Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing.

Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)-based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease-causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83%-100%). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low- and medium-income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.

Supramolecular Photocatalysis by Utilizing the Host-Guest Charge-Transfer Interaction: Visible-Light-Induced Generation of Triplet Anthracenes for [4+2] Cycloaddition Reactions.

Supramolecular photocatalysis via charge-transfer excitation of a host-guest complex was developed by use of the macrocyclic boronic ester [2+2]BTH-F containing highly electron-deficient difluorobenzothiadiazole moieties. In the presence of a catalytic amount of [2+2]BTH-F , the triplet excited state of anthracene was generated from the charge-transfer excited state of anthracene@[2+2]BTH-F by visible-light irradiation, and cycloaddition of the excited anthracene with several dienes and alkenes proceeded in a [4+2] manner in high yields.

Interactions of head-kidney leucocytes from giant grouper, Epinephelus lanceolatus, with pathogenic Streptococcus agalactiae strains from marine and terrestrial origins.

Streptococcus agalactiae (Group B Streptococcus, GBS) is emerging as a genetically diverse species infecting farmed and wild fish, including commercially and culturally important groupers. To better understand how S. agalactiae are pathogenic in fish, we investigated interactions between isolates from fish and terrestrial hosts and the cellular immune system of Queensland grouper Epinephelus lanceolatus using flow cytometry. Adherent head-kidney leucocytes (HKL) from Queensland grouper displayed two main cell populations with distinct forward and side scatter by flow cytometry. The population of smaller and less complex cells (P1) was composed of monocytes, lymphocytes and thrombocytes, while the population of primarily larger and more complex cells (P2) comprised predominantly of macrophages and neutrophils. The cells in P2 had higher phagocytic index and capacity when incubated with fluorescent latex beads. HKL were activated by phorbol myristate acetate (PMA) but were unresponsive to lipopolysaccharide (LPS) and peptidoglycan (PTG), suggesting the absence of specific receptors on the surface of these cells for these ligands or a requirement for intermediates. In in vitro phagocytosis assays, all fish isolates of GBS activated a respiratory burst in P2 indicated by significant production of intracellular reactive oxygen species (ROS). Similarly, dog and cat isolates of different serotype and sequence type also induced ROS production in grouper HKL. However, human, crocodile and bovine isolates of GBS did not elicit significant ROS in HKL although they coincided with the highest phagocytic index. This suggests that these strains are capable of quenching ROS production. Terrestrial isolates significantly increased mortality of Queensland grouper leucocytes in vitro, aligned with a more diverse repertoire of cellular toxins in these strains. Opsonisation of a marine strain and terrestrial strain of GBS with antiserum raised against the marine strain resulted in an increase in ROS production by HKL in both cases although there was low antigenic cross reactivity between the two strains by flow cytometry, reflecting their diverse serotypes (Ib vs III). However, pre-incubation of either strain with normal serum from grouper also increased ROS production of HKL suggesting other opsonins may be involved. Based on these results it appears that piscine and terrestrial GBS isolates have contrasting strategies when interacting with the cellular immune system of Queensland grouper; the former seemingly evading phagocytosis, whilst the latter are readily phagocytosed but counteract ROS production.

Microevolution of Streptococcus agalactiae ST-261 from Australia Indicates Dissemination via Imported Tilapia and Ongoing Adaptation to Marine Hosts or Environment.

Streptococcus agalactiae (group B Streptococcus [GBS]) causes disease in a wide range of animals. The serotype Ib lineage is highly adapted to aquatic hosts, exhibiting substantial genome reduction compared with terrestrial conspecifics. Here, we sequence genomes from 40 GBS isolates, including 25 isolates from wild fish and captive stingrays in Australia, six local veterinary or human clinical isolates, and nine isolates from farmed tilapia in Honduras, and compared them with 42 genomes from public databases. Phylogenetic analysis based on nonrecombinant core-genome single nucleotide polymorphisms (SNPs) indicated that aquatic serotype Ib isolates from Queensland were distantly related to local veterinary and human clinical isolates. In contrast, Australian aquatic isolates are most closely related to a tilapia isolate from Israel, differing by only 63 core-genome SNPs. A consensus minimum spanning tree based on core-genome SNPs indicates the dissemination of sequence type 261 (ST-261) from an ancestral tilapia strain, which is congruent with several introductions of tilapia into Australia from Israel during the 1970s and 1980s. Pangenome analysis identified 1,440 genes as core, with the majority being dispensable or strain specific, with non-protein-coding intergenic regions (IGRs) divided among core and strain-specific genes. Aquatic serotype Ib strains have lost many virulence factors during adaptation, but six adhesins were well conserved across the aquatic isolates and might be critical for virulence in fish and for targets in vaccine development. The close relationship among recent ST-261 isolates from Ghana, the United States, and China with the Israeli tilapia isolate from 1988 implicates the global trade in tilapia seed for aquaculture in the widespread dissemination of serotype Ib fish-adapted GBS.IMPORTANCEStreptococcus agalactiae (GBS) is a significant pathogen of humans and animals. Some lineages have become adapted to particular hosts, and serotype Ib is highly specialized to fish. Here, we show that this lineage is likely to have been distributed widely by the global trade in tilapia for aquaculture, with probable introduction into Australia in the 1970s and subsequent dissemination in wild fish populations. We report here the variability in the polysaccharide capsule among this lineage but identify a cohort of common surface proteins that may be a focus of future vaccine development to reduce the biosecurity risk in international fish trade.

Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: potential role of surface carbohydrates in successful parasitism.

In order to establish a successful relationship with their hosts, parasites must subvert or evade immune defences. Cockle Anadara trapezia and Sydney Rock oyster (SRO) Saccostrea glomerata live in the same location but only ark cockles are infected by sporocysts of hemiuroid trematode. This provides an opportunity to explore differing interactions between the parasite and the immune system of susceptible and refractive hosts. Rapid migration and encapsulation of sporocysts was observed by SRO hemocytes but not by cockle hemocytes. This migration/encapsulation was inhibited by N-acetylglucosamine or N-acetylgalactosamine but not by the other sugars, implicating specific surface carbohydrates in immune detection. Effector responses of hemocytes were investigated in vitro in terms of production of reactive oxygen production (ROS). Hemocytes of both species strongly reacted to Zymosan, but only SRO hemocytes responded to live sporocysts. Neither species' hemocytes produced ROS in the presence of dead/fixed sporocysts, and there was no suppression of Zymosan-induced respiratory burst by sporocysts. This suggests that immune escape is mediated by avoiding encapsulation, perhaps through molecular mimicry. Membrane-shaving with proteases indicated that sporocyst surface proteins are not a key factors in hemocytic detection. Surface carbohydrates of SRO and cockle hemocytes and of sporocysts were profiled with a panel of biotinylated lectins. This revealed substantial differences between cockle and SRO hemocytes, but greater similarity between cockle hemocytes and sporocysts. Results suggest that surface carbohydrates play an integral role in hemocyte immunorecognition and that surface carbohydrate molecular mimicry is a potential strategy for immune evasion in cockles by hemiuroid trematode sporocysts.

Effect of a hemiuroid trematode on the hemocyte immune parameters of the cockle Anadara trapezia.

When a trematode parasite penetrates a potential molluscan host, it has to circumvent the host's internal defense system. In molluscs, the primary effector cells of this system are the hemocytes which orchestrate many of the cellular and humoral immune functions. Survival of the parasite can occur only in the absence of a successful immune response, and continued development only if the host is physiologically suitable. This study investigated hemocytic response against asexual stages of a hemiuroid trematode by its host, the marine bivalve Anadara trapezia. Hemocyte characteristic (type, morphology) and function (mortality, phagocytosis and oxidative activity) were analyzed by flow cytometry in parasitized and non-parasitized cockles. A. trapezia possesses two types of hemocytes: amebocytes and erythrocytes. Analysis of histological section showed that there was no host hemocytic response around hemiuroid sporocysts. The infection induced a significant increase of the total circulating hemocytes with a higher proportion of erythrocytes relative to amebocytes, coupled with a lower phagocytosis rate and a statistically non-significant decrease of the intracellular oxidative activity. No significant differences were observed in hemocyte size and complexity, mortality, or phagocytic capacity. Our results indicate that in A. trapezia, hemiuroids modulate the immune response by increasing the number of circulating hemocytes and decreasing phagocytosis.

Mixed culture purple phototrophic bacteria is an effective fishmeal replacement in aquaculture.

Aquaculture is the fastest growing animal food production industry, now producing 50% of all food fish. However, aquaculture feeds remain dependent on fishmeal derived from capture fisheries, which must be reduced for continued sustainable growth. Purple phototrophic bacteria (PPB) efficiently yield biomass from wastewater with high product homogeneity, a relatively high protein fraction, and potential added value as an ingredient for fish feeds. Here we test bulk replacement of fishmeal with PPB microbial biomass in diets for Asian sea bass (Lates calcarifer), a high value carnivorous fish with high protein to energy requirement. Mixed culture PPB were grown in a novel 1 m3 attached photo-biofilm process using synthetic and real wastewater. Four experimental diets were formulated to commercial specifications but with the fishmeal substituted (0%, 33%, 66%, and 100%) with the synthetic grown PPB biomass and fed to a cohort of 540 juvenile fish divided amongst 12 tanks over 47 days. Weight and standard length were taken from individual fish at 18, 28, and 47d. No significant difference in survival was observed due to diet or other factors (94-100%). There was a negative correlation between PPB inclusion level and final weight (p = 5.94 × 10-5) with diet accounting for 4.1% of the variance over the trial (general linear model, R2 = 0.96, p = 1 × 10-6). Feed conversion ratio was also significantly influenced by diet (p = 6 × 10-7) with this factor accounting for 89% of variance. Specifically, feed conversion ratio (FCR) rose to 1.5 for the 100% replacement diet during the last sample period, approximately 1.0 for the partial replacement, and 0.8 for the nil replacement diet. However, this study demonstrates that bulk replacement of fishmeal by PPB is feasible, and commercially viable at 33% and 66% replacement.

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms.

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries.