Effects of temperature,pH, and heavy metals (copper,zinc) upon proteinase activities in digestive tract mucosa of typical and facultative piscivorous fish

Species-specific differences and effects of natural (temperature, pH) and anthropogenic (zinc, copper) factors upon activities of casein-and hemoglobinlytic proteinases have been studied in the gut mucosa in pike (Esox lucius), zander (Stizostedion lucioperca), burbot (Lota lota) and perch (Perca fluviatilis). It is revealed that the intestine mucosa proteinase activities differ considerably from those ones in the fish stomach mucosa and that studied factors affect these activities to different extents. The stomach mucosa proteinases are most tolerant to the effects of studied factors, which is especially true for pike. The least tolerant are the intestine caseinlytic proteinases (except for the enzymes in perch). The effects of temperature, pH, and heavy metals depend on fish species, factors’ combinations (mono-factor, bi-factor, or multi-factor action) and on factor variability.     

Improved tolerance against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from <Emphasis Type="Italic">Capsicum annuum</Emphasis>

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.     

Ability of extracellular proteinases of micromycetes <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>, <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>, and <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> to affect proteins of the human haemostatic system

The effect of extracellular proteinases of A. flavipes A17, A. fumigatus D1, and A. sydowii 1 on proteins of the human haemostasis system was studied. It was shown that A. fumigatus D1 proteinases are able to hydrolyze a wide range of chromogenic peptide substrates of specific human proteinases of the haemostatic system. Proteinases formed by A. flavipes A17 and A. sydowii 1 have a narrow specificity, mainly to thrombin and plasmin substrates. It was first shown that proteinase of A. flavipes A17 is capable to activate protein C and Factor X. Extracellular proteinase produced by A. sydowii 1 has greater fibrinolytic activity as compared with proteinases produced by A. flavipes A17 and A. fumigatus D1.     

Peculiarities of digestive function of proteinases in invertebrates—Inhabitants of cold seas

Digestive proteinases of various taxa of invertebrates of the boreal seas have been studied: crustaceans Paralithodes camtchaticus, Pandalus borealis; molluscs Chlamys islandicus, Buccinum undatum, Serripes groenlandicus, and echinoderms Strongylocentrotus droebachiensis, Cucumaria frondosa, Asterias rubens, and Crossaster papposus. The presence of two proteolytic activity peaks in the acidic (pH 2.5–3.5) and lower alkaline ranges (pH 7.5–8.5) and a similar proteinase spectrum have been revealed in digestive organs of the studied animals. The proteolytic activity in digestive organs of the Barents Sea invertebrates exceeds significantly that of terrestrial homoiothermal animals, which seems to be an extensive compensation for poor differentiation of the digestive system and for low substrate specificity of the enzymes as well as for cold conditions of the habitat. The principal qualitative difference between vertebrates and invertebrates consists in that the latter have no pepsin activity, but do have the cathepsin activity that is absent in vertebrate digestive organs. Contribution to the acid proteolysis is made by lysosomal cathepsins, rather than by pepsins. Activity in the alkaline and neutral pH ranges is provided by serine proteinases. In digestive cavities of invertebrates, hydrolysis of proteins and mechanical processing of food occur only in the lower alkaline pH range, whereas acid proteolysis has intracellular lysosomal localization.     

Effects of diet type,developmental stage,and gut compartment in the gut bacterial communities of two <Emphasis Type="Italic">Cerambycidae species</Emphasis> (Coleoptera)

The gut bacterial community of wood-feeding beetles has been examined for its role on plant digestion and biocontrol method development. Monochamus alternatus and Psacothea hilaris, both belonging to the subfamily Lamiinae, are woodfeeding beetles found in eastern Asia and Europe and generally considered as destructive pests for pine and mulberry trees, respectively. However, limited reports exist on the gut bacterial communities in these species. Here, we characterized gut bacterial community compositions in larva and imago of each insect species reared with host tree logs and artificial diets as food sources. High-throughput 454 pyrosequencing of bacterial 16S rRNA gene revealed 225 operational taxonomic units (OTUs) based on a 97% sequences similarity cutoff from 138,279 sequence reads, the majority of which were derived from Proteobacteria (48.2%), Firmicutes (45.5%), and Actinobacteria (5.2%). The OTU network analysis revealed 7 modules with densely connected OTUs in specific gut samples, in which the distributions of Lactococcus-, Kluyvera-, Serratia-, and Enterococcus-related OTUs were distinct between diet types or developmental stages of the host insects. The gut bacterial communities were separated on a detrended correspondence analysis (DCA) plot and by c-means fuzzy clustering analysis, according to diet type. The results from this study suggest that diet was the main determinant for gut bacterial community composition in the two beetles.     

Activities of proteinases in invertebrate animals—Potential objects of fish nutrition. Effects of temperature,pH, and heavy metals

Differences in the degree of separate and combined effects of temperature, pH, and heavy metals (zinc, copper) on the trypsin-and chymotrypsin-like proteinase activities have been established in the whole body of some invertebrate animals—potential objects of fish nutrition: pond snail Lymnaeae stagnalis, orb snail Planorbarius purpura, zebra mussel Dreissena polymorpha, oligochaetes Tubifex sp. and Lumbriculus sp. in total, chironomid larvae Chironimus sp. and Ch. riparus, as well as crustacean zooplankton. It has been shown that enzymes of the potential prey at low temperature can compensate the low activity of intestinal proteinases of fish bentho- and planktophages.     

Gene expression of serine and cysteine proteinase inhibitors during cereal leaf beetle larvae feeding on wheat: the role of insect-associated microorganisms

Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding.     

Characterization of an Insecticidal Protein from <Emphasis Type="Italic">Withania somnifera</Emphasis> Against Lepidopteran and Hemipteran Pest

Lectins are carbohydrate-binding proteins with wide array of functions including plant defense against pathogens and insect pests. In the present study, a putative mannose-binding lectin (WsMBP1) of 1124 bp was isolated from leaves of Withania somnifera. The gene was expressed in E. coli, and the recombinant WsMBP1 with a predicted molecular weight of 31 kDa was tested for its insecticidal properties against Hyblaea puera (Lepidoptera: Hyblaeidae) and Probergrothius sanguinolens (Hemiptera: Pyrrhocoridae). Delay in growth and metamorphosis, decreased larval body mass and increased mortality was recorded in recombinant WsMBP1-fed larvae. Histological studies on the midgut of lectin-treated insects showed disrupted and diffused secretory cells surrounding the gut lumen in larvae of H. puera and P. sanguinolens, implicating its role in disruption of the digestive process and nutrient assimilation in the studied insect pests. The present study indicates that WsMBP1 can act as a potential gene resource in future transformation programs for incorporating insect pest tolerance in susceptible plant genotypes.     

Degradation capacities of bacteria and yeasts isolated from the gut of <Emphasis Type="Italic">Dendroctonus rhizophagus</Emphasis> (Curculionidae: Scolytinae)

Bark beetles (Curculionidae: Scolytinae) feed on the xylem and phloem of their host, which are composed of structural carbohydrates and organic compounds that are not easily degraded by the insects. Some of these compounds might be hydrolyzed by digestive enzymes produced by microbes present in the gut of these insects. In this study, we evaluated the enzymatic capacity of bacteria (Acinetobacter lwoffii, Arthrobacter sp., Pseudomonas putida, Pseudomonas azotoformans, and Rahnella sp.) and yeasts (Candida piceae, Candida oregonensis, Cyberlindnera americana, Zygoascus sp., and Rhodotorula mucilaginosa) isolated from the Dendroctonus rhizophagus gut to hydrolyze cellulose, xylan, pectin, starch, lipids, and esters. All isolates, with the exception of C. piceae, showed lipolytic activity. Furthermore, P. putida, P. azotoformans, C. americana, C. piceae, and R. mucilaginosa presented amylolytic activity. Esterase activity was shown by A. lwoffii, P. azotoformans, and Rahnella sp. Cellulolytic and xylanolytic activities were present only in Arthrobacter sp. and P. azotoformans. The pectinolytic activity was not recorded in any isolate. This is the first study to provide evidence on the capacity of microbes associated with the D. rhizophagus gut to hydrolyze specific substrates, which might cover part of the nutritional requirements for the development, fitness, and survival of these insects.     

Fibrinolytic and collagenolytic activity of extracellular proteinases of the strains of micromycetes <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> L-1 and <Emphasis Type="Italic">Aspergillus ustus</Emphasis> 1

It was shown that extracellular proteinases produced by the strains of micromycetes A. ochraceus L-1 and A. ustus 1 differ by the activity at various pH as well as by the intensity of the effect on fibrillar proteins. It was revealed that the proteinases of A. ochraceus L-1 demonstrated maximum activity during the growth of the producer in the nitrate-free growth medium (the pH of enzyme reaction was 8.0), whereas those of A. ustus 1 showed maximal activity during the growth of the micromycete in the medium containing sodium nitrate (the pH of enzyme reaction was 6.0). Values of specific fibrinolytic and collagenolytic activities of A. ochraceus L-1 were 2.2 and 1.6 times higher than those of A. ustus 1. At the same time, A. ustus 1 showed very low values of total proteolytic (caseinolytic) activity and had a high ratio of fibrinolytic activity to total proteolytic (caseinolytic) activity (6.92). It makes the strain a promising producer of proteinases, which hydrolyze fibrin and collagen.     

Comparative bacterial survey in the gut of lepidopteran insects with different bionetwork

Insect gut is an untapped reservoir persist numerous unidentified gene responsible for the digestion of complex food material into a simpler nutrients, this activity of gut was enhanced by the microbial interaction. Insect gut-microbe interaction encodes beneficial activity to its host in several aspects, to illuminate the tactic of the interaction this study was framed and worked out in two insect species such as Spodoptera litura and Bombyx mori. The present study surveyed the comparative diversity of microbial population in the gut of a field pest Spodoptera litura and domesticated insect silkworm Bombyx mori at their active feeding larval stage. Both culture dependent and culture independent approaches exposed the dominance of Enterobacteriacae family and with majority of proteobacteria and firmicutes residing in the gut of both Spodoptera litura and Bombyx mori. Especially, the Enterococcus mundtii is a predominant affiliate in both processes of both insects.     

Trypsin-Like Proteinases and Trypsin Inhibitors in Fruiting Bodies of Higher Fungi

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).     

Discovery of a Highly Virulent Strain of <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> ssp<Emphasis Type="Italic">. akhurstii</Emphasis> from Meghalaya,India

Photorhabdus is an insect-pathogenic Gram negative enterobacterium found in the gut of Heterorhabditis nematodes. Photorhabdus is highly virulent to insects, and can kill insects rapidly upon injection at very low concentrations of one to few cells. We characterized the virulence of Photorhabdus symbionts isolated from the Heterorhabditis nematodes collected from various parts of India by injecting different concentrations of bacterial cells into fourth instar larval stage of insect Galleria mellonella. Photorhabdus luminescens ssp. akhurstii strain IARI-SGMG3 from Meghalaya was identified as the most virulent of all the tested strains on the basis of LT₅₀ and LC₅₀ values. This study forms a basis for further investigations on the genetic basis of virulence in Photorhabdus bacteria.     

Interaction between toxin crystals and vegetative insecticidal proteins of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in lepidopteran larvae

Insecticides based on crystalline toxins of Bacillus thuringiensis are very good biological plant protection products. However, the spectrum of activity of some toxins is narrow or resistance among insects has been developed. We tested the insecticidal activity of crystals of the B. thuringiensis MPU B9 strain alone and supplemented with Vip3Aa proteins against important pests: Spodoptera exigua Hübner (Lepidoptera: Noctuidae), Cydia pomonella L. (Lepidoptera: Tortricidae) and Dendrolimus pini L. (Lepidoptera: Lasiocampidae). The Cry toxins were more active for D. pini but less active against S. exigua and C. pomonella than Vip3Aa. Supplementation of Cry toxins by small amounts of vegetative insecticidal proteins demonstrated synergistic effect and significantly enhanced the toxicity of the insecticide. The results indicate the utility of Cry and Vip3Aa toxins mixtures to control populations of crops and forests insect pests.     

The effect of temperature,pH, and heavy metals (copper,zinc) upon proteinase activities in the potential preys for typical and facultative piscivores

Separate and combined action of temperature, pH, and heavy metals (copper and zinc) upon whole body activities of casein-and hemoglobinlytic proteinases were studied in perch (Perca fluviatilis), ruff (Gymnocephalus cernuus), roach (Rutilus rutilus), kilka (Clupeonella cultriventris), and common carp (Cyprinus carpio). Also, these activities were determined in cyprinids’ underyearlings (in sum) dominating the food of typical and facultative piscivores. It was revealed that, following a decrease in temperature to 0°C and in the presence of heavy metals (copper and zinc) at concentrations of 10 mg/l, casein-and hemoglobinlytic proteinase activities decreased statistically significantly in all studied fish. Both decrease and increase in pH values lead to less pronounced changes in the enzymatic activities varied in different species. As a rule, decrease in pH values resulted in considerable increase of the hemoglobinlytic activity, while pH increase caused inconsiderable enzyme activation. Combined action of natural factors and, in some cases, combined action of low temperature and metals, changed the caseinlytic activity to a greater extent than at their separate actions. Magnitude of multifactor effects in most cases is lower than of bifactor effects of temperature and metals.     

Proteinase inhibitors from <Emphasis Type="Italic">Cajanus platycarpus</Emphasis> accessions active against pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Cajanus platycarpus, a wild relative of Cajanus cajan, is an important source for various agronomically desirable traits, including resistance towards pod borer, Helicoverpa armigera. In the present study, the inhibitory activity of proteinase inhibitors (PIs) present in crude protein extracted from different accessions of C. platycarpus and cultivars of C. cajan was evaluated against H. armigera under in vitro and in vivo conditions. The PIs active against H. armigera gut trypsin-like proteinases (HGPs), referred to as ‘HGPIs’, were more pronounced in mature dry seeds of C. platycarpus accessions when compared with cultivars, which is also evident through gelatin activity staining studies. Therefore, the inhibitory activity of HGPIs was further evaluated in various plant organs of C. platycarpus accessions, such as leaves, flowers, pods, developing seeds at 8–10 days (DAP-I), 18–20 days (DAP-II), and 28–32 days after pollination (DAP-III). However, the HGPI activity was more pronounced in mature dry seeds > DAP-III > DAP-II > DAP-I > flowers > pods > leaves. The observed quantitative allocation of HGPIs closely resembled “Optimal Defense Theory”. Further, bioassays demonstrated that there was a significant reduction in the body weight of the larvae fed upon crude PI extracts of C. platycarpus accessions with concomitant increase in mortality rate and the formation of larval–pupal intermediates. Nevertheless, such changes were not observed when the larvae were fed on crude PI extracts of C. cajan cultivars. These results suggest that the PI gene(s) from C. platycarpus accessions could be exploited in the management of H. armigera by introgression into C. cajan cultivars.     

Existence of two strains of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> (Hymenoptera: Braconidae): a complex in Thailand and Japan

Habrobracon hebetor Say (Hymenoptera: Braconidae) is a cosmopolitan gregarious ectoparasitoid that attacks larvae of several species of Lepidoptera. Although there are two genetically different strains within H. hebetor, distribution of the strains has been poorly understood. In 2010, in Thailand, where H. hebetor has been known as a parasitoid of stored grain pests, it was found that H. hebetor attacked Opisina arenosella Walker (Lepidoptera: Oecophoridae), which is an invasive pest of coconut palm. For correct identification of this H. hebetor, we conducted DNA analysis and cross tests using populations collected from O. arenosella and stored grain pests in Thailand and populations in Japan known as H. hebetor. We obtained 413 bp of mitochondrial cytochrome oxidase I (COI) sequences and 414 bp of 16S rRNA gene sequences, and both indicated that there are two distinct clades within H. hebetor: one contains insects from Thailand, Spain, India, and Barbados; the other contains insects from Japan and the USA. There were no genetic differences or sexual isolation between Thai populations from different hosts. Our results also showed that populations in Thailand were sexually isolated from a H. hebetor population in Japan.