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. 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.

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. 9999A: XX–XX, 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. 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. 9999A: 1–13, 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. 9999A: 1–9, 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. 9999A: 1–9, 2013. © 2013 Wiley Periodicals, Inc.

The goal of this study was to determine the relationship between cortisol and the toadfish serotonin 2A (5-HT_2A) receptor, which is believed to be responsible for the activation of the toadfish urea transporter, tUT. We hypothesize that elevations in cortisol would play a role in the regulation of the 5-HT_2A receptor at the level of mRNA expression, ligand binding, and/or function. To test this idea, cortisol levels were manipulated by either crowding or through treatment with the cortisol synthesis blocker, metyrapone. Crowded fish had significantly higher circulating cortisol levels compared to uncrowded fish and cortisol levels in metyrapone-treated fish were significantly lower than saline-treated controls. No significant difference was measured in gill 5-HT_2A mRNA expression levels between uncrowded and crowded, control- or metyrapone-treated fish. Furthermore, no significant difference was measured in [³H]-5-HT binding kinetics or in the competitive binding of the 5-HT₂ agonist, α-methyl 5-HT, to isolated gill basolateral membranes of uncrowded or crowded toadfish. However, the binding maximum (B_max) of the 5-HT_2A receptor antagonist, [³H]-ketanserin, was significantly different between all four groups of fish (metyrapone > control > crowded > uncrowded). Furthermore, metyrapone-treated fish excreted approximately twofold more urea compared to controls when injected with α-methyl 5-HT, a 5-HT₂ receptor agonist shown to stimulate urea excretion. Our results suggest that cortisol may have differential effects on 5-HT receptor binding, which could have potential implications on the control of pulsatile urea excretion in toadfish. J. Exp. Zool. 319A:237–248, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

The mechanism of reproduction in mammals is very complex and in some cases is quite particular. For example in some bat species, the male presents a reproductive mechanism characterized by an annual testicular cycle that goes from recrudescence to regression (spermatogenesis to inactivity period, respectively). After recrudescence, the spermatozoa arrive at epididymis and wait to be expelled at the time of ejaculation during the mating period, which occurs some months later. Because serotonin (5-HT) has gained reproductive importance in the last years, the aim of the present study was to analyze the expression of this indolamine and both tryptophan hydroxylase and monoamine oxidase isoform A—enzymes involved in its metabolism—in Myotis velifer testes, a seasonal reproductive bat species that shows temporal asynchrony in its sexual cycle, across the principal periods of their reproductive cycle. By using both Falck–Hillarp histochemistry and immunofluorescence techniques, we found serotonin in vesicles of Leydig cells and probably Sertoli cells too; interestingly, both intracellular localization and concentration was variable across the different stages of the reproductive cycle, being lower during spermatogenesis phase and increasing during the mating phase. These results suggest that 5-HT is present in bat testes and it could play an important role in testicular function during their reproductive cycle. J. Exp. Zool. 319A:249–258, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

Parchment-shelled eggs are characteristic of most squamates, including the basal clades of gekkotan lizards. The majority of gekkotan lizards, however, produce rigid-shelled eggs that are highly impermeable to gas exchange; eggs are laid in dry sites and experience a net loss of water during incubation. We tested the hypothesis that the 1,000-fold lower rate of oxygen diffusion through the shells of rigid- compared to parchment-shelled eggs imposes a physiological cost on development. To do this, we contrasted species with rigid and with parchment shells with regards to (1) rates of embryonic metabolism and (2) rates and patterns of development of the yolk sac and chorioallantois, the vascularized extra-embryonic membranes that transport oxygen to embryonic tissues. Metabolic rates of embryos from the rigid-shelled eggs of Gehyra variegata did not differ from those of the parchment-shelled eggs of Oedura lesueurii. Moreover, maximum metabolic rates of gekkotans with rigid shells did not differ from those of gekkotan or scincid lizards with parchment shells. In contrast, the yolk sac covered more of the surface area of the egg at oviposition, and the chorioallantois reached its full extent earlier for the species with rigid shelled eggs (Chondrodactylus turneri, G. variegata) than for the species with parchment-shelled eggs (Eublepharis macularius, O. lesueurii). Differences in the temporal patterns of yolk sac and chorioallantois development would thus serve to compensate for low rates of oxygen diffusion through rigid shells of gekkotans. J. Exp. Zool. 319A:259–267, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

The physiological and biological properties of skeletal muscle in crustacea have not been well understood compared with those of vertebrates. The present study focused on myosin, the major protein in skeletal muscle, from shrimps. In our previous study, two full-length genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle of kuruma Marsupenaeus japonicus, black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps, and named as MHCa and MHCb. In this study, we renamed these as MHC1 and MHC2, respectively, due to the presence of various isoforms newly identified. Partial MHC sequences were identified from pleopod muscle of these shrimps. Two MHCs, named MHC3 and MHC4, were identified from pleopod muscle of kuruma shrimp, whereas two MHCs, named MHC4 and MHC5, were cloned from Pacific white shrimp pleopod. MHC3 was cloned only from black tiger shrimp pleopod. Partial MHC sequences from zoea, mysis, and postlarvae of black tiger and Pacific white shrimps were also determined. The phylogenetic tree demonstrated that most MHCs from pleopod muscle and larval MHCs formed clades with MHC1 and MHC2, respectively. These MHCs were considered to be of fast type, since MHC1 and MHC2 are fast-type MHCs according to our previous study. MHC5 obtained from pleopod muscle of Pacific white shrimp in this study was monophyletic with American lobster Homarus americanus S₂ slow tonic MHC previously reported, indicating that MHC5 from Pacific white shrimp is of slow type. J. Exp. Zool. 319A:268–276, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

The white-faced ibis Plegadis chihi Vieillot, 1817 (Pelecaniformes: Threskiornithidae) is a socially monogamous colonially breeding bird in which behavioral and ecological observations suggest the occurrence of conspecific brood parasitism (CBP). We inferred aspects of the genetic mating system of P. chihi in nature, using a genetic approach in the absence of parental information. We used five heterologous microsatellite loci and a multiple-step methodological approach to infer kinship patterns among 104 pairs of nestlings sampled inside 80 nests in a breeding colony from southern Brazil. The estimated effective population size was 69 white-faced ibises (95% CI: 50–98), enough to ensure long-term population survival. Kinship patterns were identified for 38% of the analyzed pairs: 60% of the diagnosed pairs were identified as full-siblings, 2.5% as half-siblings and 37.5% as unrelated individuals. CBP could explain the presence of unrelated nestlings within broods, in agreement with available non-genetic evidence. The presence of half-siblings within broods could indicate extra-pair paternity. Results suggest that a non-strictly monogamous genetic mating system may be present in the white-faced ibis. This study is the first molecular approach to better characterize the reproductive behavior of P. chihi in the wild. Our findings set the stage for further research to investigate the possible causes and consequences of alternative reproductive strategies in this species. J. Exp. Zool. 319A:277–284, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

In the nematode Caenorhabditis elegans, the odorant diacetyl is sensed by AWA sensory neurons in the amphid sensory organ and elicits an attractive response, whereas 2-nonanone is sensed by AWB amphid sensory neurons and elicits an avoidance response. In the present study, we report that nematodes exhibit a sensory interaction between the attractant diacetyl and repellent 2-nonanone. In the presence of food, the chemotactic response to 0.01% diacetyl in nematodes preexposed to 0.1% diacetyl was greater than that in nonexposed naive nematodes (P < 0.05). The response to diacetyl was also greater in nematodes preexposed to 3% 2-nonanone in the presence of food than that in naive nematodes (P < 0.01). In the absence of food, the response to diacetyl in nematodes preexposed to diacetyl or 2-nonanone was significantly lower than that in nonexposed control nematodes (P < 0.01). The avoidance response to 10% 2-nonanone in nematodes preexposed to each odorant in the presence or absence of food was lower than that in nonexposed nematodes (P < 0.05). To confirm the validity of our results, the chemotactic responses to diacetyl and 2-nonanone were observed using che-3, odr-4, and odr-10 mutants, which exhibited defective sensitivity to diacetyl or 2-nonanone. From the results of our experiments, we conclude that nematodes exhibit a sensory interaction between diacetyl and 2-nonanone and speculate that this interaction is driven by higher-level neuronal circuits that underlie sensory integration. J. Exp. Zool. 319A:285–295, 2013. © 2013 © 2013 Wiley Periodicals, Inc.