John Paul Matthew Domingo Guzman

Manila clam (Ruditapes philippinarum) is a commercially important bivalve in aquaculture industries for its adaptability, fast growth, and high market demand. Unfortunately, diseases cause significant economic losses in its farming. Understanding its immune response is crucial to mitigating these losses. Fibrinogen-related proteins (FREPs) are vital components of an organism’s innate immunity. In R. philippinarum, FREPs are linked to pathogen detection, agglutination, and opsonization. This study characterized RpFREP-CCP, a FREP from R. philippinarum, using in silico methods to explore its putative immune function. The mRNA sequence of RpFREP-CCP (NCBI Accession No.: XM_060697753.1) was retrieved, followed by phylogenetic analysis using homologous sequences from the NCBI database and prediction of physicochemical properties. RpFREP-CCP was predicted to be a hydrophilic and highly stable protein, exhibiting distinct phylogenetic similarities to FREPs from other mollusks. Protein structure prediction revealed regions of high confidence alongside intrinsically disordered regions while functional analysis identified a typical fibrinogen-like structure and a unique complement control protein (CCP) domain, suggesting involvement in pathogen recognition and immune regulation. Furthermore, RpFREP-CCP is predicted to interact with other FREPs, Toll-like receptors, and extracellular protein kinases, potentially enhancing immune responses through protein-protein interaction networks. These findings provide insights into the role of RpFREP-CCP in R. philippinarum immune defense, warranting further validation through in vitro and in vivo studies.

Kuruma shrimp ( Penaeus japonicus ) is an economically important shrimp perennially affected by diseases. In 2022, White Muscle Disease (WMD) was first characterised in this Penaeid species, caused by Photobacterium damselae subsp. damselae (Pdd). In this study, muscular and gut microbiome dynamics and their function in the disease progression are investigated by 16S rRNA metagenome sequencing using Illumina sequencing technologies. Alpha diversity indices showed that Pdd infection in the muscle, stomach, and intestine did not significantly change bacterial diversity between control and infected groups at all time points observed (Days 0, 1, 3, 5, 7 and 10). In the infected samples, the Shannon and Simpson indices increased starting Day 5 (D5), in congruence with the first observation of muscle whitening. Bacterial composition for the infected group at the genus level revealed that Photobacterium and Vibrio have increased their relative abundance in the muscle at Day 5 (D5) until Day 7 (D7), but declined at Day 10 (D10). As for stomach samples, Photobacterium declined in abundance and later increased significantly at Day 7 (D7). Photobacterium in the intestinal samples from the infected group increased at Day 5 (D5) but later decreased at Day 7 (D7). Meanwhile, linear discriminant analysis Effect Size (LEfSe) identified that most taxa belong to phylum Pseudomonadota, which can be potential markers for WMD. Moreover, the temporal dynamics of the amplicon sequencing variant ASV2, confirmed to be 100% homologous to the WMD‐P3 strain used in this study, were characterised. For all tissues, the logarithmic relative abundance is considered high and very apparent in infected samples collected at Day 7 (D7). Overall, our study provides an understanding of the muscle and gut microbial community, specifically at the genus level, distinguished between WMD‐infected and healthy Kuruma shrimps.

The formation of extracellular traps (ETosis) is an innate immune mechanism in shrimp against pathogens. Microorganisms are entrapped in extruded DNA fibres co‐localised with antimicrobial peptides and eventually killed. However, as a cell death mechanism, its strict regulation is essential as excessive formation of ETs may cause detrimental effects to the host by contributing to disease pathophysiology. Here, we investigated the ability of freeze‐dried Lactiplantibacillus plantarum (FD‐LAB), previously reported to enhance shrimp immunity against pathogenic infections, to regulate ETosis. In an ex vivo setup, gill cells and circulating hemocytes were pre‐exposed to FD‐LAB and were then stimulated with Vibrio parahaemolyticus . Results showed that V. parahaemolyticus alone induced ETosis in both gill cells and circulating hemocytes, while FD‐LAB alone did not. However, when cells were pre‐exposed to FD‐LAB prior to stimulation with V. parahaemolyticus , no ETosis occurred. Similarly, changes in reactive oxygen species (ROS) production coincided with the formation of ETs, thus signifying that FD‐LAB may regulate ETosis in shrimp gill cells and circulating hemocytes possibly by dampening ROS production. These results present a novel means to regulate ETosis and indicate that FD‐LAB may enhance shrimp immunity while also acting on immune regulation.

Gut bacterial communities play a key role in shrimp health; thus, their modulation has been a target of dietary supplements which also function in enhancing disease and stress resistance of shrimp. However, this also raised the question of whether immunostimulants yield distinct changes in the gut bacterial composition or whether there are consistent features across all treatments. Here, we performed a machine learning‐aided meta‐analysis of 16S rRNA gut bacterial community studies of immunostimulants for Penaeus vannamei . Sequence reads from the selected studies were obtained and processed through bioinformatics tools. While beta diversity analysis suggests similarities between the normal, infected and stimulated shrimp, alpha diversity indices showed higher species richness in the gut bacterial communities of shrimp fed with immunostimulants. Specific beneficial taxa were enriched upon immunostimulation, while potentially pathogenic taxa decreased in abundance. Random forest modelling also identified key predictor taxa which may be used to classify gut bacterial communities based on immune status, type of immunostimulant and the specific immunostimulant. Despite some shared patterns in differential abundance—having decreased relative abundances of Photobacterium and other members of Gammaproteobacteria—the influence of immunostimulation on gut bacterial community composition was type‐ and treatment‐specific, as evident in the distinct abundance profiles of the predictor taxa. Functional prediction analysis also showed distinct pathways enriched in immunostimulated shrimp, as influenced by the type of the immunostimulant. This study highlighted the specific impacts of dietary supplementation‐induced immunostimulation on gut bacterial communities and identified key features in immunostimulated shrimp which provide a novel perspective on the interplay between gut bacterial community and immunity.

Aquaculture diseases caused by pathogens such as Vibrio spp., amplified by the emergence of antibiotic resistance, threaten the aquaculture industry. Due to its critical role in regulating the expression of genes related to antibiotic resistance and virulence, quorum sensing (QS) was proved to be an ideal target in disease control. In this study, we investigated the effects of the alkaloid piperine on the QS system of a fish pathogenic Vibrio harveyi strain. In vitro assays showed that piperine inhibits biofilm formation of V. harveyi without affecting their growth. Moreover, piperine specifically reduced QS activity in V. harveyi, as evident in the inhibition of biofilm and bioluminescence, likely through the AI‐2 pathway. Molecular docking simulations showed significant binding energies between piperine and QS proteins—LuxP, LuxQ, LuxR and LuxS—revealing competitive inhibitory effects against LuxP, LuxR, and LuxS, and non‐competitive interactions with LuxQ. This study demonstrated the effects of piperine against V. harveyi and elucidated its mechanism of action against V. harveyi QS, implying its potential application in aquaculture systems.