Introduction
Shrimp farming plays a vital role in global aquaculture, with the whiteleg shrimp (Penaeus vannamei) being the most widely farmed species. However, disease outbreaks continue to cause significant economic losses, highlighting the need for reliable indicators to assess shrimp health and immune status (Flegel et al., 2019). Immune parameters in P. vannamei, including hemocyte counts, phenoloxidase (PO) activity, and antimicrobial peptide (AMP) gene expression, are commonly used to evaluate immune function (Tassanakajon et al., 2013; Xu et al., 2014). However, reported values vary widely across studies due to differences in sampling, handling, and shrimp physiological conditions. This variability limits the establishment of reliable baseline values and complicates comparisons between laboratories and production systems. Therefore, this study aims to standardize methods and establish reference values for key immune parameters in healthy P. vannamei, including total (THC) and differential hemocyte counts (DHC), PO activity, and AMP gene expression. Establishing consistent reference ranges for these parameters will support reliable health assessment and improve disease monitoring in aquaculture systems.
Materials and Methods
Shrimp (average body weight, 10 g) at the intermoult stage were selected for sampling. Hemolymph samples were collected using a well established in-house procedure under sterile conditions to minimize hemocyte activation and subsequent hemolymph coagulation. THC was determined through manual counting using a hemocytometer, while DHC was performed using optimized staining methods to differentiate hemocyte subpopulations. In parallel, high-throughput profiling was conducted using flow cytometry to characterize hemocyte subpopulations and validate manual counting results. Functional immune parameters were assessed through optimization of PO activity assays. Molecular immune responses were evaluated through RT-qPCR, targeting AMP genes.
Results and Discussion
Preliminary results showed that THC averaged approximately 107 hemocytes mL-1 hemolymph (n > 100 shrimp from different batches), which is at the upper range of values commonly reported in the literature (105–106 hemocytes mL-1 hemolymph)(Yildrim-Aksoy et al., 2022; Huang et al., 2013). Reported hemocyte counts vary widely, possibly due to differences in shrimp handling, hemolymph collection, and hemocyte sampling procedures. Lower counts observed in other studies may be mainly attributed to suboptimal hemolymph collection techniques and subsequent clotting. To reduce these sources of variation, standardized hemolymph collection and handling protocols were applied in this study. DHC showed improved morphological differentiation using optimized staining, addressing common inconsistencies in hemocyte classification. Flow cytometry results were comparable to manual counting, supporting its use as a reliable and high-throughput method for standardized immune monitoring and reducing repetitive manual labor.
PO activity measurements are frequently influenced by differences in assay parameters, such as substrate and incubation time. In this study, PO assays are being optimized under standardized conditions to improve consistency. Similarly, AMP expression can vary due to PCR efficiency and normalization methods. The verified amplification efficiency (E = 88–92%) indicates reliable assay performance, although further validation is required to establish consistent reference ranges for these functional immune parameters.
Overall, inconsistencies in immune parameter measurements highlight the need for standardized analytical procedures to ensure comparable and reproducible results. Once validated, these standardized procedures and derived immune parameter reference values will serve as practical diagnostic indicators for shrimp health monitoring. Similar to blood tests in human and veterinary medicine, routine immune assessment may enable early detection of physiological stress or immune suppression and support proactive health management in shrimp aquaculture systems, while reliable reference values for healthy shrimp would strengthen research on diseases and treatment strategies.
Acknowledgement
This research received funding from Flanders Innovation and Entrepreneurship (Belgium) through Baekeland mandate HBC.2024.0278 and acknowledges flow cytometer funding from EMBRC Belgium – FWO agreement 20151029-03 and Hercules agreement 20140910-03 for infrastructure support.
References
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