Effects of chytrid fungus and a glyphosate-based herbicide on survival and growth of wood frogs (Lithobates sylvaticus)

Anthropogenic-derived stressors in the environment, such as contaminants, are increasingly considered important cofactors that may decrease the immune response of amphibians to pathogens. Few studies, however, have integrated amphibian disease and contaminants to test this multiple-stressor hypothesis for amphibian declines. We examined whether exposure to sublethal concentrations of a glyphosate-based herbicide and two strains of the pathogenic chytrid fungus, Batrachochrytrium dendrobatidis (Bd) could: (1) sublethally affect wood frogs (Lithobates sylvaticus) by altering the time to and size at metamorphosis, and (2) directly affect survivability of wood frogs after metamorphosis. Neither Bd strain nor herbicide exposure alone significantly altered growth or time to metamorphosis. The two Bd strains did not differ in their pathogenicity, and both caused mortality in post-metamorphic wood frogs. There was no evidence of an interaction between treatments, indicating a lack of herbicide-induced susceptibility to Bd. However, the trends in our data suggest that exposure of wood frogs to a high concentration of glyphosate-based herbicide may reduce Bd-caused mortality compared to animals exposed to Bd alone. These results exemplify the complexities inherent when populations are coping with multiple stressors. In this case, the perceived stressor, glyphosate-based herbicide, appeared to affect the pathogen more than the host's immune system, relieving the host from disease-caused effects. This suggests caution when invoking multiple stressors as a cause for increased disease susceptibility and indicates that the effects of multiple stressors on disease outcome depend on the interrelationships of stressors to both the pathogen and the host.     

Interactions of an Insecticide with Larval Density and Predation in Experimental Amphibian Communities

Abstract: This study examines the effects of the short-lived insecticide carbaryl, a neurotoxin, on amphibian communities experiencing natural stresses of competition and predation. Tadpoles of three species (  Woodhouse's toad [ Bufo woodhousii ], gray treefrog [ Hyla versicolor ], and green frog [ Rana clamitans ]), representing a commonly encountered assemblage in Missouri, were reared in outdoor polyethylene pond mesocosms. We determined the effects of initial tadpole density ( low or high), predation (newts [  Notophthalmus viridescens ] absent or present), chemical exposure (0, 3.5, or 7.0 mg /L carbaryl), and their interactions on body mass, larval period, and survival to metamorphosis. Green frogs in high-density ponds did not reach metamorphosis, but metamorphs in low-density ponds and tadpoles in high-density ponds were not significantly affected by treatments or their interaction. Carbaryl reduced survival to metamorphosis in toads and treefrogs and increased mass at metamorphosis in treefrogs. Effects of carbaryl varied with predator environment and initial larval density. Interactions of carbaryl with predator and with density may result in an indirect effect of carbaryl causing increased food resources through the elimination of zooplankton populations that may compete for similar resources. Our results indicate that differences in biotic conditions influenced the potency of carbaryl and that even low concentrations induce changes that may alter community dynamics in ways not predicted from single-factor, laboratory-based studies.     

Multiple stressors in amphibian communities: effects of chemical contamination, bullfrogs, and fish.

A leading hypothesis of amphibian population declines is that combinations of multiple stressors contribute to declines. We examined the role that chemical contamination, competition, and predation play singly and in combination in aquatic amphibian communities. We exposed larvae of American toads (Bufo americanus), southern leopard frogs (Rana sphenocephala), and spotted salamanders (Ambystoma maculatum) to overwintered bullfrog tadpoles (R. catesbeiana), bluegill sunfish (Lepomis macrochirus), the insecticide carbaryl, and ammonium nitrate fertilizer in 1000-L mesocosms. Most significantly, our study demonstrated that the presence of multiple factors reduced survival of B. americanus and A. maculatum and lengthened larval periods of R. sphenocephala. The presence of bluegill had the largest impact on the community; it eliminated B. americanus and A. maculatum and reduced the abundance of R. sphenocephala. Chemical contaminants had the second strongest effect on the community with the insecticide, reducing A. maculatum abundance by 50% and increasing the mass of anurans (frogs and toads) at metamorphosis; the fertilizer positively influenced time and mass at metamorphosis for both anurans and A. maculatum. Presence of overwintered bullfrogs reduced mass and increased time to metamorphosis of anurans. While both bluegill and overwintered bullfrog tadpoles had negative effects on the amphibian community, they performed better in the presence of one another and in contaminated habitats. Our results indicate that predicting deleterious combinations from single-factor effects may not be straightforward. Our research supports the hypothesis that combinations of factors can negatively impact some amphibian species and could contribute to population declines.     

Gonadal histology and reproductive steroidogenesis in Lithobates pipiens exposed to atrazine

Abstract

Atrazine, an endocrine-disrupting compound and environmental pollutant, can have adverse effects on amphibian reproductive development and function. To determine the effects of atrazine on anuran gonadal development, Lithobates pipiens larvae were exposed from Gosner stage 25 through 1-month post metamorphosis to 0, 2, 20, or 200?μg/L atrazine or estradiol. Exposure to atrazine did not markedly alter tadpole growth, development, gonadal histology, or reproductive steroidogenesis. Testicular ovarian follicles appeared in L. pipiens metamorphs following all treatments, including controls, whereas ovotestes occurred only in positive controls. Testicular ovarian follicles may be a natural ontogenetic occurrence in this species while ovotestes indicate disruption in development. Establishing the normal pattern of reproductive development for anuran species and conducting comparisons in fully sexually differentiated animals is necessary to clarify the influence of endocrine disrupting compounds.     

Induction of metamorphosis in larvae of the oyster Crassostrea gigas using neuroactive compounds

Chemical (neuroactive compounds at different concentrations and exposure times) and physical (water agitation, light) factors with potential effects on the metamorphosis of larvae of the oyster Crassostrea gigas (Thunberg) larvae have been studied. The neurotransmitters l-dihydroxyphenylalanine (DO), epinephrine (EP), norepinephrine (NE), and acetylcholine (AC) have been identified as very active inducers of metamorphosis, whilst serotonin (SE), dopamine (DA) and potassium (K) were less effective inducers. The -aminobutyric acid (GA) and ammonium (AM) were found ineffective at the concentrations tested. Exposure to 10^-4 M EP for 15 min was sufficient to promote >80% metamorphosis within 48 h, whereas NE required 2 h to exert comparable induction. Maximum induction by DO (>50%) was achieved after 2 h exposure to 10^-4 M. However, unlike EP and NE, DO was lethal at that concentration in the long term. Maximum induction by AC (30% metamorphosis) was achieved at a concentration of 10^-4 M. In contrast to other neurotransmitters, AC induced settlement behaviour, cementation and eventual metamorphosis, yielding postlarvae which were all attached to the substratum. EP and NE triggered the morphogenetic changes, by-passing settlement and leading to postlarvae not cemented to the substratum. DO induced mostly attached spat at low concentrations (10^-5 M) and unattached spat at high concentrations (10^-4 M), and a similar pattern was apparent for the weaker inducers SE and DA. Regarding physical factors, a highly reflectant surface significantly increased the percentage of attached spat obtained, compared to a dark bottom. No consistent effect of water current or light was detected on the production of unattached spat. The three different forms of induction are discussed in relation to different regulatory pathways of settlement and metamorphosis.     

Morphological and behavioral alterations induced by endocrine disrupters in amphibian tadpoles

Developmental toxicity effects of endocrine disrupter chemicals, acephate and cypermethrin were studied in Bufo melanostictus tadpoles. Thirty developing eggs of B. melanostictus were exposed to each concentration (0.01, 0.05, 0.1, 0.5, and 1?µg?L^?1) of acephate or cypermethrin in the laboratory (temperature: 23?±?1°C; photoperiod: 11.5–12.5?h). Eggs maintained in conditioned water alone served as controls. After hatching, larvae were fed on boiled spinach until the completion of metamorphosis. In control group, larvae that hatched on 3rd day were heavily pigmented, voracious feeders, and active swimmers; in these tadpoles, hind limb and forelimb-buds emerged on 16th and 24th day and metamorphosis was complete on 32nd day. Eggs exposed to acephate also hatched on 3rd day but larvae exhibited deformities such as, (i) tail distortions, (ii) laterally crooked trunk, (iii) decreased pigmentation, (iv) inactivity, (v) peeling of the skin, and (vi) delay in emergence of limbs and completion of metamorphosis. Cypermethrin-exposed eggs exhibited a delay (4–8 days) in hatching, there was no mortality, deformities in tail, trunk and head region, delay in the emergence of limbs, and completion of metamorphosis were evident. The demonstrated data indicate that these pesticides interfere with amphibian development when present in the aquatic system.     

农药和化肥对无尾两栖类蝌蚪的毒性效应研究进展

以两栖动物研究环境污染物的毒性效应进而监测环境的变化,已成为国内外的研究热点.两栖类的胚胎发育和变态过程对水的依赖性极强,而且鳃和皮肤有很强的渗透性,当其受到污染胁迫时,在细胞、组织及生理生化水平上都会发生显著变化,进而会影响其存活和生长发育.在总结国内外相关研究基础上,综述和分析了农药、化肥(氮肥)对无尾两栖类蝌蚪的行为表现、生长发育、组织结构和生理生化变化等方面的毒性效应,以期为农药、化肥的水环境监测及合理使用提供全面的科学依据.     

Comparative sensitivity of various developmental stages of sea urchins to some chemicals

The sensitivity to some chemical agents was examined comparatively at sperm, fertilization, cleavage, blastula, gastrula, pluteus and metamorphosis stages of a sand dollar from Japanese waters (Peronella japonica) and a sea urchin from the Pacific coast of Australia (Heliocidaris erythrogramma). These agents included Cu sulphate, ABS and NH₃ chloride. Responses observed included departures from control rates of fertilization and developmental reduction at the attainment of first cleavage, gastrula, pluteus or metamorphosis. Developmental anomalies were noted at the fertilization, 2-cell, gastrula, pluteus and metamorphosis stages. Using minimum effective concentrations of the 3 chemicals at various developmental stages of P. japonica, it was found that sensitivity to chemicals varies from fertilization to metamorphosis. It seems that sperm activity is the most sensitive, and that fertilization and gastrulation are more sensitive than first cleavage, blastulation and pluteus formation. H. erythrogramma seems to show nearly the same responses to Cu, but is more sensitive at metamorphosis.Experiments conducted at the Seto Marine Biological Laboratory, Wakayama Prefecture, JapanExperiments conducted at School of Biological Sciences, the University of Sydney, Sydney, N.S.W., AustraliaContributions from the Seto Marine Biological Laboratory, No. 664     

Correlating gene expression with larval competence,and the effect of age and parentage on metamorphosis in the tropical abalone<Emphasis Type="Italic"> Haliotis asinina</Emphasis>

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P<0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.Communicated by M.S. Johnson, Crawley     

Effects of temporary starvation on larvae of the sea star Asterina miniata

Patchy food distribution may force temporary starvation conditions on planktonic larvae. This potential food limitation may affect survivorship, duration of larval period, and post-metamorphic succes. In this study, larvae of the asteroid Asterina miniata were subjected to temporary food deprivation of several durations and at different stages. Developmental effects were documented by quantification of larval stage, total length, time to metamorphosis, initial juvenile radius, range of settling times, and percent survival to metamorphosis. All starved treatments were significantly affected in settling time and most in percent survival. However, larvae starved later in development demonstrated tremendous tolerance of food deprivation (e.g. the total number of settlers in the treatment starved for 28 d was not significantly different from the fed control). Survival was lower in treatments starved earlier in development than those starved later. Food is apparently required until late in larval development to facilitate metamorphosis. The range of settling times was large; for example, the continuously-fed control treatment produced juveniles from Days 58 through 136. Temporary starvation had no effect on initial juvenile radius.     

Initiation of feeding and salinity tolerance in the pacific lamprey Lampetra tridentata

Changes in salinity tolerance were determined during metamorphosis in Lampetra tridentata. Lampreys in Phase 5 of metamorphosis were unable to withstand salinities>13.4S, while those in Phase 6 survived direct transfer to sea water (30S). This abrupt change in tolerance coincided with the opening of the foregut lumen. Parasitic feeding began at the end of Phase 7 of metamorphosis following the completion of tooth development.     

Bacteria on the surface of crustose coralline algae induce metamorphosis of the crown-of-thorns starfish Acanthaster planci

The crustose coralline alga Lithothamnium pseudosorum induces high rates of settlement and metamorphosis of larvae of the coral-eating crown-of-thorns starfish (Acanthaster planci). In cases where crustose coralline algae (CCA) induce metamorphosis of marine invertebrate larvae it is normally assumed that the inductive molecules are produced by the alga, but an alternative is that they originate from bacteria on the plant surface. Bioassays using shards of L. pseudosorum treated with several antibiotics, whereby some shards were reinfected with bacteria from the alga, showed that if bacteria populations are depleted then settlement and metamorphosis of larvae of A. planci are inhibited. This demonstrates that bacteria are necessary for induction and suggests that morphogenic substances are produced by bacteria on the surface of the alga and not directly by the alga itself. However, surface bacteria are not inductive if they are isolated from soluble algal compounds, suggesting either that they require a substrate from the alga to produce the inductive agents or, alternatively but less likely, that compounds from both the alga and bacteria are required. There is no evidence that inductive compounds derive from the alga, since algal cell debris and soluble extracts prepared from the alga do not induce metamorphosis of A. planci. This is the first time that induction of metamorphosis in a marine invertebrate by CCA has been shown to be mediated by bacteria associated with the alga.     

Effects of clotrimazole and amiodarone on early development of amphibian (Xenopus tropicalis)

Pharmaceuticals have been recognized as a continuing threat to environmental stability. Few experimental data are available for the effects of clotrimazole and amiodarone on the ecological environment. An acute test with embryos and a chronic test with larvae of amphibian (Xenopus tropicalis) were thus conducted to determine the influence of clotrimazole or amiodarone on early amphibian development. In acute test, % survival and the body length were numerically decreased by both pharmaceuticals treatments compared to control. In chronic test, the cumulative mortality was 22.2% with 0.1?µg?L^?1 clotrimazole treatment and 21.7% with 1?µg?L^?1 amiodarone. The whole body length and the biomass were significantly decreased and developmental stages significantly delayed by both pharmaceuticals. The results of our study suggest that clotrimazole exerted adverse effects on larvae of X. tropicalis at environmentally relevant concentrations.     

Changes in biochemical and fatty acid composition of the razor clam <Emphasis Type="Italic">Solen marginatus</Emphasis> (Solenidae: Bivalvia) during larval development

Aquaculture studies have revealed that polyunsaturated fatty acids are critical for maintaining substantial growth, survival and reproductive rates, and high food conversion efficiencies for a wide variety of marine and freshwater organisms. The aim of this study was to investigate the gross biochemical and fatty acid composition of both neutral and polar lipid compartments of the razor clam Solen marginatus throughout embryonic and larval development. High levels of stored reserves in S. marginatus eggs allow a short larval development, lasting only 8 days. The energy required for embryogenesis was obtained from stored proteins. During larval development from D-shaped veliger until settlement, protein, lipid, and carbohydrate reserves were indistinctly stored for metamorphosis. Although total lipids increased, fatty acids in both neutral and polar lipids decreased during embryonic development. The depots allow a short larval development in which settlement is reached with lower amounts of stored neutral and polar lipids than the contents found in the oocytes. Non-methylene-interrupted dienoic fatty acid levels were similar to those of some polyunsaturated fatty acids, with increasing percentages at the onset of metamorphosis. This study indicates that S. marginatus exhibits a different pattern in the use of gross biochemical and fatty acid reserves during larval development compared to other razor clam and bivalve species, mainly due to the large size of its eggs and the short larval development stage reported in this species.     

Food limitation stimulates metamorphosis of competent larvae and alters postmetamorphic growth rate in the marine prosobranch gastropodCrepidula fornicata

The effects of food limitation on growth rates and survival of marine invertebrate larvae have been studied for many years. Far less is known about how food limitation during the larval stage influences length of larval life or postmetamorphic performance. This paper documents the effects of food limitation during larval development (1) on how long the larvae ofCrepidula fornicata (L.) can delay metamorphosis in the laboratory after they have become competent to metamorphose and (2) on postmetamorphic growth rate. To assess the magnitude of nutritional stress imposed by different food concentrations, we measured growth rates (as changes in shell length and ash-free dry weight) for larvae reared in either 0.45-m filtered seawater or at phytoplankton concentrations (Isoehrysis galbana, clone T-ISO) of 1 × l0³, 1 × 10⁴, or 1.8 × 10⁵ cells ml^–1. Larvae increased both shell length and biomass at 1 × 10⁴ cells ml^–1, although significantly more slowly than at the highest food concentration. Larvae did not significantly increase (p > 0.10) mean shell length in filtered seawater or at a phytoplankton concentration of only 1 × 10³ cells ml^–1, and in fact lost weight under these conditions. To assess the influence of food limitation on the ability of competent individuals to postpone metamorphosis, larvae were first reared to metamorphic competence on a high food concentration ofI. galbana (1.8 × 10⁵ cells ml^–1). When at least 80% of subsampled larvae were competent to metamorphose, as assessed by the numbers of indlviduals metamorphosing in response to elevated K⁺ concentration in seawater, remaining larvae were transferred either to 0.45-m filtered seawater or to suspensions of reduced phytoplankton concentration (1 × 10³, 1 × 10⁴, or 5 × 10⁴ cells ml^–1), or were maintained at 1.8 × 10⁵ cells ml^–1. All larvae were monitored daily for metamorphosis. Individuals that metamorphosed in each food treatment were transferred to high ration conditions (1.8 × 10⁵ tells ml^–1) for four additional days to monitor postmetamorphic growth. Competent larvae responded to all food-limiting conditions by metamorphosing precociously, typically 1 wk or more before larvae metamorphosed when maintained at the highest food ration. Surprisingly, juveniles reared at full ration grew more slowly if they had spent 2 or 3 d under food-limiting conditions as competent larvae. The data show that a rapid decline in phytoplankton concentration during the larval development ofC. fornicata stimulates metamorphosis, foreshortening the larval dispersal period, and may also reduce the ability of postmetamorphic individuals to grow rapidly even when food concentrations increase.     

Evidence for a water-borne cue inducing metamorphosis in the dorid nudibranch mollusc Adalaria proxima (Gastropoda: Nudibranchia)

The dorid nudibranch Adalaria proxima (Alder & Hancock) is a specialist predator of the cheilostome bryozoan Electra pilosa (L.). Natural induction of metamorphosis of the pelagic lecithotrophic larva of A. proxima was assessed in response to solutions from sonicated prey tissue and (live) E. pilosa-conditioned seawater (Electra-CSW). We exploited the tendency of larvae to become entrapped (rafted) at the air-water interface in cultures to examine whether larvae require direct contact with the live prey for metamorphosis to proceed. Larvae metamorphosed when rafted above colonies of live E. pilosa, above plankton mesh bags isolating live E. pilosa, and in choline chloride controls; there was no metamorphosis of larvae that were rafted in filtered seawater controls. Entrapped veliger shells remained rafted throughout the experimental period in all cases. No metamorphosis occurred in treatments containing either the supernatants or pelleted particulates obtained from sonicated colonies of E. pilosa. Both one-colony and three-colony Electra-CSW induced metamorphosis of larvae. These data are at variance with previous results in showing that direct contact with the live prey is not necessary for metamorphosis to proceed. Furthermore, the fact that competent larvae metamorphosed in response to Electra-CSW in the absence of any other cue strongly suggests that the inductive cue is water-borne.     

Requirement of exogenous inducers for metamorphosis of axenic larvae and buds of Cassiopea andromeda (Cnidaria: Scyphozoa)

Planula larvae and asexually-produced buds of the rhizostome scyphozoan Cassiopea andromeda (collected throughout the year in Eilat, Israel) have the ability, under axenic conditions, to attach to a substrate and undergo morphogenetic development to form a polyp (=scyphistoma) in: (1) the presence of unidentified inducers found in the adult habitat and (2) the presence of cefined organic compounds. Axenic planulae and buds were unable to settle and complete metamorphosis in autoclaved artificial or natural seawater from the North Sea when maintained without food, but continued swimming while decreasing in size and protein content, eventually dying within three months. When maintained in autoclaved seawater from the Red Sea, between 25 and 46% of the planulae and 4 and 11% of the buds metamorphosed within 30 d. Axenic solutions of cholera toxin, thyroid stimulating hormone, and pancreatic casein hydrolysate peptides in artificial seawater induced morphogenic development of 20 to 100% of planulae and buds within 2 to 18 d. The natural inducer(s) in Red Sea seawater, though unidentified, may have characteristics similar to the large proteins and small peptide inducers used in this study. Planulae and buds older than 20 d metamorphosed sooner and responded to lower concentrations of pancreatic casein hydrolysate peptides than younger individuals. This may be a physiological mechanism for enhancing metamorphosis and survival in nature. The data show that settlement and metamorphosis can be induced by solutions of cholera toxin and thyroid stimulating hormone, suggesting that, as in mammalian systems, the mechanism of action of these chemicals may involve cyclic adenosine monophosphate (cAMP) as an intermediate messenger. However, dibutyric cAMP, which is capable of passing through membranes and functioning normally inside the cell, did not induce metamorphosis of buds, and the levels of intracellular cAMP in buds and larvae typically increased slowly during induction of metamorphosis, unlike the high and rapid increases associated with cAMP-mediated biochemical events in mammalian cells. These results suggest that the observed cAMP changes seen were associated with metamorphic development, but not with the triggering mechanism.     

敌百虫对中国林蛙蝌蚪生长发育的毒性效应

为评价水域环境中敌百虫(trichlorfon)污染对两栖类幼体的急性毒性,将中国林蛙(Rana chensinensis)28～29期(Gos-ner)蝌蚪分别暴露于10～30 mg· L-1敌百虫5个不同浓度的水体中,分别在24、48、72和96 h统计蝌蚪的死亡率,计算半致死浓度(LC50).结果显示,暴露24、48、72和96 h,敌百虫对蝌蚪的LC50分别为14250±3.23、49.19±128、25.68±2.04、1555±1.93 mg·L-1,安全浓度(SC)为156±0.19 mg· L-1.蝌蚪中毒后尾部多呈弯曲状,仰翻,外观浮肿.对死亡蝌蚪的解剖表明,其鳃腔内充水,内鳃萎缩,肝脏、肠管和肾脏呈灰白色.另外,将28～29期蝌蚪分别暴露于0.2 ～ 2.0 mg·L-1敌百虫4个不同浓度的水体中进行慢性暴露实验,检测蝌蚪暴露28和42 d时的体重和体长以及75％个体变态所需的时间.结果表明,蝌蚪在低剂量敌百虫水体中持续暴露,其生长发育受到明显抑制,并可导致蝌蚪身体扭曲、尾部强直性弯曲等畸型发生,蝌蚪的死亡率显著增高,作用强度呈现剂量和时间的累积效应.慢性暴露实验证明SC以下的敌百虫水体仍威胁着蝌蚪的生存.     

Amphibian Encounter Rates on Roads with Different Amounts of Traffic and Urbanization

Abstract: Although amphibians have relatively high rates of road mortality in urban areas, the conditions under which traffic threatens the survival of local amphibian populations remain unclear. In the Sandhills region of North Carolina (U.S.A.), we counted living and dead amphibians along two transects (total length 165 km) established on roads in areas with varying degrees of urbanization. We found 2665 individuals of 15 species, and amphibian encounter rates declined sharply as traffic and urban development increased. Regression‐tree models indicated that 35 amphibians/100 km occurred on roads with <535 vehicles/day, whereas the encounter rate decreased to only 2 amphibians/100 km on roads with >2048 vehicles/day. Although mortality rate peaked at higher traffic levels (47% dead on roads with >5200 vehicles/day), the number of dead amphibians was highest at low levels of traffic. This suggests that areas where amphibian mortality is concentrated may actually contain the largest populations remaining on a given road transect.     

Growth and differentiation during delayed metamorphosis of feeding gastropod larvae: signatures of ancestry and innovation

Extent of larval growth among marine invertebrates has potentially profound implications for performance by benthic recruits because body size influences many biological processes. Among gastropods, feeding larvae often attain larger size at metamorphic competence than non-feeding larvae of basal gastropod clades. Delay of metamorphosis can further influence size at recruitment if larvae continue to grow during the delay. Some caenogastopod larvae grow during delayed metamorphosis, but opisthobranch larvae do not. Data on larval growth of neritimorph gastropods are needed to help determine which of these growth patterns for planktotrophic gastropod larvae is more derived. We cultured planktotrophic larvae from all three major gastropod clades with feeding larvae through delays of metamorphosis of 3–10 weeks. Larvae of the caenogastropod Euspira lewisii and the euthyneurans Haminoea vesicula (Opisthobranchia) and Siphonaria denticulata (Pulmonata) conformed to previously described growth patterns for their respective major clades. Furthermore, the caenogastropod continued to lengthen the prototroch (ciliary band for swimming and feeding) and to differentiate prospective post-metamorphic structures (gill filaments and radular teeth) during delayed metamorphosis. Larvae of the neritimorph Nerita atramentosa arrested shell growth during delayed metamorphosis but the radula continued to elongate, a pattern most similar to that of non-feeding larvae of Haliotis, a vetigastropod genus. Character mapping on a phylogenetic hypothesis suggests that large larval size and capacity for continued growth during delayed metamorphosis, as exhibited by some caenogastropods, is a derived innovation among feeding gastropod larvae. This novelty may have facilitated post-metamorphic evolution of predatory feeding using a long proboscis.