Since 2009, HPM caused by EHP emerged as an economically important disease being highly prevalent in some of the major shrimp producing countries in SE Asia, and more recently the pathogen has spread to Venezuela . In SE-Asian countries, along with white spot disease (WSD) and acute hepatopancreatic necrosis (AHPND), HPM remains a major threat contributing to significant economic losses in grow-out ponds. Penaeus vannamei is an euryhaline species which is raised in a wide range of conditions including high salinities (30 ppt), estuarine environments (10-20 ppt), and low salinities (2 ppt) . In this study, we investigated the prevalence and severity of EHP infection in two salinities, high salinity (30 ppt) and low salinity (2 ppt) under laboratory challenged conditions. The fecal strings, used as a source of EHP inocula, in the challenges was sufficient to cause disease to shrimp maintained at both salinities, as confirmed by histopathology and PCR. The results from this study provide a new EHP infection method through fecal strings via fecal-oral route. Previous studies have shown some routes of infection included cohabitation [15, 16], reverse gavage, and direct hepatopancreas injection with EHP-inoculum . EHP lesions, including the presence of plasmodium in the cytoplasm of an infected cells and the mature spores within the cytoplasm or released spores in the lumen, were found by histopathology in EHP-challenged shrimp reared at both salinities. This unequivocally confirms EHP infection at a wide range of salinities as high as 2 to 30 ppt. When the initial inoculum used for the experimental challenge was low, i.e. 1x103 copies of EHP/ ng of total HP DNA, the HPM prevalence was similar (i.e. 25%) irrespective of the salinities (See Table 1). However, the prevalence of EHP infection increased at 30 ppt salinity (87.5% prevalence) compared to 2 ppt salinity (33.3% prevalence) when the inoculum level was increased from 1x103 to 1x106 copies of EHP/ ng of total HP DNA in the challenge experiment (i.e. challenge #2). Dose-dependent challenge has been well documented for other shrimp pathogens such as AHPND and Hepatobacter penaei [9, 17]. In AHPND challenge tests, an infectious dose of 2.0x106 CFU/ml is required to provoke high mortality (>90%) in immersion challenge method. In contrast, a lower dose (2.0x104 CFU/ml) does not cause mortality nor histological lesions in the challenged population . In the present study, the inoculum with low copy number (1.6x103 copies/ng HP DNA) used in the challenge #1 caused mild infections in the challenged shrimp, whereas, severe infections (Grades G3 to G4) and a higher prevalence occurred in the challenge #2 when the EHP copy number in the inoculum was higher (i.e. 1x106 copies/ng HP DNA).
The histological lesions in shrimp maintained at 30 ppt salinity were more severe. Moderate-severe grade of infection (G3-G4) were found in 50% of the shrimp affected. In contrast, only 16% of shrimp reared at salinity of 2 ppt showed grade G3 level of infection, according to the Lightner’s scale . The difference in the severity of EHP infection at two different salinities was probably due to the differential effect of salinity on spore germination. One of the critical phases in the spore germination is the increase of intra-spore osmotic pressure. It is possible that the difference in salinities led to a hypotonic environment at 2 ppt compared to hypertonic environment at 30 ppt. It is possible that the hypertonic solution enhances the germination of the spore by increasing the spore activation process. He et al  found a difference of eversion of the polar tube in different osmotic environments in Encephalitozoon intestinalis, an obligate intracellular microsporidium that causes gastrointestinal diseases in immunocompromised and immunocompetent people. Differences in polar tube germination associated to change in salinity have also been reported by other researchers. De Graff et al. showed an increase in germination of Nosema apis spores at 0.5 N NaCl concentration  and an increase of germination of Nosema algerae spores at 0.1 M NaCl concentration vs. 0.05 M NaCl [21, 22]. In our study, the NaCl concentration was about 0.5 M NaCl vs. 0.03 M at 30 ppt and 2 ppt, respectively. This difference could explain in part the effect on germination of EHP spores and resulting prevalence levels.
Hardness is another variable that was different in the two salinities used in this study and could have been a factor that affect the spore germination. The hardness in low salinity (2 ppt) was about 240 mg/L (City of Tucson https://www.tucsonaz.gov/water/water-quality-reports-and-publications), vs. the marine water artificially prepared at 30 ppt that was around 1575 mg/L (Crystal Sea, Marinex). It has been reported that calcium is an important second messenger that activates many cell events and calcium influx might be in part responsible for the activation of microsporidian spore discharge at higher salinities .
In grow-out ponds in some EHP endemic areas in Asia, the salinity conditions are found to vary widely. For example, in India there are some shrimp farming areas in high and low salinity, and the prevalence of EHP seems to be lower at lower salinities (below 5 ppt) as observed in Andhra Pradesh in 2019 (Aranguren et al., Unpublished data). Similar conditions were recorded in two major shrimp farming areas in Venezuela, i.e. Maracaibo lake where the salinities are around 4-6 ppt, and in Falcon state where the salinities varies from 36-40 ppt. In Venezuela, shrimp farming is not fully integrated, and the movement of nauplii and post-larvae between Falcon and Maracaibo lake area is a common practice. This suggests that EHP-infected PL or broodstock could have been moved between these two zones in a similar way. However, EHP has only been detected in the Falcon area where the salinities are high. In the Maracaibo’s lake where the salinities are low, EHP has not been reported yet. One possibility that has limited the EHP infection could have been the difference in water salinity.