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Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs

Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13–27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild.

Mucosal defenses are crucial in animals for protection against pathogens and predators. Host defense peptides (antimicrobial peptides, AMPs) as well as skin-associated microbes are key components of mucosal immunity, particularly in amphibians. We integrate microbiology, molecular biology, network-thinking, and proteomics to understand how host and microbially derived products on amphibian skin (referred to as the mucosome) serve as pathogen defenses. We studied defense mechanisms against chytrid pathogens

The disease chytridiomycosis caused by the fungus Bd has devastated amphibian populations worldwide. Functional genomic contributions to host susceptibility remain enigmatic and vary between species and populations. We conducted experimental Bd infections in Rana yavapaiensis, a species with intraspecific variation in chytridiomycosis susceptibility, to assess the skin and spleen transcriptomic response to infection over time. We predicted that increased immune gene expression would be associated with a positive disease outcome, but we instead found that surviving frogs had significantly reduced immune gene expression compared to susceptible frogs and to uninfected controls. MHC class IIβ gene expression was also significantly higher in susceptible frogs compared to surviving frogs. Furthermore, susceptible frogs expressed a significantly larger number of distinct class IIβ alleles, demonstrating a negative correlation between class IIβ expression, functional diversity, and survival. Expression of the MHC class IIβ locus previously associated with Bd disease outcomes was a significant predictor of Bd infection intensity at early infection stages but not at late infection stages, suggesting initial MHC-linked immune processes are important for ultimate disease outcomes. We infer through disease association and phylogenetic analysis that certain MHC variants are linked to the immune expression that was negatively associated with survival, and we hypothesize that frogs that did not express these alleles could better survive infections. Our study finds that MHC expression at early and late infection stages predicts Bd infection intensity, and suggests that generating a sustained immune response against Bd may be counterproductive for surviving chytridiomycosis in this partially susceptible species.

This chapter evaluates the detrimental effects of exotic anurans on native biota and how those provide an important justification for the ways frogs matter relative to exotic species impacts. It is clear that in the fight to save frogs, context matters. The species, its ecology, the value systems of the people living alongside them, and the audience of the message are all critical. One classic example of an invasive amphibian species is the cane toad, introduced to Australia in 1945 as a form of biological control for pests in sugarcane fields. They are highly toxic to predators like snakes, goannas, crocodiles, and quolls, and populations of these predators appear reduced in newly invaded parts of Australia where predators have not yet developed avoidance behaviors. In addition to direct impacts of invasive amphibians, some non-native species can have indirect effects on native amphibians by spreading disease.

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound. One day following probiotic treatment, the engineered Diaphorobacter and the violacein-producing pathway could be detected on the frogs but the treatment failed to improve frog survival when exposed to Bd. The second approach exposed frogs to the genetically modified bacterium mixed into a consortium with six other known anti-Bd bacteria isolated from captive A. zeteki, with no preliminary antibiotic treatment. The consortium treatment increased the frequency and abundance of three probiotic isolates (Janthinobacterium, Chryseobacterium, and Stenotrophomonas) and these persisted on the skin 4 weeks after probiotic treatment. There was a temporary increase in the frequency and abundance of three other probiotics isolates (Masillia, Serratia, and Pseudomonas) and the engineered Diaphorobacter isolate, but they subsequently disappeared from the skin. This treatment also failed to reduce frog mortality upon exposure.

Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.

Captive breeding programs are a valuable conservation strategy, particularly when integrated with research goals. Panamanian Harlequin frogs (genus Atelopus) serve as a case study for integrating captive breeding and research goals because they have experienced drastic chytridiomycosis-related declines and have large captive populations. Captive breeding efforts in Panama and the United States established secure ex-situ populations of Atelopus certus, A. glyphus, A. limosus, A. varius, and A. zeteki. Atelopus chiriquiensis is presumed to be extinct with no captive populations. The status of one undescribed species, Atelopus aff. limosus, has not been evaluated and no secure captive population has yet been established. Captive breeding efforts that produce a surplus of Atelopus are an important resource for research into collections management, disease mitigation, and adaptive management approaches for Atelopus reintroduction efforts. We reevaluated all Panamanian Atelopus species through the IUCN Redlist and compiled occurrence records for Panamanian Atelopus species to create a historical distribution map. We model Atelopus habitat suitability using Maxent and found annual mean air temperature to be the best predictor of Atelopus occurrence. The model will improve our knowledge of their likely spatial distribution and guide future conservation and reintroduction efforts. The recent proliferation of molecular tools, climate models, bio-banking, and reproductive technologies position us to address multiple applied and basic evolutionary questions such as: What factors cause differential disease outcomes? Do persisting populations have heritable traits associated with improved survivorship? Are there climatic refugia from disease? Ultimately, the answers to these questions will help us develop applied solutions and facilitate the reestablishment of self-sustaining wild populations.

Cold-adapted hellbender salamanders that inhabit cool mountain streams are expected to fare poorly under warmer projected climate scenarios. This study investigated the physiological consequences of long-term, naturalistic temperature variation on juvenile hellbenders under simulated current and warmer (+1.6 C) climates vs. controlled steady temperatures. Mean temperature and temperature variability were both important predictors of growth as indicated by monthly body mass change (%), stress as indicated by neutrophil:lymphocyte (N:L) ratio and bacteria-killing ability of blood. Cold exposure in hellbenders was associated with weight loss, increased N:L ratios and reduced Escherichia coli killing ability of blood, and these effects were less pronounced under a warmer climate scenario. These observations suggest that cold periods may be more stressful for hellbenders than previously understood. Growth rates peaked in late spring and late fall around 14–17°C. Hellbenders experiencing warmer simulated climates retained body condition better in winter, but this was counter-balanced by a prolonged lack of growth in the 3-month summer period leading up to the fall breeding season where warmer simulated conditions resulted in an average loss of −0.6% body mass/month, compared to a gain +1.5% body mass/month under current climate scenario. Hellbenders can physiologically tolerate projected warmer temperatures and temperature fluctuations, but warmer summers may cause animals to enter the fall breeding season with a caloric deficit that may have population-level consequences.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearing Atelopus varius tadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

Lambert et al . question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

A chytridiomycosis outbreak from Batrachochytrium dendrobatidis ( Bd) in a mixed-species plethodontid salamander exhibit resulted in four green salamander ( Aneides aeneus) deaths. One green salamander died before treatment, and three died during treatment with daily 0.005% itraconazole baths. All salamanders had evidence of severe Bd infections via cytology, histopathology, and/or polymerase chain reaction (PCR) at the time of death. Ten long-tailed salamanders ( Eurycea longicauda) and one two-lined salamander ( Eurycea bislineata) that shared the enclosure were initially negative for Bd on quantitative PCR but were prophylactically treated with daily 0.01% itraconazole baths for 11 days. Posttreatment testing yielded eight long-tailed salamanders and one two-lined salamander positive for Bd with low gene equivalents. All salamanders were negative after two to three treatment courses, and there were no additional mortalities. The difference in mortality and fungal load suggested that genus Aneides salamanders may be more susceptible to Bd than genus Eurycea salamanders.

Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly 10 , D-Ala 6 , Pro-NHEt 9- -GnRH 1, 2 or 4 μg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 10 6 cells/mL) followed by 4 μg/gbw GnRH-A (2.65 ± 0.21 × 10 6 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 10 6 cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7–6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50–70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.

Amphibians around the world are declining from threats that cannot currently be mitigated, making it impossible to safeguard some species in their natural habitats. Amphibians in the mountainous neotropics are one example where severe diseaserelated declines prompted calls for the establishment of captive assurance colonies to avoid extinctions. We surveyed experts in Panamanian amphibians to determine the probability of avoiding chytridiomycosis-related extinctions using captive breeding programs. We ranked Panamanian amphibian species by perceived susceptibility to chytridiomycosis, then calculated the likelihood of avoiding extinction as the product of three probabilities, which include (1) finding sufficient founder animals, (2) successfully breeding these species in captivity and (3) becoming extinct in the wild. The likelihood of finding enough animals to create a captive founding population was low for many rare species, especially for salamanders and caecilians. It was also low for frogs which were once regularly encountered, but have already disappeared including Atelopus chiriquiensis, Craugastor emcelae, C. obesus, C. punctariolus, C. rhyacobatrachus, Ecnomiohyla rabborum, Isthmohyla calypsa and Oophaga speciosa. Our results indicate that captive breeding could improve the odds of avoiding extinction for species that have severely declined or are likely to decline due to chytridiomycosis including Atelopus certus, A. glyphus, A. limosus, A. varius, A. zeteki, Anotheca spinosa, Gastrotheca cornuta, Agalychnis lemur and Hemiphractus fasciatus. Priority species that experts predicted were highly susceptible to chytridiomycosis that might also benefit from ex situ management include Craugastor tabasarae, C. azueroensis, C. evanesco, Strabomantis bufoniformis and Colostethus panamansis. In spite of high levels of uncertainty, this expert assessment approach allowed us to refine our priorities for captive amphibian programs in Panama and identify priority conservation actions with a clearer understanding of the probability of success.

The demise of amphibians? Rapid spread of disease is a hazard in our interconnected world. The chytrid fungus Batrachochytrium dendrobatidis was identified in amphibian populations about 20 years ago and has caused death and species extinction at a global scale. Scheele et al. found that the fungus has caused declines in amphibian populations everywhere except at its origin in Asia (see the Perspective by Greenberg and Palen). A majority of species and populations are still experiencing decline, but there is evidence of limited recovery in some species. The analysis also suggests some conditions that predict resilience. Science , this issue p. 1459 ; see also p. 1386

A better understanding of the factors influencing the biology of amphibian spermatozoa after release from the testis is a prerequisite to the development of sperm preservation methods. The objective of the study was to determine the effect of extracellular conditions (exposure to water and different temperatures) over time on the sperm motility and structural properties (including morphology and DNA integrity) collected from hormonally stimulated Atelopus zeteki. Following intraperitoneal injection of gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9 GnRH; 4 μg/g of body weight), human chorionic gonadotropin (hCG, 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 27 males were collected and analyzed for sperm motility, morphology and DNA integrity while maintained at room temperature (23 °C), 4 °C, or diluted in water (hypo-osmotic environment) over a period of 46 min post-collection. Percentages of sperm motility and forward progressive motility remained high (>60%) when spermic urine was kept at room temperature or at 4 °C for 46 min regardless of the hormonal stimulation method. Dilution in water at room temperature greatly reduced the percentage of motile spermatozoa and forward progression (

We engaged pet salamander owners in the United States to screen their animals for two amphibian chytrid fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). We provided pet owners with a sampling kit and instructional video to swab the skin of their animals. We received 639 salamander samples from 65 species by mail, and tested them for Bd and Bsal using qPCR. We detected Bd on 1.3% of salamanders (95% CI 0.0053–0.0267) and did not detect Bsal (95% CI 0.0000–0.0071). If Bsal is present in the U.S. population of pet salamanders, it occurs at a very low prevalence. The United States Fish and Wildlife Service listed 201 species of salamanders as “injurious wildlife” under the Lacey Act (18 U.S.C. § 42) on January 28, 2016, a precautionary action to prevent the introduction of Bsal to the U.S. through the importation of salamanders. This action reduced the number of salamanders imported to the U.S. from 2015 to 2016 by 98.4%. Our results indicate that continued precautions should be taken to prevent the introduction and establishment of Bsal in the U.S., which is a hotspot of salamander biodiversity.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 μM, respectively. Violacein showed a MIC of 15 μM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

For many threatened vertebrates, captivity may be the only option for species survival. Maintaining species in captivity prior to reintroduction presents many challenges, including the need to preserve genetic diversity and mitigation of disease risks. Recent studies suggest that captivity can alter the suite of symbiotic microbes that play important roles in host health. The Panamanian golden frog (Atelopus zeteki) has not been seen in its native habitat in Panama since 2009. Along with habitat loss and illegal collecting, the lethal disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for the severe decline of this species. Prior to the spread of Bd into golden frog habitat, conservation organizations collected golden frogs and placed them in captive survival assurance colonies. The skin of amphibians is host to a diverse resident bacterial community, which acts as a defense mechanism in some amphibians to inhibit pathogens. We characterized the cutaneous bacterial community from wild and F1 captive golden frogs originating from the same population with Illumina sequencing to assess how long-term captivity has affected this community. We found that species richness, phylogenetic diversity, and community structure of the skin microbiota was significantly different between wild and captive golden frogs. However, after approximately eight years of living in captivity, the offspring of the original captive golden frogs still shared 70% of their microbial community with wild frogs. These results demonstrate that host-associated microbial communities can be significantly altered by captive management, but most of the community composition can be preserved.

Populations of native Panamanian golden frogs (Atelopus zeteki) have collapsed due to a recent chytridiomycosis epidemic. Reintroduction efforts from captive assurance colonies are unlikely to be successful without the development of methods to control chytridiomycosis in the wild. In an effort to develop a protective treatment regimen, we treated golden frogs with Janthinobacterium lividum, a skin bacterium that has been used to experimentally prevent chytridiomycosis in North American amphibians. Although J. lividum appeared to colonize A. zeteki skin temporarily, it did not prevent or delay mortality in A. zeteki exposed to Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis. After introduction of J. lividum, average bacterial cell counts reached a peak of 1.7 × 10(6) cells per frog ~2 weeks after treatment but declined steadily after that. When J. lividum numbers declined to ~2.8 × 10(5) cells per frog, B. dendrobatidis infection intensity increased to greater than 13,000 zoospore equivalents per frog. At this point, frogs began to die of chytridiomycosis. Future research will concentrate on isolating and testing antifungal bacterial species from Panama that may be more compatible with Atelopus skin.

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

There have been surprisingly few analyses of how the international trade in amphibians for food affects the conservation status of this group. We analyzed information from the UN Commodity Trade Statistics Database and found that, by volume, Indonesia supplied nearly half of the animals entering the world's US$40 million per year international frog legs trade, and that – collectively – France, Belgium, and the US imported more than 75% of all frog legs traded internationally. Nonetheless, a close examination of available information from 1996 through 2006 revealed that most countries throughout the world participated in the frog legs trade at some level. These extensive international amphibian trade networks could facilitate the spread of pathogens, including Batrachochytrium dendrobatidis, which has been identified as a threat connected with the disappearance and possible extinction of over 90 amphibian species around the world. Given the size and extent of the international trade in frog legs, we advoca...

Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki , a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.

We surveyed fish distribution in three lagoons and adjacent forereefs in the British Virgin Islands recording about 28,000 fish from 40 families and 118 species. Canonical correspondence indicated that rock, sand, fleshy algae, gorgonians, mangroves and live hard coral were the most important habitat types influencing fish assemblage composition. About 47% of fishes occurring at more than 10 stations displayed evidence of ontogenetic partitioning between reefs and lagoons but post-settlement ontogenetic life history strategies were quite varied depending on the species. For example Chaetodon striatus juveniles occurred exclusively in lagoons and all sexually mature adults were found on reefs. Some differences were less pronounced as seen in Halichoeres bivittatus where individuals of all sizes occurred on reefs and lagoons, but when analysed it was found that reefs had larger individuals than lagoons. Some species, such as Acanthurus bahianus, were primarily reef species whose juveniles also used lagoon habitats while others, such as Gerres cinereus, were generally lagoon species whose adults occasionally moved onto reefs. Even with all this variation in life-history strategies, all the species that exhibited bay-reef partitioning used the lagoons as juveniles then moved onto reefs as adults and not vice versa, supporting the hypothesis that bays are important nursery areas for reef-dwelling fishes. These results show that a detailed review of the natural life-history strategies and habitat requirements are required before making further generalisations about the role of near-shore habitat types as nurseries for reef fishes. This is especially important given the rapid changes in tropical near-shore habitats around the world.

Fish migrations into the streams of seasonally-flooded depressions (or dambos) that cover the central plateaus of Africa are a well-known phenomenon to scientists and lay people alike. However, the factors causing seasonal migrations are poorly understood and most scientific references to them are anecdotal, lacking rigorous description. Fish migrations in two dambos in Zimbabwe were investigated during the 1999/2000 rainy season, by electrofishing and using traps. The fish fell into two categories: those that used the dambo, and those that remained in the main river. At the beginning of the rainy season Barbus paludinosus, B. lineomaculatus, Clarias gariepinus and Tilapia sparrmanii moved from the perennial river into the small dambo streams and spawned there, then moved back to the perennial river, leaving their young in the dambo, which acted as a nursery area. Barbus spp. were the most numerous migrants and they moved mainly during daylight hours. Other fish species present in the system included Opsa...

A simple habitat assessment score (HAS) was designed to assess habitat complexity across several different shallow tropical marine habitats including sandy patches, algal beds, seagrass beds and reefs. It measured rugosity, variety of growth forms, height, refuge size categories, percentage live cover and percentage hard substratum. Multiple regression models using HAS variables as predictors accounted for 71 and 22% of the variation in observed species richness and total fish abundance respectively. The two most important predictors of observed species richness were rugosity and variety of growth forms, while height was the most important predictor of total fish abundance. The HAS method worked consistently across a variety of habitat types and the complexity map closely mirrored the map of observed species richness, reflecting the patchy habitat mosaic of shallow tropical marine areas. Stations at the mouth of an enclosed lagoon, however, had a higher number of species than might have been expected judging from the habitat complexity scores. It is possible that this was linked to the preferential settling of pelagic fish larvae in this area as tidal water exchanges between the bay and the reef were funnelled through one small gap. This study highlights the need for fish biodiversity studies to take habitat complexity into account.

Sets of artificial reefs were replicated in 5 bays off Tortola in the British Virgin Islands to investigate the effects of habitat complexity on fish assemblages. Increasing percentage hard sub- strate and the number of small reef holes increased fish abundance on reefs. The observed number of species (Sobs) occurring on each reef increased with increasing rugosity, variety of growth forms, percentage hard substrate, and variety of refuge hole sizes. A rarefied or abundance-corrected spe- cies richness measure (Srare) was calculated to take the varying fish abundances into account. After this correction, rugosity was the only variable that significantly increased fish species-richness. Experimental reefs of different height (20 and 60 cm) did not have significantly different fish abun- dance or species richness. The presence of long-spined sea urchins Diadema antillarum increased Sobs and total fish abundance on artificial rock-reefs and in seagrass beds, but the effect was most pronounced in seagrass beds where shelter was a strongly limiting factor. These results indicate that complex habitats or animals such as D. antillarum that provide shelter to fish are essential for main- taining fish biodiversity at local scales. The most important aspects of complexity are rugosity, hard substrate and small refuge holes. Artificial reefs may be used to mitigate habitat damage in impacted areas, and if management objectives are to increase local fish abundance and species richness, the reefs should provide a stable substrate where this is unavailable, have a rugose surface with many small refuge holes, and have a variety of growth forms.

We compared abundance and diversity of phytoplankton, zooplankton, and fishes among limnetic (P: always without macrophytes) and littoral habitats with (L +) and without (L- ) hyacinths in Lake Chivero, a man-made hypertrophic reservoir near Harare (Zimbabwe). In addition, the littoral macrophyte community, and macro-inver- tebrates associated with hyacinth mats were inventoried. The phytoplankton commu- nity was dominated by blue-green algae (mainly Microcystis aeruginosa ), typical for a hyper-eutrophic lake. Total absolute densities were about 10 to 30 times higher at the L+ sites than at the unvegetated L- and P sites. On the basis of relative species abun- dances the L- zones were more similar to the P than to the L + zones. There was an in- creasing importance of chlorophytes ( Staurastrum sp. and Pandorina morum ) and di- atoms (Cyclotella meneghiniana and pennales) and a decreasing dominance of Mycro- cystis along the discriminant axis from L +, L- to P. The zooplankton community was most dense in the unvegetated zones. Daphnids and bosminids were more abundant in the pelagic than in both littoral zones. Calanoids and Diaphanosoma were dominantly represented in the unvegetated zones. The two littoral zones were characterised by higher densities of chydorids, while they could be discriminated by the dominance of cyclopoids in the vegetated site. Seventeen different fish species were captured by at least one of the different fishing methods. Apparent habitat preferences differed ac

A survey of the upper Manyame River catchment, middle Zambezi system, Zimbabwe, yielded a total of 22 fish species from 48 stations. The most widespread species (present at >20 stations) were Marcusenius macrolepidotus, Barbus paludinosus, Labeo cylindricus, Clarias gariepinus and Tilapia sparrmanii. The most numerous species (>10% of the total) were B. paludinosus, B. lineomaculatus and T. sparrmanii. A number of species that occurred, or formerly occurred, in the catchment were not collected and the possible reasons for their absence are discussed. Some distinctive patterns of distribution could be discerned, with the presence of exotic predators (Micropterus salmoides and Serranochromis robustus) and pollution being the most important determinants. Although the species richness of indigenous fishes was relatively high when the predators were present, their relative abundance was low. Pollution from sewage, in contrast, brought about a reduction in both species richness and biomass. Land use also influenced both species richness and abundance, which were lower on commercial farms where most streams were regulated by dams and where exotic predators were present. In contrast, both variables were highest on communal lands where subsistence farmers had not built dams, leaving the streams unregulated, and where exotic predators were generally absent. Keywords: Zimbabwe; stream fish; distribution; land use; impacts; exotic predators; conservation; species richness (Afr J Aqua Sci: 2003 28(1): 25-34)

Several biogeographic studies of salamanders have described relationships between salamander body size and climate. We specifically selected Plethodon cinereus as a widely distributed species that was well represented in museum collections to investigate the effects of warming climate on adult body size. We found a positive correlation between mean maximum July temperature and body size, and a negative correlation between precipitation of the driest month and body size. Surface-collected adult P. cinereus were 2.3% larger in warmer counties on the coastal plain compared with cooler counties in the Appalachian Mountains. We compared salamanders collected between 1950 and 1970 versus those collected between 1980 and 2000 and found that body size increased 1.8% in counties on the coastal plain where mean maximum July temperatures had also increased by 0.5–1.2 °C. Warming temperatures alone, however, did not adequately account for the observed size increases, because body size also increased 1.3% in places that experienced less than 0.25 °C warming, but that difference was not statistically significant.

We have established ex situ assurance colonies of two endangered Panamanian harlequin frogs, Atelopus certus and Atelopus glyphus, but observed that males fought with each other when housed as a group. Housing frogs individually eliminated this problem, but created space constraints. To evaluate the potential stress effects from aggressive interactions when grouping frogs, we housed male frogs in replicated groups of one, two, and eight. We measured aggressive behavioral interactions and fecal glucocorticoid metabolite (GC) concentrations as indicators of stress in each tank. In both small and large groups, frogs initially interacted aggressively, but aggressive interactions and fecal GCs declined significantly after the first 2 weeks of being housed together, reaching the lowest levels by week 4. We conclude that aggressive interactions in same-sex groups of captive Atelopus may initially cause stress, but the frogs become habituated within a few weeks and they can safely be housed in same-sex groups for longer periods of time.

Samples of fish were collected from 42 stations in streams around Harare, Zimbabwe. The total abundance of fish was lower by 50% or more at stations where the exotic predators Micropterus salmoides and Serranochromis robustus were present compared with stations where they were absent. The greatest differences in density occurred among small Barbus species, whose populations were 86% lower (S. robustus alone) and 99% lower (M. salmoides alone). Populations of small rock and sand catlets were less dense when S. robustus, but not M. salmoides, was present probably because the latter did not enter the riffles where the catlets live. These predators apparently did not affect densities of certain other species in the system that were either cryptic, bottom-dwelling forms or too large. Low Barbus diversity and abundance in streams containing exotic predators is of concern because this group of fluvial fishes is threatened throughout southern Africa by dam-building, pollution, and other factors.

Summary Two species of barred minnow, Opsaridium zambezense and O. peringueyi, occur in Zimbabwe. The former is widely distributed in the Zambezi basin and some other rivers but is now absent from Lake Kariba and possibly some of its tributaries. The latter, which was only recently recognised as a distinct species and admitted to the Zimbabwean list, is much scarcer. It occurs in the Save and Limpopo catchments and there are only four specimens in the Natural History Museum of Zimbabwe collection. These specimens are all more than 40 years old and the lack of more recent ones suggest that O. peringueyi may be extinct, or close to extinction, in Zimbabwe. This situation may have come about through habitat alteration and drought, and emphasises the need for further monitoring of the current occurrence/distribution of fish species in the country.

Summary The effect of season on the South African Scoring System (SASS) biotic index for rapid assessment of water quality using benthic macroinvertebrates was investigated using the Yellow Jacket and Mazowe Rivers in Zimbabwe as a case study. Several impacts in the area degraded water quality. These were habitat destruction, acid mine drainage and organic pollution. SASS score at all sites changed with season. At and downstream from polluted sites, water quality determined using SASS was best at the end of the rainy season and it deteriorated steadily into the dry season. SASS scores from sites upstream of pollution appeared to improve towards the end of the rainy season. Average Score Per Taxon indices did not have a significant pattern of seasonal variation. SASS was designed to monitor water quality in South African rivers, so when implementing any monitoring programme, selection of sampling season and the interpretation of results in areas with similar climate patterns should take seasonal effects in...

Summary Ectothermic species living in temperate regions can experience rapid and potentially stressful changes in body temperature driven by abrupt weather changes. Yet, among amphibians, the physiological impacts of short-term temperature variation are largely unknown. Using an ex situ population of Cryptobranchus alleganiensis, an aquatic North American salamander, we tested the hypothesis that naturally-occurring periods of temperature variation negatively impact amphibian health, either through direct effects on immune function or by increasing physiological stress. We exposed captive salamanders to repeated cycles of temperature fluctuations recorded in the population's natal stream and evaluated behavioral and physiological responses, including plasma complement activity (i.e., bacteria killing) against Pseudomonas aeruginosa, Escherichia coli and Aeromonas hydrophila. The best-fit model (ΔAICc = 0, wi = 0.9992) revealed 70% greater P. aeruginosa killing after exposure to variable temperatures and no evidence of thermal acclimation. The same model predicted 50% increased E. coli killing, but had weaker support (ΔAICc = 1.8, wi = 0.2882). In contrast, plasma defenses were ineffective against A. hydrophila, and other health indicators (leukocyte ratios, growth rates and behavioral patterns) were maintained at baseline values. Our data suggest that amphibians can tolerate, and even benefit from, natural patterns of rapid warming/cooling. Specifically, temperature variation can elicit increased activity of the innate immune system. This immune response may be adaptive in an unpredictable environment, and is undetectable by conventional health indicators (and hence considered cryptic). Our findings highlight the need to consider naturalistic patterns of temperature variation when predicting species' susceptibility to climate change.