Journal articles on the topic 'Fish virome'

While planktonic viruses have received much attention in recent decades, knowledge of the virome of marine organisms, especially fish, still remains rudimentary. This is notably the case with tuna, which are among the most consumed fish worldwide and represent considerable economic, social and nutritional value. Yet the composition of the tuna virome and its biological and environmental determinants remain unknown. To begin to address this gap, we investigated the taxonomic diversity of viral communities inhabiting the skin mucus, gut and liver of two major tropical tuna species (skipjack and yellowfin) in individuals fished in the Atlantic and Indian Oceans. While we found significant differences in the virome composition between the organs, this was totally independent of the tuna species or sex. The tuna virome was mainly dominated by eukaryotic viruses in the digestive organs (gut and liver), while bacteriophages were predominant in the mucus. We observed the presence of specific viral families in each organ, some previously identified as fish or human pathogens (e.g., Iridoviridae, Parvoviridae, Alloherpesviridae, Papillomaviridae). Interestingly, we also detected a ‘core virome’ that was shared by all the organs and was mainly composed of Caudovirales, Microviridae and Circoviridae. These results show that tuna host a mosaic of viral niches, whose establishment, role and circulation remain to be elucidated.

Much of the knowledge on viruses is focused on those that can be propagated using cell-cultures or that can cause disease in humans or in economically important animals and plants. However, this only reflects a small portion of the virosphere. Therefore, in this study, we explore by targeted next-generation sequencing, how the virome varies between Atlantic horse mackerels and gilthead seabreams from fisheries and aquaculture from the center and south regions of Portugal. Viral genomes potentially pathogenic to fish and crustaceans, as well as to humans, were identified, namely Astroviridae, Nodaviridae, Hepadnaviridae, Birnaviridae, Caliciviridae, and Picornaviridae families. Also bacteriophages sequences were identified corresponding to the majority of sequences detected, with Myoviridae, Podoviridae, and Siphoviridae, the most widespread families in both fish species. However, these findings can also be due to the presence of bacteria in fish tissues, or even to contamination. Overall, seabreams harbored viruses from a smaller number of families in comparison with mackerels. Therefore, the obtained data show that fish sold for consumption can harbor a high diversity of viruses, many of which are unknown, reflecting the overall uncharacterized virome of fish. While cross-species transmission of bonafide fish viruses to humans is unlikely, the finding of human pathogenic viruses in fish suggest that fish virome can be a potential threat regarding food safety.

ABSTRACTLymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affectedS. auratafish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of thePapillomaviridaefamily described in fish, provides evidence for a more ancient origin of this family than previously thought.IMPORTANCELymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the familyIridoviridae. In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified withinS. auratalymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

How many parasites are there on Earth? Here, we use helminth parasites to highlight how little is known about parasite diversity, and how insufficient our current approach will be to describe the full scope of life on Earth. Using the largest database of host–parasite associations and one of the world’s largest parasite collections, we estimate a global total of roughly 100 000–350 000 species of helminth endoparasites of vertebrates, of which 85–95% are unknown to science. The parasites of amphibians and reptiles remain the most poorly described, but the majority of undescribed species are probably parasites of birds and bony fish. Missing species are disproportionately likely to be smaller parasites of smaller hosts in undersampled countries. At current rates, it would take centuries to comprehensively sample, collect and name vertebrate helminths. While some have suggested that macroecology can work around existing data limitations, we argue that patterns described from a small, biased sample of diversity aren’t necessarily reliable, especially as host–parasite networks are increasingly altered by global change. In the spirit of moonshots like the Human Genome Project and the Global Virome Project, we consider the idea of a Global Parasite Project: a global effort to transform parasitology and inventory parasite diversity at an unprecedented pace.

Viruses that infect fish are understudied, yet they provide important evolutionary context to the viruses that infect terrestrial vertebrates. We surveyed gill tissue meta-transcriptomes collected from two species of native freshwater fish from Aotearoa New Zealand—Retropinna retropinna and Gobiomorphus cotidianus. A total of 64 fish were used for gill tissue meta-transcriptomic sequencing, from populations with contrasting life histories—landlocked (i.e., lacustrine) and diadromous—on the South Island and Chatham Islands. We observed that both viral richness and taxonomic diversity were significantly associated with life history and host species, with lacustrine R. retropinna characterised by higher viral alpha diversity than diadromous R. retropinna. Additionally, we observed transcripts of fish viruses from 12 vertebrate host-associated virus families, and phylogenetically placed eight novel RNA viruses and three novel DNA viruses in the Astroviridae, Paramyxoviridae, Orthomyxoviridae, Rhabdoviridae, Totiviridae, Poxviridae, Alloherpesviridae, and Adintoviridae in their evolutionary contexts. These results represent an important survey of the viruses that infect two widespread native fish species in New Zealand, and provide insight useful for future fish virus surveys.

Abstract Feed plays an important role in fish farming. Feed requirements achieve 60-70% of the cost of fish farming operations. Availability of feed with quality and sufficient quantity is aimed in increasing fish production. A very high feed prices due to artificial feed ingredients used are expensive and required in large quantities. Therefore, it is necessary to find alternatives to fish directly or indirectly obtaining appropiate and adequate nutrition needs to grow. The raw material content used for feed is coconut dregs (Cocos nucifera). The availability of the coconut dregs is abundant and potensial for fish feed ingredients. The coconut dregs is also one type or plantation waste which still has potential to be processed into the manufacture of fish feed. The coconut dregs flour can be processed by fermentation to improve quality. This study was aimed to determine the increase in crude protein content and a decrease in crude fiber content of coconut dregs fermented with Trichoderma viride; Bacillus subtilis and EM4. The research method was an experiment with Completely Randomized Design (CRD). The treatments used without the addition of microbes (P0), Bacillus subtilis 6% (P1), Trichoderma viride 6% (P2), and EM4 6% (P3) with 5 repeatitions each. The parameters observed were the content of crude protein and crude fiber after 7 days of fermentation. Data analysis used Analysis of Variance (ANOVA) and to determine the best treatments using Duncan Multiple Distance Test with 5% confidence interval. The results indicated that coconut dregs fermented with Bacillus subtilis (P1), Trichoderma viride (P2), and EM4 (P3) produced the difference of crude protein and fiber. The best treatment on the increase in crude protein content was Bacillus subtilis (P1) of 7.5564%. The best treatment on the decrease in crude fiber content was EM4 (P3) of 22.3967%. However, the results of the flour fermented coconut dregs can not be used as an alternative feed material because the fermented coconut dregs is not qualified for fish feed. This is due to a lack of crude protein and high crude fiber content.

Abstract Utilization of fish waste as an alternative feed ingredient in the effort to increase the availability of raw materials making up rations. Banana skin is one that has the potential of agricultural waste into alternative feed ingredients, but the nutritional value contained in the banana peel is still low so we need to attempt to improve nutritional content, one of which is fermentation method using Trichoderma viride and Bacillus subtilis in order to improve crude protein and lower content of crude fiber on a banana peel. This study aims to determine the influence of Trichoderma viride on crude protein and crude fiber are fermented banana skin, know the effect of Bacillus subtilis on crude protein and crude fiber are fermented banana skin and know the effect of interaction of Trichoderma viride and Bacillus subtilis of crude protein and fiber fermented banana skin rough. This research method is to experiment with Completely Randomized Design (CRD) as a factorial experimental design. The treatments used consisted of two factors, namely the effect of Trichoderma viride (factor A) and Bacillus subtilis (factor B). Factor A consisted of three dose levels: 0%, 3% and 6%, while factor B consists of three levels ie dose of 0%, 4% and 8%. Each treatment combination was replicated 3 times. The results showed that the use of Trichoderma viride and Bacillus subtilis in fermentation processes plantain skin showed significant differences on crude protein and crude fiber on dry matter (p<0.05).

Abstract Invasive crayfish are known to displace native crayfish species, alter aquatic habitat and community structure and function, and are serious pests for fish hatcheries. White River Crawfish (WRC; Procambarus acutus) were inadvertently introduced to a warm-water fish hatchery in Missouri, USA, possibly in an incoming fish shipment. We evaluated the use of chemical control for crayfish to ensure incoming and outgoing fish shipments from hatcheries do not contain live crayfish. We conducted acute (≤ 24 hr) static toxicity tests to determine potency, dose-response, and selectivity of pesticides to WRC, Virile Crayfish (VC; Orconectes virilis), and Fathead Minnow (FHM; Pimephales promelas). Testing identified a formulation of cypermethrin (Cynoff®) as the most potent of five pesticides evaluated for toxicity to crayfish. A 4-hr exposure to a cypermethrin concentration of 100 μg·L-1 was found to kill 100% of juvenile and adult WRC; however, adult VC were not consistently killed. Concentrations of cypermethrin ≤ 100 μg·L-1 did not cause significant (> 10%) mortality in juvenile FHM. Additional testing is needed to examine selectivity between crayfish and hatchery fish species. Biosecurity protocols at hatcheries that use chemical control have the potential to reliably prevent inadvertent transfers of live crayfish in fish shipments.

Ecological theory predicts that the timing of the transition between life-history stages should vary with the costs and benefits associated with each stage. For example, the timing of hatching or metamorphosis may vary with the predation risk in each stage. Predator-induced changes in hatching time are well documented in some taxa but have not been reported in fishes. We provide the first empirical evidence that a species of fish can alter its hatching time in response to predator cues. We showed that fathead minnows, Pimephales promelas Rafinesque, 1820, exposed to chemical cues from virile crayfish, Orconectes virilis (Hagen, 1870), foraging on minnow embryos hatch sooner than those exposed to a blank control. Moreover, in the presence of cues from virile crayfish feeding on minnow embryos, the hatchlings exhibited a change in morphology because they had significantly shorter total lengths. There was a significant positive correlation between hatchling size and length of the developmental period, except in the high predation threat treatment. By hatching earlier, the fathead minnows escaped predation from virile crayfish that were actively foraging on fathead minnow eggs. Hatching at a smaller size, however, may make the fry more vulnerable to other predators.

Environmental reservoirs of zooxanthellae are essential for coral larvae settlement; understanding where they occur and how they are maintained is important for coral reef ecology. This study investigated the dispersal of Symbiodinium spp. by the stoplight parrotfish Sparisoma viride , which had high mean densities of viable and cultivable Symbiodinium (3207–8900 cells ml −1 ) in faeces. Clades A, B and G were detected using amplified chloroplast ribosomal sequences (cp23S-HVR), and corresponded with diet preferences of fish and the environmental Symbiodinium diversity of the region. Cells are constantly dispersed in the water column and deposited in the substrate at a local level (86 ± 17.8 m 2 ), demonstrating that parrotfishes are vectors for short-distance dispersal of zooxanthellae. Such dispersal could constitute a key role in the maintenance of environmental Symbiodinium reservoirs.

Omega fatty acids, major importance in the prevention or treatment of a range of human diseases or disorders related with inflammation. These fatty acids are found in transgenic plants, fungi, and animals and even in microorganisms but in major amounts can be extracted from fatty fish. However, due to bioaccumulation of fat-soluble vitamins and high levels of saturated and omega-6 fatty acids, they may have deleterious health effects. It becomes necessary to search for novel and rich sources containing omega-3 fatty acids and one of the alternatives include fungi. The present study deals with production and purification of omega-3 fatty acids from Trichoderma viride and Aspergillus niger. In the present study, the main objective was to explore the beneficial effects of fungi for the maximum lipid production through optimized conditions and the results clearly showed that Trichoderma viride was the significantly highest lipid producer, with lipid production at initial pH 6.0 and incubation temperature 40℃.

Southern White River Crayfish (Procambarus zonangulus), an aquatic, potentially invasive species, is documented from New Brunswick for the first time. It was found in a small, privately owned, lake in the Saint John River system that was apparently stocked for recreational purposes with non-native fish and the crayfish. Procambarus zonangulus has successfully overwintered at the site for at least a year and, more likely, for several years. This is the third species of non-native crayfish recorded in New Brunswick, joining Spiny-cheeked Crayfish (Faxonius limosus) and Virile Crayfish (Faxonius virilis). This is also the first persisting introduction for the genus Procambarus in Canada of which we are aware.

Because herbivory is a critical component of resilient reefs, there is a need to investigate the dynamics of herbivorous fish assemblages over various spatiotemporal scales. During the period of 2011–2019, 483 belt transect surveys were conducted across 26 sites around New Providence to assess the status of parrotfish populations across different reefs. Non-metric multidimensional scaling revealed two distinct parrotfish assemblages around New Providence temporally, differing between 2019 and earlier surveys, and spatially between fore and patch reef zones. Temporal analysis showed a significant decrease (59%) in parrotfish densities across reef sites and significant changes in mean density over time for three species Scarus iseri, Sparisoma aurofrenatum and Sp. atomarium. Changes in the size frequency distribution of parrotfish—particularly reductions in individuals ≥31 cm in size and the complete loss of fish >40 cm across all sites were found. Interactions between reef zone and size class were significant with the greater frequencies of larger individuals (≥21 cm) driving patterns (positive associations) on forereefs. These patterns also appear to be partly driven by variability in the abundance and size composition of Sparisoma viride, which is one of the species primarily targeted by Bahamian fishers, as well as one of the most common parrotfish across reef zones.

AbstractAquatic prey often alter their morphology, physiology, and/or behaviour when presented with predatory chemical cues which are heavily influenced by the diet of the predator. We tested the roles that diet and prey familiarity with predators play in the ability of prey to recognize predator threats. Odours from two fish, bass and cichlid fed a vegetarian, protein, heterospecific, and a conspecific diet, were collected and presented to virile crayfish in a choice arena. Our results show that crayfish altered their behaviour in the presence of odours containing conspecific, as opposed to heterospecific diets, but only from familiar predators. A reduced anti-predator response was measured with odours from an unfamiliar predator fed conspecific crayfish. Therefore, crayfish may be able to determine different threat levels based on the different dietary cues from a potential predator, but only when the prey have familiarity with the predators.

<p class="NoParagraphStyle" align="center"><strong>ABSTRACT</strong></p><p class="NoParagraphStyle" align="center"> </p><p class="NoParagraphStyle">This research aimed at determining digestibility coefficient of cassava peel<em>(Manihot utilissim</em>a) after immersion in 3% (w/v) NaOH for three days, fermentation using combined fungi of 10% <em>Trichoderma viride</em> and <em>Phanerochaete chrysosporium</em> for seven days, and fermentation using 15% (w/w) <em>Bacillus megaterium</em> for five days as feed ingredients for Nile tilapia <em>Oreochromis niloticus</em>. Total digestibility test was conducted by mixing 30% of cassava peel and 70% of reference diet. Nile tilapia at the average weight of 16.6 g were used as experimental fish. Fish was held for 28 days in aquarium (50x50x50 cm³) at the density of 10 fish/aquarium. Fish were fed twice daily to satiation. Feces collection started after five days of adaptation to chromium oxide diets. The results showed that the three treatments had significant effects compared to control (P&lt;0.05), protein digestibility of were improved 5%, 15%, and 10%, energy digestibilitiy were 20%, 18%, 16%, and total digestibility of test cassava peel were 174%, 151%, and 164%, respectively. Cassava peel fermented with combined 10% mold showed the highest protein digestibility impliying it potency as feed ingredient for Nile tilapia diet.</p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle">Keywords: Nile tilapia, cassava peel, NaOH, mold, bacteria, digestibility</p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle" align="center"><strong>ABSTRAK</strong></p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle">Penelitian ini bertujuan untuk mengevaluasi kecernaan kulit ubi kayu (<em>Manihot utilissima</em>) setelah perendaman dengan NaOH 3% (w/v) selama tiga hari, fermentasi kapang <em>Trichoderma viride</em> dan <em>Phanerochaete chrysosporium</em> 10% (w/w) selama tujuh hari, dan fermentasi bakteri <em>Bacillus megaterium</em> 15% (w/w) selama lima hari sebagai bahan baku pakan ikan nila. Uji kecernaan total bahan dilakukan dengan mencampurkan 30% kulit ubi kayu dengan 70% pakan acuan. Ikan uji yang digunakan adalah ikan nila dengan bobot rata-rata 16,6 g. Ikan dipelihara selama 28 hari dengan kepadatan 10 ekor/akuarium berukuran 50x50x50 cm³. Pemberian pakan dilakukan dua kali sehari secara <em>at satiation</em>. Pengumpulan feses dimulai setelah lima hari adaptasi pakan uji yang diberi indikator kromium oksida. Hasil penelitian menunjukkan bahwa ketiga perlakuan memberikan pengaruh yang berbeda nyata terhadap kontrol (P&lt;0,05) dan meningkatkan nilai kecernaan protein pakan berturut-turut sebesar 5%, 15%, dan 10%, nilai kecernaan energi sebesar 20%, 18%, dan 16%, serta nilai kecernaan total bahan sebesar 174%, 151%, dan 164%. Perlakuan kulit ubi kayu yang difermentasi dengan kapang menunjukkan nilai kecernaan protein pakan yang tertinggi sehingga berpotensi sebagai bahan baku pakan ikan nila.</p><p class="NoParagraphStyle"> </p><p>Kata kunci: ikan nila, kulit ubi kayu, NaOH, kapang, bakteri, kecernaan</p>

The role of a marine reserve and fisheries regulations in restoring fish populations on reefs disturbed by climate impacts was evaluated. Eight patch reefs, divided equally between no-fishing and fished zones in the remote Glover’s Reef atoll lagoon, were studied for 22 yr: 13 yr before and 9 yr after a ban on parrotfish capture. Findings indicate that the main effect of the fisheries closure was the recovery of targeted carnivorous species, notably snappers, jacks, and groupers. Recovery continued for most of the time series, including the later period when parrotfish capture was banned. Parrotfish abundance slowly declined in both management zones and across the ban period. The loss occurred for both small non-fished species, such as the striped parrotfish Scarus iserti, and for larger fished species, such as the stoplight Sparisoma viride and red-band parrotfish Sparisoma aurofrenatum. Consequently, parrotfish abundance appeared to be controlled by the ecology of these patch reefs rather than fishing mortality. We suggest that the high and persistent cover of late-successional algae reduces the renewal rates of algae, which had negative consequences for all studied parrotfish populations. Low ocean currents and physical energy in the lagoon appeared to promote algal persistence. Thus, parrotfish bans may be more effective in promoting reef recovery in environments that promote rapid algal turnover. Fisheries regulations are unlikely to rapidly restore hard corals on these patch reefs, which have slowly transitioned to algal dominance since first described in 1970.

Avian nests are a unique and sometimes extreme environment in which fungi occur. In this study, a correlation was recorded between the breeding biology and ecology of wetland birds and the biology and ecology of fungi in nests of wetland birds. The abundance of ecophysiologically diversified fungi, i.e. saprotrophs, cellulolytic fungi, and potentially zoo- and phytopathogenic fungi, was shown to be significantly higher in large nests, while species diversity (Shannon index) of fungi in nests with similar properties was not significant. The taxonomic structure (genus and species composition) and the spatial structure (frequency) of the nest mycobiota are mainly affected by nests’ specific physical and chemical properties which depend on the breeding and feeding preferences of the birds. In dry, highly sun-exposed nests of the Grey Heron (Ardea cinerea), a species that establishes breeding colonies high in the trees, mainly feeds on fish and has young who defecate into the nest, populations of xerophilic, alkali- and thermotolerant fungi, including keratinolytic fungi (Chrysosporium tropicum), developed. In the nests of the Mute Swan (Cygnus olor), a precocial species which constructs very large, relatively low-hygiene nests at the water's edge and which feeds on plant food, populations of hydrophilic and thermotolerant fungi, including highly cellulolytic fungi such as Chaetomium globosum, were detected. Nests of other small species of wetland birds, whose nests are also located on water but contain smaller amounts of animal-derived material, did not differ significantly mycologically and were colonised mainly by species such as Trichoderma viride and Penicillium purpurogenum, ubiquitous fungi with very high water and thermotolerant requirements.

Abstract Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterized the viromes of nineteen wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterization. From this, we identified twenty-five new vertebrate-associated viruses and a further twenty-two viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, positive-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae, and Flaviviridae, and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition—viral richness, abundance, and diversity—can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi, and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

ABSTRACT Chicken is one of the economically important poultry species. Avian leucosis virus subgroup J (ALV-J) has emerged as a serious cause of mortality and suboptimal performance of domestic chickens. Changes in virome may contribute to pathogenesis. Thus, it is important to investigate the effects of ALV-J infection on the composition of the virome in chicken. In the study metagenomic sequencing was used to characterize the virome of feces collected from the AVL-J infected chickens and the controls. Our results indicated that the chicken gut virome contained a diverse range of viruses that can be found in mammal, reptile, fish, and frogs. Furthermore, at the order, family and genus levels, AVL-J infection significantly altered the chicken gut virome composition. The predominant order was Herpesvirales, accounting for more than 96% of the chicken gut virome. Furthermore, the relative abundance of Caudovirales in the controls was higher than that in the AVL-J-infected chickens. At the family level, the relative abundance of Herpesviridae, Myoviridae, Alloherpesviridae, and Genomoviridae was significantly altered in the AVL-J-infected chickens compared with that in the controls. Additionally, the relative abundance of 15 genera showed a significant difference between the AVL-J-infected chickens and controls. These results will increase our understanding of the viral diversity and changes in the virome of chicken gut, with implications in chicken health.

ABSTRACT The Cuatro Ciénegas Basin (CCB) is located in the Chihuahuan desert in the Mexican state of Coahuila; it has been characterized as a site with high biological diversity despite its extreme oligotrophic conditions. It has the greatest number of endemic species in North America, containing abundant living microbialites (including stromatolites and microbial mats) and diverse microbial communities. With the hypothesis that this high biodiversity and the geographic structure should be reflected in the virome, the viral communities in 11 different locations of three drainage systems, Churince, La Becerra, and Pozas Rojas, and in the intestinal contents of 3 different fish species, were analyzed for both eukaryotic and prokaryotic RNA and DNA viruses using next-generation sequencing methods. Double-stranded DNA (dsDNA) virus families were the most abundant (72.5% of reads), followed by single-stranded DNA (ssDNA) viruses (2.9%) and ssRNA and dsRNA virus families (0.5%). Thirteen families had dsDNA genomes, five had ssDNA, three had dsRNA, and 16 had ssRNA. A highly diverse viral community was found, with an ample range of hosts and a strong geographical structure, with very even distributions and signals of endemicity in the phylogenetic trees from several different virus families. The majority of viruses found were bacteriophages but eukaryotic viruses were also frequent, and the large diversity of viruses related to algae were a surprise, since algae are not evident in the previously analyzed aquatic systems of this ecosystem. Animal viruses were also frequently found, showing the large diversity of aquatic animals in this oasis, where plants, protozoa, and archaea are rare. IMPORTANCE In this study, we tested whether the high biodiversity and geographic structure of CCB is reflected in its virome. CCB is an extraordinarily biodiverse oasis in the Chihuahuan desert, where a previous virome study suggested that viruses had followed the marine ancestry of the marine bacteria and, as a result of their long isolation, became endemic to the site. In this study, which includes a larger sequencing coverage and water samples from other sites within the valley, we confirmed the high virus biodiversity and uniqueness as well as the strong biogeographical diversification of the CCB. In addition, we also analyzed fish intestinal contents, finding that each fish species eats different prey and, as a result, presents different viral compositions even if they coexist in the same pond. These facts highlight the high and novel virus diversity of CCB and its “lost world” status.

AbstractViruses linked to animals inhabiting Antarctic latitudes remain poorly studied. Remote environments hosting large pinniped populations may be prone to exposure of immunologically naïve animals to new infectious agents due to increasing human presence or introduction of new animal species. Antarctic fur seals (Arctocephalus gazella) inhabiting the Western Antarctic Peninsula and the South Shetland Islands are challenged because of climate change and increased anthropogenic activity. In the present study, the fecal and serum virome of A. gazella was characterized by applying target enrichment next generation sequencing. The resulting viromes were dominated by CRESS-DNA sequences. Viruses known to infect vertebrate and invertebrate hosts were also observed in fecal samples. Fur seal picornavirus was present in all the fecal pools studied suggesting it is a prevalent virus in these species. Six different viruses presenting similarities with previously described A. gazella viruses or other otariids and mammal viruses were identified as potential new A. gazella viruses. Also, diet-derived viruses such as crustacean viruses were present in fecal content. Penguin viruses, but not fish viruses, were also detected. Obtained results contribute to a better understanding of the viral community present in these species, which is relevant for its conservation.

ABSTRACT Inferred virus-host relationships, community structures of ubiquitous ecologically relevant groups, and identification of transcriptionally active populations have been achieved with our Baltic Sea study. Further, these data, highlighting the transcriptional activity of viruses, represent one of the more powerful uses of omics concerning ecosystem health. The use of omics-related data to assess ecosystem health holds great promise for rapid and relatively inexpensive determination of perturbations and risk, explicitly with regard to viral assemblages, as no single marker gene is suitable for widespread taxonomic coverage. Metagenomic and metatranscriptomic data were generated from size-fractionated samples from 11 sites within the Baltic Sea and adjacent marine waters of Kattegat and freshwater Lake Torneträsk in order to investigate the diversity, distribution, and transcriptional activity of virioplankton. Such a transect, spanning a salinity gradient from freshwater to the open sea, facilitated a broad genome-enabled investigation of natural as well as impacted aspects of Baltic Sea viral communities. Taxonomic signatures representative of phages within the widely distributed order Caudovirales were identified with enrichments in lesser-known families such as Podoviridae and Siphoviridae. The distribution of phage reported to infect diverse and ubiquitous heterotrophic bacteria (SAR11 clades) and cyanobacteria (Synechococcus sp.) displayed population-level shifts in diversity. Samples from higher-salinity conditions (>14 practical salinity units [PSU]) had increased abundances of viruses for picoeukaryotes, i.e., Ostreococcus. These data, combined with host diversity estimates, suggest viral modulation of diversity on the whole-community scale, as well as in specific prokaryotic and eukaryotic lineages. RNA libraries revealed single-stranded DNA (ssDNA) and RNA viral populations throughout the Baltic Sea, with ssDNA phage highly represented in Lake Torneträsk. Further, our data suggest relatively high transcriptional activity of fish viruses within diverse families known to have broad host ranges, such as Nodoviridae (RNA), Iridoviridae (DNA), and predicted zoonotic viruses that can cause ecological and economic damage as well as impact human health. IMPORTANCE Inferred virus-host relationships, community structures of ubiquitous ecologically relevant groups, and identification of transcriptionally active populations have been achieved with our Baltic Sea study. Further, these data, highlighting the transcriptional activity of viruses, represent one of the more powerful uses of omics concerning ecosystem health. The use of omics-related data to assess ecosystem health holds great promise for rapid and relatively inexpensive determination of perturbations and risk, explicitly with regard to viral assemblages, as no single marker gene is suitable for widespread taxonomic coverage.

ABSTRACTCane toads are a notorious invasive species, inhabiting over 1.2 million km2of Australia and threatening native biodiversity. The release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile- and amphibian-specific cluster of thePicornaviridaebasal to theKobuvirus-like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, Rhinella marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains 8 complete RMERV proviruses as well as 21 additional truncated insertions. The oldest full-length RMERV provirus was estimated to have inserted 1.9 million years ago (MYA). To screen for these viral sequences in additional toads, we analyzed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, while RMERV transcripts were observed at all six sites. Finally, we scanned the cane toad genome for nonretroviral endogenous viral elements, finding three sequences related to small DNA viruses in the familyCircoviridae. This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species.IMPORTANCECane toads are poisonous amphibians that were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threaten many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality rates, yet few cane toad viruses have been characterized. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences that were genetically similar to viruses infecting frogs, reptiles, and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies that investigate their prevalence and potential as agents for biocontrol.

Fish passage facilities for reservoir dams have been used to restore habitat connectivity within riverine networks by allowing upstream passage for native species. These facilities may also support the spread of invasive species, an unintended consequence and potential downside of upstream passage structures. We documented dam passage of the invasive virile crayfish (Orconectes virilis) at fish ladders designed for upstream passage of American eels (Anguilla rostrata) in the Shenandoah River drainage. Ladder use and upstream passage of 11 virile crayfish occurred from 2007–2014 during periods of low river discharge (<30 m3s–1) and within a wide range of water temperatures from 9.0–28.6 °C. Virile crayfish that used the eel ladders were large adults with a mean carapace length and width of 48.0 mm and 24.1 mm, respectively. Our data demonstrated the use of a species-specific fish ladder by a non-target species, which has conservation and management implications for the spread of aquatic invasive species and upstream passage facilities.

AbstractThe growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

Abstract Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray–Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to ninety per cent of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control agent. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray–Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray–Darling Basin, covering over 2,200 km of the river system. Across a total of thirty-six RNA libraries representing ten species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified eighteen vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further twenty-seven that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fish sampled here, suggesting that there is little virus transmission from common carp to native fish species, despite co-existing for over fifty years. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.

Healthy parrotfish (family Scaridae) communities fulfill the essential ecosystem process of herbivory in coral reefs, but artisanal fisheries that target parrotfish have degraded their populations. Outright bans and gear restrictions that do not allow parrotfish capture can effectively protect and restore parrotfish populations. As these management actions would be unfeasible in many places, options that allow some fishing but still encourage population rebuilding need to be considered. The life history of parrotfish complicates management decisions because they transition from a mostly female “initial phase” to an all-male “terminal phase.” Size-selective fishing on the largest fish can lead to unnaturally low proportions of males in a population, potentially leading to losses in reproduction. At the same time, these visually distinct life phases could present an opportunity to employ a type of catch restriction that would be easy to understand and monitor. We built an agent-based model of the stoplight parrotfish, Sparisoma viride, which included three possible mechanisms of life-phase transitioning, to predict how this species and others like it might react to catch restrictions based on life phase. We found that restricting catch to only terminal-phase (male) fish typically led to populations of greater abundance and biomass and less-disturbed life-phase ratio, compared to a similar fishing mortality applied to the whole population. This model result highlights a potentially important lesson for all exploited protogynous hermaphrodites: a robust population of initial-phase fish may be key to maximizing reproductive potential when the size at life-phase transition compensates for changes in population structure.

AbstractParrotfishes and surgeonfishes are major Caribbean herbivores that primarily graze reef algae and thereby play an important functional role in indirectly promoting coral recruitment and growth. Yet, an emerging body of research suggests that these nominal herbivores graze on a diverse array of other food sources and researchers have questioned whether they may target more nutrient-dense foods growing within or upon algae, such as cyanobacteria. In this study, we investigated the species-specific foraging rates of parrotfishes and surgeonfishes on Brown Chromis (Chromis multilineata) fecal pellets compared to other major dietary items. We found that almost 85% of observed fecal pellets were ingested by fishes and that over 90% of ingested fecal pellets were consumed by parrotfishes and surgeonfishes alone. While there were species-specific differences in the levels of feces consumption (coprophagy), we found that all three surgeonfishes (Acanthurus chirurgus, A. coeruleus, and A. tractus) and six of the nine of parrotfish species surveyed (Scarus coeruleus, S. iseri, S. taeniopterus, S. vetula, Sparisoma aurofrenatum, and S. viride) consumed C. multilineata feces. To better understand the nutritional value of this behavior, we analyzed the composition of proteins, lipids, carbohydrates, total calories, and micronutrients in C. multilineata fecal pellets and compared these to published values for other food sources targeted by these fishes. Our findings suggest that these fecal pellets may have higher values of proteins, carbohydrates, total calories, and important micronutrients, such as phosphorus, compared to various macroalgae and the epilithic algae matrix, though comparable or lower values compared to cyanobacteria. To our knowledge, this is the first study to document coprophagy by tropical herbivorous fishes in the Caribbean region. This research advances our understanding of the foraging ecology of nominally herbivorous fishes and highlights the importance of fish feces as a nutritional resource on coral reefs. Graphical abstract

This paper argues that illegitimacy is a concept that relates to almost all of the fundamental ways in which Western society has traditionally organised itself. Sex, family and marriage, and the power of the church and state, are all implicated in the various ways in which society reproduces itself from generation to generation. All employ the concepts of legitimacy and illegitimacy to define what is and what is not permissible. Further, the creation of the illegitimate can occur in more or less legitimate ways; for example, through acts of consent, on the one hand; and force, on the other. This paper uses the study of an English Renaissance text, Shakespeare’s Henry V, to argue that these concepts remain potent ones, regularly invoked as a means of identifying and denouncing perceived threats to the good ordering of the social fabric. In western societies, many of which may be constructed as post-marriage, illegitimate is often applied as a descriptor to unlicensed migrants, refugees and asylum seekers. In countries subject to war and conflict, rape as a war crime is increasingly used by armies to create fractures within the subject community and to undermine the paternity of a cohort of children. In societies where extramarital sex is prohibited, or where rape has been used as a weapon of war, the bastard acts as physical evidence that an unsanctioned act has been committed and the laws of society broken, a “failure in social control” (Laslett, Oosterveen and Smith, 5). This paper explores these themes, using past conceptions of the illegitimate and bastardy as an explanatory concept for problematic aspects of legitimacy in contemporary culture.Bastardy was a particularly important issue in sixteenth and seventeenth century Europe when an individual’s genealogy was a major determining factor of social status, property and identity (MacFarlane). Further, illegitimacy was not necessarily an aspect of a person’s birth. It could become a status into which they were thrust through the use of divorce, for example, as when Henry VIII illegitimised his daughter Mary after annulling his marriage to Mary’s mother, Catherine of Aragon. Alison Findlay’s study of illegitimacy in Renaissance literature lists over 70 portrayals of illegitimacy, or characters threatened with illegitimacy, between 1588 and 1652 (253–257). In addition to illegitimacy at an individual level however, discussions around what constitutes the “illegitimate” figure in terms of its relationship with the family and the wider community, are also applicable to broader concerns over national identity. In work such as Stages of History, Phyllis Rackin dissected images of masculine community present in Shakespeare’s history plays to expose underlying tensions over gender, power and identity. As the study of Henry V indicates in the following discussion, illegitimacy was also a metaphor brought to bear on issues of national as well as personal identity in the early modern era. The image of the nation as a “family” to denote unity and security, both then and now, is rendered complex and problematic by introducing the “illegitimate” into that nation-family image. The rhetoric used in the recent debate over the Scottish independence referendum, and in Australia’s ongoing controversy over “illegitimate” migration, both indicate that the concept of a “national bastard”, an amorphous figure that resists precise definition, remains a potent rhetorical force. Before turning to the detail of Henry V, it is useful to review the use of “illegitimate” in the early modern context. Lacking an established position within a family, a bastard was in danger of being marginalised and deprived of any but the most basic social identity. If acknowledged by a family, the bastard might become a drain on that family’s economic resources, drawing money away from legitimate children and resented accordingly. Such resentment may be reciprocated. In his essay “On Envy” the scientist, author, lawyer and eventually Lord Chancellor of England Francis Bacon explained the destructive impulse of bastardy as follows: “Deformed persons, and eunuchs, and old men, and bastards, are envious. For he that cannot possibly mend his own case will do what he can to impair another’s.” Thus, bastardy becomes a plot device which can be used to explain and to rationalise evil. In early modern English literature, as today, bastardy as a defect of birth is only one meaning for the word. What does “in everything illegitimate” (quoting Shakespeare’s character Thersites in Troilus and Cressida [V.viii.8]) mean for our understanding of both our own society and that of the late sixteenth century? Bastardy is an important ideologeme, in that it is a “unit of meaning through which the ‘social space’ constructs the ideological values of its signs” (Schleiner, 195). In other words, bastardy has an ideological significance that stretches far beyond a question of parental marital status, extending to become a metaphor for national as well as personal loss of identity. Anti-Catholic polemicists of the early sixteenth century accused priests of begetting a generation of bastards that would overthrow English society (Fish, 7). The historian Polydore Vergil was accused of suborning and bastardising English history by plagiarism and book destruction: “making himself father to other men’s works” (Hay, 159). Why is illegitimacy so important and so universal a metaphor? The term “bastard” in its sense of mixture or mongrel has been applied to language, to weaponry, to almost anything that is a distorted but recognisable version of something else. As such, the concept of bastardy lends itself readily to the rhetorical figure of metaphor which, as the sixteenth century writer George Puttenham puts it, is “a kind of wresting of a single word from his owne right signification, to another not so natural, but yet of some affinitie or coueniencie with it” (Puttenham, 178). Later on in The Art of English Poesie, Puttenham uses the word “bastard” to describe something that can best be recognised as being an imperfect version of something else: “This figure [oval] taketh his name of an egge […] and is as it were a bastard or imperfect rounde declining toward a longitude.” (101). “Bastard” as a descriptive term in this context has meaning because it connects the subject of discussion with its original. Michael Neill takes an anthropological approach to the question of why the bastard in early modern drama is almost invariably depicted as monstrous or evil. In “In everything illegitimate: Imagining the Bastard in Renaissance Drama,” Neill argues that bastards are “filthy”, using the term as it is construed by Mary Douglas in her work Purity and Danger. Douglas argues that dirt is defined by being where it should not be, it is “matter in the wrong place, belonging to ‘a residual category, rejected from our normal scheme of classifications,’ a source of fundamental pollution” (134). In this argument the figure of the bastard aligns strongly with the concept of the Other (Said). Arguably, however, the anthropologist Edmund Leach provides a more useful model to understand the associations of hybridity, monstrosity and bastardy. In “Animal Categories and Verbal Abuse”, Leach asserts that our perceptions of the world around us are largely based on binary distinctions; that an object is one thing, and is not another. If an object combines attributes of itself with those of another, the interlapping area will be suppressed so that there may be no hesitation in discerning between them. This repressed area, the area which is neither one thing nor another but “liminal” (40), becomes the object of fear and of fascination: – taboo. It is this liminality that creates anxiety surrounding bastards, as they occupy the repressed, “taboo” area between family and outsiders. In that it is born out of wedlock, the bastard child has no place within the family structure; yet as the child of a family member it cannot be completely relegated to the external world. Michael Neill rightly points out the extent to which the topos of illegitimacy is associated with the disintegration of boundaries and a consequent loss of coherence and identity, arguing that the bastard is “a by-product of the attempt to define and preserve a certain kind of social order” (147). The concept of the liminal figure, however, recognises that while a by-product can be identified and eliminated, a bastard can neither be contained nor excluded. Consequently, the bastard challenges the established order; to be illegitimate, it must retain its connection with the legitimate figure from which it diverges. Thus the illegitimate stands as a permanent threat to the legitimate, a reminder of what the legitimate can become. Bastardy is used by Shakespeare to indicate the fear of loss of national as well as personal identity. Although noted for its triumphalist construction of a hero-king, Henry V is also shot through with uncertainties and fears, fears which are frequently expressed using illegitimacy as a metaphor. Notwithstanding its battle scenes and militarism, it is the lawyers, genealogists and historians who initiate and drive forward the narrative in Henry V (McAlindon, 435). The reward of the battle for Henry is not so much the crown of France as the assurance of his own legitimacy as monarch. The lengthy and legalistic recital of genealogies with which the Archbishop of Canterbury proves to general English satisfaction that their English king Henry holds a better lineal right to the French throne than its current occupant may not be quite as “clear as is the summer sun” (Henry V 1.2.83), but Henry’s question about whether he may “with right and conscience” make his claim to the French throne elicits a succinct response. The churchmen tell Henry that, in order to demonstrate that he is truly the descendant of his royal forefathers, Henry will need to validate that claim. In other words, the legitimacy of Henry’s identity, based on his connection with the past, is predicated on his current behaviour:Gracious lord,Stand for your own; unwind your bloody flag;Look back into your mighty ancestors:Go, my dread lord, to your great-grandsire’s tomb,From whom you claim; invoke his warlike spirit…Awake remembrance of these valiant dead,And with your puissant arm renew their feats:You are their heir, you sit upon their throne,The blood and courage that renowned themRuns in your veins….Your brother kings and monarchs of the earthDo all expect that you should rouse yourselfAs did the former lions of your blood. (Henry V 1.2.122 – 124)These exhortations to Henry are one instance of the importance of genealogy and its immediate connection to personal and national identity. The subject recurs throughout the play as French and English characters both invoke a discourse of legitimacy and illegitimacy to articulate fears of invasion, defeat, and loss of personal and national identity. One particular example of this is the brief scene in which the French royalty allow themselves to contemplate the prospect of defeat at the hands of the English:Fr. King. ‘Tis certain, he hath pass’d the river Somme.Constable. And if he be not fought withal, my lord,Let us not live in France; let us quit all,And give our vineyards to a barbarous people.Dauphin. O Dieu vivant! shall a few sprays of us,The emptying of our fathers’ luxury,Our scions, put in wild and savage stock,Spirt up so suddenly into the clouds,And overlook their grafters?Bourbon. Normans, but bastard Normans, Norman bastards!...Dauphin. By faith and honour,Our madams mock at us, and plainly sayOur mettle is bred out; and they will giveTheir bodies to the lust of English youthTo new-store France with bastard warriors. (Henry V 3.5.1 – 31).Rape and sexual violence pervade the language of Henry V. France itself is constructed as a sexually vulnerable female with “womby vaultages” and a “mistress-court” (2.4.131, 140). In one of his most famous speeches Henry graphically describes the rape and slaughter that accompanies military defeat (3.3). Reading Henry V solely in terms of its association of military conquest with sexual violence, however, runs the risk of overlooking the image of bastards themselves as both the threat and the outcome of national defeat. The lines quoted above exemplify the extent to which illegitimacy was a vital metaphor within early modern discourses of national as well as personal identity. Although the lines are divided between various speakers – the French King, Constable (representing the law), Dauphin (the Crown Prince) and Bourbon (representing the aristocracy) – the images develop smoothly and consistently to express English dominance and French subordination, articulated through images of illegitimacy.The dialogue begins with the most immediate consequence of invasion and of illegitimacy: the loss of property. Legitimacy, illegitimacy and property were so closely associated that a case of bastardy brought to the ecclesiastical court that did not include a civil law suit about land was referred to as a case of “bastardy speciall”, and the association between illegitimacy and property is present in this speech (Cowell, 14). The use of the word “vine” is simultaneously a metonym for France and a metaphor for the family, as in the “family tree”, conflating the themes of family identity and national identity that are both threatened by the virile English forces.As the dialogue develops, the rhetoric becomes more elaborate. The vines which for the Constable (from a legal perspective) represented both France and French families become instead an attempt to depict the English as being of a subordinate breed. The Dauphin’s brief narrative of the English origins refers to the illegitimate William the Conqueror, bastard son of the Duke of Normandy and by designating the English as being descendants of a bastard Frenchman the Dauphin attempts to depict the English nation as originating from a superabundance of French virility; wild offshoots from a true stock. Yet “grafting” one plant to another can create a stronger plant, which is what has happened here. The Dauphin’s metaphors, designed to construct the English as an unruly and illegitimate offshoot of French society, a product of the overflowing French virility, evolve instead into an emblem of a younger, stronger branch which has overtaken its enfeebled origins.In creating this scene, Shakespeare constructs the Frenchmen as being unable to contain the English figuratively, still less literally. The attempts to reduce the English threat by imagining them as “a few sprays”, a product of casual sexual excess, collapses into Bourbon’s incoherent ejaculation: “Normans, but bastard Normans, Norman bastards!” and the Norman bastard dominates the conclusion of the scene. Instead of containing and marginalising the bastard, the metaphoric language creates and acknowledges a threat which cannot be marginalised. The “emptying of luxury” has engendered an uncontrollable illegitimate who will destroy the French nation beyond any hope of recovery, overrunning France with bastards.The scene is fascinating for its use of illegitimacy as a means of articulating fears not only for the past and present but also for the future. The Dauphin’s vision is one of irreversible national and familial disintegration, irreversible because, unlike rape, the French women’s imagined rejection of their French families and embrace of the English conquerors implies a total abandonment of family origins and the willing creation of a new, illegitimate dynasty. Immediately prior to this scene the audience has seen the Dauphin’s fear in action: the French princess Katherine is shown learning to speak English as part of her preparation for giving her body to a “bastard Norman”, a prospect which she anticipates with a frisson of pleasure and humour, as well as fear. This scene, between Katherine and her women, evokes a range of powerful anxieties which appear repeatedly in the drama and texts of the sixteenth and early seventeenth centuries: anxieties over personal and national identity, over female chastity and masculine authority, and over continuity between generations. Peter Laslett in The World We Have Lost – Further Explored points out that “the engendering of children on a scale which might threaten the social structure was never, or almost never, a present possibility” (154) at this stage of European history. This being granted, the Dauphin’s depiction of such a “wave” of illegitimates, while it might have no roots in reality, functioned as a powerful image of disorder. Illegitimacy as a threat and as a strategy is not limited to the renaissance, although a study of renaissance texts offers a useful guidebook to the use of illegitimacy as a means of polarising and excluding. Although as previously discussed, for many Western countries, the marital status of one’s parents is probably the least meaningful definition associated with the word “illegitimate”, the concept of the nation as a family remains current in modern political discourse, and illegitimate continues to be a powerful metaphor. During the recent independence referendum in Scotland, David Cameron besought the Scottish people not to “break up the national family”; at the same time, the Scottish Nationalists have been constructed as “ungrateful bastards” for wishing to turn their backs on the national family. As Klocker and Dunne, and later O’Brien and Rowe, have demonstrated, the emotive use of words such as “illegitimate” and “illegal” in Australian political rhetoric concerning migration is of long standing. Given current tensions, it might be timely to call for a further and more detailed study of the way in which the term “illegitimate” continues to be used by politicians and the media to define, demonise and exclude certain types of would-be Australian immigrants from the collective Australian “national family”. Suggestions that persons suspected of engaging with terrorist organisations overseas should be stripped of their Australian passports imply the creation of national bastards in an attempt to distance the Australian community from such threats. But the strategy can never be completely successful. Constructing figures as bastard or the illegitimate remains a method by which the legitimate seeks to define itself, but it also means that the bastard or illegitimate can never be wholly separated or cast out. In one form or another, the bastard is here to stay.ReferencesBeardon, Elizabeth. “Sidney's ‘Mongrell Tragicomedy’ and Anglo-Spanish Exchange in the New Arcadia.” Journal for Early Modern Cultural Studies 10 (2010): 29 - 51.Davis, Kingsley. “Illegitimacy and the Social Structure.” American Journal of Sociology 45 (1939).John Cowell. The Interpreter. Cambridge: John Legate, 1607.Greenblatt, Stephen. Renaissance Self-Fashioning: From More to Shakespeare. 1980. Chicago: University of Chicago Press, 2005.Findlay, Alison. Illegitimate Power: Bastards in Renaissance Drama. Manchester: Manchester University Press, 2009.Hay, Denys. Polydore Vergil: Renaissance Historian and Man of Letters. Oxford: Clarendon Press, 1952.Laslett, Peter. The World We Have Lost - Further Explored. London: Methuen, 1983.Laslett, P., K. Oosterveen, and R. M. Smith, eds. Bastardy and Its Comparative History. 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