The Insecticidal Activity of Rhinella schneideri (Werner, 1894) Paratoid Secretion in Nauphoeta cinerea Cocroaches.

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07µg/g) and 48 h exposure (44.07 µg/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p < 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p < 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p < 0.001, n ≥ 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p < 0.001, n ≥ 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p < 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p < 0.05, n ≥ 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

Jaburetox, a urease-derived peptide: Effects on enzymatic pathways of the cockroach Nauphoeta cinerea.

Jaburetox is a recombinant peptide derived from one of the Canavalia ensiformis urease isoforms. This peptide induces several toxic effects on insects of different orders, including interference on muscle contractility in cockroaches, modulation of UDP-N-acetylglucosamine pyrophosphorylase (UAP) and nitric oxide synthase (NOS) activities in the central nervous system of triatomines, as well as activation of the immune system in Rhodnius prolixus. When injected, the peptide is lethal for R. prolixus and Triatoma infestans. Here, we evaluated Jaburetox toxicity to Nauphoeta cinerea cockroaches, exploring the effects on the central nervous system through the activities of UAP, NOS, acid phosphatases (ACP), and acetylcholinesterase (AChE). The results indicated that N. cinerea is not susceptible to the lethal effect of the peptide. Moreover, both in vivo and in vitro treatments with Jaburetox inhibited NOS activity, without modifying the protein levels. No alterations on ACP activity were observed. In addition, the enzyme activity of UAP only had its activity affected at 18 hr after injection. The peptide increased the AChE activity, suggesting a mechanism involved in overcoming the toxic effects. In conclusion, our findings indicate that Jaburetox affects the nitrinergic signaling as well as the AChE and UAP activities and establishes N. cinerea as a Jaburetox-resistant model for future comparative studies.

Molecular mechanisms underlying milk production and viviparity in the cockroach, Diploptera punctata.

Viviparous reproduction is characterized by maternal retention of developing offspring within the reproductive tract during gestation, culminating in live birth. In some cases, a mother will provide nutrition beyond that present in the yolk; this is known as matrotrophic viviparity. While this phenomenon is best associated with mammals, it is observed in insects such as the viviparous cockroach, Diploptera punctata. Female D. punctata carry developing embryos in the brood sac, a reproductive organ that acts as both a uterus and a placenta by protecting and providing a nutritive secretion to the intrauterine developing progeny. While the basic physiology of D. punctata pregnancy has been characterized, little is known about the molecular mechanisms underlying this phenomenon. This study combined RNA-seq analysis, RNA interference, and other assays to characterize molecular and physiological changes associated with D. punctata reproduction. A comparison of four stages of the female reproductive cycle and males revealed unique gene expression profiles corresponding to each stage and between sexes. Differentially regulated transcripts of interest include the previously identified family of milk proteins and transcripts associated with juvenile hormone metabolism. RNA interference and methoprene application experiments established the potential impacts of bothbreakdown and synthesis reduction of juvenile hormone in maintaining pregnancy in D. punctata. These studies provide the comprehensive molecular mechanisms associated with cockroach viviparity, which will be a critical resource for comparative purposes among viviparity in insect systems.

Optimal conditions for the storage of German cockroach extract.

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease­rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18­26˚C, RT) or refrigerated (2­8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol NS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease­rich cockroach extract.

Assessing the toxicant effect of spontaneously volatilized 4-vinylcyclohexane exposure in nymphs of the lobster cockroach nauphoeta cinerea.

Vinylcyclohexene (VCH) is an environmental contaminant well known for its ovotoxicant effects in several organisms. However, the mechanisms underlying the toxicity of VCH as well as its harmful effects toward other organs are until unclear. In this work, we assess some endpoint signals of toxicity induced by volatilized VCH exposure using nymphs of the lobster cockroach Nauphoeta cinerea. Nymphs were exposed to VCH via inhalation for 70 days. The levels of volatilized VCH were quantified by headspace gas chromatography and the concentration varied between 3.41 and 7.03 nmol/µl. VCH inhalation caused a reduction of 35% in the survival rate of the exposed animals. Nymphs exposed to volatilized VCH for 35 and 70 days had a reduction in the body weight gain of 1.8- and 2.6-fold, respectively with a reduction in dissected head, fat body, and maturing reproductive organs. The exposure did not change water consumption, excepting on the 20th day (with a 3-fold change) and decreased the food intake significantly. Regarding biochemical markers, we found that the activity of GST from the dissected organs was increased by volatilized VCH after both 35 and 70 days of exposure. The fat body presented the most prominent GST activity especially after 35 days of exposure with 1.6-fold higher than the control group. Exposure also caused an increase in RS levels in the fat body of 1.35-fold and 1.47-fold after 35 and 70 days, respectively and did not affect the activity of the AChE from the head. Our findings support the harmful impact of volatilized VCH inhalation, highlighting the cockroach N.cinerea as a valuable insect model to investigate environmental toxicants.

The digestive system in Zygentoma as an insect model for high cellulase activity.

The digestive system of selected phytophagous insects has been examined as a potential prospecting resource for identification of novel cellulolytic enzymes with potential industrial applications. In contrast to other model species, however, limited detailed information is available that characterizes cellulolytic activity and systems in basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we have for the first time, identified species of Zygentoma that displayed the highest relative cellulase activity levels when compared to all other tested insect groups under the experimental conditions, including model species for cellulolytic systems such as termite and cockroach species in Rhinotermitidae (formerly Isoptera) and Cryptocercidae (formerly Blattodea). The goal of the present study was to provide a morphohistological characterization of cellulose digestion and to identify highly active cellulase enzymes present in digestive fluids of Zygentoma species. Morphohistological characterization supported no relevant differences in the digestive system of firebrat (Thermobia domestica) and the gray silverfish (Ctenolepisma longicaudata). Quantitative and qualitative cellulase assays identified the foregut as the region with the highest levels of cellulase activity in both T. domestica and C. longicaudata. However, T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Using nano liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS/MS) and a custom gut transcriptome we identified cellulolytic enzymes from digestive fluids of T. domestica. Among the identified enzymes we report putative endoglucanases matching to insect or arthropod enzymes and glucan endo-1,6-ß-glucosidases matching bacterial enzymes. These findings support combined activities of endogenous and symbiont-derived plant cell wall degrading enzymes in lignocellulose digestion in Zygentoma and advance our understanding of cellulose digestion in a primitive insect group.

[Molecular modification of ß-glucosidase from the midgut of Macrotermes barneyi].

Cellulose hydrolysis to glucose requires a series of cellulase enzymes, of which ß-glucosidases play a crucial role. ß-glucosidase (MbmgBG1) derived from the midgut of Macrotermes barneyi has higher glucose tolerance (maintaining more than 60% enzyme activity at 1.5 mol/L glucose). However, low enzyme activity and poor thermal stability limit the applications of ß-glucosidase in food industries. Point mutants (F167L, T176C, E347I, R354K, N393G and V425M) were obtained by site-directed mutagenesis of non-conserved amino acids near conserved amino acids. Among them, the specific activities against to substrate pNPG of two mutants (F167L and R354K) were about 2-fold and 4-fold higher than that of MbmgBG1. Kcat/Km values were also higher than that of the wild-type, reflecting stronger affinity to the substrate and higher catalytic ability of mutants than MbmgBG1. When the glucose concentration was 1.5 mol/L, the enzyme activity of MbmgBG1 was about 60% of the original activity. F167L and R354K kept 60% enzymatic activity when the glucose concentrations of was 2.0 mol/L and 3.0 mol/L, respectively. These results lay a foundation for further studies on the catalytic efficiency of ß-glucosidase.

Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation.

Protease allergens are known to enhance allergic inflammation but their exact role in initiation of allergic reactions at mucosal surfaces still remains elusive. This study was aimed at deciphering the role of serine protease activity of Per a 10, a major cockroach allergen in initiation of allergic inflammation at mucosal surfaces. We demonstrate that Per a 10 increases epithelial permeability by disruption of tight junction proteins, ZO-1 and occludin, and enhances the migration of Monocyte derived dendritic cell precursors towards epithelial layer as exhibited by trans-well studies. Per a 10 exposure also leads to secretion of IL-33, TSLP and intracellular Ca2+ dependent increase in ATP levels. Further, in vivo experiments revealed that Per a 10 administration in mice elevated allergic inflammatory parameters along with high levels of IL-33, TSLP, IL-1α and uric acid in the mice lungs. We next demonstrated that Per a 10 cleaves CD23 (low affinity IgE receptor) from the surface of PBMCs and purified B cells and CD25 (IL-2 receptor) from the surface of PBMCs and purified T cells in an activity dependent manner, which might favour Th2 responses. In conclusion, protease activity of Per a 10 plays a significant role in initiation of allergic airway inflammation at the mucosal surfaces.

Heterologous expression in Pichia pastoris and characterization of a ß-glucosidase from the xylophagous cockroach Panesthia angustipennis spadica displaying high specific activity for cellobiose.

A ß-glucosidase (BG), PaBG1b, from the xylophagous cockroach Panesthia angustipennis spadica was heterologously expressed in the methylotrophic yeast Pichia pastoris, purified, and biochemically characterized. Post-translational modification and N-terminal sequencing analysis demonstrated that the expression product was comprised of two polypeptides with different N-terminal sequences, presumably due to the presence of lysine-arginine (KR) sequence in the putative mature region. Substrate specificity analysis showed that PaBG1b hydrolyzed a broad range of substrates including cellohexaose, with the preference for aryl ß-d-fucosyl linkage and laminaribiose. Although the glucose tolerance of PaBG1b was moderate (Ki=200.3±1.1mM), PaBG1b demonstrated high specific activity and catalytic efficiency towards cellobiose with Vmax and kcat/Km values of 436.7±6.3U/mg and 109.8mM-1s-1, respectively. In addition, PaBG1b was not inhibited by cellobiose up to the highest concentration tested (100mM). Collectively, our work demonstrates that PaBG1b is a potentially valuable BG for commercial bioethanol production from cellulose.

[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Pseudoproteases: mechanisms and function.

Catalytically inactive enzymes (also known as pseudoproteases, protease homologues or paralogues, non-peptidase homologues, non-enzymes and pseudoenzymes) have traditionally been hypothesized to act as regulators of their active homologues. However, those that have been characterized demonstrate that inactive enzymes have an extensive and expanding role in biological processes, including regulation, inhibition and immune modulation. With the emergence of each new genome, more inactive enzymes are being identified, and their abundance and potential as therapeutic targets has been realized. In the light of the growing interest in this emerging field the present review focuses on the classification, structure, function and mechanism of inactive enzymes. Examples of how inactivity is defined, how this is reflected in the structure, functions of inactive enzymes in biological processes and their mode of action are discussed.

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata.

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca(2+) and cAMP, which suggests that Dippu-AstR can act through the Gαq and Gαs protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.

Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase.

A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggest that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors.

[Effect of population density on enzymatic activity of antioxidative and phenol oxidase systems of imagoes and nymphs of the marble cockroach Nauphoeta cinerea].

The work deals with effect of density of population on functional activity of components pf protective system of adult individuals and nymphs of the marble cockroach. The resistance of individuals has been noted to decrease both at individual maintenance and under conditions of overpopulation. Changes in activities of enzymes of antioxidative and phenoloxidase systems are studied ion the insect hemolymph and intestine. Possible consequences of isolation and overpopulation are discussed both for stability and for individual development.

RNAi reveals the key role of Nervana 1 in cockroach oogenesis and embryo development.

Na(+), K(+)-ATPases is a heterodimer protein consisting of α- and ß-subunits that control the ion transport through cell membranes. In insects the ß-subunit of the Na(+), K(+)-ATPase, known as Nervana, was characterized as a nervous system-specific glycoprotein antigen from adult Drosophila melanogaster heads. Nervana is expressed ubiquitously in all insect tissues, and in epithelial cells appeared located in a basolateral position as part of the septate junctions. Herein we study two Nervana isoforms from Blattella germanica, a cockroach species with panoistic ovaries. The sequencing and the phylogenetic analysis results suggest that these two isoforms are orthologs of D. melanogaster Nervana 1 and Nervana 2, respectively. Nervana 1 is highly expressed in the ovary of B. germanica, and depleting its expression results in changes in oocyte shape that do not impair oviposition. However, the resulting embryos show different defects and never hatch. These findings highlight the importance of this type of membrane pump in insect oogenesis as well as in embryo development, and its possible regulation by juvenile hormone.

Role of cockroach proteases in allergic disease.

Allergic asthma is on the rise in developed countries, and cockroach exposure is a major risk factor for the development of asthma. In recent years, a number of studies have investigated the importance of allergen-associated proteases in modulating allergic airway inflammation. Many of the studies have suggested the importance of allergen-associated proteases as having a direct role on airway epithelial cells and dendritic cells. In most cases, activation of the protease activated receptor (PAR)-2 has been implicated as a mechanism behind the potent allergenicity associated with cockroaches. In this review, we focus on recent evidence linking cockroach proteases to activation of a variety of cells important in allergic airway inflammation and the role of PAR-2 in this process. We will highlight recent data exploring the potential mechanisms involved in the biological effects of the allergen.

Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization.

BACKGROUND: The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. OBJECTIVE: The objective of this study was to investigate the cross-reactivity between a major glutathione-S transferase allergen of cockroach (Bla g 5) and the glutathione-S transferase of Wuchereria bancrofti (WbGST), a major lymphatic filarial pathogen of humans. METHODS: We compared the molecular and structural similarities between Bla g 5 and WbGST by in silico analysis and by linear epitope mapping. The levels of IgE, IgG, and IgG(4) antibodies were measured in filarial-infected and filarial-uninfected patients. Mice were infected with Heligmosomoides bakeri, and their skin was tested for cross-reactive allergic responses. RESULTS: These 2 proteins are 30% identical at the amino acid level with remarkable similarity in the N-terminal region and overall structural conservation based on predicted 3-dimensional models. Filarial infection was associated with IgE, IgG, and IgG(4) anti-Bla g 5 antibody production, with a significant correlation between antibodies (irrespective of isotype) to Bla g 5 and WbGST (P< .0003). Preincubation of sera from cockroach-allergic subjects with WbGST partially depleted (by 50%-70%) anti-Bla g 5 IgE, IgG, and IgG(4) antibodies. IgE epitope mapping of Bla g 5 revealed that 2 linear N-terminal epitopes are highly conserved in WbGST corresponding to Bla g 5 peptides partially involved in the inhibition of WbGST binding. Finally, mice infected with H bakeri developed anti-HbGST IgE and showed immediate-type skin test reactivity to Bla g 5. CONCLUSION: These data demonstrate that helminth glutathione-S transferase and the aeroallergen Bla g 5 share epitopes that can induce allergic cross-sensitization.

[Cloning and expression of fibrinolytic enzyme cDNA sequence from Eupolyphaga sinensis].

OBJECTIVE: To obtain the cDNA sequence encoding fibrinolytic enzyme from Eupolyphaga sinensis and express it in prokaryotic and eukaryotic expression system. METHOD: The primers were designed according to the cDNA of other animals'fibrinolytic enzyme. The cDNA sequence was cloned by RT-PCR and 3 RACE. RESULT: Sequence analysis revealed that the length of the cDNA fragment was 672 bp and encoded a protein of 224 amino acid residues, the N end amino acid sequence residues was IVGG in accordance with other fibrinolytic enzyme. The cDNA sequence was expressed in E. coli, inactive protein was obtained. While expressed in Pichia pastoris, recombinant protein had fibrinolytic activity. CONCLUSION: The cDNA sequence of fibrinolytic enzyme from E. sinensis Walker was cloned and expressed for the first time and it proved a good basis for further functional study of the enzyme.

Functional analysis and molecular characterization of two acetylcholinesterases from the German cockroach, Blattella germanica.

Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. With the exceptions of paraoxon and propoxur, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulphide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagen-like tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.