Isolation and characterization of 16 polymorphic microsatellite loci for the Asian green mussel Perna viridis (Mollusca, Mytilidae)

The Asian green mussel Perna viridis is an abundant and important ecological and economical species across its native range. However, outside its native range, this species has been considered invasive and concerns have been raised worldwide regarding its potential impacts. Despite this, little work has been done to investigate the genetics of native and/or introduced populations of this species. In the present study, we developed 16 new polymorphic microsatellite markers using the Illumina MiSeq Platform. Four to 15 alleles per locus were detected. There was no evidence of linkage disequilibrium between pairs of loci and all loci were in Hardy-Weinberg equilibrium

Early Vascular and Neuronal Changes in a VEGF Transgenic Mouse Model of Retinal Neovascularization

PURPOSE. To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR). METHODS. Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy. RESULTS. Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest. CONCLUSIONS. Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease. (Invest Ophthalmol Vis Sci. 2006;47:4638 -4645

Establishment of a taxonomic and molecular reference collection to support the identification of species regulated by the Western Australian Prevention List for Introduced Marine Pests

Introduced Marine Pests (IMP, = non-indigenous marine species) prevention, early detection and risk-based management strategies have become the priority for biosecurity operations worldwide, in recognition of the fact that, once established, the effective management of marine pests can rapidly become cost prohibitive or impractical. In Western Australia (WA), biosecurity management is guided by the “Western Australian Prevention List for Introduced Marine Pests” which is a policy tool that details species or genera as being of high risk to the region. This list forms the basis of management efforts to prevent introduction of these species, monitoring efforts to detect them at an early stage, and rapid response should they be detected. It is therefore essential that the species listed can be rapid and confidently identified and discriminated from native species by a range of government and industry stakeholders. Recognising that identification of these species requires very specialist expertise which may be in short supply and not readily accessible in a regulatory environment, and the fact that much publicly available data is not verifiable or suitable for regulatory enforcement, the WA government commissioned the current project to collate a reference collection of these marine pest specimens. In this work, we thus established collaboration with researchers worldwide in order to source representative specimens of the species listed. Our main objective was to build a reference collection of taxonomically vouchered specimens and subsequently to generate species-specific DNA barcodes suited to supporting their future identification. To date, we were able to obtain specimens of 75 species (representative of all but four of the pests listed) which have been identified by experts and placed with the WA Government Department of Fisheries and, where possible, in accessible museums and institutions in Australasia. The reference collection supports the fast and reliable taxonomic and molecular identification of marine pests in WA and constitutes a valuable resource for training of stakeholders with interest in IMP recognition in Australia. The reference collection is also useful in supporting the development of a variety of DNA-based detection strategies such as real-time PCR and metabarcoding of complex environmental samples (e.g. biofouling communities). ThePrevention List is under regular review to ensure its continued relevance and that it remains evidence and risk-based. Similarly, its associated reference collection also remains to some extent a work in progress. In recognition of this fact, this report seeks to provide details of this continually evolving information repository publicly available to the biosecurity management community worldwid

Molecular tools for identification of shark species involved in depredation incidents in Western Australian fisheries.

Shark depredation is an issue of concern in some Western Australian recreational and commercial fisheries where it can have economic, social and ecological consequences. Knowledge of the shark species involved is fundamental to developing effective management strategies to mitigate the impacts of depredation. Identification of the species responsible is difficult as direct observation of depredation events is uncommon and evaluating bite marks on fish has a high degree of uncertainty. The use of trace DNA techniques has provided an alternative method for species identification. We demonstrate proof of concept for a targeted DNA barcoding approach to identify shark species using trace DNA found at bite marks on recovered remains of hooked fish. Following laboratory validation, forensic analysis of swabs collected from samples of bitten demersal fish, led to the definitive identification of shark species involved in 100% of the incidences of depredation (n = 16)

Development of a real-time PCR (qPCR) method for the identification of the invasive paddle crab Charybdis japonica (Crustacea, Portunidae)

Crabs can be transported beyond their native range via anthropogenic-mediated means such as aquarium trade, live seafood trade and shipping. Once introduced into new locations, they can establish persisting populations and become invasive, often leading to negative impacts on the recipient environment and native species. Molecular techniques are increasingly being used as complementary tools in biosecurity surveillance and monitoring plans for invasive species. Molecular tools can be particularly useful for early detection, rapid identification and discrimination of closely related species, including when diagnostic morphological characters are absent or challenging, such as early life stages, or when only part of the animal is available. In this study, we developed a species-specific qPCR assay, which targets the cytochrome c oxidase subunit 1 (CO1) region of the Asian paddle crab Charybdis japonica. In Australia, as well as many parts of the world, this species is considered invasive and routine biosecurity surveillance is conducted to reduce the risk of establishment. Through rigorous testing of tissue from target and non-target species we demonstrate that this assay is sensitive enough to detect as little as two copies per reaction and does not cross amplify with other closely related species. Field samples and environmental samples spiked with C. japonica DNA in high and low concentrations indicate that this assay is also a promising tool for detecting trace amounts of C. japonica eDNA in complex substrates, making it a useful complementary tool in marine biosecurity assessments

Functional topography and integration of the contralateral and ipsilateral retinocollicular projections of ephrin-A-/- mice.

Topographically ordered projections are established by molecular guidance cues and refined by neuronal activity. Retinal input to a primary visual center, the superior colliculus (SC), is bilateral with a dense contralateral projection and a sparse ipsilateral one. Both projections are topographically organized, but in opposing anterior-posterior orientations. This arrangement provides functionally coherent input to each colliculus from the binocular visual field, supporting visual function. When guidance cues involved in contralateral topography (ephrin-As) are absent, crossed retinal ganglion cell (RGC) axons form inappropriate terminations within the SC. However, the organization of the ipsilateral projection relative to the abnormal contralateral input remains unknown, as does the functional capacity of both projections. We show here that in ephrin-A(-/-) mice, the SC contains an expanded, diffuse ipsilateral projection. Electrophysiological recording demonstrated that topography of visually evoked responses recorded from the contralateral superior colliculus of ephrin-A(-/-) mice displayed similar functional disorder in all genotypes, contrasting with their different degrees of anatomical disorder. In contrast, ipsilateral responses were retinotopic in ephrin-A2(-/-) but disorganized in ephrin-A2/A5(-/-) mice. The lack of integration of binocular input resulted in specific visual deficits, which could be reversed by occlusion of one eye. The discrepancy between anatomical and functional topography in both the ipsilateral and contralateral projections implies suppression of inappropriately located terminals. Moreover, the misalignment of ipsilateral and contralateral visual information in ephrin-A2/A5(-/-) mice suggests a role for ephrin-As in integrating convergent visual inputs

Research horizons for invasive marine species detection with eDNA/eRNA

The global marine ecosystem is changing rapidly as the result of biogeochemical cycles and ecosystem structure being altered by industrial civilization. Invasive marine species (IMS) are one of the most damaging regional consequences of human activity, and one of the most easily attributable to specific processes. This makes IMS introduction one of most tractable threats for management by appropriate policies. Once established, a different set of policies are required either to restrict IMS spread, or to attempt local eradication. The key ecosystem management tool for IMS damage mitigation is rapid, widely deployable IMS detection. Environmental Nucleic Acids (eNA), combining environmental DNA (eDNA) and environmental RNA (eRNA) analyses, have emerged as valuable tools for sensitive, cost-effective and readily deployable detection of IMS. Methods for IMS detection by eNA are still being developed through a widespread and active research community, so identifying the limitations of current processes will help prioritise eNA-based IMS detection research. We analysed and synthesised the opinions of expert marine ecosystem managers and researchers in Australia and New Zealand about the knowledge gaps and research needs for eNA-based IMS detection. This synthesis was placed in context with current research literature on what eNA technologies are currently providing as an IMS management tool; what problems exist with the current technology; and what could be done to improve this general approach. Our analyses produced a list of priorities that chart a path towards the best possible systems for IMS detection by eNA.</p

Genetic diversity of a hitchhiker and prized food source in the Anthropocene: the Asian green mussel Perna viridis (Mollusca, Mytilidae)

Insight into a species’ native and introduced range is essential in understanding the invasion process. Genetic diversity, propagule pressure and environmental conditions all have been recognised as playing a determinant role in invasion success. Here, we aimed to investigate the genetic diversity and population genetic structure (using the COI mtDNA gene region and 22 nDNA microsatellite markers) of the Asian green mussel Perna viridis within its potential native range in Asia and at introduced locations in the USA and the Caribbean. We also analyse genetic data from vessel intercepts and an incursion. By doing so, we aimed to identify genetic signatures that could allow to track vessel samples to their source and provide further insight into potential high-risk invasive populations or areas. Three top hierarchical clusters were identified using the individual-based Bayesian clustering method in STRUCTURE, corresponding to populations in three world regions: (1) USA and Caribbean, (2) India and (3) Southeast Asia. Within Southeast Asia, additional analysis indicate a shallow genetic differentiation of three subgroups consisting of (3a) Thailand, (3b) Taiwan and Hong-Kong, and (3c) a cluster of Singapore–Indonesia samples. Overall, the population structure found in this study suggests that the markers used could be useful in identifying source populations, particularly between the three mains world regions. Most surprisingly however, this study shows that the genetic diversity of samples collected from vessel intercepts and incursions did not differ significantly from established populations in Southeast Asia. In this region, in addition to the high vessel connectivity and number of P. viridis transported, all sampled populations are likely to pose a comparable risk in terms of genetic diversity. The present work represents the most comprehensive population genetic study of P. viridis, and the first to address the potential genetic introduction risk posed by populations of this species. The information and genetic markers in this study constitute a valuable addition to the tools already used to infer on potential high-risk source populations of P. viridis. They should therefore prove useful for biosecurity surveillance and management actions directed at this species