Daniel Powell

Comparison of molecular markers to detect fresh sewage in environmental waters.

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% host-specificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10(-5), 10(-4) and 10(-3) respectively. HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10(-4) whilst EVs were detected up to dilution 10(-5). The ability of the HS-HF183 marker to detect fresh sewage was 3-4 orders of magnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.

Evaluation of the host-specificity and prevalence of enterococci surface protein (esp) marker in sewage and its application for sourcing human fecal pollution.

The suitability of the enterococci surface protein (esp) marker to detect human fecal pollution was evaluated by testing 197 fecal samples from 13 host groups in Southeast Queensland, Australia. Overall, this marker was detected in 90.5% of sewage and septic system samples and could not be detected in any fecal samples from 12 animal host groups. The sensitivity of the esp primer to detect the human-specific esp marker in sewage and septic samples was 100 and 67%, respectively. The overall specificity of this marker to distinguish between human and animal fecal pollution was 100%. Its prevalence in sewage was also determined by testing samples from the raw sewage, secondary effluent, and treated effluent of a sewage treatment plant (STP) over five consecutive days. Of the 15 samples tested, 12 (80%) were found to be positive for this marker. In contrast, it was not found in three samples from the treated effluent and these samples did not contain any culturable enterococci. The PCR limit of detection of this marker in freshwater samples was up to dilution 1 x 10(-4) and the number of culturable enterococci at this dilution was 4.8 x 10(1) +/- 7.0 x 10 degrees colony forming unit (CFU). The utility of this marker was evaluated by testing water samples from three non-sewered catchments in Pine Rivers in Southeast Queensland. Of the 13 samples tested, eight were positive for this marker with the number of enterococci ranging between 1.8 x 10(3) to 8.5 x 10(3) CFU per 100 mL of water. Based on the results, it can be concluded that the esp marker appears to be sewage specific and could be used as a reliable marker to detect human fecal pollution in surface waters in Southeast Queensland, Australia.

De-novo transcriptome analysis of the banana shrimp (Fenneropenaeus merguiensis) and identification of genes associated with reproduction and development

The banana shrimp Fenneropenaeus merguiensis is a commercially important marine crustacean for world aquaculture and fisheries. Despite this, limited genetic information is available for it and many other penaeid shrimp species. Here we present the first in-depth analysis of the transcriptional content of 8 different tissues from the banana shrimp using RNA-Seq technologies. A total of over 1 million single-end and over 49 million paired-end reads were obtained from Roche 454FLX and illumina sequencing platforms, respectively, resulting in an assembly of 124,631 transcripts with an N50 of 1,332 and mean length of 514 nt. A total of 59,179 putative protein sequences obtained from the assembled transcripts were annotated using public protein sequence databases and assigned 20,430 BLAST hits, 16,866 GO terms and 13,304 KOG categories. Further analysis revealed a rich set of transcript sequences exhibiting homology with genes associated with reproduction, sex determination and development and distinguished the tissues responsible for this expression. This report adds a substantial contribution to the sequence data currently available for F. merguiensis, providing valuable resources for further research.

A real‐time polymerase chain reaction assay for quantitative detection of the human‐specific enterococci surface protein marker in sewage and environmental waters

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.

Heritability for body colour and its genetic association with morphometric traits in Banana shrimp ( Fenneropenaeus merguiensis )

BackgroundBanana shrimp Fenneropenaeus merguiensis has emerged as an important aquacultured shrimp species in South East Asia and Australia. However, the quantitative genetic basis of economically important traits in this species are currently not available, while for body colour, cooked or uncooked, there are no genetic parameter estimates for any shrimp or indeed any decapod crustacean. In this study, we report for banana shrimp genetic parameters for morphometric traits and, the first time for any shrimp, parameter estimates for body colour. Ten highly polymorphic microsatellite markers were developed from genomic sequences and used to construct a pedigree for 2000 offspring from approximately 60 female and 60 male parents that were sampled from a single routine commercial production pond.ResultsRestricted maximum likelihood method applied to a single trait mixed model was used to estimate heritabilities, while correlations were estimated using the multi-trait approach. The estimates of heritability for morphometric traits were moderate to high (h2 = 0.14 – 0.50). Body colour of uncooked shrimp showed a heritable additive genetic component (h2 = 0.03 – 0.55), and those estimates obtained for cooked shrimp were significantly different from zero. Genetic correlations among morphometric traits were all positive and very high (close to unity, rg = 0.85 – 0.99). The genetic correlations of body traits (weight, length and width) were positive with both colour after cooking (0.74 – 0.84) and body colour measured on live shrimp (0.59 to 0.70). The positive genetic correlations between the cooked body colour and uncooked body colour (0.64 ± 0.20) suggests these two traits can be simultaneously improved in practical selective breeding programs. This first ever report of genetic parameters for cooked or uncooked colour in crustacean indicates there is potential for genetic improvement of both growth and body colour through selection.ConclusionsIn the present study we demonstrated for banana shrimp that genetic parameters can be estimated from commercial samples (using pedigrees based on DNA markers), that selection for shrimp colour should be successful under such commercial conditions.

Reproductive neuropeptides that stimulate spawning in the Sydney Rock Oyster (Saccostrea glomerata)

The Sydney Rock Oyster, Saccostrea glomerata, is a socioeconomically important species in Australia, yet little is known about the molecular mechanism that regulates its reproduction. To address this gap, we have performed a combination of high throughput transcriptomic and peptidomic analysis, to identify genes and neuropeptides that are expressed in the key regulatory tissues of S. glomerata; the visceral ganglia and gonads. Neuropeptides are known to encompass a diverse class of peptide messengers that play functional roles in many aspects of an animals life, including reproduction. Approximately 28 neuropeptide genes were identified, primarily within the visceral ganglia transcriptome, that encode precursor proteins containing numerous neuropeptides; some were confirmed through mass spectral peptidomics analysis of the visceral ganglia. Of those, 28 bioactive neuropeptides were synthesized, and then tested for their capacity to induce gonad development and spawning in S. glomerata. Egg laying hormone, gonadotropin-releasing hormone, APGWamide, buccalin, CCAP and LFRFamide were neuropeptides found to trigger spawning in ripe animals. Additional testing of APGWa and buccalin demonstrated their capacity to advance conditioning and gonadal maturation. In summary, our analysis of S. glomerata has identified neuropeptides that can influence the reproductive cycle of this species, specifically by accelerating gonadal maturation and triggering spawning. Other molluscan neuropeptides identified in this study will enable further research into understanding the neuroendocrinology of oysters, which may benefit their cultivation.