Species composition and hybridisation of mussel species (Bivalvia: Mytilidae) in Australia

Mussels belonging to the Mytilus edulis species complex have been the focus of numerous studies exploring the systematics and origin of this commercially and ecologically important genus. Species have wide geographical ranges and hybridise where their distributions overlap, making identification difficult. Several molecular markers have been used to distinguish between the species within the M. edulis species complex; however, no single marker system has been found to be completely diagnostic, and a combination of markers are used. Here, we used a combination of three nuclear genes and a mitochondrial gene region to assess the species composition of Mytilus mussels collected across its geographical range in Australia. Our results show that the majority (98.5%) of individuals sampled from Australian populations are Mytilus galloprovincialis, with 56.2% of them displaying a southern hemisphere haplotype, 10.3% displaying a putatively northern hemisphere haplotype, and 32% having M. galloprovincialis genotypes consistent with either northern or southern hemisphere M. galloprovincialis lineages. The taxonomic origin of the remaining 1.5% of samples (n ¼ 3) could not be conclusively determined. Our results suggest that there have been significant introductions of non-native M. galloprovincialis lineages into both southern and northern hemisphere populations.

Molecular genetic approaches to the identification of commercial fish species

A DNA database was developed to enable the identification and discrimination of commercially important fish species. Three genetic techniques were compared for efficiency, accuracy and reliability. It is envisaged this study will aid authorities to identify mislabelled fish fillets and provide greater consumer confidence in the Australian Seafood Industry.

Identification and genetic determination of an early life risk disposition for depressive disorder : atypical stress-related behaviour in early childhood

Progress in psychiatric genetics has been slow despite evidence of high heritability for most mental disorders. We argue that greater use of early detectable intermediate traits (endophenotypes) with the highest likely aetiological significance to depression, rather than complex clinical phenotypes, would be advantageous. Longitudinal data from the Western Australian Pregnancy Cohort (Raine) Study were used to identify an early life behavioural endophenotype for atypical hypothalamic-pituitaryadrenocortical function in adolescence, a neurobiological indicator of anxiety and depression. A set of descriptors representing rigid and reactive behaviour at age 1 year discriminated those in the top 20% of the free salivary cortisol exposure at age 17 years. Genetic association analysis revealed a male-sensitive effect to variation in three specific single nucleotide polymorphisms within selected genes underpinning the overall stress response. Furthermore, support for a polygenic effect on stress-related behaviour in childhood is presented.

Population genetic structure of the Australian caddisfly Lectrides varians Mosely (Trichoptera: Leptoceridae) and the identification of cryptic species in south-eastern Australia

Lectrides varians (Mosely) is a large, ecologically-important, caddisfly found in perennial and intermittent streams throughout much of eastern Australia. We conducted a population genetic analysis to investigate the dispersal potential of L. varians, building on previous works that have assessed life-history traits associated with drought resistance. Genetic analyses of L. varians from the Grampians region of Victoria, based on mitochondrial DNA sequence data, revealed extensive gene flow and a lack of genetic structure across the sample range (ΦST = 0.04). This suggests that the species is a strong disperser and is likely to be resilient to increased drying and habitat fragmentation under climate change considering other known resistance traits. However, during this study, two divergent genotypes were identified, indicating a potential species complex. A comprehensive phylogenetic analysis of L. varians across its current range was subsequently performed, confirming the species is indeed paraphyletic, consisting of one lineage that is restricted to the Grampians National Park and the other being widespread throughout south-eastern Australia. Further analyses revealed consistent morphological differences between these lineages supporting the notion that L. varians is a species complex. We discuss the implications of these findings with regard to conservation and taxonomy of this important invertebrate group.

Automatic inspection of metallic surface defects using genetic algorithms

This paper is concerned with the problem of automatic inspection of metallic surface using machine vision. An experimental system has been developed to take images of external metallic surfaces and an intelligent approach based on morphology and genetic algorithms is proposed to detect structural defects on bumpy metallic surfaces. The approach employs genetic algorithms to automatically learn morphology processing parameters such as structuring elements and defect segmentation threshold. This paper describes the detailed procedures which include encoding scheme, genetic operation and evaluation function.

The proposed method has been implemented and tested on a number of metallic surfaces. The results suggest that the method can provide an accurate identification to the defects and can be developed into a viable commercial visual inspection system.

Manual on application of molecular tools in aquaculture and inland fisheries management. Part I : conceptual basis of population genetic approaches

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the first volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents.

Cystic fibrosis in children of the eastern Arabian peninsula : a clinical, spatial and genetic study

Aim: The aim of this thesis is to describe the process by which the inherited disease, cystic fibrosis, (CF) was recognised as an important clinical entity in the United Arab Emirates (UAE) and the Sultanate of Oman (Oman). It examines the clinical presentation of the first patients and assesses their degree of severity. Further, it describes the first studies carried out to determine the underlying CF mutations associated with the disease in the UAE and Oman. An estimate is offered of the birth frequency of the condition. Overall, the cultural, geographical and historical aspect of the societies in which the disease occurs is stressed. Methods: An initial literature search was carried out using Medline of any literature pertaining to the Arab World and CF. this was read and classified into the relevance to Arabs in general, the Middle East and then specifically the Arab (Persian) Gulf societies. Thereafter, a clinic was established at Tawam Hospital, Al Ain, UAE, for children presenting With chronic respiratory disease that could serve as a national referral centre. It was run by the Author as a service of the Paediatric Department of the UAE University Medical School. I sent a letter to every Paediatrician working in the UAE informing them of our clinic and offering our services for the diagnosis and management of chronic respiratory disease in children. This was based on the author's experience as a respiratory paediatrician in Australia and New Zealand and as the Professor of Paediatrics in the UAE. No such service then existed in the UAE. Funding was sought to establish a research programme and develop a molecular genetics laboratory in the UAE Medical School. A series of successful research applications provided the grants to commence the investigations. Once a small number of children had been identified as having CF from those referred to the respiratory clinic, the initial project was to assess and report their clinical presentation. Following this an early start was made on the identification of the mutations responsible. Once these were established an attempt was made to estimate the frequency of the condition at birth. Additional clinical studies revolved around assessing the severity of the condition that was associated with the main mutations that were identified. A clinical comparison was made with those with the mutation AF508 and the other main mutation, despite the obvious limitation of small numbers then available. Radiological assessment was made to evaluate the progression of the disease. The final aspect of the study was to assess patients from Oman and compare their findings and mutations with the neighbouring UAE. Based on information gained hypotheses are proposed regarding the spread of the gene mutation by population drift. Thesis outline: A literature review is presented in the form of a critique on the disease and a resume of the relevant aspects of the genetics of CF. Additionally, facts about the two countries' geography and history are presented. Finally, knowledge about CF mutations and population origins from other areas is presented. The second main section deals with the clinical features of the disorder as it presents in the UAE. Molecular findings are then presented and details of the common mutation found in Bedouin Arabs. Hypotheses are then presented based on the information gathered. Results: CF is not a rare disease in the Arab children of the UAE and Oman. These findings refute previous reports of CF being a rare or non-existent disease in Arabs. The condition presents with a severe clinical picture, with early colonisation of the respiratory tract with staphylococcus, haemophilus and pseudomonas organisms, even with conventional CF management practices in place. The CF mutation S549R is prevalent in Arabs of Bedouin stock, while AF508 is found in those of Baluch origin. The former may be descendants of Arabs who left southern Arabia and travelled to the Trucial Coast at the time of the destruction of the great dam at Marib. The origins of this mutation may lie in the area that corresponds to the modern Republic of Yemen. The latter groups are descendants of those who came originally from Baluchistan. It is hypothesised also that the ancestral home of the AF508 mutation may be in the geographical area now known as Baluchistan, that spans three separate modern political territories. The evidence presented supports the concept that the S549R mutation may be associated with a severe, if not the severest, clinical pattern recognised. It equates with that seen with the homozygous AF508 genotype. The absence of an additional mutation in the promoter region accounts for the different clinical pattern seen in previously described patients. Conclusions: There needs to be a major awareness of the presence of CF as a severe clinical disease in the children of the Gulf States. The clinical presentation and findings support the concept of under recognition of the disease. Climatic conditions put the children at special risk of hyponatraemia and electrolyte imbalance. The absence of surviving adults with the disease suggests premature deaths have occurred, but the high fertility rates have maintained the gene pool for this recessive disorder.

Identification and characterisation of novel genes involved in skeletal muscle hypertrophy

There is mounting evidence in support of the view that skeletal muscle hypertrophy results from the complex and coordinated interaction of numerous signalling pathways. Well characterised components integral to skeletal muscle adaptation include the transcriptional activity of the members of the myogenic regulatory factors, numerous secreted peptide growth factors, and the regenerative potential of satellite cells. Whilst studies investigating isolated components or pathways have enhanced our current understanding of skeletal muscle hypertrophy, our knowledge of how all of these components react in concert to a common stimulus remains limited. The broad aim of this thesis was to identify and characterise novel genes involved in skeletal muscle hypertrophy. We have created a customised human skeletal muscle specific microarray which contains ∼11,000 cDNA clones derived from a normalised human skeletal muscle cDNA library as well as 270 genes with known functional roles in human skeletal muscle. The first aspect of this thesis describes the production of the microarray and evaluates the robustness and reproducibility of this analytical technique. Study one aimed to use this microarray in the identification of genes that are differentially expressed during the forced differentiation of human rhabdomyosarcoma cells, an in vitro model of skeletal muscle development. Firstly using this unique model of aberrant myogenic differentiation we aimed to identify genes with previously unidentified roles in myogenesis. Secondly, the data from this study permitted the examination of the performance of the microarray in detecting differential gene expression in a biological system. We identified several new genes with potential roles in the myogenic arrest of rhabdomyosarcoma and further characterised the expression of muscle specific genes in rhabdomyosarcoma differentiation. In study two, the molecular responses of cell cycle regulators, muscle regulatory factors, and atrophy related genes were mapped in response to a single bout of resistance exercise in human skeletal muscle. We demonstrated an increased expression of MyoD, myogenin and p21, whilst the expression of myostatin was decreased. The results of this study contribute to the existing body of knowledge on the molecular regulation skeletal muscle to a hypertrophic stimulus. In study three, the muscle samples collected in study two were analysed using the human skeletal muscle specific microarray for the identification of novel genes with potential roles in the hypertrophic process. The analysis uncovered four interesting genes (TXNIP, MLP, ASB5, FLJ 38973) that have not previously been examined in human skeletal muscle in response to resistance exercise. The functions of these genes and their potential roles in skeletal muscle are discussed. In study four, the four genes identified in study three were examined in human primary skeletal muscle cell cultures during myogenic differentiation. Human primary skeletal muscle cells were derived from the vastus lateralis muscle of 8 healthy volunteers (6 males and 2 females). Cell cultures were differentiated using serum withdrawal and serum withdrawal combined with IGF-1 supplementation. Markers of the cell proliferation, cell cycle arrest and myogenic differentiation were examined to assess the effectiveness of the differentiation stimulus. Additionally, the expressions of TXNIP, MLP, ASB5 and FLJ 38973 measured in an attempt to characterise further their roles in skeletal muscle. The expression of TXNIP changed markedly in response to both differentiation stimuli, whilst the expression of the remaining genes were not altered. Therefore it was suggested that expression of these genes might be responsive to the mechanical strain or contraction induced by the resistance exercise. In order to examine whether these novel genes responded specifically to resistance type exercise, their expression was examined following a single bout of endurance exercise. The expression of TXNIP, MLP, and FLJ 38973 remained unchanged whilst ASB5 increased 30 min following the cessation of the exercise.

Shedding new light on old species identifications : morphological and genetic evidence suggest a need for conservation status review of the critically endangered bat, Saccolaimus saccolaimus

Information based on the accurate identification of species is a vital component for achieving successful outcomes of biodiversity conservation and management. It is difficult to manage species that are poorly known or that are misidentified with other similar species. This is particularly problematic for rare and threatened species. Species that are listed under endangered species classification schemes need to be identified accurately and categorised correctly so that conservation efforts are appropriately allocated. In Australia, the emballonurid Saccolaimus saccolaimus is currently listed as ‘Critically Endangered’. On the basis of new observations and existing museum specimens, we used a combination of genetic (mitochondrial DNA sequence) and morphological (pelage characteristics, dig III : phalanx I length ratio, inter-upper canine distance) analyses to identify six new geographic records for S. saccolaimus, comprising ~100 individuals. Our analyses also suggested that there are likely to be more records in museum collections misidentified as S. flaviventris specimens. The external morphological similarities to S. flaviventris were addressed and genetic, morphological and echolocation analyses were used in an attempt to provide diagnostic characters that can be used to readily identify the two species in the field. We recommend genetic testing of all museum specimens of Australian Saccolaimus to clarify species’ distributions and provide data for reassessing the conservation status for both S. saccolaimus and S. flaviventris. Museum curators, taxonomists and wildlife managers need to be aware of potential species misidentifications, both in the field and laboratory. Misidentifications that result in misclassification of both threatened and non-threatened species can have significant implications.

Zebrafish as a genetic model in pre-clinical drug testing and screening

The traditional drug discovery pipeline for the identification and development of compounds that selectively target specific molecules to ameliorate disease remains a major focus for medical research. However, the zebrafish is increasingly providing alternative strategies for various components of this pipeline. Zebrafish and their embryos are small, easily accessible and relatively low cost, making them applicable to high-throughput, small molecule screening. Zebrafish can also be manipulated by a range of forward and reverse genetics techniques to facilitate gene discovery and functional studies. Moreover, their physiological and developmental complexity provides accurate models of human disease to underpin mechanism of action and in vivo validation studies. Finally, several of these biological characteristics make zebrafish eminently suitable for toxicity testing, including eco-toxicology. Here we review the application of zebrafish to preclinical drug development and toxicity testing, including recent advances in mutant generation, drug screening and toxicology that serve to further enhance the capabilities of this valuable model organism in drug discovery.

Identification of novel therapeutics for complex diseases from genome-wide association data

Human genome sequencing has enabled the association of phenotypes with genetic loci, but our ability to effectively translate this data to the clinic has not kept pace. Over the past 60 years, pharmaceutical companies have successfully demonstrated the safety and efficacy of over 1,200 novel therapeutic drugs via costly clinical studies. While this process must continue, better use can be made of the existing valuable data. In silico tools such as candidate gene prediction systems allow rapid identification of disease genes by identifying the most probable candidate genes linked to genetic markers of the disease or phenotype under investigation. Integration of drug-target data with candidate gene prediction systems can identify novel phenotypes which may benefit from current therapeutics. Such a drug repositioning tool can save valuable time and money spent on preclinical studies and phase I clinical trials.

Attitudes towards and beliefs about genetic testing in the haemophilia community: a qualitative study

Widespread genetic testing for haemophilia has recently been introduced in Victoria, Australia. While attitudes towards predictive testing have been studied in other conditions, such as cancer, there is limited knowledge about the attitudes of members of the haemophilia community towards predictive testing. This study aimed at exploring attitudes towards, and beliefs about, genetic testing amongst members of the haemophilia community in Victoria prior to the widespread introduction of testing. The study was qualitative and descriptive. In-depth face to face interviews were held with a sample of 39 individuals, including men with haemophilia, female carriers and family members. Data were analysed thematically using cross-case analysis techniques. There was considerable knowledge about the proposed introduction of widespread genetic testing. However, not everyone thought that testing was accessible or user friendly, and there was confusion about who needed to be tested. Most thought that testing was necessary for adolescent girls to determine carrier status to help prepare families for a child with haemophilia, rather than leading them to choose to terminate a pregnancy or not to have children. A minority of women stated that if there was a history of inhibitors in a family then a termination might be considered. The study revealed strong religious beliefs among those studied, which may have influenced attitudes and approaches towards testing. Further investigation is needed into how people with a possible haemophilia genotype negotiate decisions about their further identification, and how this knowledge is placed within cultural, religious and family contexts.