Trypsin Activity Research Articles

Effects of Salinity on Growth, Digestive Enzyme Activity, and Antioxidant Capacity of Spotbanded Scat (Selenotoca multifasciata) Juveniles

As a euryhaline fish species that inhabits estuarine and coastal regions, the spotbanded scat (Selenotoca multifasciata) experiences growth influences during its larval stage due to variations in salinity. Here, we evaluated salinity required by early-stage spotbanded scat juveniles to achieve the highest growth performance, digestive enzyme activity, survival, and antioxidant capacity. We reared spotbanded scat juveniles (all 0.50 ± 0.05 g) in 0–35‰ salinity gradients for 50 days and recorded their survival rate every 10 days. After 50 experimental days, we measured morphological data, stomach and intestinal digestive enzyme activities, and liver antioxidant enzyme activities and malondialdehyde contents. In general, 5–15‰ salinity led to 100% survival. The 5‰ salinity group demonstrated the highest values for the following measures: final wet body weight; weight gain rate; specific growth rate; growth percentage; average daily gain; stomach amylase and lipase specific activities; and intestinal amylase, lipase, trypsin, and pepsin specific activities. However, stomach trypsin and pepsin activities did not demonstrate significant between-group differences (all p > 0.05). The 25‰ salinity group demonstrated the highest liver superoxide dismutase and glutathione peroxidase activities and malondialdehyde content. Finally, the 0‰ salinity group demonstrated the highest liver catalase activity. Thus, spotbanded scat juveniles demonstrate the highest survival rates, growth performance, and digestive enzyme activity at 5‰ salinity and the strongest oxidative stress responses at 25‰ salinity.

Structural, Biochemical Characterization and Molecular Mechanism of Cerastokunin: A New Kunitz-Type Peptide with Potential Inhibition of Thrombin, Factor Xa and Platelets.

The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% β-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration.

Effects of cadmium on the growth, muscle composition, digestion, gene expression of antioxidant and lipid metabolism in juvenile tilapia (Oreochromis niloticus)

Cadmium could induce various degrees of harm to aquatic organisms. A 30-day feeding trial was conducted to investigate the effects of cadmium on growth, muscle composition, digestive enzyme activity, gene expression of antioxidants and lipid metabolism in juvenile genetic improvement of farmed tilapia (GIFT, Oreochromis niloticus, Initial weight: 21.36 ± 0.24 g). Four cadmium concentrations of aquaculture water were designed: 0, 0.2, 0.4, and 0.6 mg/L Cd2+. The main results are as follows: Compared with the control group (0 mg/L Cd2+), the weight gain rate (WGR), specific growth rate (SGR), daily growth index (DGI), and spleen index (SI) of juvenile GIFT under cadmium stress were significantly decreased (p< 0.05). The contents of crude protein and crude lipid in muscle were significantly decreased (p< 0.05), and the ash was significantly increased (p< 0.05). The activities of trypsin, lipase, and α-amylase in the intestinal were significantly decreased (p< 0.05). The relative expression levels of carnitine palmityl transferase 1 (cpt-1), peroxisome proliferator-activated receptor α (pparα), pparγ, hormone-sensitive lipase (hsl), lipoprotein lipase (lpl), malate dehydrogenase (mdh), leptin (lep), fatty acid synthetase (fas), cholesterol response element binding protein 1 (srebp1), squalene cyclooxygenase (sqle), and stearoyl-CoA desaturase (scd) genes in liver were significantly decreased (p< 0.05). The relative expression levels of catalase (cat), superoxide dismutase (sod), glutathione S-transferase (gst), and glutathione peroxidase (gsh-px) genes in the liver were significantly decreased (p< 0.05). In conclusion, exposure to cadmium stress could impact growth, muscle composition, digestive enzyme activity, gene expression of antioxidant and lipid metabolism in juvenile GIFT.

Combination of herbal extracts regulates growth performance, liver and intestinal morphology, antioxidant capacity, and intestinal microbiota in Acrossocheilus fasciatus

In the present study, the herbal extracts (RPTS) composed of Rheum palmatum: Schisandra sphenanthera: Terminalia chebula: Punica granatum with a weight ratio of 1.07: 3.00: 1.18: 1.38, was evaluated through a bacteriostatic assay in vitro. Afterwards, a 56-day feeding trial was operated to assess the impact of dietary RPTS at the dose of 0, 1.0, 2.5, 5.0, and 10 g·Kg−1 feed (entitled as Con, RPTS010, RPTS025, RPTS050, RPTS100 group, respectively) on the performance of Acrossocheilus fasciatus fingerlings. The results revealed that dietary RPTS at the dose of 2.5 g·Kg−1 feed remarkably improved the specific growth rate (SGR), and the gastrointestinal trypsin activity, as well as the catalase (CAT) activities, while slightly increased the alanine aminotransferase (ALT) activity and aspartate aminotransferase (AST) activity in the fish liver. Histological examination indicated that dietary high dose of RPTS (5.0 and 10.0 g·Kg−1 feed) could induce hepatic cell vacuolization and reduce the intestinal villi density of fish. Furthermore, the analysis of intestinal microbiota showed that Bacteroidota, Firmicutes and Proteobacteria were the three dominant phylum in all experimental groups, while the Firmicutes/Bacteroidota ratio exhibited a trend of initially decreasing followed by rising, with the supplementation level of RPTS increase. Additionally, the LEfSe analysis revealed that Cetobacterium was the most impacted species, which notably increased in RPTS025 group. Collectively, this study unveiled that the impact of dietary RPTS on fish performance was in a dose-dependent manner, and dietary RPTS at the dose of 2.5 g·Kg−1 feed could potentially enhance growth performance, digestive and antioxidant capacity, and positively modulate the intestinal microflora, without significantly impact liver health of A. fasciatus.

Amylase and Trypsin Activity in an Economically Important Fish LabeoCalbasu of Tripura, India

Amylase and Trypsin Activity in an Economically Important Fish LabeoCalbasu of Tripura, India

Enzymatic response of heparin-protamine complex: Spectroscopic investigation and application for lung adenocarcinoma cells detection

Though the heparin-protamine complex (HP complex) is a crucial system utilized in clinical settings, the metabolic pathways of this complex remain inadequately understood. Herein, the enzymatic degradation of the heparin-protamine complex by trypsin and its broader implications were investigated. By utilizing fluorescent gold nanoclusters liganded with the HP complex (AuNCs-HP complex), we observed significant morphological and spectral changes during enzymatic degradation. Experiments showed that AuNCs-HP complex could be degraded and cleaved into small fragments by trypsin. Moreover, the AuNCs-HP complex demonstrated its potential as a highly sensitive spectral sensing platform, enabling precise measurement of trypsin activity with an outstanding detection limit (0.34 ng mL−1). Additionally, we explored its utility for specific tumor cell detection, focusing on lung adenocarcinoma cells, and successfully identified their presence through distinctive fluorescence changes. These remarkable findings not only contribute valuable insights into targeted degradation systems but also offer promising opportunities for cancer biomarker detection. The AuNCs-HP complex serves as an innovative tool for real-time trypsin activity monitoring, paving the way for advanced biomedical applications.

Efficiently Substituting Dietary Fish Meal with Terrestrial Compound Protein Enhances Growth, Health, and Protein Synthesis in Largemouth Bass.

Inappropriate substitution of dietary fishmeal (FM) can adversely affect the growth, health, and metabolism of carnivorous fish species. To effectively reduce the amount of dietary FM in carnivorous largemouth bass (Micropterus salmoides), a terrestrial compound protein (Cpro) with chicken meal, bone meal, and black soldier fly protein was used to formulate four isoproteic (52%) and isolipidic (12%) diets, namely T1 (36% FM), T2 (30% FM), T3 (24% FM), and T4 (18% FM), for feeding juveniles (initial weight: ~12 g) for 81 days. Results indicated that the growth performance, feed efficiency, and morphological indicators, as well as muscle texture and edible quality of fish, did not differ significantly among the four groups. However, the muscle protein contents and ATP/AMP ratio of fish in the T4 group were significantly increased in comparison with those of fish in the T1 group, while the opposite was true for muscle glycogen. Compared with the T1 group, high serum total amino acid and MDA contents, as well as low AST activities, were observed in the T3 and T4 groups, and relatively high intestinal trypsin and lipase activities were found in the T2-T4 groups. The transcripts of intestinal proinflammatory cytokines (il-1β, il-6, and tnf-α) were downregulated in the T2-T4 groups compared with T1 group, while the expression of anti-inflammatory cytokines (il-10) and tight junction (zo-1 and occludin) showed the reverse trend. The mRNA expression of positive regulators related to protein synthesis (sirt1, pgc1-α, pi3k, and akt) were significantly upregulated in the muscle of fish fed diets T3 and T4, while their negative regulators (4e-bp1) mRNA levels were downregulated. The results indicate that the dietary FM of largemouth bass could be effectively reduced to at least 18% by the Cpro, which is beneficial to health, digestion, and protein synthesis for maintaining accelerated growth.

Arecoline inhibits the growth of Spodoptera litura by inducing intestinal metabolic dysfunction

Arecoline (ACL), an active constituent derived from Areca catechu L., exerts various pharmacological effects and serves as a potential plant-based insecticide. However, the effects of ACL on Spodoptera litura, an important and widely distributed agricultural pest, remain unknown. This study aimed to elucidate the mechanism underlying ACL-induced toxicity and its inhibitory effects on larval growth and development through intestinal pathology observations, intestinal transcriptome sequencing, intestinal digestive enzyme activity analysis. The results indicated that ACL exposure leads to pathological alterations in the S. litura midgut. Furthermore, the detection of digestive enzyme activity revealed that ACL inhibits the activities of acetyl CoA carboxylase, lipase, α-amylase, and trypsin. Simultaneously, upregulation of superoxide dismutase activity and downregulation of malondialdehyde levels were observed after ACL exposure. Transcriptome analysis identified 1118 genes that were significantly differentially expressed in the midgut after ACL exposure, potentially related to ACL toxic effects. Notably, ACL treatment downregulated key enzymes involved in lipid metabolism, such as fatty acid binding protein 2-like, pancreatic triacylglycerol lipase-like, pancreatic lipid-related protein 2-like, and fatty acid binding protein 1-like. Taken together, these results suggest that ACL induces midgut damage and impedes larval growth by suppressing digestive enzyme activity in the intestine. These findings can aid in the development of environmentally friendly plant-derived insecticides, utilizing ACL to effectively combat S. litura proliferation.

Effects of Substituting Tenebrio molitor and Elodea nuttallii as Feed on Growth, Flesh Quality and Intestinal Microbiota of Red Swamp Crayfish (Procambarus clarkii).

This study aimed to evaluate the impact of substituting a portion of feed with Tenebrio molitor (TM) and Elodea nuttallii (EN) on crayfish culture. A total of 270 crayfish (5.1 ± 0.4 g) were fed three different diet combinations (A: 100% feed; B: 80% feed + 10% TM + 10% EN; C: 75% feed + 15% TM + 10% EN) for 12 weeks. The findings demonstrated that group C had an important beneficial impact on the growth performance of crayfish. This was evidenced by a rise in digestive enzyme activity (trypsin, lipase, and cellulase) in the intestinal and hepatopancreas, as well as an upregulation in the expression of growth-related genes (ghsr, igfbp7, mhc, mlc1, mef2, and pax7) in the muscle. Furthermore, the assessment of the flesh quality of crayfish muscle in group C was conducted. The findings indicated a significant increase (p < 0.05) in the energy value (moisture, crude protein, and crude lipid) within the muscle. The levels of delicious amino acids (Glu, Ala, Ser, Gly, and Tyr) and polyunsaturated fatty acids (ARA, DHA) were enhanced, resulting in an improved nutritional profile and flavor of the muscle while maintaining the Σn-3/Σn-6 ratio. The remodeling of the intestinal microbiota (abundance of Proteobacteria and ratio of Firmicutes/Bacteroidota bacteria) also revealed improved growth performance. Additional research is necessary to ascertain whether excessive use of TM or EN feed substitution can have negative effects on crayfish culture.

The impact of Neoporphyra haitanensis dietary supplement on astaxanthin esters and fatty acids accumulation associated with immune promotion in Pacific white shrimp (Litopenaeus vannamei)

To explore and develop effective feed additives for enhancing shrimp cultivation, an 8-week feeding experiment was conducted in Litopenaeus vannamei with dietary supplementation ranging from 0% to 5% Neoporphyra haitanensis. The results indicated that a diet containing 2% N. haitanensis enhanced trypsin activity, as well as the height and width of intestinal villi, leading to improved growth performance of L. vannamei. The specific growth rate (SGR), weight gain rate (WGR) and the ratio of flesh content were increased by 1.1 to 2.0 times, while the hepatosomatic index (HSI) and condition factor (CF) values were 85.6% and 91.5% of those in the control group, respectively. Additionally, the inclusion of 2% N. haitanensis in the diet led to a 1.1-fold increase in the accumulation of fatty acids, particularly DHA and EPA. The diet also exhibited elevated levels of β-carotene, zeaxanthin, α-cryptoxanthin, and β-cryptoxanthin, which correlated with the increased content of carotenoids in the shrimp. The exoskeleton was identified as the primary site for assimilating α−/β-carotenes, free astaxanthin, and its diesters (mainly EPA and DHA chains), accompanied by heightened redness (a*) value. Meanwhile, the muscle tended to accumulate astaxanthin and its monoesters (mainly C16:0 and C18:0 chains) and hepatopancreas had lowest contents of astaxanthin and its diesters (mainly DHA chain). Furthermore, the Toll and IMD immune signal pathways in L. vannamei were activated with 2% N. haitanensis in the die, up-regulating the expression of immune-related genes dorsal, relish and imd, and antibacterial peptides genes crustin, lysozyme, and penaeidin3a. Concurrently, the activities of immune-related enzymes ACP and AKP, as well as antioxidant enzymes CAT, SOD, and GPH-Px, were enhanced, while AST and ALT activities were decreased. In terms of intestinal health, the addition of 2% N haitanensis contributed to the preservation of the intestinal tissue structure and promoted the abundance of beneficial bacteria, while reducing the abundance of Vibrio in L. vannamei. The enhanced immune and antioxidant performance of L. vannamei was demonstrated to prevent intestinal structure damage and increase survival rates, as evidenced by WSSV infection experiments. These results offer valuable insights into the dose-dependent effects of dietary seaweeds in promoting shrimp cultivation.

Network pharmacology combined with experimental validation reveals the mechanism of action of erpixing granules on functional dyspepsia

Ethnopharmacological relevanceFunctional dyspepsia (FD) is a prevalent gastrointestinal disorder characterised by high incidence and recurrence rates, posing significant health risks. Erpixing Granules (EPX), approved by the National Food and Drug Administration in 2002, are known for their spleen and stomach invigorating properties, effectively treating FD. However, its mechanism of action remains unclear. Aim of the studyThis study aims to elucidate EPX's mechanism of treating FD through network pharmacology, and experimental validation using FD animal models. MethodsIn this study, the chemical composition of EPX in positive and negative ion modes was analyzed by UHPLC-Q-TOF MS. The mass spectral data were processed and analyzed using MS-DIAL software to automatically match compound fragment information and identify the known components with the compound database to obtain the active components of EPX. SwissTargetPrediction was used to obtain EPX targets, while FD-related targets were sourced from GeneCards, OMIM and DisGeNET databases. A protein-protein interaction (PPI) network was constructed using the STRING platform, and potential signalling pathways of EPX were determined through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Finally, an FD model was established in rates by administering a 0.1% iodoacetamide sucrose solution, followed by tail clamp stimulation to experimentally validate the network pharmacology findings. ResultsOur results revealed 139 effective ingredients in EPX, targeting 60 core FD-related genes. PPI network analysis identified EGFR, CTNNB1 and NFκB1 as core target genes. The KEGG pathway analysis indicated that EPX can modulate FD progression through the PI3K/AKT signalling pathway. Animal experiments demonstrated EPX's capacity to increase body mass, food intake and food utilisation efficiency in FD rats, alongside increased gastric juice secretion, pepsin activity, trypsin activity, cholesterol, bile acid and bilirubin activity. HE examination revealed that EPX improved the inflammatory infiltration of gastric mucosal cells in rats. Furthermore, EPX also promoted gastric emptying and intestinal propulsion in mice. These results suggest that EPX improves spleen and stomach function, enhances the protective effect on the spleen and stomach and promotes food digestion and absorption. Immunofluorescence studies revealed upregulated expression of PI3K, AKT and ANO1 proteins in gastric tissue following EPX administration, while Western blotting indicated increased expression of SCF and C-kit proteins. ConclusionSuggesting EPX's anti-FD effect may involve the regulation of the SCF/C-kit signalling pathway and activation of downstream PI3K/AKT signalling pathway, thereby promoting gastrointestinal motility and improving FD symptoms.

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Effects of dietary supplementation of Enterococcus faecium postbiotics on growth performance and intestinal health of growing male mink.

Recent studies have demonstrated that postbiotics possess bioactivities comparable to those of probiotics. Therefore, our experiment aimed to evaluate the effects of postbiotics derived from Enterococcus faecium on the growth performance and intestinal health of growing male minks. A total of 120 growing male minks were randomly assigned to 4 groups, each with 15 replicates of 2 minks. The minks in the 4 groups were fed a basal diet supplemented with 0 (control), 0.05, 0.1, and 0.15% postbiotics derived from E. faecium (PEF), respectively. Compared to the control, PEF improved feed/gain (F/G) during the first 4 weeks and the entire 8 weeks of the study (p < 0.05); in addition, 0.1% PEF improved average daily gain (ADG) during the first 4 weeks and the entire 8 weeks of the study (p < 0.05), while 0.15% PEF improved ADG during the first 4 weeks of the study (p < 0.05). Consequently, 0.1% PEF minks displayed greater body weight (BW) at weeks 4 and 8 (p < 0.05), and 0.15% PEF minks had greater BW at week 4 (p < 0.05) than minks in the control. Furthermore, compared to the control, both 0.05 and 0.1% PEF enhanced the apparent digestibility of crude protein (CP) and ether extract (EE) (p < 0.05) in the initial 4 weeks, while both 0.1 and 0.15% PEF enhanced the apparent digestibility of CP and DM in the final 4 weeks (p < 0.05). Additionally, trypsin activity was elevated in the 0.1 and 0.15% PEF groups compared to the control (p < 0.05). In terms of intestinal morphology, PEF increased the villus height and villus/crypt (V/C) in the jejunum (p < 0.05), and both 0.1 and 0.15% PEF decreased the crypt depth and increased the villus height and V/C in the duodenum (p < 0.05) compared to the control group. Supplementation with 0.1% PEF increased the SIgA levels but decreased the IL-2, IL-8, and TNF-α levels in the jejunum (p < 0.05). Compared to the control, E. faecium postbiotics decreased the relative abundances of Serratia and Fusobacterium (p < 0.05). In conclusion, the results indicate that the growth performance, digestibility, immunity, and intestine development of minks are considerably affected by E. faecium postbiotics. In particular, dietary supplementation with 0.1% E. faecium postbiotics provides greater benefits than supplementation with 0.05 and 0.15%.

Secretagogue-induced pancreatitis in mice devoid of chymotrypsin.

The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del × Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin, yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del × Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del × Ctrl-KO mice spontaneously progressed to chronic pancreatitis, whereas C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del × Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice; however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis; however, the minor isoform CTRL also contributes to an appreciable extent.NEW & NOTEWORTHY Chymotrypsins defend the pancreas against the inflammatory disorder pancreatitis by degrading harmful trypsinogen. This study demonstrates that mice devoid of pancreatic chymotrypsins are phenotypically normal but become sensitized to secretagogue hyperstimulation and exhibit increased intrapancreatic trypsin activation, more severe acute pancreatitis, and rapid progression to chronic pancreatitis. The observations confirm and extend the essential role of chymotrypsins in pancreas health.

Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

Trypsin-encoding gene function of efficient star polycation nanomaterial-mediated dsRNA feeding delivery system of Grapholita molesta.

Grapholita molesta is an important and harmful fruit pest worldwide, with widespread feeding hosts. Trypsin, an indispensable hydrolytic digestive protease in the insect gut, is crucial in digestion, growth and development. We analyzed the characteristics of the trypsin-encoding genes, screened for the optimal dose of RNAi mediated by nanocarriers, and investigated various indices of larval growth and development of G. molesta. Gut content (GC) and RNase A degraded double-stranded RNA (dsRNA), with a faster degradation rate at higher concentrations. Star polycation (SPc) nanomaterials protected dsGFP from degradation by anion-cation binding and did not migrate through agarose gel. The key conserved motifs of the trypsin-encoding genes were similar, exhibiting high homology with those in other lepidopteran insects. An interference efficiency of ≈70% was achieved with SPc nanomaterial-mediated RNA interference with 0.05 μg dsRNA. The efficiency of continuous interference was stable. Trypsin activity, body weight of 8-day-old larvae, pupal weight and emergence rate were significantly reduced, and the larval stage was significantly prolonged. The investigated trypsin gene is a key target gene in the growth and development of G. molesta. We investigated the efficiency and convenience of feeding SPc nanomaterials in a functional study of insects. Our results provide valuable data for the development of efficient trypsin-targeting pesticides. © 2024 Society of Chemical Industry.

Feasibility evaluation of cottonseed protein concentrate to replace soybean meal in the diet of juvenile grass carp (Ctenopharyngodon idellus): Growth performance, antioxidant capacity, intestinal health and microflora composition

An investigation into the replacement of soybean meal (SBM) with cottonseed protein concentrate (CPC) in the diet of grass carp feed (Ctenopharyngodon idellus) was carried out. During a 56-day feeding trial, 750 grass carp were fed five experimental diets (isonitrogenous and isolipidic) with different levels of CPC: SBM (control group), CPC25, CPC50, CPC75, and CPC100, corresponding to dietary SBM replacements levels of 0%, 25%, 50%, 75%, and 100%. The results showed that dietary CPC inclusion significantly increased FW, WGR, and PER (P < 0.05) except CPC100 groups, which did not differ with SBM group (P > 0.05). FCR was remarkably increased when SBM replacement levels reached up to 100% (P < 0.05). Based on broken-line regression analysis of WGR against the dietary replacement of SBM with CPC, the optimal replacement level of SBM with CPC was estimated to be 75.11% for juvenile grass carp. Muscle texture parameters hardness, gumminess and chewiness were apparently elevated (P < 0.05), demonstrated an improved flesh quality followed CPC added. The CPC inclusion groups showed significantly higher serum IgM, C3 amd C4 contents, as well as ACP and AKP activities than SBM group except CPC100 groups (P < 0.05). The hepatic antioxidant capacity was enhanced followed CPC inclusion verified at enzymatic and transcriptional levels (P < 0.05). The intestinal inflammation was relieved evidenced by the down-regulated il-β, il-6 and tnf-α expressive abundance mediated through NF-κB pathway (P < 0.05). Additionally, the intestinal mucosal barrier function was enhanced by the up-regulation of tight junction proteins (occludin, claudin12, zo-1, and zo-2). The intestinal lipase and trypsin activities in CPC50 and CPC75 and intestinal folds height in CPC25, CPC50 and CPC75 were advanced (P < 0.05). The gut microbiota revealed no significant differences in the alpha diversity indices across treatments (P > 0.05). While the key bacteria of Aeromonas in the CPC100 group was increased leading intestinal dysbacteriosis. Besides, the ME1 of the microbial network has a negative correlation with pro-inflammatory gens and a positive correlation with antioxidant gens and tight linking proteins (P < 0.05). Summarily, CPC could partially replace SBM with positive influences on growth, antioxidant capacity, immunity, and intestinal microbiota in grass carp.

Intra-cohort cannibalism and aggressive behavior in tropical gar (Atractosteus tropicus) larvae: Effects of stocking density and starvation

Cannibalism in early fish developmental stages is a biological phenomenon affecting aquaculture, so seeding density and feeding schemes play a fundamental role in its optimal control. The objective of this research was to evaluate two initial stocking densities, 0.7 larvae/L (D7) and 1.4 larvae/L (D14), which were fed (F) and not fed (NF) in tropical gar (Atractosteus tropicus) larvae on growth, attacks, survival, and digestive enzyme activity, digestive morphology, and oxidative enzymes and aggressiveness gene expression. A total of 420 larvae (0.022 ± 0.003 g; 1.52 ± 0.10 cm) were placed in 70 L tanks connected to a recirculation system. Treatment F larvae were fed a formulated food. D14F larvae had the highest final weight, total length, and specific growth rate (SGR) (p < 0.05). The highest survival percentage was in D7F larvae (46.19 ± 2.74) (p < 0.05). The highest frequency of attacks occurred in D14F larvae (26.66 ± 0.82) (p < 0.05). The coefficient of variation (CV) and size heterogeneity (SH) were higher in D7F larvae (13.56 ± 1.80; 0.81 ± 0.10) (p < 0.05). The F larvae started attacks at 8 days after hatching (DAH), while the NF larvae showed it at 10 DAH. The difference in weight between the attacker and the attacked was 16.44 ± 9.75% and in total length 12.28 ± 4.57% (p < 0.05). Acid and alkaline protease, trypsin, and lipase activities presented differences between both factors, as well as the interaction between them. D14F larvae presented the highest Glutathione peroxidase (GPX) activity (p < 0.05), while D7-NF larvae registered the highest catalase (CAT) activity (p < 0.05). Superoxide dismutase (SOD) activities only showed differences between F and NF larvae (p < 0.05). The liver presented the highest percentage of area with melanomacrophage centers (MMC) in D7-NF larvae, and hepatocytes with the highest area were recorded in D14F larvae (p < 0.05). In gut, the highest enterocytes were recorded in D14F larvae (p < 0.05). cat, cck, and gpx expression did not show difference between feeding regimes. The expression of sod1 and sod2 in D7F and D14F treatments presented the highest expressions (p < 0.05). D7F larvae presented higher expression of avp1, crh, htr1, sstr1, th and tph1 genes (p < 0.05). Our results show that A. tropicus larvae D14F presented the highest growth and morphometric parameters and decreased CV and SH, which shows a more homogeneous growth. Regarding cannibalism, fed larvae (D7F, D14F) presented cannibalism in a greater proportion than unfed larvae regardless of density, however, it is necessary to analyze other physiological and social factors that could be related to the aggressiveness and cannibalism of the species.

Optimizing winged-bean meal through oven and autoclave heating as viable alternative for plant-based aquafeed

Winged-bean meal can serve as a substitute for soybean meal in plant-based fish feed, despite its trypsin-inhibitor properties, which can be alleviated through heating processes. This study aimed to explore the potential of winged-bean meal subjected to different heating methods, namely oven (110 °C for 30 minutes) and autoclave (121 °C for 30 minutes), based on proximate analysis and trypsin activity. The study applied three treatments with four replications: oven heating (O), autoclave heating (A), and control (without heating, E). All data were analyzed using analysis of variance and Duncan’s multiple range test to determine the best treatment. Data were also compared to the nutrient requirements of several cultured freshwater fish in Indonesia. Heating treatments improved in vitro trypsin activity (1300 units/O and 1135 units/A vs. 835 units) but reduced protein (29.05±1.96%/A and 31.73±1.52%/O vs. 35.17±0.38%) and moisture contents. Additionally, the O treatment met the standards for aquafeed protein content (27-45%) and potentially fulfilled the protein requirements for catfish (25-40%), Nile tilapia (30-35%), and Java barb (30-32%). Therefore, oven heating presents a viable candidate as an alternative plant-based aquafeed ingredient. Nevertheless, the in vivo application of winged-bean meal as an aquafeed ingredient should be further evaluated to determine whether heating treatment effectively enhances digestive activity, nutrient retention, and growth performance in target fish species.

Effects of dietary Pediocuccus pentosaceus and/or ferulic acid on performance, digestive enzymes, blood biochemistry, and growth-related genes expression in rainbow trout (Oncorhynchus mykiss)

This study investigated the effects of dietary Pediocuccus pentosaceus (PP) and/or ferulic acid (FA) on the performance, digestive enzymes, and growth-related genes expression in rainbow trout (Oncorhynchus mykiss). Fish (94.30 ± 1.77 g) were fed on the control diet (T0), 108 CFU/g of PP probiotic (T1), 100 mg/kg of FA (T2), and a combination of PP and FA (T3) for 8 weeks. The present results indicated that rainbow trout fed with PP and/or FA showed better final weight, weight gain, and feed conversion ratio than the control group (P < 0.05). The activities of trypsin and chymotrypsin were higher in T2 and T3 than in the control group (P < 0.05). Meanwhile activities protease and alpha-amylase were highest in T2, lipase activity was highest in T3. Compared to the control group, total protein, total antioxidant activity, and cholesterol levels were significantly (P < 0.05) higher in T2 and T3 with no significant (P > 0.05) differences between them. Supplemented diets with FA and/or PP decreased markedly (P < 0.05) triglycerides levels and its lowest levels were obtained in T2 and T3 with no significant (P > 0.05) differences between them. The relative expression levels of GHRL and GHr genes were significantly higher in the treatments fed with FA and/or PP (T1 – T3) than those in the control group (P < 0.05). Highest upregulation of IGF-1, and IGF-II gens were detected in T2 and T3 with no significant (P > 0.05) differences between them. In conclusion, it was shown that both dietary supplements have the potential role to enhance the performance, digestive enzymes, and growth related-genes expression in rainbow trout. Feeding fish on FA-enriched diets showed superior enhancements than that fed on the PP diet only.