Current status of parasitic ciliates Chilodonella spp. (Phyllopharyngea: Chilodonellidae) in freshwater fish aquaculture.

Freshwater fish farming contributes to more than two-thirds of global aquaculture production. Parasitic ciliates are one of the largest causes of production loss in freshwater farmed fishes, with species from the genus Chilodonella being particularly problematic. While Chilodonella spp. include 'free-living' fauna, some species are involved in mortality events of fish, particularly in high-density aquaculture. Indeed, chilodonellosis causes major productivity losses in over 16 species of farmed freshwater fishes in more than 14 countries. Traditionally, Chilodonella species are identified based on morphological features; however, the genus comprises yet uncharacterized cryptic species, which indicates the necessity for molecular diagnostic methods. This review synthesizes current knowledge on the biology, ecology and geographic distribution of harmful Chilodonella spp. and examines pathological signs, diagnostic methods and treatments. Recent advances in molecular diagnostics and the ability to culture Chilodonella spp. in vitro will enable the development of preventative management practices and sustained freshwater fish aquaculture production.

Novel genomic microsatellite markers for genetic population and diversity studies of tropical scalloped spiny lobster (Panulirus homarus) and their potential application in related Panulirus species.

Fourteen polymorphic microsatellites with perfect di-, tri-, and tetra-nucleotide repeats were identified for Panulirus homarus using Roche 454 whole-genome sequencing method. Microsatellites were efficiently co-amplified in four multiplexes and a singleplex, providing consistent and easily interpretable genotypes. The number of alleles per locus ranged from 2 to 11 with the observed and expected heterozygosity ranging between 0.000-0.532 and 0.031-0.836, respectively. A significant deviation from Hardy-Weinberg equilibrium was observed for majority of the loci, probably due to homozygote excess. Genetic linkage disequilibrium analysis between all the possible pairs of the loci showed significant departure from the null hypothesis in the loci pairs Pho-G11-Pho-G33 and Pho-G33-Pho-G57. High success in primer cross-species amplification of these microsatellite markers indicates their utility for genetic studies of different Panulirus species.

Isolation, characterization, and multiplexing of novel microsatellite markers for the tropical scalloped spiny lobster (Panulirus homarus).

Of the various spiny lobster species in the tropical and subtropical Indo-West Pacific region, the tropical scalloped spiny lobster (Panulirus homarus) supports one of the most commercially valuable fishery resources in many coastal African and Asian countries. The last decade has witnessed a serious decline in the wild populations of this species. Knowledge of the genetic basis of spiny lobster population structure is a prerequisite to achieve a sustainable fisheries management for this species. Here, we describe 13 novel polymorphic microsatellite markers developed for P. homarus, using a cross-species primer design strategy based on P. ornatus Roche 454 shot-gun generated sequencing. Microsatellite polymorphisms were assessed in 96 unrelated P. homarus individuals of a natural population, with the number of alleles per locus varying from 2 to 14, the observed and expected heterozygosity from 0.00 to 0.78 and from 0.03 to 0.79, respectively, and with only four loci (Pho-G27, Pho-G32, Pho-G36, and Pho-G58) deviating from Hardy- Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Pho-G22 - Pho-G30, and Pho-G30 - Pho-G35. The successful cross amplification of these microsatellites highlights the potential of the developed microsatellites for future population genetic research within the different Panulirus species.

The gene expression response of the catadromous perciform barramundi Lates calcarifer to an acute heat stress.

The acute heat-shock response of the tropical estuarine fish species barramundi Lates calcarifer as indicated by the expression of genes within stress (hsp 90AA, hsp 90AB, hsp 70 and hsc 70), metabolic (cisy, cco II and ldh) and growth (igf1 and mstn 1) related pathways was examined following an increase in water temperature from 28 to 36° C over 30 min. Lates calcarifer were maintained at the acute stress temperature of 36° C for 1 h before being returned to 28° C and allowed to recover at this temperature for a further 2 weeks. Muscle tissue sampling over the experimental period allowed for the expression quantification of stress, metabolic and growth-related genes via quantitative real-time polymerase chain reaction (qrt-PCR) where a robust and reliable normalization approach identified both α-tub and Rpl8 as appropriate genes for the analysis of gene expression in response to an acute heat stress. hsp90AA and hsp70 of the inducible heat-shock response pathway showed a massive up-regulation of gene expression in response to heat stress, whilst the constitutive heat-shock genes hsp90AB and hsp70 showed no change over the course of the experiment and a small increase after 2 weeks of recovery, respectively. Of the three genes representing the metabolic pathway (cisy, cco II and ldh) only cco II changed significantly showing a decrease in gene expression, which may suggest a small suppression of aerobic metabolism. igf1 of the growth pathway showed no significant differences in response to an acute heat stress, whilst mstn1 increased at the beginning of the heat stress but returned to basal levels soon after. Overall, the results demonstrate that an acute heat stress in L. calcarifer caused a significant increase in the expression of genes from the stress response pathway and a possible decrease in aerobic metabolism with only relatively minor changes to the growth pathway highlighting the hardy nature of L. calcarifer and its resilience in coping with sudden temperature changes routinely encountered within its natural environment.

A robust flow-cytometric protocol for assessing growth rate of hatchery-reared barramundi Lates calcarifer larvae.

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments.

Transcriptome analysis of biomineralisation-related genes within the pearl sac: host and donor oyster contribution.

Cultured pearl production is a complex biological process involving the implantation of a mantle graft from a donor pearl oyster along with a bead nucleus into the gonad of a second recipient host oyster. Therefore, pearl production potentially involves the genetic co-operation of two oyster genomes. Whilst many genes in the mantle tissue have been identified and linked to shell biomineralisation in pearl oysters, few studies have determined which of these biomineralisation genes are expressed in the pearl sac and potentially linked to pearl biomineralisation processes. It is also uncertain whether the host or donor oyster is primarily responsible for the expression of biomineralisation genes governing pearl formation, with only two shell matrix proteins previously identified as being expressed by the donor oyster in the pearl sac. To further our understanding of pearl formation, the pearl sac transcriptome of Pinctada maxima and Pinctada margaritifera was each sequenced to an equivalent 5× genome coverage with putative molluscan biomineralisation-related genes identified. Furthermore, the host and donor contribution of these expressed genes within the pearl sac were quantified using a novel approach whereby two pearl oyster species harbouring unique genomes, P. maxima or P. margaritifera, were used to produce xenografted pearl sacs. A total of 19 putative mollusc biomineralisation genes were identified and found to be expressed in the pearl sacs of P. maxima and P. margaritifera. From this list of expressed genes, species-diagnostic single nucleotide polymorphisms (SNP) were identified within seven of these genes; Linkine, N66, Perline, N44, MSI60, Calreticulin and PfCHS1. Based on the presence/absence of species diagnostic gene transcripts within xenografted pearl sacs, all seven genes were found to be expressed by the species used as the donor oyster. In one individual we also found that the host was expressing Linkine. These results convincingly show for the first time that the donor mantle tissue is primarily responsible for the expression of biomineralisation genes in the pearl sac.

Effects of chlorpyrifos on cholinesterase activity and stress markers in the tropical reef fish Acanthochromis polyacanthus.

Tropical coastal ecosystems, including the Great Barrier Reef (GBR) of Australia are increasingly threatened by pollution; yet few studies have investigated the sensitivity of GBR species to these pollutants. Here we exposed juveniles of the tropical reef fish Acanthochromis polyacanthus (spiny damselfish) to three concentrations of the insecticide chlorpyrifos (CPF) and measured (i) muscle cholinesterase (ChE) activity; (ii) hepatic glutathione-S-transferase (GST) activity; and (iii) coenzyme Q (CoQ) redox balance, after 6h and 96h of exposure. After 96h, muscle ChE activity was significantly inhibited by 26%, 49% and 53% when fish were exposed to 1, 10 or 100µg/L CPF, respectively. Muscle ChE characterization revealed three types of ChEs, including two atypical forms. Hepatic CoQ antioxidant form significantly increased at 10µg/L after 6h of exposure, potentially demonstrating an early response to CPF-induced oxidative stress in liver. Hepatic GST was not affected by CPF exposure.

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima.

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.

A hybrid zone and bidirectional introgression between two catadromous species: Australian bass Macquaria novemaculeata and estuary perch Macquaria colonorum.

The presence and distribution of hybrid individuals and the existence of a hybrid zone between the catadromous Australian bass Macquaria novemaculeata and estuary perch Macquaria colonorum were investigated throughout the range of both species in Australia. Bayesian analyses and genotypic simulations identified 140 putative hybrids (11·5% of the total sample) with varying levels of introgression. Most hybrids were observed in an area extending from the Snowy River to the Albert River suggesting a hybrid zone in the eastern Bass Strait region. Sixteen hybrids, however, were found outside this zone, possibly reflecting the movement of hybrid offspring between estuaries or their inadvertent release during fish stocking programmes. Biparental backcrossing was found to occur suggesting that hybrids were fertile. These results have implications for the management of the extensive stocking programme in M. novemaculeata and for understanding the potential role of habitat degradation and reduced water flow in facilitating hybridization in species with migratory life histories.

Differential tissue-regulation of myostatin genes in the teleost fish Lates calcarifer in response to fasting. Evidence for functional differentiation.

Gene or genome duplication is a fundamental evolutionary mechanism leading towards the origin of new genes, or gene functions. Myostatin (MSTN) is a negative regulator of muscle growth that in teleost fish, as a result of genome duplication, is present in double copy. This study provides evidence of differentiation of MSTN paralogs in fish by comparatively exploring their tissue-regulation in the Asian sea bass (Lates calcarifer) when subjected to fasting stress. Results showed differential regulation as well as specific tissue-responses in the muscle, liver, gill and brain of L. calcarifer after nutritional deprivation. In particular, the LcMstn-1 expression increased in liver (∼4 fold) and muscle (∼3 fold) and diminished in brain (∼0.5 fold) and gill (∼0.5 fold) while that of LcMstn-2 remained stable in brain and muscle and was up regulated in gill (∼2.5 fold) and liver (∼2 fold). Differential regulation of Mstn paralogs was supported by in silico analyses of regulatory motifs that revealed, at least in the immediate region upstream the genes, a differentiation between Mstn-1 and Mstn-2. The Mstn-1 in particular showed a significantly higher conservation of regulatory sites among teleost species compared to its paralog indicating that this gene might have a highly conserved function in the taxon.

Isolation and characterization of 16 microsatellite loci in the humbug damselfish, Dascyllus aruanus (family Pomacentridae).

Here we report on 16 microsatellite loci designed for the damselfish Dascyllus aruanus. All loci were tested on 98 individuals and were polymorphic (seven to 35 alleles). Expected heterozygosity ranged from 0.705 to 0.942. Six loci showed Hardy-Weinberg disequilibrium due to the occurrence of null alleles. Cross-species amplifications conducted within the genus Dascyllus (D. carneus, D. strasburgi, D. trimaculatus) lead to polymorphic fragments in 32 out of 48 tests. These 16 loci will enable future research into the behavioural ecology and population ecology of Dascyllus aruanus throughout the Indo-Pacific.

The phylogenetic relationships of the bilby, Macrotis lagotis (Peramelimorphia: Thylacomyidae), to the bandicoots- DNA sequence evidence.

Partial sequencing of the 12S ribosomal RNA gene was used to test two competing hypotheses concerning the phylogenetic relationship of the bilby (Macrotis lagotis) to the Australian and New Guinean species of bandicoot. The first hypothesis proposes that the Australian and New Guinean bandicoots are in a monophyletic clade to the exclusion of the bilbies, whereas the second hypothesis proposes that the bilby is monophyletic with the Australian bandicoots to the exclusion of the New Guinean bandicoots. Phylogenies determined by both maximum-likelihood and neighbour-joining approaches supported the first hypothesis in which the bilby is excluded from the clade represented by the Australian and New Guinean bandicoots. Monophyly of the Australian and New Guinean bandicoots is consistent with the biogeographical scenario in which Australia and Papua New Guinea have undergone repeated connection and disconnection over the last 20 million years.

Phylogenetic relationships of Australian members of the family percichthyidae inferred from mitochondrial 12S rRNA sequence data.

Phylogenetic relationships among 20 Australian species of the family Percichthyidae were investigated from sequence data of two portions of the mitochondrial 12S rRNA gene. The molecular data indicate that Australian genera within this family cluster into three distinct clades. The first clade is composed of some species currently ascribed to the genus Macquaria, along with Nannatherina, Nannoperca, and Bostockia, the second of Maccullochella and two catadromous Macquaria species, and the third of Gadopsis. However, the positioning of Gadopsis within this family was unresolved. Monophyly within each genus was well supported, except for Macquaria, which is clearly polyphyletic. The molecular data were used to examine two hypotheses of Australian percichthyid evolution and favor a freshwater origin for the family.

Consequences of a catadromous life-strategy for levels of mitochondrial DNA differentiation among populations of the Australian bass, Macquaria novemaculeata.

The influence of a catadromous life-strategy on levels of spatial genetic structuring in fish is poorly understood. In an effort to gain a better appreciation of how this specialized life-strategy determines population genetic structuring, we assessed variation in the mitochondrial DNA (mtDNA) control region in a catadromous perciform, the Australian bass Macquaria novemaculeata. Nineteen putative haplotypes were resolved using temperature gradient gel electrophoresis from 10 geographically distinct populations. Significant heterogeneity was revealed in haplotype frequencies and their spatial distributions among many locales. Gene partitioning statistics (AMOVA) for both raw haplotype frequency data and frequency data with sequence divergences were concordant, indicating that M. novemaculeata populations were moderately genetically structured (phi ST = 0.05, 0.06; P < 0.001, respectively). Isolation by distance seems to be a strong structuring force in M. novemaculeata, culminating in no detectable phylogeographic structuring among haplotypes. Low sequence divergences were observed among many haplotypes and it is suggested that these are the result of pruning of maternal lineages by cyclical variations in female reproductive success. This study highlights the importance of life-history patterns and, in particular, spawning locality, in determining spatial structuring of mtDNA variation in catadromous species.