White Spot Syndrome Virus and the Caribbean Spiny Lobster, Panulirus argus: Susceptibility and Behavioral Immunity

The Caribbean spiny lobster Panulirus argus is susceptible to infection by Panulirus argus Virus 1 (PaV1), the only virus known to naturally infect any lobster species. However, P. argus is able to mitigate PaV1 transmission risk by avoiding infected individuals. White Spot Syndrome Virus (WSSV) has a particularly wide host range. WSSV has not been documented in wild populations of spiny lobsters, but has been experimentally transmitted to six other lobster species from the genus Panulirus spp. While WSSV has been detected intermittently in wild populations of shrimp in the Caribbean region, the risk to P. argus has not been evaluated. Potential emergence of the disease could result in fisheries losses and ecological disruption. To assess the risk to P. argus, we tested its susceptibility to WSSV via injection and waterborne transmission. We also tested whether healthy lobsters can detect and avoid conspecifics with qPCR-quantifiable WSSV infections. We found P. argus to be highly susceptible to WSSV via intramuscular injection, with mortality reaching 88% four weeks post inoculation. Panulirus argus was also susceptible to WSSV via waterborne transmission, but WSSV burden was low after four weeks via qPCR. Behavioral assays indicated that P. argus can detect and avoid conspecifics infected with WSSV and the avoidance response was strongest for the most heavily infected individuals – a response comparable to PaV1-infected conspecifics. Panulirus argus is the first spiny lobster found to be susceptible to WSSV in the Americas, but it is possible that a generalized avoidance response by healthy lobsters against infected conspecifics provides a behavioral defense and may reduce WSSV infection potential and prevalence. Such avoidance may extend to other directly transmitted pathogens in spiny lobster populations preventing them from becoming common in their population. Author Summary Erica P. Ross is a PhD candidate at the University of Florida, studying the disease ecology of the Caribbean spiny lobster, with a focus on chemosensory ecology. Donald C. Behringer is an associate professor at the University of Florida and his research focuses on disease ecology, epidemiology, and fishery ecology, with a focus on crustaceans and other marine invertebrates. Jamie Bojko received his PhD from the University of Leeds and is currently a post-doctorate associate at the University of Florida studying experimental and systemic crustacean pathology.


Introduction
238 3') was synthesized and labeled with the fluorescent dyes 5-carboxyfluroscein (FAM) on the 5' 239 end and N'-tetramethyl-6-carboxyrhodamine (TAMRA) on the 3' end. The TaqMan assay used in 240 this study was adapted from Durand and Lightner (2002) and Bateman et al. (2012) [2,21]. The 241 assay consisted of extracted total DNA template that was added to the TaqMan Fast Advanced 242 master mix (Applied Biosystems), which contained 0.3 µM of each primer and 0.15 µM of TaqMan 243 probe, with a final reaction volume of 20 µl. Amplification and detection were performed using a 244 ThermoFisher Quant Studio 5 Real-Time PCR system. The reaction mix was subjected to 95 °C 245 for 20 s, then 40 cycles at 95 °C for 1 s and 60 °C for 20 s. Quantification of the number of WSSV 246 copies were determined by measuring Ct values and using the standard curve described in 247 Durand and Lightner (2002) [21]. Each sample was analyzed in triplicate, and the mean viral 248 quantity calculated. Positive samples with WSSV virion counts under 100 viral copies were 249 excluded to decrease the chance of false positives and possible environmental contamination.

250
All animals used in these experiments were confirmed negative for PaV1 DNA using the 251 qPCR assay described in Clark et al. (2018) [22]. The primers were PaV1nucleaseF (5'-252 CGTTGTACGGAATCGTTATTAAAGC-3') and PaV1nucleaseR ( 275 aggregation was used to obtain p-values ( = 0.05) and 95% confidence intervals where the null 276 probability of choosing a conspecific shelter was equal to 0.5 (random). These data were then 277 analyzed using a two-tailed binomial test for aggregation, where the null probability of choosing a 278 conspecific shelter was 0.8, equal to the rate of aggregation found in our control trials and previous 279 published data on aggregation behavior for P. argus [39]. A contingency table and Chi Square 280 (X 2 ) analysis was used to compare differences in aggregation rates in response to healthy, PaV1 281 infected, and WSSV infected conspecifics.

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Although the avoidance cue for PaV1 is found in the urine, heavily infected individuals are 507 avoided at equal rates to dead conspecifics and heavily injured conspecifics [40,49]. A few 508 hypotheses may reveal why avoidance of conspecifics infected by PaV1, and now WSSV, is 509 similar to avoidance of dead and injured conspecifics. PaV1 and WSSV are systemic infections 510 that lead to catabolism, tissue necrosis, and a decline in total hemocyte counts [20,59,60].

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The strength of PaV1 avoidance may be similar to injured conspecific avoidance because 535 would therefore result in avoidance due to alarm cues, rather than the virus itself.

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Determining the identity of the cue that initiates disease avoidance is an ongoing effort, 538 than a pathogen-specific avoidance. Further work is needed to identify the chemical, 539 physiological, and molecular pathways driving the avoidance response. Identifying the bioactive 540 molecules responsible for avoidance would likely illuminate the source of the avoidance cue. 854 .