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This study was conducted to examine digestibility of insect meals for Pacific white shrimp (Litopenaeus vannamei) and their utilization as fish meal substitutes. The tested insect meals were mealworm, silkworm, black soldier fly, rice grasshopper, two-spotted cricket, dynastid beetle and white-spotted flower chafer. Apparent digestibility coefficients of the tested insect meals were 83-89% for protein, 91-98% for lipid, 84-90% for energy, 77-81% for dry matter, 28-36% for chitin, 76-96% for amino acids and 89-93% for fatty acids. The amino acid availability of insect meals was high in taurine (93-96%), arginine (91-95%) and lysine (90-95%). Availability of fatty acids were 89-93% for saturated fatty acids, 90-93% for monounsaturated fatty acids and 88-93% for polyunsaturated fatty acids. For a feeding trial, a control diet was formulated using 27% tuna byproduct meal as a fish meal source and seven other diets were prepared replacing 10% tuna byproduct meal in the control diet with each insect meal. Triplicate groups of shrimp (initial body weight: 0.17 g) were fed the diets for 65 days. The growth performance was significantly improved when the shrimp were fed black soldier fly or dynastid beetle included diet. Dietary supplementation of insect meals significantly improved non-specific immune responses and antioxidant enzyme activity in the shrimp. These results indicate that the tested insect meals have high potentials to be used as a protein source that could replace fish meal in diets for the shrimp., Competing Interests: The authors have declared that no competing interests exist.

To unveil the neuroendocrine-immune (NEI) mechanism of crustaceans under high ambient ammonia-N, crustacean hyperglycemic hormone (CHH) in L. vannamei was knocked down under 20 mg/L ammonia-N exposure. The results showed that the expression of CHH in the eyestalks decreased significantly when CHH was silenced. After CHH was knocked down, the levels of CHH, ACh, DA, NE, and 5-HT in the haemolymph decreased significantly. Correspondingly, the expressions of GC, ACh 7 R, DM1, DA 1 R, and 5-HT 7 R in haemocytes down-regulated significantly, while DA 4 R and α 2 AR up-regulated significantly. Besides, the expression of Toll3 reduced significantly. And significantly changes occurred in the levels of G protein effectors (AC and PLC), second messengers (cAMP, cGMP, CaM, and DAG), protein kinases (PKA, PKC and PKG), and nuclear transcription factors (CREB, Dorsal, Relish and NKRF). Furthermore, immune defense proteins (BGBP and PPO3, Crustin A, ALF, LYC, TNFα, and IL-16), phagocytosis-related proteins (Cubilin, Integrin, Peroxinectin, Mas-like protein, and Dynamin-1) and exocytosis-related proteins (SNAP-25, VAMP-2 and Syntaxin) changed significantly. Eventually, a significant decrease in the levels of THC, haemocytes phagocytosis rate, plasma PO, antibacterial and bacteriolytic activities was detected. Therefore, these results indicate that under ammonia-N stress, the combination of CHH and GC mainly affects exocytosis of shrimp through the cGMP-PKG-CREB pathway. Simultaneously, CHH stimulates the release of biogenic amines, and then activate G protein effectors after binding to their specific receptors, to regulate exocytosis mainly via the cAMP-PKA-CREB pathway and influence phagocytosis primarily by the cAMP-PKA-NF-κB pathway. CHH can enhance ACh, and then activate G protein effectors after binding to the receptors, and finally regulate exocytosis mainly through the cAMP-PKA-CREB pathway and regulate phagocytosis by the cAMP-PKA-NF-κB pathway. CHH can also promote Toll3-NF-κB pathway, thereby affecting the expressions of immune defense factors. This study contributes to a further understanding of the NEI mechanism of crustacean in response to environmental stress., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pathogen infections in shrimps trigger the release of reactive oxygen species (ROS) as a part of immune response. The excessive accumulation of ROS causes the production of oxidative stress, which leads to oxidative damage of the biomolecules in the host cells. The inclusion of dietary antioxidants is known to mitigate oxidative stress and stimulate immunity. Curcumin, a potential antioxidant was encapsulated in chitosan nanoparticles to surge its bioavailability and was administered orally to Vibrio harveyi challenged and non-challenged Litopenaeus vannamei. The non-challenged shrimps fed with curcumin-loaded chitosan nanoparticles (Cur-CSNPs) showed a significant increase (p ≤ 0.05) in the specific growth rate, daily growth coefficient and survival rate. A significant increase (p ≤ 0.05) in the phenoloxidase activity, total hemocyte count and superoxide dismutase activity was observed in both the challenged and non-challenged shrimps fed with Cur-CSNPs. Additionally, a significant increase (p ≤ 0.05) in the relative mRNA expression of lysozyme, cMnSOD and lectin was observed in the Cur-CSNPs fed shrimps. The findings of this research suggest that Cur-CSNPs reinforce the immune system of L. vannamei against V. harveyi infection. Moreover, the non-challenged shrimps showed improvement in the growth parameters in addition to immunostimulation. Thereby a routine inclusion of dietary Cur-CSNPs could mitigate the oxidative damage caused by the incidence of environmental or pathogen-mediated oxidative stress., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Invertebrates are considered completely dependent on their innate immunity to defend themselves against pathogens as they lack an adaptive immunity. However, a growing body of evidence has indicated a specific acquired immunity called 'immune priming' may exist. The Pacific white shrimp, Penaeus vannamei is one of the most economically important shrimp species in the world. In the previous research, we investigated the hepatopancreas immune response of shrimp immunized with trans -vp28 gene Synechocystis sp. PCC6803 at the protein level. In this study, on the basis of the previous research, the shrimp were then challenged with WSSV, and hepatopancreas analyzed using isobaric tags for relative and absolute quantification (i TRAQ) labeling. In total, 308 differentially expressed proteins (DEPs) were identified including 84 upregulated and 224 downregulated. Upregulated proteins such as calmodulin B and calreticulin, and downregulated proteins such as calnexin, and signaling pathways like Ras, mTOR were differentially expressed in both studies. Data from this study are more significant than previous work and indicate increased sensitivity to WSSV after immunization with trans-vp28 gene Synechocystis sp. PCC6803. In addition, selected DEPs (upregulated: A0A3R7QHH6 and downregulated: A0A3R7PEF6, A0A3R7MGX8, A0A423TPJ4, and A0A3R7QCC2) were randomly analyzed using parallel reaction monitoring (PRM). These data preliminarily confirm immune priming in P. vannamei, and show that the initial stimulation with trans -vp28 gene Synechocystis sp. PCC6803 regulate P. vannamei immune responses and they provide shrimp with enhanced immune protection against secondary stimulation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The cytosolic DNA-sensing immune response is essential for recognizing and establishing an effective host immune response to pathogens. However, the importance of the cytosolic signalling molecules responsible for facilitating an appropriate immune response following infection with a DNA virus in shrimps remains unknown. Here, we report the discovery of the Penaeus monodon stimulator of interferon gene (PmSTING) and interferon regulatory factor (PmIRF) genes and their important roles in the host defense against viral infection. High expression levels of PmSTING transcripts were detected in the midgut, hepatopancreas, and hindgut, with lower levels in foregut, while PmIRF was highly expressed in the hindgut, foregut, and hepatopancreas of P. monodon. The mRNA expression level of both PmSTING and PmIRF was up-regulated in the foregut in response to white spot syndrome virus (WSSV; dsDNA virus) infection. RNA-interference-mediated gene silencing of PmSTING and PmIRF rendered shrimps to be more susceptible to WSSV infection; suppression of PmIRF decreased the mRNA transcript level of PmSTING; and silencing of the cytosolic sensor PmDDX41 suppressed both PmSTING and PmIRF gene transcript levels. Thus, PmSTING and PmIRF are likely to be important for the antiviral innate response against the dsDNA WSSV pathogen and may mediate the antiviral immune defenses via PmDDX41/PmSTING/PmIRF signaling cascade in P. monodon., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 10 7 , 10 8 , 10 9 , and 10 10  cfu kg -1 , respectively. Shrimp fed with the diet containing probiotic at the doses of 10 9-10  cfu kg -1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 10 10  cfu kg -1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 10 7-8  cfu kg -1 . In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 10 9-10  cfu kg -1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 10 8-10  cfu kg -1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 10 10  cfu kg -1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and β-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 10 10  cfu kg -1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Disease epidemics in shrimp aquaculture increase apace with the development of aquaculture systems throughout the world. The disease caused by Vibrio spp. (vibriosis) is considered the most devastating, which has made it the most feared bacterial disease in the shrimp sector. In aquaculture, several strategies have already been applied to control Vibrio strains, including chemicals, probiotics, antibiotics, natural products from plants, including plant oils; hence, there has been considerable attention for using plants in shrimp aquaculture to provide sustainable, eco-friendly and safe compounds, such as alkaloids, saponins, terpenoids and flavonoids for replacing chemical compounds and antibiotics in current aquaculture. Medicinal plants may also have immunostimulating activity, increase growth and resistance in shrimps. The present paper aims to review the inhibition of Vibrio spp. in shrimp by medicinal plants, using both in vitro or/and in vivo techniques. Several medicinal plants appear capable of inhibiting growth of Vibrio pathogens outside living shrimp or in the body of shrimp, through enhancing growth and immune capacity when shrimps are fed or injected with them. In the current review Gracilaria spp. (Gracilariaceae family) and Sargassum spp. (family Sargassaceae) have been used most for in vitro and in vivo experiments. Among the terrestrial plants, Eucalyptus camaldulensis, Psidium guajava, Rhodomyrtus tomentosa, and Syzygium cumini (Myrtaceae family) had significant activity against Vibrio., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Many tripartite motif (TRIM) family proteins played an important role in regulating innate immune and autophagy pathway and were important for host defenses against viral pathogens. However, the role of TRIM proteins in autophagy and innate immunity during virus infection was seldom studied in crustaceans. In this study, a novel TRIM32 homolog was identified from Penaeus monodon (named PmTRIM32). PmTRIM32 was significantly upregulated by rapamycin stimulation and WSSV infection. RNA interference experiments showed that PmTRIM32 could restrict WSSV replication and lead P. monodon more resistance to WSSV challenge. Autophagy could be induced by WSSV or rapamycin challenge and has been proved to play a positive role in restricting WSSV replication in P. monodon. The autophagy activity induced by WSSV or rapamycin challenge could be obviously inhibited by silence of PmTRIM32 in P. monodon. Further studies revealed that PmTRIM32 positively regulated the expression of nuclear transcription factor (NF-κB) and it mediated antimicrobial peptides. Moreover, Pull-down and in vitro ubiquitination assay demonstrated that PmTRIM32 could interact with WSSV envelope protein and target it for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM32 restricted WSSV replication and was involved in positively regulating autophagy and NF-κB pathway during WSSV infection in P. monodon., (Copyright © 2021. Published by Elsevier Ltd.)

Prophenoloxidase (proPO)-activating system is a critical innate immune defense in invertebrates. However, the mechanisms involved in regulating the phenoloxidase (PO) activity in shrimp hemolymph remain ill-defined. Our previous studies showed that Penaeus vannamei hemocyanin (HMC) and α2-macroglobulin (α2M), two key regulators of proPO-activating system in plasma, might interact with each other, indicating that this interaction could be implicated in controlling PO activity. Herein, we further confirmed that HMC specifically bind to α2M using Pull down and Far-Western blot analyses. Further studies demonstrated that HMC could directly interact with the receptor binding domain of α2M. In addition, HMC and α2M followed similar expression pattern upon Vibrio parahaemolyticus infection, suggesting the interaction of HMC and α2M might have a role in immune response. Finally, we found that α2M, as a broad-spectrum proteinase inhibitor, suppressed the serum PO activity in vitro, while hemocyanin could partially restore this inhibitory effect. In sum, the present data indicate that HMC interacts with α2M and therefore modulates the PO activity. This finding contributes to better understanding of stable state maintenance of PO activity in shrimp., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The biogenic amines, tyramine and octopamine, in the octopaminergic synthesis pathway play critical roles in regulating physiological and immunological homeostasis in Litopenaeus vannamei. Tyrosine decarboxylase (TDC) is an enzyme catalyzing the first decarboxylation step in the biosynthesis of tyramine and octopamine. The full-length gene sequence of TDC cloned from the brain of L. vannamei (LvTDC) was predicted to encode a 779-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain in close phylogenetic relationship with arthropod TDCs. LvTDC gene expression was found to be abundant in nervous thoracic ganglia. RNA interference was used to assess the immune and physiological function of LvTDC. The LvTDC knockdown shrimp revealed significant decreases in the total haemocyte count, hyaline cells, antimicrobial peptides, respiratory bursts, gene expression, respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Furthermore, LvTDC knockdown was accompanied by decreases in octopamine deficiency. In the V. alginolyticus challenge test, the survival rate of LvTDC knockdown shrimp was lower than the shrimp injected with DEPC-water or GAPDH-dsRNA. In conclusion, the cloned LvTDC was responsible for octopaminergic synthesis, which then regulated physiological and immune responses in L. vannamei., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion., (© 2021. The Author(s).)

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp ( Penaeus monodon ) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp ( Penaeus vannamei ) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Taengchaiyaphum, Buathongkam, Sukthaworn, Wongkhaluang, Sritunyalucksana and Flegel.)

Crustacean allergy, especially to shrimp, is the most predominant cause of seafood allergy. However, due to the high flexibility of immunoglobulin E (IgE), its three-dimensional structure remains unsolved, and the molecular mechanism of shrimp allergen recognition is unknown. Here a chimeric IgE was built in silico, and its variable region in the light chain was replaced with sequences derived from shrimp tropomyosin (TM)-allergic patients. A variety of allergenic peptides from the Chinese shrimp TM were built, treated with heating, and subjected to IgE binding in silico. Amino acid analysis shows that the amino acid residue conservation in shrimp TM contributes to eliciting an IgE-mediated immune response. In the shrimp-allergic IgE, Glu98 in the light chain and other critical residues that recognize allergens from shrimp are implicated in the molecular basis of IgE-mediated shrimp allergy. Heat treatment could alter the conformations of TM allergenic peptides, impact their intramolecular hydrogen bonding, and subsequently decrease the binding between these peptides and IgE. We found Glu98 as the characteristic amino acid residue in the light chain of IgE to recognize general shrimp-allergic sequences, and heat-induced conformational change generally desensitizes shrimp allergens.

Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3-6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3-6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3-6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5-6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection., Competing Interests: APC provided support in the form of salary to JC. The specific roles of this author are articulated in the ‘author contributions’ section. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Cytokines are a class of immunoregulatory proteins that are secreted by cells. Although vertebrate cytokine, especially mammalian cytokine has been well studied for the past decades. Much less attention has been paid to invertebrate so that only some cytokines have been identified in invertebrates. We have chosen Peaneus vannamei as a model to explore novel invertebrate cytokines. To achieve this, we previously purified shrimp plasma low abundance proteins and identified more than 400 proteins with proteomics analyses. In this study, a cellular repressor of E1A-stimulated gene (CREG)-like protein, which is highly conserved from Drosophila melanogaster to Homo sapiens , was further characterized in shrimp plasma. We found that shrimp plasma CREG was a glycoprotein which was strongly induced in hemolymph at 8 h post-LPS injection. Further function experiment unveiled that recombinant shrimp CREG protein injection significantly increased phagocytic hemocyte and lysosome-high hemocyte proportion in hemolymph. After that, hemocytes from rEGFP- and rCREG-protein injected shrimps were subjected to transcriptome analyses, which revealed that shrimp CREG protein could comprehensively promote hemocyte maturation and activation. Taken together, our data clearly indicated that shrimp plasma CREG protein is a novel hemocyte activation factor, which is probably a conserved myeloid cell lineage activation factor from invertebrate to vertebrate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huang, Yang and Wang.)

Nuclear DNA-binding TCF proteins, which act as the main downstream effectors of Wnt signaling, are essential for the regulation of cell fate and innate immunity. However, their role during viral infection in shrimp remains unknown. Herein, we demonstrated that Litopenaeus vannamei TCF (LvTcf) acts independently of Lvβ-catenin to promote interferon-like protein LvVago1 production, thus mounting the response to WSSV infection. Further, we observed that WSV083, a WSSV serine/threonine protein kinase, bound to LvTcf and phosphorylated it. Phosphorylated LvTcf was then recognized and degraded via the ubiquitin-proteasome pathway. Moreover, mass spectrometry analyses indicated that the T39 and T104 residues of LvTcf were target sites phosphorylated by WSV083. Point mutation analyses suggested that additional sites of LvTcf may undergo phosphorylation via WSV083. Taken together, the current work provides valuable insights into host immunity and viral pathogenesis. LvTcf is not only a modulator of shrimp innate immunity but is also an important target for WSSV immune evasion. Thus, the current findings will help improve disease control in shrimps., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Ruan, Shi, Lin, Liu and Li.)

The cGAS-STING pathway plays essential roles in detecting cytosolic dsDNA and initiating antiviral and antibacterial responses in vertebrates. However, knowledge about its function in antiviral response of invertebrates is very limited. In the present study, a gene encoding a Mab21-containing protein, a cGAS homologue, was identified from a decapod crustacean Litopenaeus vannamei and designated as LvMab21cp. LvMab21cp was mainly distributed in intestine and hepatopancreas, showing similar expression profile with other genes in the cGAS-STING pathway, such as LvSTING and LvIRF. The expression levels of LvMab21cp, LvSTING and LvIRF were up-regulated in intestine and hepatopancreas of shrimp after white spot syndrome virus (WSSV) infection. Knockdown of LvMab21cp by dsRNA-mediated RNA interference could decrease the expression levels of its putative downstream genes, including LvSTING, LvIRF, LvVago4 and LvVago5, and enhance the in vivo propagation of WSSV in shrimp. Overexpression of LvMab21cp and LvSTING in HEK 293T cells activated the expression of mammalian IFNs upon simulation with interferon stimulatory DNA (ISD). These data suggest that LvMab21cp was a cGAS homologue, a member of the shrimp cGAS-STING pathway, and play an important role during WSSV infection. To our knowledge, this is the first report to show the role of the cGAS-STING pathway in the antiviral response of invertebrates, which will provide new insights into the innate immunity of invertebrates., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

With the rapid growth in the global demand, the shrimp industry needs integrated approaches for sustainable production. A high-quality shrimp larva is one of the crucial key requirements to maximize shrimp production. Survival and growth rates during larval development are often criteria to evaluate larval quality, however many aspects of gene regulation during shrimp larval development have not yet been identified. To further our understanding of biological processes in their early life, transcriptomic analysis of larval developmental stages (nauplius, zoea, mysis, and postlarva) were determined in the black tiger shrimp, Penaeus monodon using next-generation RNA sequencing. Gene clustering and gene enrichment analyses revealed that most of the transcripts were mainly related to metabolic processes, cell and growth development, and immune system. Interestingly, Spätzle and Toll receptors were found in nauplius stage, providing evidence that Toll pathway was a baseline immune system established in early larval stages. Genes encoding pathogen pattern-recognition proteins (LGBP, PL5-2 and c-type lectin), prophenoloxidase system (PPAE2, PPAF2 and serpin), antimicrobial peptides (crustin and antiviral protein), blood clotting system (hemolymph clottable protein) and heat shock protein (HSP70) were expressed as they developed further, suggesting that these immune defense mechanisms were established in later larval stages.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular and non-structural glycoprotein. In shrimp, a significant function of SPARC in WSSV infection remains unclear. In this study, the full-length cDNA sequence of a secreted protein acidic and rich in cysteine -like was cloned from shrimp Litopenaeus vannamei (named as LvSPARC-L). LvSPARC-L contained an open reading frame of 1002 bp, encoding 333 amino acids. Bioinformatics analysis showed that LvSPARC-L contained a SPARC Ca 2+ -binding region in the C-terminus, a Kazal-type serine protease inhibitor domain and a BUD22 domain. Tissue distribution assay indicated that LvSPARC-L generally expressed in all tissues selected with a higher expression in hemocyte, stomach and pleoplod. In hepatopancreas and intestine, the relative expression of LvSPARC-L was significantly up-regulated following the WSSV challenge. Besides, the relative expression of viral immediately early gene IE1 and a late gene VP28 was significantly increased in the LvSPARCL-silenced shrimp. Furthermore, the relative expression of LvP53 and LvCaspase3 was extremely decreased in the stomach of dsLvSPARC-L treated shrimp, while that of LvP38 was not affected significantly. All data together suggest that LvSPARC-L might play an antiviral role by regulating apoptosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Hippo signalling pathway plays a vital role in organ size control, cell proliferation, apoptosis, and immune regulation. In this study, a Hippo homologue with three isoforms (named MnHippo-a, MnHippo-b, and MnHippo-c) was isolated and characterized for the first time from the freshwater prawn Macrobrachium nipponense. The deduced amino acid sequences of MnHippo-a (698 aa), MnHippo-b (688 aa), and MnHippo-c (656 aa) were highly similar, and they all contained an N-terminal S&#95;TKc (serine/threonine protein kinase catalytic) domain and a C-terminal Mst1&#95;SARAH (Sav/Rassf/Hpo) domain. MnHippo-a and MnHippo-c were derived from alternative splicing. Phylogenetic analysis was performed, and the results revealed that MnHippo was a member of the clade containing STPK4 and Hippo of Penaeus vannamei. The expression distribution showed that MnHippo was constitutively expressed in various tissues of uninfected prawns and highly expressed in the hepatopancreas and intestine. In prawns challenged with Vibrio parahaemolyticus and Staphylococcus aureus, the expression of MnHippo in haemocytes was significantly upregulated. Furthermore, in MnHippo-knockdown prawns injected with V. parahaemolyticus or S. aureus, the transcription levels of five antimicrobial peptides were downregulated. MnHippo silencing weakened the clearance of V. parahaemolyticus and S. aureus in prawns. The survival rate of the MnHippo-dsRNA group was obviously decreased from 2 to 6 days post-injection with V. parahaemolyticus or S. aureus. Hence, MnHippo might be involved in the antibacterial immune defence of M. nipponense.

The shrimp aquaculture industry has experienced serious economic losses due to diseases caused by Vibrio species. The application of antibiotics to combat diseases has led to environmental hazards, antibiotic-resistance in pathogens and accumulation of antibiotics in tissues. This study explores the use of probiotics as an alternative to antibiotics. A probiotic consortium SFSK4 (comprising salt pan bacteria Bacillus licheniformis TSK71, Bacillus amyloliquefaciens SK27, Bacillus subtilis SK07, Pseudomonas sp. ABSK55) was used as a water additive during shrimp culture. It significantly increased shrimp (Litopenaeus vannamei) immunity i.e. total hemocyte count, phagocytosis, total plasma protein, respiratory burst and bactericidal activity as compared to the control. It also stimulated the phenoloxidase activity by two-fold. Proteomic analysis revealed the differential expression of 50 immune proteins (39 up-regulated and 11 down-regulated) in SFSK4 treated shrimps. Four major immune modulation proteins viz. Caspase2, GTPase activating protein, Hemocyanin and Glucan pattern-recognition lipoprotein involved in cell mediated immune response were identified in SFSK4 treated shrimp hemolymph. SFSK4 decreased shrimp mortality by more than 50% against pathogens. Toxicology studies revealed that administration of the highest dose of probiotic (10 12  CFU/mL) showed no adverse effect on shrimp survival (LC 50 analysis) and neither exhibited cytotoxicity. Genotoxicity study confirmed that the probiotic did not cause DNA damage in shrimps. The findings suggest that the probiotic SFSK4 is an eco-friendly water additive to enhance shrimp immunity against diseases in aquaculture, which could help curtail environmental hazards as an effective alternative to antibiotics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As a downstream interactor of β-catenin, Pangolin which is the homologous protein of the T cell factor/lymphoid enhancer factor (TCF/LEF) in vertebrates is less understood in the research field of immunity. In this study, two isoforms of Litopenaeus vannamei Pangolin (LvPangolin1 and LvPangolin2) were identified. Phylogenetic tree analysis revealed that all of the Pangolin proteins from invertebrates were represented the same lineage. The mRNA expression profiles of the LvPangolin1 and LvPangolin2 genes differed across different tissues. The expression of LvPangolin1 and the amount of LvPangolin1and LvPangolin2 combined (LvPangolinComb) were significantly increased in the haemocyte, intestine and gill but reduced in the hepatopancreas after white spot syndrome virus (WSSV) challenge. The inhibition of LvPangolin1 but not LvPangolinComb significantly reduced the survival rates of L. vannamei after WSSV infection, while significantly higher WSSV viral loads in both LvPangolin1-inhibited and LvPangolinComb-inhibited L. vannamei were observed. Knockdown of LvPangolin by RNAi could distinctly decrease the expression of antimicrobial peptide (AMP) genes and their related transcription factors. All of these results indicate that LvPangolin plays a positive role in the response to WSSV infection and that this may be mediated through regulating the immune signalling pathways which control the expression of AMPs with antiviral abilities., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Shrimp progressively gets more attention among marine invertebrates from researchers all over the world due to it being a healthy food as well as having economic importance. There were a lot of attempts to develop a continuous cell line from shrimp but none successful. In this context a novel hybrid cell line named 'PmLyO-Sf9' could be developed by fusing shrimp lymphoid organ cells with Sf9 cells after to metabolic blocking of Sf9 cells using puromycin and actinomycin D and effecting the fusion by way of PEG application. The cells are maintained and multiplied in a mixture of SCCM and TNM-FH having osmolality 550 mOsm kg -1 and pH 6.8. Transmission electron microscopy of the hybrid cells revealed the presence of two nuclei during the initial stages and a single nucleus subsequently. The cell line is with shrimp and Sf9 genomic components and shrimp specific protein and is susceptible to WSSV. Shrimp elongation factor, Sf9 beta-actin, shrimp STAT and peroxinectin could be expresses through RT-PCR in the cell line. This is the first successful report of a hybrid cell line with shrimp genomic components and envisaged to be recognized a model system for multitudes of biomedical research in vitro. The cell line is in the National Cell Line Repository of ICAR - National Bureaue of Fish Genetic Resources, Lucknow, India., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this work, a simplified dynamic digestion system was developed, and used for comparing the digestibility and potential allergenicity of raw shrimp extracts (RSE) in static and dynamic digestion systems. Protein hydrolysis was analyzed by electrophoresis, and the potential allergenicity was reflected in IgG/IgE binding ability and activation of basophils. In comparison with static digestion, protein hydrolysis indicated different kinetic behaviors, especially tropomyosin (TM) showed better digestion stability during dynamic digestion. The potential allergenicity of RSE exhibited different changing trends with digestion in the two systems. However, the apparent molecular weight (Mw) of immune fragments (>11 kDa) showed good approximation, and the IgE-binding fragment near 70 kDa revealed outstanding digestion stability than primordial protein in both systems. In conclusion, the dynamic conditions had a significant impact on the assessment of the persistence and potential allergenicity of digestion-resistant allergens, while the apparent Mw of IgG/IgE binding hydrolysate was not affected., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Shrimp aquaculture is an essential economic venture globally, but the industry faces numerous challenges, especially pathogenic infections. As invertebrates, shrimp rely mainly on their innate immune system for protection. An increasing number of studies have shown that ubiquitination plays a vital role in the innate immune response to microbial pathogens. As an important form of posttranslational modification (PTM), both hosts and pathogens have exploited ubiquitination and the ubiquitin system as an immune response strategy to outwit the other. This short review brings together recent findings on ubiquitination and how this PTM plays a critical role in immune modulation in penaeid shrimps. Key findings inferred from other species would help guide further studies on ubiquitination as an immune response strategy in shrimp-pathogen interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Aweya, Yao, Zheng, Tran, Li and Zhang.)

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pir A and pir B in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei . This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mai, Caro, Cruz-Flores, White and Dhar.)

Isoquinoline alkaloids (IQs) from Macleaya cordata are promising natural products for enhancing the growth performance and overall health condition of farmed animals. The present study aimed to investigate the effects of two formulas of IQ extract, provided in either a powdered formula (IQ-E) or a water-soluble, granulated formula (IQ-WS) and containing the main active component sanguinarine at a concentration of 0.5% and 1%, respectively, on the growth, survival, immune response, and resistance to Vibrio parahaemolyticus infection of Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the postlarvae were divided into five groups (four replicates/group and 100 shrimp/tank) and fed four times/day for 30 days with a control feed, IQ-E at 200 or 300 mg/kg of feed, or IQ-WS at 100 or 150 mg/kg of feed. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups (four treatment groups as in Experiment 1 plus the positive and negative controls with four replicates/group and 30 shrimp/tank) and challenged with V. parahaemolyticus by immersion at a concentration of 103 colony-forming units (CFU)/mL and were fed with the same diets for another 14 days. The results revealed that all IQ-fed shrimp in Experiment 1 had significantly enhanced survival rates and immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) compared to the control group, even though the growth performances were similar across all groups. In Experiment 2, all IQ-fed groups showed better growth performance and survival rates compared to the positive control. Other than in the positive control group, no histopathological lesions in the hepatopancreas and the intestine were found. In summary, the current study demonstrated the benefits of using IQs from M. cordata as feed additives for improving the growth performance, survival rate, immune responses, and resistance to vibriosis of Pacific white shrimp., Competing Interests: Phytobiotics (Thailand) Co., Ltd. provided support in the form of salary to WK. The specific roles of this author are articulated in the ’author contributions’ section. Phytobiotics also provided material support in the form of the isoquinoline extracts for the study. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates the functions of interleukin-2 (IL-2) at the levels of transcription, splicing and translation, and plays other critical roles in the immune system. ILF2 is well-documented in vertebrates, while little is currently known in crustacean species such as the Pacific white shrimp (Litopenaeus vannamei). In the present study, five cDNA for spliced isoforms of Lv-ILF2 were identified, in which four of them are the full-length long isoforms (Lv-ILF2-L1, Lv-ILF2-L2, Lv-ILF2-L3 and Lv-ILF2-L4) and one of them is a truncated short isoform (Lv-ILF2-S). The whole sequence of ILF2 gene from L. vannamei was obtained, which is 11,680 bp in length with 9 exons separated by 8 introns. All five isoforms contain a domain associated with zinc fingers (DZF). Two alternative splicing types (alternative 5' splice site and alternative 3' splice site) were identified in the five isoforms. The Lv-ILF2 mRNA showed a broad distribution in all detected tissues, and the Lv-ILF2-L transcript levels were higher than those of Lv-ILF2-S in corresponding tissues. The mRNA levels of Lv-ILF2-S in the hepatopancreas, heart, muscle and stomach, but not in the eyestalk, were significantly increased after challenges with Vibrio harveyi or lipopolysaccharide (LPS), while no significant changes were observed for the transcript levels of Lv-ILF2-L in these tissues under the same immune stimulants. On the contrary, the transcript levels of neither Lv-ILF2-S nor Lv-ILF2-L were affected by challenges of polyinosinic: polycytidylic acid [Poly (I:C)]. In addition, after knockdown of the Lv-ILF2 mRNA level by siRNA, the mortality of shrimp and the hepatopancreatic bacterial numbers were significantly increased under V. harveyi challenge, indicating that Lv-ILF2 might participate in the immune defenses against V. harveyi invasion. Collectively, our study here supplied the first evidence for a novel splicing mechanism of ILF2 transcripts, and provided a functional link between the Lv-ILF2 isoforms and the capacity against pathogenic Vibrio in penaeid shrimp., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Heat-killed Vibrio alginolyticus (HVa), formalin-inactivated V. alginolyticus (FVa), heat-killed Vibrio harveyi (HVh), formalin-inactivated V. harveyi (FVh), live V. alginolyticus (LVa), and live V. harveyi (LVh) were used in this study. White shrimp Litopenaeus vannamei receiving two mixtures (HVa + FVa) or four mixtures (HVa + FVa + HVh + FVh) served as primary exposure, and shrimp receiving LVa or LVh afterward served as secondary exposure. Shrimp receiving marine saline and then receiving either LVa or LVh served as controls. Phagocytic activity and clearance efficiency were examined in shrimp that received two mixtures after 1-8 weeks and then received LVa. Both the phagocytic activity and clearance efficiency of shrimp receiving two mixtures were significantly higher than in control shrimp after 1-8 weeks. In another experiment, phagocytic activity and clearance efficiency were examined in shrimp that received four mixtures after 1-8 weeks and then received LVa and LVh, respectively. The phagocytic activity of shrimp receiving four mixtures was significantly higher than in control shrimp after 1-8 weeks post exposure to LVa and LVh. The clearance efficiency of shrimp receiving four mixtures was significantly higher than in control shrimp after 1-6 weeks post exposure to LVa, and 1-7 weeks post exposure to LVh. In the other experiment, the survival rate of shrimp that received four mixtures after five weeks were challenged with LVa at 6.4 × 10 7  colony-forming units (cfu) shrimp -1 and LVh at 4.4 × 10 6  cfu shrimp -1 . Shrimp that received marine saline for five weeks and then challenged with LVa and LVh at a same dose served as challenged controls. The survival rate of shrimp that received four mixtures was significantly higher (90%) than that of control shrimp (67%), and significantly higher (73%) than that of control shrimp (53%) after 3-7 days post challenge with LVa and LVh. It is concluded that the mixtures have feature of adjuvant and antigen, and shrimp receiving mixtures of heat-killed and formalin-inactivated V. alginolyticus and V. harveyi even after 5-8 weeks exhibit memory recall and show increased phagocytosis and resistance to Vibrio infections., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Penaeus vannamei is one of the most economically vital shrimp globally, but infectious diseases have hampered its proper production and supply. As antibiotics pose a huge threat to the environment and humankind, it is essential to seek an alternative strategy to overcome infection and ensure proper culture and production. The present study investigates the effect of an anti-infective biosurfactant derivative lipopeptide MSA31 produced by a marine bacterium on the growth performance, disease resistance, and the gut microbiome of P. vannamei when challenged with pathogenic Vibrio parahaemolyticus SF14. The shrimp were fed with a commercial and lipopeptide formulated diet for 60 days and the growth performance was analyzed. The lipopeptide fed shrimp group showed enhanced growth performance and specific growth rate with improved weight gain than the control group. The challenge experiment showed that the survival rate was significant in the lipopeptide fed group compared to the control group. The results revealed 100% mortality in the control group at the end of 12 h of challenge, while 50% of the lipopeptide diet-fed group survived 24 h, which indicates the enhanced disease resistance in shrimp fed with a lipopeptide diet. The test group also showed higher levels of digestive and immune enzymes, which suggests that the lipopeptide diet could positively modulate the digestive and immune activity of the shrimp. The gut microbiome profiling by Illumina high-throughput sequencing revealed that the most abundant genera in the lipopeptide diet-fed group were Adhaeribacter, Acidothermus, Brevibacillus, Candidatus, Mycobacterium, Rodopila, and Streptomyces, while opportunistic pathogens such as Streptococcus, Escherichia, Klebsiella, Neisseria, Rhizobium, and Salmonella were abundant in the control diet-fed shrimp. Also, lipopeptide diet-fed shrimp were found to have a high abundance of ammonia and nitrogen oxidizing bacteria, which are essential pollutant degraders. Therefore, the study reveals that the dietary supplementation of lipopeptide in shrimp aquaculture could positively modulate the gut microbiome and enhance the shrimp's overall health and immunity in an eco-friendly manner., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The present study aims to investigate the effects of dietary synbiotics supplementation methods on growth, feed utilization, hepatopancreas and intestinal histology, non-specific immunity and microbiota community of Pacific white shrimp (Litopenaeus vannamei). A control diet was designed to contain 18% fish meal (CON), and then 3 g kg -1 synbiotics (Bioture, consisting of Bacillus subtilis, Saccharomyces cerevisiae, β-glucan and mannan oligosaccharide, etc) was supplemented to the control diet with three methods, directly adding in diets for pelleting (DAP), spraying diets after pelleting at once (SDA), spraying diets before feeding every day (SDE). Shrimp with initial body weight of 1.5 ± 0.12 g were fed one of the four diets for 56 days. The results showed that dietary synbiotics significantly increased the weight gain (WG), apparent digestibility coefficient (ADC) of crude protein (CP) and dry matter (DM), hepatopancreatic protease activity and decreased feed conversion ratio (FCR) (P < 0.05). Among the three synbiotics-added diets, SDE group showed the best growth with significantly higher WG than DAP group (P < 0.05). Serum activities of total superoxide dismutase, catalase, acid phosphatase, lysozyme and alkaline phosphatase of synbiotics-added groups were significantly higher, and serum malondialdehyde level was significantly lower than those of the control (P < 0.05). The intestinal villus width and villus number were also increased by the supplementation of synbiotics. The cumulative mortality was reduced in the three synbiotics-added groups after challenging with Vibrio parahaemolyticus (P < 0.05), and SDE group showed a significantly lower mortality than the control and DAP groups (P < 0.05). In intestinal microbiota composition, the abundance of Lactococcus tended to increase and Vibro tended to decreased in SDA and SDE groups. In conclusion, dietary synbiotics improved the growth, feed utilization, intestine health and non-specific immunity of Pacific white shrimp, and spraying synbiotics on diet presented better performance than adding synbiotics in diet for pelleting., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In the present study, a hot water crude extract from Ulva intestinalis (Ui-HWCE) was used as a dietary supplement, and the effects on growth, immune responses, and resistance against white spot syndrome virus (WSSV) and yellowhead virus (YHV) infection in Pacific white shrimp (Litopenaeus vannamei) were investigated. Chemical analyses of Ui-HWCE revealed 13.75 ± 0.41% sulfate, 37.86 ± 5.96% uronic acid, and 46.63 ± 5.16% carbohydrate contents. The monosaccharide content of Ui-HWCE contained glucose (6.81 ± 0.94%), xylose (4.15 ± 0.11%), and rhamnose (25.84 ± 0.80%). Functional group analysis of Ui-HWCE by Fourier transform infrared (FTIR) spectroscopy revealed a typical infrared spectrum of ulvan similar to the infrared spectrum of commercially purified ulvan from Ulva armoricana (77.86 ± 2.19% similarity). Ui-HWCE was added to shrimp diets via top-dressing at 0, 1, 5, and 10 g/kg diet. After 28 days, Ui-HWCE supplementation at 5 g/kg diet efficiently improved shrimp growth performance, as indicated by weight gain, average daily growth, specific growth rates, and villus height determined by observing gut morphology. Additionally, Ui-HWCE feed supplementation at 5 g/kg diet significantly increased immune responses against a pathogenic bacterium (Vibrio parahaemolyticus AHPND stain), including phagocytic activity and clearance efficiency. Furthermore, Ui-HWCE feed supplementation upregulated the expression of several immune-related genes in the hemocytes and gills. Ui-HWCE supplementation at 1 and 5 g/kg resulted in effective anti-YHV but not anti-WSSV activity, which significantly decreased the mortality rate and YHV burden in surviving shrimp. It was concluded that Ui-HWCE supplied at 5 g/kg diet exhibits growth-promoting, immune-stimulatory, and antiviral activity that could protect L. vannamei against YHV infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp (Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly or indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates., Competing Interests: The authors have declared that no competing interests exist.

Peritrophic membrane (PM) refers to a vital physical barrier enabling shrimp to resist pathogen invasion. It primarily consists of chitin and proteins, mostly chitin-binding protein (CBP). CBPs have been identified from microorganisms to higher organisms. In the present study, a CBP, designated MjCBP, was reported from Marsupenaeus japonicus. The open reading frame of MjCBP was 1854 bp, encoding a protein with 618 amino acids (MH544098). To be specific, the theoretical pI and molecular mass of mature MjCBP reached 5.43 and 66064.00 Da, respectively. MjCBP consisted of seven type Ⅱ chitin-binding domains (ChtB D2), which was up-regulated after being challenged with Vibrio anguillarum and then agglutinating several bacteria. In addition, MjCBP and the first chitin-binding domain (CBD1) could bind to several Gram-positive and Gram-negative bacteria via the binding process to lipopolysaccharides and peptidoglycans, whereas CBD1 was not capable of agglutinating bacteria. Moreover, the anterior and posterior segments of CBD1 were synthesized in vitro, and the posterior segment could bind to lipopolysaccharides. However, both segments fail to agglutinate bacteria. Furthermore, MjCBP and CBD1 facilitated the clearance of V. anguillarum in vivo, and the silencing of MjCBP via RNA interference reduced the ability of bacterial clearance. As revealed from the mentioned results, MjCBP acts as an opsonin or pattern recognition receptor to achieve antibacterial immune response in shrimp., (Copyright © 2020. Published by Elsevier Ltd.)

Intestinal microbiota are closely related to host physiology. Over the long course of evolution and interaction, both commensal bacteria and their host have evolved multiple strategies to adapt to each other. However, in invertebrates, the regulatory mechanism of intestinal microbiota homeostasis is largely unknown. In the current study, a digestive tract-specific C-type lectin, designated as CTL33, was identified because of its abundance and response to bacteria in the intestine of kuruma shrimp ( Marsupenaeus japonicus ). Silencing of CTL33 expression led directly to intestinal dysbiosis, tissue damage, and shrimp death. CTL33 could facilitate biofilm formation by the intestinal bacteria. This function originated from its unique architecture, with a lectin domain responsible for bacteria recognition and a coiled coil region that mediated CTL33 dimerization and cross-linked the bacteria into a biofilm-like complex. By mediating the formation of a biofilm, CTL33 promoted the establishment of intestinal bacteria in intestine and maintained the homeostasis of the microbiota. Thus, to our knowledge, we demonstrated a new mechanism of C-type lectin-mediated biofilm formation by intestinal bacteria, providing new insights into intestinal homeostasis regulation in invertebrates., (Copyright © 2021 by The American Association of Immunologists, Inc.)

In this study, the effect of enzymatic cross-linking of shrimp tropomyosin (TM) with tyrosinase and caffeic acid (TM-Tyr/CA) on the allergic response were assessed using in vitro and in vivo models. The RBL-2H3 and KU812 cell lines were employed to evaluate the changes in the stimulation abilities of TM-Tyr/CA that showed significant inhibition of mediators and cytokines. The digestibility of cross-linked TM was improved and the recognitions of IgG/IgE were markedly reduced, as revealed by western blotting. TM-Tyr/CA decreased anaphylactic symptoms, and hindered the levels of IgG1, IgE, histamine, tryptase and mouse mast-cell protease-1 (mMCP-1) in mice sera. Cross-linked TM downregulated the production of interleukin (IL)-4, IL-5, and IL-13 by 51.36, 12.24 and 20.55%, respectively, whereas, IL-10 and IFN-γ were upregulated by 20.71 and 19.0%. TM-Tyr/CA showed reduced allergenicity and may have preventive effect in relieving TM induced allergic response via immunosuppression and positive modulation of T-helper (Th)1/Th2 immunobalance., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Shrimp is a causative food that elicits food-dependent exercise-induced anaphylaxis (FDEIA). In this study, we sought to identify IgE-binding allergens in patients with shrimp-FDEIA. Sera were obtained from eight patients with shrimp-FDEIA and two healthy control subjects. Proteins were extracted from four shrimp species by homogenization in Tris buffer. Immunoblot analysis revealed that IgE from patient sera bound strongly to a 70-kDa and a 43-kDa protein in a preparation of Tris-soluble extracts from Litopenaeus vannamei. Mass spectrometry identified the 70-kDa and 43-kDa proteins as a P75 homologue and fructose 1,6-bisphosphate aldolase (FBPA), respectively. To confirm that the putative shrimp allergens were specifically recognized by serum IgE from shrimp-FDEIA patients, the two proteins were purified by ammonium sulfate precipitation followed by reversed-phase HPLC and/or anion-exchange hydrophobic interaction chromatography and then subjected to immunoblot analysis. Purified P75 homologue and FBPA were positively bound by serum IgE from one and three, respectively, of the eight patients with shrimp-FDEIA, but not by sera from control subjects. Thus, P75 homologue and FBPA are identified as IgE-binding allergens for shrimp-FDEIA. These findings could be useful for the development of diagnostic tools and desensitization therapy for shrimp-FDEIA patients.

Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca 2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.

Allergic asthma remains an important worldwide health issue. Animal models are valuable for understanding the pathophysiological mechanisms of asthma and the development of effective therapeutics. This study aims to develop an alternative murine model induced by shrimp tropomyosin (ST) instead of ovalbumin (OVA). To investigate responses to short-term exposure to antigens, mice were sensitized with intraperitoneal injections of ST or ST plus aluminum adjuvant on days 0, 7, 14 followed by an intranasal challenge with ST for seven consecutive days. We reveal that sensitization with ST alone or ST plus aluminum induces significant levels of serum total IgE and ST-specific IgE in mice. Challenge results show that ST causes severe eosinophilic airway inflammation. Histology analysis of the lung tissues demonstrates airway inflammation and mucus hypersecretion within the bronchi in mice exposed to ST. Analysis of the cell composition in bronchoalveolar lavage fluid (BALF) shows a significant increase in eosinophil count in ST alone and ST plus aluminum groups. We also detect increased CD4 + T lymphocytes in lung tissues and production of helper T cell type 2-associated cytokines (IL-4 and IL-5) in BALF. In addition, airway hyperresponsiveness to methacholine in ST alone and ST plus aluminum groups is much higher than that in control groups. For the chronic model, mice were sensitized by ST or ST plus aluminum adjuvant for 3weeks and challenged with ST for 6weeks. We find severe structural changes in animals upon prolonged exposure to ST, including goblet cell hyperplasia, collagen deposition, and smooth muscle thickening. In conclusion, ST-induced asthma is a simple murine model for studying pathogenesis of asthma and evaluating new therapeutic drugs., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca 2+ ) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As a kind of important economic marine crustaceans in aquaculture, shrimp can be infected by more than 20 viruses. To fight against the virus invasion, shrimp have developed the innate immunity, including RNA interference (RNAi). RNAi, mediated by short interfering RNAs (siRNAs) or microRNAs (miRNAs), plays important roles in virus-host interactions. At present, RNAi is considered to be an efficient antiviral response of shrimp. The siRNA-based RNAi, first recognized as an antiviral response of animals to resist RNA viruses, has emerged in animals as an efficient antiviral strategy against the invasion of DNA viruses and RNA viruses. In shrimp, as well as in other animals, siRNA contains a seed region (2nd-7th nt) and a supplementary region (12th-17th nt). Based on the findings in shrimp and other animals, miRNAs are essential regulators of virus-host interactions, such as virus infection/latency, and host apoptosis, autophagy and phagocytosis. Except for the seed sequence (2nd-7th), the complementary bases (to the target mRNA sequence) of a miRNA 9th-18th non-seed sequence are essential for the miRNA targeting. So far, rapidly growing evidences have supported the existence of functional RNAi machinery in shrimp. In this review, we summarize the progress of RNAi in the antiviral immune response of shrimp. The potential applications of RNAi to control shrimp diseases were also summarized., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 10 6  cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Shrimp inhabiting coasts that are frequented by humans are exposed to various pollutants. Additionally, viral infections that cause serious damage to shrimp populations have been observed in these environments. The present study sought to evaluate the immunotoxic effects of phenanthrene (Phe), a pollutant detected in coastal environments, on kuruma shrimp (Penaeus japonicus). We further examined the survival of shrimp following combined exposure to Phe (30 or 300 μg/L) and white spot syndrome virus (WSSV). Results show that exposure to Phe for seven days decreased immune system-related parameters, including total hemocyte count and phenoloxidase activity in hemolymph (p < 0.05). However, these effects were not detected after three days of exposure. Moreover, a combined exposure assay revealed that shrimp mortality increased following exposure to 300 μg/L Phe and infection with WSSV. The number of WSSV gene copies was also observed to increase in these co-exposed shrimp. Taken together, these results indicate that long-term Phe exposure impairs the immune system of P. japonicus, resulting in fatal proliferation of WSSV. Hence, considering that combined exposure to Phe and WSSV leads to increased mortality of shrimp, it is imperative that the detrimental effects elicited by multiple stresses be considered, and controlled, in areas inhabited by kuruma shrimp., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The LARK proteins containing a C2HC-type zinc finger motif and two RNA recognition motifs are conserved across vertebrates and invertebrates. Previous studies have suggested that invertebrate LARKs and their mammalian counterparts, the RBM4 proteins, regulate gene expression by affecting RNA stability and post-transcriptional processing, participating in multiple life processes. In the current study, the LARK gene from Pacific white shrimp Litopenaeus vannamei was identified and functionally explored in the context of immunity. The LARK protein was mainly present in the nucleus of its expression vector-transfected S2 cells, and the LARK mRNA was detectable in all the tested shrimp tissues. Expression of LARK in gill was up-regulated by immune stimulation with various pathogens. In vivo experiments demonstrated that LARK played positive roles in both antiviral and antibacterial responses and silencing of LARK could make shrimp more susceptible to infection with Vibrio parahaemolyticus and white spot syndrome virus (WSSV). Although silencing of LARK did not affect the phagocytic activity of hemocytes, it regulated expression of many components of the NF-κB and JAK-STAT pathways and a series of immune function proteins. These suggested that LARK could be mainly involved in regulation of humoral immunity. The current study could help reveal the roles of LARK/RBM4 in immunity and further explore the regulatory mechanisms of shrimp immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

White spot syndrome virus (WSSV) is highly virulent toward shrimp, and F1 ATP synthase β subunit (ATPsyn-β) has been suggested to be involved in WSSV infection. Therefore, in this study, interactions between Penaeus monodon ATPsyn-β (PmATPsyn-β) and WSSV structural proteins were characterized. Based on the results of yeast two-hybrid, co-immunoprecipitation, and protein pull-down assays, WSSV VP51B and VP150 were identified as being able to interact with PmATPsyn-β. Membrane topology assay results indicated that VP51B and VP150 are envelope proteins with large portions exposed outside the WSSV virion. Cellular localization assay results demonstrated that VP51B and VP150 co-localize with PmATPsyn-β on the membranes of transfected cells. Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA results demonstrated that VP51B and VP150 bound to PmATPsyn-β in a dose-dependent manner, which could be competitively inhibited by the addition of WSSV virions. In vivo neutralization assay results further showed that both recombinant VP51B and VP150 could delay mortality in shrimp challenged with WSSV., (Copyright © 2020. Published by Elsevier Ltd.)

Outbreaks of infectious disease in shrimp pose a serious threat to shrimp agriculture worldwide. Shrimp lack adaptive immunity and depend only on innate immunity as a defense system against infectious disease. Toll-like receptors (TLR) are reported to play a critical role in the innate immune system. In this study, we identified a Toll-like receptor gene of a species of freshwater shrimp, Macrobrachium nipponense, designated MnToll, for the first time. The sequence of MnToll encoded 935 residues arranged as 10 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR CT) domain and a Toll/interleukin-1 receptor (TIR) domain and displayed 90% amino acid similarity to previously identified TLRs (Toll 1 and 2) of Macrobrachium rosenbergii. We additionally evaluated mRNA expression of MnToll in various tissues, including heart, gills, stomach, digestive gland, ventral nerve cord, antennal gland and muscle. Following infection with a viral pathogen, white spot syndrome virus (WSSV), MnToll expression was significantly upregulated between 12 and 72 h. Our data collectively suggest that the newly identified MnToll gene belongs to the TLR family in shrimp and is potentially involved in innate host defense, especially against WSSV., (Copyright © 2020 Elsevier Ltd. All rights reserved.)