78 SEPTEMBER • WORLD AQUACULTURE • WWW.WAS.ORG PHOTO 1. DNA extraction for pathogen detection via qPCR. Photo by Pipitpan Lerdpipitpon al. 2020). In a broodstock facility, animals are typically not tested individually. For example, in the case of EHP, detection is challenging on a per-animal basis since samples are collected from feces, making it difficult to trace a positive result back to a specific animal. However, for viral diseases, if each animal is individually tested, it becomes much more effective in ensuring the stock is free from viral pathogens. That said, not every broodstock facility is willing to bear the cost of testing each and every animal, which remains a limitation in implementing such stringent biosecurity measures. To address this, protocols must embrace statistically significant sample sizes, especially for high-value broodstock and larval batches. Additionally, sampling should be representative, covering various tanks, batches, and times, and stratified based on production stage. The required sample size depends on the estimated prevalence of the disease. For WSSV testing in PL, the sample size should reflect an estimated prevalence of 1% to 2% at a 95% confidence level, which corresponds to testing approximately 150–300 PL shrimp per ≥100,000 individuals, with sampling distributed across five distinct locations in the pond. For broodstock shrimp, all individuals should be tested. Alternatively, the broodstock sample size may be calculated based on an estimated prevalence of 5%, using statistical methods that achieve the required confidence level. For juvenile shrimp, the estimated disease prevalence may range from 2% to 10%, and the sampling plan should be adjusted accordingly to meet surveillance objectives and ensure diagnostic reliability (National Bureau of Agricultural Commodity and Food Standards 2007). Detectability at Low Viral Loads Another challenge is the detection threshold of qPCR. Many pathogens, especially in broodstock or early-stage larvae, may exist in subclinical or low-copy forms. These carriers may not display symptoms but can still transmit disease vertically. Traditional qPCR may miss these infections, especially in pooled samples. Digital PCR (dPCR) offers a solution, with greater sensitivity and precision for detecting low-copy viral loads (Hindson et al. 2011).Tissue type also plays a role. Sampling non-target or low-infection tissues can lead to undetected infections. For example, WSSV is best detected Shrimp farming has rapidly evolved into a multibillion-dollar global industry, playing a vital role in food production, employment, and economic development in many coastal nations. Historically dominated by Penaeus monodon (black tiger shrimp), the industry underwent a pivotal transformation with the widespread adoption of Litopenaeus vannamei (Pacific white shrimp). The switch was primarily due to L. vannamei’s adaptability to intensification, rapid growth cycle, and the commercial availability of Specific Pathogen Free (SPF) broodstock (Wyban and Sweeney 1991). This species now accounts for over 75% of the world’s farmed shrimp production. However, with this transition have come new challenges. Disease outbreaks remain a persistent threat, often wiping out entire crops and causing economic devastation. The increasing frequency and complexity of disease emergence, despite the use of SPF broodstock and rigorous PCR testing, suggest that some foundational assumptions in shrimp farming may be flawed. Core practices such as diagnostic screening, broodstock management, and feed formulation are overdue for reevaluation. This paper presents a call to the shrimp aquaculture industry to critically examine and revise these core components. We explore the overreliance on PCR diagnostics, the role of broodstock handling in vertical transmission, and the subtle but significant impact of nutrition on disease resistance. A paradigm shift toward more integrative, scientifically grounded practices is urgently needed. Reevaluating the Reliability of PCR Diagnostics Limitations in Sampling Strategy PCR, particularly quantitative real-time PCR (qPCR), has become the mainstay of disease screening in hatcheries and growout farms (Lightner 2011, Tang and Lightner 2005). However, the method’s reliability hinges on appropriate sampling strategy. In most hatcheries, only a fraction of animals are tested, often 30–150 samples from populations numbering in the tens or hundreds of thousands. This creates a high risk of false-negative results if pathogen prevalence is low. Statistically, such small samples provide a very narrow window into population health (Corbel et Rethinking the Foundations of Shrimp Farming: A Call for Change in Understanding and Practice Farshad Shishehchian, Wiphada Mitbumrung and Erika Chong
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