Impact of temperature on the immune response of WSSV-infected Indian white shrimp (Penaeus indicus): Insights from flow cytometry

White spot syndrome virus (WSSV) is one of the most virulent pathogens of cultured shrimp. Water temperature is known to have a profound effect on disease expression and viral pathogenesis. This study evaluated the effect of temperature variations on the virus replication and host response. Flow cytometry method was used to analyse Penaeus indicus immune parameters (apoptosis, respiratory burst, cytoplasmic free calcium concentration (cf-Ca²⁺), cell cycle analysis, and phagocytosis) against WSSV infection at 27 °C, 30 °C and 33 °C temperatures at different time points 12hpi, 24 hpi, and 48 hpi. Effect of WSSV replication at different temperatures was quantified by qPCR showing the shrimps exposed to higher temperatures (33 °C) with reduced viral load. At this higher temperature of 33 °C, shrimp responded with elevated percentages of early apoptosis (11.77 ± 2.11 %) and late apoptosis (19.88 ± 4.99), and higher percentage of cf-Ca²⁺ activity (80.10 %) at 48 hpi. Phagocytosis analysis revealed maximum percentage (27 %) of semi granulocytes at 33 °C at 48 hpi. WSSV infected shrimps responded with an increased respiratory burst from 12 hpi to 48 hpi exposed to 33 °C when the virus replication was observed to be low. Decreased percentage of G1 phase in WSSV infected shrimp samples at all time points and temperature, except for minimal increase at 12 hpi at 33 °C indicated cell cycle is deregulated by WSSV infections. The flow cytometric based immune analysis of WSSV infected shrimps in this study suggests elevated temperatures plays a significant role to induce host defense reaction and in inhibiting replication of WSSV. The differentially expressed gene (DEGs) analysis of WSSV infected shrimps at 12, 24 and 48 hpi resulted in 846, 854, 1291 DEGs respectively, offering insights into WSSV infection and shrimp immunity under varying temperature conditions at molecular level.