Phenoloxidase Activity Research Articles

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<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<0.05). Additionally, hydrolyzable tannin contributed to increased intestinal wall thickness after the 28-day feeding trial (p <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.

Synergistic effects of combined probiotics Bacillus pumilis D5 and Leuconostoc mesenteroide B4 on immune enhancement and disease resistance in Litopenaeus vannamei

This study investigated the effects of two distinct probiotics, Leuconostoc mesenteroides B4 (B4) and Bacillus pumilus D5 (D5), along with their combination, on the diet of white shrimp (Litopenaeus vannamei) during an eight-week feeding trial. The diets tested included B4 + dextran at 107 CFU/g feed (the B4 group), D5 alone at 107 CFU/g feed (the D5 group), and a combination of B4 + dextran and D5 at 5 × 106 CFU/g feed each (the B4+dextran + D5 group). Relative to the control group, those administered probiotics exhibited moderate enhancements in growth. By the eighth week, the weight gain for the B4, D5, and B4+D5 groups was 696.50 ± 78.15%, 718.53 ± 130.73%, and 693.05 ± 93.79%, respectively, outperforming the control group's 691.66 ± 31.10% gain. The feed conversion ratio was most efficient in the B4 group (2.16 ± 0.06), closely followed by B4+D5 (2.21 ± 0.03) and D5 (2.22 ± 0.06), with the control group having the highest ratio (2.27 ± 0.03). While phenoloxidase activity was somewhat elevated in the B4 and D5 groups, no significant differences were noted in respiratory burst activity or total hemocyte count across all groups. Challenge tests at weeks 4 and 8 showed that the B4 + D5 combination offered superior protection against AHPND-causing Vibrio parahaemolyticus. The 4-week cumulative survival rate was highest in shrimp treated with B4 + dextran + D5 (56.25%), followed by B4 + dextran (31.25%), control (18.75%), and lowest in D5 (12.5%). By week 8, the B4 + dextran + D5 (43.75%) and B4 + dextran (37.5%) groups significantly outperformed the control group (6.25%, p < 0.05), with no significant difference observed between the D5 group (37.5%) and the control group at day 56. Analysis of the shrimp's foregut microbiota revealed an increase in unique OTUs in the B4 and B4 + D5 groups. Compared to the control, Proteobacteria abundance was reduced in all probiotic groups. Potential pathogens like Vibrio, Bacteroides, Neisseria, Botrytis, Clostridioides, and Deltaentomopoxvirus were detected in the control but were reduced or absent in probiotic groups. Beneficial microbes such as Methanobrevibacter and Dictyostelium in the B4+D5 group, and Sugiyamaella in the B4 group, showed significant increases.Probiotics also led to higher transcript levels of nitric oxide synthase in the hemocytes, and lysozyme and transglutaminase in the midgut, along with lysozyme and α2-macroglobulin in the foregut. Notably, the combined B4 + D5 probiotics synergistically enhanced the expression of superoxide dismutase and prophenoloxidase in the foregut, indicating an improved immune response. In summary, this study demonstrates that the probiotics evaluated, especially when used in combination, significantly boost the expression of specific immune-related genes, enhance the bacterial diversity and richness of the intestine, and thus prevent the colonization and proliferation of Vibrio spp. in L. vannamei.

Shrinkage and desiccation: evaluating the streambed bacterial responses to intermittent water deficit

Hydrological drought, characterized by a significant deficiency in rainfall events, promotes natural desiccation processes. The reduction in water recharge and the loss of freshwater ecosystems pose urgent challenges in the face of increasing global population and the escalating demand for water resources. The variability in the duration of no-flow periods can have profound implications for ecosystem functioning, specifically impacting the microbiota residing in streambed sediments and the vital processes they perform. The streambed serves as an ecotone where microorganisms form biofilms, playing critical roles in in-stream biogeochemical cycles and greenhouse gas emissions. Consequently, prolonged desiccation periods and subsequent rewetting episodes can disrupt, limit, or alter the functions and structure of microbial communities, thereby compromising the overall functioning of aquatic ecosystems. Understanding how streambed microbial communities respond to prolonged desiccation events is crucial. This revision manuscript provides a synthesis of recent empirical and experimental studies that have explored various aspects of streambed microbial communities in the context of diverse drying-rewetting periods. A special focus is placed on highlighting key microbial community structural and functional responses that could be used as endpoints of responses to desiccation. In particular, we showed the greater expression of the phenol-oxidase activity among the intermittent streambeds submitted to long-term drought. This result suggested a potential begin of transition from freshwater to terrestrial systems. Additionally, we observed a tendency of decreasing bacterial diversity in the dry conditions together with a change in the relative abundance of certain microbial taxa and a general shift from Gram-negative to Gram-positive bacteria. All of these evidences strengthened the similarity between the dry streambed systems studied and a (dry)-soil environment, suggesting that prolonged and unusual dry periods could boost the terrestrial transition of the aquatic intermittent ecosystem. By summarizing these findings, we aim to enhance our understanding of the responses exhibited by streambed microbiota in the face of desiccation events. The synthesized results may further provide potential diagnosis and/or management tools for intermittent freshwater ecosystems functioning.

Role of serpin-25 in prophenoloxidase activation and expression of antimicrobial peptide genes in the silkworm Bombyx mori

Serine protease inhibitors (Serpins) are involved in diverse physiological and developmental processes, constituting one of the largest superfamilies of protease inhibitors. They are widely distributed across various organisms such as plants, animals, prokaryotes, and viruses. In insects, serpins exert their inhibitory effects on serine proteinase cascades, thereby regulating important pathways such as the Toll pathway and prophenoloxidase (proPO) in hemolymph. In this study, we cloned and analyzed the serpin-25 gene in Bombyx mori (Bmserpin-25). Bmserpin-25 mRNA was detected in all examined tissues, with high expression levels observed in the fat body, midgut, and epidermis. Upon injection of B. mori with four different microorganisms, including Gram-positive microbes (Micrococcus luteus), Gram-negative microbes (E. coli), fungi (Beauveria bassiana), and the B. mori nuclear polyhedrosis virus (BmNPV), the significant changes in Bmserpin-25 expression were observed specifically in response to E. coli and BmNPV treatments. Furthermore, the recombinant Bmserpin-25 was successfully expressed in Escherichia coli and purified for subsequent functional analysis. In vitro experiments showed that the recombinant Bmserpin-25 protein inhibited proPO activation but did not affect phenoloxidase activity. Additionally, injection of recombinant Bmserpin-25 protein into B. mori larvae resulted in a significant downregulation of antimicrobial peptide gene expression in the fat body. These findings emphasize the crucial role of serpin-25 in prophenoloxidase activation and the regulation of antimicrobial peptide gene expression in B. mori.

The biotic effects of lignite on humic acid components conversion during chicken manure composting

Targeted regulation of composting to convert organic matter into humic acid (HA) holds significant importance in compost quality. Owing to its low carbon content, chicken manure compost often requires carbon supplements to promote the humification progress. The addition of lignite can increase HA content through biotic pathways, however, its structure was not explored. The Parallel factor analysis revealed that lignite can significantly increase the complexity of highly humified components. The lignite addition improved phenol oxidase activity, particularly laccase, during the thermophilic and cooling phases. The abundance and transformation functions of core bacteria also indicated that lignite addition can influence the activity of microbial transformation of HA components. The structural equation model further confirmed that lignite addition had a direct and indirect impact on enhancing the complexity of HA components through core bacteria and phenol oxidase. Therefore, lignite addition can improve HA structure complexity during composting through biotic pathways.

Effect of biochar on soil microbial community, dissipation and uptake of chlorpyrifos and atrazine

For the application of biochar in restoring pesticide-contaminated soils and minimizing the risk associated with their uptake in plants, it is crucial to understand the biochar impact on soil biological activities and dissipation and accumulation of pesticides in plant and soil systems. In this study, the effect of Mentha-distilled waste-derived biochar was investigated on chlorpyrifos and atrazine contaminated sandy loam soil. The four application rates of atrazine (2, 4, 6, and 8 mg kg−1) and chlorpyrifos (2, 4, 6, and 12 mg kg−1) and a single application rate of biochar (4%) were used in this study. The degradation of pesticides, the diversity of the bacterial community, and enzymatic activities (alkaline phosphatase, dehydrogenase, arylsulfatase, phenol oxidase, urease activity and N-acetyl glucosaminidase) were examined in soil. The uptake of two pesticides and their effect on growth and stress parameters were also investigated in plants (A. paniculata). The dissipation of chlorpyrifos and atrazine followed simple first-order kinetics with a half-life of 6.6–74.6 and 21–145 days, respectively. The presence of deisopropyl atrazine desethyl atrazine (metabolites of atrazine) and 3,5,6-trichloro-2-pyridinol (a metabolite of chlorpyrifos) was observed in soil and plant tissues. Biochar application significantly (p = 0.001) enhanced the degradation rate of chlorpyrifos and atrazine leading to the lower half-life of chlorpyrifos and atrazine in soil. A significant reduction (p = 0.001) in the uptake of chlorpyrifos and atrazine and alteration in their binding affinity and uptake rate in plant tissues was observed in biochar treatments. The incorporation of biochar improved chlorpyrifos/atrazine degrader and plant growth-promoting bacterial genera such as Balneimonas, Kaistobacter, Rubrobacter, Ammoniphilus, and Bacillus. The upregulation of functional genes associated with nucleotide, energy, carbohydrate, amino acid metabolism, xenobiotic biodegradation, and metabolism: atrazine degradation was observed in biochar treatments. The biochar amendments significantly (p = 0.001) reduced the plant’s uptake velocity (Vmax) and affinity (Km) of chlorpyrifos and atrazine. These results delineated that Mentha-distilled waste-derived biochar can potentially remediate chlorpyrifos and atrazine contaminated soils and ensure the safety of plants for consumption.Graphical

Combined hemicellulolytic and phenoloxidase activities of Thermobacillus xylanilyticus enable growth on lignin-rich substrates and the release of phenolic molecules

Major challenge in biorefineries is the use of all lignocellulosic components, particularly lignins. In this study, Thermobacillus xylanilyliticus grew on kraft lignin, steam-exploded and native wheat straws produced different sets of phenoloxidases and xylanases, according to the substrate. After growth, limited lignin structural modifications, mainly accompanied by a decrease in phenolic acids was observed by Nuclear Magnetic Resonance spectroscopy. The depletion of p-coumaric acid, vanillin and p-hydroxybenzaldehyde combined to vanillin production in the culture media indicated that the bacterium can transform some phenolic compounds. Proteomic approaches allowed the identification of 29 to 33 different hemicellulases according to the substrates. Twenty oxidoreductases were differentially expressed between kraft lignin and steam-exploded wheat straw. These oxidoreductases may be involved in lignin and aromatic compound utilization and detoxification. This study highlights the potential value of Thermobacillus xylanilyticus and its enzymes in the simultaneous valorization of hemicellulose and phenolic compounds from lignocelluloses.

Comparative immunotoxicity of bees, apis mellifera (hymenoptera: apidae), exposed to natural and synthetic xenobiotics

The objective of this study was to compare the effects of natural and synthetic chemical pesticides, thereby examining the supposed selectivity of these natural compounds on Apis mellifera bees. The LC50 values used in the bioassays were obtained from the research by Souza et al. (2023) and are as follows: Karate® (13.4 µL/100 mL), Limonene compound (1,440 µL/100 mL), and Roundup® (712,290 µL/100 mL). However, as reported by these authors, a concentration of 250 µL/100 ml was used for Azamax®. These LC50 values, along with a concentration of 250 µL/100 mL of Azamax®, were employed in immunohistochemical analyzes using the TUNEL method and PCNA in the midgut of bees. Immunological assessments (nitric oxide, phenoloxidase, and oxidative stress markers TBARS and GSH) were also conducted on adult worker bees. The xenobiotic treatments did not reveal apoptosis or cell proliferation. Nonetheless, we observed epithelial degeneration, marked by the presence of vacuolated cells, suggesting a necrotic process. Except for the Limonene compound, all substances induced oxidative stress, leading to increased levels of TBARS. Although there were no differences in GSH levels, we observed alterations in the immune system of these insects, characterized by increased phenoloxidase activity and NO2 levels. Based on the acquired results, it is possible to conclude that caution should be exercised when using chemical pesticides in agriculture, whether they are of synthetic or natural origin, as they have the potential to cause irreversible histopathological and immunological damage. This study also underscores the importance of conducting more comprehensive investigations into the impact of natural products on the physiology of pollinator insects.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans.

Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Dietary N-carbamylglutamate (NCG) supplementation improves the survival, digestive and immunological parameters by promoting ammonia excretion in Chinese mitten crab (Eriocheir sinensis), under ammonia nitrogen stress

Ammonia nitrogen is one of the most common environmental stress factors in aquaculture. The aim of this experiment was to evaluate the effects of ammonia nitrogen stress on ammonia metabolism, digestion and immune function as well as the protective effects of dietary N-carbamylglutamate (NCG) in the Chinese mitten crab (Eriocheir sinensis). Two feeds were used, namely a control feed (regular commercial feed) and an NCG feed (NCG (302.96 ± 4.07) mg/kg added). Subsequently, animals fed by these two feeds were exposed to 30 mg/L or 60 mg/L ammonia nitrogen stress (named A30 or A60) for 21 days, respectively. The rate of hemolymph ammonia excretion was significantly increased in NCG group compared to C (control) group (P < 0.05). Hemolymph ammonia levels were significantly lower in NCG A30 (NCG feed and A30) and NCG A60 (NCG feed and A60) groups compared to A30 (control feed and A30) and A60 (control feed and A60) groups (P < 0.05). Glutamate dehydrogenase and glutamine synthetase activities in the gills and hepatopancreas of NCG A60 group were significantly increased compared with the A60 group, which promoted glutamine synthesis (P < 0.05). The cumulative mortality rate of Eriocheir sinensis in NCG A30 and NCG A60 groups were effectively reduced (P < 0.05). The arginine levels of hepatopancreas were significantly increased in NCG A60 group (P < 0.05). Under ammonia nitrogen stress, NCG significantly increased the activities of trypsin, pepsin, lipase and α-Amylase in NCG feed group, and promoted feeding (P < 0.05). In hemolymph, hepatopancreas and intestine, NCG significantly increased nitric oxide synthase activity, lysozyme and phenol oxidase activities compared to the ammonia nitrogen stress group (P < 0.05). The results indicated that, NCG added in feed could decelerate the toxic effects of ammonia on Eriocheir sinensis by promoting ammonia excretion and accelerating glutamine and endogenous arginine synthesis by metabolism. Moreover, the activity of digestive enzymes and immune-related enzymes is improved.

The identification of a serpin with immune defense role in oriental river prawn Macrobrachium nipponense

Serpins are a protein superfamily of serine protease inhibitors. One of their functions is to participate in immune responses by inhibiting the activation of prophenoloxidase. To elucidate the immune role of serpin in Macrobrachium nipponense, a serpin gene (Mnserpin) was cloned from M. nipponense in this study. Mnserpin protein has an N-terminal signal peptide and a serpin domain that contains a hinge region, a signature sequence of serpin and a P1(arginine)-P1’ scissile bond, and evolutionally closely related to the crustacean serpins. Mnserpin highly expressed in the hepatopancreas and gill. Mnserpin expression increased first and then decreased after Vibrio parahaemolyticus and Aeromonas hydrophila infection, and was knocked down by dsMnserpin injection with a maximum knockdown efficiency of 92 %. Mnserpin knockdown increased the expression of the clip domain serine protease and prophenoloxidase genes and phenoloxidase activity of M. nipponense as well as its mortality rate after V. parahaemolyticus and A. hydrophila infection. The recombinant Mnserpin (rMnserpin) showed bacteria-binding and bacteriostatic activity in vitro. Moreover, rMnserpin injection decreased the bacterial number and the mortality rate of M. nipponense post V. parahaemolyticus and A. hydrophila infection. These results suggested that Mnserpin plays a major role in the innate immune response of M. nipponense.

Investigation of how gate residues in the main channel affect the catalytic activity of Scytalidium thermophilum catalase.

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.

MicroRNA Targets PAP1 to Mediate Melanization in Plutella xylostella (Linnaeus) Infected by Metarhizium anisopliae.

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the molecular biology of this pest, our knowledge regarding the role of miRNAs in regulating key immunity-related genes remains limited. In this study, we leveraged whole transcriptome resequencing data from Plutella xylostella infected with Metarhizium anisopliae to identify specific miRNAs targeting the prophenoloxidase-activating protease1 (PAP1) gene and regulate phenoloxidase (PO) cascade during melanization. Seven miRNAs (pxy-miR-375-5p, pxy-miR-4448-3p, pxy-miR-279a-3p, pxy-miR-3286-3p, pxy-miR-965-5p, pxy-miR-8799-3p, and pxy-miR-14b-5p) were screened. Luciferase reporter assays confirmed that pxy-miR-279a-3p binds to the open reading frame (ORF) and pxy-miR-965-5p to the 3' untranslated region (3' UTR) of PAP1. Our experiments demonstrated that a pxy-miR-965-5p mimic significantly reduced PAP1 expression in P. xylostella larvae, suppressed PO activity, and increased larval mortality rate. Conversely, the injection of pxy-miR-965-5p inhibitor could increase PAP1 expression and PO activity while decreasing larval mortality rate. Furthermore, we identified four LncRNAs (MSTRG.32910.1, MSTRG.7100.1, MSTRG.6802.1, and MSTRG.22113.1) that potentially interact with pxy-miR-965-5p. Interference assays using antisense oligonucleotides (ASOs) revealed that silencing MSTRG.7100.1 and MSTRG.22113.1 increased the expression of pxy-miR-965-5p. These findings shed light on the potential role of pxy-miR-965-5p in the immune response of P. xylostella to M. anisopliae infection and provide a theoretical basis for biological control strategies targeting the immune system of this pest.

Growth and longevity modulation through larval environment mediate immunosenescence and immune strategy of Tenebrio molitor

BackgroundThe Disposable Soma Theory of aging suggests a trade-off between energy allocation for growth, reproduction and somatic maintenance, including immunity. While trade-offs between reproduction and immunity are well documented, those involving growth remain under-explored. Rapid growth might deplete resources, reducing investment in maintenance, potentially leading to earlier or faster senescence and a shorter lifespan. However, rapid growth could limit exposure to parasitism before reaching adulthood, decreasing immunity needs. The insect immunity’s components (cellular, enzymatic, and antibacterial) vary in cost, effectiveness, and duration. Despite overall immunity decline (immunosenescence), its components seem to age differently. We hypothesize that investment in these immune components is adjusted based on the resource cost of growth, longevity, and the associated risk of parasitism.ResultsWe tested this hypothesis using the mealworm beetle, Tenebrio molitor as our experimental subject. By manipulating the larval environment, including three different temperatures and three relative humidity levels, we achieved a wide range of growth durations and longevities. Our main focus was on the relationship between growth duration, longevity, and specific immune components: hemocyte count, phenoloxidase activity, and antibacterial activity. We measured these immune parameters both before and after exposing the individuals to a standard bacterial immune challenge, enabling us to assess immune responses. These measurements were taken in both young and older adult beetles. Upon altering growth duration and longevity by modifying larval temperature, we observed a more pronounced investment in cellular and antibacterial defenses among individuals with slow growth and extended lifespans. Intriguingly, slower-growing and long-lived beetles exhibited reduced enzymatic activity. Similar results were found when manipulating larval growth duration and adult longevity through variations in relative humidity, with a particular focus on antibacterial activity.ConclusionThe impact of growth manipulation on immune senescence varies by the specific immune parameter under consideration. Yet, in slow-growing T. molitor, a clear decline in cellular and antibacterial immune responses with age was observed. This decline can be linked to their initially stronger immune response in early life. Furthermore, our study suggests an immune strategy favoring enhanced antibacterial activity among slow-growing and long-lived T. molitor individuals.

Higher temperature accelerates the aging-dependent weakening of the melanization immune response in mosquitoes.

The body temperature of mosquitoes, like most insects, is dictated by the environmental temperature. Climate change is increasing the body temperature of insects and thereby altering physiological processes such as immune proficiency. Aging also alters insect physiology, resulting in the weakening of the immune system in a process called senescence. Although both temperature and aging independently affect the immune system, it is unknown whether temperature alters the rate of immune senescence. Here, we evaluated the independent and combined effects of temperature (27°C, 30°C and 32°C) and aging (1, 5, 10 and 15 days old) on the melanization immune response of the adult female mosquito, Anopheles gambiae. Using a spectrophotometric assay that measures phenoloxidase activity (a rate limiting enzyme) in hemolymph, and therefore, the melanization potential of the mosquito, we discovered that the strength of melanization decreases with higher temperature, aging, and infection. Moreover, when the temperature is higher, the aging-dependent decline in melanization begins at a younger age. Using an optical assay that measures melanin deposition on the abdominal wall and in the periostial regions of the heart, we found that melanin is deposited after infection, that this deposition decreases with aging, and that this aging-dependent decline is accelerated by higher temperature. This study demonstrates that higher temperature accelerates immune senescence in mosquitoes, with higher temperature uncoupling physiological age from chronological age. These findings highlight the importance of investigating the consequences of climate change on how disease transmission by mosquitoes is affected by aging.

Dietary effect of botanical blend (Phyto AquaNity™) on growth, immunity and survival of Pacific White shrimps challenged against WSSV

The present study was performed to assess the effect of a commercial product Phyto AquaNity™ (Artepillin C and curcuminoids) supplementation on growth performance, immune parameters, and disease resistance of whiteleg shrimps (Litopenaeus vannamei) against White Spot Syndrome Virus (WSSV) infection. Shrimps of initial weight of 0.9–1 g were fed Phyto AquaNity™ at three dosages (0.0, 0.5 and 1.0 g kg−1) over 42 days. After the feeding period, growth performance parameters were significantly improved in the group of 1.0 g kg−1. The highest specific growth rate and feed efficiency were observed in the group 1.0 g kg−1 (P < 0.05). At the end of the feeding period, all groups were challenged against WSSV by recording daily mortalities over 14 days and measuring immune parameters. Shrimps fed with Phyto AquaNity™ (1.0 g kg−1) showed a significant reduction of mortalities with the lowest rate compared to other groups (0.5 and 0.0 g kg−1). Moreover, this dosage enhanced all immunological parameters in challenging conditions such as total hemocyte counts, granular cell, hyaline cell, phenoloxidase activity and phagocytic activity (p > 0.05). Analysis of lysozyme gene expression was improved in groups fed with Phyto AquaNity™ and the lowest value was observed for the positive group (challenged & without Phyto AquaNity™ supplement). Overall, 1.0 g kg−1 of Phyto AquaNity™ showed better results in improving growth performance, immune responses, and survival in shrimps challenged with WSSV.