Participation of shrimp pva-miR-166 in hemocyte homeostasis by modulating apoptosis-related gene
PvProsaposin
during white spot syndrome virus infection
Authors: Wongdontri Chantaka , Luangtrakul Waruntorn , Boonchuen Pakpoom , Sarnow Peter , Somboonviwat Kulwadee , Jaree Phattarunda , Somboonwiwat Kunlaya 
Published: July 2024

ABSTRACT

Tiny controllers referred to as microRNAs (miRNAs) impede the expression of genes to modulate biological processes. In invertebrates, particularly in shrimp as a model organism, it has been demonstrated that miRNAs play a crucial role in modulating innate immune responses against viral infection. By analyzing small RNAs, we identified 60 differentially expressed miRNAs (DEMs) in
Penaues vannamei
hemocytes following infection with white spot syndrome virus (WSSV). We predicted the target genes of WSSV-responsive miRNAs, shedding light on their participation in diverse biological pathways. We are particularly intrigued by pva-miR-166, which is the most notably elevated miRNA among 60 DEMs. At 24 h post-infection (hpi), the negative correlation between the expression of pva-miR-166 and its target gene,
PvProsaposin
, was evident and their interaction was confirmed by a reduction in luciferase activity
in vitro
. Suppression of
PvProsaposin
in unchallenged shrimp led to decreased survival rates, reduced total hemocyte count (THC), and increased caspase 3/7 activity, suggesting its significant role in maintaining hemocyte homeostasis. In WSSV-infected shrimp, a lower number of hemocytes corresponded to a lower WSSV load, but higher shrimp mortality was observed when
PvProsaposin
was suppressed. Conformingly, the introduction of the pva-miR-166 mimic to WSSV-infected shrimp resulted in decreased levels of
PvProsaposin
transcripts, a significant loss of THC, and an increase in the hemocyte apoptosis. Taken together, we propose that pva-miR-166 modulates hemocyte homeostasis during WSSV infection by suppressing the
PvProsaposin
, an anti-apoptotic gene.
PvProsaposin
inhibition disrupts hemocyte homeostasis, rendering the shrimp’s inability to withstand WSSV invasion.

IMPORTANCE

Gene regulation by microRNAs (miRNAs) has been reported during viral infection. Furthermore, hemocytes serve a dual role, not only producing various immune-related molecules to combat viral infections but also acting as a viral replication site. Maintaining hemocyte homeostasis is pivotal for the shrimp’s survival during infection. The upregulated miRNA pva-miR-166 could repress
PvProsaposin
expression in shrimp hemocytes infected with WSSV. The significance of
PvProsaposin
in maintaining hemocyte homeostasis via apoptosis led to reduced survival rate, decreased total hemocyte numbers, and elevated caspase 3/7 activity in
PvProsaposin
-silenced shrimp. Additionally, the inhibitory ability of pva-miR-166-mimic and dsRNA-
PvProsaposin
on the expression of
PvProsaposin
also lowered the THC, increases the hemocyte apoptosis, resulting in a lower WSSV copy number. Ultimately, the dysregulation of the anti-apoptotic gene
PvProsaposin
by pva-miR-166 during WSSV infection disrupts hemocyte homeostasis, leading to an immunocompromised state in shrimp, rendering them incapable of surviving WSSV invasion.