Environmental conditions during early development can profoundly influence an individual's phenotype. Development requires simultaneous maturation and orchestration of multiple physiological systems creating the potential for interaction among key systems and requiring substantial resources. We investigated the influence of thermoregulation on immunocompetence in Wood Duck ducklings (Aix sponsa). At both 1 and 2 days post hatch (dph) we evaluated ducklings' abilities to thermoregulate during a thermal challenge at one of four temperatures (36 [thermoneutral controls], 20, 10, or 5°C). At 3 dph, ducklings received a superficial wound, which was monitored until full recovery to quantify wound healing ability, an ecologically relevant, integrative measure of immune function. We demonstrated that duckling body temperature decreased with increasing thermal challenge severity, thermoregulatory ability increased with age, and thermoregulation had temperature-dependent effects on the immune system. Specifically, a more severe thermal challenge (5°C) resulted in decreased immune performance when compared to a mild challenge (20°C). We conclude that early thermoregulatory experiences are influential in shaping immune responses early in development. Furthermore, our results emphasize that future studies of environmental stressors need to consider multiple physiological endpoints since interaction among systems can result in competing physiological demands. J. Exp. Zool. 9999A: 1–8, 2013. © 2013 Wiley Periodicals, Inc.

Almost the whole life cycle of the grass puffer (Takifugu niphobles) occurs in seawater (SW), but it is also sometimes found in fresh water (FW) rivers. This study aims to evaluate the effects of FW exposure on the stress, osmoregulatory, and physiological responses of the grass puffer. The grass puffers were captured from a local wetland and acclimated to SW (35‰) or FW in the laboratory. In the stress responses, plasma glucose concentrations and the abundances of hepatic and branchial heat shock proteins were higher in the FW group than in the SW group. FW acclimation led to a significant increase in the protein abundance and the specific activity of branchial Na⁺/K⁺-ATPase (NKA). Immunochemical staining showed that the NKA immunoreactive (NKIR) cells of the FW and SW puffer were distributed mainly in gill filaments. Although the number of NKIR cells was similar in the two groups, the protein levels of proliferating cell nuclear antigen (PCNA) of nuclear fractions were elevated in the gills of the FW puffer. The induction of gill PCNA might contribute to cell proliferation which would maintain the amount of NKIR cells or repair DNA when exposed to FW, an osmotically stressful environment. Hence, activation of stress responses would provide the osmoprotection associated with FW adaptation of the grass puffer. Changes of branchial NKA expression and activity for osmoregulatory adjustment were required for stable blood osmolality and muscle water content. Based on our findings, the grass puffer was suggested to be a euryhaline teleost with SW preference. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

Freshwater mussels have decreased dramatically in Iberia over the last decades. These animals are responsible for important ecosystem services such as recycling nutrients and improving water clarity. Under this view a better knowledge on the biological features of these animals is extremely important for future conservation and management actions. In this study the reproductive and gametogenic cycle of Anodonta anatina were studied during 2 years in one population as well as the sex ratio and hermaphroditism in six distinct populations, using standard histology. Gametogenesis was continuous in both sexes and germinal epithelium in early stages of development. Gametes were present throughout the reproductive cycle. Oogenesis and spermatogenesis occurred mainly between January and May. Larvae brooding occurred between September and March and main glochidia discharge occurred over a short period (2–3 weeks) in March. For the sex-ratio and hermaphroditism assessments a variable number of individuals were collected from several populations from lakes and rivers. Previous studies described A. anatina as mainly dioecious with only a few populations presenting occasional hermaphroditism. However, the present study indicates that A. anatina sexual behavior is influenced by environmental conditions, being mainly dioecious in rivers with increased hermaphroditism in standing waters. Although self-fertilization was not confirmed, additional studies with molecular characterization of larvae using fast evolving markers should be used in future studies to enlighten this process. Overall, this study indicates that for more efficient conservation actions and management plans, freshwater mussel reproductive biology should be studied at the population level mainly in the subfamily Anodontinae. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

In Daphnia pulex, juveniles form “neckteeth” a defensive structure on their heads, in response to predatory kairomones released by Chaoborus larvae. This phenomenon provides a model experimental system for the study of developmental mechanisms and evolutionary processes in predator-induced polyphenisms. Although it is thought that kairomone signals are sensed and converted into physiological signals resulting in morphological changes, little is known about the endocrine and physiological mechanisms of this process. Juvenile hormones and related chemicals, that is, juvenoids, are key hormones responsible for various physiological events in insects, including polyphenisms. In some crustaceans, methyl farnesoate (MF) is known to act as a juvenoid. In order to investigate the functions of juvenoids in defense morph formation, we treated daphnids with MF as well as JHIII (Juvenile Hormone III, an insect juvenoid) and fenoxycarb (a synthetic juvenile hormone analog) during their developmental stages. Strikingly, in the first-instar juveniles, all examined juvenoids stimulated the formation of neckteeth only in the presence of kairomones, not by themselves. This juvenoid effect on the neckteeth formation might be due to disturbance of the JH pathway. Juvenoid treatments reduced tail-spine length, whereas predatory kairomones are known to elongate tail spine. These results suggest that other physiological factors are responsible for the tail-spine elongation. J. Exp. Zool. 9999A: 1–11, 2013. © 2013 Wiley Periodicals, Inc.

Bony fish primarily excrete ammonia as adults however the persistence of urea cycle genes may reflect a beneficial role for urea production during embryonic stages in protecting the embryo from toxic effects of ammonia produced from a highly nitrogenous yolk. This study aimed to examine the dynamic scope for changes in rates of urea synthesis and excretion in one such species (zebrafish, Danio rerio) by manipulating the intrinsic developmental rate (by alteration of light:dark cycles), as well as by direct chemical manipulation via ammonia injection (to potentially activate urea production) and nicotinamide exposure (to potentially inhibit urea production). Continuous dark exposure delayed development in embryos as evidenced by delayed appearance of hallmark anatomical features (heartbeat, eye pigmentation, body pigmentation, lateral line, fin buds) at 30 and 48 hr post-fertilization, as well by a lower hatching rate compared to embryos reared in continuous light. Both ammonia and urea excretion were similarly effected and were generally higher in embryos continuously exposed to light. Ammonia injection resulted in significant increases (up to fourfold) of urea N excretion and no changes to ammonia excretion rates along with modest increases in yolk ammonia content during 2–6 hr post-injection. Nicotinamide (an inhibitor of urea synthesis in mammals) reduced the ammonia-induced increase in urea excretion and led to retention of ammonia in the yolk and body of the embryo. Our results indicate that there is a relatively rapid and large scope for increases in urea production/excretion rates in developing embryos. Potential mechanisms for these increases are discussed. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

Limited information is available on effects of growth hormone transgene and nutrition on growth and development of aquatic animals. Here, we present a study to test these effects with growth-enhanced transgenic common carp under two nutritional conditions or feeding rations (i.e., 5% and 10% of fish body weight per day). Compared with the nontransgenic fish, the growth rates of the transgenic fish increased significantly in both feeding rations. The shape of the pharyngeal bone was similar among treatments, but the transgenic fish had relatively smaller and lighter pharyngeal bone compared with the nontransgenic fish. Calcium content of the pharyngeal bone of the transgenic fish was significantly lower than that of the nontransgenic fish. Feeding ration also affected growth rate but less of an effect on bone development. By manipulating intrinsic growth and controlling for both environment (e.g., feeding ration) and genetic background or genotype (e.g., transgenic or not), this study provides empirical evidence that the genotype has a stronger effect than the environment on pharyngeal bone development. The pharyngeal bone strength could be reduced by decreased calcium content and calcification in the transgenic carp. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

Female sperm storage can influence male reproductive success and may favour males that produce sperm that remain viable across several ovarian cycles. Here we show that sperm are viable in the female reproductive tract across ovarian cycles in the mallee dragon, Ctenophorus fordi. Based on experimental mating trials, we show that stored sperm were generally less likely to fertilize eggs than recently inseminated sperm. The fertilization success of stored sperm increased with male body size relative to rivals. This may be due to differences in ejaculate volume or sperm number transferred by males of different sizes. However, there was no evidence that copulation time, which is correlated with ejaculate volume, contributed to fertilization success. We suggest that sperm storage across ovarian cycles may be common in small, multi-clutched lizards and that its impact on selection on male phenotypes could contribute to the evolution of lizard mating systems. J. Exp. Zool. 9999A: 1–5, 2013. © 2013 Wiley Periodicals, Inc.

Continuous presentation of the odorant diacetyl to the nematode Caenorhabditis elegans causes a decrease in the level of chemotactic response to diacetyl. This decline in response is caused by diacetyl adaptation. When wild-type nematodes were maintained at 15°C after pre-exposure to diacetyl, diacetyl adaptation did not continue up to 2 hr. Adaptation continued up to 6 hr in nematodes bred at 20°C, and it continued beyond 12 hr in nematodes bred at 25°C. These results indicate that the retention time of diacetyl adaptation is dependent on the environmental breeding temperature and suggest that moderate oxygen signals are required for maintaining the attenuated response to diacetyl because of the correlation between breeding temperature and production of oxygen intermediates. When isp-1 and clk-1 mutants, which show reduced rates of oxygen intermediate production, were maintained at 20 and 25°C after pre-exposure to diacetyl, the mutants showed a shorter retention time of diacetyl adaptation compared with that of wild-type nematodes. When gas-1 and mev-1 mutants, which have a hypersensitive response to oxidative stress, were maintained at 15 and 20°C, they showed a longer retention time of adaptation, that is, adaptation continued beyond 2 and 12 hr, respectively. When wild-type nematodes were maintained on plates that included 0.05% α-lipoic acid, which suppresses production of oxygen intermediates, the retention time of adaptation did not continue up to 6 hr in nematodes bred at 20°C and up to 12 hr in nematodes bred at 25°C. These results support the possibility that oxygen intermediates contribute to retention time for diacetyl adaptation in the nematode C. elegans. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

Melatonin (N-acetyl-5-methoxy-tryptamine) was first discovered from the bovine pineal gland extract in 1958. Since then, its synthesis, metabolism, physiological, and patho-physiological functions are well studied in vertebrates; there is an increasing recognition of melatonin in invertebrates and especially in crustaceans. The presence of melatonin in crustaceans is now well documented and some functional aspects in the framework of crustacean biology have been demonstrated. This review aims at giving a comprehensive overview of the various physiological events regulated by this pleiotropic hormone. Topics include: glucose homeostasis, regulation of reproduction, molting, limb regeneration, and antioxidant properties. Finally, perspectives on current and possible research are offered. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages. J. Exp. Zool. 319A:297–309, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

The impact of temperature during incubation and gestation has been tested in various reptiles; the postnatal period has been rarely investigated however. Three groups of newborn aspic vipers (Vipera aspis) were placed under contrasted thermal regimes during 7 months: (1) a cool 23°C constant regime, (2) a warm 28°C constant regime, and (3) an optimal regime with free-access to a wide range of temperatures. Later, all the snakes were placed under hibernation conditions (6°C) during 3 months. Finally all the snakes were placed in the optimal thermal regime during 2 additional months. The total duration of the experiment was of 12 months. Body mass and feeding rates were recorded weekly, body size was measured monthly. We also assessed locomotor performance and recorded several behavioral traits (e.g., defensive and predatory behaviors). As expected, snakes raised under cool temperatures exhibited low feeding rate, growth rate, body condition, and they exhibited poor locomotor performance; they also displayed marked defensive behaviors (e.g., high number of defensive bites) whilst hesitating during longer periods to bite a prey. Such behavioral effects were detected at the end of the experiment (i.e., 5 months after exposure to contrasted thermal treatments [3 months of hibernation plus 2 months of optimal regime]), revealing long term effects. Surprisingly, growth rate and locomotor performance were not different between the two other groups, warm constant 28°C versus optimal regimes (albeit several behavioral traits differed), suggesting that the access to a wide range of ambient temperatures was not a crucial factor. J. Exp. Zool. 319A:310–318, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

Animal momentum and heat transfer analysis has historically used direct animal measurements or approximations to calculate drag and heat transfer coefficients. Research can now use modern 3D rendering and computational fluid dynamics software to simulate animal–fluid interactions. Key questions are the level of agreement between simulations and experiments and how superior they are to classical approximations. In this paper we compared experimental and simulated heat transfer and drag calculations on a scale model solid aluminum African elephant casting. We found good agreement between experimental and simulated data and large differences from classical approximations. We used the simulation results to calculate coefficients for heat transfer and drag of the elephant geometry. J. Exp. Zool. 319A:319–327, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

Amphibians are known to better tolerate and endure adverse environmental conditions such as redox imbalances conferred by reactive oxygen species (ROS), compared to mammals. Interestingly, the exact adaptation strategies and signaling mechanisms mediating these effects have not been fully elucidated. Therefore, in the present study, we probed into the molecular response of the isolated perfused Rana ridibunda heart to curcumin, in the context of mitogen-activated protein kinases (MAPKs) phosphorylation patterns and apoptotic markers occurrence. In particular, this polyphenol was found to exert a pro-oxidant effect in our model and to significantly upregulate p38-MAPK and JNKs phosphorylation (thus activation). The early apoptosis observed, substantiated by poly(ADP-ribose) polymerase (PARP) cleavage, was established to be JNKs- and ROS-mediated, while no involvement of p38-MAPK was detected. Subsequently, the pro-oxidative activity of curcumin was confirmed to mimic H₂O₂. Furthermore, NADPH oxidase as well as Na⁺/K⁺-ATPase were found to mediate JNKs phosphorylation as well as PARP proteolytic cleavage. Curcumin exerts pleiotropic actions, both beneficial and detrimental and is currently the subject of intense scientific research. Being a low-molecular-weight antioxidant, it is intriguing to investigate curcumin's role in redox homeostasis in the amphibian heart, under conditions that apparently favor its pro-oxidative properties. Comparative studies of its multifaceted role in different species may contribute to the clarification of the signaling mechanisms it triggers and the terminal physiological response it confers. Collectively, this is to our knowledge, the first time that the signal transduction pathways stimulated by curcumin have been assessed in a non-mammalian species. J. Exp. Zool. 319A:328–339, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

Previous studies examining behavioral responses to CO₂ revealed that high [CO₂] acts as a natural repellent in a concentration dependent manner for crayfish. Physiologically, CO₂ can rapidly block the autonomic responses in heart rate, as well as, inhibit an escape tail flip reflex in crayfish. Here, we demonstrate that the behavioral observations can be mechanistically explained by CO₂ blocking glutamate receptors at the neuromuscular junction and through inhibition of recruiting motor neurons within the CNS. The effects are not mimicked with a lower pH in the bathing solution. Since spontaneous and sensory-evoked activities in the sensory root and motor neurons are reduced by CO₂, this is an anesthetic effect. We propose this is due to blockage of electrical synapses, as well as, some of the central glutamatergic-drive. We used agonists and antagonists (glutamate, nicotine, domoic acid, cadmium, heptanol) to various synaptic inputs, which are possibly present in the ventral nerve cord (VNC). Results from these chemicals supported the idea that there is electrical as well as chemical drive within the circuit that can modulate intrinsic as well as sensory evoked activity in the motor neurons. We have documented that CO₂ has actions in the periphery as well as in the CNS, to account for the behavioral responses previously shown. Furthermore, we document that gap junctions as well as glutamatergic synapses are potential targets. This study also aids in the dissection of a neural circuitry within the VNC that drives spontaneous and sensory evoked activity of the superficial flexor motor neurons. J. Exp. Zool. 319A:340–354, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

In bufonids, the Bidder's organ (BO), located in the anterior pole of the testis, is sometimes referred to as a rudimentary ovary because of the presence of previtellogenic follicles. In males of Rhinella arenarum it has been demonstrated that some follicles are vitellogenic and also express several steroidogenic enzymes in follicular cells. The purpose of this study is to describe seasonal variations in plasma estradiol (E₂) and in aromatase activity of the BO, and to determine the capacity of the BO to synthesize E₂ from cholesterol in males of R. arenarum. E₂ was determined by radioimmunoassay and aromatase activity was measured by transformation of radioactive substrates into products. Results indicate that plasma E₂ reached the highest concentration in April and the lowest one in animals captured in June, showing a progressive increase to the end of the year. Plasma E₂ and total activity of aromatase in the BO were significantly lower during the pre-reproductive season than during the reproductive and post-reproductive seasons. It was also demonstrated that the BO is able to produce E₂ from endogenous substrates throughout the year. No correlation was found between plasma E₂ and total BO weight, while there was a significant correlation between plasma E₂ and total activity of aromatase, and between plasma E₂ and E₂ produced in vitro. Taken together, these results demonstrate seasonal variations in plasma E₂, in bidderian total activity of aromatase and, that the BO of adult males of R. arenarum is able to produce E₂ from endogenous substrates. J. Exp. Zool. 319A:355–364, 2013. © 2013 © 2013 Wiley Periodicals, Inc.