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Background: The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major potato (Solanum tuberosum) pest, infesting over 16 million km 2 and causing substantial economic losses. The insect cuticle forms an apical extracellular matrix (ECM) envelope covering exposed organs to direct morphogenesis and confer structural protection. While select chitinase (Cht) genes have proven essential for larval development, their potential activities directing ECM remodeling underlying adult wing maturation remain undefined., Results: We investigated the expression patterns and performed an oral RNA interference (RNAi) screen targeting 19 LdChts in late-instar L. decemlineata larvae. Subsequently, we assessed their effects on adult eclosion and wing characteristics. Knockdown of LdCht5, LdCht7, LdCht10, LdIDGF2, and LdIDGF4, as well as others from Group IV (LdCht15, LdCht12, LdCht17, and LdCht13) and Groups VII-X (LdCht2, LdCht11, LdCht1, and LdCht3), resulting in shrunken, misshapen elytra with reduced areal density, as well as transverse wrinkling and impaired wing-tip folding in hindwings. Scanning electron micrographs revealed eroded elytral ridges alongside thinned, ruptured hindwing veins, indicative of mechanical fragility post-LdCht suppression. Spectroscopic analysis uncovered biomolecular alterations underlying the elytral anomalies, including decreases in peaks representing chitin, proteins, and lipids. This loss of essential ECM components provides evidence for the fragility, wrinkling, and shrinkage observed in the RNAi groups., Conclusion: Our findings elucidate the crucial role of chitinases in the turnover of chitinous cuticles on beetle wings, offering insights into RNAi-based control strategies against this invasive pest. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Aedes albopictus is highly prevalent in the northern part of West Bengal and is considered to be responsible for the recent dengue outbreaks in this region. Control of this vector is largely relied on the use of synthetic pyrethroids, which can lead to the development of resistance. In the present study, larvae of three wild Ae. albopictus populations from the dengue-endemic regions were screened for deltamethrin resistance, and the role of cytochrome P450 monooxygenases (CYPs) was investigated in deltamethrin exposed and unexposed larvae. Two populations were incipient resistant, and one population was completely resistant against deltamethrin. Monooxygenase titration assay revealed the involvement of CYPs in deltamethrin resistance along with an induction effect of deltamethrin exposure. Gene expression studies revealed differential expression of five CYP6 family genes, CYP6A8, CYP6P12, CYP6A14, CYP6N3 and CYP6N6, with high constitutive expression of CYP6A8 and CYP6P12 in all the populations before and after deltamethrin exposure. From these findings, it was evident that CYPs play an important role in the development of deltamethrin resistance in the Ae. albopictus populations in this region., (© 2024 Royal Entomological Society.)

Morphological attributes and chemical composition of host plants shape growth and development of phytophagous insects via influences on their behavior and physiological processes. This research delves into the relationship between Eriogyna pyretorum and various host plants through studuying how feeding on different host tree species affect growth, development, and physiological enzyme activities. We examined E. pyretorum response to three distinct host plants: Camphora officinarum , Liquidambar formosana and Pterocarya stenoptera . Notably, larvae feeding on C. officinarum and L. formosana displayed accelerated development, increased pupal length, and higher survival rates compared to those on P. stenoptera . This underlines the pivotal role of host plant selection in shaping the E. pyretorum 's life cycle. The activities of a-amylase, lipase and protective enzymes were the highest in larvae fed on the most suitable host L. formosana which indicated that the increase of these enzyme activities was closely related to growth and development. Furthermore, our investigation revealed a relationship between enzymatic activities and host plants. Digestive enzymes, protective enzymes, and detoxifying enzymes exhibited substantial variations contingent upon the ingested host plant. Moreover, the total phenolics content in the host plant leaves manifested a noteworthy positive correlation with catalase and lipase activities. In contrast, a marked negative correlation emerged with glutathione S-transferase and α-amylase activities. The total developmental duration of larvae exhibited a significant positive correlation with the activities of GST and CarE. The survival rate of larvae showed a significant positive correlation with CYP450. These observations underscore the insect's remarkable adaptability in orchestrating metabolic processes in accordance with available nutritional resources. This study highlights the interplay between E. pyretorum and its host plants, offering novel insights into how different vegetation types influence growth, development, and physiological responses. These findings contribute to a deeper comprehension of insect-plant interactions, with potential applications in pest management and ecological conservation., Competing Interests: The authors declare there are no competing interests., (©2024 Lin et al.)

Botanical insecticides are preferred for their environment and user-friendly nature. Eugenol is a plant-based monoterpene having multifarious biocidal activities. To understand whether eugenol would persistently work against Aedes aegypti, we performed larvicidal bioassays on thirty successive generations and determined median lethal concentration (LC50) on each generation. Results showed no apparent differences between LC50 at F0 (63.48 ppm) and F30 (64.50 ppm) indicating no alteration of susceptibility toward eugenol. To analyze, if eugenol has any effect on metabolic detoxification-associated enzymes, we measured esterases (alpha and beta), cytochrome P450, and GST activities from the survived larvae exposed to LC50 concentration from F0-F30. Results revealed a decrease of esterases, GST, and cytochrome P450 activities at the initial 4-8 generations and then a gradual increase as the generations progressed. GST activity remained significantly below the control groups. Synergists (TPP, DEM, and PBO) were applied along with eugenol at F30 and LC50 concentration, and the said enzyme activities were recorded. Results showed a noticeable decrease in LC50 and enzyme activities indicating effective inhibitions of the respective enzymes. Overall, present results inferred that eugenol would effectively work as a larvicide for a longer period in successive generations without initiating rapid resistance and therefore could be advocated for controlling A. aegypti., (© 2022. The Author(s).)

The effects of two drugs containing the synthetic thyroid hormone levothyroxine (LEV) and an anti-thyroid drug containing propylthiouracil (PTU) on the three early life stages of Xenopus laevis were evaluated with the Frog Embryo Teratogenesis Assay-Xenopus, Tadpole Toxicity Test, and Amphibian Metamorphosis Assay using biochemical and morphological markers. Tested drugs caused more effective growth retardation in stage 8 embryos than stage 46 tadpoles. Significant inhibition of biomarker enzymes has been identified in stage 46 tadpoles for both drugs. AMA test results showed that LEV-I caused progression in the developmental stage and an increase in thyroxine level in 7 days exposure and growth retardation in 21 days exposure in stage 51 tadpoles. On the other hand, increases in lactate dehydrogenase activity for both drugs in the AMA test may be due to impacted energy metabolism during sub-chronic exposure. These results also show that the sensitivity and responses of Xenopus laevis at different early developmental stages may be different when exposed to drugs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Palm weevil, Rhynchophorus palmarum L., is an important pest of palm trees (Elaeis guineensis) around the tropical regions. Characterization of their digestive enzymes could be an important stage to develop appropriate pest control strategies. Study of these enzymes could also be of interest in different biotechnological applications. Among digestive enzymes, there is β-glucosidase which hydrolytically catalyzes the β-glycosidic linkage of glycosides. In the present work, the catalytic activity of β-glucosidase in the digestive juice of last larval instar of R. palmarum L. (Rpbgl) has been investigated using p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate. The "classical" physico-chemical properties for purified Rpbgl have been determined by the help of enzymatic activity modeling. Thus, the values of (325.4 ± 0.2) K, 5.28 ± 0.07 and (37.9 ± 0.6) kJ mol -1 were obtained for optimum temperature, optimum pH and activation energy, respectively. The pK values for enzyme-substrate complex are 4.25 ± 0.07 and 6.20 ± 0.07 for nucleophile and the proton donor, respectively. Enzyme kinetics study was also performed and the values of (127 ± 6) U mg -1 and (0.78 ± 0.08) mM were obtained for V max and K m , respectively. Using the Equilibrium model (EM), the thermal inactivation data were analyzed. ΔH eq , T eq , ΔG inact ∗ and ΔG cat ∗ were found to be (222 ± 4) kJ mol -1 , (323.0 ± 0.1) K, (101.9 ± 0.2) kJ mol -1 and (53.37 ± 0.02) kJ mol -1 , respectively. These results show that Rpbgl is less stable with a narrow temperature tolerance compared to other β-glucosidases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The antibiosis effect of gallic acid on Spodoptera litura F. (Lepidoptera: Noctuidae) and its parasitoid evaluated by feeding six days old larvae on artificial diet incorporated with different concentrations (5 ppm, 25 ppm, 125 ppm, 625 ppm, 3125 ppm) of the phenolic compound revealed higher concentration (LC 50 ) of gallic acid had a negative impact on the survival and physiology of S. litura and its parasitoid Bracon hebetor (Say) (Hymenoptera:Braconidae). The mortality of S. litura larvae was increased whereas adult emergence declined with increasing concentration of gallic acid. The developmental period was delayed significantly and all the nutritional indices were reduced significantly with increase in concentration. Higher concentration (LC 50 ) of gallic acid adversely affected egg hatching, larval mortality, adult emergence and total development period of B. hebetor. At lower concentration (LC 30 ) the effect on B. hebetor adults and larvae was non-significant with respect to control. Gene expression for the enzymes viz., Superoxide dismutase, Glutathione peroxidase, Peroxidase, Esterases and Glutathione S transferases increased while the total hemocyte count of S. litura larvae decreased with treatment. Our findings suggest that gallic acid even at lower concentration (LC 30 ) can impair the growth of S. litura larvae without causing any significant harm to its parasitoid B. hebetor and has immense potential to be used as biopesticides.

Honey bee populations in North America are an amalgamation of diverse progenitor ecotypes experiencing varying levels of artificial selection. Genetic differences between populations can result in variable susceptibility towards environmental stressors, and here we compared pesticide tolerances across breeding stocks using a mixture of seven pesticides frequently found in colonies providing pollination services. We administered the pesticide mixture chronically to in vitro reared larvae at four concentrations of increasing Hazard Quotient (HQ, or cumulative toxicity) and measured mortality during larval development. We found that different stocks had significantly different tolerances to our pesticide mixture as indicated by their median lethal toxicity (HQ 50 ). The intensively selected Pol-Line stock exhibited the greatest pesticide sensitivity while Old World (progenitor) and putatively feral stocks were the most pesticide-tolerant. Furthermore, we found that activity of the detoxification enzyme esterase was positively correlated with pesticide tolerance when measured using two different substrate standards, and confirmed that larvae from the Pol-Line stock had generally lower esterase activity. Consistent with an increased pesticide tolerance, the Old World and putatively feral stocks had higher esterase activities. However, esterases and other detoxification enzymes (CYP450s and GSTs) were found in similar abundances across stocks, suggesting that the differences in enzyme activity we observed might arise from stock-specific single nucleotide polymorphisms or post-translational modifications causing qualitative variation in enzyme activity. These results suggest that selective breeding may inadvertently increase honey bees' sensitivity to pesticides, whereas unselected, putatively feral and Old World stocks have larvae that are more tolerant., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Amphibian larvae are plastic organisms that can adjust their growth and developmental rates to local environmental conditions. The consequences of such developmental alterations have been studied in detail, both at the phenotypic and physiological levels. While largely unknown, it is of great importance to assess how developmental alterations affect the pigmentation pattern of the resulting metamorphs, because pigmentation is relevant for communication, mate choice, and camouflage and hence influences the overall fitness of the toads. Here we quantify the variation in several aspects of the pigmentation pattern of juvenile spadefoot toads experimentally induced to accelerate their larval development in response to decreased water level. It is known that induced developmental acceleration comes at the cost of reduced size at metamorphosis, higher metabolic rate, and increased oxidative stress. In this study, we show that spadefoot toads undergoing developmental acceleration metamorphosed with a less complex, more homogeneous, darker dorsal pattern consisting of continuous blotches, compared to the more contrasted pattern with segregated blotches and higher fractal dimension in normally developing individuals, and at a smaller size. We also observed a marked effect of population of origin in the complexity of the pigmentation pattern. Complexity of the post-metamorphic dorsal pigmentation could therefore be linked to pre-metamorphic larval growth and development.

Bifenthrin is a second generation synthetic pyrethroid insecticide that is widely used in Australia and worldwide. It is frequently found in urban freshwater sediments at concentrations likely to impact biota as it is highly toxic to fish and macroinvertebrates, such as chironomids. Our main goal was to evaluate if oxidative stress and hydrolase enzymes are useful biomarkers of effect of synthetic pyrethroids exposure under different scenarios. Chironomus tepperi larvae (5 days old) were exposed to sub-lethal sediment concentrations of bifenthrin for 5 days under controlled laboratory conditions. A field-based microcosm exposure with bifenthrin-spiked sediments (using the same concentrations as the laboratory exposure) was carried out at a clean field site for four weeks to allow for colonization and development of resident chironomid larvae. At the end of both experiments, Chironomus larvae (C. tepperi in the laboratory exposures and C. oppositus in the microcosm exposures) were collected and oxidative stress enzymes (Glutathione-s-Transferase, Glutathione Reductase and Glutathione Peroxidase) and hydrolase enzymes (Acetylcholinesterase and Carboxylesterase) were measured. Only the Glutathione Peroxidase activity was significantly impacted in larvae from the laboratory exposure. On the contrary, significant changes were observed in all the measured enzymes from the field-based microcosm exposure. This is likely because exposure was throughout the whole life cycle, from egg mass to fourth instar, showing a more realistic exposure scenario. Furthermore, this is the first time that changes in oxidative stress and hydrolase enzymes have been shown to occur in Australian non-biting midges exposed under field-based microcosm conditions. Thus, this study demonstrated the usefulness of these enzymes as biomarkers of effect following bifenthrin exposure in microcosms. It also highlights the importance of using a range of different biochemical endpoints to get a more holistic understanding of pesticide effects and the pathways involved., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report sublethal effects of environmentally relevant concentrations of chlorpyrifos at the individual (swimming alterations) and biochemical (esterase activities and antioxidant enzymes) levels in the Montevideo tree frog Boana pulchella larvae. The 50% lethal concentration at 96 h (LC50-96h) for chlorpyrifos in stage-29 B. pulchella tadpoles was 0.98 mg/L, which was close to the 65th percentile of published anuran species sensitivity. In B. pulchella, chlorpyrifos disrupted biochemical processes: tadpoles showed a significant inhibition of esterase activity and a significant induction of antioxidant enzymes, indicating a response to an environmental challenge causing oxidative stress. Using principal components analysis, we could associate chlorpyrifos reduction in esterase activity with swimming alterations at 0.5 mg/L of the toxicant. The biochemical biomarkers reported in the present study respond at levels 20 times lower than the LC50-96h and were associated with a biologically important response-swimming behavior. The link of responses across different levels of biological organization was demonstrated. The species is suitable as a model for ecotoxicological studies at different levels, including the individual and biochemical levels, and may be considered a good reference organism in environmental control programs. Environ Toxicol Chem 2020;39:834-841. © 2020 SETAC., (© 2020 SETAC.)

Proteolytic processing of Bacillus thuringiensis (Bt) Cry protoxins by insect midgut proteases is critical to their insecticidal activities against target insects. Although transgenic Bt cotton expressing Cry1Ac and Cry2Ab proteins have been widely used for control of the cotton bollworm (Helicoverpa armigera) in the field, the proteolytic cleavage sites in the two protoxins targeted by H. armigera midgut proteases are still not clear. In this study, the proteolysis of Cry1Ac and Cry2Ab protoxins by midgut juice prepared from midgut tissue of H. armigera larvae was investigated. Cleavage of Cry1Ac protoxin by midgut proteases formed a major protein fragment of ~65 kDa, and N-terminal sequencing revealed that cleavage occurred at Arg28 in the fore-end of helix α-1 in domain I of Cry1Ac. Cleavage of Cry2Ab protoxin by midgut juice proteases produced a major protein fragment of ~50 kDa, and the cleavage occurred at Arg139 between helices α-3 and α-4 in domain I of Cry2Ab. The amino acids Arg28 of Cry1Ac and Arg139 of Cry2Ab were predicted as putative trypsin cleavage sites. Bioassay data showed that the toxicities (LC50s) of Cry1Ac and Cry2Ab protoxins were equivalent to those of their respective midgut juice-activated toxins in the susceptible SCD strain of H. armigera. Identification of the exact sites of N-terminal activation of Cry1Ac and Cry2Ab protoxins will provide a basis for a better understanding of the mode of action and resistance mechanisms based on aberrant activation of these protoxins in H. armigera., Competing Interests: The authors have declared that no competing interests exist.

Dimethoate (D) are among the most commonly used organophosphates insecticides in the world. To evaluate the toxicity of two D formulations were selected as test organisms tadpoles of Rhinella arenarum. This toad species has an extensive neotropical distribution and is easy to handle and acclimate to laboratory conditions. The tadpoles were exposed in an acute assay for 48 h to D soluble concentrates (DSC) and emulsifiable concentrates (DEC). The 48 h-LC 50 (95% confidence limits) value of DSC was 57.46 mg L -1 (40.52-81.43) and to DEC was 12.76 mg L -1 (10.39-15.68). These differences in toxicity were statistically significant (p < 0.05). In both formulations, acetylcholinesterase), carboxylesterase, and glutathione-S-transferases enzyme activities varied significantly respect to those of control group (p < 0.05). The DEC formulation was the most toxic. These results would allow the assessment and characterization of potential ecological risks following the application of those formulations.

Methylmercury (MeHg) is a highly toxic contaminant in coastal environments and poses threats to marine fish in early life stages (ELSs). However, MeHg toxicity to fish embryos and larvae is not well investigated. This study investigated the antioxidant defenses and immune responses of flounder Paralichthys olivaceus larvae exposed to waterborne MeHg (0, 0.1, 1.0 and 10.0 μg L - 1 ) for 35 days, from embryogenesis to settlement. The results revealed that metal accumulation in the larvae was positively correlated with MeHg concentration, reduced larval growth and survival. The activities of catalase and glutathione reductase were significantly increased at 10.0 μg L - 1 , while glutathione peroxidase activity and lipid peroxidation level were significantly increased at concentrations over 1.0 μg L - 1 . The corresponding antioxidant-related genes were upregulated under MeHg exposure (cat and gpx at 10.0 μg L - 1 ; gr over 1.0 μg L - 1 ). Lysozyme content was significantly increased, but immunoglobulin M content was significantly decreased at 10.0 μg L - 1 . The immune-related genes were significantly upregulated (hsp70 at 0.1 and 10.0 μg L - 1 ; lzm and il-1β over 1.0 μg L - 1 ; tnf-α and il-6 at 10.0 μg L - 1 ) or downregulated (igm, over 0.1 μg L - 1 ). Overall, MeHg exposure induced oxidative stress and caused immunotoxicity at concentrations over 1.0 μg L - 1 and 10.0 μg L - 1 , respectively. The transcription of selected genes correlated with the corresponding biochemical markers in response to MeHg toxicity. These findings improve our knowledge to better understand the mechanisms by which marine fish at ELSs cope with oxidative stress and immunotoxicity induced by MeHg., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To identify the effects of poplar secondary metabolites on Lymantria dispar, six poplar secondary metabolites (i.e., caffeic acid, salicin, rutin, quercetin, flavone, and catechol) and three mixtures containing characteristic secondary metabolites in poplar were selected. Mixture 1 contained flavone and salicin, mixture 2 contained salicin, caffeic acid, and catechol, and mixture 3 contained flavone, catechol, and caffeic acid. Mixtures were added to artificial diets used to feed 2nd instar L. dispar larvae. The effects of different secondary metabolites on larval growth and development, antifeedant activity, nutrient utilization, and detoxifying enzymatic activity were investigated. Results revealed that there were different influences on L. dispar larvae. The maximum antifeedant rate of flavone was 87.58%. Larvae treated with mixture 2 had a significantly longer development time of 5.61 d with a survival rate of 38.75% for 15 d, which is lower than a single secondary metabolite. No L. dispar larvae survived on feeding diets containing flavone for 7 d. An increase in GST and P450 activities in larvae was significantly induced during the 72 h feeding on artificial diets containing experimental secondary metabolites. After treatment containing salicin and flavone for 24-72 h, P450 activity increased at first then decreased. These results provide a foundation for further investigation on the host selection and underlying adaptation mechanisms in L. dispar., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo . According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

The larvae of the black soldier fly ( Hermetia illucens L., BSFL) have received increased industrial interest as a novel protein source for food and feed. Previous research has found that insects, including BSFL, are capable of metabolically converting aflatoxin B 1 (AFB 1 ), but recovery of total AFB 1 is less than 20% when accounting for its conversion to most known metabolites. The aim of this study was to examine the conversion of AFB 1 by S9 extracts of BSFL reared on substrates with or without AFB 1 . Liver S9 of Aroclor-induced rats was used as a reference. To investigate whether cytochrome P450 enzymes are involved in the conversion of AFB 1 , the inhibitor piperonyl butoxide (PBO) was tested in a number of treatments. The results showed that approximately 60% of AFB 1 was converted to aflatoxicol and aflatoxin P 1 . The remaining 40% of AFB 1 was not converted. Cytochrome P450s were indeed responsible for metabolic conversion of AFB 1 into AFP 1 , and a cytoplasmic reductase was most likely responsible for conversion of AFB 1 into aflatoxicol.

In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis., (© 2019 Wiley Periodicals, Inc.)

Serine protease (SPs) is one of the immune enzyme's molecules that play a main role in the variation of a physiological process by controlling protease actions in vertebrates. For example, signaling cells, protector and improvement, which are included in melanization, are utilized to cascade with the meddling pathogens and defense the harmed tissue in insects. In this study, we explore the biochemical process of (SP-22) from Bombyx mori. Reverse-transcription polymerase chain reaction (RT-PCR) discloses that BmSP-22 is expressed in all tissues including the fat body. The formative expression profile of BmSP-22 reveal that BmSP-22 messenger RNA is expressed constitutively in larvae. Injection of recombinant BmSP-22 into B. mori larvae reduces significantly the transcript levels of antimicrobial peptides in the fat body. Our results suggest that BmSP-22 plays an important role in the innate immunity of B. mori and possibly in other insects., (© 2019 Wiley Periodicals, Inc.)

The insecticidal and antifeedant activities of five 7-chloro-4-(1 H -1,2,3-triazol-1-yl)quinoline derivatives were evaluated against the maize armyworm, Spodoptera frugiperda (J.E. Smith). These hybrids were prepared through a copper-catalyzed azide alkyne cycloaddition (CuAAC, known as a click reaction) and displayed larvicidal properties with LD 50 values below 3 mg/g insect, and triazolyl-quinoline hybrid 6 showed an LD 50 of 0.65 mg/g insect, making it 2-fold less potent than methomyl, which was used as a reference insecticide (LD 50 = 0.34 mg/g insect). Compound 4 was the most active antifeedant derivative (CE 50 = 162.1 μg/mL) with a good antifeedant index (56-79%) at concentrations of 250-1000 μg/mL. Additionally, triazolyl-quinoline hybrids 4 - 8 exhibited weak inhibitory activity against commercial acetylcholinesterase from Electrophorus electricus (electric-eel AChE) (IC 50 = 27.7 μg/mL) as well as low anti-ChE activity on S. frugiperda larvae homogenate (IC 50 = 68.4 μg/mL). Finally, molecular docking simulations suggested that hybrid 7 binds to the catalytic active site (CAS) of this enzyme and around the rim of the enzyme cavity, acting as a mixed (competitive and noncompetitive) inhibitor like methomyl. Triazolyl-quinolines 4 - 6 and 8 inhibit AChE by binding over the perimeter of the enzyme cavity, functioning as noncompetitive inhibitors. The results described in this work can help to identify lead triazole structures from click chemistry for the development of insecticide and deterrent products against S. frugiperda and related insect pests.

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% β-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8 , Caspase-9 , Ec-R , E74A , and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.

Odontoprynus americanus tadpoles were used to determine the safety concentration of pyriproxyfen (PPF) insecticide by acute and sublethal toxicity tests (nominal range tested 0.01 to 10 [± 15%] PPF mg/L). Median lethal concentration (LC 50 ) and no, and lowest-observed-effect concentrations (NOEC and LOEC, respectively) were calculated. We also assessed the effect on the activities of glutathione S-transferse (GST), acetylcholinesterase (AChE), and carboxylesterase (CbE) and compared to control (CO) tadpoles. Based on the 48-h NOEC value, two sublethal concentrations of PPF (0.01 and 0.1 mg/L) were assayed to detect effects on enzymes activities (GST and CbE), thyroid hormone's levels (triiodothyronine; T3 and thyroxine; T4), heart function, and tadpoles swimming behaviour. The results showed that the LC 50 values of O. americanus tadpoles were 3.73 PPF mg/L and 2.51 PPF mg/L at 24-h and 48-h, respectively (NOEC = 0.1 mg/L; LOEC = 1 mg/L, for both times). PPF concentrations at 48 h, induced enzymatic activities such as GST (212.98%-242.94%), AChE (142.15%-165.08%), and CbE (141.86%-87.14%) significantly respect to COs. During the 22 days of chronic PPF exposure, GST (0.01 mg/L 88%-153% NOEC), AChE (177.82% NOEC), and T4 (70% NOEC) also significantly increased respect to COs. Similarly, heart rate (fH) and ventricular cycle length (VV interval) in CO tadpoles were significantly higher than PPF treated. Finally, at NOEC tadpoles exhibited significant effects on the behavioral endpoint (swimming distance, mean speed, and global activity; P < 0.05)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vector-borne diseases and especially malaria are responsible for more than half million deaths annually. The increase of insecticide resistance in wild populations of Anopheles malaria vectors emphasises the need for novel vector control strategies as well as for identifying novel vector targets. Venus kinase receptors (VKRs) constitute a Receptor Tyrosine Kinase (RTK) family only found in invertebrates. In this study we functionally characterized Anopheles VKR in the Gambiae complex member, Anopheles coluzzii. Results showed that Anopheles VKR can be activated by L-amino acids, with L-arginine as the most potent agonist. VKR was not required for the fecundity of A. coluzzii, in contrast to reports from other insects, but VKR function is required in both Anopheles males and females for development of larval progeny. Anopheles VKR function is also required for protection against infection by Plasmodium parasites, thus identifying a novel linkage between reproduction and immunity in Anopheles. The insect specificity of VKRs as well as the essential function for reproduction and immunity suggest that Anopheles VKR could be a potentially druggable target for novel vector control strategies.

Kresoxim-methyl (KM) is a broad spectrum strobilurin fungicide that has been used widely on crops around the world. In the present study, we aimed to investigate the toxic effects of KM using various sublethal endpoints during zebrafish (Danio rerio) larval development. Results showed that the LC 50 values of KM to zebrafish at multiple life stages (embryo, larvae, juvenile and adult) were 0.340, 0.224, 0.328 and 0.436 mg/L, respectively. The transcription patterns of 45 genes involved in hypothalamic-pituitary-thyroid/gonadal (HPT/HPG) axis, oxidative stress and apoptosis revealed KM could affect zebrafish larval development at multiple pathways. The activities of aromatase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), caspase 3 (Cas3) and caspase 9 (Cas9), and the levels of estradiol (E 2 ), vitellogenin (VTG), thyroid hormones (T 3 and T 4 ), reactive oxygen species (ROS) and ATP after embryos exposed to KM for 3 d, 6 d and 10 d were correlated well with the transcription of the corresponding molecules involved in these pathways. In addition to providing the first description of the toxic effects induced by KM during larval development, the results of present study also provided the potential mechanisms of KM on multi-level biomarker responses in larval zebrafish., (Copyright © 2018. Published by Elsevier Ltd.)

The Cry1Ac protein is the most active insecticidal toxin from the bacterium Bacillus thuringiensis (Bt) to members of the heliothinae subfamily in Lepidoptera, which includes some of the most devastating pests of corn and cotton worldwide. However, there are wide discrepancies in susceptibility among members of this subfamily in the US. Specifically, susceptibility to Cry1Ac in Helicoverpa zea (Hz) is >100-fold lower when compared to Heliothis virescens (Hv) larvae. The biochemical properties and Cry1Ac protoxin processing activity of gut digestive fluids from larvae of Hz and Hv were compared to test their role in differential susceptibility to Cry1Ac. Comparatively lower protease activity, associated with slower Cry1Ac proteolytic processing, was detected in digestive fluids of Hz compared to Hv. Moreover, Cry1Ac toxin processed by Hz digestive fluids displayed significantly lower toxicity in vitro against cultured insect cells compared to toxin activated by Hv proteases. These data support a contributing role for gut proteases in differential susceptibility to Cry1Ac in heliothine larvae., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

The resistance status of Selangor Aedes aegypti (Linnaeus) larvae against four major groups of insecticides (i.e., organochlorines, carbamates, organophosphates and pyrethroids) was investigated. Aedes aegypti were susceptible against temephos (organophosphate), although resistance (RR 50  = 0.21-2.64) may be developing. The insecticides susceptibility status of Ae. aegypti larvae were found heterogeneous among the different study sites. Results showed that Ae. aegypti larvae from Klang, Sabak Bernam and Sepang were susceptible against all insecticides tested. However, other study sites exhibited low to high resistance against all pyrethroids (RR 50 = 1.19-32.16). Overall, the application of synergists ethacrynic acid, S.S.S.- tributylphosphorotrithioate and piperonyl butoxide increased the toxicity of insecticides investigated. However, the application failed to increase the mortality to susceptible level (>97%) for certain populations, therefore there are chances of alteration of target site resistance involved. Biochemical assays revealed that α-esterase, (Gombak, Kuala Langat, Kuala Selangor and Sabak Bernam strains) β-esterase (Klang and Sabak Bernam strains), acetylcholinesterase (Kuala Selangor and Sabak Bernam strains), glutathione-S-transferase (Kuala Selangor and Sabak Bernam strains) and mono-oxygenases (Gombak, Hulu Langat, Hulu Selangor and Kuala Langat strains) were elevated. Spearman rank-order correlation indicated a significant correlation between resistance ratios of: DDT and deltamethrin (r = 0.683, P = 0.042), cyfluthrin and deltamethrin (r = 0.867, P =0.002), cyflyuthrin and lambdacyhalothrin (r = 0.800, P =0.010), cyfluthrin and permethrin (r = 0.770, P =0.015) deltamethrin and permethrin (r = 0.803, P =0.088), propoxur and malathion (r = 0.867, P = 0.002), malathion and temephos (r = 0.800, P = 0.010), etofenprox and MFO enzyme (r = 0.667, P =0.050). The current study provides baseline information for vector control programs conducted by local authorities. The susceptibility status of Ae. aegypti should be monitored sporadically to ensure the effectiveness of current vector control strategy in Selangor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We assessed the effectiveness of a biofortified maize line (4BtxHC) which accumulates high levels of antioxidant carotenoids that also expressed the insecticidal Cry1Ac Bacillus thuringiensis (Bt) gene against the European corn borer Ostrinia nubilalis. This line had been previously engineered to accumulate carotenoids specifically in the seed endosperm, whereas the Bt gene was expressed constitutively. The concentrations of Bt toxin (Cry 1Ac) in the leaves of the 4Bt and 4BtxHC lines were not significantly different at 47±6 μg/g of fresh weight (FW); neither were they in the kernels of both lines (35±3 μg/g FW). The kernels and leaves were toxic to the larvae of O. nubilalis. However, the insecticidal activity was substantially lower (ca. 20%) than that of lines that expressed only Bt in spite that the two lines showed a quantity of toxin not significantly different in kernels or in leaves. Although the reduced effectiveness of Cry1Ac in kernels may not be entirely surprising, the observation of the same phenomenon in vegetative tissues was unexpected. When semi-artificial diets containing kernels from 4Bt supplemented with different levels of β-carotene were used in insect bioassays, the β-carotene moderated the effectiveness of the Bt similarly to the plant material with carotenoid enrichment. To elucidate the biochemical basis of the reduced effectiveness of Bt toxin in the carotenoid-enriched plants, we measured the activity of three enzymes known to be implicated in the detoxification defence, namely, catalase, superoxide dismutase and glutathione S-transferase. Whereas Cry1Ac expression significantly increased SOD and CAT enzymatic activity in the absence of carotenoids, carotenoids, either in 4BtxHC or in artificial diets enriched with β-carotene, significantly lowered CAT activity. Carotenoids can therefore moderate the susceptibility of the maize borer O. nubilalis to Cry1Ac, and we hypothesize that their role as antioxidants could explain this phenomenon via their scavenging of reactive oxygen species produced during Cry1Ac detoxification in the larvae. The involvement of this mechanism in the decreased mortality caused by Cry1Ac when carotenoids are present in the diet is discussed., Competing Interests: We would also like to clarify that the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Project was partially funded by RecerCaixa a philanthropic Catalan Foundation funding academic research through competitive projects selected in open competition by the public scientific agency AGAUR, which is part of the Catalan Government. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Insects have developed tolerance against mycoses caused by entomopathogenic fungi through several humoral and cellular mechanisms. Antioxidant enzymes such as superoxide dismutase, lipid peroxidase, and peroxidase can play a role in defense against mycosis, but the physiological interactions between the fungus and the insect are not well characterized. In this study, the effects of infection by entomopathogenic fungus, Aspergillus flavus on the antioxidant defense system of Spodoptera litura, were investigated. The fungi, A. flavus exposure resulted in modification of the levels of antioxidant enzymes, as well as significant decline in phenoloxidase titers and the total hemocyte count 48 h post exposure. A significant increase was observed in detoxifying enzymes. All these results suggest that A. flavus infects S. litura by directly acting on the immune system, resulting in decreased immune function. Bioassay results showed that A. flavus affects third and fourth instar larvae of S. litura. This report supports the importance of A. flavus as a candidate for biological control of S. litura., (Copyright © 2018. Published by Elsevier Inc.)

Exposure of Culex quinquefasciatus larvae to hypothermia or hyperthermia in relation to ecological temperature has resulted in alteration of enzyme activities related to maintenance of free amino acid pool and protein degradation. Sudden changes in water temperature have led to elevation of protein content particularly in molecular weight between 66 and 97.4 kDa. MALDI TOF analysis revealed the presence of putative uncharacterized protein and Phospholipase A2 activating protein in larvae subjected to 40°C for 1 hour which was not found in control and other temperature treated larvae. These proteins may be playing a role in survival of larvae at higher temperatures. Thermal shock has resulted in channeling of free amino acids for protein synthesis and elevation of amino acid catabolism through increased deamination and altered transamination, which resulted in a sharp decrease in free amino acid pool. Inhibition of Leucine amino peptidase and elevation of Cathepsin D, under the influence of thermal shock may be an adaptive response of larvae to prevent unnecessary degradation of protein and at the same time facilitated rapid internal re-organization which may accelerate pupation. Elevated activity of Phenol oxidase and its co-stimulator Trypsin -like Serine protease, along with increased expression of Phospholipase A2 activating protein is a well co-ordinated defense mechanism leading to increased immune response in stressed state. This study assumes significance because variation in environmental conditions such as global warming and their impact on mosquito physiology can influence efficiency of disease vector., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A novel Kunitz-type inhibitor from Platypodium elegans seeds (PeTI) was purified and characterized. The mass spectrometry analyses of PeTI indicated an intact mass of 19 701 Da and a partial sequence homologous to Kunitz inhibitors. PeTI was purified by ion exchange and affinity chromatographies. A complex with a 1:1 ratio was obtained only for bovine trypsin, showing a K i = 0.16 nM. Stability studies showed that PeTI was stable over a wide range of temperature (37-80 °C) and pH (2-10). The inhibitory activity of PeTI was affected by dithiothreitol (DTT). Bioassays of PeTI on Spodoptera frugiperda showed negative effects on larval development and weight gain, besides extending the insect life cycle. The activities of digestive enzymes, trypsin and chymotrypsin, were reduced by feeding larvae with 0.2% PeTI in an artificial diet. In summary, we describe a novel Kunitz inhibitor with promising biotechnological potential for pest control.

Aedes aegypti mosquitoes are responsible for dengue, chikungunya, and Zika virus transmission in tropical and subtropical areas around the world. Due to the absence of vaccines or antiviral drugs for human treatment, the majority of control strategies are targeted at Ae. aegypti elimination. Our research on mosquito control insecticidal agents has previously shown that the alkaloid girgensohnine and its analogues (α-aminonitriles) present in vitro acetylcholinesterase inhibition and in vivo insecticidal activity against Ae. aegypti. However, acetylcholinesterase inhibition may not be the only mechanism of action behind these effects. On this basis, the principal aim of this study was to elucidate the possible action mode of four α-aminonitriles on Ae. aegypti by studying other important enzymatic targets, such as mitochondrial electron transport chain complexes, catalase, and superoxide dismutase, key oxidative stress enzymes. Mitochondria were isolated from Ae. aegypti larvae by differential centrifugation, stored at -70°C, and fragmented using ultrasound for 10min. The effects of α-aminonitriles (1 to 4) over enzymatic activities were evaluated using concentrations of 8nM, 2μM, 8μM, and 40μM. Results indicated that α-aminonitriles caused significant NADH dehydrogenase and succinate oxidase inhibition (~44% at the highest concentration tested). Succinate dehydrogenase and cytochrome c oxidase activities were found to increase (162% and 106% at 40μM, respectively). It was also observed that these compounds produced catalase inhibition and thus prevented H 2 O 2 reduction, which induced the formation of reactive oxygen species (ROS). Moreover, NBT assay showed that compounds 3 and 4 (with 2-(pyrrolidin-1-yl) acetonitrile as substituent) increased by approximately 50% the O 2 ●- concentration in the mitochondrial respiratory chain. It was concluded that the tested compounds act as complex I inhibitors by blocking electron transport and causing electron leak, possibly between complex I and III. Furthermore, α-aminonitriles inhibited catalase activity; compounds 1 and 2 (with piperidine fragment) inhibited glutathione reductase activity and further promoted the accumulation of ROS, which probably induced oxidative stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Organisms react to environmental changes through plastic responses that often involve physiological alterations with the potential to modify life-history traits and fitness. Environmentally induced shifts in growth and development in species with complex life cycles determine the timing of transitions between subsequent life stages, as well as body condition at transformation, which greatly determine survival at later stages. Here we show that spadefoot toad larvae surviving pond drying and predators experienced marked alterations in growth and development, and in their fat reserves, oxidative stress, and relative telomere length. Tadpoles accelerated development but reduced growth and consumed more fat reserves when facing pond drying. However, oxidative stress was buffered by increased antioxidant enzyme activity, and telomeres remained unchanged. Predators caused opposite effects: they reduced larval density, hence relaxing competition and allowing faster development and enhanced growth of survivors. Tadpoles surviving predators metamorphosed bigger and had larger fat bodies, increasing their short-term survival odds, but showed signs of oxidative stress and had shorter telomeres. Developmental acceleration and enhanced growth thus seemed to have different physiological consequences: reduced fat bodies and body size compromise short-term survival, but are reversible in the long run, whereas telomere shortening is non-reversible and could reduce long-term survival.

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.)

Alimentary products of medicinal Lucilia sericata larvae are studied to determine their mechanisms of action, particularly in the contexts of wound debridement and disinfection. Furthermore, the larvae can be applied to patients in contained medical devices (such as the BioBag; BioMonde). Here, we tested the materials and larval content of the most commonly used debridement device (the "BB-50") to explore the possibility that endotoxins may be contributing to the bio-activity of the product, given that endotoxins are potent stimulants of cellular activation. Using standardised protocols to collect larval alimentary products (LAP), we proceeded to determine residual endotoxin levels in LAP derived from the device, before and after the neutralisation of interfering enzymatic activity. The debridement device and its associated larval content was not a significant source of lipopolysaccharide (LPS) activity. However, it is clear from these experiments that a failure to remove the confounding serine proteinase activity would have resulted in spuriously high and erroneous results. The residual LPS levels detected are unlikely to be active in wound healing assays, following cross-referencing to publications where LPS at much higher levels has been shown to have positive and negative effects on processes associated with wound repair and tissue regeneration., (© 2017 by the Wound Healing Society.)

In-vivo effects of polybrominated diphenyl ethers (PBDEs) containing 3, 4 and 5 bromine atoms were tested on fourth-instar larvae of Chironomus sancticaroli (Diptera: Chironomidae) after 48h of exposure, by measuring the activity of the acetyl cholinesterase, alpha and beta esterases and glutathione S-transferase. The PBDE congeners 2,2',4-triBDE (BDE-17), 2,2',4,4'-tetraBDE (BDE-47) and 2,2',4,4',5-pentaBDE (BDE-99) were evaluated at 0.5, 1.0, 2.0 and 3.0ngmL -1 . Acetyl cholinesterase activity decreased significantly (p≤0.05) at all evaluated concentrations of the three PBDE congeners, except for larvae exposed to BDE-17 at 1.0 and 2.0ngmL -1 . The significant inhibition of acetyl cholinesterase activity ranged from 18% (BDE-47 at 0.5ngmL -1 ) to 72% (BDE-47 at 2.0ngmL -1 ). The enzymes alpha and beta esterase were also affected by the three congeners, reducing their activity from 14% (BDE-99 at 1.0ngmL -1 ) to 52% (BDE-47 at 2.0ngmL -1 ) and from 7% (BDE-99 at 2.0ngmL -1 ) to 34% (BDE-47 at 3.0ngmL -1 ) respectively. Substantial increments in glutathione S-transferase activity were similarly observed, varying from 138% (BDE-99 2.0 at ng mL -1 ) to 346% (BDE-17 at 1.0ngmL -1 ). DNA strand breaks were detected exclusively in larvae exposed to BDE-99 at 2.0 and 3.0ngmL -1 (H=11.7, p=0.019). These results showed that C. sancticaroli larvae were sensitive to the PBDEs treatments under the experimental conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Athetis lepigone was a new lepidopteran pest and caused severe damage to maize crops in China. We have detected that Cry1Ac protoxin and toxin were highly active against the larvae of A. lepigone. However, there is no report about the mode of action of Bt Cry1Ac toxin against this pest until now. A 110 kDa APN5 protein from BBMV of A. lepigone was identified as the binding receptor of Cry1Ac toxin using Ligand blotting. The Cry1Ac receptor APN5 was cloned from A. lepigone larval midgut mRNA and named as AlAPN5 (GenBank accession no.: KU950745). AlAPN5 had a GATEN motif and been classified to Class 5 APNs. 79.2% reduction in mortality was observed when A. lepigone larvae were injected with siRNA of the AlAPN5 gene and treated with Cry1Ac toxin. These data demonstrate that AlAPN5 is a putative functional receptor and maybe the only receptor of Cry1Ac in A. lepigone.

Caspase-1 is one of the effector caspases in mammals that plays a central role in apoptosis. However, the lepidopteran caspase-1, especially the Bombyx mori caspase-1 (Bm-caspase-1), has not been investigated in detail. In this study, Bm-caspase-1 was identified from an expressed sequence tag database in B. mori by BLAST search. The open reading frame of Bm-caspase-1 contained 879 nucleotides and encoded 293 amino acids with a predicted molecular mass of 33 kDa. Bm-caspase-1 contained two consensus amino acid motifs of caspase cleavage sites, DEGDA and TETDG. Caspase activity assays revealed significant proteolytic activity of the Ac-DEVD-pNA substrate. Bm-caspase-1 can be detected in all tissues and developmental stages by a semi quantitative polymerase chain reaction assay. More importantly, the expression level of Bm-caspase-1 is increased upon baculovirus infection and up-regulated in BmNPV-resistant silkworms. Taken together, these results indicate that Bm-caspase-1 plays an important role during baculovirus infection.

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of V max and K m of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

Biomarkers have been widely employed in ecotoxicology as early warning indicators of exposure to toxicants. Very often, they are used to compare reference and polluted sites, or to analyse time trends. However, very few studies focus on the natural variability range of biomarkers in the environment, which is pivotal to understand if the detected differences are actually determined by any adverse effects due to pollution. This work assesses the natural spatio-temporal variability of some enzymatic levels, frequently used as biomarkers, in freshwater benthic invertebrates. The influence of some environmental parameters on the enzymatic levels was also evaluated. Three families of insect larvae (Perlidae, Baetidae, and Heptageniidae) were sampled in three pristine streams and in eight different dates. Four enzymes (acetylcholinesterase, glutathione-S-transferase, alkaline phosphatase, and catalase) were measured. The natural variability of enzymatic levels was often significant in all considered species across both space and time. The observed pattern was poorly explained by the monitored environmental parameters. The results of this work show that great care should be paid when interpreting monitoring data in which biomarker levels are measured and compared among sites or dates. Presuming that measured differences are due to anthropogenic factors can be misleading, when other potentially influencing factors have not been accounted for.

An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125μgL -1 chlorpyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost two-fold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity - measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) - was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue- and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42-78% of controls) compared with pellet-fed tadpoles (<60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency.

Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2 h, 100% in 48 h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated.

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonahm) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonahm reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pterodon emarginatus Vogel is a Brazilian species that belongs to the family Fabaceae, popularly known as sucupira. Its oil has several biological activities, including potent larvicidal property against Aedes aegypti. This insect is the vector of dengue, a tropical disease that has been considered a critical health problem in developing countries, such as Brazil. Most of dengue control methods involve larvicidal agents suspended or diluted in water and making active lipophilic natural products available is therefore considered a technological challenge. In this context, nanoemulsions appear as viable alternatives to solve this major problem. The present study describes the development of a novel nanoemulsion with larvicidal activity against A. aegypti along with the required Hydrophile Lipophile Balance determination of this oil. It was suggested that the mechanism of action might involve reversible inhibition of acetylcholinesterase and our results also suggest that the P. emarginatus nanoemulsion is not toxic for mammals. Thus, it contributes significantly to alternative integrative practices of dengue control, as well as to develop sucupira based nanoproducts for application in aqueous media.

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

This study evaluated the larvicidal activity of 12 Apiaceae plant essential oils and their components against the Asian tiger mosquito, Aedes albopictus, and the inhibition of acetylcholine esterase with their components. Of the 12 plant essential oils tested, ajowan (Trachyspermum ammi), caraway seed (Carum carvi), carrot seed (Daucus carota), celery (Apium graveolens), cumin (Cuminum cyminum), dill (Anethum graveolens), and parsley (Petroselinum sativum) resulted in >90% larval mortality when used at 0.1 mg/mL. Of the compounds identified, α-phellandrene, α-terpinene, p-cymene, (-)-limonene, (+)-limonene, γ-terpinene, cuminaldehyde, neral, (S)-+-carvone, trans-anethole, thymol, carvacrol, myristicin, apiol, and carotol resulted in >80% larval mortality when used at 0.1 mg/mL. Two days after treatment, 24.69, 3.64, and 12.43% of the original amounts of the celery, cumin, and parsley oils, respectively, remained in the water. Less than 50% of the original amounts of α-phellandrene, 1,8-cineole, terpinen-4-ol, cuminaldehyde, and trans-antheole were detected in the water at 2 days after treatment. Carvacrol, α-pinene, and β-pinene inhibited the activity of Ae. albopictus acetylcholinesterase with IC50 values of 0.057, 0.062, and 0.190 mg/mL, respectively. A spherical microemulsion of parsley essential oil-loaded poly(vinyl alcohol) (PVA) was prepared, and the larvicidal activity of this formulation was shown to be similar to that of parsley oil.

This study assessed the effect of the pesticides on activity and expression of glutathione S-transferases (GSTs) in the midgut of the silkworm after intense selections by phoxim and fenpropathrin for five and four generations, respectively. GSTs activity towards cumene hydroperoxide (CHP), a substrate of GSH-dependent peroxidase, was significantly increased in the crude homogenate of midguts after long term exposure to the insecticides. An epsilon-class GST gene (BmGSTe2) was identified and showed elevated expression in the midguts of phoxim- and fenpropathrin-selected strains. Expression of BmGSTe2 was only detected in the midgut at transcriptional and translational levels. The recombinant BmGSTE2 possessed peroxidase activity and significantly inhibited by fenpropathrin, phoxim and chlorpyrifos in vitro. These results indicated that BmGSTE2 might be one of the enzymes involved in enhancing larval tolerance to the insecticides used. Furthermore, GST activity and expression level of BmGSTe2 might be used as biomarkers of organophosphorus and pyrethroid insecticide exposures., (Copyright © 2015 Elsevier Inc. All rights reserved.)