Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish, Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.

Seasonal and diet comparisons of the diets of four dominant fish species within the main channel and flood-zone of a small intermittently open estuary in south-eastern Australia

The diets of four highly-abundant, dominant fish species within the Surrey River, a small intermittently open estuary in south-east Australia, were examined from specimens collected between July 2004 and June 2005. These four, similar-sized species (Atherinosoma microstoma, Galaxias maculatus, Philypnodon grandiceps and Pseudogobius olorum) have limited ability to spatially segregate along the length of the estuary owing to its small size relative to other estuarine habitats. All four species fed on a variety of prey items including crustaceans, insects and detritus. Despite this parity, the four species were demonstrated to occupy differing dietary niches that were concluded to be responsible for reducing interspecific feeding competition. Seasonal variations in the diets were observed for A. microstoma and Philypnodon grandiceps, with these species also exhibiting contrasting diel feeding behaviours. The closure of the estuary mouth led to the flooding of its margins, resulting in an increase in the size of the estuary and providing alternative food resources for the fish to exploit. It appears the inundation of the flood-zone facilitated further significant divergence in the diets of the fish and is likely to be of high ecological value to the estuary.

Differential effects of arginine, glutamate and phosphoarginine on Ca2+-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.

Functional morphology of the gastric mills of carnivorous, omnivorous, and herbivorous land crabs

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth.

Control of ovarian development in the yabby (Cherax destructor)

A study under controlled conditions of ovarian development and rematuration in the yabby (Cherax destructot) was undertaken. The purpose of the study was to improve fundamental understanding of the reproductive biology of the species and provide a basis for application to hatchery management in culture. A review was made of the current status of yabby culture in Australia and the present understanding of reproductive biology of decapod Crustacea. The review emphasised factors controlling several aspects of ovarian development, in particular the processes of vitellogenesis. The subsequent study was designed within the context of current hatchery practice and was based on existing knowledge of decapod reproduction, The sexual differentiation of the yabby after hatching was investigated by serial histological sections, and experiments were carried out to investigate the possibility of sex reversal of males. Most of this Investigation was concerned with removing the influence of the androgenic gland in directing male development, with the intent of observing the development of the elementary gonadal tissue into ovary. It was found that in contrast to other crustacean species, the sex of the yabby becomes fixed before the development of external secondary sexual characteristics, and before the androgenic gland can be discerned. Ovarian tissue developed in females at less than 8 weeks after hatching. A preliminary examination was undertaken for feminising parasites in gonadal tissue of a hermaphrodite yabby. Investigation of the ovary after spawning demonstrated that whilst the female was held under constant conditions of temperature and photoperiod, little rematuration occurred. Except for generation of previtellogenic oocytes during the first two days, the gonaciosomatic index remained low for up to 5 months after spawning. If the temperature of the female was reduced to 10°C and maintained constant, the previtellogenic oocytes were partially resorbed over a three week period. Rematuration then commenced, albeit at a low rate because of the reduced temperature, A method for standardising gonadosomatic indices was developed which took into account differences in hepatopancreatic nutrient reserves of individuals and loss of one or more appendages. This part of the study also considered constraints to rematuration and developed a method of accounting for differences in the ability of females to remature after spawning. Experiments were carried out to investigate the effect of crowding and temperature manipulation on initiating ovarian rematuration and to determine the rate of rematuration at 22°C once initiated. The duration of low temperature had no effect on rematuration; an overnight cooling was sufficient to initiate the process, Rematuration to the end of stage 2 vltellogenesis was substantially complete within 10 days. Crowding of females suppressed rematuration, but less than ideal water quality was not found to have any effect. The presence of a male initiated rematuration at a similar rate, but also led to stage 3 vitetlogenesis and spawning. A study was made of the pheromonal influence of the male through water borne factors without success. Rematuration could not be induced in ovigerous females. The literature review indicated that ovarian rematuration was under the control of an ovary stimulating hormone produced by the thoracic nerve ganglia. Attempts were therefore made to stimulate ovarian rematuration by incorporating the thoracic nerve into the diet of females. Attempts were also made to induce the release of ovary stimulating hormone from the thoracic nerve with 5-hydroxytryptamine, and also with octopamine. No effects were found, but a significant difference between the neurophysiology of the yabby and northern hemisphere crayfish was observed, and the implications of this finding are discussed. The study did not produce any conclusive evidence of an ovary stimulating hormone for the yabby. A model of ovarian rematuration which collects the findings of the experimental investigations was developed, and was used to suggest a hatchery broodstock management protocol. This model differs from existing models in that rematuration triggers and nutritional status are considered.

n-3 Polyunsaturated fatty acid-rich vegetable oils and blends

α-Linseed, camelina. perilla, and echium oils are n-3 C₁₈ polyunsaturated fatty acid (PUFA)-rich vegetable oil sources viewed as favorable replacements to fish oil in aquaculture feed (aquafeed) production in consideration of their high (α-linolenic acid (ALA, 18:3n-3) and/or stearidonic acid (SDA, 18:4n-3) contents and potential for subsequent bioconversion to n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in farmed aquatic species. While the total production of these oils is currently low in comparison with that of other terrestrial oil sources, their distinct fatty acid composition and high n-3 to n-6 ratio deliver a unique substitute to fish oil in aquafeeds, presently unparalleled in other alternative terrestrial oil sources. The dietary inclusion of these oil sources has therefore attracted significant research attention, resulting in a multitude of investigations across a broad range of aquatic species (finfish and crustaceans). Generally, providing that the essential fatty acid (EFA) requirements of the species under investigation were met and an adequate level of fish meal was present in the diet, it was found possible to replace 100% and 60-70% of the dietary fish oil component for freshwater and marine species, respectively, with minimal impact on growth performance indices. However, the substitution of fish oil with n-3-rich vegetable oils and/or vegetable oil blends resulted in substantially reduced concentrations of health-promoting eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in the edible portion of the farmed species. This chapter provides an overview of the use of n-3 PUFA-rich vegetable oils and/or vegetable oil blends for use in aquafeeds. In particular, key aspects of oil production, processing, and refinement will be presented, and individual differences pertaining to the physical, chemical, and nutritional characteristics of the oil types will be highlighted. Following on from this, a summary of the key findings relevant to n-3 PUFA-rich vegetable oil inclusion in aquafeeds will be discussed, with particular emphasis placed on growth performance and nutritional modification.

Fish oil replacement in starter, grow-out, and finishing feeds for farmed aquatic animals

As aquaculture production continues to grow, there will be an increased use of lipid resources (oils and fats) alternative to fish oil for feed production. The potential for the use of these alternatives varies depending on the feeds in which they are included according to the production phase of the animals to which they are being fed. In starter feeds, where rapid growth, high survival, and normal development are critical priorities, there will remain a need for the use of lipid resources high in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Fish in this starter phase have a critical requirement for the n-3 LC-PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and fish oils remain the only cost-effective source of these nutrients in the volumes required. However, the greatest demand for lipids is in those diets for the grow-out phase. Most studies on alternative lipid use with animals in this part of the production phase show positive outcomes, in that there are few studies where all the added fish oil cannot be replaced. There are some species, however, where potential replacement levels are suggested to be more conservative, and a general substitution level in this production phase of 75% has been suggested. One of the key effects noted across the grow-out phase is that all alternatives affect the flesh fatty acid characteristics by reducing the level of n-3 LC-PUFA. This issue has provoked the concept of finisher diets, whereby a high n-3 LC-PUFA content diet is fed in order to restore the desired meat fatty acid profiles. Studies examining this concept have found that the tissue triacylglycerol fatty acids were greatly modified and responded in a simple dilution process to the added oil fatty acid composition, whereas the fatty acids of tissue phospholipids were less influenced by dietary fatty acid makeup.

The last piece in the cellulase puzzle : the characterisation of β-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis

A 160 kDa enzyme with β-glucosidase activity was purified from the midgut Gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although β-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose<cellotriose=cellotetraose). It was able to synthesise some cellotetraose by the transglycosylation of smaller substrates – another common feature of glucohydrolases. The interaction between the glucohydrolase described here and the endo-β-1,4-glucanases described previously for G. natalis provides a complete model for cellulose hydrolysis in crustaceans and possibly in other invertebrates. After mechanical fragmentation by the gastric mill, multiple endo-β-1,4-glucanases would initially cleave β-1,4-glycosidic bonds within native cellulose, releasing small oligomers, including cellobiose, cellotriose and cellotetraose. The glucohydrolase would then attach to these oligomers, progressively releasing glucose. The glucohydrolase might also attach directly to crystalline cellulose to release glucose from free chain ends. This two-enzyme system differs from the traditional model, which suggests that total cellulose hydrolysis requires the presence an endo-β-1,4-glucanse, a cellobiohydrolase and a β-glucosidase

Early Triassic (early Olenekian) life in the interior of East Gondwana: Mixed marine-terrestrial biota from the Kockatea Shale, Western Australia

A new terrestrial-marine assemblage from the lower beds of a thin outcrop section of the Kockatea Shale in the northern Perth Basin, Western Australia, contains a range of fossil groups, most of which are rare or poorly known from the Lower Triassic of the region. To date, the collection includes spinose acritarchs, organic-cemented agglutinated foraminifera, lingulids, minute bivalves and gastropods, ammonoids, spinicaudatans, insects, austriocaridid crustaceans, actinopterygians, a temnospondyl-like mandible, plant remains, and spores and pollen. Of these groups, the insects, crustaceans and macroplant remains are recorded for the first time from this unit. Palynomorphs permit correlation to nearby sections where conodonts indicate an early Olenekian (Smithian) age. The locality likely represents the margin of an Early Triassic shallow interior sea with variable estuarine-like water conditions, at the southwestern end of an elongate embayment within the East Gondwana interior rift-sag system preserved along the Western Australian margin. Monospecific spinose acritarch assemblages intertwined with amorphous organic matter may represent phytoplankton blooms that accumulated as mats, and suggest potentially eutrophic surface waters. The assemblage represents a mixure of marine and terrestrial taxa, suggesting variations in water conditions or that fresh/brackish-water and terrestrial organisms were transported from adjacent biotopes. Some of the lower dark shaly beds are dominated by spinicaudatans, likely indicating periods when the depositional water body was ephemeral, isolated, or subjected to other difficult environmental conditions. The biota of the Kockatea Shale is insufficiently known to estimate biotic diversity and relationships of individual taxa to their Permian progenitors and Triassic successors, but provides a glimpse into a coastal-zone from the interior of eastern Gondwana. Specialist collecting is needed to clarify the taxonomy of many groups, and comparisons to other Lower Triassic sites are required to provide insights into the pattern of biotic decline and recovery at the end-Permian crisis.

Changes in the nutritional composition of captive early-mid stage Panulirus ornatus phyllosoma over ecdysis and larval development

Elucidation of the key nutritional requirements for complete larval development of the tropical spiny rock lobster, Panulirus ornatus, presents a major challenge for the development of robust commercial aquaculture for this crustacean. As a foundation study in this area, the chemical composition of early-mid stage P. ornatus phyllosoma (Stages I-VI) receiving a novel formulated diet was analysed immediately prior and post-ecdysis to provide insight into the crude nutritional trends during the larval development cycle. From the onset of moulting, cyclical patterns were evident in the proximate composition of phyllosoma, resulting in substantial restructuring between the pre- and post-moult stages of the moult cycle. Proportions of protein, lipid and ash were high at the premoult stage, reflecting growth and nutrient accumulation over the intermoult period, and reduced at the post-moult stage, reflecting the large uptake of water to facilitate subsequent growth. Polar lipid was the dominant lipid class, accounting for >. 90% of the total lipid content. Conversely, triacylglycerol concentrations were low (<. 5%), despite being the principal lipid class available in the formulated diet. Likewise, despite receiving high concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) (9.2 and 7.6% of the dietary lipid source, respectively), levels of these fatty acids were comparatively low in phyllosoma (3.4 and 4.7%, respectively). In contrast, there is selective deposition of these fatty acids in wild caught phyllosoma. This finding suggests a poor assimilation of triacylglycerols by captive larvae and highlights the importance of future investigations into alternative sources of EPA and DHA. Ultimately, this study provides insight into the nutritional requirements of phyllosoma, providing valuable knowledge on diet formulation for commercially viable hatchery production of spiny rock lobsters. © 2014 Elsevier B.V.

Invertebrate diversity of the unexplored marine western margin of Australia: taxonomy and implications for global biodiversity

However derived, predictions of global marine species diversity rely on existing real data. All methods, whether based on past rates of species descriptions, on expert opinion, on the fraction of undescribed species in samples collected, or on ratios between taxa in the taxonomic hierarchy, suffer the same limitation. Here we show that infaunal macrofauna (crustaceans and polychaetes) of the lower bathyal depth range are underrepresented among available data and documented results from Australia. The crustacean and polychaete fauna (only partially identified) of the bathyal continental margin of Western Australia comprised 805 species, representing a largely novel and endemic fauna. Overall, 94.6% of crustacean species were undescribed, while 72% of polychaete species were new to the Australian fauna, including all tanaidaceans, amphipods, and cumaceans, as well as most isopods. Most species were rare, and the species accumulation rate showed no sign of reaching an asymptote with increasing area sampled. Similar data are likely for the largely unexplored bathyal regions. This leads us to conclude that the numbers upon which extrapolations to larger areas are based are too low to provide confidence. The Southern Australian and Indo-West Pacific deep-sea regions contribute significantly to global species diversity. These regions and bathyal and abyssal habitats generally are extensive, but are so-far poorly sampled. They appear to be dominated by taxonomically poorly worked and species-rich taxa with limited distributions. The combination of high species richness among infaunal taxa-compared to better known taxa with larger individuals, higher endemism than presently acknowledged because of the presence of cryptic species, the low proportion of described species in these taxa, and the vast extent of unexplored bathyal and abyssal environments-will lead to further accumulation of new species as more and more deep sea regions are explored. It remains to be tested whether ratios of 10 or more undescribed to described species, found in this study for the dominant taxa and for the deep Southern Ocean and the Indo-West Pacific, are replicable in other areas. Our data and similar figures from other remote regions, and the lack of faunal overlap, suggest that Appeltans et al.'s (Current Biology 22:1-14, 2012) estimate that between one-third and two-thirds of the world's marine fauna is undescribed is low, and that Mora et al.'s (PLoS Biol 9(8):e1001127. doi:10.1371/journal.pbio.1001127, 2011) of 91% is more probable. We conclude that estimates of global species, however made, are based on limited data. © 2014 Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg.

Resource distribution influences positive edge effects in a seagrass fish

According to conceptual models, the distribution of resources plays a critical role in determining how organisms distribute themselves near habitat edges. These models are frequently used to achieve a mechanistic understanding of edge effects, but because they are based predominantly on correlative studies, there is need for a demonstration of causality, which is best done through experimentation. Using artificial seagrass habitat as an experimental system, we determined a likely mechanism underpinning edge effects in a seagrass fish. To test for edge effects, we measured fish abundance at edges (0-0.5 m) and interiors (0.5-1 m) of two patch configurations: continuous (single, continuous 9-m2 patches) and patchy (four discrete 1-m2 patches within a 9-m2 area). In continuous configurations, pipefish (Stigmatopora argus) were three times more abundant at edges than interiors (positive edge effect), but in patchy configurations there was no difference. The lack of edge effect in patchy configurations might be because patchy seagrass consisted entirely of edge habitat. We then used two approaches to test whether observed edge effects in continuous configurations were caused by increased availability of food at edges. First, we estimated the abundance of the major prey of pipefish, small crustaceans, across continuous seagrass configurations. Crustacean abundances were highest at seagrass edges, where they were 16% greater than in patch interiors. Second, we supplemented interiors of continuous treatment patches with live crustaceans, while control patches were supplemented with seawater. After five hours of supplementation, numbers of pipefish were similar between edges and interiors of treatment patches, while the strong edge effects were maintained in controls. This indicated that fish were moving from patch edges to interiors in response to food supplementation. These approaches strongly suggest that a numerically dominant fish species is more abundant at seagrass edges due to greater food availability, and provide experimental support for the resource distribution model as an explanation for edge effects.

Estimating animal populations and body sizes from burrows: marine ecologists have their heads buried in the sand

Most ecological studies require knowledge of animal abundance, but it can be challenging and destructive of habitat to obtain accurate density estimates for cryptic species, such as crustaceans that tunnel deeply into the seafloor, beaches, or mudflats. Such fossorial species are, however, widely used in environmental impact assessments, requiring sampling techniques that are reliable, efficient, and environmentally benign for these species and environments.2.Counting and measuring the entrances of burrows made by cryptic species is commonly employed to index population and body sizes of individuals. The fundamental premise is that burrow metrics consistently predict density and size. Here we review the evidence for this premise. We also review criteria for selecting among sampling methods: burrow counts, visual censuses, and physical collections.3.A simple 1:1 correspondence between the number of holes and population size cannot be assumed. Occupancy rates, indexed by the slope of regression models, vary widely between species and among sites for the same species. Thus, 'average' or 'typical' occupancy rates should not be extrapolated from site- or species specific field validations and then be used as conversion factors in other situations.4.Predictions of organism density made from burrow counts often have large uncertainty, being double to half of the predicted mean value. Whether such prediction uncertainty is 'acceptable' depends on investigators' judgements regarding the desired detectable effect sizes.5.Regression models predicting body size from burrow entrance dimensions are more precise, but parameter estimates of most models are specific to species and subject to site-to-site variation within species.6.These results emphasise the need to undertake thorough field validations of indirect census techniques that include tests of how sensitive predictive models are to changes in habitat conditions or human impacts. In addition, new technologies (e.g. drones, thermal-, acoustic- or chemical sensors) should be used to enhance visual census techniques of burrows and surface-active animals.