Temperature-Associated Differences in Mortality Timing in WSSV-Exposed Penaeus monodon Under Additional AHPND and WSSV Challenge

Abstract

Water temperature can affect the clinical expression of white spot syndrome virus (WSSV) disease. However, mortality patterns under the influence of directional temperature change remain unclear. Here, we evaluated 576 specimens of Penaeus monodon from a WSSV-positive cohort in a 4 × 4 factorial experiment combining four challenge conditions (no additional challenge, acute hepatopancreatic necrosis disease (AHPND) challenge, additional-WSSV challenge, or combined AHPND/WSSV challenge) with four temperature regimes: T1 (23 °C constant), T2 (23 → 33 °C), T3 (33 °C constant), and T4 (33 → 23 °C). Challenges were administered daily from Days 0 to 10. Tank-level analysis showed that additional challenge increased Day-10 mortality (p < 0.001), whereas the effects of temperature (p = 0.406) and challenge × temperature interaction (p = 0.482) were not significant. Under the reference Day-10 assignment, temperature regime was significantly associated with mortality timing in exploratory, individual-level log-rank analyses that did not account for tank-level clustering (WSSV, p < 0.001; AHPND/WSSV, p = 0.0346). In the WSSV group, T4 differed significantly from T2 and T3 after Holm correction. Tank-level survival-curve comparisons were nonsignificant, although restricted mean survival time showed a nominal temperature difference in the AHPND/WSSV group (p = 0.0381). Sensitivity analyses showed that the statistical significance of some temperature-associated survival comparisons changed when the 103 deaths with unknown timing were assigned to earlier days. Additional challenge was associated with increased cumulative mortality, while temperature-associated temporal findings remain exploratory.