Phenoloxidase Activity Research Articles

Assessment of fish protein hydrolysate as a substitute for fish meal in white shrimp diets: Impact on growth, immune response, and resistance against Vibrio parahaemolyticus infection

This study investigated the effects of fish protein hydrolysate derived from barramundi on growth performance, muscle composition, immune response, disease resistance, histology and gene expression in white shrimp (Penaeus vannamei). In vitro studies demonstrated FPH enhanced mRNA expressions of key immune-related genes and stimulated reactive oxygen species (ROS) production and phagocytic activity in shrimp hemocytes. To evaluate the effects of substituting fish meal with FPH in vivo, four isoproteic (43 %), isolipidic (6 %), and isoenergetic diets (489 kcal/100 g) were formulated with fish meal substitution levels of 0 % (control), 30 % (FPH30), 65 % (FPH65), and 100 % (FPH100). After 8-week feeding, the growth performance of FPH65 and FPH100 were significantly lower than that of control and FPH30 (p < 0.05). Similarly, the midgut histological examination revealed the wall thickness and villi height of FPH100 were significantly lower than those of control (p < 0.05). The shrimps were received the challenge of AHPND + Vibrio parahaemolyticus at week 4 and 8. All FPH-fed groups significantly enhanced resistance against Vibrio parahaemolyticus at week 4 (p < 0.05). However, this protective effect diminished after long-period feeding. No significant difference of survival rate was observed among all groups at week 8 (p > 0.05). The expressions of immune-related genes were analyzed at week 4 before and after challenge. In control group, V. parahaemolyticus significantly elevated SOD in hepatopancreas and Muc 19, trypsin, Midline-fas, and GPx in foregut (p < 0.05). Moreover, hepatopancreatic SOD of FPH65 and FPH100 were significantly higher than that of control before challenge (p < 0.05). Immune parameters were measured at week 8. Compared with control, the phagocytic index of FPH 30 was significantly higher (p < 0.05). However, dietary FPH did not alter ROS production, phenoloxidase activity, phagocytic rate, and total hemocyte count (p > 0.05). These findings suggest that FPH30 holds promise as a feed without adverse impacts on growth performance while enhancing the immunological response of white shrimp.

Combined intestinal microbiota and transcriptomic analysis to investigate the effect of different stocking densities on the ability of Pacific white shrimp (Litopenaeus vannamei) to utilize Chlorella sorokiniana

Aiming to investigate the impact of different stocking densities on the ability of Pacific white shrimp (Litopenaeus vannamei) to utilize Chlorella sorokiniana (CHL), a 3 × 2 factorial design stocking experiment was used in this study. Specifically, shrimp was fed with two dietary protein sources (fishmeal [FM] and CHL) at low (LSD; 100 per m3), medium (MSD; 200 per m3) and high (HSD; 300 per m3) stocking densities for 8 weeks. The growth performance and resistance to Vibrio parahaemolyticus (1.0 × 107 CFU/mL) of shrimp decreased with the increase of stocking density, but dietary CHL improved this result. Differences between the CHL and FM groups for V. parahaemolyticus resistance were significant only under high-density conditions (P < 0.05). Significant interactions between stocking density and protein source were found on the activities of catalase (CAT), superoxide dismutase (SOD) and phenol oxidase (PO), and the contents of malondialdehyde (MDA) in the hepatopancreas and the activities of intestinal amylase, most of which were significantly different between CHL and FM groups only at high stocking density (P < 0.05). Analysis of 16S rDNA sequencing showed that dietary CHL increased the alpha diversity of intestinal microbiota, inhibited the colonization of pathogenic bacteria and enhanced the abundance of beneficial bacteria. Transcriptomic results showed that at high stocking densities, differentially expressed genes (DEGs) in the FM vs CHL group were mostly upregulated and primarily enriched in immune and metabolic related pathways including Toll, immune deficiency (Imd) and glycolysis–gluconeogenesis pathways. Pearson correlation analysis revealed significant correlation between the top ten intestinal bacteria at the genus level and markedly enriched DEGs, also more were detected under high density situations. In conclusion, CHL has great potential as a novel protein source in the intensive farming of shrimp.

Next-generation neonicotinoid: The impact of cycloxaprid on the crustacean decapod Penaeus vannamei

Cycloxaprid, a new neonicotinoid pesticide, poses ecological risks, particularly in aquatic environments, due to its unique action and environmental dispersal. This study investigated the ecotoxicological effects of various concentrations of cycloxaprid on Penaeus vannamei over 28 days. High cycloxaprid levels significantly altered shrimp physiology, as shown by changes in the hepatosomatic index and fattening. Indicators of oxidative stress, such as increased serum hemocyanin, respiratory burst, and nitric oxide, as well as decreased phenol oxidase activity, were observed. Additionally, elevated activities of lactate dehydrogenase, succinate dehydrogenase, and isocitrate dehydrogenase indicated disrupted energy metabolism in the hepatopancreas. Notably, analyses of the nervous system revealed marked disturbances in neural signaling, as evidenced by elevated acetylcholine, octopamine, and acetylcholinesterase levels. Transcriptomic analysis highlighted significant effects on gene expression and metabolic processes in the hepatopancreas and nervous system. This study demonstrated that cycloxaprid disrupts neural signaling and oxidative balance in P. vannamei, potentially affecting its growth, and provides key insights into its biochemical and transcriptomic toxicity in aquatic systems.

Response of Soil CO2 Emission to Addition of Biochar and Dissolved Organic Carbon along a Vegetation Restoration Gradient of Subtropical China

Biochar, as a soil amendment, has been widely confirmed to increase soil carbon sequestration. However, how biochar addition affects soil carbon changes during the vegetation restoration process is still unclear, which constrains our ability to explore biochar’s application in the technology of soil carbon sequestration in forests. We conducted an incubation experiment on biochar and dissolved organic matter (DOM) addition to soil at three stages of revegetation (degraded land (DS), plantation forest (PS), and secondary natural forest (NS) in Changting County in Fujian province, China) to investigate the effects of vegetation restoration, biochar, DOM, and their interaction on soil CO2 emission and its relative mechanisms. We found that the accumulative release of CO2-C in the NS and PS soils was 7.6 and 6.8 times higher, respectively, in comparison to that from the DS soil. In the DS, biochar addition significantly increased the accumulative release of CO2-C, soil pH, NH4+-N content, qCO2, phenol oxidase, and peroxidase activities. Peroxidase activities were positively correlated with the accumulative release of CO2-C, and oxidase was the most important direct factor influencing the accumulative release of CO2-C in the DS. However, the accumulative release of CO2-C, soil NH4+-N content, qCO2, β-glucosidase, and N-acetylglucosaminidase activities was significantly reduced after the application of biochar in the PS and NS. These two hydrolases were positively associated with the accumulative release of CO2-C, and hydrolase was the most vital direct factor influencing the accumulative release of CO2-C from the PS and NS soils. The positive effect of DOM addition on CO2 emission under biochar application declined with a vegetation restoration age increase. Our results indicated that biochar could alter microbial physiological processes, inhibit qCO2 and hydrolase activities, and further decrease CO2 emission in relatively fertile soil from the PS and NS; but in the relatively barren soil from the DS, biochar might promote CO2 emission by stimulating microorganisms to enhance qCO2 and oxidase activities.

Differential responses of soil cellulase enzymes and oxidative enzymes to soil erosion

The activities of soil carbon-degrading enzymes play an important role in determining carbon mineralization. Soil erosion is the most common form of soil degradation worldwide, but the responses of cellulase enzymes and oxidative enzymes to erosion remain elusive. Here, we studied the effects of erosion on β-1,4-Glucosidase (BG) and phenol oxidase (PHO) activities as well as their contributions to carbon mineralization at three erosion hotspots in China. We found that erosion did not change SOC-normalized BG activity in both eroded plots and depositional plots, but significantly increased SOC-normalized PHO activity in eroded plots. The SOC-normalized PHO activity explained more variance in carbon mineralization than BG. Further analysis showed that the lignocellulose index (the ratio of PHO to the sum of BG and PHO) was closely related to microbial stoichiometric balance rather than the carbon composition under the impact of erosion. These results indicate that erosion may stimulate the utilization of recalcitrant carbon by microbial oxidase in eroded areas, potentially exacerbating the loss of soil carbon in slopes.

Dietary Poly-β-Hydroxybutyrate Improved the Growth, Non-specific Immunity, Digestive Enzyme Activity, Intestinal Morphology, Phagocytic Activity, and Disease Resistance Against Vibrio parahaemolyticus of Pacific White Shrimp, Penaeus vannamei.

This study assessed the effects of dietary supplementation of poly-β-hydroxybutyrate (PHB) on growth performance, feed efficiency, non-specific immunity, digestive enzyme capacity, phagocytic activity, hemocyte count, intestinal morphology, and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp (Penaeus vannamei). Six diets were prepared by supplementing graded levels of PHB at 0.00, 0.25, 0.50, 1.00, 2.00, and 4.00% (Con, P0.25, P0.5, P1.0, P2.0, and P4.0, respectively). Triplicate groups of 90 shrimps (initial body weight 0.25 ± 0.01g) per treatment were randomly assigned and fed an experimental diet for 56days. The growth performance of shrimp was significantly improved by 1% dietary PHB supplementation. PHB-included diets fed shrimp showed significantly improved hepatopancreatic trypsin, chymotrypsin, and pepsin activities. Villus height was significantly increased with dietary PHB supplementation, and villus width was increased at a 1% inclusion level. P0.25, P0.5, and P4.0 groups significantly increased phenoloxidase activity, and the P2.0 group significantly increased anti-protease activity compared to the Con group. The survival of shrimp challenged against V. parahaemolyticus was higher in P0.5, P1.0, and P2.0 groups than in the Con diet. Dietary PHB supplementation improved weight gain, digestive enzyme activity, intestinal morphology, non-specific immunity, and disease resistance against V. parahaemolyticus of shrimp. According to the above observations, the optimal dietary PHB supplementation level for maximum weight gain would be 1% for Pacific white shrimp.

Entomopathogenic fungi-based silver nanoparticles: a potential substitute of synthetic insecticides to counter behavioral and physiological immunity in Aedes aegypti mosquito (Diptera: Culicidae).

Excessive use of synthetic insecticides has resulted in environmental contamination and adverse effects on humans and other non-target organisms. Entomopathogenic fungi offer eco-friendly alternatives; however, their application for pest control requires significant advancement owing to limitations like slow killing time and effectiveness only when applied in higher amounts, whereas exposure to UV radiation, high temperature, and humidity can also reduce their viability and shelf-life. The nanoparticles synthesized using fungal extracellular extracts provide a new approach to use fungal pathogens. Our study focused on the synthesis of Metarhizium anisopliae-based silver nanoparticles (AgNPs) and evaluation of their efficiency on various physiological and behavioral parameters of the mosquito Aedes aegypti. The synthesis, size (27.6 d.nm, PDI = 0.209), zeta potential (- 24.3 mV), and shape of the AgNPs were determined through dynamic light scattering, scanning and transmission electron microscopic, and UV-visual spectroscopic analyses (432 nm). Our results showed significantly reduced survival (100% decrease in case of 3.2 and 1.8 μL/cm2 volumes, and 60% decrease in case of 0.8 μL/cm2 volume), phenoloxidase activity (t = 39.91; p = 0.0001), and gut microbiota, with increased oxidative stress and cell apoptosis in AgNPs-challenged mosquitoes. Furthermore, the AgNPs-exposed mosquitoes presented a concentration-specific decrease in flight locomotor activity (F = 17.312; p < 0.0001), whereas no significant changes in antifungal activity, self-grooming frequencies, or time spent were found. These findings enhance our understanding of mosquito responses to AgNPs exposure, and offer a more efficient mosquito control strategy using entomopathogenic fungi.

Exposure to entomopathogenic fungus and high larval density induce a strong immune response and life‐history costs in black soldier fly, a commercially important insect

AbstractPathogen infection and conspecific density may considerably affect key life‐history traits of organisms. For naturally aggregating species, even low concentrations of pathogens or high larval densities may have detrimental effects. However, the detailed influence of these factors, particularly their interaction effect, is often overlooked in ecological and life‐history studies. To investigate the effects of conspecific density and pathogen infection on life‐history traits, we explored the influence of larval density (1 and 5 larvae cm−2, i.e., low and high density, respectively) on phenoloxidase (PO) activity, body mass, and development time of Hermetia illucens L. (Diptera: Stratiomyidae) larvae in the presence of two strains of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.‐Criv.) Vuill. We observed higher PO activity in EPF‐treated larvae than in the untreated control and a pronounced difference in PO activity between the two EPF‐strain treatments. Larvae reared at high density and treated with EPF showed higher PO activity than untreated larvae at low density. The EPF‐treated larvae and larvae reared at high density had longer larval periods than untreated larvae and larvae reared at low density, respectively. Larvae reared at high density also achieved reduced prepupal and pupal masses compared to conspecifics at low density. Interestingly, untreated larvae only achieved higher prepupal and pupal masses at low density, whereas at high density, the pattern was reversed (treated individuals had higher pupal masses). Overall, our results demonstrate that high density and fungal pathogens both induce a higher immune response compared to low density and pathogen‐free environments, but this comes with a cost of a longer larval period and reduced body mass.

ZnO nanoparticles: A key ingredient of sunscreen shows absence of adverse effects on Drosophila melanization pathway

The rapid development of zinc oxide nanoparticle (ZnO NP) based sunscreens, shampoos and other skin care products has raised a concern about their safety at the cellular level. The melanization pathway, being an important pathway of skin cells, is prone to being affected by ZnO NPs. Therefore it is necessary to understand the ZnO NP mediated effect on the melanin synthetic pathway. Our previous study on bulk zinc oxide has shown that large sized zinc oxide particles did not have any adverse effect on melanization in Drosophila, hence it would be interesting to study the behavior of ZnO when present in the nano-form by virtue of the changed size and thereby changed properties. The present study was undertaken to investigate ZnO NP mediated effects on key metabolites of the Drosophila melanin pathway. Spectrophotometric analysis of melanin was performed. followed by a study of the enzyme activity of phenol oxidase and HPLC quantification of dopa and tyrosine. FTIR spectroscopy was carried out to examine the interaction of the ZnO NPs and precursor amines of the melanin synthetic pathway. Pigmentation mutants of Drosophila were also investigated for validation of the results under a different genetic background. The ZnO NPs did not show any decrement in the levels of melanin after the treatment. Moreover there was no adverse effect on the phenol oxidase activity, Tyrosine and L-Dopa. The FTIR studies showed minimal interaction between the ZnO NPs and the metabolites of the melanin pathway. The results of the present study have clearly indicated the absence of any adverse effects on key metabolites of the melanin pathway after ZnO NP treatment in both wild-type and mutant Drosophila. This is the first report of a detailed analysis of ZnO NP mediated effects on key constituents of the Drosophila melanization pathway, providing information about its safety for the usage in cosmetics.

Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus)

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL −1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Poria cocos polysaccharides alleviate cyclophosphamide-induced immunosuppression effects in silkworm and their metabolic profiling analysis

The present study investigated the immune regulatory mechanisms of Poria cocos polysaccharides (PS) in a cyclophosphamide (CTX)-induced immunosuppression silkworm model. We found that PS increased the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, as well as reactive oxygen species (ROS) levels in the fat body or haemocytes of CTX-treated silkworms. PS also strongly stimulated the immunity of silkworms by increasing haemocyte counts and PO activity and upregulating the mRNA expression of immune-related genes. We further investigated the metabolic profile of the fat body after PS treatment using liquid chromatography–massspectrometry/mass spectrometry (LC–MS/MS) and identified 236 differentially expressed metabolites. Among them, nine metabolites related to amino acid metabolism were present at high levels in the PS-treated group, whereas the contents of metabolites involved in sphingolipid metabolism and alpha-linolenic acid metabolism were significantly decreased after PS treatment. The amino acid metabolism pathways and lipid metabolism pathways were found to be significantly altered in the PS-treated group, which indicates that PS may regulate the immune system of silkworms by reprogramming lipid and amino acid metabolism. This study provides new insight into the immune regulatory effect of PS in animals.

Pathogenicity, infection process, physiological and biochemical effects of Metarhizium rileyi against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae

BackgroundMetarhizium rileyi is an important entomopathogenic fungus which has a wide range of host pests. In this work, the virulence of M. rileyi EFNL-08 against 3rd instar larvae of the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), was quantified. Additionally, the infection process and aspects of the host immunological response were described in the laboratory.ResultsBioassay results proved that M. rileyi was highly virulent to 3rd instar S. frugiperda larvae with 98.33% mortality at 1 × 108 conidia/ml. Scanning electron microscopy was used to monitor morphological aspects of the infection process. When hyphal bodies (Hbs) of M. rileyi were injected into the hemocoel of host, they were not recognized by the host’s immune system as invaders. There were nonsignificant differences in the hemocytes and phenoloxidase activity between the infected and control larvae at the initial 30 h, indicating that fungus successfully avoids the attack from the immune system. However, it inhibited the enzyme activity at the latest stage when Hbs reached a high density. After infection with M. rileyi, the activity of glutathione-S-transferase (GST) of S. frugiperda larvae presented a significant upward trend, while the activity of acetylcholinesterase (AchE) initially increased, followed by a decrease, and eventually stabilized. However, the activity of both detoxification enzymes in the control group showed slight fluctuations in the initial stage, and then stabilized at a low level.ConclusionObtained results provided new insights into the virulence and potential physiological responses of M. rileyi. This provides ideas for further development of novel and efficient entomopathogenic fungi (EPF) for pest control.

Chemical Recalcitrance Rather Than Soil Microbial Community Determined Short-Term Biochar Stability in a Poplar Plantation Soil

The stability of biochar is fundamental to its soil carbon (C) sequestration potential. The relative importance of chemical recalcitrance and the soil microbial community on biochar stability is still unclear. To unveil the question, we conducted a 60-day incubation to explore the stability of two rice-straw-derived biochars pyrolyzed at 300 and 500 °C (denoted as BS300 and BS500), as well as the relative contribution of the soil microbial community and biochar chemical recalcitrance to biochar stability in a poplar plantation soil. Biochar-derived cumulative carbon dioxide (CO2) emission was estimated to be 41.3 and 6.80 mg C kg−1, accounting for 0.73 and 0.11% of the amended biochar-derived organic C (OC) in BS300 and BS500 treatments, respectively. The mean retention time (MRT) estimated by double-exponential model fitting was 49.4 years for BS300 and 231 years for BS500. Compared to control, BS300 and BS500 decreased β-D-glucosidase activity by 20.9 and 18.0%, while they decreased phenol oxidase activity by 31.8 and 18.9%, respectively. Furthermore, BS300 increased the soil microbial metabolic quotient (qCO2) by 155%, but BS500 decreased it by 13.4%. In addition, BS300 resulted in a 520% higher biochar-derived hot-water-extractable OC than BS500. Partial least-squares path modeling (PLSPM) showed that the path efficients of biochar’s chemical recalcitrance and microbial qCO2 were 0.52 and 0.25, respectively, and that of the soil microbial activity is neglected. We conclude from this short-term study that chemical recalcitrance imposed a greater effect than soil microbial community on biochar stability.

Soil dependence of biochar composts in mitigating greenhouse gas emissions: An overlooked biophysical mechanism

Biochar-manure co-compost (BM) has garnered considerable attention as a promising soil amendment for improving nutrient retention and mitigating the emission of greenhouse gases (GHGs). However, its efficacy often varies largely from one soil to another. By comparing soil CO2 and N2O effluxes, solution chemistry, enzyme activities, and the abundances of N-cycle genes between BM and manure compost (M) across three soils of different texture classes through microcosm incubations, we aimed to develop biophysical mechanics to untangle the soil-dependent efficacy of BM in mediating soil carbon and nitrogen transformation processes. Compared to M addition, BM addition significantly reduced soil CO2 and N2O emissions, but its effectiveness was soil texture-dependent, being strongest for CO2 and N2O in fine-textured clay loam and coarse-textured sand, respectively. Such soil texture-dependent effects of BM versus M were also observed in soil enzyme activities and gene copy numbers of ammonia-oxidizing bacteria and denitrifiers. Our datasets suggest that BM interacted strongly with soil texture to modulate the pore scale-based diffusion of oxygen, thereby creating divergence in the aeration status among soils. Sequentially, soil phenol oxidase activity was greater in BM than in M under more aerobic conditions but no difference between BM and M in oxygen-limited soils. The balance between oxygen depletion due to microbial respiration of organic amendments and oxygen diffusion through soil/biochar pores also shaped the activities of nitrifying and denitrifying prokaryotes differently between coarse- and fine-textured soils. This logic model well-explained the magnitude of change in soil-dependent CO2 and N2O emissions from organic amendments. The knowledge gained in this work will likely have profound practical ramifications for optimizing BM efficacy in mitigating the GHGs emission.

Dietary supplementation of micelle silymarin enhances the antioxidant status, innate immunity, growth performance, resistance against Vibrio parahaemolyticus infection, and gut morphology in Pacific white shrimp (Litopenaeus vannamei)

Silymarin, a flavonoid compound derived from the milk thistle plant (Silybum marianum) is well-known for its hepatoprotective, antioxidant, immunomodulatory, and anti-inflammatory properties. Therefore, this study sought to evaluate the effect of silymarin and micelle silymarin (MS) supplementation on Pacific white shrimp (Litopenaeus vannamei). The experimental diets were formulated to include MS at levels of 0.25, 0.5, 1.0, 2.0, and 4.0 g/kg or silymarin at levels of 1.0 and 2.0 g/kg in the basal diet (Con, M0.25, M0.5, M1, M2, M4, S1 and S2, respectively). Juvenile shrimp (0.98 g) were fed with experimental diets (four replicates) for 42 days. The growth metrics were significantly (P < 0.05) increased in M0.25, M0.5, and M1 groups compared to the Con group. Feed conversion ratio decreased (P < 0.05) in M0.5, M1, and M2 groups, whereas the protein efficiency ratio increased (P < 0.05) in M0.5 and M1 groups compared to the Con group. Phenoloxidase, lysozyme, glutathione peroxidase, and superoxide dismutase activities were enhanced (P < 0.05), with the highest levels observed in the M0.5 group. Malondialdehyde level decreased (P < 0.05) in M1 and M2 groups compared to the Con group. Total protein increased (P < 0.05) in M0.25, M0.5 and S1 groups compared to the Con group. AST levels were significantly reduced (P < 0.05) in shrimp fed M0.5, M1, M2, M4, and S2 groups, and ALT levels decreased (P < 0.05) in M1 and S2 groups compared to Con group. Wall thickness was significantly higher (P < 0.05) in the M0.5 group, and villi length increased (P < 0.05) in M0.25, M0.5, and S1 groups. The resistance against Vibrio parahaemolyticus increased; however, no significant differences in cumulative survival were observed (P ˃ 0.05). Significantly higher (P < 0.05) proPO levels were observed in M0.5, M1, M2, M4, and S2 groups, whereas higher (P < 0.05) TLR-3 levels were observed in M1 and M2 groups compared to the Con group. TNF-α expression decreased (P < 0.05) in all other dietary treatments compared to the Con group. The findings suggest that dietary silymarin and MS can enhance growth, immune response, antioxidant capacity, gut morphology, and immune gene expression, while reducing lipid peroxidation and inflammation in Pacific white shrimp. The shrimp performance shows no significant difference between silymarin and MS groups at 1% and 2% levels. The optimal suggested inclusion level of MS in shrimp feed is 0.5–1.0 g/kg.

Влияние ингибиторов фенолоксидаз на эффективность деколоризации малахитового зеленого бактериями рода Azo spirillum

Synthetic dyes are widely used in various branches of light industry. Due to the insufficient efficiency of industrial painting processes, a large percentage of dyes end up in the wastewater of enterprises in an unmodified form, which creates a huge risk of environmental pollution with these compounds. Triphenylmethane dyes, in particular malachite green, are toxic, allergenic and carcinogenic compounds. To date, biodegradability of triphenylmethane dyes has been shown for some bacteria and fungi producing phenol oxidase complex enzymes, including soil associative bacteria of the genus Azospirillum. Many factors are capable of inducing and inhibiting the biodegradation efficiency, in particular the enzymatic systems that are involved in bleaching processes. In the present work we studied the effects of typical deactivating agents of phenol oxidases, such as H2O2, EDTA, SDS-Na, β-mercaptoethanol, dithiothreitol, Tween, and sodium azide, on the azospirilla’s phenol oxidases activity and the ability to decolorize malachite green. It was found that Tween and sodium azide do not have an inhibitory effect on azospirillum enzymes and exhibit a total stabilizing effect on the entire complex. An inhibitory effect from 60 to 100% was noted for laccase and Mn-peroxidase activity under the action of β-mercaptoethanol, dithiothreitol and EDTA, which is directly proportional to the decolorization rate of malachite green. Compared with the latest issues on classical phenol-oxidizing enzymes, our data revealed non-typical properties of the phenol oxidase complex enzymes of azospirillum.

Construction of suspended bagasse bioflocs and evaluation of their effectiveness in shrimp (Penaeus vannamei) aquaculture systems

This experiment was carried out to improve the operability of bioflocs technology and to better the application of bagasse, a sugarcane by-product or fibrous pulp of other plant materials, in aquaculture. Firstly, a generator device for suspended-bagasse was designed, and indigenous efficient nitrogen-reducing bacteria were screened and combined. Then a suspended-bagasse bioflocs Penaeus vannamei (initial body weight of 0.78 ± 0.33 g, density of 350 shrimp/m2) culture system was constructed with bagasse and indigenous probiotics (single probiotic or composite probiotic from the previous step) as components through the generator device. The commercial shrimp culture model with commercial probiotics applied was used as a control group with three replicates in each group. The experiment lasted for 50 days. The effects of suspended-bagasse bioflocs on the water quality, shrimp growth, and immune response in the shrimp culture system were evaluated. All experimental results were analyzed using one-way variance or linear estimation. The suspended-bagasse generator could promote rapid biofilm formation on the surface of the bagasse, and the hydraulic retention parameter can be set to 12 h. Besides, three efficient nitrogen-reducing strains were screened and identified (Halomonas alkalihila AC3–2, Rugerella sp BD15–5, and Halomonas venusta BD15–8). They were non-hemolytic, sensitive to most antibiotics, and non-pathogenic to shrimp. Their combination with Bacillus megaterium BZ5 (a previously isolated probiotic) in a 1:1:1:1 ratio resulted in the highest degradation efficiencies of 97.1% and 51.4% for ammonia and nitrite nitrogen, respectively. Furthermore, application experiments in shrimp aquaculture systems showed that suspended-bagasse flocs (with single bacteria and combined bacteria) reduced ammonia and nitrite nitrogen levels and promoted nitrification in the water of the shrimp aquaculture system. This suspended bagasse floc (with combined bacteria) system reduced water usage by 37.6% per kg of shrimp produced compared to the common culture system. Moreover, the suspended-bagasse flocs (with combined bacteria) improved hepatopancreatic digestive enzyme (trypsin, lipase, and α-amylase) and serum immune-related enzyme (lysozyme and phenol-oxidase) activity. This study indicates that it is feasible to cultivate suspended-bagasse flocs in shrimp aquaculture systems using a generator device with bagasse and probiotic bacteria as components. The flocs based on bagasse and indigenous complex bacteria can effectively reduce water exchange during shrimp aquaculture and improve shrimp production. Imperfectly, the microbial community and function of the biofilm on the surface of bagasse, which is very important for the interpretation of water quality variations, was not revealed in this experiment and needs to be refined in future work. Additionally, it is also worthwhile to evaluate the effectiveness of this device in other aquatic animal culture systems.

An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli, Candida albicans, Sporothrix schenckii, Sporothrix globosa, and Sporothrix brasiliensis. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.

Dietary Supplementation with Hydrolyzable Tannin Improves Nonspecific Immune Responses, Intestinal Morphology, and Disease Resistance against Vibrio alginolyticus in Whiteleg Shrimp

This research aimed to evaluate the impact of hydrolyzable tannin on immune responses, intestinal morphology, and resistance to Vibrio alginolyticus in whiteleg shrimp (Penaeus vannamei). In preliminary in vitro experiments, shrimp hemocytes were exposed to different concentrations of hydrolyzable tannin. The findings indicated that hydrolyzable tannin exhibited nontoxic characteristics and could stimulate respiratory burst activity. For the subsequent in vivo trial, shrimp (11.83 ± 0.98 g) were subjected to a 28-day oral administration of diets containing diverse hydrolyzable tannin concentrations (0, 1, 2, 4, and 8 g/kg denoted as control, T1, T2, T4, and T8, respectively). Immune parameters, antimicrobial peptide genes, and intestinal morphology were measured. Significant enhancements were observed in total hemocyte count (THC), phagocytic activity, respiratory burst activity, and phenoloxidase activity (PO) across all treatment groups compared to the control group, particularly in T2 and T4 groups (p&lt;0.05). The gene expressions of Penaeidin-2, Penaeidin-3, Penaeidin-4, crustin, and lysozyme were markedly higher in the treatment groups, especially in T2 (p&lt;0.05). Additionally, hydrolyzable tannin contributed to increased intestinal wall thickness after the 28-day feeding trial (p &lt;0.05). In a separate challenge experiment, shrimps (11.36 ± 0.66 g) were exposed to V. alginolyticus after 7 days of culture. Notably, T2 and T4 exhibited enhanced resistance, resulting in survival rates of 66.67% and 69.70%, respectively. To conclude, administering 4 g of hydrolyzable tannin per kilogram of feed proved effective in enhancing nonspecific immune responses, improving intestinal morphology, and augmenting resistance to V. alginolyticus in whiteleg shrimp.

A C-type lectin (CTL2) mediated both humoral and cellular immunity against bacterial infection in Tribolium castaneum

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.