Migratory Shorebird Gut Microbes are not Associated with Bivalve Prey in Monsoon Tropical Australia.

Migratory animals can carry symbionts over long distances. While well-studied for parasite and pathogen transmission, less is known about use of this route by other symbiotic taxa, particularly those non-pathogenic. Here we ask the question of whether gut bacteria can be spread between continents by long-distance bird migration, although gut microbiomes in birds may not be as stable or persistent as those of non-volant animals. We used amplicon sequencing of both bacterial 16S rRNA gene and Vibrio-centric hsp60 gene to determine whether the faecal bacteria of migratory great knots (Calidris tenuirostris) also occur in their main food source in Northern Australia or in nearby sand, comparing samples before and after the birds' long-distance migration. Our data suggest that there is little connectivity among the bacterial microbiomes, except in the bivalve prey. Our results are consistent with previous studies finding that bird faecal microbiomes were not host-specific and contrast with those showing an influence of diet on bird faecal bacteria. We also found little connectivity among Vibrio spp. However, although faecal sample sizes were small, the dominance of different individual Vibrio spp. suggests that they may have been well-established in knot guts and thus capable of moving with them on migration. We suggest that the physiological impacts of a long-distance migration may have caused shifts in the phyla comprising great knot faecal communities.

Geographic variation in bacterial assemblages on cane toad skin is influenced more by local environments than by evolved changes in host traits.

Bacterial assemblages on amphibian skin may play an important role in protecting hosts against infection. In hosts that occur over a range of environments, geographic variation in composition of bacterial assemblages might be due to direct effects of local factors and/or to evolved characteristics of the host. Invasive cane toads (Rhinella marina) are an ideal candidate to evaluate environmental and genetic mechanisms, because toads have evolved major shifts in physiology, morphology, and behavior during their brief history in Australia. We used samples from free-ranging toads to quantify site-level differences in bacterial assemblages and a common-garden experiment to see if those differences disappeared when toads were raised under standardised conditions at one site. The large differences in bacterial communities on toads from different regions were not seen in offspring raised in a common environment. Relaxing bacterial clustering to operational taxonomic units in place of amplicon sequence variants likewise revealed high similarity among bacterial assemblages on toads in the common-garden study, and with free-ranging toads captured nearby. Thus, the marked geographic divergence in bacterial assemblages on wild-caught cane toads across their Australian invasion appears to result primarily from local environmental effects rather than evolved shifts in the host.

Microbial communities associated with mounds of the Orange-footed scrubfowl Megapodius reinwardt.

Megapodius reinwardt, the orange-footed scrubfowl, belongs to a small family of birds that inhabits the Indo-Australian region. Megapodes are unique in incubating their eggs in mounds using heat from microbial decomposition of organic materials and solar radiation. Little is known about the microorganisms involved in the decomposition of organic matter in mounds. To determine the source of microbes in the mounds, we used 16S and 18S rRNA gene sequencing to characterize the microbial communities of mound soil, adjacent soil and scrubfowl faeces. We found that the microbial communities of scrubfowl faeces were substantially different from those of the mounds and surrounding soils, suggesting that scrubfowls probably do not use their faeces to inoculate their mounds although a few microbial sequence variants were present in both faeces and mound samples. Further, the mound microbial community structure was significantly different to the adjacent soils. For example, mounds had a high relative abundance of sequence variants belonging to Thermomonosporaceae, a thermophilic soil bacteria family able to degrade cellulose from plant residues. It is not clear whether members of Thermomonosporaceae disproportionately contribute to the generation of heat in the mound, or whether they simply thrive in the warm mound environment created by the metabolic activity of the mound microbial community. The lack of clarity in the literature between designations of heat-producing (thermogenic) and heat-thriving (thermophilic) microbes poses a challenge to understanding the role of specific bacteria and fungi in incubation.

Physiological control of water exchange in anurans.

Research on water exchange in frogs has historically assumed that blood osmotic potential drives water exchange between a frog and its environment, but here we show that the "seat patch" (the primary site of water exchange in many anurans), or other sites of cutaneous water uptake, act as an anatomic "compartment" with a water potential controlled separately from water potential of the blood, and the water potential of that compartment can be the driver of water exchange between the animal and its environment. We studied six frog species (Xenopus laevis, Rana pipiens, R. catesbeiana, Bufo boreas, Pseudacris cadaverina, and P. regilla) differing in ecological relationships to environmental water. We inferred the water potentials of seat patches from water exchanges by frogs in sucrose solutions ranging in water potential from 0 to 1000-kPa. Terrestrial and arboreal species had seat patch water potentials that were more negative than the water potentials of more aquatic species, and their seat patch water potentials were similar to the water potential of their blood, but the water potentials of venters of the more aquatic species were different from (and less negative than) the water potentials of their blood. These findings indicate that there are physiological mechanisms among frog species that can be used to control water potential at the sites of cutaneous water uptake, and that some frogs may be able to adjust the hydric conductance of their skin when they are absorbing water from very dilute solutions. Largely unexplored mechanisms involving aquaporins are likely responsible for adjustments in hydric conductance, which in turn, allow control of water potential at sites of cutaneous water uptake among species differing in ecological habit and the observed disequilibrium between sites of cutaneous water uptake and blood water potential in more aquatic species.

Environmental resistance and habitat quality influence dispersal of the saltwater crocodile.

Landscape genetics commonly focuses on the effects of environmental resistance on animal dispersal patterns, but there is an emerging focus on testing environmental effects on emigration and settlement choices. In this study, we used landscape genetics approaches to quantify dispersal patterns in the world's largest crocodilian, the saltwater crocodile (Crocodylus porosus), and demonstrated environmental influences on three processes that comprise dispersal: emigration, movement and settlement. We found that both environmental resistance and properties of the source and destination catchments (proportion of breeding habitat) were important factors influencing observed dispersal events. Our habitat quality variables related to hypotheses about resource competition and represented the ratio of breeding habitat (which limits carrying capacity), suggesting that competition for habitat influences emigration and settlement choices, together with the strong effect of environmental resistance to movement (where high-quality habitat was associated with greatest environmental permeability). Approximately 42% of crocodiles were migrants from populations other than their sampling locations and some outstandingly productive populations had a much higher proportion of emigration rather than immigration. The distance most commonly travelled between source and destination was 150-200 km although a few travelled much longer distances, up to 600-700 km. Given the extensive dispersal range, individual catchments or hydrographic regions that combine two or three adjacent catchments are an appropriate scale for population management.

Pearl Oyster Bacterial Community Structure Is Governed by Location and Tissue-Type, but Vibrio Species Are Shared Among Oyster Tissues.

Diseases of bivalves of aquacultural importance, including the valuable Australian silver-lipped pearl oyster (Pinctada maxima), have been increasing in frequency and severity. The bivalve microbiome is linked to health and disease dynamics, particularly in oysters, with putative pathogens within the Vibrio genus commonly implicated in oyster diseases. Previous studies have been biased toward the Pacific oyster because of its global dominance in oyster aquaculture, while much less is known about the microbiome of P. maxima. We sought to address this knowledge gap by characterizing the P. maxima bacterial community, and we hypothesized that bacterial community composition, and specifically the occurrence of Vibrio, will vary according to the sampled microenvironment. We also predicted that the inside shell swab bacterial composition could represent a source of microbial spillover biofilm into the solid pearl oyster tissues, thus providing a useful predictive sampling environment. We found that there was significant heterogeneity in bacterial composition between different pearl oyster tissues, which is consistent with patterns reported in other bivalve species and supports the hypothesis that each tissue type represents a unique microenvironment for bacterial colonization. We suggest that, based on the strong effect of tissue-type on the pearl oyster bacterial community, future studies should apply caution when attempting to compare microbial patterns from different locations, and when searching for disease agents. The lack of association with water at each farm also supported the unique nature of the microbial communities in oyster tissues. In contrast to the whole bacterial community, there was no significant difference in the Vibrio community among tissue types nor location. These results suggest that Vibrio species are shared among different pearl oyster tissues. In particular, the similarity between the haemolymph, inside shell and solid tissues, suggests that the haemolymph and inside shell environment is a source of microbial spillover into the oyster tissues, and a potentially useful tool for non-destructive routine disease testing and early warning surveillance. These data provide important foundational information for future studies identifying the factors that drive microbial assembly in a valuable aquaculture species.

First line of defence: Skin microbiota may protect anurans from infective larval lungworms.

Functional roles of the rich microbiota of the skin are not fully understood, but include protection against microbial diseases and other environmental challenges. In experimental studies, we show that reducing the microbiota from cane toad (Rhinella marina) skin by gently wiping with absorptive gauze resulted in threefold higher rates of infection by lungworms (Rhabdias pseudosphaerocephala) following standardised exposure to infective skin-penetrating larvae. Higher concentrations of microbial DNA were associated with lower rates of lungworm entry. Our data suggest that microbial activity on the anuran skin comprises an important line of defence against attack by macroparasites as well as by fungi and other microbes.

Skin resistance to water gain and loss has changed in cane toads (Rhinella marina) during their Australian invasion.

The water-permeable skin of amphibians renders them highly sensitive to climatic conditions, and interspecific correlations between environmental moisture levels and rates of water exchange across the skin suggest that natural selection adapts hydroregulatory mechanisms to local challenges. How quickly can such mechanisms shift when a species encounters novel moisture regimes? Cutaneous resistance to water loss and gain in wild-caught cane toads (Rhinella marina) from Brazil, USA (Hawai'i) and Australia exhibited strong geographic variation. Cutaneous resistance was low in native-range (Brazilian) toads and in Hawai'ian populations (where toads were introduced in 1932) but significantly higher in toads from eastern Australia (where toads were introduced in 1935). Toads from recently invaded areas in western Australia exhibited cutaneous resistance to water loss similar to the native-range populations, possibly because toads are restricted to moist sites within this highly arid landscape. Rates of rehydration exhibited significant but less extreme geographic variation, being higher in the native range than in invaded regions. Thus, in less than a century, cane toads invading areas that impose different climatic challenges have diverged in the capacity for hydroregulation.

Disease Exposure and Antifungal Bacteria on Skin of Invasive Cane Toads, Australia.

Cane toads, an invasive species in Australia, are resistant to fungal pathogens affecting frogs worldwide (Batrachochytrium dendrobatidis). From toad skin swabs, we detected higher proportions of bacteria with antifungal properties in Queensland, where toad and pathogen distributions overlap, than in other sites. This finding suggests that site-specific pathogen pressures help shape skin microbial communities.

Dehydration enhances innate immunity in a semiaquatic snake from the wet-dry tropics.

Dehydration is considered a physiological challenge, and many organisms live in environments that undergo periods of reduced water availability that can lead to dehydration. Recent studies have found a positive relationship between dehydration and innate immune function in animals adapted to xeric or semixeric environments. To explore the generality of this relationship, we examined the impact of dehydration on innate immune performance in water pythons (Liasis fuscus), a semiaquatic snake from the wet-dry tropics of Australia. We collected blood samples from male and female water pythons held in the laboratory without food and water for 4 weeks. We also collected blood from free-ranging snakes throughout the Austral dry-season. We evaluated plasma osmolality and innate immune function (agglutination, lysis, and bacterial-killing ability) and found that increased osmolality, whether manipulated in the laboratory or as a result of natural water limitation, resulted in enhanced aspects of innate immune performance. Counter-intuitively, snakes in the wild became more hydrated as the dry season progressed, suggesting the dehydrated snakes move to water sources periodically to rehydrate. Comparing our data with those from previous studies, we suspect species divergence in the level of dehydration (i.e., hyperosmolality) that triggers enhanced immune capabilities.

Cane toads (Rhinella marina) rely on water access, not drought tolerance, to invade xeric Australian environments.

The invasion of habitats with novel environmental challenges may require physiological tolerances not seen in conspecifics from the native range. We used a combination of field and laboratory-based experiments to assess physiological tolerance to limited water access at four sites distributed across the historical invasion path of cane toads (Rhinella marina) in Australia that, from east to west, alternated between mesic and seasonally xeric habitats. Toads from all locations were well hydrated at the time of capture. However, experimental dehydration caused greater mass loss, higher plasma osmolality, and inhibition of lytic ability in toads from xeric compared to mesic locations. These results suggest somewhat surprisingly that toads from xeric environments are physiologically more vulnerable to water loss. In contrast, bactericidal ability was not sensitive to hydric state and was greater in toads from eastern (long-colonized) areas. Similar patterns in lytic ability in hydrated toads and agglutination ability in wild toads suggest that toads along the invasion front face a tradeoff between enhanced dispersal ability and physiological responses to dehydration. The ability of this invasive species to spread into drier environments may be underpinned by a combination of phenotypic plasticity and evolved (heritable) traits.

Skin bacterial diversity is higher on lizards than sympatric frogs in tropical Australia.

Animal skin acts as a barrier between the organism and its environment and provides the first line of defense against invading pathogens. Thus, skin surfaces harbor communities of microbes that are interacting with both the host and its environment. Amphibian skin bacteria form distinct communities closely tied to their host species, but few studies have compared bacterial communities between amphibians and other, non-amphibian sympatric animals. Notably, skin microbes on reptiles have gained little attention. We used next-generation sequencing technology to describe bacterial communities on the skin of three lizard species and compared them to bacteria on six cohabiting frog species in the Northern Territory of Australia. We found bacterial communities had higher richness and diversity on lizards than frogs, with different community composition between reptiles and amphibians and among species. Core bacteria on the three lizard species overlapped by over 100 operational taxonomic units. The bacterial communities were similar within species of frogs and lizards, but the communities tended to be more similar between lizard species than between frog species and when comparing lizards with frogs. The diverse bacteria found on lizards invites further questions on how and how well reptiles interact with microorganisms through their scaly skin.

Ecological patterns in the skin microbiota of frogs from tropical Australia.

The microbiota of frog skin can play an important role in protecting against diseases and parasites. The frog skin microbial community represents a complex mix of microbes that are promoted by the chemical environment of the frog skin and influenced by the animal's immediate past environment. The microbial communities of six species of frogs sampled from the campus of Charles Darwin University (CDU) were more similar within species than between species. The microbiota of the introduced cane toad (Rhinella marina) was most dissimilar among the species. Pairwise comparisons showed that the microbial communities of each species were different, except for the terrestrial Litoria nasuta and the arboreal L. rothii. The microbial communities of the six species were not related to ecological habit (arboreal or terrestrial), and neither was the alpha diversity of the microbes. The core microbes (defined as being on ≥90% of individuals of a species or group) were significantly different among all species, although 89 microbial operational taxonomic units (OTUs) were core microbes for all six species at CDU. Two species, Rhinella marina and Litoria rothii, were sampled at additional sites approximately 10 and 30 km from CDU. The microbial communities and the core OTU composition were different among the sites, but there were nevertheless 194 (R. marina) and 181 (L. rothii) core OTUs present at all three sites. Thus, the core microbiota varied with respect to geographic range and sample size.

The thermal dependency of locomotor performance evolves rapidly within an invasive species.

Biological invasions can stimulate rapid shifts in organismal performance, via both plasticity and adaptation. We can distinguish between these two proximate mechanisms by rearing offspring from populations under identical conditions and measuring their locomotor abilities in standardized trials. We collected adult cane toads (Rhinella marina) from invasive populations that inhabit regions of Australia with different climatic conditions. We bred those toads and raised their offspring under common-garden conditions before testing their locomotor performance. At high (but not low) temperatures, offspring of individuals from a hotter location (northwestern Australia) outperformed offspring of conspecifics from a cooler location (northeastern Australia). This disparity indicates that, within less than 100 years, thermal performance in cane toads has adapted to the novel abiotic challenges that cane toads have encountered during their invasion of tropical Australia.

Differences in structure of northern Australian hypolithic communities according to location, rock type, and gross morphology.

Hypolithic communities (under translucent rocks) were compared between a semi-arid site (Wave Hill) and a site with considerably higher rainfall (Lake Bennett) to test the hypothesis that the communities at the higher rainfall site would be more diverse. A total of 153 cyanobacteria operational taxonomic units (OTUs) were identified, and only 50 of those were found at both sites. Of these, only two were core OTUs, as defined as being present in ≥90% of samples, highlighting the extreme differences in the cyanobacterial communities at the two sites. At Wave Hill, we compared the composition of the cyanobacterial components under two different rock types (quartz and prehnite) to determine if the different minerals would result in different hypolithic communities, but no differences were found. Of the 42 core OTUs found at Wave Hill, 22 (52%) were shared between the two rock types. As hypothesised, the diversity of both cyanobacteria and eukaryotes in the hypolithic communities was significantly higher at Lake Bennett. Some hypolithic communities were thin and tightly adhered to the rock surface, but others were thicker and could be peeled off the rock in sheets. However, the two types were not significantly different in OTU composition. Metazoans, primarily nematodes, were ubiquitous, raising the possibility that nematodes may act as vectors to transport the components of hypolithic communities from rock to rock as a mechanism of colonization.

Spatial patterns of hypolithic cyanobacterial diversity in Northern Australia.

Photosynthetic microbial communities under translucent rocks (hypolithic) are found in many arid regions. At the global scale, there has been little intercontinental gene flow, and at a local scale, microbial composition is related to fine-scale features of the rocks and their environment. Few studies have investigated patterns of hypolithic community composition at intermediate distances. We examined hypolithic cyanobacterial diversity in semi-arid Australia along a 10-km transect by sampling six rocks from four adjacent 1 m2 quadrats ("distance zero") and from additional quadrats at 10, 100, 1,000, and 10,000 m to test the hypothesis that diversity would increase with the number of rocks sampled and distance. A total of 3,108 cyanobacterial operational taxonomic units (OTUs) were detected. Most were neither widespread nor abundant. The few that were widespread tended to be abundant. There was no difference in the community composition between the four sites at distance zero, but the samples 10 m away were significantly different, as were those at all other distances compared to distance zero. Many additional OTUs were recorded with increasing distance up to 100 m. These patterns of distribution are consistent with a colonization model involving dispersal from rock to rock. Our results indicate that distance was a significant factor that can be confounded by interrock differences. Most diversity was represented in the first 100 m of the transect, with an additional 1.5% of the total diversity added by the sample at 1 km, but only 0.2% added with the addition of the 10-km site.

Locomotor performance of cane toads differs between native-range and invasive populations.

Invasive species provide a robust opportunity to evaluate how animals deal with novel environmental challenges. Shifts in locomotor performance-and thus the ability to disperse-(and especially, the degree to which it is constrained by thermal and hydric extremes) are of special importance, because they might affect the rate that an invader can spread. We studied cane toads (Rhinella marina) across a broad geographical range: two populations within the species' native range in Brazil, two invasive populations on the island of Hawai'i and eight invasive populations encompassing the eastern, western and southern limits of the toad invasion in Australia. A toad's locomotor performance on a circular raceway was strongly affected by both its temperature and its hydration state, but the nature and magnitude of those constraints differed across populations. In their native range, cane toads exhibited relatively low performance (even under optimal test conditions) and a rapid decrease in performance at lower temperatures and hydration levels. At the other extreme, performance was high in toads from southern Australia, and virtually unaffected by desiccation. Hawai'ian toads broadly resembled their Brazilian conspecifics, plausibly reflecting similar climatic conditions. The invasion of Australia has been accompanied by a dramatic enhancement in the toads' locomotor abilities, and (in some populations) by an ability to maintain locomotor performance even when the animal is cold and/or dehydrated. The geographical divergences in performance among cane toad populations graphically attest to the adaptability of invasive species in the face of novel abiotic challenges.

Frogs host faecal bacteria typically associated with humans.

Tree frogs commonly access drinking water tanks; this may have human health implications. Although amphibians might not be expected to host mammalian faecal indicator bacteria (FIB), it is possible that they may have human FIB on their skin after exposure to human waste. We collected faeces and skin wash from green tree frogs (Litoria caerulea) from a natural environment, a suburban site, and a suburban site near a creek occasionally contaminated with sewage effluent. We used molecular techniques to test for FIB that are routinely used to indicate human faecal contamination. Enterococci colonies were isolated from both faecal and skin wash samples, and specific markers (Enterococcus faecium and Bacteroides thetaiotaomicron) were found in frog faeces, demonstrating that these markers are not human- or mammalian-specific. Bacteroides thetaiotaomicron was detected in frogs from both natural and urban sites, but E. faecium was only associated with the sewage impacted site.

Temperature rise and parasitic infection interact to increase the impact of an invasive species.

Invasive species often detrimentally impact native biota, e.g. through predation, but predicting such impacts is difficult due to multiple and perhaps interacting abiotic and biotic context dependencies. Higher mean and peak temperatures, together with parasites, might influence the impact of predatory invasive host species additively, synergistically or antagonistically. Here, we apply the comparative functional response methodology (relationship between resource consumption rate and resource supply) in one experiment and conduct a second scaled-up mesocosm experiment to assess any differential predatory impacts of the freshwater invasive amphipod Gammarus pulex, when uninfected and infected with the acanthocephalan Echinorhynchus truttae, at three temperatures representative of current and future climate. Individual G. pulex showed Type II predatory functional responses. In both experiments, infection was associated with higher maximum feeding rates, which also increased with increasing temperatures. Additionally, infection interacted with higher temperatures to synergistically elevate functional responses and feeding rates. Parasitic infection also generally increased Q10 values. We thus suggest that the differential metabolic responses of the host and parasite to increasing temperatures drives the synergy between infection and temperature, elevating feeding rates and thus enhancing the ecological impact of the invader.