In shrimp farms, salinity drop in the water due to excessive rainfall has been mentioned to be a risk factor for WSSV outbreaks (Thuong et al., 2016a). It was hypothesized that when WSSV is introduced into the rearing water and a sudden lowering of the salinity occurs, this could lead to an uptake of water through the nephropores into the antennal gland, as shrimp attempt to regulate their haemolymph osmolarity and urinary ion excretion . Once the cells of the antennal gland become infected, the virus infection can spread further into the body. An experimental WSSV immersion challenge mimics a natural water-borne WSSV transmission. Thuong et al. (2016a), performed an experiment in which shrimp were immersed in sea water containing 10 5.5 SID50 mL-1 of WSSV . Subsequently, these shrimp were subjected to a salinity change from 35 to 5 g l-1. After 5 hours, the salinity was restored to 35 g l-1 . The mortality due to WSSV infection was 53%. There was no mortality in the control group without a salinity drop . This suggested an important role of a salinity drop in the WSSV infectivity during an immersion challenge . However, WSSV is also reported to be transmitted through consumption of infected tissues (Wang et al., 1999). In the current study, we examined the effect of a salinity change on infection and mortality during a per os WSSV challenge, because it simulates natural WSSV infections through cannibalism. By testing these conditions, we aim to investigate if salinity change is also a risk factor for WSSV infection during an oral WSSV challenge in L. vannamei . These results could then be used in future work to further elucidate WSSV transmission dynamics.
Materials and Methods
Virus stock production: specific pathogen free (SPF) Litopenaeus vannamei were imported as postlarvae (PLs) from the United States of America (USA) . Shrimp were housed in artificial seawater at 20 ppt salinity and 27°C ± 1°C. They were injected intramuscularly with t he WSSV Thai-1 strain (Escobedo-Bonilla et al., 2005). WSSV positive solid inoculum was prepared from the resulting infected carcasses .
WSSV challenge and salinity drop: The inoculum was used to infect PL76 shrimp through oral route. Briefly, during the experiment, shrimp were randomly divided into three challenge groups (A, B, C) consisting of three replicates of 10 shrimp . Ten shrimp were assigned to a first negative control (Mock1- 15 ppt drop ). Another group of ten shrimp served as the second negative control (Mock2 – 30 ppt drop ). Shrimp were housed individually in 10L tanks. Shrimp from groups A, B, and Mock1 were acclimatised to 20 ppt salinity, while shrimp from groups C and Mock2 were acclimatised to 35 ppt salinity. The oral infection trial followed a procedure adapted from Van Thuong , et al. (2016b). Group A remained at a salinity of 20ppt during and after the oral WSSV challenge. I ndividual shrimp from groups B, C, Mock1, and Mock2, were transferred into seawater with a 5ppt salinity. Groups A, B, and C received WSSV positive inoculum, while Mock1 and Mock 2 received negative solid inoculum. After a period of 5 hours, the salinity in the individual tanks from groups B, and Mock1 was restored to 20ppt, while the salinity in the tanks of groups C and Mock2 was restored to 35ppt. The animals were observed in the following days and the experiment ended when no mortality was observed for 48hours. WSSV infection presence or absence in the tissues of collected shrimp was confirmed by qPCR. The survival/mortality data were analysed statistically using the Log-rank (Mantel-Cox) test.
At the end of the challenge trial, cumulative mortality rates in the WSSV-challenged groups A, B, and C were respectively 21%, 33% and 40%. The differences in mortality rates showed a trend between group A, that was not subjected to a drop in salinity, and group C, that was subjected to a 30ppt drop (from 35 to 5 ppt) (p-value = 0.0952). In the t wo control groups, Mock1 and Mock2, that were subjected to a salinity drop of respectively 15 and 30ppt, all shrimp survived. WSSV infection was confirmed by qPCR in a sample of the dead shrimp. WSSV was absent in sampled survivors and negative controls.
Discussion and conclusion
The results of the experiment showed that the probability or risk of infection in the population increased when the animals were subjected to a salinity drop during an oral WSSV challenge. This result was similar to the results obtained by Thuong et al. (2016a) during their WSSV immersion experiments with the same change in salinity. It suggests that salinity change could indeed be a risk factor for WSSV infection in the field, where natural WSSV transmission occurs both by water-borne or cannibalism routes . De Gryse et al. (2020) argued that this could be explained, because sudden salinity drop during, e.g., heavy monsoon rains, aggression, establishment of social dominance, and feed intake (cannibalism) are conditions where frequent urination , and thus frequent opening of the nephropore, takes place. Subsequently, this could create a window of opportunity for WSSV invasion, making entry via the antennal gland possible (de Gryse et al., 2020).
This research received funding from Flanders Innovation and Entrepreneurship (VLAIO, Belgium).