Inhibitory Effects of Kojic Acid on Phenoloxidase of Diamondback Moth Plutella xylostella

The effects of kojic acid on phenoloxidase (PO) of Plutella xylostella were investigated, when it had been partially purified by 40% saturated [(NH4)2SO4] and Sephadex G-100 gel filtration. Kojic acid showed inhibitory effects on both monophenolase and o-diphenolase activity of the PO. The inhibitor concentrations leading to 50% (IC50) activity lost were estimated to be 0.07 mmol L-1 for monophenolase and 1 mmol L-1 for diphenolase, respectively. Kojic acid can also prolong the lag time of PO for oxidation of L-tyrosine. The inhibition kinetics were analyzed by Lineweaver-Burk plots and kojic acid was found to be a reversible competitive inhibitor with the Ki of 0.47 mmol L-1. The ability of kojic acid to inhibit the enzyme activity may be associated with its ability to chelate copper at the active site. In addition, the iron ion was found to shorten the lag time obviously, but have no important effect on the monophenolase activity.

Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species

Phenoloxidases(POs)are a group of copper proteins including tyrosinase,catecholase and laccase,which play crucial roles in the innate immune response of mollusks.In this research,POs were studied in cultured mollusk species,including scallop Chla-mys farreri,abalone Haliotis discus hannai and clam Scapharca subcrenata.The POs were isolated from hemocytes using linear-gradient native-PAGE combined with catechol staining.The PO activities and their characters were investigated.The molecular mass of PO in C.farreri was 576 kDa,and it was 228 kDa in H.discus hannai.In S.subcrenata,four POs were detected and their mole-cular masses were 391 kDa,206 kDa,174 kDa and<67 kDa,which were named as 391-PO,206-PO,174-PO and s-PO,respectively.Ki-netic analyses indicated that all of the POs,except for 391-PO had higher affinity to L-DOPA and catechol than to hydroquinone and dopamine.However,all of the POs failed to oxidize tyrosine.The effects of divalent metal ions on POs’activities were assayed,in-cluding Fe^(2+),Mg^(2+),Zn^(2+),Mn^(2+),Cu^(2+)and Ca^(2+)from FeCl_(2),MgSO_(4),ZnSO_(4),MnCl_(2),CuSO_(4)and CaCl_(2).The POs were inhibited by Fe^(2+)at all determined concentrations.Additionally,the inhibition assay showed that all of the POs were inhibited by cysteine,ascorbic acid,sodium sulfite,citric acid,ethylenediaminetetraacetic acid disodium(EDTA)and sodium diethyldithiocarbamate(DETC).The inhibition effects of critric acid and EDTA are dose-dependent.H.discus hannai PO and 391-PO were slightly inhibited by sodium azide,and H.discus hannai PO,391-PO and 174-PO were slightly inhibited by thiourea.In conclusion,the POs in the three cultured mollusks are copper-containing laccase-type phenoloxidases with similar biochemical characteristics even though their molecular masses are different.

A silkworm hemolymph protein is a prophenoloxidase activation blocker

Melanization in insect hemolymph is triggered by the recognition of pathogen-associated molecular patterns via pattern recognition receptors. The signal transduction leads to the activation of the prophenoloxidase and hence the generation of melanin. The proPO activation process must be tightly controlled to minimize the host damage caused by reactive intermediates during melanin synthesis. The full-length cDNA sequence of a 20 kDa hemolymph protein from Bombyx (Bmhp20) was determined. Bmhp20 gene was expressed in larval fat body, integument, trachea, and ovary and was induced by the challenge of B. bombyseptieus. Binding of recombinant Bmhp20 to microbial cell wall components as well as gram-positive bacteria and fungi was confirmed. Phenoloxidase activity assay indicated that recombinant Bmhp20 blocked the proPO activation in hemolymph that was triggered by peptidoglycan or beta-1, 3-glucan. Our data suggest that Bmhp20 plays bifunctional roles in silkworm humoral responses: to participate in pattern recognition and to block the activation of proPO.

Effect of the endoparasitoid Campoletis chlorideae on phenoloxidase activity in Helicoverpa armigera hemo-lymph

Endoparasitoid wasps can develop inside permissive host due to their ability to overcome or to evade the host’s cellular and humoral immune response. Oviposition of Campoletis chlorideae (Hymenoptera: Ichneumonidae) in larvae of Helicoverpa armigera (Lepidoptera) was accompanied by inhibition of phenoloxidase (PO) activity and melanization reaction in host hemolymph in vitro. PO activity in host plasma was decreased about 83% 48 h post para-sitization. A similar result was found when the host insect was injected with 0.5 wasp equivalent calyx fluid. This indicated that the calyx fluid was concerned with suppression of PO activity after parasitization. Furthermore, the prophen-oloxidase (proPO) in host haemocytes could be activated by bovine trypsin in unparasitized insects, while it could not be activated in parasitized or calyx fluid-injected host. The results suggested that inhibition of PO activity by parasitization was related to the calyx fluid of Campoletis chlorideae, and the components of calyx

Enzymatic properties of phenoloxidase from Pieris rapae （Lepidoptera） larvae

The kinetic parameters of partially purified phenoloxidase （PO, EC. 1.14.18.1） from the 5th instar larvae of Pieris rapae （Lepidoptera） were determined, using L-3, 4- dihydroxyphenylalanine （L-DOPA） as substrate. The optimal pH and temperature of the enzyme for the oxidation of L-DOPA were determined to be at pH 7.0 and at 42℃, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37℃. At pH 6.8 and 37℃, the Michaelis constant （Kin） and maximal velocity （V） of the enzyme for the oxidation of L-DOPA were determined to be 0.80 mmol/L and 1.84 μmol/ L/min, respectively. Tetra-hexylresorcinol and 4-dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC50 of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47μmol/L, respectively.

Effects of 4-hexylresorcinol on the phenoloxidase from Hyphantria cunea （Lepidoptera： Arctiidae）： In vivo and in vitro studies

Insecticidal effects of 4-hexylresorcinol, a phenoloxidase （PO） inhibitor, were determined on Hyphantria cunea （Drury） under laboratory conditions. The LC50 for the 15- d-old larvae was estimated to be 2.95 g/L after 96 h exposure. The activities of glutathione S-transferase （GST） and PO showed a decrease in larvae treated with 4-hexylresorcinol, and the IC50 of GST and PO were estimated to be 0.8 and 0.43 g/L, respectively, 24 h after treatment. The PO from the hemolymph of fall webworm was purified by ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography, and then enzymatic characteristics and the mechanism of inhibition were determined using L-dihydroxyphenylalanine （L-DOPA） as the substrate. The purified PO showed a single band on SDS-PAGE with a molecular weight of about 70 kDa. The optimum pH for PO activity was observed at pH 7.0, optimum temperature was found to be 45 ℃, and PO activity was strongly inhibited by Zn2＋. IC50 values were estimated to be 8.2, 19.14, and 24.04/zmol/L for 4-hexylresorsinol, kojic acid, and quercetin, respectively. The inhibitory potencies （i.e., 150 of each compound/Is0 of 4-hexylresorcinol） ofkojic acid and quercetin on H. cunea PO were estimated to be 1.87 and 2.89, respectively. 4-hexylresorcinol was determined to be a competitive inhibitor, and kojic acid and quercetin were determined to be mixed inhibitors. PO is one of the most important enzymes in an insect＇s immune system, and the use ofPO inhibitors seems to be a promising approach for pest control due to their potential safety for humans.

Hosting certain facultative symbionts modulates the phenoloxidase activity and immune response of the pea aphid Acyrthosiphon pisum

The pea aphid Acyrthosiphon pisum hosts different facultative symbionts(FS)which provide it with various benefits,such as tolerance to heat or protection against natural enemies(e.g.,fungi,parasitoid wasps).Here,we investigated whether and how the presence of certain FS could affect phenoloxidase(PO)activity,a key component of insect innate immunity,under normal and stressed conditions.For this,we used clones of A.pisum of difTerent genetic backgrounds(LLOl,YR2 and T3-8V1)lacking FS or harboring one or two(Regiella insecticola,Hamiltonella defensa,Serratia symbiotica Rickettsiella viridis).Gene expression and proteomics analyses of the aphid hemolymph indicated that the two A.pisum POs,PPOl and PP02,are expressed and translated into proteins.The level of PPO genes expression as well as the amount of PPO proteins and phenoloxidase activity in the hemolymph depended on both the aphid genotype and FS species.In particular,H.defensa and R.insecticola,but not S.symbiotica-h R.viridis,caused a sharp decrease in PO activity by interfering with both transcription and translation.The microinjection of different types of stressors(yeast,Escherichia coli,latex beads)in the YR2 lines hosting different symbionts affected the survival rate of aphids and,in most cases,also decreased the expression of PPO genes after 24 h.The amount and activity of PPO proteins varied according to the type of FS and stressor,without clear corresponding changes in gene expression.These data demonstrate that the presence of certain FS influences an important component of pea aphid immunity.

Degradation of Phenolic Compounds in Coal Gasification Wastewater by Biofilm Reactor with Isolated Klebsiella sp.

This study was conducted to evaluate the degradation of phenolic compounds by one strain isolated from coal gasification wastewater( CGW). 16S rRNA gene sequences homology and phylogenetic analysis showed that the isolate is belonged to the genus Klebsiella sp. The effect of different phenolic compounds on the isolate was investigated by determining OD600and phenoloxidase activity,of which the results showed that the isolate can utilize phenol,4-methyl phenol,3,5-dimethyl phenol and resorcinol as carbon resources. The biofilm reactor( formed by the isolate) can resist the influent concentration of phenolic compounds as high as750 mg /L when fed with synthetic CGW and incubated at optimum conditions. The capacity of improving the biodegradability of CGW through degrading phenolic compounds was testified with fed the biofilm reactor with real CGW. Thus,it might be an effective strain for bioaugmentation of CGW treatment.

Comparison of White Spot Syndrome Virus Infection Resistance Between Exopalaemon carinicauda and Litopenaeus vannamei Under Different Salinity Stresses

Exopalaemon carinicauda is one of the important economic shrimp species in China, and can tolerate a wide range of salinities. However, its disease resistance remains to be unclear in comparison with other shrimp species under salinity stress. In this study, the resistance to white spot syndrome virus(WSSV) of E. carinicauda and Litopenaeus vannamei was determined by comparing their hemocyanin(Hc) and phenoloxidase(PO) activities under different salinity stresses. In E. carinicauda, the PO activity and Hc gene transcript abundance showed a coherent pattern of increase and decrease while Hc content showed a slightly decrease with Vibrio anguillarum and WSSV infections. For both E. carinicauda and L. vannamei under salinity stress, the PO activity showed a positive correlation with the salinity while the Hc content and expression level of its gene increased significantly in salinities of 5, 15 and 25 g L^(-1). The survival rate of E. carinicauda with WSSV infection was higher than that of L. vannamei in the first 24 h under different salinity stresses. Drastic mortality of E.carinicauda and L. vannamei appeared at 48 h and 3 h post-injection, respectively. Furthermore, compared with L. vannamei, E. carinicauda displayed higher PO activity, Hc content and abundance of Hc gene m RNA. The results collectively indicated that Hc and PO have obviously functional connection in resisting pathogens and tolerating salinity stress, and PO activity and Hc gene m RNA abundance may reflect the resistance of shrimp to disease. E. carinicauda has higher level of immune potential than L. vannamei, suggesting its greater capacity in resisting pathogens under salinity stresses.

Cloning and Functional Analysis of Calcineurin Subunits A and B in Development and Fecundity of Nilaparvata lugens(Stål)

Serine/threonine phosphatase calcineurin(CN)is a unique but confounding calcium/calmodulin-mediated enzyme,which is composed of a catalytic subunit A(CNA)and a regulatory subunit B(CNB).We cloned six transcripts for CNA named from NlCNA-X1 to NlCNA-X6,one CNB named NlCNB1 and one CNB homologous gene NlCNBH1 from Nilaparvata lugens.All of them are constitutively transcripted in various tissues and developmental stages.The primary structure of the six isoforms showed obvious differences in the length and composition of the amino acid sequence between the two binding domains of Ca^(2+)/calmodulin(CaM)and CNB.Ca^(2+)-binding EF-hand motifs were found in NlCNB1 and NlCNBH1.The specific gene silencing of NlCNA,NlCNB1 and NlCNBH1 respectively by RNAi resulted in drastical reduction in survival rate,female weight,eclosion rate and fecundity of N.lugens.These results showed that NlCNA,NlCNB1 and NlCNBH1 were required for N.lugens growth and reproduction.The negative effects of NlCNB1 silence on nymph mortality(97%),molting malformation(90%)and female sterile(50%)were more serious than those of NlCNA or NlCNBH1.qRT-PCR and enzyme-linked immunosorbent assay(ELISA)analyses indicated that the nymphs with silenced NlCNA,NlCNB1 or NlCNBH1 showed impaired hormone and energy metabolism.In nymphs,the contents of 20-hydroxyecdysone(20E)after NlCNB1 RNAi and phenoloxidase after NlCNA RNAi were particularly decreased.These results suggested that NlCNA is involved in immunity of N.lugens by regulation of phenoloxidase,while NlCNB1 may control the growth and development of N.lugens by 20E signaling pathway in addition to interact with CNA.Injection of 70 ng/μL dsNlCNB1 resulted in 77.0%down regulation of NlCNB1,and the nymph mortality was up to 57.9%at 10 d after injection.Therefore,NlCNB1 could be a potential candidate target used for strategy design in control of N.lugens.Our results revealed the importance of CN in the regulation of the growth and development of N.lugens,which provided a basis for further study of the molecular mechanism of CN.

The selective regulation of immune responses by matrix metalloproteinase MMP14 in Ostrinia furnacalis

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune-related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection-responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune-related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double-stranded RNA and bacterial infection.

The long-term immunological effects of alloferon and its analogues in the mealworm Tenebrio mofitor

The subject of this article is a search for the long-term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro-apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe（p-NH2）^1 ]- and [Phe（p-OMe） ^1 ]-alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria-challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide-untreated bacteria-challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide-induced separation ofhemocyte fragments may resemble the formation ofplatelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half-life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N-terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.

Host permissiveness to baculovirus in flue nces time-dependent immune responses and fitness costs

Insects possess specific immune responses to protect themselves from different types of pathogens.Activation of immune cascades can inflict significant developmental costs on the surviving host.To characterize infection kinetics in a surviving host that experiences baculovirus inoculation,it is crucial to determine the timing of immune responses.Here,we investigated time-dependent immune responses and developmental costs elicited by inoculations from each of two wild-type baculoviruses,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)and Helicoverpa zea single nucleopolyhedrovirus(HzSNPV),in their common host H.zea.As H.zea is a semi-permissive host of AcMNPV and fully permissive to HzSNPV,we hypothesized there are differential immune responses and fitness costs associated with resisting infection by each virus species.Newly molted 4th-instar larvae that were inoculated with a low dose(LD15)of either virus showed signify icantly higher hemolymph FAD-glucose dehydrogenase(GLD)activities compared to the corresponding control larvae.Hemolymph phenoloxidase(PO)activity,protein concentration and total hemocyte numbers were not increased,but instead were lower than in control larvae at some time points post-inoculation.Larvae that survived either virus inoculation exhibited reduced pupal weight;survivors inoculated with AcMNPV grew slower than the control larvae,while survivors of HzSNPV pupated earlier than control larvae.Our results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.

Effect of associated fungi on the immunocompetence of red turpentine beetle larvae, Dendroctonus valens （Coleoptera： Curculionidae： Scolytinae）

Abstract Dendroctonus-fungus symbioses are often considered as the ideal model sys- tems to study the development and maintenance ofectosymbioses, and diverse interactions, including antagonism, commensalism and mutualism, have been documented between these organisms. The red turpentine beetle, Dendroctonus valens LeConte （Coleoptera： Curculionidae： Scolytinae） is a pine-killing invasive beetle in northern China. Fungi species Ophiostoma minus, Leptographium sinoprocerum, L. terebrantis and L. procerum were associated with this bark beetle. Antagonistic interactions between D. valens and its as- sociated fungi, such as O. minus and L. sinoprocerum, have been demonstrated, but the underlying causes of this phenomenon are unknown. Here, we first found the two tested fungi species retarded the net weight gain of D. valens larvae after completing 3-day feeding on their media. Furthermore, we provide direct evidence indicating the effect of associated fungi on the immunocompetence olD. valens larvae to explain the documented antagonism. Our results showed that the activity of phenoloxidase and total phenoloxi- dase in D. valens larvae were significantly upregulated by two strains of associated fungi, O. minus and L. sinoprocerum as compared with the controls. The phenoloxidase ratio increased significantly in the larvae which had fed for 3 days on media inoculated with O. minus. Because insect immtme defenses are costly to be deployed, these results could be explored as one of the underlying mechanisms of the documented antagonism.