Phenoloxidase Enzyme Activity Research Articles

Photooxidation of pyrogenic organic matter reduces its reactive, labile C pool and the apparent soil oxidative microbial enzyme response

The surface chemistry of pyrogenic organic matter (PyOM) is altered by a variety of abiotic and biotic oxidative and sorption/desorption processes in the environment. Exposure of PyOM to high energy light prior to addition to soil or sediment, or while entrained in the atmosphere, may induce significant surface photooxidation, i.e., photochemical weathering, altering its environmental reactivity. We report on a 30-day soil incubation experiment testing the effects of the photochemical weathering of a 13C-enriched ponderosa pine PyOM, produced by pyrolysis at 450°C, on PyOM and soil organic carbon (SOC) mineralization. PyOM C mineralization was measured for both the photochemically weathered (i.e. UV treated PyOM or PyOMUV) and PyOM not exposed to high-energy light (i.e. PyOMW serving as a dark control). PyOMW exhibited a 3.7 times faster C mineralization rate across the 30-d study, which was driven by a large early mineralization of accessible/labile C during the first 6d. In contrast, PyOMUV had faster C mineralization rates in the later part of the experiment (days 11–30). Overall, PyOMUV had a 13% lower net C mineralization than the untreated PyOMW where the MRT of accessible PyOMUV-C and PyOMW-C was calculated at 25.7±6.8d and 1.7±0.2d, respectively. Both forms of PyOM promoted a similar net reduction in native SOC mineralization (i.e., negative priming) of approximately 50% relative to the unamended, control soil. Addition of either PyOM form resulted in an equivalent minor decrease in the concentration of extractable soil lignin phenols and substituted fatty acids chemistry with respect to the unamended soil. At 30d, soil phenol oxidase and peroxidase enzyme activities were higher with additions of either form of PyOM compared with the unamended control soils with PyOMUV exhibiting lower activities than PyOMW. Our results indicate that PyOM photochemical weathering can impart important changes to short-term PyOM reactivity and soil microbial activity, which could have important implications for soil systems by ultimately lowering turnover rates for both NSC and PyOM-C.

Ảnh hưởng của hỗn hợp dược liệu lên tăng trưởng, tỷ lệ sống và đáp ứng miễn dịch của tôm thẻ chân trắng (Litopenaeus vannamei) nuôi thâm canh trong bể

This study is aimed to determine the effects of herbal mixture concentrations on growth, survival rate and immune responses of white leg shrimp in tank culture system. The experiment consisted of four treatments with different concentration of herbal mixtures as follow: (i) 20 mL/kg of feed, (ii) 40 mL/kg of feed, (iii) 60 mL/kg of feed, and (iv) control (no herbal extracts). After three months, the shrimp in control treatment showed the lowest results in body weight (11.8±1.1 g), body length (12.2±0.5 cm), productivity (1,278±149 g/m3) and significant difference (p 0.05) among treatments. Some indicators of shrimp immune response including the total leukocyte count, granulocytes and phenoloxidase enzyme activity (PO) in 20 mL/kg treatment were increased and significantly different (p

Characterization and pathogenicity assessment of gut-associated microbes of muga silkworm Antheraea assamensis Helfer (Lepidoptera: Saturniidae)

Antheraea assamensis Helfer (muga silkworm) is an economically important endemic insect species of North Eastern Region of India. The silkworm is often susceptible to infection by pathogenic bacteria, leads to a disease commonly known as flacherie which causes 40% loss per annum to the silk industry. In this study, we have reported isolation, characterization and pathogenicity assessment of gut-associated bacteria of healthy and diseased muga silkworms. Thirty five bacterial isolates were screened from the gut of healthy and diseased silkworms by morphological observation and biochemical tests. 11 and 5 strains from healthy and diseased silkworm respectively were identified by 16S rRNA gene sequencing and analyzed. Pseudomonas aeruginosa (DRK1), Ornithinibacillus bavariensis (DRK2), Achromobacter xylosoxidans (KH3) and Staphylococcus aureus (FLG1) strains were commonly found in healthy as well as diseased larvae whereas, Bacillus thuringiensis (MK1) was found only in diseased larvae. Survivability analysis was performed with the identified strains by injection and oral administration (104CFU/ml). The immune response of the silkworm against the pathogen was also studied by phenoloxidase and lysozyme enzyme activity assay, total and differential hemocyte count and phagocytic activity of hemocytes. It was observed that S. aureus, P. aeruginosa and B. thuringiensis significantly reduced the survivability of silkworm (p<0.001) hence found highly pathogenic. The lethal concentrations (LC50) values of the pathogenic strains were calculated at different time intervals (24, 48, 72 and 96h) within the range from 1.38×102 to 3.63×107CFU/ml. The pathogenic groups demonstrated inhibition of phenoloxidase activity and decreased in total hemocyte count after 48h of infection. However, the lysozyme activity increased significantly in the pathogenic groups compared to the control (p<0.05). Granulocytes and plasmatocytes showed phagocytosis whereas; other types of cells did not show any phagocytic activity. Increasing granulocytes and plasmatocytes counts corroborates the results of phagocytic activity. The present study might be helpful in understanding the disease prognosis and colonization of bacteria causing the disease in muga silkworm.

Effects of T-2 toxin on growth, immune function and hepatopancreas microstructure of shrimp (Litopenaeus vannamei)

T-2 toxin (T-2) is a trichothecene-A-type mycotoxin produced by the fungus Fusarium spp., which can contaminate animal feed. It is toxic to many living cells especially the hepatocytes and bone marrow cells. The effects of dietary T-2 on growth, biochemical and immunity parameters, and histopathological changes in the hepatopancreas of white shrimp (Litopenaeus vannamei) were studied. Shrimp with an initial body weight of 3.5±0.5g were fed five diets (n=50/group) containing 0, 0.5, 1.2, 2.4, or 4.8mgkg−1 T-2. Dietary exposure to T-2 significantly decreased shrimp growth rate and specific growth rate (P<0.5), but not the fat content, compared with the control group. Exposure to T-2 damaged the hepatopancreas microstructure in a dose-dependent manner and this may have contributed to a lower hemolymph albumin concentration and lower phenoloxidase activity. Total hemocyte counts and activity of hemolymph glutamic-pyruvic transaminase>glutamic-oxaloacetic transaminase and alkaline phosphatase enzymes increased initially in a dose-dependent manner but declined thereafter. This study showed that T-2 is a multi-organ toxin with effects on the hemolymph, immune system, and hepatopancreas of shrimp. Collectively, these effects may have contributed to the inhibition of growth and decreased survival rate leading to a lower yield and quality of shrimp meat with resultant economic loss. Statement of relevanceIt is proposed that hemolymph phenoloxidase enzyme activity could be used as a cheap non-specific biomarker for initial screening of shrimp exposed to even low T-2 toxin concentrations, instead of more expensive LC-MS/MS T-2 analysis — although a dose response was not evident at high exposure concentrations.

Response of the insect immune system to three different immune challenges

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD50 dose of LPS from Escherichia coli elicited a more rapid response than an LD50 dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2hpi, followed by an increase by 7dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system’s response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.

In vitro effects of the nonsteroidal anti-inflammatory drug, ibuprofen, on the immune parameters of the colonial ascidian Botryllus schlosseri

In this study, in vitro effects of ibuprofen (IBU) on the immune parameters of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed for 1h to 0 (control), 100 and 1000μg IBU/L and the effects on haemocyte viability and morphology (shape factor), lysosomal membrane stability (Neutral Red Retention Assay), phagocytic activity, apoptosis (TUNEL reaction), hydrolytic (acid phosphatase) and oxidative (phenoloxidase and peroxidase) enzyme activities were evaluated. The exposure of haemocytes to IBU did not affect significantly their viability, but increased the percentage of cells with round shape. IBU caused a significant reduction in both phagocytic activity and lysosomal membrane stability. The percentage of haemocytes positive to TUNEL reaction (indicative of DNA fragmentation) increased significantly after IBU exposure. Significant decreases in the percentage of haemocytes positive to acid phosphatase were recorded at 1000μg/L of IBU. Conversely, no significant variations were recorded in the percentage of haemocytes positive to phenoloxidase and peroxidase. Results obtained indicate that exposure of ascidian haemocytes to IBU induces marked alterations in cell functionality. Immunomarkers measured in this study are sensitive, rapid and reproducible. However, their responsiveness and biological relevance will need to be verified for in vivo exposure.

Effects of metal pollution on survival and physiological responses in Carabus (Chaetocarabus) lefebvrei (Coleoptera, Carabidae)

The aim of this study was to evaluate the effects of heavy metal pollution on life cycle traits and physiological responses of Carabus (Chaetocarabus) lefebvrei Dejean 1826. The bioaccumulation factor revealed that As and Hg were bioaccumulated in adults collected in the field close to an urban solid waste site, a source of heavy metal pollution. The ultrastructure of Malpighian tubules was investigated as a marker of adult detoxification capability. The metal contents recorded in adults emerging from laboratory rearing indicate that the uptake of metals or their transfer along the food chain is related to the concentrations in soil and food. The levels of elements such as B, Cr and Cu in the beetle body are closely related to their amounts in the soil. Some elements are accumulated with respect to the levels in food, in the following rank order: Hg > Cr > B > Be > Pb > V = Zn = As. Larval growth and survival do not vary significantly with the metal concentrations recorded in soil and food. The immune response, indicated by phenoloxidase enzyme activity, is highly sensitive to environmental pollution in the preimaginal stages and thus can be used as an early warning parameter to assess the sublethal effects of heavy metal pollution.

Maturation of the immune system of the male house cricket, Acheta domesticus

The immune system functions to counteract the wide range of pathogens an insect may encounter during its lifespan, ultimately maintaining fitness and increasing the likelihood of survival to reproductive maturity. In this study, we describe the maturation of the innate immune system of the male house cricket Acheta domesticus during the last two nymphal stages, and during early and late adulthood. Total hemolymph phenoloxidase enzyme activity, lysozyme-like enzyme activity, the number of circulating hemocytes, and encapsulation ability were all determined for each developmental stage or age examined. The number of circulating hemocytes and lysozyme-like enzyme activity were similar for all developmental stages examined. Nymphs and newly molted adult males, however, had significantly lower total phenoloxidase activity than later adult stages, yet nymphs were able to encapsulate a nylon thread just as well as adults. Encapsulation ability would thus appear to be independent of total phenoloxidase activity.

Effect of Plant Based Essential Oil on Immune Response of Silkworm, Antheraea assama Westwood (Lepidoptera: Saturniidae)

Botanicals have been regarded as sustainable solution of pest control because of their nominal effect on nontarget pest, biodegradability and other environment friendly properties. However, study on impact of botanicals against beneficial insects is less. In this investigation effort has been made to study the effect of essential oils of Ocimum sanctum, Ocimum gratissimum and Ageratum conyzoides against non-mulberry silkworm Antheraea assama in terms of percent mortality, total haemocyte count (THC) and phenol oxidase (PO) enzyme activity. After application of essential oil of the plants by contact residual film technique, age dependent decrease of percent mortality was recorded. Topical treatment of oil at higher dosages (<TEX>$1{\mu}lg^{-1}$</TEX> and <TEX>$0.5{\mu}lg^{-1}$</TEX>) caused 100% mortality of late instar larvae at 24 hour but the lowest dose (<TEX>$0.25{\mu}lg^{-1}$</TEX>) did not cause any mortality till 48 hour of treatment. Initial increase of total haemocyte count (THC) was recorded at early hours of treatment (0.25 hour) at all dosages (1, 0.5, <TEX>$0.25{\mu}lg^{-1}$</TEX>) after topical treatment. A dose dependent increase was observed in phenol oxidase enzyme activity and this might indicate ability of the essential oils to cause induction of immune response in Antheraea assama larvae.

Bacterial community response to tillage and nutrient additions in a long-term wheat cropping experiment

The effects of agricultural management practices on bacterial community structure and function are not well defined, yet are of concern for long-term soil resilience. In this study, soil microbial biomass, bacterial community structure (determined by TRFLP), and function, (determined by enzyme assays and Biolog assays), and soil physicochemical properties were investigated in a wheat cropping system subjected to long-term tillage (20 years) and short-term nutrient addition treatments. Samples were taken over a full cropping cycle. Tillage, nutrient addition, and time all significantly affected bacterial community structure (rDNA and rRNA), which showed considerable shifts across the sampling period. Microbial biomass and Biolog profiles changed significantly with time, but were not affected by treatment. With regard to specific enzyme assays, there were significant main effects of treatment and time on glucosidase, phosphatase and phenol oxidase enzyme activity, while for cellobioside and peroxidase, treatment and time had significant interactive effects. For the hydrolases significant effects were observed between nutrient treatments, while for the oxidases they were observed between tillage treatments. Overall, however, we found little evidence of major detrimental long-term effects of tillage on the soil bacterial communities or their important functions in the dryland wheat system studied. The bacterial communities showed both long and short-term trajectories that could be disentangled with appropriate experimental design. Concerns over significant long-term detrimental impacts of tillage on the soil bacterial communities appear unfounded, at least under systems similar to those studied here.

Bacterial and enchytraeid abundance accelerate soil carbon turnover along a lowland vegetation gradient in interior Alaska

Boreal wetlands are characterized by a mosaic of plant communities, including forests, shrublands, grasslands, and fens, which are structured largely by changes in topography and water table position. The soil associated with these plant communities contain quantitatively and qualitatively different forms of soil organic matter (SOM) and nutrient availability that drive changes in biogeochemical cycling rates. Therefore different boreal plant communities likely contain different soil biotic communities which in turn affect rates of organic matter decomposition. We examined relationships between plant communities, microbial communities, enchytraeids, and soil C turnover in near-surface soils along a shallow topographic soil moisture and vegetation gradient in interior Alaska. We tested the hypothesis that as soil moisture increases along the gradient, surface soils would become increasingly dominated by bacteria and mesofauna and have more rapid rates of C turnover. We utilized bomb radiocarbon techniques to infer rates of C turnover and the 13C isotopic composition of SOM and respired CO2 to infer the degree of soil humification. Soil phenol oxidase and peroxidase enzyme activities were generally higher in the rich fen compared with the forest and bog birch sites. Results indicated greater C fluxes and more rapid C turnover in the surface soils of the fen sites compared to the wetland forest and shrub sites. Quantitative PCR analyses of soil bacteria and archaea, combined with enchytraeid counts, indicated that surface soils from the lowland fen ecosystems had higher abundances of these microbial and mesofaunal groups. Fungal abundance was highly variable and not significantly different among sites. Microbial data was utilized in a food web model that confirmed that rapidly cycling systems are dominated by bacterial activity and enchytraeid grazing. However, our results also suggest that oxidative enzymes play an important role in the C mineralization process in saturated systems, which has been often ignored.

Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricketGryllus texensis

In many parts of the world, climate change is increasing the frequency and severity of heat waves. How do heat waves impact short-lived poikilotherms such as insects? In the cricket, Gryllus texensis, 6 days of elevated temperatures (i.e. 7°C above the average field temperature and 5°C above their preferred temperature) resulted in increased egg laying, faster egg development and greater mass gain. The increased temperature also increased activity of phenoloxidase and lysozyme-like enzymes, two immune-related enzymes, and enhanced resistance to the Gram-negative bacterium Serratia marcescens. When given a sublethal S. marcescens infection, G. texensis maintained increased reproductive output at the elevated temperature (33°C). These data suggest that heat waves could result in more numerous, disease resistant, crickets. However, resistance to the Gram-positive bacterium, Bacillus cereus was lower at temperatures above or below the average field temperature (26°C). A sublethal infection with B. cereus reduced egg laying at all temperatures and suppressed the increase in egg laying induced by higher temperatures. These results suggest that for some species-pathogen interactions, increased temperatures can induce trade-offs between reproduction and disease resistance. This result may partly explain why G. texensis prefers temperatures lower than those that produce maximal reproductive output and enhanced immune function.

Characterization of trapped lignin-degrading microbes in tropical forest soil.

Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition.

Bacterial Metabolites of an Entomopathogenic Bacterium, Xenorhabdus nematophila, Inhibit a Catalytic Activity of Phenoloxidase of the Diamondback Moth, Plutella xylostella

A monoterpenoid compound, benzylideneacetone (BZA), is identified from bacterial metabolites synthesized by an entomopathogenic bacterium, Xenorhabdus nematophila. It inhibits phospholipase A2 of target insects to shut down biosynthesis of various eicosanoids, which play significant roles in insect immunity. This study discovered another novel activity of BZA that directly inhibited phenoloxidase (PO) activity required for immune-associated melanization. When it was injected into larvae of Plutella xylostella, it suppressed PO activity in the plasma by inhibiting its activation from inactive proPO. However, BZA did not influence on gene expression of PO, which was analyzed by RT-PCR using gene-specific primers designed from a partial cDNA sequence of PO of the P. xylostella identified in this study. To test a direct inhibitory activity of BZA against PO, the activated PO of P. xylostella was prepared from the hemolymph collected from the larvae challenged by bacteria. When the activated PO was incubated in vitro with BZA, it was inhibited in a dose-dependent manner. The inhibition of PO by BZA was recovered by addition of increasing amounts of substrate, L-3,4-dihydroxyphenylalanine. Three other known bacterial metabolites containing a benzene propane core structure synthesized by X. nematophila also inhibited the PO enzyme activity. However, modification of the core structure by hydroxylation of BZA lost its strong inhibitory activity against the activated PO.

Inhibitors of oxidative enzymes affect water uptake and vase life of cut Acacia holosericea and Chamelaucium uncinatum stems

Cut Acacia holosericea (Velvet Leaf Wattle) foliage has a short vase life, possibly because of blockage in xylem vessels. We indirectly investigated a hypothesised role for peroxidase and phenoloxidase enzyme activities in xylem occlusion of Acacia stems by using their inhibitors. We also tested these inhibitors with cut Chamelaucium uncinatum (Geraldton waxflower), another woody stemmed cut flower.The peroxidase inhibitors used were 3-amino-1,2,4-triazole (AT), catechol (CH), hydroquinone (HQ), p-phenylene diamine (PD) and copper sulphate (CS, Chamelaucium only). The catechol oxidase inhibitors were tropolone (TP), 4-hexylresorcinol (HR) and 2,3-dihydroxynaphthalene (DN). A laccase inhibitor, cetyltrimethylammonium bromide (CM), was also used. Other phenoloxidase inhibitors tested were p-chlorophenol (CP), p-nitrocathechol (NC), p-nitrophenol (NP) and sodium metabisulphite (SM). 2-Mercaptoethanol (ME), phenyl hydrazine (PH) and salicylhydroxamic acid (SH) were used as inhibitors of both peroxidase and phenoloxidase.Twelve inhibitors (CH, HQ, DN, HR, TP, CM, CP, NC, NP, SM, PH and SH) significantly improved water uptake, maintained relative fresh weight and increased vase life of Acacia at least once in two experiments. In Chamelaucium, six inhibitors had significant positive effects on water relations (CH, PD, CS, CM, CP and PH) and vase life (AT, CH, PD, CS, ME and PH), while four of them (DN, TP, NC and NP) were phytotoxic at applied concentrations. Only one of the 46 inhibitor treatments inhibited transpiration and increased fresh weight, suggesting that the inhibitors mainly acted by increasing water uptake. Overall, results indicate that oxidative enzyme activities, potentially through phenolic deposition, contribute to xylem occlusion in woody cut stems of Acacia and Chamelaucium.

Dredging influenced sediment toxicity of endosulfan and lindane on black tiger shrimp ( Penaeus monodon Fabricius) in Chantaburi River estuary in Thailand

Abstract Series of experiments, including background sediment elutriate, spiked-sediment elutriate and spiked-water toxicity tests, were carried out to study the effect of dredging on survivability and ecological competence of commercially important shrimp postlarvae of Penaeus monodon Fabricius. The test sediment represented the most contaminated from endosulfan and lindane in the Chantaburi river mouth estuary. The maximum sediment contamination levels were 49.4 μg/kg of endosulfan and 58.1 μg/kg of lindane in dry weight basis. Dose-response studies in static, non-renewal spiked-sediment elutriate toxicity tests revealed that LC 50 (96 h) values for survival effect based on measured concentrations were 0.50 (0.26–0.95) μg/L for endosulfan and 0.92 (0.63–1.34) μg/L for lindane. The LOECs for endosulfan and lindane were 0.141 and 0.429 μg/L, respectively. The NOECs were estimated to be the dissoluted endosulfan and lindane concentrations from the Chantaburi estuary sediment. The effects on phenoloxidase enzyme activity in shrimp postlarvae exposed for sub-lethal to lethal doses in sediment elutriate were not significant (p>0.05) at all tested dose levels. The worst case dissolution concentrations of endosulfan and lindane in the water column upon simulatory dredging in the Chantaburi estuary were found to be toxic to shrimp postlarvae on lethal and sub-lethal effects. The NOECs of respective chemicals (i.e., 0.034 and 0.16 μg/L) shared the same orders of magnitude for corresponding NOECs established for whole salt water aquatic life by the USEPA and “safe” levels calculated by 0.01 safety factor approach. In addition, the significance of multiple pollutant assessments for ecological toxicity testing is highlighted.

A clip domain serine proteinase plays a role in antibacterial defense but is not required for prophenoloxidase activation in shrimp

The clip domain serine proteinases (clip-SPs) play critical roles in the signaling processes during embryonic development and in the innate immunity of invertebrates. In the present study, we identified a homolog of the clip-SP, designated as PmClipSP1, by searching the Penaeus monodon EST database (http://pmonodon.biotec.or.th), and using RACE-PCR to obtain the complete gene which contained a 1101bp open reading frame encoding 366 amino acids with a 25 amino acid signal peptide. The deduced PmClipSP1 protein sequence, which shares a predicted structural similarity to the clip-SPs of other arthropod species, appears to possess a clip domain at the N-terminus and an enzymatically active serine proteinase domain at the C-terminus. Tissue distribution analyses revealed that, at the transcript level, PmClipSP1 is mainly expressed in shrimp hemocytes, whilst temporal gene expression analyses showed that the hemocyte PmClipSP1 transcript levels were upregulated at 3h and downregulated at 6–48h following systemic Vibrio harveyi infection. RNAi-mediated silencing of the PmClipSP1 gene, by injection of double-stranded RNA (dsRNA) corresponding to the PmClipSP1 gene into shrimp, significantly reduced PmClipSP1 transcript levels, but neither significantly altered the other clip-SP and clip-SPH transcript levels nor reduced the total phenoloxidase (PO) enzyme activity in shrimp hemocytes, compared to the levels seen in the GFP dsRNA control, suggesting that PmClipSP1 is not involved in the proPO system. However, suppression of the PmClipSP1 gene led to a significant increase in the number of viable bacteria in the hemolymph (∼2.4-fold) and in the mortality rate (59%) of shrimp systemically infected with V. harveyi. These findings suggest that PmClipSP1 plays a role in the antibacterial defense mechanism of P. monodon shrimp.

The innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvae

BackgroundThere is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of social immunity, yet some specific immune factors are up-regulated in response to infection. To explore the response to infection more broadly, we employ mass spectrometry-based proteomics in a quantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, but become progressively more resistant with age. We used this host-pathogen system to probe not only the role of the immune system in responding to a highly evolved infection, but also what other mechanisms might be employed in response to infection.ResultsUsing quantitative proteomics, we compared the hemolymph (insect blood) of five-day old healthy and infected honey bee larvae and found a strong up-regulation of some metabolic enzymes and chaperones, while royal jelly (food) and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO), lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidence suggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymph that is proteolytically activated upon infection. Phenoloxidase (PO) enzyme activity was undetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling very closely the age-related ability of larvae to resist infection.ConclusionWe propose a model for the host response to infection where energy stores and metabolic enzymes are regulated in concert with direct defensive measures, such as the massive enhancement of PO activity.

Interactive effects of wildfire and permafrost on microbial communities and soil processes in an Alaskan black spruce forest

AbstractBoreal forests contain significant quantities of soil carbon that may be oxidized to CO2 given future increases in climate warming and wildfire behavior. At the ecosystem scale, decomposition and heterotrophic respiration are strongly controlled by temperature and moisture, but we questioned whether changes in microbial biomass, activity, or community structure induced by fire might also affect these processes. We particularly wanted to understand whether postfire reductions in microbial biomass could affect rates of decomposition. Additionally, we compared the short‐term effects of wildfire to the long‐term effects of climate warming and permafrost decline. We compared soil microbial communities between control and recently burned soils that were located in areas with and without permafrost near Delta Junction, AK. In addition to soil physical variables, we quantified changes in microbial biomass, fungal biomass, fungal community composition, and C cycling processes (phenol oxidase enzyme activity, lignin decomposition, and microbial respiration). Five years following fire, organic surface horizons had lower microbial biomass, fungal biomass, and dissolved organic carbon (DOC) concentrations compared with control soils. Reductions in soil fungi were associated with reductions in phenol oxidase activity and lignin decomposition. Effects of wildfire on microbial biomass and activity in the mineral soil were minor. Microbial community composition was affected by wildfire, but the effect was greater in nonpermafrost soils. Although the presence of permafrost increased soil moisture contents, effects on microbial biomass and activity were limited to mineral soils that showed lower fungal biomass but higher activity compared with soils without permafrost. Fungal abundance and moisture were strong predictors of phenol oxidase enzyme activity in soil. Phenol oxidase enzyme activity, in turn, was linearly related to both 13C lignin decomposition and microbial respiration in incubation studies. Taken together, these results indicate that reductions in fungal biomass in postfire soils and lower soil moisture in nonpermafrost soils reduced the potential of soil heterotrophs to decompose soil carbon. Although in the field increased rates of microbial respiration can be observed in postfire soils due to warmer soil conditions, reductions in fungal biomass and activity may limit rates of decomposition.

Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon

Phenoloxidase (PO) plays an important role in arthropod melanization. Previously, a prophenoloxidase ( PmproPO1) gene was cloned and characterized from the hemocytes of the black tiger shrimp, Penaeus monodon. In the present study, we report a novel proPO gene ( PmproPO2) belonging to the proPO family identified from the P. monodon EST database ( http://pmonodon.biotec.or.th). The full-length sequence of PmproPO2 consists of 2513 bp encoding a predicted 689 amino acid residues with a calculated molecular mass and p I of 79.21 kDa and 6.69, respectively. It is predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues and a thiol ester-like motif, sharing 67% amino acid sequence identity with PmproPO1. Tissue distribution analyses revealed that the two proPO genes are primarily expressed in the hemocyte. Gene silencing of either PmproPO1 or PmproPO2 or both by RNA interference (RNAi) resulted in a significant decrease in the respective endogenous proPO mRNA level in hemocytes and a reduction of total PO enzyme activity by 75, 73 and 88%, respectively. Experimental infection of P. monodon with the pathogenic bacterium, Vibrio harveyi, revealed that PmproPO silenced shrimps were more susceptible to bacterial infection than the control GFP injected shrimps, and suggesting that the two proPOs are important components in the shrimp immune defense.