# Innovative use of squid (Loligo spp.) ink powder as a potent immunostimulant for vannamei shrimp (Litopenaeus vannamei) in the treatment of infectious myonecrosis

## TL;DR

Squid ink powder boosts shrimp immunity against a deadly virus, offering a natural alternative to chemicals in aquaculture.

## Contribution

Squid ink powder is shown to be a novel, effective immunostimulant for vannamei shrimp infected with infectious myonecrosis virus.

## Key findings

- A 500 mg/kg dose of squid ink powder significantly increased immune parameters like THC, RB, SOD, PO, and phagocytic activity in shrimp.
- Squid ink powder reduced ribonucleotide reductase expression, indicating effective suppression of the virus.
- The treatment improved water quality and feed protein content without adverse effects.

## Abstract

Infectious myonecrosis virus (IMNV) is a significant pathogen affecting Litopenaeus vannamei, causing high mortality and substantial economic losses in shrimp aquaculture. Conventional chemotherapeutics have limited efficacy and raise environmental concerns. This study explores the immunostimulatory potential of squid (Loligo spp.) ink powder as a natural dietary supplement to enhance the nonspecific immune responses in L. vannamei and mitigate IMNV-associated pathology.

A completely randomized design was employed, with five groups: a negative control (healthy shrimp), a positive control (IMNV-infected), and three treatment groups that received squid ink powder at 400, 500, and 600 mg/kg feed, respectively. The feed was administered before and after IMNV immersion challenge. Immune parameters assessed included total hemocyte count (THC), differential hemocyte count (DHC), respiratory burst (RB), phenoloxidase (PO), superoxide dismutase (SOD), phagocytic activity, and ribonucleotide reductase (RR) expression. Statistical analysis was conducted using a one-way analysis of variance with Duncan’s post hoc test.

The 500 mg/kg dose of squid ink powder significantly enhanced shrimp immunity post-IMNV challenge. This treatment yielded the highest THC (6 × 105 cells/mL), RB (1.13 optical density [OD]), SOD (0.98 units/mL), PO (0.619 OD), and phagocytic activity. A marked reduction in RR enzyme expression was observed, indicating effective viral suppression. DHC analysis revealed elevated granulocyte and semi-granulocyte counts, suggesting heightened immunological activity. Water quality parameters remained within acceptable aquaculture limits, and proximate analysis confirmed an improvement in protein content in the feed following supplementation.

Squid ink powder at 500 mg/kg feed significantly enhances the non-specific immune system in L. vannamei and reduces IMNV-induced pathology. This natural additive offers a promising, sustainable alternative to synthetic immunostimulants in shrimp aquaculture.