Developmental Exposure to Trace Concentrations of Chlorpyrifos Results in Nonmonotonic Changes in Brain Shape and Behavior in Amphibians.

Despite regulations and improved design, pesticides remain ubiquitous in the environment at relatively low, trace concentrations. To understand how prolonged exposure to trace pesticide concentrations impacts vertebrate brain development and behavior, we raised larval amphibians (northern leopard frogs, Lithobates pipiens) in 0, 1, or 10 µg/L of the organophosphorus pesticide chlorpyrifos (CPF) from hatching to metamorphosis. Tadpoles exposed to 1 µg/L CPF, but not 10 µg/L CPF, had changes in relative brain mass, relative telencephalon shape, and behavioral responses to a novel visual cue. Tadpoles exposed to 10 µg/L CPF had altered behavioral responses to predator-associated olfactory cues. After metamorphosis, frogs raised in 1 µg/L CPF, but not 10 µg/L CPF, had changes in the shape of their optic tectum and medulla. Thus, we provide robust evidence that even trace, yet ecologically realistic, concentrations of CPF have neurodevelopmental and behavioral effects that carry over to later life-history stages, further emphasizing the potent effects of trace levels of CPF on vertebrate development. Also, some but not all effects were nonmonotonic, meaning that effects were evident at the lowest but not at the higher concentration of CPF.

External nasal gland morphology of Eurycea bislineata (Amphibia, Urodela, Plethodontidae).

Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands. External nasal glands are paired, branched tubular glands that extend from excretory ducts dorsal to the nares to terminal secretory units posterior to the eyes. We found that the glands hypertrophy and stain/react more intensely with histochemical procedures during the mating season. Hypertrophy of the glands is more pronounced in males, and seasonal variation in epithelial height of external nasal glands of males is significantly correlated to that of seasonal variation in mental gland epithelial height, a known courtship gland found in males, when compared throughout the year. This correlation was not as strong in females, confirming sexual dimorphism of external nasal glands in terms of seasonal variation. We found no ultrastructural differences between male and female external nasal glands. In all specimens, the glandular tubules were lined by a simple, columnar epithelium that was packed with secretory granules that often obscured other cytoplasmic contents.

Water-borne corticosterone assay is a valid method in some but not all life-history stages in Northern Leopard Frogs.

There is a particular need to develop conservation tools for use in amphibian populations, which are declining rapidly. Glucocorticoid hormones like corticosterone (CORT) are often used as biomarkers of amphibian stress. A relatively new method of assessing CORT in amphibians is to measure CORT concentrations in water that has held amphibians (water-borne (WB) CORT). Here, we tested whether WB CORT is a valid measure of CORT in larval and metamorphic Northern Leopard Frogs (Lithobates pipiens). We assessed whether levels of WB CORT are different among groups of animals that should have different levels of CORT due to a handling challenge, a pharmacological challenge (ACTH), or developmental stage. We also assessed whether WB CORT was correlated with plasma CORT within individuals. Results indicated that measurement of WB CORT is valid in prometamorphic tadpoles because injection with ACTH increased WB CORT, and WB CORT and plasma CORT levels were correlated within an animal in most cases. However, were unable to fully validate the use of WB CORT in metamorphic frogs (metamorphs) because although injection with ACTH elevated levels of WB CORT, WB CORT was not correlated with plasma CORT within individual metamorphs. Also, there was no correlation between WB CORT and plasma CORT in early stage (premetamorphic) tadpoles or tadpoles undergoing metamorphic climax, indicating that WB CORT is not sensitive enough to detect natural variation of organismal CORT in these groups. Together, results indicated that WB CORT is a valid method of assessing plasma CORT in Northern Leopard Frogs, but only for some life-history stages. Our results illustrate the importance of carefully validating the use of WB CORT for appropriate interpretation of results.

Insecticide-induced changes in amphibian brains: How sublethal concentrations of chlorpyrifos directly affect neurodevelopment.

Widespread use of pesticides often contaminates natural habitats, exposing nontarget organisms to pesticides that were designed to control pest populations. Even low levels of pesticides can affect aquatic communities both directly and indirectly. Previous work has shown that trace amounts of the pesticide chlorpyrifos altered tadpole morphology and neurodevelopment in artificial ponds (mesocosms). To determine whether effects resulted from direct chlorpyrifos exposure or from disruption of the food web due to a pesticide-induced decline in zooplankton, we examined the impacts of chlorpyrifos on amphibian development in the presence of chlorpyrifos-resistant zooplankton, a key component of the aquatic trophic community. Northern leopard frog (Lithobates pipiens) tadpoles were reared through metamorphosis in mesocosms containing either 0 or 1 µg/L chlorpyrifos and either chlorpyrifos-resistant or chlorpyrifos-sensitive Daphnia pulex zooplankton. Developmental exposure to chlorpyrifos resulted in metamorphs with a relatively wider optic tectum, medulla, and diencephalon compared with controls, and this result was found regardless of the zooplankton population within the mesocosm. Thus, chlorpyrifos directly impacted brain development, independent of the effects on the trophic community. With respect to body shape, chlorpyrifos had no effect on body shape of metamorphs reared in mesocosms with chlorpyrifos-sensitive zooplankton, but body shape was sensitive to zooplankton population in the absence of chlorpyrifos. To conclude, low, ecologically relevant doses of organophosphorous pesticides can directly impact neurodevelopment in a vertebrate model. Environ Toxicol Chem 2018;37:2692-2698. © 2018 SETAC.

Microvascular patterns in the blubber of shallow and deep diving odontocetes.

Blubber, a specialized form of subdermal adipose tissue, surrounds marine mammal bodies. Typically, adipose tissue is perfused by capillaries but information on blubber vascularization is lacking. This study's goals were to: 1) describe and compare the microvasculature (capillaries, microarterioles, and microvenules) of blubber across odontocete species; 2) compare microvasculature of blubber to adipose tissue; and 3) examine relationships between blubber's lipid composition and its microvasculature. Percent microvascularity, distribution, branching pattern, and diameter of microvessels were determined from images of histochemically stained blubber sections from shallow-diving bottlenose dolphins (Tursiops truncatus), deeper-diving pygmy sperm whales (Kogia breviceps), deep-diving beaked whales (Mesoplodon densirostris; Ziphius cavirostris), and the subdermal adipose tissue of domestic pigs (Sus scrofa). Tursiops blubber showed significant stratification in percent microvascularity among the superficial, middle, and deep layers and had a significantly higher percent microvascularity than all other animals analyzed, in which the microvasculature was more uniformly distributed. The percent microvasculature of Kogia blubber was lower than that of Tursiops but higher than that of beaked whales and the subdermal adipose tissue of domestic pigs. Tursiops had the most microvascular branching. Microvessel diameter was relatively uniform in all species. There were no clear patterns associating microvascular and lipid characteristics. The microvascular characteristics of the superficial layer of blubber resembled the adipose tissue of terrestrial mammals, suggesting some conservation of microvascular patterns in mammalian adipose tissue. The middle and deep layers of blubber, particularly in Tursiops, showed the greatest departure from typical mammalian microvascular arrangement. Factors such as metabolics or thermoregulation may be influencing the microvasculature in these layers.