Oviposition site selection by mosquitoes is affected by cues from conspecific larvae and anuran tadpoles

Abstract The oviposition site that a female mosquito selects will influence the fitness of her larvae. We conducted a series of artificial pond experiments to compare the oviposition responses of two species of mosquitoes with the presence of tadpoles, conspecifics and chemical cues from these organisms. The two mosquito species differ markedly in larval ecology. The larvae of one species, Culex quinquefasciatus, co‐occur with numerous freshwater organisms, including tadpoles of Linmodynastes peronii (the striped marsh frog). Larvae of the other mosquito, Ochlerotatus australis, inhabit small brackish rock ponds where the main potential competitors are tadpoles of Crinia signifera (the common eastern froglet). In field trials, females of both mosquito species oviposited significantly more often in water that contained (or had previously contained) conspecific larvae. However, these superficially similar responses were mediated via different pathways: fungicide abolished the response by C. quinquefasciatus but not by O. australis. The two mosquito species also responded differently to cues associated with syntopic tadpoles. The presence of tadpoles did not influence oviposition by C. quinquefasciatus, but O. australis oviposited less often if tadpoles were present. These interspecific differences in oviposition behaviour may be adaptive to differences in larval ecology: competition with tadpoles is likely to be more significant for O. australis than for C. quinquefasciatus. Our findings thus support the hypothesis that mosquitoes oviposit selectively to avoid potential anuran larval competitors.     

Temperature modulates the effects of predation and competition on mosquito larvae

1. Predation risk affects interspecific competition by decreasing foraging activity and relative competitive ability. Predation risk is determined by predators' prey choice and prey responses, both of which can be influenced by temperature. Temperature is especially important for larval prey and can result in a trade‐off between predator‐induced decreases in foraging activity and growth. Interspecific competition must also be examined in relation to intraspecific density‐dependent competition; weaker interspecific competition leads to coexistence of competitors. 2. This study explored how temperature (15 and 25 °C) could affect a focal species, larvae of the mosquito Culex quinquefasciatus, by examining prey choice in a shared predator (mosquitofish; Gambusia holbrooki) and the effects of predation risk on interspecific competition with Limnodynastes peronii tadpoles. Intraspecific density‐dependent competition in C. quinquefasciatus at these temperatures was also examined. 3. At 25 °C, G. holbrooki consumption of both C. quinquefasciatus and L. peronii increased; however, the effects of interspecific competition on mosquito survival did not decrease with L. peronii exposure to predation risk. The relationship between intraspecific density‐dependent competition and interspecific competition was temperature‐dependent, with competitive dominance of L. peronii at 25 °C. Male and female mosquitoes had different temperature‐dependent responses, indicating sex‐specific intrinsic responses to starvation and differential selection pressures. At 25 °C, females were susceptible to interspecific competition by L. peronii, while males were susceptible to intraspecific competition. 4. The use of competitors as biological controls has implications for mosquito disease transmission, and these results suggest that control effectiveness may be modified by climate change.     

Competition between tadpoles and mosquito larvae

Tadpoles and mosquito larvae often co-occur, and may compete for scarce resources. However, competition between such distantly related organisms has attracted less scientific attention than have interactions among closely related taxa. We examined ecological interactions in two tadpole-mosquito systems in southeastern Australia, one from freshwater ponds (Limnodynastes peronii and Culex quinquefasciatus) and one from brackish-water habitats (Crinia signifera and Ochlerotatus australis). Diets of these tadpoles and mosquito larvae overlap considerably, potentially leading to competition for food. Laboratory experiments show that, in both study systems, mosquitoes reduced the growth rates of tadpoles, and tadpoles reduced the growth rates and survival of mosquito larvae. These negative effects were seen even at high food levels. Thus, our study suggests that tadpoles and mosquito larvae affect each other strongly, and do so via pathways other than simple consumptive competition. Because mosquitoes are important vectors for human diseases, the global decline in amphibian populations may have more impact on human health than has generally been anticipated.     

Comparison of frog assemblages between urban and non‐urban habitats in the upper Blue Mountains of Australia

1. World wide, and in Australia, many frog populations have declined over the last two decades. The present study was undertaken to determine whether urbanization has affected frog diversity and abundance. 2. Five urban sites were paired with non‐urban sites. Urban sites were in Katoomba and Blackheath, and were subject to physical environmental disturbance and impacted by storm water pollution due to urban runoff. Non‐urban sites were in the Blue Mountains National Park and were effectively subject to no human impact. 3. Water quality at urban sites was typical of sites polluted with sewage, while non‐urban sites exhibited water quality typical of ‘pristine’ natural bushland streams. 4. Six species were found at urban sites (Litoria peronii, Litoria dentata, Litoria verreauxii, Limnodynastes dumerilii, Limnodynastes peronii, Crinia signifera), with up to four species present at a site. Only one species (C. signifera) was recorded at non‐urban sites, and frogs were absent from most non‐urban sites. 5. The situation in non‐urban sites mirrors the trend of decline observed in other montane regions. Surprisingly, frog abundance and diversity were higher in urban habitats, running counter to this trend. 6. We hypothesize that the salts, detergents and other chemicals in urban wastewaters provide frogs with a level of protection against disease, particularly chytridiomycosis.     

Predator‐mediated phenotypic plasticity in tadpoles of the striped marsh frog,Limnodynastes peronii

Abstract We tested the phenotypic responses of larval striped marsh frogs (Limnodynastes peronii) to the odonate nymph predator, Aeshna brevistyla. When reared in the presence of dragonfly nymphs feeding upon conspecifics of L. peronii larvae the tadpoles showed a strong change in morphology. Morphological changes included an increase in total tail height, but also an unexpected marked change in head‐body shape. In addition, we examined how tadpole development, as well as mass and length at metamorphosis, was affected by exposure to dragonfly nymphs. Larval development of L. peronii was strongly influenced by exposure to the predatory behaviour of dragonfly nymphs. Predator‐induced tadpoles had significantly slower developmental rates than control larvae. Although metamorphs of non‐exposed L. peronii were approximately 33% lighter than predator‐exposed metamorphs and possessed lower jump distances, after adjusting for mass there was no difference in jump distance. The newly described morphological response may assist in more accurately relating morphological plasticity to fitness.     

Unraveling microbe-mediated interactions between mosquito larvae in a laboratory microcosm

Interspecies interactions have important impacts on communities and when multiple trophic levels are involved, effects can be complex and indirect. For mosquitoes, interactions experienced as larvae affect adult attributes such as survivorship, reproductive output, and longevity, factors that can affect their ability to vector disease. We examined how larvae of two ecologically distinct mosquito species, Aedes japonicus japonicus and Culex quinquefasciatus, interact at different temperatures (17 and 27 °C) and at different relative densities. We also quantified abundances of bacteria and protozoan flagellates to uncover how changes in the microbial community affect the outcome of the two mosquitoes’ interaction. At 17 °C, survival and size of both mosquito species were not affected by the other’s presence. Cx. quinquefasciatus was strongly affected by intraspecific, but not interspecific, competition at both temperatures. At 27 °C, Ae. j. japonicus larvae experienced 100 % mortality in treatments by themselves and treatments where Cx. quinquefasciatus was abundant, surviving only in the presence of low densities of Cx. quinquefasciatus. Both the total bacteria count and counts of a protozoan flagellate identified as Spumella spp. decreased with increasing numbers of Cx. quinquefasciatus. We postulate that at 27 °C, the survival of Ae. j. japonicus depends on the interaction between Cx. quinquefasciatus and the microbial community. This study demonstrates that one mosquito species may alter the microbial community in ways that indirectly influence another mosquito species’ larval survival, and by extension adult abundance and potential disease transmission.     

Biological warfare in the garden pond: tadpoles suppress the growth of mosquito larvae

Abstract. 1. Although tadpoles and mosquito larvae may compete for scarce resources in natural freshwater systems, the mechanisms involved in such competition remain largely unstudied.
2. Replicated artificial ponds were set up to examine the role of pathogenic interference (water-borne growth inhibitors) in two tadpole–mosquito systems from south-eastern Australia. One system comprised taxa that are commonly sympatric in freshwater ponds (tadpoles of Limnodynastes peronii and larvae of Culex quinquefasciatus ) while the other comprised species that co-occur in brackish water ponds (tadpoles of Crinia signifera and larvae of Ochlerotatus australis ).
3. Water that had previously contained tadpoles suppressed the rates of survival and pupation of mosquito larvae in both systems. Fungicide reduced or eliminated this effect, suggesting that the growth inhibitors may be fungal organisms (possibly the yeast Rhodotorula glutinis ) from tadpole faeces. Fungicide also enhanced growth rates of tadpoles.
4. These results suggest that interference competition between tadpoles and mosquito larvae is mediated by other organisms in some ecological systems.     

Tadpoles of three common anuran species from Thailand do not prey on mosquito larvae

Tadpoles are often considered to be predators of mosquito larvae and are therefore beneficial for the control of certain disease vectors. Nevertheless, only a few species have actually been recorded to prey on mosquito larvae. The mosquito larvae predation rates of tadpoles of three common Thai anuran species (Bufo melanostictus, Kaloula pulchra and Hylarana raniceps) were experimentally tested. Tadpoles in varying developmental stages were used to assess a size/age effect on the predation rate. In addition, different instars of Culex quinquefasciatus were used in order to assess a prey size effect on the predation rates. All three species failed to show any evidence of mosquito larvae predation. Neither small nor large tadpoles fed on mosquito larvae. Prey size also did not affect predation. Although tadpoles do not feed on mosquito larvae, there may be other direct or indirect inter‐specific interactions that adversely impact the development of larvae in shared habitats with tadpoles.     

Drought and the organization of tree-hole mosquito communities

Summary In southeastern North America (North Florida, USA), the duration, frequency, and timing of drought differentially affect the survivorship of pre-adult tree-hole mosquitoes. Drought affects survivorship both by the direct action of dehydration on developing larvae and pupae and by the indirect modulation of predation. The drought-susceptible species, Toxorhynchites rutilus, Orthopodomyia signifera, and Anopheles barberi co-occur in more permanent holes that are larger, with larger, more vertical openings, lower down in larger trees, and contain darker water with higher conductivity, pH, and tannin-lignin content than the holes occupied by Aedes triseriatus that has drought-resistant eggs and rapid larval development. Ovipositing mosquitoes cue on physical and chemical attributes of tree holes independently of host tree species. These same attributes differ among drought-prone and drought-resistant holes but mosquitoes track these attributes more faithfully than the attributes predict tree-hole stability.     

A field study of potential ecological costs of resistance by ‘stem ducking’ in tall goldenrod,Solidago altissima

Culex quinquefasciatus Say (Diptera: Culicidae) is an abundant urban mosquito that is the vector of filariasis. Breeding in septic tanks, where there are very high levels of bacterial food, it is likely to have a different reaction to crowding compared with other mosquitoes. To test for the presence and type of crowding effects, four larval densities of C. quinquefasciatus varying from 0.4 to 3.2 larvae ml^?1 of water were reared in tubes. Mortality was found to greatly increase at densities above 0.8 larvae, whereas larval duration increased even above 0.4 larvae ml^?1. Changing the water in the tubes daily gave a small (but significant) response in reducing mortality and larval duration. However, when larvae kept at a low density shared the same water with larvae at high density, there was no chemical influence on their growth rate and mortality. The effect of crowding was primarily due to physical disturbances between larvae. When larvae were kept at a high density in the same volume of water, but in shallow trays with a large surface area and therefore much less contact between them, mortality was the same as for the lowest density. There was still, however, a significant increase in larval duration from 8.6 days at 0.4 larvae ml^?1 to 12.1 days at 3.2 larvae ml^?1. It is therefore concluded that the larvae respond to physical rather than chemical factors by prolonging larval development and having some increase in mortality.     

Understanding the mechanism of Chikungunya virus vector competence in three species of mosquitoes

Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE‐06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non‐permissiveness of these mosquitoes to infection.     

Evaluation of collection methods for Culex quinquefasciatus,Aedes aegypti,and Aedes simpsoni in northeastern Tanzania

In East Africa, significant morbidity and mortality are caused by infections spread by Culex quinquefasciatus and Aedes aegypti. Sticky traps have been shown to be effective tools for sampling populations of Aedes mosquitoes and have been found to catch Cx. quinquefasciatus. Thus, they could potentially be used to sample populations of this species. This study compared Sticky ovitraps (SO) and MosquiTraps (MQT) with the CDC Gravid trap (CDC‐GT) for collection of Culex and Aedes mosquito populations in Tanzania. A follow‐up experiment was carried out using traps set for a 24‐h period to accommodate the oviposition habits of Aedes aegypti and Ae. simpsoni s.l. mosquitoes. The results showed that the CDC‐GT caught significantly more Cx. quinquefasciatus and Ae. aegypti than the SO or MQT, but there was no significant difference in the number of mosquitoes caught between the two sticky traps or of Ae. simpsoni s.l. caught among the three trap types. The results suggest that CDC‐GTs are the most appropriate in sampling of Cx. quinquefasciatus. Although CDC‐GTs collected more Ae. aegypti than the sticky traps, the simplicity and cost benefit of sticky traps facilitates large scale studies. All three trap types should be considered for monitoring Aedes mosquitoes.     

Larval habitat for the avian malaria vector Culex quinquefasciatus (Diptera: Culicidae) in altered mid‐elevation mesic‐dry forests in Hawai'i

Effective management of avian malaria (Plasmodium relictum) in Hawai'i's endemic honeycreepers (Drepanidinae) requires the identification and subsequent reduction or treatment of larval habitat for the mosquito vector, Culex quinquefasciatus (Diptera: Culicidae). We conducted ground surveys, treehole surveys, and helicopter aerial surveys from 2001–2003 to identify all potential larval mosquito habitat within two 100+ ha mesic‐dry forest study sites in Hawai'i Volcanoes National Park, Hawai'i; ‘Ainahou Ranch and Mauna Loa Strip Road. At ‘Ainahou Ranch, anthropogenic sites (43%) were more likely to contain mosquitoes than naturally occurring (8%) sites. Larvae of Cx. quinquefasciatus were predominately found in anthropogenic sites while Aedes albopictus larvae occurred less frequently in both anthropogenic sites and naturally‐occurring sites. Additionally, moderate‐size (≈20–22,000 liters) anthropogenic potential larval habitat had >50% probability of mosquito presence compared to larger‐ and smaller‐volume habitat (<50%). Less than 20% of trees surveyed at ‘Ainahou Ranch had treeholes and few mosquito larvae were detected. Aerial surveys at ‘Ainahou Ranch detected 56% (95% CI: 42–68%) of the potential larval habitat identified in ground surveys. At Mauna Loa Strip Road, Cx. quinquefasciatus larvae were only found in the rock holes of small intermittent stream drainages that made up 20% (5 of 25) of the total potential larval habitat. The volume of the potential larval habitat did not influence the probability of mosquito occurrence at Mauna Loa Strip Road. Our results suggest that Cx. quinquefasciatus abundance, and subsequently avian malaria, may be controlled by larval habitat reduction in the mesic‐dry landscapes of Hawai'i where anthropogenic sources predominate.     

Interspecific variation in the phenology of advertisement calling in a temperate Australian frog community

Spatial and temporal partitioning of resources underlies the coexistence of species with similar niches. In communities of frogs and toads, the phenology of advertisement calling provides insights into temporal partitioning of reproductive effort and its implications for community dynamics. This study assessed the phenology of advertisement calling in an anuran community from Melbourne, in southern Australia. We collated data from 1432 surveys of 253 sites and used logistic regression to quantify seasonality in the nightly probability of calling and the influence of meteorological variables on this probability for six species of frogs. We found limited overlap in the predicted seasonal peaks of calling among these species. Those shown to have overlapping calling peaks are unlikely to be in direct competition, due to differences in larval ecology (Crinia signifera and Litoria ewingii) or differences in calling behavior and acoustics (Limnodynastes dumerilii and Litoria raniformis). In contrast, closely related and ecologically similar species (Crinia signfera and Crinia parinsignifera; Litoria ewingii and Litoria verreauxii) appear to have staggered seasonal peaks of calling. In combination with interspecific variation in the meteorological correlates of calling, these results may be indicative of temporal partitioning of reproductive activity to facilitate coexistence, as has been reported for tropical and temperate anurans from other parts of the globe.     

Characterisation of Culex quinquefasciatus (Diptera: Culicidae) larval habitats at ground level and temporal fluctuations of larval abundance in Córdoba, Argentina

The aims of this study were to characterise the ground-level larval habitats of the mosquito Culex quinquefasciatus, to determine the relationships between habitat characteristics and larval abundance and to examine seasonal larval-stage variations in Córdoba city. Every two weeks for two years, 15 larval habitats (natural and artificial water bodies, including shallow wells, drains, retention ponds, canals and ditches) were visited and sampled for larval mosquitoes. Data regarding the water depth, temperature and pH, permanence, the presence of aquatic vegetation and the density of collected mosquito larvae were recorded. Data on the average air temperatures and accumulated precipitation during the 15 days prior to each sampling date were also obtained. Cx. quinquefasciatus larvae were collected throughout the study period and were generally most abundant in the summer season. Generalised linear mixed models indicated the average air temperature and presence of dicotyledonous aquatic vegetation as variables that served as important predictors of larval densities. Additionally, permanent breeding sites supported high larval densities. In Córdoba city and possibly in other highly populated cities at the same latitude with the same environmental conditions, control programs should focus on permanent larval habitats with aquatic vegetation during the early spring, when the Cx. quinquefasciatus population begins to increase.     

Identification of Metarhizium strains highly efficacious against Aedes,Anopheles and Culex larvae

Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, have been shown to be efficacious in killing mosquito larvae of different mosquito species. The current study compared the pathogenicity and efficacy of two formulations of three fungal strains against different instars of three mosquito species with the aim of identifying the most virulent strain for use under field conditions. Three strains of Metarhizium, ARESF 4556, ARSEF 3297 and V275, were assayed against early (L_2?3) and late (L_3–4) instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Two formulations of the fungi were tested, dry conidia and aqueous suspensions (i.e. ‘wet’ conidia). Effects of all combinations of conidia, mosquito species, instar, fungal strain and concentration on mosquito mortality were analysed using Cox regression and Kaplan–Meier analyses. Strain ARSEF 4556 was more virulent than ARSEF 3297 and V275, with LT₅₀ values ranging from 0.3 to 1.1 days, with Anopheles and Culex being more susceptible than Aedes. Early and late instars were equally susceptible independent of species. Although the formulation did influence mortality rates, both ‘wet’ and ‘dry’ conidia applications were highly effective in killing mosquito larvae. Viable spores were more efficacious than heat killed spores. The latter did cause mortality but only at high concentrations. Metarhizium sp. has proved to be effective in reducing survivability of all larval stages of Aedes, Anopheles and Culex under laboratory conditions. Aedes larvae were generally more tolerant than Anopheles and Culex irrespective of fungal strain.     

Aquatic microfauna alter larval food resources and affect development and biomass of West Nile and Saint Louis encephalitis vector Culex nigripalpus (Diptera: Culicidae)

Ciliate protists and rotifers are ubiquitous in aquatic habitats and can comprise a significant portion of the microbial food resources available to larval mosquitoes, often showing substantial declines in abundance in the presence of mosquito larvae. This top‐down regulation of protists is reported to be strong for mosquitoes inhabiting small aquatic containers such as pitcher plants or tree holes, but the nature of these interactions with larval mosquitoes developing in other aquatic habitats is poorly understood. We examined the effects of these two microbial groups on lower trophic level microbial food resources, such as bacteria, small flagellates, and organic particles, in the water column, and on Culex larval development and adult production. In three independent laboratory experiments using two microeukaryote species (one ciliate protist and one rotifer) acquired from field larval mosquito habitats and cultured in the laboratory, we determined the effects of Culex nigripalpus larval grazing on water column microbial dynamics, while simultaneously monitoring larval growth and development. The results revealed previously unknown interactions that were different from the top‐down regulation of microbial groups by mosquito larvae in other systems. Both ciliates and rotifers, singly or in combination, altered other microbial populations and inhibited mosquito growth. It is likely that these microeukaryotes, instead of serving as food resources, competed with early instar mosquito larvae for microbes such as small flagellates and bacteria in a density‐dependent manner. These findings help our understanding of the basic larval biology of Culex mosquitoes, variation in mosquito production among various larval habitats, and may have implications for existing vector control strategies and for developing novel microbial‐based control methods.     

Evaluation of four fresh-water unicellular cyanobacteria as potential hosts for mosquitocidal toxins

Summary For biocontrol of mosquitoes, mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus have been cloned into a number of cyanobacteria. However, little is known about the persistence of such recombinant cyanobacteria in mosquito larval habitats. Four fresh water unicellular cyanobacteria, Synechococcus PCC6301, PCC7425, PCC7942 and Synechocystis PCC 6803, were evaluated under laboratory conditions related to mosquito breeding environments. Results indicated that Synechococcus PCC6301 was potentially the most suitable organism for use in the natural mosquito habitat as it could tolerate a wide range of temperatures, salinities, and biological and chemical insecticides. Moreover, strain PCC6301 could be ingested and digested by Culex quinquefasciatus larvae and could support the development of larvae to full insect maturity.     

Aedes species in treeholes and fruit husks between dry and wet seasons in southeastern Senegal

During the dry season in February, 2010 and the wet season in September, 2011 we sampled mosquito larvae and eggs from treeholes of seven native hardwood species and the husks of Saba senegalensis in 18 sites in the PK‐10 forest in southeastern Senegal. Larvae were reared to adults for species identification. In the dry season, we recovered 408 Aedes mosquitoes belonging to seven species. Aedes aegypti s.l. comprised 42.4% of the collection, followed by Ae. unilineatus (39%). In contrast to reports from East Africa, both Ae. aegypti aegypti and Ae. aegypti formosus were recovered, suggesting that both subspecies survive the dry season in natural larval habitats in West Africa. In the wet season, 455 mosquitoes were collected but 310 (68.1%) were the facultatively predaceous mosquito Eretmapodites chrysogaster. The remaining 145 mosquitoes consisted of ten Aedes species. Aedes aegypti s.l. comprised 55.1% of these, followed by Ae. apicoargenteus (15.2%) and Ae. cozi (11.7%). Similar to East Africa, most (90%) of Ae. aegypti s.l. in the wet season were subspecies formosus.     

Heavy metal pollution negatively correlates with anuran species richness and distribution in south‐eastern Australia

Heavy metal pollution has likely played an important role in global biodiversity decline, but there remains a paucity of information concerning the effects of metals on amphibian diversity. This study assessed anuran species richness and distribution in relation to sediment metal content and water chemistry in wetlands located along the Merri Creek corridor in Victoria, south‐eastern Australia. Anurans were present in 60% (21/35) of study sites, with a total of six species detected: the eastern common froglet (Crinia signifera), the eastern sign‐bearing froglet (Crinia parinsignifera), the southern brown tree frog (Litoria ewingii), the growling grass frog (Litoria raniformis), the eastern banjo frog (Limnodynastes dumerilii) and the spotted marsh frog (Limnodynastes tasmaniensis). Mean species richness was 1.77 ± 0.32 per site, and species richness ranged from zero to six species per site. Across sites, species richness correlated negatively with sediment concentrations of six heavy metals: copper, nickel, lead, zinc, cadmium and mercury. Species richness also correlated negatively with wetland water electrical conductivity (a proxy for salinity) and concentrations of orthophosphate. Distributions of the three most commonly observed frog species (C. signifera, L. tasmaniensis and L. ewingii) were significantly negatively associated with the total level of metal contamination at individual sites. The study is the first to provide evidence for an association between metal contamination and anuran species richness and distribution in the southern hemisphere, adding to a small but growing body of evidence that heavy metal pollution has contributed to global amphibian decline.