A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia.

Tilapia farming is one of the most important sectors in aquaculture worldwide and of major importance to global food security. Infectious spleen and kidney necrosis virus (ISKNV) has been identified as an agent of high morbidity and mortality, threatening tilapia aquaculture. ISKNV was detected in Lake Volta, Ghana, in September 2018 and spread rapidly, with mortality rates between 60 and 90% and losses of more than 10 tonnes of fish per day. Understanding the spread and evolution of viral pathogens is important for control strategies. Here, we developed a tiled-PCR sequencing approach for the whole-genome sequencing of ISKNV, using long read sequencing to enable field-based, real-time genomic surveillance. This work represents the first use of tiled-PCR for whole genome recovery of viruses in aquaculture, with the longest genome target (>110 kb dsDNA) to date. Our protocol was applied to field samples collected from the ISKNV outbreaks from four intensive tilapia cage culture systems across Lake Volta, between October 2018 and May 2022. Despite the low mutation rate of dsDNA viruses, 20 single nucleotide polymorphisms accumulated during the sampling period. Droplet digital PCR identified a minimum requirement of template in a sample to recover 50% of an ISKNV genome at 275 femtograms (2410 viral templates per 5 µL sequencing reaction). Overall, tiled-PCR sequencing of ISKNV provides an informative tool to assist in disease control in aquaculture.

High-resolution app data reveal sustained increases in recreational fishing effort in Europe during and after COVID-19 lockdowns (preprint)

It is manifest that COVID-19 lockdowns extensively impacted human interactions with natural ecosystems. One example is recreational fishing, an activity which involves nearly 1 in 10 people in developed countries. Fishing licence sales and direct observations at popular angling locations suggest that recreational fishing effort increased substantially during lockdowns. However, the extent and duration of this increase remain largely unknown due to a lack of objective data. We used four years (2018 to 2021) of anonymous, high-resolution data from a personal fish-finder device to explore the impact of COVID-19 lockdowns on recreational fishing effort in four European countries (Lithuania, the Czech Republic, Denmark, and Germany). We show that device use and, by extension, angling effort increased 1.2-3.8 fold during March-May 2020 and remained elevated even at the end of 2021 in all countries except Denmark. Fishing during the first lockdown also became more frequent during weekdays. Statistical models with the full set of fixed (weekdays, lockdown, population) and random (season, year, administrative unit) factors typically explained 50-70% of the variation, suggesting that device use and angling effort were relatively consistent and predictable through space and time. Our study demonstrates that recreational fishing behaviour can change substantially and rapidly in response to societal shifts, with profound ecological, human well-being and economic implications. We also show the potential of angler devices and smartphone applications to supply data for high-resolution fishing effort analysis and encourage more extensive science and industry collaborations to take advantage of this information. Significance statementRecreational fishing is a popular and widespread activity with ecological, social and economic impacts, though problematic to assess and manage due to a paucity of information regarding effort and catch. Here, we use high-resolution data from a personal angler sonar device to show how the COVID-19 pandemic changed angler behaviour and fishing effort across Europe. We demonstrate that angling effort doubled and remained higher at the end of 2021 than before the first lockdowns. Such rapid and profound changes could have significant consequences for aquatic ecosystems, possibly requiring new management approaches. We encourage the adoption of novel data from angler devices, citizen science, and more active science-industry collaborations to improve recreational fishing assessment and management.

Impact of Covid-19 lockdown on trap fishery in Olaikuda Region of Palk Bay, South India (preprint)

The present work analysed the fish catches and associated prices of trap fisheries during the regular and COVID-19 lockdown along the Olaikuda fishing village, Palk Bay region. The main target fishery in this region is coral associated fishes. A total of 713 kg of fishes harvested from the trap for eight months of study. The higher and lower average catch was recorded in May and April during 2020 lockdown respectively. But in the regular fishing months, the higher and lower average catch was recorded in July and March 2020 respectively. The total value of harvested fishes during the regular fishing months was ₹ 66,137 and in lockdown months ₹ 54,583 with the cumulative value of ₹ 1,20,720 as against the operation cost of ₹70,750 excluding the cost of craft. Thus, COVID-19 lockdown influenced the price of export quality fishes belonging to the family Scaridae due to which the fishes were sold at lower prices leading to low income of trap fishers. Data on this type of work is not available to compare the present situation. Therefore, long term study will be conducted in different trap fishing areas to compare the catch data along with market value for better understanding.

Impact of climate-driven temperature increase on inland aquaculture: Application to land-based production of common carp (Cyprinus carpio L.).

Climate change will expose the food-producing sector to a range of challenges. Inland aquaculture farms are particularly vulnerable, due to the difficulty in changing their location, and therefore require specific tools to predict the influence of direct and indirect effects on production, environment and economic feasibility. The objective of our study was to apply a simple set of models to produce a set of growth, risk and suitability maps for stakeholders within the common carp sector in Poland, to assist decision-making under two different scenarios of climate change: a moderate situation (RCP 4.5) and an extreme situation (RCP 8.5). We used present (2000-2019) and future projections (2080-2099) for water surface temperature based on land surface temperature data from regionally downscaled climate models to draw maps to: (i) show optimal temperature conditions for carp growth, (ii) assess risk of disease outbreak caused by three important common carp pathogens: Cyprinid herpesvirus 3 (CyHV-3), carp oedema virus (CEV) and spring viremia of carp (SVCV) and (iii) predict potential suitability changes of carp farming in Poland. The study identified areas with the most and least favourable temperature conditions for carp growth, as well as those areas with the highest/lowest number of days with suitable temperatures for virus infection. These suitability maps showed the combined effect of direct and indirect effects of climate change projections under RCP 8.5 and RCP 4.5 scenarios. The approach applied herein will be of use worldwide for analysing the risks of temperature increase to land-based aquaculture, and the results presented are important for carp farmers in Poland and elsewhere, industry in general, and government stakeholders, to understand the direct and indirect effects of climate change on the triple bottom line of people, planet, and profit.

Comparative transcriptome analysis of long non coding RNA (lncRNA) in RTG-2 cells infected by infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is the vital pathogen that has caused the great economic loss in salmonid fisheries. To date, there is limited information concerning the changes of lncRNAs in RTG-2 cells infected by IHNV. In this study, a comparative transcriptome analysis of lncRNAs was performed in RTG-2 cells with and without IHNV infection to determine their changes and the effects on IHNV infection. The results showed that IHNV infection significantly changed the expression levels of lncRNAs and mRNAs, including 3693 differentially expressed lncRNAs (DE-lncRNAs) and 3503 differentially expressed mRNAs (DE-mRNAs) respectively. These DE-lncRNAs and DE-mRNAs induced by IHNV were mostly associated with immune response, RNA processing, and viral diseases related pathways. Further analysis found that some DE-lncRNAs might participate in the regulation of extracellular matrix metabolism, apoptosis, lipid synthesis, autophagy, and immune responses referring to the functions of their target genes. Afterwards, 349 co-expression relationships were constructed by 223 DE-lncRNAs and 271 DE-mRNAs, of which LTCONS_00146935 was the pivotal node in the interaction networks, and was together with its target genes modulated the immune responses under the IHNV infection. RT-qPCR results showed that the changes of the selected immune-related DEGs were in consistent with the RNA-seq data, suggesting that the sequencing data was relatively reliable. In summary, this is the first study to determine the changes and interactions of lncRNA-mRNA in RTG-2 cells under the IHNV infection. The results provided the valuable information concerning the lncRNAs in salmonid fish, which will benefit for future study on uncovering the roles of lncRNAs-mRNAs during the viral infection.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Developing a Virus-Binding Bacterium Expressing Mx Protein on the Bacterial Surface to Prevent Grouper Nervous Necrosis Virus Infection.

Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.

Identification of coronavirus sequences in carp cDNA from Wuhan, China.

Severe acute respiratory syndrome (SARS)-like coronavirus sequences were identified in two separate complementary DNA (cDNA) pools. The first pool was from a Carassius auratus (crusian carp) cell line and the second was from Ctenopharyngodon idella (grass carp) head kidney tissue. BLAST analysis suggests that these sequences belong to SARS-like coronaviruses, and that they are not evolutionarily conserved in other species. Investigation of the submitting laboratories revealed that two laboratories from the Institute of Hydrobiology at the Chinese Academy of Sciences in Wuhan, China performed the research and submitted the cDNA libraries to GenBank. This institution is very close in proximity to the Wuhan South China Seafood Wholesale Market where SARS-CoV-2 first amplified in the human population. It is possible that these sequences are an artifact of the bioinformatics pipeline that was used. It is also possible that SARS-like coronaviruses are a common environmental pathogen in the region that may be in aquatic habitats.