The stakes are considerable. Shrimp aquaculture supplies more than half of the global shrimp market, and L. vannamei alone accounts for roughly 80 percent of farmed shrimp output, thanks to its rapid growth and tolerance of salinities ranging from 0.5 to 40 parts per thousand. But the industry is chronically undermined by environmental stress. Rising ammonia in culture water impairs growth, invites disease, and kills animals; temperature swings disrupt hormones and enzymes, which function best between 28 and 32 degrees Celsius; and ammonia competes with oxygen for binding sites on the oxygen-carrying pigment haemocyanin, choking respiration and suppressing immunity. Biofloc technology promises a way out by converting toxic nitrogenous waste into microbial biomass that shrimp can themselves consume.

To understand what biofloc actually does inside the animal, the team first ran an optimization experiment using a Taguchi L9 orthogonal array and grey relational analysis, testing stocking densities of 100 to 300 shrimp per cubic meter, carbon-to-nitrogen ratios of 10 to 20, and probiotic doses of 7 to 21 milliliters per liter. Two winning configurations emerged as treatments for the main trial: GRG1, with 200 shrimp per cubic meter, a C/N ratio of 10:1, and 14 milliliters per liter of the probiotic Bacillus tropicus; and GRG2, with the same density but a C/N ratio of 20:1 and half the probiotic dose. Both were run inside the IMTAS facility, a closed-loop system combining shrimp culture, cyclone-based flocculation with UV treatment, and a phytoremediation unit where Caulerpa seaweed strips nutrients from recirculating water, all monitored through an Internet of Things control network.

The water chemistry alone told a compelling story. Over the twelve-week culture period, both biofloc treatments maintained significantly lower total ammonia nitrogen, nitrite, and phosphate than the control, which relied on a modest 2.5 percent daily water exchange and no microbial management. Dissolved oxygen was significantly higher in the biofloc tanks, as were pH levels, while conductivity, total dissolved solids, and salinity were all lower. In other words, the microbial flocs were doing precisely what biofloc advocates claim: assimilating inorganic nitrogen into microbial biomass and stabilizing the culture environment without external water input.

The shrimp responded in kind. Mean final weight climbed from 10.50 grams in the control to 21.45 grams in GRG1, more than a doubling. Specific growth rate rose from 3.96 to 4.79 percent per day, feed conversion ratio improved from 1.39 to 1.05, and survival jumped from 49.08 percent to 75.91 percent. GRG1 outperformed every other treatment on every metric measured, a phenotypic signal that the transcriptomic data would soon help explain.

For the molecular analysis, the researchers dissected midgut tissue at harvest, flash-froze it in liquid nitrogen, and sequenced RNA on an Illumina NovaSeq 6000, mapping 150-base-pair paired-end reads against the L. vannamei reference genome. Differential expression analysis with DESeq2, using strict thresholds of a log2 fold change of at least 1 and an adjusted p-value of 0.05 or below, revealed 1,245 differentially expressed genes in GRG1 versus the control, 1,145 in GRG2 versus the control, and 681 between the two biofloc treatments themselves. Hierarchical clustering and principal component analysis confirmed that both biofloc groups clustered apart from the control, with treatment-specific expression signatures. The smaller number of differences between GRG1 and GRG2 makes sense: both animals were living in biofloc, so their shared transcriptomic response dwarfed the differences between the two floc recipes.

The pathway-level results were the most revealing. In GRG1 versus the control, three KEGG pathways were significantly enriched: the proteasome, autophagy, and phagosome pathways. Proteasome subunits across both the 26S regulatory particles and the 20S core were strongly upregulated, indicating accelerated degradation of ubiquitinated and misfolded proteins and tighter cellular quality control. The autophagy pathway showed four-fold or greater upregulation of TSC1, AMPK, ULK1/2, LC3, ATG2, and ATG9L, pointing to vigorous autophagosome formation and cellular component recycling, while BNIP3, a hypoxia-responsive gene, rose 3.2-fold, suggesting improved tolerance of environmental stress. In the phagosome pathway, tubulin genes surged up to ten-fold, actin and integrins rose five-fold, and thrombospondin increased a remarkable 22-fold, together implying enhanced cytoskeletal dynamics, immune cell adhesion, and intracellular pathogen clearance.

GRG2 shrimp told a different molecular story. Their enriched pathways were aminoacyl-tRNA biosynthesis and protein processing in the endoplasmic reticulum, reflecting a shift in how protein was built rather than broken down. Alanyl-tRNA synthetase was upregulated, favoring alanine incorporation, while synthetases for glutamine, aspartate, and glycine were downregulated, hinting at selective modulation of amino acid use under the higher carbon-to-nitrogen regime. In the endoplasmic reticulum pathway, heat shock protein 70 rose four-fold and ER-associated degradation genes such as p97 and ufd1 climbed, while calnexin, BiP, and eIF2α fell, suggesting a reduced burden from the unfolded protein response. Comparing the two biofloc treatments directly, oxidative phosphorylation emerged as the distinguishing pathway: NADH dehydrogenase subunits were upregulated in GRG1, while cytochrome c oxidase subunits were downregulated, indicating treatment-specific differences in mitochondrial energy metabolism.

The convergence of molecular and phenotypic data is what gives the study its punch. GRG1, the treatment with the cleanest water and the strongest growth, was also the one with the greatest enrichment of protein turnover, immune, and energy pathways, suggesting that biofloc conditions triggered a systemic reprogramming that favored metabolic efficiency and resilience. Proteasome activity kept cellular proteins in order, autophagy recycled components under culture stress, and phagosome activation plausibly bolstered defense against pathogens, all of which created an internal environment conducive to the doubled weight gain and 76 percent survival observed. The authors are careful to frame the work as exploratory: the control group had only two usable RNA libraries after one degraded, physiological immune markers such as total hemocyte count and phenoloxidase activity were not measured, and the control’s different water exchange regime makes it hard to fully separate biofloc effects from water management effects. No qPCR validation was performed, so the expression patterns await independent confirmation.

Even with those caveats, the study delivers something the biofloc field has lacked: a tissue-specific, genome-wide account of what microbial flocs actually do to the animals farmed among them. With the sequencing data deposited in the NCBI Sequence Read Archive under BioProject PRJNA1468551, the researchers have laid a molecular foundation for optimizing biofloc protocols, potentially identifying gene expression markers that predict which floc configurations will produce the healthiest, fastest-growing shrimp. As global demand for shrimp keeps climbing and freshwater and coastal resources come under pressure, farming systems that let microbes do the water treatment, while the shrimp’s own genes do the rest, may prove to be one of aquaculture’s most important technological turns.

Subject of Research: Transcriptomic responses of Pacific white shrimp to biofloc technology in indoor aquaculture

Article Title: Transcriptomic adaptation of Litopenaeus vannamei to biofloc conditions in an indoor aquaculture system

Article References: Transcriptomic adaptation of Litopenaeus vannamei to biofloc conditions in an indoor aquaculture system. (n.d.). https://doi.org/10.1007/s44338-026-00257-0

Image Credits: AI Generated

DOI: 10.1007/s44338-026-00257-0

Keywords: biofloc technology, Litopenaeus vannamei, transcriptomics, aquaculture, gene expression, RNA sequencing, immune response, protein turnover, autophagy, phagosome, oxidative phosphorylation, water quality