A.R. Lopes

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.

Effects of soybean proteinase inhibitor on development, survival and reproductive potential of the sugarcane borer, Diatraea saccharalis

One approach that can be employed in integrated pest management is the use of proteins with antinutritional effects on insect metabolism and development. The antimetabolic properties of soybean proteinase inhibitor (SPI) on growth of neonate larvae of the sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) have been evaluated. When incorporated into an artificial diet at 0.5% (w/w), SPI retarded growth rate and development of larvae when compared with larvae fed on artificial diet alone. However, larval survival was not significantly affected. The purpose of our research was to calculate demographic statistics for the sugarcane borer reared on diet either with or without semi‐purified extract of SPI. Net reproductive rate (R0), instantaneous rate of increase (rm), combined age‐specific survivorship (lx) and age specific fecundity (mx) provide information about population growth potential. These parameters were measured in order to determine the effects of the proteinase inhibitor on the insects population dynamics. The observed differences would potentially translate into large reductions in population growth, indicating a potential value of using SPI for protecting sugarcane plants against damage by the sugarcane borer.

Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors.

Abstract The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.

Digestion in larvae of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on α-amylases and oligosaccharidases

Abstract Determinations of carbohydrases, aminopeptidases and acid phosphatase in the larval midgut cells and in the luminal contents of Callosobruchus maculatus and Zabrotes subfasciatus have been carried out. The results showed that larvae of both species displayed similar distribution of digestive enzymes in the intestinal compartments. Most larval digestive enzyme activities were found in the luminal contents. Of the activities found in the midgut tissue, only aminopeptidase is predominant in the membrane fraction. Comparisons of activities recovered from a seed flour mass equivalent to the midgut mass showed that a high percentage of the luminal aminopeptidase activity and, to a lesser extent, α-galactosidase activity, can be derived from the seeds, whereas the other enzymes are produced by the insects. Activities against starch, maltose and maltodextrins were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on differences in elution profiles on hydrophobic chromatography and banding patterns in mildly denaturing electrophoresis, both species showed a multiplicity of glycosidases. The data suggest that the majority of carbohydrate digestion occurs in the midgut lumen, whereas protein digestion should take place partly in the lumen and partly at the cell surface. Larvae of Z. subfasciatus can modulate the levels of α-amylases and α-glucosidase in response to different diets. The complex of carbohydrases found is qualitatively appropriate to digest the free oligosaccharides and oligomaltodextrins produced by α-amylases from the starch granules of host seeds.

Purification, properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.

Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants.

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Four insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera, and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization using different colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe p-nitroanilide). Chloromethyl ketones (TPCK, N- alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N- carbobenzoxy-Gly-Gly-Phe-CK) inactivated all chymotrypsins tested. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1) s(-1); Z-GGF-CK, 150 to 450 M(-1) s(-1)) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1) s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa value. This is proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed to be an adaptation to the presence of dietary ketones.

Dimorfismo sexual alar em Aedes scapularis (Diptera: Culicidae)

A deteccao do sexo de mosquitos da familia Culicidae e importante em estudos faunisticos e epidemiologicos, pois somente as femeas possuem competencia vetora para patogenos. O dimorfismo sexual de genitalia e de apendices cefalicos e, em geral, facilmente visivel em culicideos. As asas tambem podem ser dimorficas e assim poderiam complementar o procedimento de sexagem. No entanto, tal distincao nao e facilmente notavel a observacao direta. Visando descrever formalmente o dimorfismo sexual alar em Aedes scapularis, um culicideo vetorialmente competente para arbovirus e filarias, asas de machos e femeas foram comparadas usando-se metodos de morfometria geometrica e analise estatistica multivariada. Nestas analises, populacoes dos municipios Sao Paulo e Pariquera-Acu (Estado de Sao Paulo) foram amostradas. A forma das asas mostrou evidente dimorfismo sexual, o que permitiu um indice de acuracia de 100% em testes-cegos de reclassificacao, independentemente da origem geografica. Ja o tamanho alar foi sexualmente dimorfico apenas na populacao de Sao Paulo. Aparentemente, a forma alar e evolutivamente mais estavel que o tamanho, interpretacao que esta de acordo com a teoria de Dujardin (2008b), de que a forma alar de insetos seria composta por caracteres geneticos quantitativos e pouco influenciada por fatores nao-geneticos, enquanto que o tamanho alar seria predominantemente determinado por plasticidade decorrente de influencias ambientais.

Transcriptome sequencing and developmental regulation of gene expression in Anopheles aquasalis.

Background Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome. Methodology/Principal Findings A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed. Conclusions/Significance This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx.