Arsenic Concentrations in Ground and Surface Waters across Arizona Including Native Lands.

Parts of the Southwestern United States report arsenic levels in water resources that are above the United States Environmental Protection Agency's current drinking water limits. Prolonged exposure to arsenic through food and drinking water can contribute to significant health problems including cancer, developmental effects, cardiovascular disease, neurotoxicity, and diabetes. In order to understand exposure risks, water sampling and testing has been conducted throughout Arizona. This information is available to the public through often non-overlapping databases that are difficult to access and in impracticable formats. The current study utilized a systemic compilation of online databases to compile a spreadsheet containing over 33,000 water samples. The reported arsenic concentrations from these databases were collected from 1990-2017. Using ArcGIS software, these data were converted into a map shapefile and overlaid onto a map of Arizona. This visual representation shows that arsenic levels in surface and ground water exceed the United States Environmental Protection Agency's drinking water limits for many sites in several counties in Arizona, and there is an underrepresentation of sampling in several tribal jurisdictions. This information is useful for water managers and private well owners throughout the State for determining safe drinking water sources and limiting exposure to arsenic.

Endosulfan exposure disrupts pheromonal systems in the red-spotted newt: a mechanism for subtle effects of environmental chemicals.

Because chemicals introduced into the environment by humans can affect both long-term survivorship and reproduction of amphibians, discovering the specific mechanisms through which these chemicals act may facilitate the development of plans for amphibian conservation. We investigated the amphibian pheromonal system as a potential target of common environmental chemicals. By treating female red-spotted newts, Notophthalmus viridescens, to a commonly used insecticide, endosulfan, we found that the pheromonal system is highly susceptible to low-concentration exposure. The impairment of the pheromonal system directly led to disrupted mate choice and lowered mating success. There were no other notable physiologic or behavioral changes demonstrated by the animals at the insecticide concentrations administered. Our findings suggest that the amphibian pheromonal system is one of the systems subject to subtle negative effects of environmental chemicals.

Repellent function of male pheromones in the red-spotted newt.

Pheromones act as attractants and sexual stimulants in most vertebrates. For example, in red-spotted newts, Notophthalmus viridescens, female pheromones attract males, and male pheromones increase female receptivity. However, no studies have determined whether male vertebrates produce a pheromone that repels competing males. Through a series of olfactory mate selection tests, we found that sexually motivated male red-spotted newts produce a pheromone that functions to repel other approaching males. Our finding is the first report of a repelling function for pheromones in male vertebrates. The pheromones may act to increase both the sender's and receiver's mating success when the operational sex ratio (OSR) is male biased.

Effects of dehydration on plasma osmolality, thirst-related behavior, and plasma and brain angiotensin concentrations in Couch's spadefoot toad, Scaphiopus couchii.

Under dehydrating conditions, many terrestrial vertebrates species exhibit increases in plasma osmolality and their drinking behavior. Under some circumstances, this behavioral change is accompanied by changes in plasma and central angiotensin concentrations, and it has been proposed that these changes in angiotensin levels induce the thirst-related behaviors. In response to dehydration, the spadefoot toad, Scaphiopus couchii, exhibits thirst-related behavior in the form of cutaneous drinking. This behavior has been termed water absorption response (WR) behavior. Spadefoot toads live in harsh desert environments and are subject annually to dehydrating conditions that may induce thirst-related behavior. We tested the hypothesis that an increase in WR behavior is associated with both an increase in plasma osmolality and an increase in plasma and brain angiotensin concentrations. First, we determined the degree of dehydration that was necessary to initiate WR behavior. Animals dehydrated to 85% of their standard bladder-empty weight via deprivation of water exhibited WR behavior more frequently than control toads left in home containers with water available. Next, using the same dehydration methods, we determined the plasma osmolality and sodium concentrations of dehydrated toads. Toads dehydrated to 85% standard weight also had a significant increase in plasma osmolality, but exhibited no overall change in plasma sodium concentrations, indicating that while an overall increase in plasma osmolality appears to be associated with WR behavior in S. couchii, changes in sodium concentrations alone are not sufficient to induce the behavior. Finally, plasma and brain angiotensin concentrations were measured in control toads and toads dehydrated to 85% standard weight. Plasma and brain angiotensin concentrations did not increase in dehydrated toads, indicating that dehydration-induced WR behavior that is associated with changes in plasma osmolality may not be induced by changes in endogenous angiotensin concentrations in S. couchii.

Intra- and extracellular dehydration has no effect on plasma levels of angiotensin II in an amphibian.

Previous studies have demonstrated that both dehydration (intra and extracellular) and treatment with angiotensin II (A-II) induce changes in thirst-related behavior in the spadefoot toad, Scaphiopus couchii. One of the steps in determining a causal relationship between a hormone and a behavior is to determine that there is association between an animal's performance of the behavior and changes in endogenous hormonal concentrations. The hypothesis tested that plasma levels of the peptide hormone A-II would change as a result of dehydration known to induce water absorption response (WR) behavior in the spadefoot toad. Plasma samples were taken from toads dehydrated intracellularly by injection of hypertonic solutions of NaCl or sucrose at levels known to induce WR behavior. As an osmotic control, a group of animals was injected with urea, which has been demonstrated to not induce WR behavior. In order to determine the effects of extracellular dehydration on plasma, A-II levels in toads dehydrated by plasma volume depletion via cardiac puncture were compared to sham-punctured controls. None of the treatments in any experiment resulted in significant differences in plasma levels of angiotensin II among groups sampled at the time when WR behavior occurs. These results do not support the hypothesis that dehydration-induced thirst is stimulated by changes in plasma A-II concentrations at the onset of WR behavior. J. Exp. Zool. 286:343-349, 2000.

Cloning of proopiomelanocortin from the brain of the african lungfish, Protopterus annectens, and the brain of the western spadefoot toad, Spea multiplicatus.

A degenerate primer, specific for the opioid core sequence YGGFM, was used to clone and sequence proopiomelanocortin (POMC) cDNAs from the brain of the African lungfish, Protopterus annectens, and from the brain of the western spadefoot toad, Spea multiplicatus. In addition, the opioid-specific primer was used to clone and sequence a 3'RACE product corresponding to a portion of the open reading frame of S. multiplicatus proenkephalin. For both species, cDNA was made from a single brain and a degenerate opioid-specific primer provided a reliable probe for detecting opioid-related cDNAs. The African lungfish POMC cDNA was 1,168 nucleotides in length, and contained regions that are similar to tetrapod POMCs and fish POMCs. The African lungfish POMC encodes a tetrapod-like gamma-MSH sequence that is flanked by sets of paired basic amino acid proteolytic cleavage sites. The gamma-MSH region in ray-finned fish POMCs either has degenerate cleavage sites or is totally absent in some species. However, the African lungfish gamma-MSH sequence does contain a deletion which has not been observed in tetrapod gamma-MSH sequences. The beta-endorphin region of lungfish POMC has the di-amino acid sequence tryptophan-aspartic acid in the N-terminal region and an additional glutamic acid residue in the C-terminal region of beta-endorphin - features found in fish beta-endorphin, but not tetrapod beta-endorphins. The western spadefoot toad POMC was 1,186 nucleotides in length, and exhibited an organizational scheme typical for tetrapod POMCs. However, the toad POMC did lack a paired basic amino acid proteolytic cleavage site N-terminal to the beta-MSH sequence. Thus, like rat POMC, it is doubtful that beta-MSH is an end product in either the toad brain or intermediate pituitary. At the amino acid level, the toad POMC had 76% sequence identity with Xenopus laevis POMC and 68% sequence identity with Rana ribidunda POMC. The use of these POMC sequences to assess phylogenetic relationships within anuran amphibians will be discussed. With respect to the fragment of S. multiplicatus proenkephalin cDNA, two metenkephalin sequences and the metenkephalin-RF sequence were found encoded in this fragment. As seen for X. laevis and R. ridibunda proenkephalin, a leuenkephalin sequence was not detected in the C-terminal region of the S. multiplicatus proenkephalin. The absence of a leuenkephalin sequence may be a common feature of anuran amphibian proenkephalins.

Intra- and extracellular dehydration-induced thirst-related behavior in an amphibian.

The behavioral response to dehydration is critical to an animal's survival. Because of their permeable skin, amphibians are particularly sensitive to dehydrating conditions. We tested the hypothesis that different forms of dehydration induce water absorption response (WR) behavior in the desert spadefoot toad, Scaphiopus couchii. First, we determined the behavioral response to intracellular dehydration by treating fully hydrated toads with increasing concentrations of hypertonic solutions of NaCl or sucrose via intraperitoneal injection (i.p.). Animals that were treated to induce intracellular dehydration with either solute exhibited a significant increase in WR behavior compared to vehicle-treated controls. To distinguish that the response was a result of an increased osmotic gradient between the intra- and extracellular compartments, we treated fully hydrated animals i.p. with urea, which freely passes into the intracellular compartment and increases overall animal osmolarity. Urea treatment did not induce WR behavior. To determine the response to extracellular dehydration, the blood volume of fully hydrated toads was reduced via cardiac puncture, and the WR behavior was measured. Animals who had a reduction in blood volume exhibited a significant increase in WR behavior compared to sham-punctured controls. Our results are the first to demonstrate that multiple forms of dehydration can induce thirst-related behavior in amphibians.

Unilateral ovariectomy influences hypothalamic monoamine asymmetries in a lizard (Anolis) that exhibits alternation of ovulation.

The lizard Anolis carolinensis alternates ovulation, and the resultant ovarian asymmetry correlates with alternating asymmetry of hypothalamic catecholamines. Unilateral and bilateral ovariectomies of cycling females were performed to determine if ovarian manipulations influence hypothalamic catecholamine asymmetries. During the middle of the ovarian cycle, we removed the larger ovary, i.e., the next one to ovulate an egg (N = 9), the smaller ovary with its corpus luteum (N = 8), or both ovaries (N = 5). A sham-operated control group was included (N = 9). The diameter of the largest ovarian follicle in each ovary was measured at the beginning and end of the experiment. After 12 days, the hemihypothalami from the sides of the initial smaller ovary (SO) and larger ovary (LO) were dissected and frozen for determination of monoamines and their metabolites using HPLC and electrochemical detection. Monoamine and metabolite concentrations at the end of the experiment in the original SO and LO sides of each hypothalamus were compared with an asymmetry ratio, or AR, of (SO side - LO side)/(SO side + LO side). No female ovulated during the experiment. Unilateral ovariectomy caused compensatory growth of the largest follicle in the remaining ovary. Removal of the SO or LO caused the AR of DOPAC to favor the brain side ipsilateral to that of the ovarian removal. Removal of the LO switched the NE AR from the SO to the LO side. Removal of the LO or SO caused the MHPG AR to favor the LO side. Ovariectomy of any kind caused 5-HT, which in the sham-operated animals favored the SO side, to become symmetric, and removal of the LO caused the 5-HIAA AR to favor the LO side. We conclude that the ovaries influence hypothalamic catecholamine asymmetries in Anolis via direct neural (as well as hormonal) pathways and that sensory input from the ovaries to the hypothalamus could be involved in control of ovarian alternation via both neural and hormonal efferent mechanisms.

Effects of androgens on male sexual behavior and secondary sex characters in the explosively breeding spadefoot toad, Scaphiopus couchii.

In male Scaphiopus couchii, plasma elevations in androgens are associated with the seasonal expression of sexual behavior. To test the hypothesis that androgens are necessary for the expression of one aspect of sexual behavior, amplexus, and for the development of secondary sex characteristics in male S. couchii, the effects of testosterone (T) and dihydrotestosterone (DHT) on clasping behavior and thumb pad growth were investigated. Forty toads were divided into five treatment groups as follows: (1) intact, with no hormonal or surgical treatment; (2) sham-castrated, with silastic implants of cholesterol; (3) sham-castrated, with implants of T and DHT; (4) castrated, with implants of cholesterol; and (5) castrated, with implants of T and DHT. Displays of sexual behavior (amplexus) for each individual were counted during a 3-hr observation period at 2, 4, and 7 weeks after the surgical procedure. For each observation trial, there was no significant difference in the amount of clasping among treatment groups: virtually all individuals from each group displayed vigorous clasping. No calling was noted at any time. Thumb pad darkness of each individual was assessed at the 4- and 7-week time intervals. At both time periods, the thumb pads of androgen-treated males were significantly darker than those of cholesterol-treated males, while intact males remained intermediate. Measurement of plasma steroid levels from the treatment groups indicated that androgens were basal in the castrate group and higher in the implant treatment. These results suggest that in S. couchii, although androgens are associated temporally with the onset of reproductive behavior, they are not necessary for its expression. The elevation in androgens during the reproductive period may be instead important in the development and maintanence of secondary sex characteristics and possibly other reproductive functions.

Reproductive endocrinology of the explosively breeding desert spadefoot toad, Scaphiopus couchii.

The spadefoot toad, Scaphiopus couchii, is an explosively breeding, desert dwelling amphibian that shows two characteristics hypothesized to select for a dissociated breeding pattern: it lives in a harsh environment and it has a very short, but predictable breeding period. We tested the hypothesis that these factors select for a dissociated breeding cycle by measuring plasma steroid hormones and the gametogenic cycles in a free-living population of S. couchii. Blood and tissue samples were obtained from prebreeding, calling (males only), amplexed, and postbreeding toads. In males, plasma testosterone (T), dihydrotestosterone (DHT), and corticosterone (B) differed significantly among reproductive groups, with calling and amplexing toads having the highest levels of each of these steroids. In females, plasma T, DHT, B, progesterone (P), and estradiol (E2) were measured; all steroids except B changed significantly among mating groups. Plasma T and E2 were greatest in prebreeding and amplexed females, while P and DHT were highest only in amplexed females. Primary spermatogonia (SPG I), secondary spermatogonia (SPG II), spermatocytes, spermatids (SPT), and spermatozoa embedded in Sertoli cells (SPS) all changed significantly among male breeding groups. Although all stages were present in all breeding groups, early stages of spermatozoa (SPG I and SPG II) were greatest in postbreeding males, while late stages (SPT and SPS) were highest in prebreeding, calling, and amplexing males. In females, oocyte stages I, II, III, V, and VI and atretic follicles also differed significantly among mating groups. Prebreeding female ovaries were dominated by late-stage (V and VI) oocytes which were subsequently ovulated during amplexus. In postbreeding females, oogenesis was well under way as indicated by the rising numbers of stage II and III oocytes. These results indicate that aspects of gametogenesis occur during and after breeding in both sexes. Although the results demonstrate that gonadal activity was high during the period of sexual activity, suggesting that S. couchii most closely fits an associated pattern of reproduction as defined by Crews (1984, Horm. Behav. 18, 22-28), we also found that gonadal activity continued past the breeding season. For this reason, S. couchii does not completely conform to an associated pattern.

Differential effects of arginine vasotocin and gonadotropin-releasing hormone on sexual behaviors in an anuran amphibian.

Both arginine vasotocin (AVT) and gonadotropin-releasing hormone (GnRH) are known to influence sexual behavior in many vertebrate taxa. We investigated the effects of both of these peptides on two different sexual behaviors, calling and amplexus, in the Great Plains toad (Bufo cognatus). AVT, at a dosage of 100 microg/100 g toad, significantly increased both the amount of calling behavior per individual and the probability that an individual would call. GnRH, however, had no effect on calling behavior. There was a reciprocal effect of these peptides on amplexus: AVT did not induce amplexus, while GnRH significantly induced this behavior. Furthermore, AVT-induced calling could be inhibited by Manning compound (an arginine vasopressin receptor antagonist). This is the first report of GnRH influencing sexual behavior in an anuran amphibian. These results suggest that specific sexual behaviors in B. cognatus may be under the regulation of independent peptide control.

Central angiotensin II induces thirst-related responses in an amphibian.

Angiotensin II (A-II), a potent inducer of thirst-related behavior in many vertebrate species, was injected into the third ventricle of the brain of the spadefoot toad, Scaphiopus couchii. Following injection of 10 ng A-II the animals demonstrated a significant increase in water absorption response (WR) behavior, in which toads press their ventral skin to a moist surface and absorb water by osmosis. This increase in the frequency of WR behavior was positively correlated with an increase in water gain during a 2-hr period indicating that centrally injected A-II stimulates water intake by this amphibian species. We have previously demonstrated that WR behavior is also induced by intraperitoneal (i.p.) injections of A-II in several anuran species, including S. couchii. Thus, amphibians, like other vertebrates, demonstrate an increase in water intake in response to either centrally administered or circulating A-II. A second series of experiments was conducted to determine whether the above response to A-II might be secondary to increases in the circulating levels of aldosterone (ALDO) or antidiuretic hormone because the release of both of these hormones has been shown by others to be stimulated by A-II. Scaphiopus couchii injected i.p. with either ALDO or arginine vasotocin in dosages of 1, 10, and 100 micrograms/100 g body weight showed no increase in WR behavior relative to toads injected with saline alone. These results suggest that A-II acts directly on the brain of S. couchii to induce WR behavior.

Angiotensin II induces water absorption behavior in two species of desert anurans.

The octapeptide, angiotensin II (A-II), induces drinking behavior in several vertebrate species; however, relatively little is understood about A-II-induced thirst in amphibians. Scaphiopus couchii and Bufo cognatus were dehydrated to 90% of their ad libitum weight. This level of dehydration was sufficient to induce water absorption response (WR) behavior in both species. Fully hydrated toads injected intraperitoneally with A-II exhibited a significant amount of WR behavior. The minimum effective dose for inducing WR behavior was 10 micrograms/100 g-animal for S. couchii and 100 micrograms/100 g-animal for B. cognatus. When dehydrated toads were treated with the A-II receptor antagonist, Thr8-saralasin, S. couchii, exhibited a significant increase in WR behavior, while B. cognatus did not respond behaviorally. Finally, treatment of dehydrated toads with captopril, a compound that inhibits conversion of angiotensin I to A-II, did not significantly affect WR behavior in either species. These results support other findings that A-II may be involved in WR behavior in amphibians. However, the failure of Thr2-saralasin or captopril to inhibit WR behavior in dehydrated toads suggests that the receptor mechanisms involved in thirst regulation in toads may be different from those in mammals, and the renin-angiotensin system may not be the only potential mediator of WR behavior in these species.

Arginine vasotocin concentrations in the supraoptic nucleus of the lizard Anolis carolinensis are associated with reproductive state but not oviposition.

Arginine vasotocin (AVT) is a neuropeptide involved in reproductive function in many nonmammalian vertebrates. We determined brain and plasma AVT concentrations during the estrous cycle and oviposition in the lizard Anolis carolinensis. There were no differences in AVT concentrations in the plasma or any brain region during the ovipositional sequence. However, we found that females with an egg in each oviduct and a large pre-ovulatory follicle (diameter > 4.5 mm) in one-ovary had significantly higher AVT concentrations in the supraoptic nucleus (SON) of the hypothalamus than did females with small pre-ovulatory follicles in both ovaries. In a second study, females with an egg in each oviduct and a large pre-ovulatory follicle had significantly greater AVT concentrations in the SON than females with only one oviductal egg and a large pre-ovulatory follicle or females with an egg in each oviduct and a small pre-ovulatory follicle in each ovary. Concentrations of AVT in other brain regions and in the plasma did not differ among these groups. Changes in steroid profiles during estrous and/or direct neural communication between the uterus, ovary, and brain may account for the changes in AVT concentrations seen in the supraoptic nucleus during the estrous cycle of Anolis carolinensis.

Distribution of arginine vasotocin in the brain of the lizard Anolis carolinensis.

The distribution of immunoreactive arginine vasotocin (AVT-ir) was determined in the brain of the lizard Anolis carolinensis. Cells and fibers containing AVT-ir were found in the medial septal region, lamina terminalis, lateral forebrain bundle, preoptic area, supraoptic nucleus, anterior hypothalamus, paraventricular nucleus, periventricular nucleus, arcuate nucleus, and ventromedial nucleus of the thalamus. Occasional AVT-ir cells were found in the interpeduncular nucleus. Fibers containing AVT-ir were found in the cortex, around the olfactory ventricle, in the diagonal band of Broca, amygdala area, dorsal ventricular ridge, striatum, nucleus accumbens, septum, ventromedial hypothalamus, lateral hypothalamus, medial forebrain bundle, median eminence, pars nervosa, nucleus of the solitary tract, locus coeruleus, cerebellar cortex (granular layer), dorsal part of the nucleus of the lateral lemniscus, substantia nigra, and myelencephalon. The intensity of AVT-ir staining was, in general, greater in males than in females. Comparison of AVT-ir distribution in A. carolinensis with those previously published for other reptilian species revealed species-specific differences in distribution of AVT.

Effects of courtship on brain gonadotropin hormone-releasing hormone and plasma steroid concentrations in a female amphibian (Taricha granulosa).

Courtship-induced changes in plasma steroid and brain gonadotropin-releasing hormone (GnRH) concentrations in Taricha granulosa were determined with respect to changes in female sexual receptivity. Females were sacrificed at several times after courtship initiation. Concentrations of GnRH (determined by RIA) in the anterior telencephalon were high at courtship initiation (females unreceptive), but decreased by sperm transfer (females receptive). Courtship had no affect on GnRH concentrations in any other brain region examined. Furthermore, courted, receptive females had higher plasma levels of estradiol than did uncourted controls, and estradiol levels remained elevated above control levels 24 hr after courtship initiation. Courtship had no influence on plasma progesterone or corticosterone levels. To determine if the observed changes in GnRH concentrations in the telencephalon were localized to the nervus terminalis, courted females and controls were sacrificed after 5, 20, or 60 min of courtship. Nervus terminalis GnRH concentrations were higher in courted females than in uncourted controls. These results may represent the first documentation of a naturally occurring physiological change in the nervus terminalis.

Neuroendocrine, behavioral, and morphological changes associated with the termination of the reproductive period in a natural population of male rough-skinned newts (Taricha granulosa).

Male rough-skinned newts (Taricha granulosa) were collected from the same natural population every second week from early April to mid-June. They were either field-tested for their sexual responsiveness or used to measure the plasma concentrations of androgens and corticosterone, the brain concentrations of immunoreactive (ir) gonadotropin-releasing hormone (GnRH) and arginine vasotocin (AVT), and morphological parameters. During the experimental period, the percentage of sexually responsive males gradually declined from 100 to 4%, concurrent with a decrease of plasma concentrations of androgens, but not corticosterone. Concentrations of irGnRH in two brain regions (medial septum; ventral telencephalon containing the nervus terminalis) did not change significantly during this time. In the infundibulum, irGnRH concentrations increased from the end of May to mid-June, which coincided with an increase in plasma androgen concentrations, a marked increase in testis weights, and a decrease of the proportion of males with spermatozoa in their vas deferens. During this period, no changes in irAVT concentrations in four brain regions (infundibulum; pars distalis of the pituitary; interpeduncular nucleus; cerebrospinal fluid) were detected, but significant changes were observed for irAVT in the dorsal preoptic area that were not correlated with the seasonal changes in behavior. Also, during this period, there were decreases in mean body weight and tail height, and in the proportion of males with smooth skin and dark nuptial pads. These results are discussed in view of our current knowledge of the endocrine mechanisms that regulate sexual behaviors and secondary sex characteristics in male amphibians.