone structure during realimentation facilitated CG in previously food-restricted turtles. Restoration of bone lengths was prioritized over restoration of bone widths during CG. Furthermore, restoration of trabecular number, connectivity density, and bone volume fraction was prioritized over restoration of cortical bone volume fraction. Finally, diaphyseal bone mineralization was partially restored, whereas no restoration of epiphyseal bone mineralization occurred during CG. Shifts in nutrient allocation away from certain bone attributes during food restriction that were not rectified when food availability increased probably provided an energy surplus that enhanced the conversion of food to growth and thus powered the CG response. Our study revealed how resource allocation to various bone attributes is prioritized as nutritional conditions change during development. These "priority rules" may have detrimental consequences later in life, indicating that conservation of green turtle foraging grounds should be given high priority.

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory.

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade "Environmental Estrogens" (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ERα) and/or beta (ERß). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-ß-D-galactopyranoside (ONPG) or chlorophenol red-ß-D-galactopyranoside (CPRG)) that are cleaved by ß-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

Body size and symbiotic status influence gonad development in Aiptasia pallida anemones.

Pale anemones (Aiptasia pallida) coexist with dinoflagellates (primarily Symbiodinium minutum) in a mutualistic relationship. The purpose of this study was to investigate the role of these symbionts in gonad development of anemone hosts. Symbiotic and aposymbiotic anemones were subjected to light cycles that induced gametogenesis. These anemones were then sampled weekly for nine weeks, and gonad development was analyzed histologically. Anemone size was measured as mean body column diameter, and oocytes or sperm follicles were counted for each anemone. Generalized linear models were used to evaluate the influence of body size and symbiotic status on whether gonads were present and on the number of oocytes or sperm follicles produced. Body size predicted whether gonads were present, with larger anemones being more likely than smaller anemones to develop gonads. Both body size and symbiotic status predicted gonad size, such that larger and symbiotic anemones produced more oocytes and sperm follicles than smaller and aposymbiotic anemones. Overall, only 22 % of aposymbiotic females produced oocytes, whereas 63 % of symbiotic females produced oocytes. Similarly, 6 % of aposymbiotic males produced sperm follicles, whereas 60 % of symbiotic males produced sperm follicles. Thus, while gonads were present in 62 % of symbiotic anemones, they were present in only 11 % of aposymbiotic anemones. These results indicate that dinoflagellate symbionts influence gonad development and thus sexual maturation in both female and male Aiptasia pallida anemones. This finding substantiates and expands our current understanding of the importance of symbionts in the development and physiology of cnidarian hosts.

Integrative and comparative reproductive biology: From alligators to xenobiotics.

Dr. Louis J. Guillette Jr. thought of himself as a reproductive biologist. However, his interest in reproductive biology transcended organ systems, life history stages, species, and environmental contexts. His integrative and collaborative nature led to diverse and fascinating research projects conducted all over the world. He doesn't leave us with a single legacy. Instead, he entrusts us with several. The purpose of this review is to highlight those legacies, in both breadth and diversity, and to illustrate Dr. Guillette's grand contributions to the field of reproductive biology. He has challenged the field to reconsider how we think about our data, championed development of novel and innovative techniques to measure endocrine function, helped define the field of endocrine disruption, and lead projects to characterize new endocrine disrupting chemicals. He significantly influenced our understanding of evolution, and took bold and important steps to translate all that he has learned into advances in human reproductive health. We hope that after reading this manuscript our audience will appreciate and continue Dr. Guillette's practice of open-minded and passionate collaboration to understand the basic mechanisms driving reproductive physiology and to ultimately apply those findings to protect and improve wildlife and human health.

Endogenous and exogenous estrogens during embryonic development affect timing of hatch and growth in the American alligator (Alligator mississippiensis).

Prenatal exposure to estrogenic endocrine disrupting chemicals (EDCs) can affect length of gestation and body mass and size of offspring. However, the dose, timing, and duration of exposure as well as sex and strain of the experimental animals determine the direction and magnitude of these effects. In this study, we examined the effects of a one-time embryonic exposure to either 17 ß-estradiol (E2) or bisphenol A (BPA) on rate of development and growth in American alligators (Alligator mississippiensis). Our results indicate that BPA and E2-treated alligators hatched approximately 1.4 days earlier than vehicle-treated (control) alligators, suggesting that estrogenic chemicals hasten hatching in these animals. We assessed growth rates, growth allometry, and body condition for 21 weeks after hatching and found that BPA-treated alligators grew more quickly shortly after hatching but more slowly thereafter compared to control alligators. Conversely, E2-treated alligators grew more slowly shortly after hatching but more quickly thereafter compared to control alligators. As a result of differences in growth rate, BPA-treated alligators were heavier, longer, and fatter than control alligators at age 5 weeks but were similar in size and leaner than control alligators at age 21 weeks. Biochemical analytes were examined at the end of the 21-week study to assess overall metabolic condition. We found that E2-treated alligators had significantly higher circulating plasma concentrations of cholesterol and triglycerides than control alligators while BPA-treated alligators had blood profiles comparable to control alligators. Our results provide important insights into the effects of exogenous estrogens on morphology and metabolism in an oviparous, semi-aquatic reptile.

Bridging developmental boundaries: lifelong dietary patterns modulate life histories in a parthenogenetic insect.

Determining the effects of lifelong intake patterns on performance is challenging for many species, primarily because of methodological constraints. Here, we used a parthenogenetic insect (Carausius morosus) to determine the effects of limited and unlimited food availability across multiple life-history stages. Using a parthenogen allowed us to quantify intake by juvenile and adult females and to evaluate the morphological, physiological, and life-history responses to intake, all without the confounding influences of pair-housing, mating, and male behavior. In our study, growth rate prior to reproductive maturity was positively correlated with both adult and reproductive lifespans but negatively correlated with total lifespan. Food limitation had opposing effects on lifespan depending on when it was imposed, as it protracted development in juveniles but hastened death in adults. Food limitation also constrained reproduction regardless of when food was limited, although decreased fecundity was especially pronounced in individuals that were food-limited as late juveniles and adults. Additional carry-over effects of juvenile food limitation included smaller adult size and decreased body condition at the adult molt, but these effects were largely mitigated in insects that were switched to ad libitum feeding as late juveniles. Our data provide little support for the existence of a trade-off between longevity and fecundity, perhaps because these functions were fueled by different nutrient pools. However, insects that experienced a switch to the limited diet at reproductive maturity seem to have fueled egg production by drawing down body stores, thus providing some evidence for a life-history trade-off. Our results provide important insights into the effects of food limitation and indicate that performance is modulated by intake both within and across life-history stages.

Gene-environment interactions: the potential role of contaminants in somatic growth and the development of the reproductive system of the American alligator.

Developing organisms interpret and integrate environmental signals to produce adaptive phenotypes that are prospectively suited for probable demands in later life. This plasticity can be disrupted when embryos are impacted by exogenous contaminants, such as environmental pollutants, producing potentially deleterious and long-lasting mismatches between phenotype and the future environment. We investigated the ability for in ovo environmental contaminant exposure to alter the growth trajectory and ovarian function of alligators at five months after hatching. Alligators collected as eggs from polluted Lake Apopka, FL, hatched with smaller body masses but grew faster during the first five months after hatching, as compared to reference-site alligators. Further, ovaries from Lake Apopka alligators displayed lower basal expression levels of inhibin beta A mRNA as well as decreased responsiveness of aromatase and follistatin mRNA expression levels to treatment with follicle stimulating hormone. We posit that these differences predispose these animals to increased risks of disease and reproductive dysfunction at adulthood.

Compensatory responses to food restriction in juvenile green turtles (Chelonia mydas).

The purpose of this study was to assess the compensatory responses to food restriction and subsequent increased food availability in juvenile green turtles (Chelonia mydas). Turtles were fed an ad libitum ration for 12 weeks (AL), a restricted ration for 12 weeks (R), or a restricted ration for 5 weeks and an ad libitum ration for 7 weeks (R-AL). Analysis of covariance was used to test the relationships between (1) growth and body size, (2) intake and body size, and (3) growth and intake for each of the three treatment groups. Body composition of turtles in each group was also evaluated at the beginning of the study and after weeks 5 and 12. After the switch to ad libitum feeding, R-AL turtles consumed comparable amounts of food and grew faster than AL turtles on a size-adjusted basis, but mean body sizes did not converge, although the overlap in their size ranges increased with time. The R-AL turtles also converted food to growth more efficiently and allocated proportionally more nutrients to protein accretion, thereby restoring body composition (except mineral content) to AL levels by the end of the study. Thus, accelerated size-specific growth without hyperphagia restored body condition but not size. These results indicate that (1) intake in juvenile green turtles is maximal when food is readily available and cannot be increased to compensate for a previous period of food limitation, (2) growth rates of ad libitum-fed turtles are only mildly plastic in response to past nutritional history, and (3) priority rules for nutrient allocation favor the attainment of an optimal condition rather than an optimal size. Nutritional setbacks experienced during the vulnerable juvenile stage could therefore have long-lasting consequences for wild turtles in terms of size-specific mortality risk, but these risks may be mitigated by the potential benefits of maintaining sufficient body stores.

Metabolic rate depression is induced by caloric restriction and correlates with rate of development and lifespan in a parthenogenetic insect.

Caloric restriction (CR) extends lifespan in most animals, but the mechanisms underlying this phenomenon are the subject of much debate. We investigated the association between longevity and resting metabolic rate (RMR) in Indian stick insects (Carausius morosus) by (i) determining the appropriate scaling coefficient for calculating mass-corrected RMR of insects throughout development, (ii) quantifying the response of RMR to diet history, and (iii) correlating RMR in multiple life-history stages with adult and total lifespan. Over a range of body sizes, whole-body RMR (measured as oxygen consumption rate) scaled linearly with body mass. Mass-specific RMR decreased in response to CR, particularly when food was restricted during juvenile stages. With one exception, RMR of insects in different life-history stages matched current feeding level and was not substantially affected by intake history. Total lifespan was affected by intake, with insects that experienced CR early in development living longer than insects that were fed ad libitum. Although CR was associated with extended total lifespan and decreased RMR, it was also associated with shortened adult lifespan. Thus, we found limited evidence that decreased RMR plays a causative role in determining longevity. Instead, CR and decreased RMR were associated with slower progression through pre-reproductive life-history stages.

Biochemical indices as correlates of recent growth in juvenile green turtles (Chelonia mydas).

Nucleic acid and protein concentrations and their ratios are increasingly used as correlates of nutritional condition and growth in marine species. However, their application in studies of reptile growth has not yet been validated. The green turtle (Chelonia mydas) is an endangered marine reptile for which assessing population health requires knowledge of demographic parameters such as individual growth rates. The purpose of this study was to evaluate a number of biochemical indices ([DNA], [RNA], RNA:DNA ratio, [protein], protein:DNA ratio, and RNA:protein ratio) in liver, heart, and blood as potential predictors of recent growth rate in juvenile green turtles under controlled feeding conditions. Intake of juvenile green turtles was manipulated over twelve weeks to obtain a range of growth rates. With the exception of [RNA](blood), [DNA](heart), and [protein]:[DNA](liver), all biochemical indices demonstrated significant linear relationships with growth rate during the last 1.5 weeks of the study. The best single predictors of recent growth were hepatic [RNA] and [RNA]:[protein], which explained 66% and 49%, respectively, of the variance in growth. Contrary to expectations, these two indices were negatively correlated with growth rate. To investigate the possibility that hepatic [RNA] was higher in slow-growing turtles because of elevated expression of antioxidant genes, we quantified glutathione peroxidase activity and total antioxidant potential. Both measures of antioxidant function were affected by intake and growth histories, but these effects did not explain our results for hepatic RNA and protein concentrations. We developed a model that predicted 68% of the variance in specific growth rate (SGR) with the equation SGR = -0.913(ln[RNA](liver)) + 17.689(Condition Index) + 4.316. In addition, our findings that [DNA] and [RNA]:[DNA] for blood were significantly correlated with SGR demonstrate the potential utility of minimally invasive tissue sampling that could facilitate instantaneous population monitoring.