Using a weight of evidence approach to identify sources of microbiological contamination in a shellfish-growing area with "Restricted" classification.

Shellfish-growing areas in rural catchments are occasionally affected by elevated faecal contamination from diffuse sources and may be subject to frequent harvest closures/classification downgrades. We combined traditional risk management methods based on sanitary surveys and monitoring of Escherichia coli in seawater and shellfish with faecal source tracking, bacterial source apportionment, and hydrometeorological modelling to determine the causes of elevated E. coli concentrations contributing to harvest closures in Papanui Inlet (Aotearoa New Zealand). These multiple lines of evidence were used to inform a weight of evidence assessment of bacterial contamination in the inlet. Ruminant livestock was estimated to contribute 80% of the faecal coliform loading. Concentrations of E. coli in seawater were low (≤ 87 MPN 100 ml-1) whilst concentrations in tuaki/cockles/little neck clams (Austrovenus stutchburyi) occasionally exceeded the "Approved" classification limit (230 MPN 100 g-1). The most frequent positive genetic markers in seawater were the seagull (Catellicoccus marimammalium) (54% of seawater samples), and in shellfish, the bovine and seagull markers (both 12.5% of shellfish samples). Solar radiation was negatively correlated with E. coli in tuaki. We found that the growing area is affected by faecal inputs from animal and, to a lesser extent, human (septic tank discharges) sources which elevate contamination to levels detectable in tuaki but not in seawater, particularly in the summer months. The innovative approach can enhance the management of shellfish-growing areas affected by intermittent contamination and enables more targeted action to reduce pollution to improve shellfish water quality.

New gregarine species (Apicomplexa) from tunicates show an evolutionary history of host switching and suggest a problem with the systematics of Lankesteria and Lecudina.

Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts.

PCR and histology identify new bivalve hosts of Apicomplexan-X (APX), a common parasite of the New Zealand flat oyster Ostrea chilensis.

Apicomplexan-X (APX) is a significant pathogen of the flat oyster Ostrea chilensis in New Zealand. The life cycle and host range of this species are poorly known, with only the zoite stage identified. Here, we report the use of molecular approaches and histology to confirm the presence of APX in samples of green-lipped mussels Perna canaliculus, Mediterranean mussels Mytilus galloprovincialis and hairy mussels Modiolus areolatus collected from widely distributed locations in New Zealand. The prevalence of APX infection estimated by PCR was 22.2% (n = 99) and 50% (n = 30) in cultured green-lipped mussels from Nelson and Coromandel, respectively; 0.8% (n = 258), 3.3% (n = 150) and 35.3% (n = 17) in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively; and 46.7% (n = 30) in wild hairy mussels from Foveaux Strait. Histology detected all cases of PCR that were positive with APX and appeared to be more sensitive. The prevalence of APX estimated by histology in green-lipped mussels from Coromandel was 60% versus 50% by PCR, and 4.3%, 10.7% and 52.9% by histology versus 0.8%, 3.3% and 35.3% by PCR in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively. The specific identity of the parasite found in mussels was determined by sequencing PCR products for a portion (676 bp) of the 18S rRNA gene; the resulting sequences were 99-100% similar to APX found in flat oysters. Phylogenetic analyses also confirmed that all isolates from green-lipped, Mediterranean and hairy mussels grouped with APX isolates previously identified from flat oysters. This study indicates the wide geographical distribution of APX and highlights the potentially multi-host specific distribution of the parasite in commercially important bivalve shellfish.

PCR test to specifically detect the apicomplexan 'X' (APX) parasite found in flat oysters Ostrea chilensis in New Zealand.

Described here is a polymerase chain reaction (PCR) test to detect the apicomplexan-X (APX) parasite of a flat oyster species, Ostrea chilensis, endemic to New Zealand. The test primers target sequences in the in situ hybridisation probes identified to bind specifically to APX 18S rRNA and amplify a 723 bp DNA product. The test did not amplify 18S rRNA gene sequences of other apicomplexan species, including Toxoplasma gondii, Neospora caninum, Selenidium spp., Cephaloidophorida spp., Lecudina spp. and Thiriotia sp. Of 73 flat oysters identified by histology to be infected with APX at different severities, 69 (95%) tested PCR-positive. Failure to amplify an internal control indicated the presence of PCR inhibitors in the 4 PCR-negative samples. The high analytical sensitivity, specificity and speed of the PCR test should make it a useful tool for detecting APX.

Early detection of eukaryotic communities from marine biofilm using high-throughput sequencing: an assessment of different sampling devices.

Marine biofilms are precursors for colonization by larger fouling organisms, including non-indigenous species (NIS). In this study, high-throughput sequencing (HTS) of 18S rRNA metabarcodes was used to investigate four sampling methods (modified syringe, sterilized sponge, underwater tape and sterilized swab) for characterizing eukaryotic communities in marine biofilms. Perspex™ plates were sampled in and out of water. DNA collected with tape did not amplify. Otherwise, there were no statistical differences in communities among the remaining three sampling devices or between the two environments. Sterilized sponges are recommended for ease of use underwater. In-depth HTS analysis identified diverse eukaryotic communities, dominated by Metazoa and Chromoalveolata. Among the latter, diatoms (Bacillariophyceae) were particularly abundant (33% of reads assigned to Chromalveolata). The NIS Ciona savignyi was detected in all samples. The application of HTS in marine biofilm surveillance could facilitate early detection of NIS, improving the probability of successful eradication.

The phylogeography of Adelie penguin faecal flora.

The gut of animals changes quickly from a totally sterile environment before birth to a numerous and highly diverse microbial community shortly after birth. However, few studies have examined the source of the bacteria colonizing the gut. We used a genetic based approach to investigate the distribution and acquisition of faecal microbial communities by Adelie penguins, Pygoscelis adeliae, breeding in the Ross Sea region of Antarctica by cloning a portion of the 16S rRNA gene and by automated ribosomal intergenic spacer analysis (ARISA). We hypothesized that bacteria were either acquired from the penguins' neighbours or inherited from their ancestors. Samples were collected from Adelie penguin breeding colonies at the north-western edge of the Ross Sea coast through to the southernmost Adelie penguin colonies on Ross Island. Most of the bacterial sequences we obtained were only distantly homologous with previously published sequences. Bacterial taxa appear to have a restricted distribution as the majority of 16S rRNA clones were isolated from only one or two hosts. Faecal bacterial community similarity was strongly negatively correlated with the genetic distance between hosts suggesting that bacterial communities are inherited. There was little support for a correlation between distance between collection sites and community similarity.

Biotic interactions are an unexpected yet critical control on the complexity of an abiotically driven polar ecosystem.

Abiotic and biotic factors control ecosystem biodiversity, but their relative contributions remain unclear. The ultraoligotrophic ecosystem of the Antarctic Dry Valleys, a simple yet highly heterogeneous ecosystem, is a natural laboratory well-suited for resolving the abiotic and biotic controls of community structure. We undertook a multidisciplinary investigation to capture ecologically relevant biotic and abiotic attributes of more than 500 sites in the Dry Valleys, encompassing observed landscape heterogeneities across more than 200 km2. Using richness of autotrophic and heterotrophic taxa as a proxy for functional complexity, we linked measured variables in a parsimonious yet comprehensive structural equation model that explained significant variations in biological complexity and identified landscape-scale and fine-scale abiotic factors as the primary drivers of diversity. However, the inclusion of linkages among functional groups was essential for constructing the best-fitting model. Our findings support the notion that biotic interactions make crucial contributions even in an extremely simple ecosystem.

Multi-host parasite species in cophylogenetic studies.

Cophylogenetic studies examine the relationship between host and parasite evolution. One aspect of cophylogenetic studies that has had little modern discussion is parasites with multiple definitive hosts. Parasite species with multiple host species are anomalous as, under a codivergence paradigm, speciation by the hosts should cause speciation of their parasites. We discuss situations such as cryptic parasite species, recent host switching or failure to speciate that may generate multi-host parasites. We suggest methods to identify which of the mechanisms have led to multi-host parasitism. Applying the suggested methods may allow multi-host parasites to be integrated more fully into cophylogenetic studies.

Phylogeny of the parasitic microgastroid subfamilies (Hymenoptera: Braconidae) based on sequence data from seven genes, with an improved time estimate of the origin of the lineage.

The microgastroid complex of braconid wasps is a widely recognized and biologically coherent lineage of endoparasitoids of lepidopteran larvae (caterpillars). The complex has received significant phylogenetic attention in recent years due in part to the taxons' association with mutualistic polydnaviruses, with which they compromise host immune systems. A number of previous attempts using a variety of morphological and molecular approaches have not unequivocally resolved relationships amongst the main subfamilies. This work represents a more extensive attempt to resolve the microgastroid relationships, using seven genes (16S rRNA, cytochrome oxidase I (CO1), 28S rRNA, arginine kinase (ArgK), long wavelength rhodopsin (Ops), elongation factor 1 alpha (EF1a) and wingless (Wg)) and a greater taxonomic representation. Bayesian, likelihood and parsimony phylogenetic reconstructions of this improved data set has determined that the chelonines diverged first from the remainder of the microgastroids, however the relationships amongst the other subfamilies are still unclear, suggesting a greater nucleotide sample is required to resolve them. Examination of the contribution of individual gene trees to the phylogeny demonstrates why the relationships between subfamilies are still unclear, with not all groups monophyletic for all trees. Filtered supernetworks demonstrate that monophyly of all subfamilies is only recovered when splits found in only one or two genes are excluded, but this also results in little remaining structure left in the deep nodes to resolve inter-subfamily relationships. By increasing the breadth of the study we were also able to re-evaluate previous attempts at dating the lineage and, therefore the origin of the polydnavirus association. Previous attempts used a much reduced data set and fewer fossil calibrations. Thorough literature searches have revealed a substantial increase in the fossil calibrations and these, combined with more sophisticated molecular dating analysis, have substantially increased the age of the microgastroid lineage from previous estimates of approximately 73MYA to approximately 100MYA. Examination of the resultant linearized clock tree also allows an insight into the evolution of the more species rich subfamilies. The chelonines appear to have had a steady rate of evolution, whilst the microgastrines and cardiochilines appear to have undergone a more significant "burst" of evolution. It is hypothesized that the different parasitism strategies of subfamilies (Chelonines are egg parasitoids and the remainder are larval parasitioids) may have influenced the evolutionary rates of the groups.

Genomic and morphological features of a banchine polydnavirus: comparison with bracoviruses and ichnoviruses.

Many ichneumonid and braconid endoparasitoids inject a polydnavirus (PDV) into their caterpillar hosts during oviposition. The viral entities carried by wasps of these families are referred to as "ichnoviruses" (IVs) and "bracoviruses" (BVs), respectively. All IV genomes characterized to date are found in wasps of the subfamily Campopleginae; consequently, little is known about PDVs found in wasps of the subfamily Banchinae, the only other ichneumonid taxon thus far shown to carry these viruses. Here we report on the genome sequence and virion morphology of a PDV carried by the banchine parasitoid Glypta fumiferanae. With an aggregate genome size of approximately 290 kb and 105 genome segments, this virus displays a degree of genome segmentation far greater than that reported for BVs or IVs. The size range of its genome segments is also lower than those in the latter two groups. As reported for other PDVs, the predicted open reading frames of this virus cluster into gene families, including the protein tyrosine phosphatase (PTP) and viral ankyrin (ank) families, but phylogenetic analysis indicates that ank genes of the G. fumiferanae virus are not embedded within the IV lineage, while its PTPs and those of BVs form distinct clusters. The banchine PDV genome also encodes a novel family of NTPase-like proteins displaying a pox-D5 domain. The unique genomic features of the first banchine virus examined, along with the morphological singularities of its virions (IV-like nucleocapsids, but enveloped in groups like some of the BVs), suggest that they could have an origin distinct from those of IVs and BVs.

Dissecting the ancient rapid radiation of microgastrine wasp genera using additional nuclear genes.

Previous estimates of a generic level phylogeny for the ubiquitous parasitoid wasp subfamily Microgastrinae (Hymenoptera) have been problematic due to short internal branches deep in the phylogeny. These short branches might be attributed to a rapid radiation among the taxa, the use of genes that are unsuitable for the levels of divergence being examined, or insufficient quantity of data. We added over 1200 nucleotides from four nuclear genes to a dataset derived from three genes to produce a dataset of over 3000 nucleotides per taxon. While the number of well-supported short branches in the phylogeny increased, we still did not obtain strong bootstrap support for every node. Parametric and nonparametric bootstrap simulations projected that an enormous, and likely unobtainable, amount of data would be required to get bootstrap support greater than 50% for every node. However, a marked increase in the number of well-supported nodes was seen when we conducted a Bayesian analysis of a combined dataset generated from morphological characters added to the seven gene dataset. Our results suggest that, in some cases, combining morphological and genetic characters may be the most practical way to increase support for short branches deep in a phylogeny.