Climate change, multiple stressors, and the decline of ectotherms.

Climate change is believed to be causing declines of ectothermic vertebrates, but there is little evidence that climatic conditions associated with declines have exceeded critical (i.e., acutely lethal) maxima or minima, and most relevant studies are correlative, anecdotal, or short-term (hours). We conducted an 11-week factorial experiment to examine the effects of temperature (22 °C or 27 °C), moisture (wet or dry), and atrazine (an herbicide; 0, 4, 40, 400 µg/L exposure as embryos and larvae) on the survival, growth, behavior, and foraging rates of postmetamorphic streamside salamanders (Ambystoma barbouri), a species of conservation concern. The tested climatic conditions were between the critical maxima and minima of streamside salamanders; thus, this experiment quantified the long-term effects of climate change within the noncritical range of this species. Despite a suite of behavioral adaptations to warm and dry conditions (e.g., burrowing, refuge use, huddling with conspecifics, and a reduction in activity), streamside salamanders exhibited significant loss of mass and significant mortality in all but the cool and moist conditions, which were closest to the climatic conditions in which they are most active in nature. A temperature of 27 °C represented a greater mortality risk than dry conditions; death occurred rapidly at this temperature and more gradually under cool and dry conditions. Foraging decreased under dry conditions, which suggests there were opportunity costs to water conservation. Exposure to the herbicide atrazine additively decreased water-conserving behaviors, foraging efficiency, mass, and time to death. Hence, the hypothesis that moderate climate change can cause population declines is even more plausible under scenarios with multiple stressors. These results suggest that climate change within the noncritical range of species and pollution may reduce individual performance by altering metabolic demands, hydration, and foraging effort and may facilitate population declines of amphibians and perhaps other ectothermic vertebrates.

Exposure, postexposure, and density-mediated effects of atrazine on amphibians: breaking down net effects into their parts.

Most toxicology studies focus on effects of contaminants during exposure. This is disconcerting because subsequent survival may be affected. For instance, contaminant-induced mortality can be later ameliorated by reduced competition among the survivors, a concept we refer to as "density-mediated compensation." Alternatively, it can be exacerbated by toxicant effects that persist or appear after exposure, a phenomenon we term "carryover effects." We developed a laboratory framework for testing the contribution of exposure, density-mediated, and carryover effects to net survival, by exposing embryos and larvae of the streamside salamander (Ambystoma barbouri) to atrazine (0, 4, 40, 400 ppb; 3 ppb is the U.S. drinking water maximum) and quantifying survival during and 14 months after exposure. Atrazine is the most commonly used herbicide in the United States and a documented endocrine disruptor. We show that atrazine-induced mortality during exposure was ameliorated by density-dependent survival after exposure, but complete density-mediated compensation was precluded by significant carryover effects of atrazine. Consequently, salamanders exposed to >or=4 ppb of atrazine had significantly lower survival than did control animals 14 months postexposure. The greatest change in survival occurred at low exposure concentrations. These nonlinear, long-term, postexposure effects of atrazine have similarities to effects of early development exposure to other endocrine disruptors. Together with evidence of low levels of atrazine impairing amphibian gonadal development, the results here raise concerns about the role of atrazine in amphibian declines and highlight the importance of considering persistent, postexposure effects when evaluating the impact of xenobiotics on environmental health.

Aquatic herbicide exposure increases salamander desiccation risk eight months later in a terrestrial environment.

Contaminants and climate change may be factors in amphibian declines. However, few studies have explored their joint impacts on postmetamorphic amphibians, a life stage of great importance to amphibian population dynamics. Here, we examine the effects of premetamorphic exposure (mean exposure of 64 d) to ecologically relevant concentrations of the globally common herbicide atrazine (0, 4, 40, 400 microg/L) on the behavior and water retention of lone and grouped postmetamorphic, streamside salamanders, Ambystoma barbouri. Salamanders exposed to > or = 40 microg/L of atrazine exhibited greater activity, fewer water-conserving behaviors, and accelerated water loss four and eight months after exposure compared to controls. No recovery from atrazine exposure was detected and its effects were independent of the presence of conspecifics. These results are consistent with the hypothesis that adverse climatic conditions and contaminants can interact to harm post-metamorphic amphibians; however, they suggest that these two stressors need not be experienced simultaneously to do so. These results emphasize the importance of considering both latent and cumulative effects of temporally linked stressors in ecotoxicology.

Androgen receptor in the oviduct of the turtle, Trachemys scripta.

Circulating androgens reach high concentrations in females of some reptiles and amphibians. We are testing the hypothesis that androgens can act directly in female reptilian reproductive tissues, via the androgen receptor. In this study, we sought to determine if androgen receptors are present in the oviduct of the turtle, Trachemys scripta, using radioligand-binding assays and immunological assays. An androgen-binding site was detected in turtle oviductal cytosol and oviductal nuclear extract by radioligand binding assay, using (3)H-dihydrotestosterone (DHT) as the ligand. This site was saturable (B(max)=11 pmol/g tissue), had a high affinity (10(-10) M), and showed specificity typical of androgen receptors (DHT>testosterone, progesterone>>estradiol, cortisol). Western blotting using an anti-androgen receptor antibody revealed a band of immunoreactivity in oviductal cytosol at approximately 115 kDa, and a more prominent band at 50 kDa, possibly indicating a truncated form of the androgen receptor. Immunohistochemistry revealed crossreactivity of the androgen receptor antibody against oviductal glandular cells but not against oviductal luminal epithelial or muscularis cells. The presence of androgen receptor in the turtle oviduct suggests that androgens have a role in female reproduction and that their action can be mediated directly by androgen receptor.

Lethal and sublethal effects of atrazine, carbaryl, endosulfan, and octylphenol on the streamside salamander (Ambystoma barbouri).

Agricultural contaminants may be contributing to worldwide amphibian declines, but little is known about which agrichemicals pose the greatest threat to particular species. One reason for this is that tests of multiple contaminants under ecologically relevant conditions are rarely conducted concurrently. In this study, we examined the effects of 37-d exposure to the agrichemicals atrazine (4, 40, and 400 micrograms/L), carbaryl (0.5, 5, and 50 micrograms/L), endosulfan (0.1, 1, and 10 micrograms/L for 31 d and 0.1, 10, and 100 micrograms/L for the last 6 d), and octylphenol (5, 50, and 500 micrograms/L) and to a solvent control on streamside salamanders (Ambystoma barbouri) in the presence and absence of food. We found that none of the agrichemicals significantly affected embryo survival, but that hatching was delayed by the highest concentration of octylphenol. In contrast to embryos, larval survival was reduced by the highest concentrations of carbaryl, endosulfan, and octylphenol. Growth rates were lower in the highest concentrations of endosulfan and octylphenol than in all other treatments, and the highest concentration of endosulfan caused respiratory distress. Significantly more carbaryl, endosulfan, and octylphenol tanks had larvae with limb deformities than did control tanks. Refuge use was independent of chemical exposure, but 10 micrograms/L of endosulfan and 500 micrograms/L of octylphenol decreased larval activity. Systematically tapping tanks caused a greater activity increase in larvae exposed to 400 micrograms/L of atrazine and 10 micrograms/L of endosulfan relative to solvent controls, suggesting underlying nervous system malfunction. Hunger stimulated a decrease in refuge use and an increase in activity, but this response was least pronounced in larvae exposed to the highest concentration of any of the four agrichemicals, possibly because these larvae were the most lethargic. More studies are needed that concurrently examine the effect of multiple contaminants on amphibians so we can better identify effective mitigating measures.

Effects of larval exposure to triphenyltin on the survival, growth, and behavior of larval and juvenile Ambystoma barbouri salamanders.

We exposed 10 sibships of the streamside salamander, Ambystoma barbouri, to two concentrations of triphenyltin (TPT) (1 and 5 microg/L) and an acetone carrier control for the entirety of the larval period. We measured effects on larval feeding rates, escape behavior, growth rates, and survival to, days to, and size at metamorphosis. Postmetamorphosis, we monitored feeding rates, growth rates, and survival of juvenile A. barbouri in order to investigate carryover effects. The 5-microg/L TPT concentration resulted in 93% mortality of the larvae. Exposure to 1 microg/L TPT had no mortality effect and no effect on the escape behavior of larvae. However, larvae exposed to this TPT concentration had significantly lower feeding rates and growth rates and therefore metamorphosed later than the controls but at the same mass. We detected a direct effect of TPT on growth rates beyond the effect through depressed feeding rates. We also found significant evidence for variation among sibships in their sensitivity to TPT toxicity. Once exposure was terminated at metamorphosis. we observed no residual effects of TPT on juveniles. Survival, feeding, and growth rates of juveniles exposed to TPT as larvae were not significantly different from those exposed only to the acetone carrier.

Cyclic histological changes of the oviductal-cloacal junction in the viviparous snake Toluca lineata.

The structure and seasonal changes of the oviductal-cloacal junction remain poorly understood in most squamates. This study was undertaken to describe the histology of the oviductal-cloaca junction of a female viviparous snake Toluca lineata, during gestation, previtellogenesis, and vitellogenesis. The oviductal-cloacal junction exhibits a wider lumen and thicker layers of connective tissue, smooth muscle layers, and total wall width compared to the posterior vagina. The lining is characterized by thick, short longitudinal mucosal folds. The luminal epithelia differ morphologically from anterior to posterior portions of the oviductal-cloacal junction. The anterior portion is lined with a simple columnar epithelium composed of nonciliated cells. The middle portion is lined with stratified epithelium that contains an apical columnar cell layer that undergoes morphological changes coincident with the reproductive cycle. The posterior portion is lined with a stratified squamous epithelium. The connective tissue underlying the epithelium contains numerous ovoid cells having abundant acidophilic cytoplasmic granules-eosinophils. Copulation occurs during the previtellogenic stage, as evidenced by the presence of abundant spermatozoa in the lumen of the anterior portion and of a copulatory plug in the middle and posterior portion of the oviductal-cloacal junction. J. Morphol. 237:91-100, 1998. © 1998 Wiley-Liss, Inc.

Histology and functional morphology of the oviduct of an oviparous snake, Diadophis punctatus.

Oviductal functional morphology remains poorly understood in oviparous snakes, particularly in regard to oviductal formation of albumen and the eggshell and to sperm storage. The oviduct of Diadophis punctatus was examined using histology and scanning electron microscopy to determine oviductal functional morphology throughout the reproductive cycle. The oviduct is composed of four morphologically distinct regions: infundibulum, uterine tube, uterus, and vagina. The infundibulum is thin, flaccid, and lined with simple ciliated cuboidal epithelial cells. The tube contains ciliated and secretory epithelial cells, which reach a maximum height and hypertrophy during early gravidity and produce glycosaminoglycans. The posterior portion of the tube contains temporary sperm storage receptacles. The uterus retains eggs throughout gestation and secretes the eggshell constituents. The endometrial glands of the uterus hypertrophy during vitellogenesis and become depleted of the secretory granules during gravidity. The functional morphology of the oviduct therefore shows cyclical changes that are correlated with eggshell formation. The vagina consists of thick longitudinal and circular smooth muscle layers, which may serve in retention of eggs during gestation. Furthermore, the vagina contains long furrows in the mucosa that serve as sperm storage receptacles. These receptacles store sperm following fall mating and overwintering, whereas the receptacles in the tube are utilized briefly during vitellogenesis just prior to ovulation. © 1996 Wiley-Liss, Inc.

Oviductal morphology and eggshell formation in the lizard, Sceloporus woodi.

Despite a great deal of work in recent years on the structure of reptilian eggshells, few studies have examined the structure and regulation of the female reproductive tract in the formation of eggshell components, and none have examined the entire process from ovulation to oviposition. In this study, we examined oviductal structure in the oviparous lizard, Sceloporus woodi, followed changes in oviductal structure during gravidity, and determined uterine function in the formation of eggshell components. The endometrial glands of the uterus produce the proteinaceous fibers of the eggshell membrane mainly during the first 24 hours following ovulation, and the fibers are secreted intact and subsequently wrapped around the in utero eggs. Eggshell fibers of different thicknesses are layered around each egg, ranging from an inner layer of thick fibers that gradually become thinner medially and finally forms an outer layer of densely packed particulate matter. These changes in the fibrous layer are reflected by the thickness and length of fibers released from the endometrial glands. Calcium deposition occurs from 3 days following ovulation through day 14 (oviposition) and is accompanied by cellular changes in the luminal epithelium suggestive of secretory activity. Deposition of the eggshell components within the uterus occurs on all eggs simultaneously, rather than sequentially. © 1993 Wiley-Liss, Inc.

Morphological changes in the oviductal endometrium during the reproductive cycle of the tortoise, Gopherus polyphemus.

The oviducts of 24 tortoises (Gopherus polyphemus) were examined using histological techniques and scanning electron microscopy to determine endometrial morphology. Measurements of endometrial characteristics (epithelial cell height, cilia length, thickness of endometrial glandular layer, and glandular diameter) in the uterus and tube (tuba uterina) were obtained to determine changes during the reproductive cycle. Epithelial cell height increases in both the uterus and the tube during vitellogenesis and remains hypertrophied during gravidity. Cilia length increases in the uterus during late vitellogenesis and gravidity, but the length of tubal cilia does not change during the reproductive cycle. The ratio of secretory to ciliated epithelial cells in the oviduct increases from quiescence to gravidity. The thickness of the glandular endometrial layer increases in both the uterus and tube during vitellogenesis. In the uterus, the glandular layer decreases in thickness during gravidity. The diameter of the uterine glands increases throughout vitellogenesis and gravidity; however, following ovulation glandular cells become depleted of secretory granules and cell height diminishes. The diameter of the tubal glands is unchanged during the reproductive cycle. Oviductal hypertrophy during vitellogenesis coincides with elevated circulating estradiol, whereas during gravidity progesterone concentrations peak (Taylor, '82, PhD Dissertation, University of Florida, Gainesville) and may induce secretion of albumen and eggshell components.

Oviductal morphology and egg shelling in the oviparous lizards Crotaphytus collaris and Eumeces obsoletus.

Morphological changes occurring in the oviduct and epithelial cells of the lizards Crotaphytus collaris and Eumeces obsoletus during the natural reproductive cycle were examined and quantified. Additionally, development of the eggshell at different stages of gravidity was described. The anterior uterus of each species has a distinct glandular type which differs between species: in E. obsoletus, the glands are tubular and in C. collaris, branched saccular. The branched saccular glands in the anterior uterus of C. collaris produce collagen-like material that forms the fibers of the shell membranes. However, fibers from the eggshell of E. obsoletus did not stain for collagen. The shell of both species is composed of a multilayered inner boundary covered externally by fibers of varying thickness. Initial layers are composed of thick fibers all lying along the same general axis. Outer layers of fibers are progressively thinner and an external surface layer composed of glycosaminoglycans (GAGs) is also present. In C. collaris, calcium, which is deposited in relatively small amounts on the shell surface, appears to be secreted by the epithelium of the anterior uterus. The nonciliated secretory epithelial cells covering the villi-like folds of the posterior infundibulum secrete GAGs. Epithelial cell height of the infundibular villi is greatest during early gravidity. A functional relationship may exist between luteal activity and oviductal secretory activity because the activity of the glandular epithelium varied as gravidity progressed.