Complete mitochondrial DNA sequences of the decapod crustaceans pseudocarcinus gigas (Menippidae) and macrobrachium rosenbergii (Palaemonidae)

The complete mitochondrial DNA sequence was determined for the Australian giant crab Pseudocarcinns gigas (Crustacea: Decapoda: Menippidae) and the giant freshwater shrimp Macrobrachium rosenbergii (Crustacea: Decapoda: Palaemonidae). The Pse gigas and Mrosenbergii mitochondrial genomes are circular molecules, 15,515 and 15,772 bp in length, respectively, and have the same gene composition as found in other metazoans. The gene arrangement of M. rosenbergii corresponds with that of the presumed ancestral arthropod gene order, represented by Limulus polyphemus, except for the position of the tRNA^Leu(UUR) gene. The Pse. gigas gene arrangement corresponds exactly with that reported for another brachyuran, Portunus trituberculatus, and differs from the M. rosenbergii gene order by only the position of the tRNA^His gene. Given the relative positions of intergenic nonoding nucleotides, the “duplication/random loss” model appears to be the most plausible mechanism for the translocation of this gene. These data represent the first caridean and only the second brachyuran complete mtDNA sequences, and a source of information that will facilitate surveys of intraspecific variation within these commercially important decapod species.

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.

Comparative distribution of a putative egg-laying hormone in neural and reproductive tissues of four Decapoda crustaceans

Evidence for the presence of a putative egg-laying (ELH) hormone has been previously described in the black tiger shrimp, Penaeus monodon, so a further investigation was carried out to detect its presence in a range of Decapoda crustaceans prior to a full molecular analysis. The crustaceans were represented by the Australian fresh water yabbie, Cherax destructor, the Australian southern rock lobster, Jasus edwardsii, the snow crab, Chionoecetes opilio, and the blue swimmer crab, Portunus pelagicus. Female cerebral ganglia, ventral nerve cords and gonads were investigated in a comparative study of the distribution of the immunoreactive hormone using immunoenzyme and immunofluorescence techniques. Immunoreactivity was detected in all tissues of interest, and the distribution patterns showed similarity within the four species, as well as that of P. monodon reported in the earlier study. There were minor variations. These data indicate that a putative ELH-like neuropeptide is widespread in crustaceans, and supports its previous identification in a range of molluscs and other invertebrates. Elucidation of the molecular structure of the peptide hormone and its encoding gene, as well as its involvement in spawning behaviour of crustaceans, is now fully under investigation.

Cellulose and hemicellulose digestion by herbivorous terrestrial crustaceans

Cellulose, the main component of plant cell walls, is insoluble and difficult to digest enzymatically. This research discovered that herbivorous land crabs have an efficient gastric mill in the stomach which disrupts this insoluble material, and a range of highly specialised enzymes that can then break down the cellulose.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098bp for G. natalis and 1095bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyrisplatycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

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.

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.

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.