Jawahar G. Patil

Development and evaluation of a PCR-based test for detection of Asterias (Echinodermata : Asteroidea) larvae in Australian plankton samples from ballast water

The northern Pacific seastar, Asterias amurensis, spread to Tasmania in the 1980s from its native range in the North Pacific. The seastar has subsequently established in Port Phillip Bay on mainland Australia. Transportation of larvae in ballast water is one vector for these introductions and is likely to contribute to additional range expansion of this species. Larval identification methods are critical to assess risks of further transport to uninvaded ecosystems; however, morphological identification of larval asteroids is impractical and unreliable. Therefore, we have developed a sensitive PCR-based method that specifically detects Asterias DNA. The method works on isolated seastar larva, mixed plankton and ballast water samples. Trials using uninfected ballast water samples spiked with known numbers of A. amurensis larvae indicate that the technique can detect single larva in 200 mg of plankton. The test also detects other Asterias species; therefore, discrimination between seastars within the genus Asterias was accomplished using denaturing gradient gel electrophoresis (DGGE). Currently, this method is being used to facilitate research on ballast water transport, distribution and the ecology of A. amurensis larvae. The general approach can be expanded to provide a rapid and cost-effective approach for detecting a suite of marine species in ballast water and environmental samples.

Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation

Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000 or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one- to twofold by electroporation. Fertilization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non-electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non-mammalian sperm as in mammalian sperm.

Tissue and sexually dimorphic expression of ovarian and brain aromatase mRNA in the Japanese medaka (Oryzias latipes): Implications for their preferential roles in ovarian and neural differentiation and development

Cytochrome P450 aromatase (CYP19) catalyzes conversion of testosterone to estrogen, and is thought to influence neural and reproductive development in vertebrates. Unlike higher vertebrates, many teleost fish, including the medaka (Oryzias latipes) have two aromatase genes, one expressed predominantly in the ovary (cyp19a) and the other in the brain (cyp19b). However, the exact roles of the two aromatase genes in neural or ovarian development in fish are unclear. The primary objective of this study was to determine the pattern of expression of each of the genes in developing and adult medaka. Real-time PCR analysis indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. cyp19a was expressed at significantly higher levels in ovaries than in testes, whereas cyp19b was expressed at higher levels in the adult brain of females than males. Ontogenic expression showed that neither of the aromatase transcripts is inherited maternally, with onset of zygotic expression of both isoforms occurring just prior to hatching (stage 39). Also the expression of the ovarian, but not the brain, isoform was significantly higher in genetically female individuals than in males of similar developmental stage. This coincided with the known increased proliferation of germ cells in XX genotypes, suggesting a possible role for cyp19a in ovarian differentiation. Differential expression of both isoforms in adults and during early larval development suggests that the genes have distinctly different roles: cyp19a contributing predominantly to ovarian differentiation and development; and cyp19b contributing towards neural development and perhaps sexual behavior in adults.

Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): Sexual dimorphism and onset of ontogenic expression

Cytochrome P450 aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen, and therefore is thought to influence gonadal sex differentiation. In an effort to understand the role of this enzyme in ovarian differentiation, we isolated cDNA encoding the two distinct isoforms, ovarian and brain (termed cyp19a and cyp19b, respectively) of adult common carp, Cyprinus carpio. The cloned cDNA for cyp19a had an open reading frame (ORF) of 518 amino acid residues, in contrast to cyp19b with an ORF of 511 amino acids. Sequence and phylogenetic analysis showed that these CYP19 isoforms were orthologous with previously described cyp19a and cyp19b from other teleosts. Quantitative real-time PCR indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. The major aromatase expressing tissue was found to be the brain, with greatest cyp19b expression in the anterior quarter (telencephalon) in both sexes. The gonad showed sexually dimorphic expression of both genes and dimorphic expression of cyp19a was observed in the cerebellum and the liver. Ontogenic expression showed that only the ovarian aromatase transcript is inherited maternally, with lower expression observed through early larval development under warmer rearing conditions. The differential and overlapping expression suggests these two aromatase genes have different roles in reproductive physiology.

Development and evaluation of a PCR based assay for detection of the toxic dinoflagellate, Gymnodinium catenatum (Graham) in ballast water and environmental samples

Gymnodinium catenatum is a bloom forming dinoflagellate that has been known to cause paralytic shellfish poisoning (PSP) in humans. It is being reported with increased frequency around the world, with ballast water transport implicated as a primary vector that may have contributed to its global spread. Major limitations to monitoring and management of its spread are the inability for early, rapid, and accurate detection of G. catenatum in plankton samples. This study explored the feasibility of developing a PCR-based method for specific detection of G. catenatumin cultures and heterogeneous ballast water and environmental samples. Sequence comparison of the large sub unit (LSU) ribosomal DNA locus of several strains and species of dinoflagellates allowed the design of G. catenatum specific PCR primers that are flanked by conserved regions. Assay specificity was validated through screening a range of dinoflagellate cultures, including the morphologically similar and taxonomically closely related species G. nolleri. Amplification of the diagnostic PCR product from all the strains of G. catenatum but not from other species of dinoflagellates tested imply the species specificity of the assay. Sensitivity of the assay to detect cysts in ballast water samples was established by simulated spiked experiments. The assay could detect G. catenatum in all ‘blank’ plankton samples that were spiked with five or more cysts. The assay was used to test environmental samples collected from the Derwent river estuary, Tasmania. Based on the results we conclude that the assay may be utilized in large scale screening of environmental and ballast water samples.

Specific amplification of mt-COI gene of the invasive gastropod Maoricolpus roseus in planktonic samples reveals a free-living larval life-history stage

The New Zealand screwshell Maoricolpus roseus was unintentionally introduced to south-eastern Tasmania in the 1920s. It has colonised more habitat than any other high-impact benthic marine pest in Australia and its wide temperature and depth tolerance makes further spread likely. We developed three sets of genetic probes, each targeting a unique region in the mitochondrial COI locus, for the rapid detection of this species in mixed plankton samples. In particular, we wanted to know whether this species has a planktonic life-history stage that could lead to its dispersal in ships’ ballast water. All probe sets were tested against as many closely related species as could be obtained and the reaction conditions were optimised for maximum sensitivity and specificity of M. roseus. Plankton samples collected in the Derwent Estuary between August 2003 and June 2004 were tested with the probes using a nested polymerase chain reaction. Maoricolpus roseus was detected in the plankton samples especially in the spring–summer period. The presence of M. roseus in the plankton and water available for ships’ ballast indicates that the risk of this species being spread by shipping needs to be managed.

Exploiting seasonal habitat use of the common carp, Cyprinus carpio, in a lacustrine system for management and eradication.

The control of invasive species is a major challenge to the preservation of native ecosystems, both terrestrial and aquatic. A population of the invasive common carp, Cyprinus carpio, was detected in Lakes Sorell and Crescent, Tasmania, Australia, in the mid-1990s. Early detection allowed for the containment of their population to this lacustrine system. Radio-telemetry was subsequently used over a 5-year period to understand seasonal distribution of carp in relation to environmental parameters to guide efficient eradication strategies. The present study found that fish displayed increased mobility during spring–summer periods, moving into shallow habitat rich in macrophytes, particularly during years of high lake levels. During years of low lake levels, this pattern was altered with frequent use of a rocky ‘secondary’ habitat in Lake Sorell. During winter, carp congregated in deeper habitat in Lake Sorell, whereas no habitat-specific winter aggregations were found in Lake Crescent. The increased knowledge of spatio-temporal preferences of carp provided a basis for deploying barriers to facilitate the capture of fish and sabotage spawning events. The results have led to the eradication of carp from Lake Crescent and a significant reduction of their population in Lake Sorell.

Effects of exposure to oestrogenic compounds on aromatase gene expression are gender dependent in the rainbowfish, Melanotaenia fluviatilis.

This study investigated the influence of two endocrine disrupting chemicals (EDCs)-an exogenous oestrogen 17β-estradiol (E2) and the oestrogen mimic 4-n-nonylphenol (NP) on the expression of aromatase transcripts in both sexes of adult Murray river rainbowfish. Reproductively active mature fish were exposed to 1, 3, and 5 μg/L E2 or 100 and 500μg/L NP for 24, 48, 72 and 96 h. The results show a significant reduction in the expression of cyp19a1a isoform in ovarian tissues with complete inhibition at the higher concentrations (3 and 5 μg/L E2; 500μg/L NP between 24 and 72 h) and at all concentrations after 96 h. There was no expression of the cyp19a1a isoform in female brain, male brain or testes in any treatment. E2 significantly increased expression of cyp19a1b in female brain except at 5 μg/L after 24h exposure. In male brain tissue E2 exposure decreased cyp19a1b expression except at 1 and 5 μg/L at 24h. NP significantly upregulated cyp19a1b in the female brain (except with 500 μg/L at 72 h) and in testes tissues. NP downregulated expression of cyp19a1b in the male brain tissue. Collectively, these observations support the hypothesis that the expression of cyp19a1b is regulated via both positive and negative feedback mechanisms, with differential modulation based on the type and concentration of the exposed oestrogens, duration of exposure, fish tissue and gender of the fish. The results also imply that exogenous oestrogens can have a disruptive effect on the steroidogenic pathway and may lead to effects on sex differentiation, sexual behaviour and reproductive cycles in this fish.

Human liver-derived cells stably modified for regulated proinsulin secretion function as bioimplants in vivo

Insulin deficiency is currently treated with pharmacological insulin secretagogues, insulin injections or islet transplants. Secondary failure of pharmacological agents is common; insulin injections often fail to achieve euglycemic control; and islet transplants are rare. Non‐β cells capable of regulated insulin secretion in vivo could be a functional cure for diabetes. Hepatocytes are good candidates, being naturally glucose‐responsive, protein‐secreting cells, while the liver is positioned to receive direct nutrient signals that regulate insulin production.

Preventing Genetic Pollution and the Establishment of Feral Populations: A Molecular Solution

Aquaculture animals that escape from farms have the potential to create major environmental problems. These include establishment of potentially destructive feral populations (e.g., Pacific oysters [Crassostrea gigas] in Australia, Atlantic salmon [Salmo salar] in British Columbia) and genetic contamination of wild stocks. The latter includes introgression of foreign genes into natural populations from both hatchery-reared fish and genetically modified fish and invertebrates. Concern about these environmental and genetic effects has already led to restrictions on aquaculture industry development and is likely to grow as demand for genetically improved stocks escalates to fulfill production objectives. To circumvent these problems, we have developed a genetic construct that, when properly integrated into production-line fish or invertebrates, should render individuals functionally sterile outside of hatchery conditions. In the hatchery, however, provision of a simple repressor compound at a particular life-history stage allows the animals to be bred and reared as normal. We Theresa M. Bert (ed.), Ecological and Genetic Implications of Aquaculture Activities, 103–114. 2007 Springer. 103 are developing this ‘‘Sterile Feral’’ technology for both invertebrate and fish species, and we anticipate practical commercial application within a few years.