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.

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.

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.

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.

TPC2 controls pigmentation by regulating melanosome pH and size.

Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

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.

Weaver ant role in cashew orchards in Vietnam.

Cashew (Anacardium occidentale L.) is a very important source of income for more than 200,000 farmer households in Vietnam. The present cashew productivity in Vietnam is low and unstable, and pest damage is partly responsible for this. Cashew farmers rely on pesticides to minimize the damage, resulting in adverse impacts on farm environment and farmers' health. Weaver ants (Oecophylla spp) are effective biocontrol agents of a range of cashew insect pests in several cashew-growing countries, and these ants are widely distributed in Vietnam. The aim of this study is to evaluate the potential of weaver ants in cashew orchards in Vietnam. Field surveys and field experiment were conducted in five cashew orchards from July 2006 to January 2008 in Binh Phuoc, Dong Nai, and Ba Ria Vung Tau provinces, Vietnam. Based on the field surveys, the most important pests that damage flushing foliar and floral shoots and young cashew fruits and nuts were mosquito bugs, brown shoot borers, blue shoot borers, and fruit-nut borers. The damage caused by each of these pests was significantly lower on trees with weaver ants compared with trees without the ants, showing that the ants were able to keep these pest damages under the control threshold. Regular monitoring of the field experiment showed that weaver ants were similar to insecticides for controlling mosquito bugs, blue shoot borers, fruit-nut borers, leaf rollers, and leaf miners. Aphids did not become major pests in plot with weaver ants. To manage insect pest assemblage in cashew orchards, an integrated pest management using weaver ants as a major component is discussed.

Cutaneous shedding in amphibians causes shifts in bacterial microbiomes.

Considerable research has focused on microbes on amphibian skin, as they act as the first line of defense against invading pathogens. This effort has generated substantial data on patterns across species, space, time, and ontogeny, alongside a growing list of beneficial antifungal symbionts. Though there is evidence of stability in amphibian skin microbial communities, there is also an indication that regular skin shedding reduces cultivable bacteria, with regrowth and recolonization in the period between sheds. This suggests that skin communities are in constant flux, and we lack an understanding of how the membership and structure of those communities are affected by shedding events. In this study, we conducted experiments on cane toads (Rhinella marina) to investigate the influence of shedding on skin microbiomes. We first used quantitative PCR to verify a positive correlation between bacterial loads and time in the days after shedding. We then resampled individuals over time to describe changes in community composition in the 38 h after shedding using amplicon sequencing. Similar to trends of bacterial loads, we found increases in alpha diversity over time after shedding, suggesting that shedding reduces bacterial diversity as it knocks down bacterial loads. During the 38-h period, community structure became similar to pre-shed communities in some individuals, but there was no consistent pattern in structural changes among individuals. In light of the amphibian chytridiomycosis pandemic, understanding how physiological events such as skin shedding affect beneficial bacteria and communities on amphibians would provide important insight into amphibian ecology.

Do weaver ant (Hymenoptera: Formicidae) marks affect mango internal quality and storage life?

In implementing the integrated pest management mango (Mangifera indica L.) program using weaver ants (Oecophylla smaragdina F.) as a major component in the Northern Territory of Australia, we received a number of questions from farmers and extension officers asking whether weaver ant marks reduce internal fruit quality and induce fruit rot, which is economically important. However, this issue has not been investigated. Soluble sugar content in fruits with and without weaver ant marks and observations on the storage of fruits with the ant marks were recorded in 2002 and 2003. The sugar contents were, in general, higher on the side of the fruit with a relatively large area of ant marks than on the side with a smaller area of ant marks, irrespective of whether fruits were exposed to the sun or not. The field experiment showed that sugar content was generally higher in the treatment with weaver ants plus soft chemicals than in the insecticide treatment. For the storage of fruits bearing weaver ant marks, no signs of diffuse rot from any ant marks were observed, but fruit skin without the ant marks developed a considerable number of dark open lenticels, most of which developed to rot marks and patches. These results suggest that weaver ant marks are positively correlated with internal fruit quality, do not induce fruit rot and can be used as an indicator of better fruit quality and safety.

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.

Condensation onto the skin as a means for water gain by tree frogs in tropical Australia.

Green tree frogs, Litoria caerulea, in the wet-dry tropics of northern Australia remain active during the dry season with apparently no available water and temperatures that approach their lower critical temperature. We hypothesized that this surprising activity might be because frogs that are cooled during nighttime activity gain water from condensation by returning to a warm, humid tree hollow. We measured the mass gained when a cool frog moved into either a natural or an artificial hollow. In both hollows, water condensed on cool L. caerulea, resulting in water gains of up to 0.93% of body mass. We estimated that the water gained was more than the water that would be lost to evaporation during activity. The use of condensation as a means for water gain may be a significant source of water uptake for species like L. caerulea that occur in areas where free water is unavailable over extended periods.

Removing the rubbish: frogs eliminate foreign objects from the body cavity through the bladder.

During the course of a telemetry study on three species of Australian frogs (Litoria caerulea, Litoria dahlii and Cyclorana australis), we found that many of the surgically implanted transmitters had migrated into the bladder. We subsequently implanted small beads into L. caerulea and they were expelled from the body in 10-23 days. Beads implanted into cane toads (Rhinella marina) to document the process were either expelled or were enveloped into the bladder. This appears to be a unique pathway for expulsion of foreign objects from the body, and suggests that caution should be employed in telemetry studies when interpreting the separation of some animals from their transmitters as a mortality event.

Changes in body fluids of the cocooning fossorial frog Cyclorana australis in a seasonally dry environment.

We investigated changes in the lymph (equivalent to plasma) and urine of the cocooning frog Cyclorana australis during the dry season in monsoonal northern Australia. Frogs in moist soil for two days were fully hydrated (lymph 220 mOsm kg(-1), urine 49 mOsm kg(-1)). From five weeks onwards the soil was dry (matric potential <-8000 kPa). Aestivating frogs at three and five months formed cocoons in shallow (<20 cm) burrows and retained bladder fluid (25-80% of standard mass). After three months, urine but not lymph osmolality was elevated. After five months, lymph (314 mOsm kg(-1)) and urine (294 mOsm kg(-1)) osmolality and urea concentrations were elevated. Urea was a major contributing osmolyte in urine and accumulated in lymph after five months. Lymph sodium concentration did not change with time, whereas potassium increased in urine after five months. Active animals had moderate lymph osmolality (252 mOsm kg(-1)), but urea concentrations remained low. Urine was highly variable in active frogs, suggesting that they tolerate variation in hydration state. Despite prolonged periods in dry soil, osmolality increase in C. australis was not severe. Aestivation in a cocoon facilitates survival in shallow burrows, but such a strategy may only be effective in environments with seasonally reliable rainfall.

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.

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.

Microclimate and limits to photosynthesis in a diverse community of hypolithic cyanobacteria in northern Australia.

Hypolithic microbes, primarily cyanobacteria, inhabit the highly specialized microhabitats under translucent rocks in extreme environments. Here we report findings from hypolithic cyanobacteria found under three types of translucent rocks (quartz, prehnite, agate) in a semiarid region of tropical Australia. We investigated the photosynthetic responses of the cyanobacterial communities to light, temperature and moisture in the laboratory, and we measured the microclimatic variables of temperature and soil moisture under rocks in the field over an annual cycle. We also used molecular techniques to explore the diversity of hypolithic cyanobacteria in this community and their phylogenetic relationships within the context of hypolithic cyanobacteria from other continents. Based on the laboratory experiments, photosynthetic activity required a minimum soil moisture of 15% (by mass). Peak photosynthetic activity occurred between approximately 8 degrees C and 42 degrees C, though some photosynthesis occurred between -1 degrees C and 51 degrees C. Maximum photosynthesis rates also occurred at light levels of approximately 150-550 micromol m(-2) s(-1). We used the field microclimatic data in conjunction with these measurements of photosynthetic efficiency to estimate the amount of time the hypolithic cyanobacteria could be photosynthetically active in the field. Based on these data, we estimated that conditions were appropriate for photosynthetic activity for approximately 942 h (approximately 75 days) during the year. The hypolithic cyanobacteria community under quartz, prehnite and agate rocks was quite diverse both within and between rock types. We identified 115 operational taxonomic units (OTUs), with each rock hosting 8-24 OTUs. A third of the cyanobacteria OTUs from northern Australia grouped with Chroococcidiopsis, a genus that has been identified from hypolithic and endolithic communities from the Gobi, Mojave, Atacama and Antarctic deserts. Several OTUs identified from northern Australia have not been reported to be associated with hypolithic communities previously.

Not just small, wet, and cold: effects of body size and skin resistance on thermoregulation and arboreality of frogs.

We used simulations from a biophysical model that integrates interlinked exchanges of energy and water between frogs and their environments to address questions about the limits to thermoregulation and about adaptations for arboreality. Body size and cutaneous resistance (Rc) both significantly affected body temperature (Tb) and the time to desiccate to 70% of standard mass (an ecologically relevant metric of desiccation). Cutaneous resistances < 25 s/cm allow basking frogs to elevate their Tb several degrees above ambient, but Rc above 25 had little additional effect on Tb. Small frogs (<10 g) are able to elevate their Tb above ambient while basking, even with small Rc. Large frogs must have greater skin resistances to be able to elevate body temperatures above ambient, yet large frogs take longer to desiccate to 70% of their standard mass. Frogs can avoid rapid desiccation with high Rc, a large body size, or some combination of these traits. Our literature survey indicates that frogs with a combination of Rc and body size that would result in long times to desiccate to 70% of standard mass tend to be arboreal, suggesting that those species may be selectively favored in a niche that often requires frogs to be away from water sources for extended periods of time.