Phenoloxidase Research Articles

Dietary β-Glucan Alleviates Antibiotic-Associated Side Effects by Increasing the Levels of Antioxidant Enzyme Activities and Modifying Intestinal Microbiota in Pacific White Shrimp (Litopenaeus vannamei).

Antibiotics and their secondary metabolites are commonly found in aquatic ecosystems, leading to the passive exposure of many aquatic animals to low doses of antibiotics, which can affect their health. However, there is limited information available on how to mitigate the side effects of antibiotics on normal aquatic animals. This study aimed to investigate the potential of dietary β-glucan to alleviate the side effects induced by antibiotics in Pacific white shrimp (Litopenaeus vannamei) (0.37 ± 0.02 g). A six-week feeding trial was conducted with four dietary treatments including a control, 1 g/kg β-glucan (β-glucan), 50 mg/kg oxytetracycline (OTC), and a combination of 50 mg/kg OTC and 1 g/kg β-glucan (Mix) groups. At the end of the trial, the growth performance, intestinal microbial composition, antioxidant capacity, and immune response of the shrimp were assessed. There were no significant differences in growth performance among the groups, but the condition factor of the shrimp in the Mix group was significantly decreased when compared to the control and β-glucan groups. The activities of hepatopancreas catalase (CAT) and serum phenol oxidase in the OTC group were significantly lower than those in the control group. On the other hand, the activities of hepatopancreas superoxide dismutase and CAT enzymes in the β-glucan group were significantly higher than those in the OTC group. The supplementation of β-glucan in combination with antibiotics significantly increased the CAT activity and bacteriolytic activity compared to the OTC and control groups, respectively. Moreover, an analysis of the intestinal microbiota revealed that the Observed_species estimator in the Mix group was significantly higher than that in the control group. Dietary antibiotics significantly increased the abundance of Actinobacteria at the phylum level, but the Mix group showed no significant difference. The supplementation of β-glucan in combination with antibiotics also significantly increased the relative abundance of Meridianimaribacter compared to the control group. Additionally, the synergistic influence of β-glucan with antibiotics increased the beta diversity of intestinal microbiotas. These findings suggest that the supplementation of β-glucan in combination with antibiotics on Pacific white shrimp can alleviate the low antioxidant capacity and immune response caused by antibiotics while enhancing the intestinal microbial composition. This provides a potential solution to mitigate the negative impacts of antibiotics in aquaculture.

An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila.

Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in Drosophila melanogaster in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in Drosophila by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.

The immune response of the whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) when parasitized by Eretmocerus eremicus (Hymenoptera: Aphelinidae).

In insects, the innate immune system is subdivided into cellular and humoral defenses. When parasitoids attack insects, both reactions can be activated and notably, the phenoloxidase (PO) cascade and lytic activity are part of both cellular and humoral defenses. However, to our knowledge, no study has characterized any immune response of the whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) to the attack of Eretmocerus eremicus (Hymenoptera: Aphelinidae). Therefore, the first objective of the present study was to determine whether whitefly nymphs recently parasitized by E. eremicus exhibit any immune response. For this, we estimate the level of prophenoloxidase (proPO), phenoloxidase (PO), and lytic activity by colorimetric assays. A second objective was to assess whether the observed whitefly immune response could be related to a previously reported preference of the predator Geocoris punctipes (Hemiptera: Lygaeidae) for non-parasitized nymphs. We therefore offered non-parasitized and recently parasitized nymphs to the predator. Our results show that parasitism of whitefly nymphs by E. eremicus induced a highly estimated level of proPO and PO, and a lower level of lytic activity. In addition, we found that G. punctipes did not show a preference for non-parasitized over recently parasitized nymphs. The nymphs of T. vaporariorum activated the PO pathway against E. eremicus; however, the increase in proPO and PO levels was traded-off with decreased lytic activity. In addition, the previously reported preference for non-parasitized nymphs was not seen in our experiments, indicating that the induced immune response did not affect predator behavior by G. punctipes.

Metarhabditis amsactae: A potential biopesticide isolated from Punjab (India) with potent insecticidal activity and immunomodulatory effects against Galleria mellonella (Lepidoptera: Pyralidae)

A survey was undertaken to isolate entomopathogenic nematodes from Amritsar district of Punjab, India. Out of 20 soil samples collected, two were found positive for the presence of nematodes. 18S and ITS rDNA gene sequencing revealed their identity as Metarhabditis amsactae. To assess its biocontrol potential, Galleria mellonella larvae were treated with concentrations of 20, 40, 80 and 160 IJs/L (infective juveniles/larva) and mortality was recorded from 24 h up to 96 h of nematode exposure. Distilled water without nematodes was used as an untreated control. M. amsactae showed potent larvicidal activity against G. mellonella that was found to be concentration and time dependent. Nematode infection caused 93.33 % larval mortality at 80 IJs/L after 72 h of treatment. 100 % mortality was observed after 96 h. No mortality was observed in control. To evaluate the immunomodulatory effects of M. amsactae, G. mellonella larvae were infected with 100 IJs/L and activities of antioxidant and detoxifying enzymes viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), phenol oxidase (PO), glutathione-S-transferase (GST) and acetylcholine esterase (AChE) were appraised after 12, 24, 36 and 48 h of nematode exposure. Malondialdehyde content was also determined. The results obtained demonstrated a significant elevation in all the enzyme activities at all time intervals in treated larvae when compared with untreated control. MDA levels were also enhanced in response to nematode infection. Thus, the present study revealed high insecticidal potential and immunomodulatory effects of M. amsactae on G. mellonella that should be further explored on other insect pests as well.

Effects of geniposide on innate immunity and antiviral activity of Scyllaparamamosain

In this study, we examined the impact of geniposide on the innate immunity of the mud crab Scylla paramamosain, specifically in relation to WSSV infection. Through the use of in vitro cell culture experiments, we assessed the effects of geniposide on various parameters of hemocyte activity in S. paramamosain. Our findings revealed that high doses of geniposide inhibited hemocyte growth, with an optimal dose of 100 mg/kg determined. Additionally, we observed that geniposide increased the total hemocyte counts in S. paramamosain following WSSV infection. Geniposide also enhanced the enzymatic activities in hemolymph following treatment. The enzymes affected by geniposide encompassed ACP (acid phosphatase), POD (phenol oxidase catalase), PO (phenoloxidase), SOD (superoxide dismutase), CAT (catalase), and LZM (lysozyme). Furthermore, the activities of ACP, POD, PO, and LZM were also observed to increase subsequent to infection with WSSV. Notably, geniposide was found to enhance the phagocytosis of V. alginolyticus within the hemocytes. Geniposide can reduce hemocyte apoptosis rates after treatment, as well as hemocytes infected with WSSV. Furthermore, geniposide treatment significantly up-regulated the expression level of Myosin, but expression levels of Astakine, C-type lectin (CTL), STAT, JAK, proPO, minichromosome maintenance protein (MCM7), caspase-3 and crustin were down-regulated in the hemocytes. Additionally, geniposide treatment inhibited WSSV replication in hemocytes of S. paramamosain, and enhanced the survival rates of mud crabs following WSSV infection. These experimental results provide evidence that geniposide can improve the immune response by regulating humoral immunity and cellular immunity, and enhance pathogen resistance in S. paramamosain.

Fluorometric assay for phenol oxidase activity in soils and its controlling variables

Phenolic compounds constitute the major component of recalcitrant carbon pools in soils and undergo degradation by enzymes such as phenol oxidases. Despite their significance, a common, appropriate assay to determine its activity is yet to be found. Until recently, only colorimetric assays, using substrates such as L-DOPA, pyrogallol, and ABTS, have been dominantly employed to measure phenol oxidase activity, despite their drawbacks, such as interference of soil background color, unavailability of a commercial product, and low sensitivity. Therefore, this study aimed to identify the optimal conditions for the novel fluorometric Amplex Red assay, compare the effectiveness and reproducibility of this assay with the conventional colorimetric L-DOPA assay, and reveal the key controlling variables of phenol oxidase activity by applying it to various soil types. Results showed that the optimal phenol oxidase activity can be determined at a substrate concentration of 500 μM and when the soil suspension is prepared with sonication only, using deionized water as a solvent. The comparison of the Amplex Red and L-DOPA assay indicated that the fluorometric assay has greater reproducibility, with a significantly smaller coefficient of variation (19.9 % and 107 %, respectively). For the L-DOPA assay, there was substantial variability between replicates and strong dependence on soil suspension preparation methods. Although this study reveals that the Amplex Red assay is more suitable for measuring phenol oxidase activity, additional research on other soil types is important to confirm its applicability, and adjustments to incubation conditions may be necessary for specific soil types. The application of the Amplex Red assay on various soil types uncovered soil pH and organic matter as the two most important factors controlling phenol oxidase activity, which aligned with previous findings. A positive linear correlation between phenol oxidase activity and soil pH (R = 0.734, P ≤ 0.000) but a negative exponential correlation with soil organic matter content (R = −0.547, P ≤ 0.01).

Whole genome structural predictions reveal hidden diversity in putative oxidative enzymes of the lignocellulose-degrading ascomycete Parascedosporium putredinis NO1.

Economic valorization of lignocellulose is paramount to realizing a true circular bioeconomy; however, this requires the development of systems and processes to expand the repertoire of bioproducts beyond current renewable fuels, chemicals, and sustainable materials. Parascedosporium putredinis NO1 is an ascomycete that thrived at the later stages of a wheat-straw composting community culture, indicating a propensity to degrade recalcitrant lignin-enriched biomass, but exists within an underrepresented and underexplored fungal lineage. This strain has been proven to be an exciting candidate for the identification of new enzymes targeting recalcitrant components of lignocellulose following the recent discovery of a new lignin β-ether linkage cleaving enzyme. The first genome for the genus Parascedosporium for P. putredinis NO1 genome was sequenced, assembled, and annotated. The genome is 39 Mb in size, consisting of 21 contigs annotated to contain 9.998 protein-coding sequences. The carbohydrate-active enzyme (CAZyme) repertoire was compared to 2570 ascomycete genomes and in detail with Trichoderma reesei, Fusarium oxysporum, and sister taxa Scedosporium boydii. Significant expansion in the oxidative auxiliary activity class of CAZymes was observed in the P. putredinis NO1 genome, resulting from increased sequences encoding putative lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes acting within LPMO redox systems, and lignin-degrading laccases. P. putredinis NO1 scored above the 95th percentile for AA gene density across the ascomycete phylum, suggesting a primarily oxidative strategy for lignocellulose breakdown. Novel structure-based searching approaches were employed, revealing 17 new sequences with structural similarity to LPMO, laccase, and peroxidase sequences and which are potentially new lignocellulose-degrading enzymes.IMPORTANCEAn annotated reference genome has revealed P. putredinis NO1 as a useful resource for the identification of new lignocellulose-degrading enzymes for biorefining of woody plant biomass. Utilizing a "structure-omics"-based searching strategy, we identified new potentially lignocellulose-active sequences that would have been missed by traditional sequence searching methods. These new identifications, alongside the discovery of novel enzymatic functions from this underexplored lineage with the recent discovery of a new phenol oxidase that cleaves the main structural β-O-4 linkage in lignin from P. putredinis NO1, highlight the underexplored and poorly represented family Microascaceae as a particularly interesting candidate worthy of further exploration toward the valorization of high value biorenewable products.

An insect sclerotization-inspired antifouling armor on biomedical devices combats thrombosis and embedding

Thrombus formation and tissue embedding significantly impair the clinical efficacy and retrievability of temporary interventional medical devices. Herein, we report an insect sclerotization-inspired antifouling armor for tailoring temporary interventional devices with durable resistance to protein adsorption and the following protein-mediated complications. By mimicking the phenol-polyamine chemistry assisted by phenol oxidases during sclerotization, we develop a facile one-step method to crosslink bovine serum albumin (BSA) with oxidized hydrocaffeic acid (HCA), resulting in a stable and universal BSA@HCA armor. Furthermore, the surface of the BSA@HCA armor, enriched with carboxyl groups, supports the secondary grafting of polyethylene glycol (PEG), further enhancing both its antifouling performance and durability. The synergy of robustly immobilized BSA and covalently grafted PEG provide potent resistance to the adhesion of proteins, platelets, and vascular cells in vitro. In ex vivo blood circulation experiment, the armored surface reduces thrombus formation by 95 %. Moreover, the antifouling armor retained over 60 % of its fouling resistance after 28 days of immersion in PBS. Overall, our armor engineering strategy presents a promising solution for enhancing the antifouling properties and clinical performance of temporary interventional medical devices.

Serine protease homolog pairs CLIPA4-A6, A4-A7Δ, and A4-A12 act as cofactors for proteolytic activation of prophenoloxidase-2 and -7 in Anopheles gambiae

Phenoloxidase (PO) catalyzed melanization and other insect immune responses are mediated by serine proteases (SPs) and their noncatalytic homologs (SPHs). Many of these SP-like proteins have a regulatory clip domain and are called CLIPs. In most insects studied so far, PO precursors are activated by a PAP (i.e., PPO activating protease) and its cofactor of clip-domain SPHs. Although melanotic encapsulation is a well-known refractory mechanism of mosquitoes against malaria parasites, it is unclear if a cofactor is required for PPO activation. In Anopheles gambiae, CLIPA4 is 1:1 orthologous to Manduca sexta SPH2; CLIPs A5−7, A12–14, A26, A31, A32, E6, and E7 are 11:4 orthologous to M. sexta SPH1a, 1b, 4, and 101, SPH2 partners in the cofactors. Here we produced proCLIPs A4, A6, A7Δ, A12, and activated them with CLIPB9 or M. sexta PAP3. A. gambiae PPO2 and PPO7 were expressed in Escherichia coli for use as PAP substrates. CLIPB9 was mutated to CLIPB9Xa by including a Factor Xa cleavage site. CLIPA7Δ was a deletion mutant with a low complexity region removed. After PAP3 or CLIPB9Xa processing, CLIPA4 formed a high Mr complex with CLIPA6, A7Δ or A12, which assisted PPO2 and PPO7 activation. High levels of specific PO activity (55−85 U/μg for PO2 and 1131−1630 U/μg for PO7) were detected in vitro, indicating that cofactor-assisted PPO activation also occurs in this species. The cleavage sites and mechanisms for complex formation and cofactor function are like those reported in M. sexta and Drosophila melanogaster. In conclusion, these data suggest that the three (and perhaps more) SPHI-II pairs may form cofactors for CLIPB9-mediated activation of PPOs for melanotic encapsulation in A. gambiae.

OYSTER BIO DEPOSITION ENHANCED THE FUNCTIONAL POTENTIAL OF LABILE AND RECALCITRANT CARBON IN SHENZHEN BAY’S SEDIMENTS

Oysters are typical aquatic animals with their special biological property of filter feeding in the water column. However, the biodeposition of oysters may change the sediment chemistry and also may affect the biogeochemical processes. We conducted research work to determine the effect of Oyster Biodeposition (OBD) on the sediments microbes, and their metabolic abilities on carbon at the site rich with oyster culture area of Shenzhen Bay China. After biochemical and Geochip5 analysis, we found that the genes coding the metabolic enzymes pullulanase, glucoamylase, exoglucanase, cellobiase, xylanase, etc. were highly abundant in oyster farming place compared to reference place. Meanwhile, the functional genes cda (6%), pectinase (5%), and xyla (5%) were greatly abundant at the surface oyster area and they were showing significantly higher ( P= <0.05) normalized gene intensities compared to bottom sediment site. These genes were mostly involved in the degradation of labile and recalcitrant forms of carbon. These detected genes were mainly involved in the carbon degradation process and they were showing more tendency towards the degradation of labile carbon. Small abundance of enzymes such as glyoxal oxidase and phenol oxidase were also found which were involved in the degradation of recalcitrant carbon. Taken together, our results indicated that the OBD enhanced the degrading capacity of both labile and recalcitrant forms of carbon, it has a positive stimulating effect on sediment carbon metabolism.

Infection with a non-lethal fungal parasite is associated with increased immune investment in the ant Myrmica scabrinodis

Social insects, such as ants, are preferred host organisms of pathogens and parasites because colonies are densely populated, and the number of potential hosts is high in the same place and time. Within a colony, individuals are exposed differentially to risks according to their function and age. Thus, older individuals forage and are therefore the most exposed to infection, predation, or physical stress, while young workers mostly stay inside the sheltered nest being less exposed. Immune investment is considered to be dependent on an individual’s age and pathogen pressure. Long-term exposure to a parasite could affect the immune activity of individuals in an intriguing way that interferes with the age-dependent decline in immunocompetence. However, there are only few cases in which such interferences can be studied. The myrmecopathogenic fungus Rickia wasmannii, which infects entire colonies without killing the workers, is a suitable candidate for such studies. We investigated the general immunocompetence of Myrmica scabrinodis ant workers associated with non-lethal fungal infection by measuring the levels of active phenoloxidase (PO) and total PO (PPO) (reflecting the amount of both active and inactive forms of the enzyme) in two age classes. The level of PO proved to be higher in infected workers than in uninfected ones, while the level of PPO increased with age but was not affected by infection. Overall, these results indicate that a long-term infection could go hand in hand with increased immune activity of ant workers, conferring them higher level of protection.

Effects of hydroxy methionine zinc on growth performance, immune response, antioxidant capacity, and intestinal microbiota of red claw crayfish (Procambarus clarkii)

This study aimed to evaluate the effects of varying zinc (Zn) levels on the growth performance, non-specific immune response, antioxidant capacity, and intestinal microbiota of red claw crayfish (Procambarus clarkii (P. clarkii)). Adopting hydroxy methionine zinc (Zn-MHA) as the Zn source, 180 healthy crayfish with an initial body mass of 6.50 ± 0.05 g were randomly divided into the following five groups: X1 (control group) and groups X2, X3, X4, and X5, which were fed the basal feed supplemented with Zn-MHA with 0, 15, 30, 60, and 90 mg kg−1, respectively. The results indicated that following the addition of various concentrations of Zn-MHA to the diet, the following was observed: Specific growth rate (SGR), weight gain rate (WGR), total protein (TP), total cholesterol (TC), the activities of alkaline phosphatase (AKP), phenoloxidase (PO), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and catalase (CAT), the expression of CTL, GPX, and CuZn-SOD genes demonstrated a trend of rising and then declining—with a maximum value in group X4—which was significantly higher than that in group X1 (P < 0.05). Zn deposition in the intestine and hepatopancreas, the activity of GSH-PX, and the expression of GSH-PX were increased, exhibiting the highest value in group X5. The malonaldehyde (MDA) content was significantly reduced, with the lowest value in group X4, and the MDA content of the Zn-MHA addition groups were significantly lower than the control group (P < 0.05). In the analysis of the intestinal microbiota of P. clarkii, the number of operational taxonomic units in group X4 was the highest, and the richness and diversity indexes of groups X3 and X4 were significantly higher than those in group X1 (P < 0.05). Meanwhile, the dietary addition of Zn-MHA decreased and increased the relative abundance of Proteobacteria and Tenericutes, respectively. These findings indicate that supplementation of dietary Zn-MHA at an optimum dose of 60 mg kg−1 may effectively improve growth performance, immune response, antioxidant capacity, and intestinal microbiota richness and species diversity in crayfish.

Depolymerisation of Kraft Lignin by Tailor-Made Alkaliphilic Fungal Laccases.

Lignins released in the black liquors of kraft pulp mills are an underutilised source of aromatics. Due to their phenol oxidase activity, laccases from ligninolytic fungi are suitable biocatalysts to depolymerise kraft lignins, which are characterised by their elevated phenolic content. However, the alkaline conditions necessary to solubilise kraft lignins make it difficult to use fungal laccases whose activity is inherently acidic. We recently developed through enzyme-directed evolution high-redox potential laccases active and stable at pH 10. Here, the ability of these tailor-made alkaliphilic fungal laccases to oxidise, demethylate, and depolymerise eucalyptus kraft lignin at pH 10 is evidenced by the increment in the content of phenolic hydroxyl and carbonyl groups, the methanol released, and the appearance of lower molecular weight moieties after laccase treatment. Nonetheless, in a second assay carried out with higher enzyme and lignin concentrations, these changes were accompanied by a strong increase in the molecular weight and content of β-O-4 and β-5 linkages of the main lignin fraction, indicating that repolymerisation of the oxidised products prevails in one-pot reactions. To prevent it, we finally conducted the enzymatic reaction in a bench-scale reactor coupled to a membrane separation system and were able to prove the depolymerisation of kraft lignin by high-redox alkaliphilic laccase.

BmPxt1 mediated immune response by regulating PGE2 in silkworm, Bombyx mori

Prostaglandins (PGs) mediates the immune response of insects to multiple stimuli. Mammalian cyclooxygenase (COXs) is a key enzyme in the synthesis of PGs, and peroxinectin (Pxt) may have similar functions in some sequenced insect genomes. As a representative of Lepidoptera, the silkworm also contains PGs, but its synthetic pathway is not clear. We cloned a full-length cDNA encoding a Pxt, designated as BmPxt1, from silkworm. Sequence alignment analysis showed that the protein encoded by BmPxt1 has a conserved domain similar to Pxts, and its catalytic site is shared with the Pxt of Manduca sexta, which also produces PGs. The expression of BmPxt1 gene was the highest in the hemocytes and was induced by Nuclear Polyhedrosis Virus (NPV) challenge in the detected tissues. Moreover, we found that dsPxt1 treatment deficiency down-regulated BmPxt1 transcript levels and efficiently inhibiting hemocyte-spreading and nodule formation in silkworm. Hemocyte-spreading, nodule formation, phenoloxidase (PO) and AMP genes (attacin, defencin and moricin) were also inhibited by aspirin, a COX inhibitor. Treatment by PGE2 but not arachidonic acid (AA) rescued the immunosuppression; PGs concentrations was also inhibited by aspirin. PGE2, but not AA, treatment rescued the PGs concentrations. The COX inhibitor, aspirin, impaired the innate immune response including nodulation, encapsulation, and melanization in silkworm, while PGE2, but not arachidonic acid (AA), partially reversed these effects of aspirin. Recombinant BmsPxt1 significantly induced PO activation in larvae hemolymph, PGs concentrations and encapsulation of agarose beads. Injection of recombinant BmsPxt1 into larvae resulted in increased transcript levels of AMP genes. Our results confirmed that BmPxt1 was involved in the synthesis of PGs in the innate immune response of silkworm larvae, and provided new information for the role of BmsPxt1 secreted by silkworm in activating PO and antimicrobial peptides.

Effects of environmental pollutant benzop[α]yrene on the innate immunity of Scylla paramamosain and its mechanism

Benzo[α]pyrene (BaP), a polycyclic aromatic hydrocarbon, is present in the aquatic environment and may be harmful to aquatic animals. We exposed the mud crab Scylla paramamosain to BaP for 7 days, the of superoxide dismutase (SOD), catalase (CAT), phenoloxidase (PO), lysozyme (LZM), glutathione (GSH), glutathione-S-transferase (GST), and acid phosphatase (ACP) activities in the hemolymph of mud crab were reduced. Additionally, the reactive oxygen species content was increased in mud crabs after exposed to BaP. When BaP concentration was increased, the total hemocyte count (THC), the survival rate of hemocytes and their proliferation were decreased. Histopathology analysis revealed damaged hepatopancreas cells, which indicating that BaP exposure is cytotoxic to crab hemocytes. However, the degree of DNA damage did not worsen with increasing BaP concentration. The expression levels of p53, MCM7, Caspase-3, and Myosin were changed with increasing concentration of BaP, which indicated that BaP exposure may affect apoptosis and phagocytosis in mud crabs. As BaP concentration was increased, the apoptosis rate of hemocytes was increased and the phagocytosis was decreased. These results confirmed that BaP exposure inhibited the innate immune response of mud crabs. A possible explanation for this effect is that BaP reduces the antioxidant enzyme activity and increases the reactive oxygen species content in mud crabs, thereby oxidizing and damaging hemocytes, which stimulates phagocytosis and apoptosis and negatively affects the innate immunity of S. paramamosain.

Power Bars: Mormon Crickets Get Immunity Boost from Eating Grasshoppers.

In addition to feeding on plants, Mormon crickets Anabrus simplex Haldeman, 1852 predate on invertebrates, including one another, which effectively drives their migration. Carnivory derives from lack of dietary protein, with Mormon crickets deprived of protein having less phenoloxidase (PO) available to combat foreign invaders, such as fungal pathogens. Because Mormon crickets commonly occur with grasshoppers that feed on the same plants, we investigated interactions between grasshoppers and Mormon crickets, and hypothesized that if Mormon crickets are predatory on grasshoppers, grasshopper abundance would influence the protein available to Mormon crickets and their immunity. In a field setting, we varied densities of Mormon crickets (0, 10, or 20 per cage) and grasshoppers Melanoplus borealis (0, 15, 30, or 45) in 68 1-m2 cages. After one month, we measured Mormon cricket dietary preferences and PO activity. As predicted, artificial diet consumption shifted away from protein as grasshopper density increased, and immunocompetence, as measured by PO activity, also increased with grasshopper availability. Although nitrogen availability in the vegetation decreased with increasing insect density, predation became an important source of protein for Mormon crickets that enhanced immunity. Grasshoppers can be an important source of dietary protein for Mormon crickets, with prey availability affecting Mormon cricket immunity to diseases.

Identification of an essential role against shrimp pathogens of prophenoloxidase activating enzyme 1 (PPAE1) from Fenneropenaeus merguiensis hemocytes

Prophenoloxidase (proPO) activating enzymes, known as PPAEs, are pivotal in activating the proPO system within invertebrate immunity. A cDNA encoding a PPAE derived from the hemocytes of banana shrimp, Fenneropenaeus merguiensis have cloned and analyzed, referred to as FmPPAE1. The open reading frame of FmPPAE1 encompasses 1392 base pairs, encoding a 464-amino acid peptide featuring a presumed 19-amino acid signal peptide. The projected molecular mass and isoelectric point of this protein stand at 50.5 kDa and 7.82, respectively. Structure of FmPPAE1 consists of an N-terminal clip domain and a C-terminal serine proteinase domain, housing a catalytic triad (His272, Asp321, Ser414) and a substrate binding site (Asp408, Ser435, Gly437). Expression of the FmPPAE1 transcript is specific to hemocytes and is heightened upon encountering pathogens like Vibrio parahaemolyticus, Vibrio harveyi, and white spot syndrome virus (WSSV). Using RNA interference to silence the FmPPAE1 gene resulted in reduced hemolymph phenoloxidase (PO) activity and decreased survival rates in shrimp co-injected with pathogenic agents. These findings strongly indicate that FmPPAE1 plays a vital role in regulating the proPO system in shrimp. Furthermore, upon successful production of recombinant FmPPAE1 protein (rFmPPAE1), it became evident that this protein exhibited remarkable abilities in both agglutinating and binding to a wide range of bacterial strains. These interactions were primarily facilitated through the recognition of bacterial lipopolysaccharides (LPS) or peptidoglycans (PGN) found in the cell wall. This agglutination process subsequently triggered melanization, a critical immune response. Furthermore, rFmPPAE1 exhibited the ability to actively impede the growth of pathogenic bacteria harmful to shrimp, including V. harveyi and V. parahaemolyticus. These findings strongly suggest that FmPPAE1 not only plays a pivotal role in activating the proPO system but also possesses inherent antibacterial properties, actively contributing to the suppression of bacterial proliferation. In summary, these results underscore the substantial involvement of FmPPAE1 in activating the proPO system in F. merguiensis and emphasize its crucial role in the shrimp's immune defense against invading pathogens.

Quality and shelf life of strawberry fruit as affected by edible coating by moringa leaf extract, aloe vera gel, oxalic acid, and ascorbic acid

ABSTRACT In the current study, the most effective concentrations of moringa leaf extract (MLE), aloe vera gel (AV gel), ascorbic acid (1 mM) and oxalic acid (5 mM) were used. Physically mature fruits harvested from a commercial orchard were dipped in aqueous solutions of different edible coatings and kept at ambient conditions (25±2ºC & 55-60% RH) for evaluation of the quality and enzymatic parameters on daily basis till marketability. Current results revealed that 5 mM ascorbic acid treatment resulted in 57% reduced fruit weight loss; while 20% AV gel coating increased firmness by 60% over a 5-day ambient storage period. Application 6% MLE exhibited 50.7% and 49.7% reduced respiration rate and ethylene production respectively. Fruit biochemical quality parameters were also improved with the lowest accumulation of total soluble solids as a result of slow respiration, higher titratable acidity, ascorbic acid contents, carotenoids and anthocyanin contents in all treated fruit. While, total phenolic contents and total antioxidants along with superoxide dismutase, catalase, peroxidase, and anthocyanins were higher in ascorbic acid treated fruits. The activity of poly phenol oxidase enzyme was lowest in fruits treated with 5 mM ascorbic acid solution. Conclusively, the application of different edible coatings was proved to be beneficial as numerous fruit biochemical and antioxidative attributes were improved by ascorbic acid treatment; furthermore, MLE, AV gel and oxalic acid treatments were also helpful in maintaining fruit quality of strawberry fruits over a 5-days ambient storage period.

Mitigating decomposition in agricultural peatlands: Influence of copper and polyphenol on C N P dynamics and enzyme activities in two contrasting soils

Drained agricultural peatlands are subject to degradation through decomposition, compaction, and erosion. This study aimed to (1) revisit an existing recommendation to systematically apply copper at a rate exceeding crop needs to slow the decomposition of agricultural peatlands, and (2) explore lignosulfonate, a polyphenol, as an alternative method to copper. Two peat soils with contrasting levels of humification (sapric and hemic) and differing Al and Fe contents were studied under controlled conditions. CO2 emissions cumulated in the two unamended soils differed importantly, representing 0.7 and 7.3 t C-CO2 ha−1 yr−1 (0–25 cm) in the sapric and hemic soil, respectively. In the sapric soil, copper did not affect CO2 emissions, N and P parameters, or the activity of most of the measured enzymes. In the same soil, lignosulfonate increased CO2 emissions and phenol oxidase activity, suggesting that the product acted as a microbial substrate. In the hemic soil, copper and lignosulfonate reduced cumulated CO2 emissions by 37 % and 61 %, respectively, as well as N parameters and phosphatase activity. The influence of both treatments on CO2 emissions was, however, temporary. This study suggests limited efficiency of copper and lignosulfonate when applied to soils where underlying mechanisms, such as organic matter recalcitrance and Al and Fe stabilization effect, already regulate decomposition rates. The divergent and temporary effects of copper found in this study and in the literature suggest that routine applications of copper at a rate exceeding crop needs is not consistently effective in slowing decomposition in agricultural peatlands. Lignosulfonate appears to be a promising alternative to copper. The inhibitory effect of polyphenol applications has been demonstrated in natural peatlands but, to our knowledge, this is the first time it has been reported for drained agricultural peatlands. Evaluating the influence of lignosulfonate on other drained agricultural peat soils and under field conditions is necessary to determine the potential of this approach.

Larvicidal activity and biochemical effect of some essential oils and indoxacarb against peach fruit fly, Bactrocera zonata (Diptera: Tephritidae)

Bactrocera zonata (Saunders) (Diptera: Tephritidae) is an important insect pest of fruits in Egypt. The desire for fresh fruits free of insecticides drives the adoption of environmentally friendly biopesticides to manage pests. The current study sought to estimate the toxicity and biochemical alterations of 3rd larval instar of B. zonata affected by clove, neemazal T/S, garlic, ginger and orange oils compared with indoxacarb at LC50 under laboratory conditions. Results after 24 h of treatment indicated that neemazal T/S is more toxic than other tested oils compared with higher toxicity of indoxacarb with LC50 values of 86.09, 174.72, 4514.38, 7224.01, 9387.73 and 2590.2 μg/mL for indoxacarb, neemazal T/S, ginger, clove, garlic and orange oils, respectively. Moreover, after 24 h of treatment, the larval content of digestive enzymes drastically decreased. All compounds caused a remarkable reduction in protease (except clove oil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), phenol oxidase enzyme, α-esterase (except indoxacarb and neemazal T/S) and β-esterase (except ginger oil and neemazal T/S) activity. Also, total carbohydrates, total lipids and total proteins take the same trend. All treatments at LC50 increased the activity of cytochrome p450 and glutathione-s-transferase against 3rd larval instar of B. zonata compared with control. Our findings recommended that the tested botanicals could be an effective substitute for conventional insecticides as well as their safety for human beings and beneficial organisms.