Phenoloxidase Research Articles

Nucleoprotein of a Rice Rhabdovirus Serves as the Effector to Attenuate Hemolymph Melanization and Facilitate Viral Persistent Propagation in its Leafhopper Vector.

Melanization in the hemolymph of arthropods is a conserved defense strategy against infection by invading pathogens. Numerous plant viruses are persistently transmitted by insect vectors, and must overcome hemolymph melanization. Here, we determine that the plant rhabdovirus rice stripe mosaic virus (RSMV) has evolved to evade the antiviral melanization response in the hemolymph in leafhopepr vectors. After virions enter vector hemolymph cells, viral nucleoprotein N is initially synthesized and directly interacts with prophenoloxidase (PPO), a core component of the melanization pathway and this process strongly activates the expression of PPO. Furthermore, such interaction could effectively inhibit the proteolytic cleavage of the zymogen PPO to active phenoloxidase (PO), finally suppressing hemolymph melanization. The knockdown of PPO expression or treatment with the PO inhibitor also suppresses hemolymph melanization and causes viral excessive accumulation, finally causing a high insect mortality rate. Consistent with this function, microinjection of N into leafhopper vectors attenuates melanization and promotes viral infection. These findings demonstrate that RSMV N serves as the effector to attenuate hemolymph melanization and facilitate viral persistent propagation in its insect vector. Our findings provide the insights in the understanding of ongoing arms race of insect immunity defense and viral counter-defense.

Molecular and functional characterization of an astakine cDNA from the giant freshwater prawn Macrobrachium rosenbergii

Astakine is a cytokine of invertebrates that promotes the proliferation and differentiation of hematopoietic stem cells, which regulates hematopoietic and immune responses. In this study, we have identified an astakine homolog (MrAst) from the freshwater prawn Macrobrachium rosenbergii. The MrAst cDNA consists of 408 bp with a 390 bp open reading frame (ORF) encoding 129 amino acids. MrAst contains a prokineticin domain, wherein the mature peptide consist of 10 conservative cysteine residues. Multiple sequence and phylogenetic analyses revealed that MrAst shares a close evolutionary relationship with astakines from crustaceans. MrAst was highly expressed in the hemocytes and nerve tissues. Both Vibrio parahemolyticus, lipopolysaccharide (LPS), and Decapod iridescent virus 1 (DIV1) infection upregulated MrAst expression in hemocytes and gills. Shrimps were more vulnerable to V. parahemolyticus and DIV1 infection upon MrAst knockdown, resulting in a lower survival rate. Additionally, inhibition of MrAst resulted in a significant reduction in total hemocyte count (THC) and phenoloxidase (PO) activity. However the lysozyme activity was increased and the superoxide dismutase (SOD) activity was unaffected. Moreover, knockdown of MrAst significantly inhibited the expression of hemocyanin, STAT, prophenoloxidase (proPO), crustacean hematopoietic factor (CHF), and Crustin and enhanced the expression of Toll-like receptor (TLR), IMD, Relish, and anti-lipopolysaccharide factor (ALF). The reduction in MrAst also increased the level of hemocytes apoptosis and significantly upregulated the apoptosis-related genes, such as Caspase 9, Caspase 3, p53, cytochrome C (Cyt C), Endonuclease G (Endo-G), and Cathepsin B. The results indicated that MrAst may play an important role in response to V. parahemolyticus and DIV1 infection and involved in the apoptosis process to avoid DNA damage for maintenance of hemocytes homeostasis.

Combined application of glycerol monolaurate and tributyrin improves the growth performance, antioxidant capacity, nonspecific immunity, gene expression and intestinal health of juvenile Litopenaeus vannamei

This study aimed to investigate the effects of the glycerol monolaurate (GML) and tributyrin (TB) and their interaction on the growth performance, body composition, nonspecific immunity, gene expression and intestinal microbiota of Litopenaeus vannamei (initial weight 0.24 ± 0.00 g). The feeding trial comprised a completely randomized 3 × 2 factorial design and six formulated diets with two different GML levels (0.10% and 0.15%) and three different TB levels (0.10%, 0.15%, and 0.20%) named G10T10, G10T15, G10T20, G15T10, G15T15, and G15T20, respectively. Results showed that three groups (G10T15, G15T10, and G15T15) had significantly higher final mean weight, weight gain rate, specific growth rate (P < 0.05) and lower feed conversion ratio than the other groups (P < 0.05). Catalase at low and medium addition levels of TB (G10T10, G10T15, G15T10, and G15T15) was promoted by GML (P < 0.05), and malondialdehyde was inhibited by GML (P < 0.05). Glutathione in G10T15 and G15T10 had the highest values among the groups (P < 0.05). Acid phosphatase was greatly influenced by GML, TB, and their interaction, and its maximum value was found in G15T10 (P < 0.05). Alkaline phosphatase was influenced by TB and was significantly higher in G10T15 and G15T15 than in the other groups (P < 0.05). The mRNA expression of lysozyme (LZM), heat shock protein 70 (HSP70), and phenol oxidase (PO) were significantly affected by GML, TB, and their interaction (P < 0.05). At the phylum level, Firmicutes and Firmicutes/Bacteroidetes ratio increased and then decreased at low level of GML (0.10%) and gradually decreased at high level of GML (0.15%), with minimum values in the G10T15 and G15T10 groups (P < 0.05). In conclusion, 0.15% GML and 0.10% TB were the optimal dietary addition on the basis of growth performance, nonspecific immunity, and intestinal health of the juvenile shrimp (Litopenaeus vannamei).

Effects of Byproduct Amendments and Nitrogen on Carbon Mineralization and Soil Enzyme Activities in Acidic Brown Soil from Jiaodong Peninsula of China

ABSTRACT It has been proposed strategy that acidic soil quality can be improved by addition of alkaline byproduct amendments containing silicon, calcium, magnesium and potassium. The effects of amendments and three different forms of nitrogen [(NH2)2CO, NaNO3 and NH4Cl] on quality of acidic soil were investigated in Jiaodong Peninsula of China. A 120-day incubation test was carried out to assess the effect of amendments and nitrogen on soil physicochemical properties, soil carbon (C) mineralization and enzyme activities. The results showed that the addition of alkaline amendments can increase soil pH from 5.93 to 6.99 at the beginning of cultivation, while when amendments combined with urea or NH4Cl, soil pH decreased with incubation time. The response of C mineralization was inversely proportional to soil pH value. The addition of amendments could improve soil C content via decreasing soil C-CO2 emissions, and the mineralization rate was reduced by 26%. Addition of amendments in combination with urea or NH4Cl, which could decrease soil pH, stimulated soil C mineralization and increased soil microbial biomass carbon (MBC) and dissolved organic carbon (DOC) fractions in acidic brown soil. However, when amendments were applied with NaNO3, the soil C mineralization could be inhibited. The addition of amendments or combination of amendments and nitrogen increased the soil invertase and phenol oxidase activities, and inhibited soil urease activity. Therefore, the chemical and biological properties of the acidic brown soil in Jiaodong Peninsula of China might be better improved by combination of amendments and NaNO3.

Combined transcriptomic and proteomic analysis of developmental features in the immune system of Plutella xylostella during larva-to-adult metamorphosis

Diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is considered one of the most destructive worldwide agricultural pests and has developed various defence mechanisms to fight against the available pesticides. Understanding the host-defence system of P. xylostella is vital for developing biocontrol-based pest management strategies. Although there are several studies on P. xylostella, little is known about the changes in the immune system during the larva-to-adult metamorphosis. RNA-seq and iTRAQ investigations of P. xylostella from 2-day-old fourth instar larvae (L4D2), pupa (P0), and adult (A0) were done to understand these alterations at a molecular level. A total of 412/ 584 up-regulated and 1430/ 757 down-regulated genes/proteins between larva and pupa, 813/ 589 up-regulated and 1206/ 846 down-regulated genes/proteins between pupa and adult were identified. It was shown that the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) expression were up-regulated during the pupation and emergence of metamorphosis. The pathway enrichment analysis demonstrated that DEGs and DEPs were mainly associated with the energy generation and metabolism and innate immunity of the insect. The expression of immune-related and developmental-related genes were significantly different during the developmental process of P. xylostella. Moreover, the expression of four focused genes, i.e., serine proteinase inhibitor (Serpin-15), prophenoloxidase activating proteinase 1 (PAP-1) and 3a (PAP-3a), Gram-negative bacteria-binding protein (GNBP-6), was different in developmental stages and after Bacillus thuringiensis HD73 and Metarhizium anisopliae infection. The phenoloxidase (PO) activity in plasma was also significantly up-regulated during the pathogen infection. Recombinant proteins PAP-1, PAP-3a, GNBP-6 could significantly trigger the PO activity in vitro, Serpin-15 could suppress the PO activity. Taken together, these results indicate that Serpin-15, PAP-1, PAP-3a, and GNBP-6 might have the potential for co-regulation of immunity and development in P. xylostella. In conclusion, this study provided the immune system dynamics in the developmental process of P. xylostella and identified four candidate genes that can serve as potential targets for pest control strategies.

Effects of different forms of nitrogen addition on microbial extracellular enzyme activity in temperate grassland soil

BackgroundNitrogen (N) deposition alters litter decomposition and soil carbon (C) sequestration by influencing the microbial community and its enzyme activity. Natural atmospheric N deposition comprises of inorganic N (IN) and organic N (ON) compounds. However, most studies have focused on IN and its effect on soil C cycling, whereas the effect of ON on microbial enzyme activity is poorly understood. Here we studied the effects of different forms of externally supplied N on soil enzyme activities related to decomposition in a temperate steppe. Ammonium nitrate was chosen as IN source, whereas urea and glycine were chosen as ON sources. Different ratios of IN to ON (Control, 10:0, 7:3, 5:5, 3:7, and 0:10) were mixed with equal total amounts of N and then used to fertilize the grassland soils for 6 years.ResultsOur results show that IN deposition inhibited lignin-degrading enzyme activity, such as phenol oxidase (POX) and peroxidase (PER), which may restrain decomposition and thus induce accumulation of recalcitrant organic C in grassland soils. By contrast, deposition of ON and mixed ON and IN enhanced most of the C-degrading enzyme activities, which may promote the organic matter decomposition in grassland soils. In addition, the β-N-acetyl-glucosaminidase (NAG) activity was remarkably stimulated by fertilization with both IN and ON, maybe because of the elevated N availability and the lack of N limitation after long-term N fertilization at the grassland site. Meanwhile, differences in soil pH, soil dissolved organic carbon (DOC), and microbial biomass partially explained the differential effects on soil enzyme activity under different forms of N treatments.ConclusionsOur results emphasize the importance of organic N deposition in controlling soil processes, which are regulated by microbial enzyme activities, and may consequently change the ecological effect of N deposition. Thus, more ON deposition may promote the decomposition of soil organic matter thus converting C sequestration in grassland soils into a C source.

Condition‐dependent immune function in a freshwater snail revealed by stable isotopes

Abstract The immune system is costly to maintain and use because it requires a lot of energy. This can make parasite resistance dependent on host nutritional state. The dependence of immune function on host condition can have broad ecological (e.g., disease dynamics) and evolutionary (e.g., expression of trade‐offs related to parasite resistance) consequences. Research on the dependence of immune function on host condition is typically conducted in laboratory experiments that manipulate either the quantity or composition of available resources. Such studies are essential in establishing conceptual frameworks, but their results are difficult to generalise to natural populations because the experimental treatments may deviate from natural variation in host resource level and use. We examined the condition dependence of immune function in a generalist freshwater snail Lymnaea stagnalis by relating the activity of two immune parameters of snail haemolymph, phenoloxidase (PO)‐like and antibacterial activity, to snail resource level and use using field‐collected individuals. We measured several variables that reflect the snails’ quality based on their recent (i.e., within the past few days; amount and stable isotope composition [δ15N and δ13C] of produced faeces) and longer‐term (i.e., weeks to months; stable isotope composition of tissues) resource use. The PO‐like activity of the snails’ haemolymph was condition‐dependent. Snails that had recently consumed food from higher trophic levels, presumably including more animal protein (high δ15N values of faeces), had a stronger defence. Additionally, snails with low energy (i.e., lipid) reserves in their tissues (high δ13C values of tissues) showed higher PO‐like activity. The antibacterial activity of the snails’ haemolymph was not condition dependent. The finding of the importance of the composition of recently consumed food on immune function (namely PO‐like activity) suggests that the parasite resistance of snails may change rapidly depending on the type of resources available in their environment. Thus, environmental variation may influence the outcome of host–parasite interactions on fine spatial and temporal scales. Furthermore, the negative relationship between the snails’ energy reserves and PO‐like activity suggests substantial energetic costs of immune activity in L. stagnalis.

Effects of the removal of invasive Moso bamboo on soil microbial biomass and enzyme activities in subtropical forests

Removal of invasive plant species is the first step to restoring the invaded ecosystems. The soil microbial biomass and extracellular enzyme activities were measured in Moso bamboo (Phyllostachys edulis) pure forest (completely invasion), invasive P. edulis removal forest (secondary succession 5 years after clear cutting), and the evergreen broadleaved forest (no invasion) in Tianmu Mountain. The results showed that compared with P. edulis pure forest, invasive P. edulis removal significantly increased the contents of soil organic carbon (SOC), nitrate nitrogen, available phosphorus and potassium, as well as microbial biomass carbon (MBC) and microbial biomass phosphorus (MBP), while significantly decreased microbial biomass nitrogen (MBN). The activities of α-glucosidase (AG), β-glucosidase (BG), leucine aminopeptidase (LAP) and phenol oxidase (POX) in the forest with removal of invasive P. edulis were significantly higher than those in P. edulis pure forest, while invasive P. edulis removal did not change the activities of cellodisaccharide hydrolase (CBH), β-N-acetyl-glucosaminopeptidase (NAG), acid phosphatase (ACP) and peroxidase (PER). Furthermore, the activities of AG, BG and LAP were positively correlated with SOC and MBC, while the increase in POX activity was positively correlated with soil nitrate content. In addition, MBC, MBN and MBP, and activities of AG, BG, NAG, LAP and ACP in P. edulis removal forest forest were significantly higher than those in evergreen broadleaved forests. Taken together, the removal of invasive P. edulis could increase soil nutrient contents, microbial biomass and extracellular enzyme activities, thus could be considered as an effective way to restore the invaded forests. Our results provide important theoretical basis for controlling P. edulis invasion in subtropical forests.

Optimization for the determination of phenol oxidase activity in subtropical forest soils developed on sandstone

Phenol oxidase plays an important role in the degradation of soil organic matter. There was no standard method to determine soil phenol oxidase activity. To fill such knowledge gap, we investigated the effects of substrate type, pH, soil storage conditions, storage time, substrate concentration, water-soil ratio, incubation time and incubation temperature on soil phenol oxidase activity in three different subtropical forest soils developed on sandstone. The pH of extraction buffers significantly affected the phenol oxidase activity. Using 2,2'-azinobis-(-3-ethylbenzo-thiazoline-6-sulfononic acid)-diammonium salt (ABTS) as substrate acquired higher oxidase activity and was applicable to wider pH range than using 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA) as substrate, indicating that ABTS was more suitable as a substrate for measuring phenol oxidase activity in acidic soils of subtropical forests. The storage condition significantly affected phenol oxidase activity. The phenol oxidase activity declined with time in all the three types of soil. The decreasing rate was air-dried > 4 ℃ refrigerated > -20 ℃ frozen > -80 ℃ frozen, suggesting that the frozen storage method was better than others in maintaining soil phenol oxidase activity if the determination of phenol oxidase activity in fresh soil samples cannot be immediately done. Substrate concentration, water-soil ratio, and incubation time and temperature all affected the activity of soil phenol oxidase. The condition of soil: buffer ratio of 1:100, 2 mmol·L-1 concentration of ABTS with an incubation time of 4 h at 25-30 ℃ was optimal for measuring phenol oxidase activity in acidic soils of subtropical forests, with high repeatability and sensitivity.

Effects of combined application of biochar with organic amendments on enzyme activity and microbial metabolic function of carbon sources in infertile red soil

To improve carbon (C) sequestration and soil fertility of red soil, a two-year (2017 and 2018) field experiment was conducted to investigate the effects of two organic amendments (i.e., corn straw and sheep manure) applied alone or combined with biochar on soil nutrient content, enzyme activities involved in C cycling, and microbial substrate utilization rate in infertile red soil. There were six treatments, including control (non-amendment), corn straw, sheep manure and across biochar treatments (without and with biochar amendment, respectively). The organic amendments and biochar were applied in 2017 and 2018. The results showed that, compared with the control, organic amendments significantly increased soil pH, organic C, total nitrogen, available phosphorus and potassium contents. Compared with straw and manure alone, the biochar co-application with straw or manure significantly increased the contents of soil organic C, available potassium, and available nitrogen, without any significant interactive effects. Application of organic amendments significantly increased the activities of soil β-glucosidase (BG), cellobiohydrolase (CB), β-xylosidase (XYL), and peroxidase (PERO). The combined application of biochar and straw significantly reduced the activity of phenol oxidase (PHOX) by 28.6% and PERO by 22.2% in comparison with straw addition alone, respectively, while the combined application of biochar and manure significantly reduced the activities of α-glucosidase (AG) by 46.1%, BG by 50.9%, XYL by 41.6%, and PERO by 31.3% compared with manure addition alone, respectively. Compared with the control, the application of organic amendments significantly enhanced soil basal respiration and microbial utilization rates of carbohydrates, whereas biochar co-application significantly decreased microbial utilization rates of carbohydrates and carboxylic acids. Microbial C source utilization rates were significantly and positively correlated with the activities of BG and PERO. Thus, biochar co-application with organic amendments can enhance nutrient content and reduce enzymatic and microbial metabolic activities, thereby may facilitate C sequestration and fertility of infertile red soil.

Metabolic and immunological responses of Drosophila melanogaster to dietary restriction and bacterial infection differ substantially between genotypes in a population.

To respond to changing environmental conditions, a population may either shift toward better‐adapted genotypes or adapt on an individual level. The present work aimed to quantify the relevance of these two processes by comparing the responses of defined Drosophila melanogaster populations to different stressors. To do this, we infected two homogeneous populations (isofemale lines), which differ significantly in fitness, and a synthetic heterogeneous population with a specific pathogen and/or exposed them to food restriction. Pectobacterium carotovorum was used to infect Drosophila larvae either fed standard or protein‐restricted diet. In particular, the two homogeneous groups, which diverged in their fitness, showed considerable differences in all parameters assessed (survivorship, protein and lipid contents, phenol‐oxidase (PO) activity, and antibacterial rate). Under fully nutritious conditions, larvae of the homogeneous population with low fitness exhibited lower survivorship and protein levels, as well as higher PO activity and antibacterial rate compared with the fitter population. A protein‐restricted diet and bacterial infection provoked a decrease in survivorship, and antibacterial rate in most populations. Bacterial infection elicited an opposite response in protein and lipid content in both isofemale lines tested. Interestingly, the heterogeneous population showed a complex response pattern. The response of the heterogeneous population followed the fit genotype in terms of survival and antibacterial activity but followed the unfit genotype in terms of PO activity. In conclusion, our results show that defined genotypes exhibit highly divergent responses to varying stressors that are difficult to predict. Furthermore, the responses of heterogeneous populations do not follow a fixed pattern showing a very high degree of plasticity and differences between different genotypes.

Keep oxygen in check: Contrasting effects of short-term aeration on hydrolytic versus oxidative enzymes in paddy soils

Enzymes produced by microorganisms and plants are very sensitive to variations in soil microclimate, yet most enzyme assays are conducted under oxic conditions irrespective of the origin of environmental samples. It remains unclear how short-term aeration (minutes to hours) affects the hydrolytic and oxidative enzymes in anoxic systems. This key gap in current methods was addressed by measuring the kinetics of hydrolytic phosphomonoesterase, β-glucosidase, and leucine aminopeptidase and the activities of oxidative phenol oxidases and peroxidases by fluorogenic substrates under oxic (+O2) and anoxic conditions (–O2). Aeration effects were tested in a flooded paddy soil with growing rice (research task 1: moderate O2 limitation) and without rice (research task 2: strong O2 limitation). We tested two hypotheses explaining possible effects of short-term aeration on hydrolytic versus oxidative enzymes. (1) Aeration promotes Fe(II) oxidation, which leads to the accumulation of phenolics through the “iron-gate” mechanism, thus suppressing the activities of hydrolytic enzymes compared to the anoxic conditions. (2) Aeration stimulates phenol oxidases that degrade phenolics according to the “enzyme latch” concept, thus eliminating the suppression of hydrolytic enzymes. The activities of hydrolytic enzymes were lower by 5–43% in both experiments under + O2 compared to –O2. In contrast, the activities of peroxidases and phenol oxidases were 2–14 times higher under + O2 than under –O2. Thus, the activation of oxidative enzymes under + O2 was uncoupled from the hydrolytic activities. This contradicts both the “iron gate” and the “enzyme latch” mechanisms. We explain the short-term suppressive effect of O2 in assays by increased concentrations of reactive oxygen species, which decreased microbial activity. We conclude that our modification of enzyme assays under anoxic conditions is required for samples taken from low-oxygen environments to avoid underestimation due to rapid suppression of hydrolytic enzyme activities by O2.

Activities of Antioxidant and Proteolytic Systems and Biomarkers in the Fat Body and Hemolymph of Young Apis mellifera Females.

Simple SummaryThe proteolytic system consists of compounds that, similar to “scissors”, cut proteins found in bee cells (e.g., to activate these proteins) or released by pathogens. During these reactions, reactive oxygen species are created and then removed by antioxidants. The actions of the proteolytic and antioxidant systems are enhanced by biomarkers. These compounds are produced mainly in the fat body and then released into the hemolymph. We determined the activities of these compounds in various localizations/segments of the fat body and in the hemolymph in females with increased reproductive potential, i.e., queens and rebels, and in normal (sterile non-rebel) workers. Rebels are workers who resemble the queen in terms of anatomical, behavioural, and physiological features. It was revealed that the activities of these compounds in the rebels were between those of queens and normal workers. Normal workers had higher activities of the proteolytic and antioxidant systems in the fat body and hemolymph than the other females. These results are important for understanding the functioning of the fat body, the stress ecology, and the formation of the different castes of Apis mellifera females.The proteolytic and antioxidant systems are important components of humoral immunity, and these biomarkers indicate the immune status. These compounds are synthesized in the bees’ fat body and released into the hemolymph. Their functions maintain the organism’s homeostasis and protect it against adverse environmental factors (including pathogens). We determined the activities of acidic, neutral, and alkaline proteases and their inhibitors, as well as superoxide dismutase (SOD), catalase (CAT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the level of total antioxidant potential (TAC). These compounds were investigated in the fat body and hemolymph in the females with increased reproductive potential, i.e., queens and rebels, and in normal (non-reproductive sterile non-rebel) workers. The phenoloxidase (PO) activities were determined in the hemolymph. The normal workers had higher activities of proteases and their inhibitors, SOD and CAT, in the fat body and hemolymph, compared to the queens and rebels. The protease inhibitors were not usually active in the queens. As we predicted, the rebels revealed values between those of the queens and normal workers. The highest activities of proteases and antioxidants were identified in the fat body from the third tergite in comparison with the sternite and the fifth tergite. These results are important for oxidative stress ecology and give a better understanding of the functioning of the fat body and the division of labor in social insects.

Improving wheat (Triticum aestivum L.) antioxidative defense mechanisms against salinity stress by exogenous application of potassium silicate

The primary objective of this study was to investigate the beneficial effects of seed priming and foliar spray of potassium silicate on antioxidant activities under different salinity levels, thereby potentially improving wheat growth. Seeds were soaked into solutions containing potassium silicate (K2SiO3, 1.5 mM) for 6 h, while foliar spray with K2SiO3 (4 mM) was applied at the early and the late stages of tillering. Lake Urmia water was used to prepare salinity levels of 0, 3, 5, 8, 10, 12, and 14 dS m − 1. For such traits as anthocyanin, catalase, ascorbate peroxidase, guaiacol peroxidase, and superoxide dismutase activity, an initial increase was observed at lower salinity levels; higher salinities subsequently decreased these traits or they remained mostly constant. Salinity also increased phenol, malondialdehyde, hydrogen peroxide, and polyphenol oxidase, but decreased flavonoid, nitrate content, and nitrate reductase activity. Seed priming and foliar spray provided effective approaches to reduce reactive oxygen species (ROS) manifestation in wheat grown under saline conditions. The improved antioxidant defense abilities by seed priming and foliar spray alleviated the oxidative damage of proteins and lipids and improved nitrate content and nitrate reductase activity.

Rhodnius prolixus Hemolymph Immuno-Physiology: Deciphering the Systemic Immune Response Triggered by Trypanosoma cruzi Establishment in the Vector Using Quantitative Proteomics.

Understanding the development of Trypanosoma cruzi within the triatomine vector at the molecular level should provide novel targets for interrupting parasitic life cycle and affect vectorial competence. The aim of the current study is to provide new insights into triatomines immunology through the characterization of the hemolymph proteome of Rhodnius prolixus, a major Chagas disease vector, in order to gain an overview of its immune physiology. Surprisingly, proteomics investigation of the immunomodulation of T. cruzi-infected blood reveals that the parasite triggers an early systemic response in the hemolymph. The analysis of the expression profiles of hemolymph proteins from 6 h to 24 h allowed the identification of a broad range of immune proteins expressed already in the early hours post-blood-feeding regardless of the presence of the parasite, ready to mount a rapid response exemplified by the significant phenol oxidase activation. Nevertheless, we have also observed a remarkable induction of the immune response triggered by an rpPGRP-LC and the overexpression of defensins 6 h post-T. cruzi infection. Moreover, we have identified novel proteins with immune properties such as the putative c1q-like protein and the immunoglobulin I-set domain-containing protein, which have never been described in triatomines and could play a role in T. cruzi recognition. Twelve proteins with unknown function are modulated by the presence of T. cruzi in the hemolymph. Determining the function of these parasite-induced proteins represents an exciting challenge for increasing our knowledge about the diversity of the immune response from the universal one studied in holometabolous insects. This will provide us with clear answers for misunderstood mechanisms in host–parasite interaction, leading to the development of new generation strategies to control vector populations and pathogen transmission.

Biochemical indexes and gut microbiota testing as diagnostic methods for Penaeus monodon health and physiological changes during AHPND infection with food safety concerns.

Severe shrimp disease outbreaks have a destructive impact on shrimp aquaculture and its associated downstream food processing industries. Thus, it is essential to develop proper methods for shrimp disease control, which emphasizes the importance of food safety. In this study, we performed biochemical tests and gut microbiome analysis using uninfected control and Vp AHPND‐infected Penaeus monodon samples. Biochemical tests were performed to assess the phenoloxidase (PO) activity, respiratory Burst (RB) activity, nitrite concentration, superoxide dismutase (SOD) activity, total hemocyte count (THC), and total protein concentrations. Overall, upregulations were detected in these biochemical tests, which showed the activation of the immune response in P. monodon during acute hepatopancreatic necrosis disease (AHPND) infection, especially at 6 hpi and 12 hpi. Besides that, shrimp gut samples were collected and pooled (n = 3), followed by DNA extraction, PCR amplification targeting the V3/V4 16S ribosomal RNA (rRNA) region, next‐generation sequencing (NGS), and bioinformatics analysis. Proteobacteria was the most abundant phylum in both samples. The Rhodobacteraceae family and Maritimibacter genus were proposed to be vital forshrimp health maintenance. Vp AHPND bacterial colonization and secondary Vibrio infections were postulated to have occurred based on the higher abundances of Vibrionaceae family and Vibrio genus in the Vp AHPND‐infected sample. Firmicutes phylum together with Photobacterium and Aliiroseovarius genera were inferred to be pathogenic or related factors of AHPND infections. In conclusion, physiology (immune response activation) and gut microbiome changes of disease tolerant P. monodon during AHPND infection were identified. Both biochemical tests and 16S rRNA analysis are proposed as a combined strategy for shrimp health diagnosis for ensuring shrimp health maintenance, disease control, and food safety.

The effects of bamboo leaf flavonoids on growth performance, immunity, antioxidant status, and intestinal microflora of Chinese mitten crabs (Eriocheir sinensis)

Bamboo leaf flavonoids (BLF) are the mainly bioactive constituents of bamboo leaves and have caused considerable attention for antibacterial, anti-inflammatory, antioxidant, antitumor, and anti-aging activities. This research evaluated dietary (commercial pellet feed form) addition of Phyllostachys BLF effects on growth, non-specific immunity, antioxidant function, and intestinal microflora of Chinese mitten crabs. A total of 216 crabs were randomly sorted into 18 tanks with 6 replicates for each group and 12 crabs per replicate. Crabs were fed with a basal diet supplemented with 0 (control), 500, and 1000 mg/kg BLF, respectively. Results shown that crabs fed diet supplemented with 1000 mg/kg BLF improved the growth performance, enhanced serum phenoloxidase (PO), lysozyme, total antioxidant capacity (T-AOC), and glutathione peroxidase (GPx) levels, increased hepatopancreas T-AOC and total superoxide dismutase (T-SOD) levels, upregulated hepatopancreas Tube and Dif mRNA expression levels compared with the crabs in the control (P < 0.05). The inclusion of 500 mg/kg BLF only increased the serum lysozyme content (P < 0.05). Dietary 500 or 1000 mg/kg BLF addition increased serum T-SOD and catalase activities, enhanced hepatopancreas GPx activity, reduced the serum malondialdehyde content, upregulated the mRNA expression levels of hepatopancreas GPx, toll-like receptors 1, myeloid differentiation factor 88, lysozyme, and prophenoloxidase of crabs (P < 0.05). Crabs in 1000 mg/kg BLF addition group had higher PO activity than that crabs in the 500 mg/kg BLF addition group (P < 0.05). The separated principal co-ordinates analysis revealed that the three groups had different microbiota composition. Both 500 and 1000 mg/kg BLF addition decreased the shannon and chao1 indexes, reduced the abundance of phyla Bacteroidota, enhanced the abundance of phyla Firmicutes and genus Candidatus_Bacilloplasma in intestine microbiota of Chinese mitten crabs (P < 0.05). In conclusion, dietary BLF inclusion improved the growth performance and intestinal microflora, enhanced the non-specific immunity and antioxidant function, activated the Toll/NF-κB pathway in Chinese mitten crabs.

The injection of one recombinant C-type lectin (LvLec) induced the immune response of hemocytes in Litopenaeus vannamei

To explore the immune function of C-type lectin in shrimp, one recombinant C-type lectin (LvLec) was injected into Litopenaeus vannamei. There were four treatments in the experiment: saline group (as control group), recombinant C-type lectin group (LvLec, 1 mg mL−1), Vibrio harveyi group (V. harveyi, 106 cfu mL−1) and recombinant C-type lectin combined with Vibrio harveyi group (LvLec + V. harveyi, 1 mg mL−1 + 106 cfu mL−1). The sampling time was set at 0, 3, 6, 9, 12, 24 h after the injection. The results showed that the total hemocyte count decreased significantly and the phagocytic activity improved notably after the injection of LvLec, V. harveyi or LvLec + V. harveyi. Prophenoloxidase (proPO) activity decreased, while phenoloxidase (PO) activity increased and the changing degree of each group exhibited a significant difference. The hemagglutinating activity and bacteriolytic activity improved significantly, while the antimicrobial activity did not show a remarkable change in all of the groups. There were also changes that occurred in the levels of second messengers (cAMP, cGMP) and protein kinase (PKA, PKG). After the injection of LvLec, V. harveyi or LvLec + V. harveyi, the concentration of cGMP and PKA increased significantly, while the concentration of cAMP and PKG did not change remarkably. The results above suggested that rLvLec could induce nonspecific immune response, including phagocytosis, release of PO, hemagglutination and bacteriolysis through cGMP-PKA pathway in vivo.

Cleavage activation and functional comparison of Manduca sexta serine protease homologs SPH1a, SPH1b, SPH4, and SPH101 in conjunction with SPH2

Phenoloxidase (PO) is a crucial component of the insect immune response against microbial infection. In the tobacco hornworm Manduca sexta, PO is generated from its precursor proPO by prophenoloxidase activating proteases (PAPs) in the presence of two noncatalytic serine protease homologs (SPHs). cDNA cloning and genome analysis indicate that SPH1a (formerly known as SPH1), SPH1b, SPH4, SPH101, and SPH2 contain a clip domain, a linker, and a protease-like domain (PLD). The first 22 residues of the SPH1b, SPH4, and SPH101 PLDs are identical, and differ from SPH1a only at position 4, Thr154 substituted with Asn154 in SPH1a. While the sequence from Edman degradation was used to establish PAP cofactor as a high Mr complex of SPH1a and SPH2, this assignment needed further validation, especially because SPH1b mRNA levels are much higher than SPH1a′s and better correlate with SPH2 transcription. Thus, here we determined expression profiles of these SPH genes in different tissues from various developmental stages using highly specific primers. High levels of SPH1b and SPH2 proteins, low SPH4, and no SPH1a or SPH101 were detected in hemolymph from larvae in the feeding, wandering and bar stages, pupae, and adults by targeted LC-MS/MS analysis, based on unique peptides from the trypsin-treated SPHs. We expressed the five proSPHs in baculovirus-infected Sf9 cells for use as standards to identify and quantify their counterparts in plasma samples. Moreover, we tested their cleavage by PAP3 and efficacy of the SPH1a, 1b, 4, and 101 as SPH2 partners in PAP3-mediated proPO activation. PAP3 processed proSPH1b and 101 more readily than proSPH1a and 4; PAP3 activated proPO more efficiently in the presence of SPH2 with SPH101 or SPH1b than with SPH1a or SPH4. These results generally agree with their order of appearance or sequence similarity: SPH101 > SPH1b (98%) > SPH1a (90%) > SPH4 (83%). In other words, likely due to positive selection, products of the newly duplicated genes (SPH1b and SPH101) are more favorable substrates of PAP3 and better SPH2 partners in forming a high Mr cofactor than SPH1a or SPH4 is. Electrophoresis on native gel and immunoblot analysis further indicated that SPH101 or 1b form high Mr complexes more readily than SPH1a or 4 does. In comparison, SPH2 showed a small mobility decrease and then increase on native gel after PAP3 cleavage at the first site. Since the natural cofactor in bar-stage hemolymph is complexes of SPH1 and 2 with an average Mr of 790 kDa, PAP3-activated SPH2 may associate with the higher Mr SPH1b scaffolds to form super-complexes. Their structures and formation in relation to cleavage of SPH1b at different sites await further exploration.

HMG-Like DSP1 Mediates Immune Responses of the Western Flower Thrips (Frankliniella occidentalis) Against Beauveria bassiana, a Fungal Pathogen.

Western flower thrips, Frankliella occidentalis, is a serious pest by directly infesting host crops. It can also give indirect damage to host crops by transmitting a plant virus called tomato spotted wilt virus. A fungal pathogen, Beauveria bassiana, can infect thrips. It has been used as a biopesticide. However, little is known on the defense of thrips against this fungal pathogen. This study assessed the defense of thrips against the fungal infection with respect to immunity by analyzing immune-associated genes of F. occidentalis in both larvae and adults. Immunity-associated genes of western flower thrips were selected from three immunity steps: nonself recognition, mediation, and immune responses. For the pathogen recognition step, dorsal switch protein 1 (DSP1) was chosen. For the immune mediation step, phospholipase A2 (PLA2) and prostaglandin E2 synthase were also selected. For the step of immune responses, two phenoloxidases (PO) genes and four proPO-activating peptidase genes involved in melanization against pathogens were chosen. Dual oxidase gene involved in the production of reactive oxygen species and four antimicrobial peptide genes for executing humoral immune responses were selected. All immunity-associated genes were inducible to the fungal infection. Their expression levels were induced higher in adults than in larvae by the fungal infections. However, inhibitor treatments specific to DSP1 or PLA2 significantly suppressed the inducible expression of these immune-associated genes, leading to significant enhancement of fungal pathogenicity. These results suggest that immunity is essential for thrips to defend against B. bassiana, in which DSP1 and eicosanoids play a crucial role in eliciting immune responses.