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Red palm weevil (RPW) Rhynchophorus ferrugineus is the most destructive insect pests of numerous palm species in the world. The introduction of botanical extract(s) as integral part of an integrated pest management (IPM) programs against RPW will reduce the use of chemical insecticides. Polyphenol oxidase (PPO) is one of the RPW innate immune mechanisms and inhibition of such enzyme could result in a disorder of the insect's immune system. A one single PO isoenzyme has been purified from the hemolymph of the 12th instar larvae of RPW. Using L-DOPA as substrate, R. ferrugineus PPO exhibited specific activity 428 Units/mg proteins with 8.3-fold purification, optimum pH and temperature for activity at 7.5 and 40 °C, respectively and is enhanced by Cu 2+ with 1.76-fold. The rank order for oxidizing R. ferrugineus PPO different substrates is catechol > pyrogallol > L-DOPA > pyrocatechuic acid and not tyrosine. The kinetic parameters Km, Vmax and Vmax/Km for L-DOPA are 3.3 mM, 1.3 μmol/ml/min, and 0.39, respectively. The catalytic efficiency of the enzyme towards catechol is 5.3-fold higher than that for L-DOPA. The enzyme completely inhibited by thiourea, ascorbic acid, dithiothreitol, and SDS. R. ferrugineus PPO is a catechol oxidase di-phenol: O 2 oxidoreductase. Based on the toxicological studies of various botanical extracts, the IC 50 ranged from 20 to 90 mg/ml. The enzyme completely inhibited by 50 mg/ml Cinnamomum camphora. Gallic acid, the major phenolic compound, has IC 50 0.8 mM and competitively inhibited the enzyme with Ki 0.54 mM. C. camphora could be a useful natural RPW-controlling agent and used as integral part in IPM programs. This interpretation can be validated in future through an in vivo investigation., Competing Interests: Declaration of competing interest The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR 176 *IIGG. HP5 then cleaves proHP6 at a unique site of LDLH 112 *ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar., Competing Interests: The authors declare no competing interest.

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi-dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi-dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated 32 P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues., (© 2020 Wiley Periodicals, Inc.)

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin β-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO - had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into β-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal β-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr 394 and Ile 395 . Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As an invasive pest, the complete and effective innate immune system is crucial for the nipa palm hispid beetle Octodonta nipae (Maulik) to adjust to new environments. C-type lectins (CTLs) are large families of carbohydrate-binding proteins that possess one or more characteristic carbohydrate-recognition domains (CRD) and function as pattern-recognition receptors, which play important roles in mediating humoral and cellular immunity. In the present study, for the first time, we report two CTL-Ss (single-CRD CTLs) from O. nipae (Maulik) (designated OnCTL1 and OnCTL2). The two CTL-Ss share high identity at conserved amino acids associated with conserved carbohydrate binding sites Gln-Pro-Asp (QPD) motifs and clearly show a 1:1 orthologous relationship in insects, which endow them with functional conservation and diversification. mRNA abundance analysis showed that OnCTL1 was upregulated upon Staphylococcus aureus and Escherichia coli challenge at 6 and 12 h, while OnCTL2 underwent no changes upon E. coli challenge and was even downregulated after S. aureus infection. Knockdown of OnCTL1 significantly decreased the transcripts of two key serine proteases (prophenoloxidase activating factors), OnPPAF1 and OnPPAF3, followed by the reduction of haemolymph phenoloxidase activity; it also increased the expression of Defensin2B. In contrast, silencing of OnCTL2 significantly decreased the expression of Defensin2B and Attacin3C, the encapsulation index, and the phagocytosis rate compared to the dsEGFP group. The spreading results showed that more irregularly shaped plasmatocytes and lower levels of aggregation were found in OnCTL2-silenced pupae than in the dsOnCTL1 and dsEGFP groups. We can infer from the results of this study that the two OnCTLs play important roles in the immune system and generate a functional division: OnCTL1 seems to function more in humoral immunity including mediating bacterial recognition and activating the phenoloxidase cascade, and OnCTL2 plays a greater role in enhancing cellular immunity. These observations could replenish information on the functional diversification of insect CTLs, and also provide valuable information to unravel the immunity in O. nipae., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Parasite traits associated with transmission success, such as the number of infective stages released from the host, are expected to be optimized by natural selection. However, in the trematode parasite Schistosoma mansoni, a key transmission trait, i.e. the number of cercariae larvae shed from infected Biomphalaria spp. snails, varies significantly within and between different parasite populations and selection experiments demonstrate that this variation has a strong genetic basis. In this study, we compared the transmission strategies of two laboratory schistosome population and their consequences for their snail host., Methods: We infected inbred Biomphalaria glabrata snails using two S. mansoni parasite populations (SmBRE and SmLE), both isolated from Brazil and maintained in the laboratory for decades. We compared life history traits of these two parasite populations by quantifying sporocyst growth within infected snails (assayed using qPCR), output of cercaria larvae and impact on snail host physiological response (i.e. hemoglobin rate, laccase-like activity) and survival., Results: We identified striking differences in virulence and transmission between the two studied parasite populations. SmBRE (low shedder (LS) parasite population) sheds very low numbers of cercariae and causes minimal impact on the snail physiological response (i.e. laccase-like activity, hemoglobin rate and snail survival). In contrast, SmLE (high shedder (HS) parasite population) sheds 8-fold more cercariae (mean ± SE cercariae per shedding: 284 ± 19 vs 2352 ± 113), causes high snail mortality and has strong impact on snail physiology. We found that HS sporocysts grow more rapidly inside the snail host, comprising up to 60% of cells within infected snails, compared to LS sporocysts, which comprised up to 31%. Cercarial production is strongly correlated to the number of S. mansoni sporocyst cells present within the snail host tissue, although the proportion of sporocyst cells alone does not explain the low cercarial shedding of SmBRE., Conclusions: We demonstrated the existence of alternative transmission strategies in the S. mansoni parasite consistent with trade-offs between parasite transmission and host survival: a "boom-bust" strategy characterized by high virulence, high transmission and short duration infections and a "slow and steady" strategy with low virulence, low transmission but long duration of snail host infections.

This study examined the effect of long-term exposure to environmentally relevant concentrations of dietary fluoranthene (6.7 and 67 ng / g dry food weight) on defense mechanisms of the polyphagous forest insects Lymantria dispar L. and Euproctis chrysorrhoea L. The activities and expression of isoforms of superoxide dismutase (SOD) and catalase (CAT), the activities of glutathione S-transferase (GST) and glutathione reductase (GR), and total glutathione content (GSH) were determined in the whole midgut and midgut tissue, while SOD and CAT activities were assessed in hemolymph of the larvae. The results showed significant changes of enzyme activities, with more pronounced responses in larval midgut tissues, and between-species differences in patterns of response. Significantly increased activity of SOD was recorded in the whole midgut and midgut tissue of L. dispar larvae, as well as in midgut tissue of E. chrysorrhoea larvae. Fluoranthene increased CAT activity in midgut tissue of L. dispar larvae, and in the whole midgut and midgut tissue of E. chrysorrhoea larvae. Different expression patterns were detected for enzyme isoforms in tissues of larvae exposed to dietary fluoranthene. Total GSH content and GST activity increased in E. chrysorrhoea larval midgut tissue. Significantly decreased SOD activity in hemolymph of L. dispar larvae, and opposite changes in CAT activity were recorded in the hemolymph of larvae of two insect species. The tissue-specific responses of enzymes to dietary fluoranthene, recorded in each species, enabled the larvae to overcome the pollutant induced oxidative stress, and suggest further assessment of their possible use as early-warning signals of environmental pollution., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Susceptibility to the fungus Metarhizium robertsii and changes in host defences were evaluated in different stages of the intermoult period (4-6 h, 34-36 h and 84-86 h post moult in IV larval instars) of the Colorado potato beetle. A significant thickening of the cuticle during larval growth was accompanied by decreases in cuticle melanization, phenoloxidase activity and epicuticular hydrocarbon contents (C28-C32). At the same time, a decrease in the conidial adhesion rate and an increase in resistance to the fungus were observed. In addition, we recorded significant elevation of the encapsulation rate and total haemocyte counts in the haemolymph during the specified period. The activity of detoxification enzymes decreased in the haemolymph but increased in the fat body during larval growth. No significant differences in the fatty acid content in the epicuticle were observed. The role of developmental disorders in susceptibility to entomopathogenic fungi is also discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Active skeletal muscles produce lactate. H+ is generated during lactate neutralization in the Cori cycle, which leads to muscle acidosis and soreness (the so-called Delayed Onset Muscle Soreness, DOMS) in vertebrates. The aim of the study was to determine the activities/concentrations of compounds involved in the Cori cycle in worker and forager bees. Muscles, fat bodies, and hemolymph from 1- and 14-day-old workers and foragers were collected and assayed for the protein, lactate, glucose, NAD+, and NADH concentrations and lactate dehydrogenase (LDH) activity. Both lactate concentration and LDH activity in the hemolymph, muscles, and fat bodies increased with age. The concentrations of NAD+ and NADH in the tissues decreased with ageing/senescence, whereas protein concentrations increased until day 14 of bee's life and then decreased in foragers. The concentration of glucose decreased in the hemolymph and muscles and increased in the fat bodies. Elevated lactate concentrations in foragers may indicate transition from the aerobic to the anaerobic phase and development of metabolic acidosis that may eventually lead to muscle damage/soreness and shorter lifespan. When analyzing flight dynamics, load mass, and bee behavior, changes in the concentrations of Cori cycle compounds should be taken into account.

Objective: Hemolymph plays many important roles in the physiology of an insect throughout its lifetime; however, for small-bodied insects, studies are lacking because of the difficulties encountered while collecting hemolymph. The objective of our study was to develop a method to collect hemolymph plasma from various stages of Tribolium castaneum and to evaluate phenoloxidase activity in the plasma samples. We first designed a procedure for easily and quickly collecting clear hemolymph plasma from T. castaneum., Results: By using this method, we collected approximately 5 µl plasma from 30 individuals at the larval, pupal or adult stages. And then, we studied the expression of phenoloxidase by performing western blot analysis of the plasma samples and found that phenoloxidase is present in hemolymph in each developmental stage. We also measured phenoloxidase activity in control plasma and plasma treated with Gram-positive bacteria, Micrococcus luteus. Phenoloxidase activity was greater in some of the M. luteus-treated plasma samples compared with control samples. Thus, we developed a method to collect hemolymph plasma that is suitable for studies of phenoloxidase activity.

Dual oxidases (DUOXs) were originally identified as NADPH oxidases (NOXs), found to be associated with the reactive oxygen species (ROS) hydrogen peroxide (H 2 O 2 ) production at the plasma membrane and crucial in host biological processes. In this study, SpDUOX1 and SpDUOX2 of mud crab (Scylla paramamosain) were identified and studied. Both SpDUOX1 and SpDUOX2 are transmembrane proteins, including an N-signal peptide region and a peroxidase homology domain in the extracellular region, transmembrane regions, and three EF (calcium-binding region) domains, a FAD-binding domain, and a NAD binding domain in the intracellular region. The SpDUOXs were expressed in all tissues examined, but mainly in hepatopancreas, heart, and mid-intestine. The expression of the SpDUOXs in the hemolymph of mud crabs was up-regulated after challenge with Vibrio parahemolyticus or LPS. RNA interference (RNAi) of the SpDUOXs resulted in reduced ROS production in hemolymph. The bacterial count increased in the hemolymph of mud crabs injected with SpDUOX1 or SpDUOX2-RNAi, while the bacterial clearance ability of hemolymph significantly reduced. At the phylum level, the phyla Bacteroidetes and Actinobacteria were significantly increased, while Proteobacteria were significantly reduced following SpDUOX2 knockdown. There was a significant increase in the relative abundance of the genera Marinomonas, Pseudoalteromonas, Shewanella, and Hydrogenoph in SpDUOX2 depleted mud crabs compared with the controls. Our current findings therefore indicated that SpDUOXs might play important roles in maintaining the homeostasis in the hemolymph microbiota of mud crab., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated M r of 44.8 kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4 h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT 50 ) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p < 0.01). A 3.5-fold increase in E. coli colony forming units occurred in larval hemolymph of the OfPAP knockdown larvae, as compared with that of the control larvae not injected with dsRNA. There were notable decreases in PO and IEARase activities in hemolymph of the OfPAP knockdown larvae. In summary, we have demonstrated that OfPAP is a component of the PPO activation system, likely by functioning as a PPO activating protease in O. furnacalis larvae., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bivalves are worldwide sentinels of anthropogenic pollution. The inclusion of biomarker responses in chemical monitoring is a recommended practise that has to overcome some difficulties. One of them is the time frame between sample collection and sample processing in order to ensure the preservation of enzymatic activities. In the present study, three bivalve species of commercial interest (mussel, Mytilus galloprovincialis, razor shell, Solen marginatus, and cockle, Cerastoderma edule) were processed within <2 h after being retrieved from their natural habitat, and 24 h after being transported in air under cold conditions (6-8 °C) to laboratory facilities. The enzymatic activities were compared in the three species submitted to both conditions revealing no differences in terms of carboxylesterase dependent activities (CEs) using different substrates: p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), 1-naphthyl acetate (1-NA), 1-naphthyl butyrate (1-NB) and 2-naphthyl acetate (2-NA). In mussels, three tissues were selected (haemolymph, gills and digestive gland). For comparative purposes, in razor shell and cockle only digestive gland was considered as it is the main metabolic organ. Baseline enzymatic activities for CEs were characterised in the digestive gland of the three bivalves using four out of the five selected CE substrates as well as the kinetic parameters (V max and K m ) and catalytic efficiency. The in vitro sensitivity to the organophosphorus metabolite chlorpyrifos oxon was also calculated. IC 50 values (pM-nM range) were lower than those obtained for vertebrate groups which suggest that bivalves have high protection efficiency against this pesticide as well as species dependent particularities., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phenoloxidases (POs) play a crucial role in melanization of crustaceans. There are at least two types of POs characterized in crustaceans: the conventional type (POα here) that is expressed in hemocytes and POβ, a secreted protein synthesized in the hepatopancreas. We investigated the source of PO activity in the hemolymph of a lobster and determined the kinetic parameters of mono- and di-PO activities. In the lobster hemolymph, POα, which formed a hexamer similar to both POβ and hemocyanin, contributed to PO activity, whereas the amount of POβ was low. Kinetic analyses using purified prophenoloxidase of crustaceans showed that lobster POα has a higher rate constant, while shrimp POβ has higher specificity in both mono- and di-PO reactions, when tyramine and dopamine were employed as substrates. There should be at least two types of PO molecules in crustacean hemolymph, but the dominant PO molecule type varies among species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Organophosphate pesticide diazinon is a specific inhibitor of acetylcholinesterase (AChE), which is a common neurotoxicity biomarker in environmental studies. In honeybees, AChE exists in two forms having different physiological roles, one existing as a soluble form and the other as membrane-bound. In most studies AChE activity has been analysed without paying considerable attention to different forms of AChE. In this study, we exposed honeybees Apis mellifera carnica for 10days to diazinon via oral exposure and analysed the total AChE activities in salt soluble (SS) and detergent soluble (DS) fractions. We assumed that SS fraction would preferentially contain the soluble AChE, but the DS fraction would contain only membrane AChE. On the contrary, our results showed that SS and DS fractions both contain soluble and membrane AChE and the latter has considerably higher activity. Despite this we obtained a differential response of AChE activity in SS and DS fractions when exposed to diazinon. The head/thorax AChE activity in DS fraction decreased, while the head/thorax AChE activity in SS fraction increased at sublethal concentrations. The AChE activity in honeybee hemolymph shown here for the first time is a salt soluble enzyme. Its activity remained unaltered after diazinon treatment. In conclusion, we provide evidence that varying results regarding AChE activity alterations upon stressor exposure are obtained when extracted through different procedures. In further environmental studies with honeybees this differential response of AChE activity should be given considerable attention because this affects the outcome of ecotoxicity study., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A typical characteristic of the insect innate immune system is the activation of the serine protease cascade in the hemolymph. As being the terminal component of the extracellular serine protease cascade in the prophenoloxidase (proPO) activating system, proPO-activating factors (PPAFs) activated by the upstream cascade may generate active phenoloxidase, which then induces downstream melanization. In the present study, we reported three PPAFs from the nipa palm hispid beetle Octodonta nipae (Maulik) (designated as OnPPAF1, OnPPAF2, OnPPAF3). All three OnPPAFs contained a single clip domain at the amino-terminus followed by a trypsin-like serine protease domain at the carboxyl-terminus, except the Ser in the active sites of OnPPAF2 and OnPPAF3 was substituted with Gly. Transcript expression analysis revealed that all OnPPAFs were highly expressed in hemolymph, whereas OnPPAF2 showed an extremely low mRNA abundance compared with that of OnPPAF1 and OnPPAF3, and that the abundance of all three OnPPAFs was dramatically increased upon bacterial challenge. Knockdown of OnPPAF1 or OnPPAF3 resulted in a reduction of hemolymph phenoloxidase activity and an inhibition of hemolymph melanization, whereas the knockdown of OnPPAF2 did not affect the proPO cascade. Our work thus implies that the three OnPPAFs may have different functions and regulation during immune responses in O. nipae., (© 2017 Wiley Periodicals, Inc.)

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme of great importance in several physiological processes. Due to its physiological importance and sensitivity to various pollutants, CA activity has been used as biomarker of aquatic contamination. Considering that in bivalves the sensitivity of CA to pollutants seems to be tissue-specific, we proposed here to analyze CA activity of hemolymph, gill and mantle of Crassostrea rhizophorae collected in two tropical Brazilian estuaries with different levels of anthropogenic impact, in dry and rainy season. We found increased carbonic anhydrase activity in hemolymph, gill and mantle of oysters collected in the Paraíba Estuary (a site of high anthropogenic impact) when compared to oysters from Mamanguape Estuary (inserted in an area of environmental preservation), especially in the rainy season. CA of hemolymph and gill were more sensitive than mantle CA to aquatic contamination. This study enhances the suitability of carbonic anhydrase activity for field biomarker applications with bivalves and brings new and relevant information on hemolymph carbonic anhydrase activity as biomarker of aquatic contamination., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Host innate immunity is one of the factors that determines the resistance of insects to their entomopathogens. In the research reported here we studied whether or not phenoloxidase (PO), a key enzyme in the melanogenesis component of humoral immunity of insects, plays a role in the protection of Lymantria dispar larvae from infection by L. dispar multiple nucleopolyhedrovirus. We studied two types of viral infection: overt and covert. The following lines of investigation were tested: i) the intravital individual estimation of baseline PO activity in haemolymph plasma followed by virus challenging; ii) the specific inhibition of PO activity in vivo by peroral treatment of infected larvae with phenylthiourea (PTU), a competitive inhibitor of PO; iii) the evaluation of PO activity in the haemolymph plasma after larval starvation. Starvation is a stress that activates the covert infection to an overt form. All of these experiments did not show a relationship between PO activity in haemolymph plasma of L. dispar larvae and larval susceptibility to baculovirus. Moreover, starvation-induced activation of covert viral infection to an overt form occurred in 70 percent of virus-carrying larvae against the background of a dramatic increase of PO activity in haemolymph plasma in the insects studied. Our conclusion is that in L. dispar larvae PO activity is not a predictor of host resistance to baculovirus.

This study investigated the possible biological, biochemical and histological changes in Bradybaena similaris(Gastropoda: Pulmonata) infected by Heterorhabditis indica (Rhabditida: Heterorhabditidae), strain LPP1. Two groups of 16 snails were formed: the control group (unexposed) and the treated group, which was exposed for three weeks to infective juveniles (J 3 ) of H. indica LPP1. The experiment was conducted in duplicate, using a total of 64 snails. After the exposure period, the snails were dissected to collect the hemolymph and tissues, for evaluation of the physiological changes caused by the infection. The number of eggs laid/snail and the viability of these eggs were also assessed as indicators of the reproductive activity of B. similaris. Intense glycogenolysis was accompanied by a significant reduction (p < 0.05) in the glucose content of the hemolymph of the exposed snails, indicating that infection by H. indica induces breakdown of the host's glycemic homeostasis. Significant variations (p < 0.05) in the lactate dehydrogenase activity occurred together with changes in the concentration of pyruvic and lactic acid in the hemolymph of the infected B. similaris snails, corroborating the transition from aerobic to anaerobic metabolism in the hosts. These metabolic alterations reflect the parasitic castration process in this interface. The results suggest that the use of H. indica LPP1 is a potential alternative for biological control of B. similaris., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Anthropogenic activity in industrial development has imposed great threats to the environment and wildlife in the form of persistent organic pollutants. Polycyclic aromatic hydrocarbons (PAH) tend to accumulate in vegetation foliage which is the main food source of polyphagous insect species Lymantria dispar L. Origin and multigenerational adaptation of L. dispar population to environmental challenges strongly condition the enzymes' sensitivity to pollutants. In this study, our aim was to investigate response of the superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) to the chronic dietary exposure of benzo[a]pyrene in the midgut tissues and hemolymph of two L. dispar populations originating from unpolluted and polluted forest habitat. Midgut tissue of the larvae from the polluted forest showed significant increase in SOD, CAT and GST activity, while in unpolluted forest's larvae SOD and CAT showed elevated activities in hemolymph. L. dispar populations adapted to different level of pollution in their environment and expressed distinct tissue-dependent antioxidative enzyme sensitivity to benzo[a]pyrene diet, implying high potential for further elucidation of these enzymes as molecular biomarkers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Allatotropins (AT) are neuropeptides with pleotropic functions on a variety of insect tissues. They affect processes such as juvenile hormone biosynthesis, cardiac rhythm, oviduct and hindgut contractions, nutrient absorption and circadian cycle. The present work provides experimental evidence that AT elicits immune responses in two important mosquito disease vectors, Anopheles albimanus and Aedes aegypti. Hemocytes and an immune-competent mosquito cell line responded to AT by showing strong morphological changes and increasing bacterial phagocytic activity. Phenoloxidase activity in hemolymph was also increased in Ae. aegypti mosquitoes treated with AT but not in An. albimanus, suggesting differences in the AT-dependent immune activation in the two species. In addition, two important insect immune markers, nitric oxide levels and expression of antimicrobial peptide genes, were increased in An. albimanus guts after AT treatment. AT conjugated to quantum dot nanocrystals (QDots) specifically labeled hemocytes in vivo in both mosquito species, implying molecular interactions between AT and hemocytes. The results of our studies suggest a new role for AT in the modulation of the immune response in mosquitoes.

In this study, we discovered that shrimp miR-100 was up-regulated at 24 h after WSSV or Vibrio alginolyticus infection, confirming its participation in the innate immune system of shrimp. The anti-miRNA oligonucleotide (AMO-miR-100) was applied to inhibit the expression of miR-100. After AMO-miR-100 treatment, the shrimp was challenged with WSSV or V. alginolyticus. The knockdown of miR-100 expression decreased the mortality of WSSV-infected shrimp from 24 h to 72 h post-infection and enhanced the mortality of V. alginolyticus-infected shrimp significantly. The knockdown of miR-100 affected phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) after the infection with WSSV or V. alginolyticus, indicating a regulative role of miR-100 in the immune potential of shrimp in the response to WSSV or V. alginolyticus infection. The knockdown of miR-100 induced the apoptosis of shrimp hemocytes, and V. alginolyticus + AMO-miR-100 treatment caused more hemocyte apoptosis than V. alginolyticus treatment. The miR-100 influenced also the morphology of shrimp hemocytes and regulated the phagocytosis of WSSV or V. alginolyticus. Thus, we concluded that miR-100 may promote the anti-Vibrio immune response of shrimp through regulating apoptosis, phagocytosis and PO activity and affects the progression of WSSV infection at a certain level., Competing Interests: The authors declare no competing financial interests.

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology., (© 2017 Wiley Periodicals, Inc.)

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

Gemifloxacin mesylate (GEM) is a synthetic, fourth-generation fluoroquinolone antibacterial antibiotic that has a broad spectrum of activity against bacteria. GEM inhibits DNA synthesis by inhibiting DNA gyrase and topoisomerase IV activities. Recent research into insect nutrition and mass-rearing programs, in which antibiotics are incorporated into the culture media to maintain diet quality, raised a question of whether clinical antibiotics influence the health or biological performance of the insects that ingest these compounds. Because some antibiotics are pro-oxidant compounds, we addressed the question with experiments designed to assess the effects of GEM (mesylate salt) on oxidative stress indicators, using Galleria mellonella larvae. The insects were reared from first-instar larvae to adulthood on artificial diets amended with GEM at 0.001, 0.01, 0.1, or 1.0%. Feeding on the 1% diets led to significantly increased hemolymph contents of the lipid peroxidation product, malondialdehyde and protein oxidation products, protein carbonyl. All GEM concentrations led to increased hemolymph glutathione S-transferase activity. We inferred that although it was not directly lethal to G. mellonella larvae, dietary exposure to GEM exerts measurable oxidative damage, possibly on insects generally. Long-term, multigenerational effects remain unknown., (© 2016 Wiley Periodicals, Inc.)

Angiostrongylus cantonensis is considered the main agent responsible for human eosinophilic meningoencephalitis. This parasite has low specificity for mollusk hosts and it can also use aquatic snails as auxiliary hosts. Studies based on the metabolic profile of Biomphalaria spp. infected by A. cantonensis have been conducted to observe parasite-host interactions. In the present study, the glucose content in the hemolymph and glycogen content in the digestive gland and cephalopedal mass of Biomphalaria tenagophila and Biomphalaria straminea experimentally infected by A. cantonensis were evaluated, along with the activity of LDH. The snails were dissected from 6 to 21days after infection to collect the hemolymph and separate the tissues. Decreases of 96% and 6.4% in the glucose content triggered a transition from aerobic to anaerobic metabolism in the two infected snail species, B. straminea and B. tenagophila, respectively. That finding was confirmed by high-performance liquid chromatography. These results indicate that when infected, these snails are able to change their metabolic profile, suggesting a strategy to maintain their homeostatic balance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The xenoestrogen 4-nonylphenol (NP) is a ubiquitous aquatic pollutant and has been shown to impair reproduction, development, growth and, more recently, immune function in marine invertebrates. We investigated the effects of short-term (7 d) exposure to low (2 μg l -1 ) and high (100 μg l -1 ) levels of NP on cellular and humoral elements of the innate immune response of Crassostrea gigas to a bacterial challenge. To this end, we measured 1) total hemocyte counts (THC), 2) relative transcript abundance of ten immune-related genes (defh1, defh2, bigdef1, bigdef2, bpi, lysozyme-1, galectin, C-type lectin 2, timp, and transglutaminase) in the hemocytes, gill and mantle, and 3) hemolymph plasma lysozyme activity, following experimental Vibrio campbellii infection. Both low and high levels of NP were found to repress a bacteria-induced increase in THC observed in the control oysters. While several genes were differentially expressed following bacterial introduction (bigdef2, bpi, lysozyme-1, timp, transglutaminase), only two genes (bpi in the hemocytes, transglutaminase in the mantle) exhibited a different bacteria-induced expression profile following NP exposure, relative to the control oysters. Independently of infection-status, exposure to NP also altered mRNA transcript abundance of several genes (bpi, galectin, C-type lectin 2) in naïve, saline-injected oysters. Finally, plasma lysozyme activity levels were significantly higher in low dose NP-treated oysters (both naïve and bacteria challenged) relative to control oysters. Combined, these results suggest that exposure to ecologically-relevant (low) and extreme (high) levels of NP can alter both cellular and humoral elements of the innate immune response in C. gigas, an aquaculture species of global economic importance., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Ubiquitination is an important post-translational protein modification and plays a crucial role in various processes such as cell cycle, signal transduction, and transcriptional regulation. In the present study, a novel ubiquitin (Ub)-protein ligase E3 (designed as CgE3Rv1) was identified from Crassostrea gigas, and its ubiquitination regulation in the immune response against lipopolysaccharide (LPS) stimulation was investigated. The open reading frame of CgE3Rv1 gene was of 1455 bp encoding a polypeptide of 484 amino acids with the predicted molecular mass of 54.89 kDa. There were two transmembrane regions and a RING-variant (RINGv) domain identified in CgE3Rv1. CgE3Rv1 shared similar C4HC3 zinc-finger-like motif with those RINGv domain Ub-protein ligases E3s identified from vertebrates and invertebrates, and it was closely clustered with the membrane-associated RING-CH2 (MARCH2) Ub-protein ligases E3s in the phylogenetic tree. The mRNA transcript of CgE3Rv1 was highest expressed in gonads and hemolymph (p < 0.05), and its mRNA expression level in hemocytes was significantly increased at 6 h (p < 0.01) after the stimulation of LPS, while the up-regulated mRNA expression was significantly decreased (p < 0.01) after acetylcholine stimulation. No significant changes of CgE3Rv1 expression were observed after peptidoglycan or mannan stimulation. Immunohistochemistry and in situ hybridization assays revealed that CgE3Rv1 protein and mRNA were dominantly distributed in the gonad. In the hemocytes, CgE3Rv1 was mainly located around the nucleus, and slightly distributed in the cytoplasm and on the cell membrane. Recombinant CgE3Rv1 RINGv domain protein (rCgE3Rv1-RINGv) was confirmed to activate the Ub reaction system in vitro with the aid of Ub-activating enzyme E1 and Ub-conjugating enzyme E2. These results demonstrated that CgE3Rv1 was an Ub-protein ligase E3, which was involved in the immune response against LPS and the interaction with cell surface signal molecules of neuroendocrine-immune system in oysters., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To evaluate the genotoxic and physiological effects of acute hypoxia on the pacific white shrimp (L. vannamei), shrimps were treated firstly with three dissolved oxygen levels 6.5 ppm (control), 3.0 ppm and 1.5 ppm for 24 h, respectively, and then reoxygenated (6.5 ppm) for 24 h. The changes of superoxide dismutase (SOD) activity, glutathione peroxidases (GPX) activity, malondialdehyde (MDA) concentration and DNA damage in the tissues of gill, hepatopancreas and hemolymph were examined during the period of hypoxia and reoxygenation. The results indicated SOD activity, GPX activity, MDA concentration and DNA damage all increased basically compared with the control during the period of hypoxia except for MDA concentrations in the gill at 12 h and 24 h hypoxia (3.0 ppm), and these parameters were recovered to some degree during the period of reoxygenation. Moreover, the comet assays in the tissues of gill and hepatopancreas showed an obvious time- and dose-dependent response to hypoxia, which indicated comet assay in the two tissues could be used as sensitive biomarker to detect the occurrence of hypoxia. We conclude that acute hypoxia can induce oxidative stress, DNA damage and lipid peroxidation in the tissues of gill, hepatopancreas and hemolymph of L. vannamei and the DNA damage may come from hypoxia-induced oxidative stress., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Biomphalaria snails are the intermediate host of the blood fluke Schistosoma mansoni, which infect more than 67 million people in tropical areas. Phenoloxidase enzymes (POs), including tyrosinases, catecholases, and laccases, are known to play a role in the immune defenses of arthropods, but the PO activity present in Biomphalaria spp. hemolymph has not been characterized. This study was designed to characterize substrate specificity and reaction optima of PO activity in Biomphalaria spp. hemolymph as a starting point to understand the role of this important invertebrate enzyme activity in snail biology and snail-schistosome interactions., Methods: We used spectrophotometric assays with 3 specific substrates (L-tyrosine for tyrosinase, L-DOPA for catecholase, and PPD for laccase) and diethylthiocarbarmate (DETC) as specific PO inhibitor to characterize PO activity in the hemolymph of uninfected snails from two Biomphalaria species, and to determine the impact of the parasite Schistosoma mansoni on the PO activity of its B. glabrata vector., Results: We identified laccase activity in hemolymph from uninfected B. glabrata and B. alexandrina. For both species, the activity was optimal at 45 °C and pH 8.5, and located in the plasma. The K m and V max of PO enzymes are 1.45 mM and 0.024 OD.min(-1) for B. glabrata, and 1.19 mM and 0.025 OD.min(-1) for B. alexandrina. When the snail vector is parasitized by S. mansoni, we observed a sharp reduction in laccase activity seven weeks after snail infection., Conclusions: We employed a highly specific spectrophotometric assay using PPD substrate which allows accurate measurement of laccase activity in Biomphalaria spp. hemolymph. We also demonstrated a strong impact of the parasite S. mansoni on laccase activity in the snail host.

Umbelliferone has potential value as it has an inhibitory effect on tumor cells; however, its impact on an animal's circulatory system and hematopoietic function has not been reported. In this study, 4-methylumbelliferon (4-MU), an umbelliferone derivative, was used as a model drug, and its potential toxicity on hemocytes and hematopoietic organs (HOs) was investigated using an invertebrate animal model, the silkworm, Bombyx mori. The results showed that the level of reactive oxygen species in HOs increased when larvae (third day of the fifth instar) were orally exposed to 4 mM 4-MU for 8 min, followed by the induction of improved antioxidative metabolism of coenzymes in hemolymph. Exposure to 4-MU also significantly upregulated the expression levels of several genes in the hemolymph and fat body (a detoxification tissue similar to the liver in mammals) including antimicrobial peptide gene cecropinA and moricin, and a phagocytosis-related gene, tetraspanin E, suggesting an increased antioxidant level and antimicrobial ability of the circulatory system. However, the percentage of dead hemocytes increased and hematopoiesis significantly decreased in HOs, indicating the toxic effect of 4-MU on hemocytes and hematopoiesis, despite it inducing enhanced antioxidant and antimicrobial activity in the circulatory system.

The study was conducted to investigate the effect of Lactobacillus rhamnosus (a commercial probiotic) and inulin (a prebiotic) on the survival rates of honeybees infected and uninfected with Nosema ceranae, the level of phenoloxidase (PO) activity, the course of nosemosis, and the effect on the prevention of nosemosis development in bees. The cells of L. rhamnosus exhibited a high rate of survival in 56.56 % sugar syrup, which was used to feed the honeybees. Surprisingly, honeybees fed with sugar syrup supplemented with a commercial probiotic and a probiotic + prebiotic were more susceptible to N. ceranae infection, and their lifespan was much shorter. The number of microsporidian spores in the honeybees fed for 9 days prior to N. ceranae infection with a sugar syrup supplemented with a commercial probiotic was 25 times higher (970 million spores per one honeybee) than in a control group fed with pure sucrose syrup (38 million spores per one honeybee). PO activity reached its highest level in the hemolymph of this honeybee control group uninfected with N. ceranae. The addition of probiotics or both probiotics and prebiotics to the food of uninfected bees led to the ~2-fold decrease in the PO activity. The infection of honeybees with N. ceranae accompanied an almost 20-fold decrease in the PO level. The inulin supplemented solely at a concentration of 2 μg/mL was the only administrated factor which did not significantly affect honeybees' survival, the PO activity, or the nosemosis infection level. In conclusion, the supplementation of honeybees' diet with improperly selected probiotics or both probiotics and prebiotics does not prevent nosemosis development, can de-regulate insect immune systems, and may significantly increase bee mortality.

Bloom forming algae and hypoxia are considered to be two main co-occurred stressors associated with eutrophication. The aim of this study was to evaluate the interactive effects of harmful algae Microcystis aeruginosa and hypoxia on an ecologically important mussel species inhabiting lakes and reservoirs, the triangle sail mussel Hyriopsis cumingii, which is generally considered as a bio-management tool for eutrophication. A set of antioxidant enzymes involved in immune defence mechanisms and detoxification processes, i.e. glutathione-S-transferases (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), lysozyme (LZM) in mussel haemolymph were analyzed during 14days exposure along with 7days depuration duration period. GST, GSH, SOD, GPX and LZM were elevated by toxic M. aeruginosa exposure, while CAT activities were inhibited by such exposure. Hypoxia influenced the immune mechanisms through the activation of GSH and GPX, and the inhibition of SOD, CAT, and LZM activities. Meanwhile, some interactive effects of M. aeruginosa, hypoxia and time were observed. Independently of the presence or absence of hypoxia, toxic algal exposure generally increased the five tested enzyme activities of haemolymph, except CAT. Although half of microcystin could be eliminated after 7days depuration, toxic M. aeruginosa or hypoxia exposure history showed some latent effects on most parameters. These results revealed that toxic algae play an important role on haemolymph parameters alterations and its toxic effects could be affected by hypoxia. Although the microcystin depuration rate of H. cumingii is quick, toxic M. aeruginosa and/or hypoxia exposure history influenced its immunological mechanism recovery., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Several biomarkers were measured to evaluate the effects of Trimethoprim (TMP; 300, 600 and 900 ng/L) in the clam Ruditapes philippinarum after exposure for 1, 3 and 7 days. The actual TMP concentrations were also measured in the experimental tanks. The total haemocyte count significantly increased in 7 day-exposed clams, whereas alterations in haemocyte volume were observed after 1 and 3 days of exposure. Haemocyte proliferation was increased significantly in animals exposed for 1 and 7 days, whereas haemocyte lysate lysozyme activity decreased significantly after 1 and 3 days. In addition, TMP significantly increased haemolymph lactate dehydrogenase activity after 3 and 7 days. Regarding antioxidant enzymes, only a significant time-dependent effect on CAT activity was recorded. This study demonstrated that environmentally realistic concentrations of TMP affect haemocyte parameters in clams, suggesting that haemocytes are a useful cellular model for the assessment of the impact of TMP on bivalves., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this study was to assess the intensity of enzymatic antioxidative parameters [catalase (CAT), glutathione peroxidase (GSTPx), glutathione reductase (GR), total antioxidant capacity (TAC)] and percentage of high granularity cells as well as low to medium granularity cells in haemolymph of wolf spiders Xerolycosa nemoralis exposed to starvation and dimethoate under laboratory conditions. Only in starved males, haemolymph included a lower percentage of high granularity cells, accompanied by high activity of CAT and GSTPx, than in the control. Exposure of males to dimethoate increased CAT activity, after single application, and significantly enhanced GR activity, after five-time application. In females, five-time contact with dimethoate elevated the percentage of high granularity cells. As in comparison to females, male X. nemoralis were more sensitive to the applied stressing factors, it may be concluded that in natural conditions both food deficiency and chemical stress may diminish the immune response of their organisms., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Haemolymph norepinephrine (NE); total haemocyte count (THC); respiratory bursts (RBs); superoxide dismutase (SOD), phenoloxidase (PO), and phagocytic activity; and prophenoloxidase (proPO)-system-related genes (lipopolysaccharide- and β-1,3-glucan-binding protein: LGBP, proPO, peroxinectin: PE, and α2-macroglobulin: α2-M) in haemocytes of Macrobrachium rosenbergii were investigated after transferring them from 28 °C to 22 °C, 28 °C, and 34 °C respectively. The results revealed that haemolymph NE, hyaline cells (HCs), and PO activity per granulocyte increased from 30 to 120 min of exposure, and however, RBs and phagocytic activity significantly decreased from 30 to 120 min of exposure as well as granular cells (GCs), semigranular cells (SGCs), and SOD activity decreased from 60 to 120 min of exposure for the prawns subjected to temperature stress. The proPO-system-related gene expression markedly increased with 60-120 min of exposure for the prawns transferred from 28 °C to 22 °C and 34 °C, except α2M at 120 min. These results provide a first insight into the effects of temperature stress on haemolymph NE level and immune functions in prawns and suggest that temperature-stress-induced acute modulation in immunity is associated with the release of haemolymph NE in M. rosenbergii., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 °C, and PO activity was strongly inhibited by Zn(2+) . IC50 values were estimated to be 8.2, 19.14, and 24.04 μmol/L for 4-hexylresorsinol, kojic acid, and quercetin, respectively. The inhibitory potencies (i.e., I50 of each compound/I50 of 4-hexylresorcinol) of kojic 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 of PO inhibitors seems to be a promising approach for pest control due to their potential safety for humans., (© 2014 Institute of Zoology, Chinese Academy of Sciences.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one-seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses., (© 2015 Wiley Periodicals, Inc.)

Two cDNA sequences of Kazal-type serine protease inhibitors (KSPIs) in Nasonia vitripennis, NvKSPI-1 and NvKSPI-2, were characterized and their open reading frames (ORFs) were 198 and 264 bp, respectively. Both NvKSPI-1 and NvKSPI-2 contained a typical Kazal-type domain. Real-time quantitative PCR (RT-qPCR) results revealed that NvKSPI-1 and NvKSPI-2 mRNAs were mostly detected specifically in the venom apparatus, while they were expressed at lower levels in the ovary and much lower levels in other tissues tested. In the venom apparatus, both NvKSPI-1 and NvKSPI-2 transcripts were highly expressed on the fourth day post eclosion and then declined gradually. The NvKSPI-1 and NvKSPI-2 genes were recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvKSPI-1 and GST-NvKSPI-2 to three serine protease inhibitors (trypsin, chymotrypsin, and proteinase K) were tested and results showed that only NvKSPI-1 could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvKSPI-1 and GST-NvKSPI-2 on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by both recombinant proteins; however, there was no significant inhibition on the PO activity. Our results suggested that NvKSPI-1 and NvKSPI-2 could inhibit PPO activation in host hemolymph and trypsin activity in vitro.

As an ectothermic animal, crayfish immunity and their resistance to pathogen can be significantly affected by environmental factors such as light and temperature. It has been found for a long time that multiple immune parameters of animals and human are circadian-regulated by light-entrained circadian rhythm. Whether temperature also affects the immune rhythm of animals still remains unclear. In the present study, we investigated the effect of temperature cycles on the rhythm of crayfish immunity and their resistance. Survival experiments demonstrated that temperature cycles of 24 °C and 18 °C effectively entrained the circadian rhythm of crayfish resistance to Aeromonas hydrophila in constant dark. After being exposed to temperature cycles, the crayfish injected at different time points exhibited significant difference in resistance to A. hydrophila. Bacterial growth and total hemocyte count (THC) also showed circadian variation in crayfish subjected to temperature cycles, but phenoloxidase (PO) activity didn't show rhythmic change under the same conditions. Quantitative real-time PCR revealed that basal expression of crustin1 and astacidin in crayfish subjected to temperature cycles was circadian-rhythmic, but induced expression by A. hydrophila didn't show the same rhythm. In contrast, crayfish maintained at constant temperature showed completely arrhythmic in bacterial resistance, immune parameters mentioned above and the expression of antimicrobial peptides. The results present here collectively indicated that temperature cycles entrained circadian rhythm of some immune parameters and shaped crayfish resistance to bacteria., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Detecting impacts of organophosphorus biocides (OP) is facilitated by analysing "biomarkers" - biological responses to environmental insults. Understanding is hampered by studying biomarkers in isolation at different levels of biological response and limited work on ecologically-important species. We tested the relevance of esterases as biomarkers of OP-exposure in limpets (Patella vulgata), abundant prosobranchs that structure the assemblages on rocky shores through their grazing. We characterized esterases in haemolymph and tissue, and quantified their dose-dependent inhibition by chlorfenvinphos (0.1-3.0 mM) in vitro. To determine whether esterases are useful biomarkers we exposed limpets to chlorfenvinphos (0-10 μg L(-1)). Despite reduced tenacity (ability to stick to a surface) and haemocyte-viability, esterases remained unaffected. Tenacity was reduced by >50% at 5 μg L(-1) and by 95% at 10 μg L(-1), whilst haemocyte-viability was more sensitive with >40% reductions at concentrations of 0.5 μg L(-1) and above. We discuss results in relation to linking sub-lethal and ecological impacts at contaminated sites., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae., (Copyright © 2015 Elsevier Ltd. All rights reserved.)