Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection.

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein-protein docking modelling suggests the helveticin-J homolog may be able to block VP28-PmRab7 interactions and interrupt WSSV infection.

Transgenic Microalgae Expressing Double-Stranded RNA as Potential Feed Supplements for Controlling White Spot Syndrome in Shrimp Aquaculture.

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture.

Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics.

RNA interference (RNAi) is an effective way of combating shrimp viruses by using sequence-specific double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp offering viral protection. In this proof of concept, we engineered the chloroplast genome of the green microalga Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head viral gene. We used a previously described chloroplast transformation approach that allows for the generation of stable, marker-free C. reinhardtii transformants without the supplementation of antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding trial to evaluate the efficiency of the algal RNAi-based vaccine against the virus. Shrimps treated with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection (dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR using viral specific primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp (55.6 ± 11.1%) compared to control groups (88.9 ± 11.1% and 100.0 ± 0.0%, respectively). Our results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral protective feedstock in shrimp aquaculture.

Applications of Microalgal Biotechnology for Disease Control in Aquaculture.

Aquaculture industries, and in particular the farming of fish and crustaceans, are major contributors to the economy of many countries and an increasingly important component in global food supply. However, the severe impact of aquatic microbial diseases on production performance remains a challenge to these industries. This article considers the potential applications of microalgal technology in the control of such diseases. At the simplest level, microalgae offer health-promoting benefits as a nutritional supplement in feed meal because of their digestibility and high content of proteins, lipids and essential nutrients. Furthermore, some microalgal species possess natural anti-microbial compounds or contain biomolecules that can serve as immunostimulants. In addition, emerging genetic engineering technologies in microalgae offer the possibility of producing 'functional feed additives' in which novel and specific bioactives, such as fish growth hormones, anti-bacterials, subunit vaccines, and virus-targeted interfering RNAs, are components of the algal supplement. The evaluation of such technologies for farm applications is an important step in the future development of sustainable aquaculture.

Feasibility of dsRNA treatment for post-clearing SPF shrimp stocks of newly discovered viral infections using Laem Singh virus (LSNV) as a model.

Using post-larvae derived from specific pathogen free (SPF) stocks in penaeid shrimp farming has led to a dramatic increase in production. At the same time, new pathogens of farmed shrimp are continually being discovered. Sometimes these pathogens are carried by shrimp and other crustaceans as persistent infections without gross signs of disease. Thus it is that a 5-generation stock of Penaeus monodon SPF for several pathogens was found, post-stock-development, to be persistently-infected with newly-discovered Laem Singh virus (LSNV). In this situation, the stock developers were faced with destroying their existing stock (developed over a long period at considerable cost) and starting the whole stock development process anew in order to add LSNV to its SPF list. As an alternative, it was hypothesized that injection of complementary dsRNA into viral-infected broodstock prior to mating might inhibit replication of the target virus sufficiently to reduce or eliminate its transmission to their offspring. Subsequent selection of uninfected offspring would allow for post-clearing of LSNV from the existing stock and for conversion of the stock to LSNV-free status. Testing this hypothesis using the LSNV-infected stock described above, we found that transmission was substantially reduced in several treated broodstock compared to much higher transmission in buffer-injected broodstock. Based on these results, the model is proposed for post-clearing of SPF stocks using dsRNA treatment. The model may also be applicable to post-clearing of exceptional, individual performers from grow-out ponds for return to a nucleus breeding center.

A novel and inexpensive application of RNAi technology to protect shrimp from viral disease.

Large-scale production of long dsRNA is needed if antiviral applications of RNAi are to succeed in shrimp farm operations. A novel hairpin-RNA expression vector was developed based on the RNA-dependent RNA polymerase (RdRp) gene of yellow head virus (YHV), the cause of a lethal shrimp disease. Using transformed RNase-deficient Escherichia coli, large amounts (approximately 5 mg dsRNA from 130 ml bacterial culture) of long dsRNA (>300 nt) were produced. Large-scale in vivo dsRNA production was approximately one-fourth the cost of production of a commercial in vitro transcription kit. The hairpin-RNA consisted of the target RdRp sequence ("forward") and a 100-base shortened version of its inverted repeat ("reverse") to introduce a loop and bypass the difficulty of including a small "loop" connector into the "carrier" vector. A test group of whiteleg shrimp Penaeus (Litopenaeus) vannamei (approximately 10-15 g) was injected with 25 microg of this dsRNA 1-day prior to YHV challenge while control groups were injected with NaCl solution or similarly prepared dsGFP-RNA. The group injected with YHV-specific dsRNA did not develop yellow head disease during 14-day of observation after YHV challenge, whereas the control groups injected with NaCl and dsGFP-RNA developed gross signs of yellow head disease and died within 7-10 days after challenge. Quantitative RT-PCR and immunohistochemistry revealed that both viral mRNA and viral proteins were suppressed in the protected shrimp.