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

Abstract

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.

Figure 1

(a) Construction procedure of convergent promoter cassette expressing dsRNA. The cloning of fragment-of-interest (FOI) linked with an inverted psaA promoter (pUC-YHV_RdRp-psaAin) (I) into pSRSap I generating recombinant plasmid for chloroplast transformation (pSRSapI-FOI-psaAin) (II). (b) PCR analysis confirming insertion and direction of FOI-psaA in pSRSap I using primers Y1 and rbcl illustrated on the left whereas lane M is 2-log DNA marker, lane T represents pSRSapI-FOI-psaAin (pSR-PYP), and lane E represents pSRSapI.

Figure 2

(a) Schematic diagram of chloroplast transformation. pSR-PYP containing psbH gene and convergent psaA promoters covering RdRp fragment was integrated into chloroplast genome (I) through double homologous recombination (left and right HR) replacing spectinomycin resistance gene (aadA) resulting spectinomycin sensitive and photosynthesis-restoration transgenic strain (II). (b) PCR analysis for selecting transformant containing dsRNA-expressed cassette; (I) qualification of chloroplast genome using Rubisco-large subunit (not shown in the diagram a) as internal control, (II) integration of dsRNA-expressed cassette using Y1 and Y2 primers, and (III) confirmation of homoplasmic status using W1 and W2, whereas lane M is 2-log DNA marker, lane TN72 and PYP represent amplicons resulting from genomic DNA extracted from TN72 and PYP, respectively.

Figure 3

Growth rate according to absorbance at wavelength 750 nm of dsRNA-expressing C. reinhardtii strain (PYP,--) comparing to the control strain (SR, -). Bar represents standard deviation.

Figure 4

Confirmation of dsRNA by RT-qPCR by *represents melting temperature (T_m) of dsRNA-YHV expressed from E. coli HT115 and dsRNA-YHV extracted from PYP strain while **represents negative reaction using ddH₂O as a template.

Figure 5

Post-larval shrimp survival percentage from feeding trial comparing between observed group (no YHV challenge) and normal feed (YHV challenge), SR supplement prior YHV challenge, and PYP supplement prior YHV oral challenge. Bar represents standard error. *and **Represent significant difference between experiment groups within observation day at confidential level p < 0.05 and p < 0.01, respectively.

Figure 6

RT-PCR analysis detecting YHV infection of survival shrimps at 8 d.p.i. Actin specific primers were use as internal control. Lane M is 2-log DNA marker. Each lane represents infection level of individual PL shrimp. Solid lines separate 3 individual shrimps in each tank. Dash lines separate 3 individual shrimp in the same tank. Noted that there were more shrimps remaining in negative group while all remaining shrimp in the other groups was analyzed and presented in this figure.