Molecular tools for studying puberty in the grey mullet, Mugil cephalus: cloning of cDNAs that regulate reproductive function

The cDNAs coding for the brain GnRHs (AY373449-51), pituitary GH, SL and PRL, and liver IGFs (AY427954-5) were isolated. Partial cDNA sequences of the brain (Cyp19b) and gonadal (Cyp19a) aromatases have also been obtained. These tools would be utilized to study the endocrine regulation of puberty in the grey mullet.

Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus.

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2.

Investigations into the potential for mixed cultures of banana prawns Penaeus merguiensis, sea mullet Mugil cephalus and rabbitfish Siganus nebulosus for bioremediation of aquaculture waste

To experimentally investigate the potential of mixed species polycultures for bioremediation of nutrient rich prawn farm effluent, a series of experiments was performed with banana prawns Penaeus (Fenneropenaeus) merguiensis, sea mullet Mugil cephalus and rabbitfish Siganus nebulosus to determine their compatibilities during particular life stages. Rabbitfish demonstrated a high tendency to prey upon banana prawn juveniles when no other food was available. Mullet of various sizes did not appear to prey upon banana prawn postlarvae (PL16) or juveniles in a fed or unfed environment. The study confirms the good potential for mullet and banana prawn polycultures.

New SNPs for population genetic analysis reveal possible cryptic speciation of eastern Australian sea mullet (Mugil cephalus)

Sustainable management of sea mullet (Mugil cephalus) fisheries needs to account for recent observations of regional-scale differentiation. Population genetic analysis is sought to assess the situation of this ecologically and economically important fish species in eastern Australian waters. Here, we report (i) new population genetic markers [single nucleotide polymorphisms (SNPs) and potential microsatellites], (ii) first estimates of spatial genetic differentiation and (iii) prospective power tests for designing more comprehensive studies. Six DNA samples from three sampling regions (North Queensland, South Queensland and central New South Wales) on the eastern coast of Australia were used to prepare restriction site associated DNA (RAD) tag libraries from genomic DNA digested with EcoRI and MseI. A pooled sample of regional RAD tag libraries was sequenced using the Roche GS-FLX Titanium platform. A total of 172837 raw reads (17.4Mbp) were retrieved, 95500 of which were used to discover 1267 SNPs and 1417 microsatellites. A subset of 161 SNPs was validated based on 63 additional DNA samples genotyped using the Sequenom MassArray (iPLEX Gold chemistry). Altogether 92 SNPs (57%) were confirmed, with 40% of these marking fixed variants between northern and southern sampling regions. Our preliminary findings indicate a multispecies fishery stock of M. cephalus in eastern Australian waters, but suggest that strong genetic differentiation occurs north of major fishing grounds. Low potential differentiation within major fishing grounds (e.g. FST=0.0025) can be resolved with a likely power 67% by using standard sample sizes of 50 and validated subsets of available markers.

Hazard Function Models to Estimate Mortality Rates Affecting Fish Populations with Application to the Sea Mullet (Mugil cephalus) Fishery on the Queensland Coast (Australia)

Fisheries management agencies around the world collect age data for the purpose of assessing the status of natural resources in their jurisdiction. Estimates of mortality rates represent a key information to assess the sustainability of fish stocks exploitation. Contrary to medical research or manufacturing where survival analysis is routinely applied to estimate failure rates, survival analysis has seldom been applied in fisheries stock assessment despite similar purposes between these fields of applied statistics. In this paper, we developed hazard functions to model the dynamic of an exploited fish population. These functions were used to estimate all parameters necessary for stock assessment (including natural and fishing mortality rates as well as gear selectivity) by maximum likelihood using age data from a sample of catch. This novel application of survival analysis to fisheries stock assessment was tested by Monte Carlo simulations to assert that it provided unbiased estimations of relevant quantities. The method was applied to the data from the Queensland (Australia) sea mullet (Mugil cephalus) commercial fishery collected between 2007 and 2014. It provided, for the first time, an estimate of natural mortality affecting this stock: 0.22±0.08 year −1 .

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

This study identified Gram-positive bacteria in three sub-tropical marine fish species: Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warned, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warned were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body. E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.

Effects of secondary crops on bacterial growth and nitrogen removal in shrimp farm effluent treatment systems

Secondary crops provide a means of assimilating some effluent nitrogen from eutrophic shrimp farm settlement ponds. However, a more important role may be their stimulation of beneficial bacterial nitrogen removal processes. In this study, bacterial biomass, growth and nitrogen removal capacity were quantified in shrimp farm effluent treatment systems containing vertical artificial substrates and either the banana shrimp Penaeus merguiensis (de Man) or the grey mullet, Mugil cephalus L. Banana shrimp were found to actively graze biofilm on the artificial substrates and significantly reduced bacterial biomass relative to a control (24.5 ± 5.6mgCm−2 and 39.2 ± 8.7mgCm−2, respectively). Bacterial volumetric growth rates, however, were significantly increased in the presence of the shrimp relative to the control 45.2±11.3mgCm−2 per day and 22.0±4.3mgCm−2 per day, respectively). Specific growth rate, or growth rate per cell, of bacteria was therefore appreciably stimulated by the banana shrimp. Nitrate assimilation was found to be significantly higher on grazed substrate biofilm relative to the control (223±54 mgNm−2 per day and 126±36 mg Nm−2 per day, respectively), suggesting that increased bacterial growth rate does relate to enhanced nitrogen uptake. Regulated banana shrimp feeding activity therefore can increase the rate of newbacterial biomass production and also the capacity for bacterial effluent nitrogen assimilation. Mullet had a negligible influence on the biofilm associated with the artificial substrate but reduced sediment bacterial biomass (224 ± 92 mgCm−2) relative to undisturbed sediment (650 ± 254 mgCm−2). Net, or volumetric bacterial growth in the sediment was similar in treatments with and without mullet, suggesting that the growth rate per cell of bacteria in grazed sediments was enhanced. Similar rates of dissolved nitrogen mineralisation werefound in sediments with and without mullet but nitrificationwas reduced. Presence of mullet increased water column suspended solids concentrations, water column bacterial growth and dissolved nutrient uptake. This study has shown that secondary crops, particularly banana shrimp, can play a stimulatory role in the bacterial processing of effluent nitrogen in eutrophic shrimp effluent treatment systems.

Treatment of shrimp farm effluent with omnivorous finfish and artificial substrates

Long-term environmental sustainability and community acceptance of the shrimp farming industry in Australia requires on-going development of efficient cost-effective effluent treatment options. In this study, we aimed to evaluate the effectiveness of a shrimp farm treatment system containing finfish and vertical artificial substrates (VAS). This was achieved by (1) quantifying the individual and collective effects of grey mullet (Mugil cephalus L.) and VASs on water and sediment quality, and (2) comparing the retention of N in treatment systems with and without the presence of finfish (M. cephalus and the siganid Siganus nebulosus (Quoy & Gaimard)), where light was selectively removed. Artificial substrates were found to significantly improve the settlement of particulate material, regardless of the presence of finfish. Mullet actively resuspended settled solids and reduced the production of nitrate when artificial substrates were absent. However, appreciable nitrification was observed when mullet were present together with artificial substrates. The total quantity of N retained by the mullet was found to be 1.8– 2.4% of the incoming pond effluent N. It was estimated that only 21% of the pond effluent N was available for mullet consumption. When S. nebulosus was added, total finfish N retention increased from 1.8% to 3.9%, N retention by mullet also improved (78±16 to 132±21-mg N day−1 before and after siganid addition respectively). Presence of filamentous macroalgae (Enteromorpha spp.) was found to improve the removal of N from pond effluent relative to treatments where light was excluded. Denitrification was also a significant sink for N (up to 24% N removed). Despite the absence of algal productivity and greater availability of nitrate, denitrification was not higher in treatments where light was excluded. Mullet were found to have no effect on the rates of denitrification but significantly reduced macroalgal growth on the surface of the water. When mullet were absent, excessive macroalgal growth led to reduced dissolved oxygen concentrations and nitrification. This study concludes that the culture of mullet alone in shrimp farm effluent treatment systems does not result in significant retention of N but can contribute to the control of macroalgal biomass. To improve N retention and removal, further work should focus on polyculturing a range of species and also on improving denitrification.

First Report of a Toxic Nodularia spumigena (Nostocales/Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in Isolated Populations of Mullet (Mugilidae)

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8-47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 mu g/kg, range 32.3-56.8 mu g/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.