Crustacean Aquaculture Research Articles

RNA Interference Applied to Crustacean Aquaculture

RNA interference (RNAi) is a powerful tool that can be used to specifically knock-down gene expression using double-stranded RNA (dsRNA) effector molecules. This approach can be used in aquaculture as an investigation instrument and to improve the immune responses against viral pathogens, among other applications. Although this method was first described in shrimp in the mid-2000s, at present, no practical approach has been developed for the use of dsRNA in shrimp farms, as the limiting factor for farm-scale usage in the aquaculture sector is the lack of cost-effective and simple dsRNA synthesis and administration procedures. Despite these limitations, different RNAi-based approaches have been successfully tested at the laboratory level, with a particular focus on shrimp. The use of RNAi technology is particularly attractive for the shrimp industry because crustaceans do not have an adaptive immune system, making traditional vaccination methods unfeasible. This review summarizes recent studies and the state-of-the-art on the mechanism of action, design, use, and administration methods of dsRNA, as applied to shrimp. In addition, potential constraints that may hinder the deployment of RNAi-based methods in the crustacean aquaculture sector are considered.

Transcriptome Profile and Gene Expression During Different Ovarian Maturation Stages of Macrobrachium rosenbergii (De Man, 1879).

Macrobrachium rosenbergii, or giant river prawn, is the most economically crucial cultured freshwater crustacean. A predominant challenge in developing crustacean aquaculture is reproduction management, particularly ovary maturation, where identifying regulative mechanisms at the molecular level is critical. Ovary is the primary tissue for studying gene and protein expressions involved in crustacean growth and reproduction. Despite significant interest in M. rosenbergii, its gene discovery has been at a relatively small scale compared to other genera. In this study, comprehensive transcriptomic sequencing data for different maturation stages of the ovary of M. rosenbergii were observed. The 20 female M. rosenbergii samples evaluated were categorised into four maturation stages, 1 to 4. A total of 817,793,14, 841,670,70, 914,248,78 and 878,085,88 raw reads were obtained from stages 1, 2, 3 and 4, respectively. The assembled unique sequences (unigenes) post-clustering (n = 98013) was 131,093,546 bp with an average size of 1,338 bp. The BLASTX unigene search against National Centre for Biotechnology Information (NCBI), non-redundant (NR), nucleotide sequence (NT), Kyoto Encyclopaedia of Genes and Genomes Orthology (KO), Swiss-Prot, Protein Family (PFAM), Gene Ontology (GO), and euKaryotic Orthologous Groups (KOG) databases yielded 27,680 (28.24%), 7,449 (7.59%), 13,026 (13.29%), 22,606 (23.06%), 29,907 (30.51%), 30,025 (30.63%) and 14,368 (14.65%) significant matches, respectively, totalling to 37,338 annotated unigenes (38.09%). The differentially expressed genes (DEG) analysis conducted in this study led to identifying cyclin B, insulin receptor (IR), oestrogen sulfotransferase (ESULT) and vitellogenin (Vg), which are critical in ovarian maturation. Nevertheless, some M. rosenbergii ovarian maturation-related genes, such as small ubiquitin-like modifier (SUMO)-activating enzyme subunit 1, E3 ubiquitin-protein ligase RNF25, and neuroparsin, were first identified in this study. The data obtained in the present study could considerably contribute to understanding the gene expression and genome structure in M. rosenbergii ovaries throughout its developmental stage.

A coumarin derivative C7 exhibits antiviral activity against WSSV by reducing phosphatidylcholine content in shrimp

The white spot syndrome virus (WSSV) causes white spot disease (WSD), a severe condition in crustacean aquaculture, leading to significant economic losses. Our previous study demonstrated that C7 is an effective therapeutic agent against WSSV infection in aquaculture. It specifically blocked viral horizontal transmission and reduced shrimp mortality in a dose- and time-dependent manner. Here, we report the potential antiviral mechanism of C7 in shrimp. C7 regulated abnormal glycerophospholipid metabolism caused by WSSV and inhibited phosphatidylcholine (PC) synthesis by more than twofold, potentially enhancing shrimp resistance to viral infection. As the primary phospholipid in the cell membrane, PC is one of the main reactants in lipid peroxidation. Our results indicated that C7 significantly reduced the levels of lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) induced by WSSV, whereas PC had the opposite effect. Accumulation of lipid peroxidation products inhibits stimulator of interferon genes (STING) signaling. Further evidence showed that C7 promoted STING transport from the endoplasmic reticulum to the Golgi apparatus, significantly activating the expression of the shrimp interferon analogue Vago4 gene. In contrast, PC suppressed Vago4 expression. Our results demonstrated that C7 inhibited PC synthesis, reduced the degree of lipid peroxidation, promoted STING translocation, activated Vago4 expression, and ultimately exerted antiviral effects. Therefore, C7 exhibits immunoregulatory activity as a preventative agent for enhancing the innate immunity of shrimp, making it potentially useful for future immunomodulatory approaches.

Effects of dietary hot water extracts of Chlorella vulgaris on muscle component, non-specific immunity, antioxidation, and resistance to non-ionic ammonia stress in Pacific white shrimp Litopenaeus vannamei

Hot water extract of Chlorella vulgaris (CVE) is a biologically substance that enhances organism’s immune function and antioxidative capacity. This study evaluated the effect of supplementation with various concentrations of CVE on muscle nutritional components, non-specific immunity, antioxidation, and resistance to non-ionic ammonia (NH3-N) stress in Litopenaeus vannamei over 45 days using diets supplemented with CVE at five different concentrations (0%, 0.5%, 1%, 5%, 10%, and 15%). Specifically, fresh and sweet amino acids (Asp, Glu) significantly increased (P < 0.05) in shrimp fed the 1% CVE diet, reaching 18.12 g/kg and 33.08 g/kg, respectively. Bitter amino acids (Leu) and Hypoxanthine (Hx) significantly decreased (P < 0.05) in shrimp fed the 1% CVE diet, at 10.56 g/kg and 10.56 ug/g. CVE supplementary enhanced the activities of acid phosphatase, alkaline phosphatase, and nitric oxide synthase while decreasing malondialdehyde levels. Shrimp fed with a 1% CVE diet exhibited significantly higher enzyme activity than the control group under NH3-N conditions (P < 0.05). Overall, this study demonstrated that 1% CVE as a feed additive significantly improved the muscle mass, boosted immunity and reduced the stress response to NH3-N in L. vannamei. This research provides a valuable reference for the application of CVE as a feed additive in crustacean aquaculture.

Cardiovascular physiology of decapod crustaceans: from scientific inquiry to practical applications.

Until recently, the decapod crustacean heart was regarded as a simple, single ventricle, contraction of which forces haemolymph out into seven arteries. Differential tissue perfusion is achieved by contraction and relaxation of valves at the base of each artery. In this Review, we discuss recent work that has shown that the heart is bifurcated by muscular sheets that may effectively divide the single ventricle into 'chambers'. Preliminary research shows that these chambers may contract differentially; whether this enables selective tissue perfusion remains to be seen. Crustaceans are unusual in that they can stop their heart for extended periods. These periods of cardiac arrest can become remarkably rhythmic, accounting for a significant portion of the cardiac repertoire. As we discuss in this Review, in crustaceans, changes in heart rate have been used extensively as a measurement of stress and metabolism. We suggest that the periods of cardiac pausing should also be quantified in this context. In the past three decades, an exponential increase in crustacean aquaculture has occurred and heart rate (and changes thereof) is being used to understand the stress responses of farmed crustaceans, as well as providing an indicator of disease progression. Furthermore, as summarized in this Review, heart rate is now being used as an effective indicator of humane methods to anaesthetize, stun or euthanize crustaceans destined for the table or for use in scientific research. We believe that incorporation of new biomedical technology and new animal welfare policies will guide future research directions in this field.

Study on the mechanism of miR-7562 regulating ATG5 and ATG12 genes in Penaeus monodon under Vibrio harveyi infection

MicroRNAs (miRNAs) play a fundamental role in the post-transcriptional regulation of genes and are pivotal in modulating immune responses in marine species, particularly during pathogen assaults. This study focused on the function of miR-7562 and its regulatory effects on autophagy against Vibrio harveyi infection in the black tiger shrimp (Penaeus monodon), an economically important aquatic species. We successfully cloned and characterized two essential autophagy-related genes (ATGs) from P. monodon, PmATG5 and PmATG12, and then identified the miRNAs potentially involved in co-regulating these genes, which were notably miR-7562, miR-8485, and miR-278. Subsequent bacterial challenge experiments and dual-luciferase reporter assays identified miR-7562 as the principal regulator of both genes, particularly by targeting the 3′UTR of each gene. By manipulating the in vivo levels of miR-7562 using mimics and antagomirs, we found significant differences in the expression of PmATG5 and PmATG12, which corresponded to alterations in autophagic activity. Notably, miR-7562 overexpression resulted in the downregulation of PmATG5 and PmATG12, leading to a subdued autophagic response. Conversely, miR-7562 knockdown elevated the expression levels of these genes, thereby enhancing autophagic activity. Our findings further revealed that during V. harveyi infection, miR-7562 continued to influence the autophagic pathway by specifically targeting the ATG5-ATG12 complex. This research not only sheds light on the miRNA-dependent mechanisms governing autophagic immunity in shrimp but also proposes miR-7562 as a promising target for therapeutic strategies intended to strengthen disease resistance within the crustacean aquaculture industry.

Molecular mechanisms of sex determination and differentiation in decapod crustaceans for potential aquaculture applications: An overview

AbstractCrustacean aquaculture, especially monosex culture, requires a comprehensive knowledge of the mechanisms regulating sex determination and differentiation. Decapod crustaceans display either female heterogamety (ZW/ZZ) or male heterogamety (XY/XX) as their mode of genotypic sex determination. However, some decapod species exhibit sexual plasticity via removing the androgenic gland, knockdown insulin receptor transcripts, administrating exogenous sex steroids, and altering environmental factors. Although the master sex‐determining gene in decapods remains elusive, various sexual genes, hormones, and neurotransmitter are emerging as important factors in sex differentiation. This review looks into recent advancements in studies related to the genetic sex determination system and factors that trigger sex reversal. Moreover, the elucidation of the role of genetic factors in sex differentiation in both females and males, as well as their underlying interrelated regulatory mechanisms, provides justification for three levels of application: the induction of monosex populations, detection of precocious sex and genotype, and prevention of uncontrolled reproduction. Finally, we proposed future directions of bioinformatics analysis and genome editing coupled with sex control to increase the yield for economic decapods. In conclusion, this review provides a foundation for future research on the reproductive biology and sexual regulation of decapods, with the aim of aiding practitioners in advancing the crustacean aquaculture industry.

Metagenomic dissection of the intestinal microbiome in the giant river prawn Macrobrachium rosenbergii infected with Decapod iridescent virus 1

Microbiome in the intestines of aquatic invertebrates plays pivotal roles in maintaining intestinal homeostasis, especially when the host is exposed to pathogen invasion. Decapod iridescent virus 1 (DIV1) is a devastating virus seriously affecting the productivity and success of crustacean aquaculture. In this study, a metagenomic analysis was conducted to investigate the genomic sequences, community structure and functional characteristics of the intestinal microbiome in the giant river prawn Macrobrachiumrosenbergii infected with DIV1. The results showed that DIV1 infection could significantly reduce the diversity and richness of intestinal microbiome. Proteobacteria represented the largest taxon at the phylum level, and at the species level, the abundance of Gonapodya prolifera and Solemya velum gill symbiont increased significantly following DIV1 infection. In the infected prawns, four metabolic pathways related to purine metabolism, pyrimidine metabolism, glycerophospholipid metabolism, and pentose phosphate pathway, and five pathways related to nucleotide excision repair, homologous recombination, mismatch repair, base excision repair, and DNA replication were significantly enriched. Moreover, several immune response related pathways, such as shigellosis, bacterial invasion of epithelial cells, Salmonella infection, and Vibrio cholerae infection were repressed, indicating that secondary infection in M. rosenbergii may be inhibited via the suppression of these immune related pathways. DIV1 infection led to the induction of microbial carbohydrate enzymes such as the glycoside hydrolases (GHs), and reduced the abundance and number of antibiotic-resistant ontologies (AROs). A variety of AROs were identified from the microbiota, and mdtF and lrfA appeared as the dominant genes in the detected AROs. In addition, antibiotic efflux, antibiotic inactivation, and antibiotic target alteration were the main antibiotic resistance mechanisms. Collectively, the data would enable a deeper understanding of the molecular response of intestinal microbiota to DIV1, and offer more insights into its roles in prawn resistance to DIVI infection.

Elimination of Decapod iridovirus 1 (DIV1) infection at high water temperature: a new environmental control strategy

AbstractDecapod iridovirus 1 (DIV1) poses a major challenge to sustainable shrimp farming and poses a serious hazard to aquaculture industry. This study investigated the complex interaction between DIV1 infection and water temperature, focusing on the effect of high temperature on DIV1 infection due to Penaeus monodon. Using models of latent and acute infection, the study revealed the response of P. monodon to DIV1 under different conditions. In the experimental set-up, the effect of high water temperature (34 ± 1 °C) compared with room temperature (26 ± 1 °C) was investigated. DIV1 replication was significantly inhibited in the high-temperature group (H), resulting in complete viral elimination within 15 days. DIV1 did not resurface even after return to room temperature (26 ± 1 °C), indicating sustained antiviral effects. Compared with the room temperature (26 ± 1 °C) group (N), the H group showed a 100% reduction in the incidence of latent and acute infection. Exposure to high water temperature directly impaired the viability of DIV1, enhancing the immune system of P. monodon, and expediting metabolic processes for efficient DIV1 clearance. The study highlights the significant inhibitory effects of high water temperature (34 ± 1 °C) on DIV1 infection in P. monodon, resulting in viral eradication. This discovery offers a potential strategy for mitigating DIV1 infections in shrimp aquaculture, prompting further investigation into underlying mechanisms. Optimising parameters and protocols for high-temperature treatment is crucial for viral control. Exploring the broader implications of the findings on other viral infections in crustacean aquaculture could provide valuable insights for comprehensive disease prevention and control.

A newly isolated strain of Bacillus subtilis W2Z exhibited probiotic effects on juvenile red claw crayfish, Cherax quadricarinatus

Aquaculture traditionally used antibiotics, leading to environmental contamination, food residue, and antibiotic-resistant superbugs. Probiotics offer a promising antibiotic alternative. In this study, a strain of Bacillus subtilis W2Z was isolated from aquaculture environments and can inhibit pathogens in aquaculture food production through chemical compounds. Eight-weeks C. quadricarinatus cultivation experiments revealed that B. subtilis W2Z, as a probiotic, contributes to increasing the yield of red claw crayfish. The supplementation of strain W2Z in the feed enhanced the crayfish's resistance to Aeromonas hydrophila by increasing immune-related enzyme activity and improving intestinal microbiota. B. subtilis W2Z has significant potential as a bioresource for exploring probiotics in crustacean aquaculture. Adding B. subtilis W2Z to C. quadricarinatus feed at a concentration of 1 × 108 to 1 × 109 colony forming unit (CFU)/g is recommended.

A protein chimera for dsRNA binding and delivery into oocytes for large-scale gene silencing in crustacean aquaculture

RNA interference (RNAi) is one of the most intensively used gene silencing techniques for gene function studies and biotechnology applications. However, the gonads, specifically the ovary, of arthropods appear to be intractable to efficient gene silencing. In this article, we report the development of a tool for specific binding and delivery of double-stranded (ds) RNA to the crustacean oocyte for gene silencing. The tool, an oocyte-specific silencing protein chimera, which we designated OSSCot, is composed of a dsRNA-binding domain retrieved from a specific Macrobrachium rosenbergii dsRNA binding protein and 24-amino acid peptide derived from the yolk protein, vitellogenin. The results of the in vitro and in vivo examinations carried out in this study indicate that an OSSCot-piggybacked dsRNA specifically enters the oocytes. The results also indicate that the OSSCot protein protects the dsRNA from degradation by endonucleases in the ovary. When OSSCot was linked to the specific PAX6-like dsRNA of M. rosenbergii (where PAX6 is the master gene for eye development) and injected into the hemolymph of vitellogenic females, the expression of the RNAi enzyme machinery was enhanced, and eye development was impaired in the embryos of the treated females. These findings validate the concept of exploiting OSSCot to piggyback a functional dsRNA into crustacean oocytes for gene silencing. The newly developed tool will find application for oocyte-specific gene silencing in other crustacean species and for research and biotechnology applications.

Interspecific hybridization of decapod crustacean species with commercial interest—A review

AbstractInterspecies hybridization has been widely used in the development of aquaculture in fish species but there is not much impact in crustacean species. Several species of crustaceans that have been successfully crossed still have low hatching rates and the hybrids obtained have not met expectations. This review tries to summarize and analyse interspecific hybridization of cultured crustacean species (shrimps, lobsters, crayfish and crabs) and its potential for development of crustacean aquaculture production. The success of cross‐breeding on several species of shrimp and lobster has been supported by artificial insemination technology. In crabs, artificial insemination cannot be applied and cross‐breeding still relies on natural mating with an unstable success rate. In addition to cross‐breeding in captivity, cross‐breeding of crustacean species (shrimp, lobster, crayfish and crab) in nature has also been found. Interbreeding of crustacean species in nature cannot always be distinguished morphologically, hybrids between them can be known with certainty after carrying out molecular analysis. Even though the level of reproductive performance in cross‐breeding of shrimp, lobster, crayfish and crab species is still low, cross‐breeding efforts must be continued to obtain more information so that it can later be mapped which species have the potential to develop hybrids for cultivation. Furthermore, growth, reproductive performance, monosex hybridity and disease resistance are all hybrid parameters that must be evaluated.

Evaluation of the antiviral activity of chlorogenic acid against white spot syndrome virus

The rapid development of crustacean aquaculture is threatened by white spot syndrome virus (WSSV), which is one of the deadliest pathogens affecting various crustacean species. Based on antiviral plant screening, we found that chlorogenic acid (CGA), a polyphenolic component of Arctium lappa Linn., possessed strong activity against WSSV infection in a red swamp crayfish (Procambarus clarkii) model. The results revealed that CGA dose-dependently inhibited WSSV proliferation in the gill and haemocytes with the inhibition rates (50 mg/kg) were 94.7% and 95.03%, respectively. Through a serial of mechanical experiments, we found that CGA arrested WSSV replication by regulating innate immune defense to reduce viral gene transcription, inducing apoptosis to restrict viral spread, and enhancing anti-inflammatory and antioxidant activities to alleviate oxidative and inflammatory damage caused by WSSV. Significant improvements in survival of WSSV infected crayfish have been made through injection and dietary administration of CGA compared to control group. In a static cohabitation model, pre-incubation of CGA suppressed the horizontal transmission of WSSV between infected donor crayfish and health crayfish. Thus, we have identified CGA as a potent WSSV inhibitor with high potential for further development as prophylaxis and therapy agents against WSSV outbreak in crustacean aquaculture.

Antiviral, antioxidant, and anti-inflammatory activities of rhein against white spot syndrome virus infection in red swamp crayfish (Procambarus clarkii).

Infection caused by white spot syndrome virus (WSSV) leads to massive mortality and huge economic losses in the crustacean aquaculture industry; however, to date, no effective control means are available to control the disease. Thus, to better understand the pathological aspects of WSSV and to find effective compounds that have the potential to be developed as anti-WSSV agents are imperative. Herein, based on screening of plants with antiviral activity, we found that rhein, an anthraquinone compound in Rheum palmatum L., exhibited strong activity against WSSV infection in a red swamp crayfish (Procambarus clarkii) model. The results showed that rhein could dose dependently inhibit transcription of WSSV ie1, DNApol, and Vp28 genes, reduce viral load in vivo, and significantly improve the survival rate of WSSV-infected crayfish. Through investigation of the mechanisms of action, we found that rhein did not affect the infectivity of viral particles in vitro as well as the entry and release process in vivo. Alternatively, rhein could activate JAK/STAT and NF-κB pathways, significantly decrease the expression of crustin-1 and BAF but increase the expression of C-type lectin and proPO to arrest WSSV replication. Moreover, rhein also attenuated WSSV-induced oxidative and inflammatory stresses by regulating the expression of antioxidant and anti-inflammatory-related genes and enhanced innate immunity by reducing total protein levels while increasing the phosphatase activity. Taken together, our study shows rhein as a potent WSSV inhibitor, which provides great potential for the development of prophylaxis and therapy agents for controlling WSSV infection in crustacean aquaculture.IMPORTANCEAquaculture is essential for ensuring global food security by providing a significant source of animal protein. However, the spread of the white spot syndrome virus (WSSV) has resulted in considerable economic losses in crustacean industries. In this study, we evaluated the antiviral activity of rhein, the primary bioactive component of Rheum palmatum L., against WSSV infection, and many pathological aspects of WSSV were also described for the first time. Our mechanistic studies indicated that rhein effectively arrested the replication of WSSV in crayfish by modulating innate immunity to inhibit viral gene transcription. Furthermore, we observed that rhein attenuated WSSV-induced oxidative and inflammatory stresses by regulating the expression of antioxidant and anti-inflammatory-related genes while enhancing innate immunity by reducing total protein levels and increasing phosphatase activity. Our findings suggest that rhein holds great promise as a potent antiviral agent for the prevention and treatment of WSSV in aquaculture.

Effect of feeding astaxanthin with different E/Z‐isomer ratios on astaxanthin accumulation in Pacific white shrimp Litopenaeus vannamei

AbstractAstaxanthin is traditionally utilized in crustacean aquaculture to enhance the characteristic pink‐red color of these species. It has been reported that the bioavailability and body accumulation efficiency of astaxanthin differ based on its E/Z‐isomer species, and these trends vary widely among animal species. This study investigated the influences of feeding astaxanthin having different E/Z‐isomer proportions on astaxanthin accumulation in Pacific white shrimp Litopenaeus vannamei. Z‐Isomer‐rich astaxanthin was prepared from the synthetic (all‐E)‐astaxanthin standard by thermal treatment, and the prepared all‐E‐isomer‐rich astaxanthin pellets (E‐AST‐P; total Z‐isomer proportion = 4.7%) and Z‐isomer‐rich astaxanthin pellets (Z‐AST‐P; total Z‐isomer proportion of astaxanthin = 81.8%) were fed to L. vannamei for 8 weeks. The feeding of Z‐AST‐P significantly improved the body astaxanthin concentration compared to those fed the control pellets without adding astaxanthin isomers and E‐AST‐P; the astaxanthin concentrations after the 8‐week treatments of the control pellets, E‐AST‐P, and Z‐AST‐P were 7.3 ± 0.5, 6.2 ± 1.8, and 12.3 ± 1.3 µg g–1, respectively. This result suggested that astaxanthin Z‐isomers have higher bioavailability and/or body accumulation efficiency than (all‐E)‐astaxanthin in L. vannamei.Practical Applications: The bioavailability of astaxanthin is generally very low, which is a critical issue for its use in crustacean aquaculture. In this study, we found that feeding a pellet rich in astaxanthin Z‐isomers showed a greater accumulation efficiency of astaxanthin in the whole body of L. vannamei than that fed a pellet rich in the all‐E‐isomer. Thus, the use of Z‐isomer‐rich astaxanthin feeds would contribute to improving the quality of crustacean and reducing the feed costs (i.e., reduction of the use amount of astaxanthin).

Correlation of Atmospheric Temperature with the Prevalence of White Spot Disease (WSD) and Acute Hepatopancreatic Necrosis Disease (AHPND) in the Philippines

The growing crustacean aquaculture industry in the Philippines is a significant contributor to the seafood production sector. However, several problems hinder the growth and overall market production, the main factor being diseases. Previous studies observed that temperature changes affect shrimp’s physiological and immunological responses to different pathogens and parasites; hence, this meta-analysis examined the correlation of white spot disease (WSD) and acute hepatopancreatic necrosis disease (AHPND) with atmospheric temperature changes. The analysis was conducted using the available data from the Quarterly Aquatic Animal Disease report of the Network of Aquaculture Centres in Asia Pacific, the Bureau of Fisheries and Aquatic Resources, and the Philippine Atmospheric, Geophysical, and Astronomical Services Administration. Results indicated that temperatures ranging from 27–28 °C generally cause higher disease occurrences in the Philippines, as it facilitates optimum conditions for the infectivity of the causative agents of both diseases. Statistical analysis also has shown a positive correlation of temperature to the disease occurrence, indicating that temperature monitoring is crucial, especially in the culture of shrimps in a controlled environment. This information will serve as a guide to the fish health and aquaculture industry in mitigating possible disease outbreaks.

The role of pattern recognition receptors in crustacean innate immunity

AbstractCrustacean species are among the most valued seafood with socioeconomic and ecological implications in the global fisheries sector. Microbial infections to the farming stock due to continued expansion and aquaculture puts global crustacean production at risk. Understanding and reviewing the complex innate immune system of crustaceans would be pivotal in formulating informed decisions for the control and management of epidemic diseases, thus making the global crustacean aquaculture industry sustainable. Under the purview of innate immunity in crustaceans, pattern recognition receptors (PRRs) represent the sensors capable of discriminating non‐self pathogenic modules and invoking the activation of immune responses. The immunity to infections can be manifested at cellular (via phagocytosis, apoptosis, nodulation and encapsulation) and humoral (effected by prophenoloxidase cascade, lectins, antimicrobial proteins) levels leading to the elimination of pathogens or subversion of the pathogenic implications. In this review, we discuss the participatory role of 12 types of PRRs identified from crustacean species with an exhaustive focus on structural and functional specificities in binding to pathogens and possible execution of the immune activity. Moreover, we have elaborated on the PRRs‐associated or possible signalling cascades (Toll‐NFκB, IMD‐NFκB and JAK‐STAT) in crustacean species that could bridge our understanding of the resistance or susceptibility of the host to varied pathogenic infections thus providing clues to the regulatory mechanisms involved in pathogenesis of infectious diseases and mechanisms of immunity.

Insect feed in sustainable crustacean aquaculture

Aquaculture is a growing global food production sector that aims to meet the increasing demand for dietary protein. Crustaceans are an important and predominantly high-priced aquaculture segment that could support the transfer of sustainable new technologies to other sectors. Areas of interest include disease management and compound feeds, both of which have the potential to improve both the profitability and sustainability of aquaculture. Modern compound feeds are largely composed of fishmeal and/or terrestrial plant materials, the production of which is unsustainable, leading to the depletion of finite resources. Insects are a promising protein-rich alternative to fishmeal that can reduce the environmental footprint of aquafeeds and crustacean aquaculture. First research data have shown that insect meal has a favourable nutritional composition with positive health effects, and is environmentally sustainable with a strong economic potential, particularly supporting circular economy by valorising otherwise unused side-streams. In this article, we discuss the current state of crustacean aquaculture and highlight the benefits of insect meal compared to today’s compound aquafeeds in terms of health and growth-promoting properties as well as environmental benefits. We then consider the molecular mechanisms that confer immunity and disease resistance in crustacean aquaculture and show how insect feeds can support disease management and thus consumer health. Next, we assess the environmental sustainability of crustacean aquaculture and insect farming, the legal framework for insect-based feeds and consumer acceptance issues. Lastly, we identify research gaps, socioeconomic considerations and the potential of insect-based sustainable crustacean aquaculture for the global market.

CRISPR/Cas9 establishment-mediated targeted mutagenesis in Macrobrachium nipponense.

Introduction: CRISPR/Cas9 is a gene-editing technology which could specifically cleave dsDNA and induce target gene mutation. CRISPR/Cas9 has been widely used in gene functional studies in many fields, such as medicine, biology, and agriculture due to its simple design, low cost, and high efficiency. Although it has been well developed in model fish and freshwater fish for gene function analysis, it is still novel in the studies dealing with economic crustacean species. Methods: In this study, we established a CRISPR/Cas9 system based on microinjection for M. nipponense, an important economic crustacean aquaculture species. The vitellogenin (Vg) gene and the eyeless (Ey) gene were selected as the targeted genes for mutation. Two sgRNAs were designed for Mn-Vg and Mn-Ey gene editing, respectively. Results and Discussion: For sg-Vg-1, the gastrula survival ratio was 8.69%, and the final hatching ratio was 4.83%. The blastula mutant ratio was 10%, and the hatching individual mutant ratio was 30%. For sg-Vg-2, the gastrula survival ratio was 5.85%, and the final hatching ratio was 3.89%. The blastula mutant ratio was 16.67%, and no mutant sequences were detected in hatching individuals. For sg-Ey-1, the gastrula survival ratio was 6.25%, and the final hatching ratio was 2.34%. The blastula mutant ratio was 10.00%, and the hatching individual mutant ratio was 66.67%. For sg-Ey-2, the gastrula survival ratio was 6.00%, and the final hatching ratio was 2.67%. No mutant sequence was detected in both blastula stage and hatching individuals. There were no significant morphological changes observed in the Mn-Vg group. Two deformed types were detected in sg-Ey-1-injected embryos. An evident developmental delay of the compound eye was detected in Ey-sg1-H1 in the zoea stage. The compound eyes of the Ey-sg1-H2 embryo could not form well-defined spheres, and the whole compound eye appeared to diffuse at the end of the late zoea stage. The establishment of a gene-editing platform based on CRISPR/Cas9 will not only provide an efficient and convenient method for gene function analysis but also provide a powerful tool for molecular-assisted breeding of Macrobrachium nipponense.

Reduced protein intake in Procambarus clarkii fed diets containing graded levels of purslane: Effects on growth and quality

AbstractIn this study, 144 red swamp crayfish (Procambarus clarkii, initial mean weight 3.9 ± 0.3 g) juveniles were fed diets containing graded levels of purslane (Portulaca oleracea), with a decreasing percentage of proteins (from 17.3% to 1.0%) for 10 weeks. Six diets were administered: four fishmeal diets containing 5%, 15%, 25%, and 50% purslane, a diet with 100% purslane, and a commercial diet. Growth performance and the amounts of fatty acids and carotenoids were measured. Crayfish fed 25% and 50% purslane diets showed optimal contents in fatty acids (FAs) and astaxanthin, and their growth was comparable to that of the control group. Significant differences in FA content of the crayfish groups were observed for saturated FAs, monounsaturated FAs (MUFAs), and polyunsaturated FAs (PUFAs). The highest levels of n − 3 α‐linolenic acid (ALA) and n − 6 linoleic acid (LA) were found in the groups fed 25%, 50%, and 100% purslane diets. This study shows that the best purslane content to be included in the crayfish diet should range between 25% and 50%. Although the nutritional value of purslane is low, its inclusion at up to 50% in crayfish diets seems to improve the carotenoid content and FA quality, while reducing the daily intake of animal protein, without affecting growth performance, thus contributing to the goal of more sustainable crustacean aquaculture.