Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 mu M); CAL2 (17 kDa, pHo 5.5, km 11 mu M Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM L-Leucine-p-nitroanilide), alpha-glucosidase (90 kDa, pHo 5.0, km 5 mM with p-nitrophenyl alpha-D-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans. (C) 2012 Elsevier Ltd. All rights reserved.

Characterization of a Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) - derived chitinase and its potential for pest control

The larval endoparasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) has a toolbox of biological weapons to secure for host colonization and the successful parasitization of its host Heliothis virescens (F.) (Lepidoptera: Noctuidae). The cDNA of a putative chitinase has been previously isolated and initially characterized from teratocytes of this parasitoid among the plethora of molecules available in the venom and calyx fluids injected by females, oral and/or anal secretions released by the parasitoid larvae and/or produced by the expression of genes of the symbiotic associated polydnavirus. This putative chitinase has been initially associated with the host cuticle digestion to allow for parasitoid egression and with the asepsis of the host environment, acting as an antimicrobial. As chitinases are commonly expressed in plants against plant pathogens, the chitinase derived from the teratocytes of T. nigriceps is a potential tool for the development of insect pest control methods based on the disruption of the perithrophic membrane of herbivores. Therefore, we aimed to characterize the activity of the putative chitinase from teratocytes of T. nigriceps (Tnchi) produced using the Escherichia coli expression system and its potential to control H. virescens larvae when expressed into transgenic tobacco plants. The purified E. coli-produced Tnchi protein showed no chitinolitic activity, but was active in binding with colloidal and crystalline chitins in water and with colloidal chitin in buffered solution (pH = 6.74). Transgenic tobacco plants showed no enhanced chitinolitic activity relative to control plants, but survival of three-day old larvae of H. virescens was severely affected when directly fed on transgenic tobacco leaves expressing the recombinant Tnchi protein. Some properties of the Tnchi protein and the potential use of Tnchi-transgenic plants to control plant pests are discussed. (c) 2012 Elsevier Inc. All rights reserved.

Plastic pellets as oviposition site and means of dispersal for the ocean-skater insect Halobates

Microplastics are omnipresent in the oceans and generally have negative impacts on the biota. However, flotsam may increase the availability of hard substrates, which are considered a limiting resource for some oceanic species, e.g. as oviposition sites for the ocean insect Halobates. This study describes the use of plastic pellets as an oviposition site for Halobates micans and discusses possible effects on its abundance and dispersion. Inspection of egg masses on stranded particles on beaches revealed that a mean of 24% (from 0% to 62%) of the pellets bore eggs (mean of 5 and max. of 48 eggs per pellet). Most eggs (63%) contained embryos, while 37% were empty egg shells. This shows that even small plastic particles are used as oviposition site by H. micans, and that marine litter may have a positive effect over the abundance and dispersion of this species. (C) 2012 Elsevier Ltd. All rights reserved.

Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self- half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.

Advanced oxidation process H2O2/UV combined with anaerobic digestion to remove chlorinated organics from bleached kraft pulp mill wastewater

This study investigated the application of an advanced oxidation process combining hydrogen peroxide with ultraviolet radiation (H2O2/UV) to remove recalcitrant compounds from Kraft bleaching effluent. Anaerobic pre-treatment was performed to remove easily degraded organics using a horizontal-flow anaerobic immobilized biomass (HAIB) reactor. Bleaching plant effluent was treated in the HAIB reactor processed over 19 h of hydraulic retention time (HRT), reaching the expected removal efficiencies for COD (61 +/- 3%), TOC (69 +/- 9%), BOD5 (90 +/- 5%) and AOX (55 +/- 14%). However, the anaerobic treatment did not achieve acceptable removal of UV254 compounds. Furthermore, there was an increase of lignin, measured as total phenols. The H2O2/UV post-treatment provided a wide range of removal efficiencies depending on the dosage of hydrogen peroxide and UV irradiation: COD ranged from 0 to 11%, UV254 from 16 to 35%, lignin from 0 to 29% and AOX from 23 to 54%. All peroxide dosages applied in this work promoted an increase in the BOD5/COD ratio of the wastewater. The experiments demonstrate the technical feasibility of using H2O2/UV for post-treatment of bleaching effluents submitted to anaerobic pre-treatment.

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification.

Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp

Recent studies on the obligate interaction between fig trees and their pollinating agaonid wasps have focused on population aspects and wasp-seed exploitation at the level of the inflorescence. Detailed studies on larval and gall development are required to more fully understand how resources are exploited and adaptations fine-tuned by each partner in nursery pollination mutualisms. We studied the larval development of the active pollinating fig wasp, Pegoscapus sp., and the galling process of individual flowers within the figs of its monoecious host, Ficus citrifolia, in Brazil. The pollinator development is strongly dependent on flower pollination. Figs entered by pollen-free wasps were in general more likely to abort. Retained, unpollinated figs had both higher larval mortality and a lower number of wasps. Pegoscapus sp. larvae are adapted to plant development, with two contrasting larval feeding strategies proceeding alongside gall development. The first two larval stages behave as ovary parasites. Later larval stages feed on hypertrophied endosperm. This indicates that a successful galling process relies on endosperm, and also reveals why pollination would be a prerequisite for the production of high-quality galls for this Pegoscapus species.

Sugarwin: A Sugarcane Insect-Induced Gene with Antipathogenic Activity

In sugarcane fields, colonization of the stalk by opportunistic fungi usually occurs after the caterpillar Diatraea saccharalis attacks the sugarcane plant. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN), sugarcane wound-inducible proteins SUGARWIN1 and SUGARWIN2, have been identified in sugarcane by an in silico analysis. Antifungal properties have been described for a number of BARWIN homologues. We report that a SUGARWIN:green fluorescent protein fusion protein is located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of sugarwin transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment. The accumulation of transcripts is late induced and is restricted to the site of the wound. Although the transcripts of sugarwin genes were strongly increased following insect attack, the protein itself did not show any effect on insect development; rather, it altered fungal morphology, leading to the apoptosis of the germlings. These results suggest that, in the course of evolution, sugarwin-encoding genes were recruited by sugarcane due to their antipathogenic activity. We rationalize that sugarcane is able to induce sugarwin gene expression in response to D. saccharalis feeding as a concerted plant response to the anticipated invasion by the fungi that typically penetrate the plant stalk after insect damage.

Comparing assemblages of Asteraceae and their insect herbivores under different land-use regimens

Accelerated tropical landscape changes occurring over recent decades have produced environmental mosaics comprising remaining isolated green areas and mixed land-use types. Our objective was to study the effects of alterations in the natural landscape on the species composition and structure of assemblages of Asteraceae and their endophagous insects through comparisons between cerrado (savanna), pastures and Eucalyptus stands. We first investigated whether similarities between assemblages of Asteraceae and their insects varied among land uses or localities. Secondly, we asked whether assemblages of Eucalyptus stands and pastures are subsets of those within the cerrado. We sampled within randomly deployed transects in 15 areas. Land use was found to be an important factor in determining plant composition similarity; however, locality did not exert any significant influence. Pastures were less similar to one another, suggesting high beta diversity. Similarities among insect assemblages were correlated with plant assemblage composition, but not with land use or locality. Species of Tephritidae were distributed along localities independently of land use. High beta diversity in Asteraceae assemblages among cerrados and pastures was supported by nestedness analysis. Plant assemblages in Eucalyptus stands were subsets of cerrado, but pasture assemblages were only partial subsets. A higher degree of nestedness in insect assemblages than in plant assemblages indicated lower beta diversity within these herbivores. Our data indicate that many herbivores are specialized on widely distributed plant genera. Conservation of Asteraceae species and their flower head insects depends not only on maintenance of landscape fragments but also on the correct matching of management form and land use. Such management may contribute to reducing isolation of plant and insect species by increasing the connectivity of remaining cerrado tracts, allowing population maintenance even at low abundances.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 degrees C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K-m = 37.53 mM S-1), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K-i = 0.196 nM), indicating that this protease is a potential target for pest control. (C) 2011 Elsevier Ltd. All rights reserved.

Analysis of Insect Cuticular Compounds by Non-lethal Solid Phase Micro Extraction with Styrene-Divinylbenzene Copolymers

Insect cuticular hydrocarbons including relatively non-volatile chemicals play important roles in cuticle protection and chemical communication. The conventional procedures for extracting cuticular compounds from insects require toxic solvents, or non-destructive techniques that do not allow storage of subsequent samples, such as the use of SPME fibers. In this study, we describe and tested a non-lethal process for extracting cuticular hydrocarbons with styrene-divinylbenzene copolymers, and illustrate the method with two species of bees and one species of beetle. The results demonstrate that these compounds can be efficiently trapped by ChromosorbA (R) (SUPELCO) and that this method can be used as an alternative to existing methods.

Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Genes involved in thoracic exoskeleton formation during the pupal-to-adult molt in a social insect model, Apis mellifera

Background: The insect exoskeleton provides shape, waterproofing, and locomotion via attached somatic muscles. The exoskeleton is renewed during molting, a process regulated by ecdysteroid hormones. The holometabolous pupa transforms into an adult during the imaginal molt, when the epidermis synthe3sizes the definitive exoskeleton that then differentiates progressively. An important issue in insect development concerns how the exoskeletal regions are constructed to provide their morphological, physiological and mechanical functions. We used whole-genome oligonucleotide microarrays to screen for genes involved in exoskeletal formation in the honeybee thoracic dorsum. Our analysis included three sampling times during the pupal-to-adult molt, i.e., before, during and after the ecdysteroid-induced apolysis that triggers synthesis of the adult exoskeleton. Results: Gene ontology annotation based on orthologous relationships with Drosophila melanogaster genes placed the honeybee differentially expressed genes (DEGs) into distinct categories of Biological Process and Molecular Function, depending on developmental time, revealing the functional elements required for adult exoskeleton formation. Of the 1,253 unique DEGs, 547 were upregulated in the thoracic dorsum after apolysis, suggesting induction by the ecdysteroid pulse. The upregulated gene set included 20 of the 47 cuticular protein (CP) genes that were previously identified in the honeybee genome, and three novel putative CP genes that do not belong to a known CP family. In situ hybridization showed that two of the novel genes were abundantly expressed in the epidermis during adult exoskeleton formation, strongly implicating them as genuine CP genes. Conserved sequence motifs identified the CP genes as members of the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Furthermore, 28 of the 36 muscle-related DEGs were upregulated during the de novo formation of striated fibers attached to the exoskeleton. A search for cis-regulatory motifs in the 5′-untranslated region of the DEGs revealed potential binding sites for known transcription factors. Construction of a regulatory network showed that various upregulated CP- and muscle-related genes (15 and 21 genes, respectively) share common elements, suggesting co-regulation during thoracic exoskeleton formation. Conclusions: These findings help reveal molecular aspects of rigid thoracic exoskeleton formation during the ecdysteroid-coordinated pupal-to-adult molt in the honeybee.

Digestion of feed fractions and intake of heifers fed hydrolyzed sugarcane stored for different periods

The objective of this study was to evaluate, in Nellore heifers, intake and digestibility of hydrolyzed sugarcane stored for different periods. The experimental design used was a 4 × 4 Latin square, four diets, four Nellore heifers with ruminal cannulas (initial body weight 285.4±23.08 kg and average initial age 14 months) and four periods of 21 days. The diets were composed by fresh sugarcane (time zero) or hydrolyzed sugarcane with addition of 0.5% of hydrated lime, stored for 24, 48 or 72 hours, as the unique forage. Intake and digestibility of feed fractions, nitrogen balance, microbial synthesis efficiency, total number of ruminal protozoans and ammoniacal nitrogen did not significantly change by storing sugarcane with addition of 0.5% of hydrated lime. Sugarcane pH varied quadratically for storage time, with maximum pH of 7.02 after 24 hours from lime addition. Ruminal liquid pH values were higher for heifers fed fresh sugarcane, in comparison with those fed hydrolyzed sugarcane. Sugarcane treated with 0.5% of hydrated lime stored for up to 72 hours does not change ruminal digestion to alter the amount of feed consumed by pubescent Nellore heifers. Thus, lime is a viable technology, once it allows long-duration storage and bee control on treated forage, which contributes to animal feeding logistics.