Structural Characterization and Spatial Mapping of Tetrodotoxins in Australian Polyclads.

Tetrodotoxin (TTX) is a potent marine neurotoxin that occurs in several Australian phyla, including pufferfish, toadfish, gobies, and the blue-ringed octopus. These animals are partially immune, and TTX is known to bioaccumulate and subject to trophic transfer. As such, it could be more ubiquitously distributed in animals than is currently known. Flatworms of the order Polycladida are commonly occurring invertebrates in intertidal ecosystems and are especially diverse in Australian waters. While TTX has been identified in polyclads from Japan and New Zealand, Australian species have yet to be tested. In this study, several eastern Australian polyclad flatworm species from the suborders Cotylea and Acotylea were tested for TTX and analogs by HILIC-HRMS to understand the distribution of this toxin within these suborders. Herein, we report the detection of TTX and some known analogs in polyclad species, one of which is a pest to shellfish aquaculture. We also report, for the first time, the application of MALDI mass spectrometry imaging utilized to map TTX spatially within the intestinal system of polyclads. The identification of TTX and its analogs in Australian flatworms illustrates a broader range of toxic flatworms and highlights that analogs are important to consider when studying the distributions of toxins in animals.

Spawning and maturity traits of coexisting Platycephalidae (Platycephalus caeruleopunctatus, Platycephalus grandispinis, Platycephalus richardsoni) from southeast Australia.

Examination of the spawning and maturity traits of coexisting Platycephalus caeruleopunctatus, Platycephalus grandispinis and Platycephalus richardsoni (Pisces: Platycephalidae) in coastal waters of southeastern Australia identified many commonalities. Each species was gonochoristic, reproductively active for a prolonged period each year, displayed asynchronous oocyte development with indeterminate fecundity and thus likely spawned multiple times throughout each spawning season. Males of all three species matured at smaller total lengths and younger ages than females, with skewed sex ratios reflecting divergent growth characteristics between sexes. Reproductive isolation among species is likely maintained through behavioural and morphological factors as well as species-specific depth-related separation of reproductively active individuals. General similarities in the reproductive strategies of each species and with other sympatric coastal teleosts suggest similar ecological adaptations to a variable coastal environment.

Patterns of mother-embryo isotope fractionation in batoids vary within and between species.

Patterns of mother-embryo fractionation of 13 C and 15 N were assessed for their predictability across three species of batoids caught as by-catch in south-eastern Australia. Stable isotope analysis of 24 mothers and their litters revealed that isotope ratios of embryos were significantly different from their corresponding mothers and that the scale and direction of the difference varied within and across species. The range of variation across species was 3.5‰ for δ13 C and 4‰ for δ15 N, equivalent to a difference in trophic level. In one species (Urolophus paucimaculatus) litters could be significantly enriched or depleted in 13 C and 15 N relative to their mothers' isotope signatures. These results suggest that patterns of mother-embryo isotope fractionation vary within and between species and that these patterns may not be explained only by developmental mode. Contrasting patterns of fractionation between and within species make it difficult to adjust mother-embryo fractionation with broad-scale correction factors.

Trophic niche of Australian cownose rays (Rhinoptera neglecta) and whitespotted eagle rays (Aetobatus ocellatus) along the east coast of Australia.

Australian cownose rays (Rhinoptera neglecta) and whitespotted eagle rays (Aetobatus ocellatus) are large myliobatiform rays that co-occur off temperate eastern Australia. Here, we performed stable-isotope analyses (δ13 C, δ15 N and δ34 S) on fin clips of both species to gain insights into their trophic interactions and isotopic niches, and assess the effect of preservation (ethanol-stored versus frozen) on isotopic values of fin-clip tissue of R. neglecta. Linear mixed models identified species as the main factor contributing to variation among δ15 N and δ34 S values, and disc width for δ13 C. Bayesian ecological niche modelling indicated a 57.4% to 74.5% overlap of trophic niches, with the niche of R. neglecta being smaller and more constrained. Because values of δ13 C were similar between species, variation in isotopic niches were due to differences in δ15 N and δ34 S values. Linear mixed models failed to detect differences in isotopic values of ethanol-stored and frozen fin tissue of R. neglecta. This study provides the first examination of the trophic ecology of R. neglecta and the comparison of isotopic niche with A. ocellatus, which will facilitate future research into the trophic interactions of these species and aid better resource management.

Can the Dynamic Colouration and Patterning of Bluelined Goatfish (Mullidae; Upeneichthys lineatus) Be Perceived by Conspecifics?

Bluelined goatfish (Upeneichthys lineatus) exhibit dynamic body colour changes and transform rapidly from a pale, buff/white, horizontally banded pattern to a conspicuous, vertically striped, red pattern when foraging. This red pattern is potentially an important foraging signal for communication with conspecifics, provided that U. lineatus can detect and discriminate the pattern. Using both physiological and behavioural experiments, we first examined whether U. lineatus possess visual pigments with sensitivity to long ("red") wavelengths of light, and whether they can discriminate the colour red. Microspectrophotometric measurements of retinal photoreceptors showed that while U. lineatuslack visual pigments dedicated to the red part of the spectrum, their pigments likely confer some sensitivity in this spectral band. Behavioural colour discrimination experiments suggested that U. lineatuscan distinguish a red reward stimulus from a grey distractor stimulus of variable brightness. Furthermore, when presented with red stimuli of varying brightness they could mostly discriminate the darker and lighter reds from the grey distractor. We also obtained anatomical estimates of visual acuity, which suggest that U. lineatus can resolve the contrasting bands of conspecifics approximately 7 m away in clear waters. Finally, we measured the spectral reflectance of the red and white colouration on the goatfish body. Visual models suggest that U. lineatus can discriminate both chromatic and achromatic differences in body colouration where longer wavelength light is available. This study demonstrates that U. lineatus have the capacity for colour vision and can likely discriminate colours in the long-wavelength region of the spectrum where the red body pattern reflects light strongly. The ability to see red may therefore provide an advantage in recognising visual signals from conspecifics. This research furthers our understanding of how visual signals have co-evolved with visual abilities, and the role of visual communication in the marine environment.

Natural Products in Polyclad Flatworms.

Marine invertebrates are promising sources of novel bioactive secondary metabolites, and organisms like sponges, ascidians and nudibranchs are characterised by possessing potent defensive chemicals. Animals that possess chemical defences often advertise this fact with aposematic colouration that potential predators learn to avoid. One seemingly defenceless group that can present bright colouration patterns are flatworms of the order Polycladida. Although members of this group have typically been overlooked due to their solitary and benthic nature, recent studies have isolated the neurotoxin tetrodotoxin from these mesopredators. This review considers the potential of polyclads as potential sources of natural products and reviews what is known of the activity of the molecules found in these animals. Considering the ecology and diversity of polyclads, only a small number of species from both suborders of Polycladida, Acotylea and Cotylea have been investigated for natural products. As such, confirming assumptions as to which species are in any sense toxic or if the compounds they use are biosynthesised, accumulated from food or the product of symbiotic bacteria is difficult. However, further research into the group is suggested as these animals often display aposematic colouration and are known to prey on invertebrates rich in bioactive secondary metabolites.

Using cone beam CT scans to reveal headfirst ingestion and possible prey manipulation tactics in sawsharks.

Prey manipulation through headfirst ingestion is a common foraging tactic in predatory taxa. Sawsharks possess a toothed rostrum that is thought to assist in prey capture, but the process from prey contact to ingestion is unknown. This study provides evidence of headfirst ingestion and possible prey orientation in situ through the use of cone beam CT scans in the common sawshark (Pristiophorus cirratus). CT scans provide an efficient method for assessing ingestion and proposing plausible behavioural tactics for food manipulation in a species difficult to observe in the wild or maintain in captivity.

Seasonal and developmental diet shifts in sympatric and allopatric intertidal gobies determined by stomach content and stable isotope analysis.

Resource partitioning facilitates the coexistence of sympatric species through spatial, temporal and/or trophic strategies. Fishes living in the intertidal zone demonstrate highly adaptive plastic behaviour, including resource partitioning, through spatial and temporal shifts in diet and microhabitat. Although intertidal fish assemblages are influenced by inter- and intraspecific competition, few studies have compared the extent of resource partitioning between sympatric species in the context of trophic niche plasticity. Here we used complementary approaches, stomach content and stable isotope (δ13 C and δ15 N) analyses, to evaluate seasonal and developmental shifts in trophic niche position in two sympatric (Favonigobius lentiginosus and Bathygobius krefftii) and one allopatric (Bathygobius cocosensis) species of intertidal goby. The results indicate that resource partitioning in the two sympatric species varied with season, with almost no trophic niche overlap in summer to about ~30% overlap in winter. Also, evidence of dietary changes was found in B. cocosensis, which is likely associated with a shift in microhabitat and intraspecific competition. The findings highlight the temporal range of behavioural plasticity in trophic niche position of intertidal gobies, which likely has high adaptive value in the dynamic intertidal zone.

Resource use of great hammerhead sharks (Sphyrna mokarran) off eastern Australia.

Great hammerhead sharks Sphyrna mokarran are the largest member of Sphyrnidae, yet the roles of these large sharks in the food webs of coastal ecosystems are still poorly understood. Here we obtained samples of muscle, liver and vertebrae from large S. mokarran (234-383 cm total length; LT ) caught as by-catch off eastern Australia and used stable-isotope analyses of δ15 N, δ13 C and δ34 S to infer their resource use and any associated ontogenetic patterns. The results indicated large S. mokarran are apex predators primarily relying on other sharks and rays for their diet, with a preference for benthic resources such as Australian cownose rays Rhinoperon neglecta during the austral summer. Teleosts, cephalopods and crustaceans were not significant components of S. mokarran diets, though some conspecifics appeared to rely on more diverse resources over the austral summer. Ontogenetic shifts in resource use were detected but trajectories of the increases in trophic level varied among individuals. Most S. mokarran had non-linear trajectories in ontogenetic resource-use shifts implying size was not the main explanatory factor. Stable isotope values of δ13 C and δ34 S in muscle suggest S. mokarran span coastal, pelagic and benthic food webs in eastern Australia.

Genetic and historical evidence of common sawsharks Pristiophorus cirratus in the waters of southern Queensland.

In 2011, a male pristiophorid was caught by a prawn trawler north east of Cape Moreton, Queensland, Australia. Molecular analyses confirmed the specimen to be the common sawshark Pristiophorus cirratus. Historical catch data indicate the occurrence of the species in the region but this is the first verified record of P. cirratus occurring in the waters of southern Queensland. Together, these records extend the recognised northern limit of P. cirratus by c. 500 km, which suggests that further investigation of its distribution is warranted.

Barcoding and multi-locus phylogeography of the globally distributed calcareous tubeworm genus Hydroides Gunnerus, 1768 (Annelida, Polychaeta, Serpulidae).

Hydroides is a large and diverse group of calcareous tubeworms (Serpulidae, Annelida) recognised by a distinctive but variable two-tiered operculum. Despite considerable research using several species of Hydroides as models in ecological and biofouling studies, phylogenetic and biogeographic relationships within the genus are still poorly understood. Using combined mitochondrial (COI, cytochrome b) and nuclear (18S, 28S and ITS) gene markers for 284 individuals of 45 morphospecies of Hydroides, we investigated the global phylogenetic and biogeographic relationships within the genus. Phylogenetic topologies were well supported and indicated high genetic diversity within Hydroides, revealing potential cryptic species. Present results also include the first COI barcoding data enabling rapid and effective species identification of Hydroides on a global scale. Phylogenetic relationships within Hydroides were more concordant with geographical distributions than morphological similarity of their opercula. Molecular divergence estimates suggested the origin and subsequent diversification in the western Tethys Sea followed by a shift of the historical centre of diversity from the Indo-Mediterranean region to the central Indo-Pacific during the last 50 million years. Further studies on population genetics of species consisting of multiple lineages would provide a better understanding on the status of potential cryptic species. Furthermore, paleogeographic studies based on fossil Hydroides tubes would provide evidence to test this biogeographic hypothesis.

Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point.

Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH -0.3 (35% reduction) and ΔpH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification.

A fish can change its stripes: investigating the role of body colour and pattern in the bluelined goatfish.

Bluelined goatfish (Upeneichthys lineatus) rapidly change their body colour from a white horizontally banded pattern to a seemingly more conspicuous vertically banded red pattern, often when foraging. Given the apparent conspicuousness of the pattern to a range of observers, it seems unlikely that this colour change is used for camouflage and instead may be used for communication/signalling. Goatfish often drive multispecies associations, and it is possible that goatfish use this colour change as a foraging success signal to facilitate cooperation, increase food acquisition, and reduce predation risk through a 'safety in numbers' strategy. Using a novel approach, we deployed 3D model goatfish in different colour morphs-white without bands, white with black vertical bands, and white with red vertical bands-to determine whether the red colouration is an important component of the signal or if it is only the vertical banding pattern, regardless of colour, that fish respond to as an indicator of foraging success. Use of remote underwater video allowed us to obtain information without the influence of human observers on the communities and behaviours of other fish in response to these different colours exhibited by goatfish. We found that conspecifics were more abundant around the black- and red-banded model fish when compared with the white models. Conspecifics were also more likely to forage around the models than to pass or show attraction, but this was unaffected by model colour. No difference in the abundance and behaviour of associated heterospecifics around the different models was observed, perhaps due to the static nature of the models. Some species did, however, spend more time around the red- and black-banded fish, which suggests the change in colour may indicate benefits in addition to food resources. Overall, the results suggest that the body colour/pattern of U. lineatus is likely a signalling tool but further work is required to explore the benefits to both conspecifics and heterospecifics and to further determine the behavioural functions of rapid colour change in U. lineatus.

Dynamic colour change as a signalling tool in bluelined goatfish (Upeneicthtys lineatus).

Many animal species can rapidly change their body colouration and patterning, but often the ecological drivers of such changes are unknown. Here, we explored dynamic colour change in the bluelined goatfish, Upeneichthys lineatus, a temperate marine teleost species. Upeneichthus lineatus can change in a matter of seconds, from a uniform white colour to display prominent, vertical, dark red stripes. Initial observations suggested that rapid colour change in U. lineatus was associated with feeding and may act as a signal to both conspecifics and heterospecifics that are frequently observed to follow feeding goatfish. Field observations of the colour and behaviour of individual U. lineatus were collected to (1) document the repertoire of behaviours that U. lineatus displays and categorise associated colour patterns; (2) quantify the speed of dynamic colour change; (3) establish the context in which U. lineatus changes colour and pattern; and (4) test whether the behaviour of follower fishes is influenced by colour patterning or specific behaviours of the focal goatfish. We found that U. lineatus changed colouration from white to the red banded pattern in less than 10 s. The key driver of rapid colour change in U. lineatus was feeding, particularly when the fish fed with its head buried in sediment. Conspecific followers were most likely to be white in colour and adopt searching behaviour, regardless of the focal fish colour or behaviour. Other species of follower fish spent significantly more time following U. lineatus that were displaying dark red stripes when searching or eating, implying the red stripes may be an interspecific signalling mechanism. Our findings indicate that rapid colour change in teleost fish may be used for social communication and may provide U. lineatus with increased protection from predation when feeding via a safety-in-numbers approach.

It pays to cheat: tactical deception in a cephalopod social signalling system.

Signals in intraspecific communication should be inherently honest; otherwise the system is prone to collapse. Theory predicts, however, that honest signalling systems are susceptible to invasion by cheats, the extent of which is largely mediated by fear of reprisal. Cuttlefish facultatively change their shape and colour, an ability that evolved to avoid predators and capture prey. Here, we show that this ability is tactically employed by male mourning cuttlefish (Sepia plangon) to mislead conspecifics during courtship in a specific social context amenable to cheating 39 per cent of the time, while it was never employed in other social contexts. Males deceive rival males by displaying male courtship patterns to receptive females on one side of the body, and simultaneously displaying female patterns to a single rival male on the other, thus preventing the rival from disrupting courtship. The use of tactical deception in such a complex communication network indicates that sociality has played a key role in the cognitive evolution of cephalopods.

Biodiversity of intertidal marine flatworms (Polycladida, Platyhelminthes) in southeastern Australia.

Flatworms of the Order Polycladida are a group of free-living invertebrates found in a diversity of marine habitats, with over 800 species described worldwide. Marine flatworms are a conspicuous component of Australias marine fauna yet have received little attention. Less than 30 scientific articles have been published on Australian marine flatworms since 1855, of which only nine include species from southeastern Australia. Here, the biodiversity and distribution of species belonging to the Order Polycladida inhabiting intertidal rocky beaches in southeastern Australian waters were identified and analysed. Sampling was conducted at low tide along the coasts of New South Wales and Victoria. Collected samples were serially sectioned for comparative anatomical studies, and tissue was removed from each individual for molecular sequencing and analyses. Both nuclear and mitochondrial DNA sequences were obtained and used as an additional source of evidence for the description of new species as well as providing further insight into the phylogenetic relationships between them. A total of 20 species, six of which are new (e.g., Eulatocestus australis sp. nov.), and a new genus (Parabolia gen. nov.) have been described, as well as two new records for Australia (e.g., Stylochoplana clara Kato, 1937) have been identified increasing our knowledge of this important component of the Australian marine biota.

Hook-shaped enterolith and secondary cachexia in a free-living grey nurse shark (Carcharias taurus, Rafinesque 1810).

The carcass of a critically endangered, juvenile female grey nurse shark (Carcharias taurus, Rafinesque 1810) was recovered from a south-eastern Australian beach and subjected to necropsy. The 1.98-m-long shark exhibited advanced cachexia with its total weight (19.0 kg) and liver weight (0.37 kg) reduced by 60% and 89%, respectively, compared with a healthy individual of the same length. Marked tissue decomposition was evident preventing histopathology and identification of a definitive cause of death. At necropsy, the abdominal organs were abnormally displaced and showed marked reductions in size compared with a healthy individual of the same size. Importantly, a hook-shaped enterolith (HSE), with a rough surface and cream in colour, was found within the spiral valve of the intestine and is to the authors' knowledge, the first description of such in any marine animal. X-ray diffractometry showed that the HSE comprised the minerals monohydrocalcite (Ca[CO3].H2O; ~70 wt%) and struvite (Mg [NH4 ] [PO4 ]. [H2 O]6 ; ~30 wt%). A CT scan showed concentric lamellate concretions around a 7/o offset J-hook that formed the nidus of the HSE. Nylon fishing line attached to the hook exited the HSE and was evident in the abdominal cavity through a perforation in the intestinal wall where the posterior intestinal artery merges. The most parsimonious reconstruction of events leading to enterolithiasis and secondary cachexia in this shark was the consumption of a hooked fish and subsequent hook migration causing perforations of the cardiac stomach wall followed by the thin, muscular wall of the apposed, sub-adjacent intestine.

Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia.

Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to ninety per cent of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control agent. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of thirty-six RNA libraries representing ten species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified eighteen vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further twenty-seven that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fish sampled here, suggesting that there is little virus transmission from common carp to native fish species, despite co-existing for over fifty years. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.

Erratum: Virome composition in marine fish revealed by meta-transcriptomics.

[This corrects the article DOI: 10.1093/ve/veab005.].