Molecular cloning and characterization of a diapause-specific peptide in the beet armyworm,Spodoptera exigua

The leaf beetle, Gastrophysa atrocyanea, diapause-specific peptide (DSP), which plays a role in diapause, inhibits Ca^2 + channels and has antifungal activity. Here, we show the molecular cloning and characterization of a diapause-specific peptide in the beet armyworm Spodoptera exigua. The S. exigua diapause-specific peptide (SeDSP) gene consists of only one exon encoding 63 amino acid residues. A comparative analysis showed that mature SeDSP consists of 40 amino acid residues including six cysteines, which are similar to those of S. littoralis Spodomicin and G. atrocyanea DSP. The SeDSP was expressed as a 4.5 kDa peptide in baculovirus-infected insect cells. SeDSP was constitutively expressed in the epidermis of S. exigua larvae and pupae after molting and metamorphosis. In addition, recombinant SeDSP showed antibacterial activity against Bacillus megaterium.     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

An adjustable action threshold using larval parasitism of Helicoverpa armigera (Lepidoptera: Noctuidae) in IPM for processing tomatoes

An adjustable action threshold that uses estimates of larval parasitism of Helicoverpa armigera (Hübner) in individual fields was assessed over three consecutive years in processing tomatoes in Hawke’s Bay, New Zealand. Tomato fields were monitored weekly for levels of infestation by H. armigera larvae. When infestation levels became of concern, either approaching or exceeding the standard action threshold of one larva per plant, collections of 30+ representative larvae were made. These larvae were measured and reared individually at 30 °C, and after 4 days rates of parasitism were estimated visually. From these data, spray recommendations were made using a formula that adjusted the action threshold to allow for mortality from parasitism. At harvest, damage assessments were made to validate these recommendations. Results showed that estimates of parasitism after 4 days were accurate predictors of final assessments of parasitism. Overall parasitism during the three seasons was 71%, confirming that the original threshold, which relies on about 50% parasitism, needed revision. The dominant parasitoid was Cotesia kazak, reared from 91% of all parasitized larvae over the 3 years. Microplitis croceipes and the self introduced polyphagous parasitoid Meteorus pulchricornis were reared from 4% and 5% of the parasitized larvae respectively. In 16 of 17 fields examined, the adjustable threshold kept fruit damage at harvest below the tolerated level of 5%. The single crop with excessive damage had only 0.5% fruit damage above this level. This adjustable threshold, which varied in this study from 1–8.3 larvae per plant, has been incorporated into an updated IPM programme and contributed to a 95% reduction in insecticide use.     

Interactions between Phaedrotoma scabriventris Nixon (Hymenoptera: Braconidae) and Diglyphus isaea Walker (Hymenoptera: Eulophidae), parasitoids of Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae)

Liriomyza leafminer flies represent a serious threat to horticultural production in East Africa. Total field parasitism rates recorded in Kenya are below 5%, with the indigenous ectoparasitoid Diglyphus isaea Walker being one of the key parasitoid species. The International Centre of Insect Physiology and Ecology (icipe), in collaboration with the International Potato Centre (CIP), imported into Kenya the endoparasitoid Phaedrotoma scabriventris Nixon to improve natural control of leafminers. The objective of this study was to investigate the interactions between D. isaea and P. scabriventris when used together for the biological control of Liriomyza species. These interactions were studied under laboratory conditions, using treatments that involved single, simultaneous and sequential releases of the different parasitoid species onto plants infested by L. huidobrensis larvae. While used separately, parasitism rates of D. isaea and P. scabriventris were 30.4 ± 10.9% and 63.6 ± 7.7% respectively. However, when used simultaneously, the total parasitism rate increased to 77.0 ± 5.3%. Although P. scabriventris had no effect on D. isaea, the presence of D. isaea reduced the specific parasitism rate of P. scabriventris. In addition, both parasitoids induced leafminer mortality through larval-feeding and stinging. However, feeding and stinging mortality induced by D. isaea (41.9 ± 9.1%) was significantly higher compared to P. scabriventris (11.9 ± 8.7). Similarly, pupal mortality due to feeding and stinging activity was 49.1 ± 6.5% and 21.6 ± 1.9% when exposed to D. isaea and P. scabriventris respectively. The implication for simultaneous use of both parasitoids in East Africa is discussed.     

Natural parasitism of Romanomermis iyengari (Welch) (Nematoda: Mermithidae) on various species of mosquitoes breeding in rice fields in Pondicherry,India

Natural habitat of Romanomermis iyengari, an entomophilic mermithid nematode, is rice field. Parasitism of this nematode on mosquito larvae breeding in rice fields in Pondicherry, India, has been studied in 1979. The present study was to find out its persistence in the same habitat, after many years of agricultural practices. Evaluation of parasitism on mosquito larvae was carried out during two rice cultivation seasons at a schedule of 1 week prior, and 1 and 2 weeks after transplantation of paddy. Larval stages of mosquito species such as Culex tritaeniorhynchus, Culex vishnui, Anopheles vagus, and Anopheles subpictus showed parasitism of R. iyengari. Parasitism was the highest on C. tritaeniorhynchus with a maximum of 15.38% during season I and 17.85% during season II. Followed by this, it was on A. vagus (10.82% and 9.43%), C. vishnui (7.37% and 8.69%), and A. subpictus (3.70% and 6.36%). The overall density of mosquito larvae and level of parasitism of R. iyengari on them during season II was significantly higher than that of season I. The parasitism was maximum at 1 week before transplantation and showed a decreasing trend towards 2 weeks after transplantation, during both the seasons. The level of natural parasitism of R. iyengari during the present investigation was higher than that observed in the same ecosystem almost 35 years ago. This is clearly an indication towards the capability of the nematode to withstand several years of agricultural practices, including pesticide and agrichemical application. Hence, if applied at sufficient level and frequency, this nematode could act as a successful biocontrol agent against mosquitoes breeding in rice fields.     

Rapid phosphorylation of MAP kinase-like proteins in two species of Arctic kelps in response to temperature and UV radiation stress

Mitogen-activated protein kinases (MAPKs) are a group of cytoplasmic phosphoproteins that constitute the central core of the signalling network to respond to stress in most organisms. Their role in stress responses has been extensively studied in organisms from yeast to humans, and recently, their presence has also been described in higher plants as well as in micro- and macroalgae. In this study, we demonstrate via short experiments (1 h in duration), the rapid activation of two MAPKs similar to p38 and JNK of mammalian cells, in the Arctic kelps Laminaria solidungula and Saccharina latissima exposed to temperature and UV stress. The molecular mass of p38 is 40 kDa in L. solidungula and 42 kDa in S. latissima, while two JNKs were detected in both species, of 36 and 42 kDa in L. solidungula, and 36 and 40 kDa in S. latissima. These MAPKs are highly phosphorylated in response to temperature and UV light. In S. latissima, both p38 and the JNK showed higher phosphorylation at 2 °C than at 7 °C, while the reverse response was shown for L. solidungula. In addition, a significant increase in phosphorylation of both kinases was found following exposure to UV radiation (UVR). Exposure to PAR + UVA + UVB induced higher phosphorylation than PAR + UVA in L. solidungula, especially at 7 °C. In S. latissima, this response occurred only with JNK, and no differences in p38 phosphorylation between PAR + UVA and PAR + UVA + UVB at any temperature were observed. These results indicate the possible participation of MAPK-like proteins in response to stress in Arctic kelps, and that their activation is species-specific.     

Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri,and its biocontrol potential

Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50 °C and retains most of its activity between 20 and 60 °C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag⁺ and Zn²⁺. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC₅₀) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57 μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduced the LC₅₀ (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera.     

Mosquito larvicidal activity of Aloe vera (Family: Liliaceae) leaf extract and Bacillus sphaericus,against Chikungunya vector,Aedes aegypti

The bio-efficacy of Aloe vera leaf extract and bacterial insecticide, Bacillus sphaericus larvicidal activity was assessed against the first to fourth instars larvae of Aedes aegypti, under the laboratory conditions. The plant material was shade dried at room temperature and powdered coarsely. A. vera and B. sphaericus show varied degrees of larvicidal activity against various instars larvae of A. aegypti. The LC₅₀ of A. vera against the first to fourth instars larvae were 162.74, 201.43, 253.30 and 300.05 ppm and the LC₉₀ 442.98, 518.86, 563.18 and 612.96 ppm, respectively. B. sphaericus against the first to fourth instars larvae the LC₅₀ values were 68.21, 79.13, 93.48, and 107.05 ppm and the LC₉₀ values 149.15, 164.67, 183.84, and 201.09 ppm, respectively. However, the combined treatment of A. vera + B. sphaericus (1:2) material shows highest larvicidal activity of the LC₅₀ values 54.80, 63.11, 74.66 and 95.10 ppm; The LC₉₀ values of 145.29, 160.14, 179.74 and 209.98 ppm, against A. aegypti in all the tested concentrations than the individuals and clearly established that there is a substantial amount of synergist act. The present investigation clearly exhibits that both A. vera and B. sphaericus materials could serve as a potential larvicidal agent. Since, A. aegypti is a container breeder vector mosquito this user and eco-friendly and low-cost vector control strategy could be a viable solution to the existing dengue disease burden. Therefore, this study provides first report on the mosquito larvicidal activity the combined effect of A. vera leaf extract and B. sphaericus against as target species of A. aegypti.     

Occurrence of Diadegma semiclausum,a parasitoid of diamondback moth in lowlands of Syria

The parasitoid, Diadegma semiclausum (Hymenoptera: Ichneumonidae) is one of the most effective parasitoids of diamondback moth (DBM) in the highlands (> 1600 m above sea level) of Asia. A Diadegma population from the lowland areas of Homs, Syria (about 203–487 m above sea level) was examined to determine if it differs at the species-level from the D. semiclausum and other Diadegma populations present in different countries using molecular diagnostic tools. Phylogenetic analysis based on the neighbor-joining method using the mitochondrial cytochrome oxidase I (COI) and the nuclear internal transcribed spacer 2 (ITS2) sequences grouped the Homs (Syria) Diadegma population with D. semiclausum populations from other countries. The results suggest that D. semiclausum occurs in the lowland conditions in Homs (Syria), where the temperature is higher. The Homs (Syria) strain did not show any variations in the parasitism when the parasitized host (DBM) larvae were exposed to varying temperatures for 24 h. It could not survive when the parasitized DBM larvae were continuously reared at 35 °C; however it inflicted significantly higher parasitism when the parasitized DBM larvae were reared at day and night temperatures of 35 °C and 20 °C, respectively. Preliminary results indicate that the D. semiclausum strain from Homs (Syria) possesses some level of heat tolerance, which could be exploited for successful management of DBM in the tropical lowlands.     

Climatic dependency of soil organic carbon isotopic composition along the S–N Transect from 34°N to 52°N in central-east Asia

We explored the relationships between surface-soil (1–20 cm) organic carbon isotopic signatures and associated climatic factors in central-east Asia in an attempt to develop transfer functions that can be used to retrieve the paleoclimatic information stored in the thick eolian–paleosol sequences within the area. Our analysis shows that the negative correlation between the surface-soil organic δ¹³C values and the mean annual precipitation is robust (R² = 0.453; n = 196; p < 0.05) and the negative correlation with the growing-season (April–September) precipitation is more significant (R² = 0.4966; n = 196; p < 0.05). Our study further shows that the positive correlation between the surface-soil organic δ¹³C values and mean growing-season aridity is most significant (R² = 0.5805; n = 196; p < 0.05). We have smoothed both the organic δ¹³C values and the mean growing-season aridity values using a 3-point moving-window average-filter method in an attempt to remove some of random errors and found that the positive correlation between the two is further increased (R² =  0.7784; n =  192; p < 0.05). These robust linear relationships demonstrate their value in reconstructing paleoclimate changes in the study area. The documented climatic dependency of the surface-soil carbon isotopic composition in the study area might have resulted both from the humidity-related isotopic enrichment processes of the dominant C₃ plants (stomatal conductance and photosynthetic discrimination) and from the aridity-related abundance of C₄ plants (mainly Chenopodiaceae species) along the S–N bioclimatic gradient.     

Benzylideneacetone suppresses both cellular and humoral immune responses of Spodoptera exigua and enhances fungal pathogenicity

An entomopathogenic fungus, Beauveria bassiana, had significant insecticidal activity against the beet armyworm, Spodoptera exigua. However, it took almost one week to cause significant mortality. This study used a mixture treatment with an immunosuppressant to enhance the fungal pathogenicity. A bacterial metabolite, benzylideneacetone (BZA), had a significant synergistic effect on the fungal pathogenicity against S. exigua, although it had little insecticidal activity by itself. The mixture treatment shortened median lethal time of B. bassiana by approximately 2 days. The synergistic activity of BZA on the pathogenicity of B. bassiana was induced by its immunosuppressive effects on both cellular and humoral antifungal responses of S. exigua. In response to B. bassiana, S. exigua larvae can form hemocytic nodules. Nodules were significantly suppressed by BZA treatment. Moreover, BZA inhibited expression of some antimicrobial peptide genes of S. exigua in response to fungal challenge. The immunosuppressive condition induced by BZA allowed B. bassiana to easily colonize and multiply in the hemocoel of treated larvae, which resulted in significant enhancement of the pathogenicity of B. bassiana.     

cis-Jasmone indirect action on egg parasitoids (Hymenoptera: Scelionidae) and its application in biological control of soybean stink bugs (Hemiptera: Pentatomidae)

In many plants, the secondary metabolite cis-jasmone activates the metabolic pathway that produces volatile organic compounds attractive to natural enemies and, sometimes, repellent to herbivores. Previous studies indicate that the feeding damage caused by the herbivore Euschistus heros or the exogenous application of cis-jasmone in soybean plants induces the release of herbivore-induced plant volatiles (HIPVs) with a similar chemical profile and these compounds can attract the stink bug egg parasitoid Telenomus podisi (Scelionidae). Herein we tested in field conditions the effect of exogenous application of cis-jasmone in soybean plants on the parasitoid and stink bug community and on stink bug egg parasitism. In two areas, one within a soybean and another within a Crotalaria matrix, we randomly distributed 2 m² plots, with soybean plants induced (treatment, n = 5) or not induced by cis-jasmone (control, n = 5) in the field. We sampled the parasitoid community weekly with yellow sticky traps (n = 3/plot) and monitored parasitism with sentinel eggs of E. heros (n = 150/plot). We also monitored the population of stink bugs weekly, by sampling each plot with shake-cloth technique. The abundance of Scelionidae was highest overall and also in treated plots during the first four weeks in the area with a soybean matrix, but decreased thereafter. The richness of parasitoid families was similar between treatment and control plots in the area with a soybean matrix, but higher in control plots in the area with a Crotalaria matrix. Evenness was higher in control plots in the area with soybean matrix, whereas the reverse occurred in the area with a Crotalaria matrix. Results suggest that treatment with cis-jasmone effectively attracted and enhanced the population of scelionid parasitoids, but had no effect on the occurrence and intensity of parasitism and in the number of stink bugs.     

Purification,cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd,Benincasa hispida

Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 μg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.     

Development response of Spodoptera exigua to eight constant temperatures: Linear and nonlinear modeling

Temperature-dependent development of Spodoptera exigua (Hübner) were evaluated at eight constant temperatures of 12, 15, 20, 25, 30, 33, 34 and 36 °C with a variation of 0.5 °C on sugar beet leaves. No development occurred at 12 °C and 36 °C. Total developmental time varied from 120.50 days at 15 °C to 14.50 days at 33 °C. As temperature increased from 15 °C to 33 °C, developmental rate (1/developmental time) of S. exigua increased but declined at 34 °C. The lower temperature threshold (T_min) was estimated to be 12.98 °C and 12.45 °C, and the thermal constant (K) was 294.99 DD and 311.76 DD, using the traditional and Ikemoto–Takai linear models, respectively. The slopes of the Ikemoto–Takai linear model for different immature stages were different, violating the assumption of rate isomorphy. Data were fitted to three nonlinear models to predict the developmental rate and estimate the critical temperatures. The T_min values estimated by Lactin-2 (12.90 °C) and SSI (13.35 °C) were higher than the value estimated by Briere-2 (8.67 °C). The estimated fastest development temperatures (T_fast) by the Briere-2, Lactin-2 and SSI models for overall immature stages development of S. exigua were 33.4 °C, 33.9 °C and 32.4 °C, respectively. The intrinsic optimum temperature (T_Φ) estimated from the SSI model was 28.5 °C, in which the probability of enzyme being in its native state is maximal. The upper temperature threshold (T_max) values estimated by these three nonlinear models varied from 34.00 °C to 34.69 °C. These findings on thermal requirements can be used to predict the occurrence, number of generations and population dynamics of S. exigua.     

Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma,Plasmodium falciparum parasites and malaria mosquitoes

The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW–AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW–AgNP showed plasmon resonance reduction in UV–vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW–AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW–AgNP were toxic to Anopheles stephensi larvae and pupae, LC₅₀ were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW–AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW–AgNP IC₅₀ were 49.3 μg/ml (CQ-s) and 55.5 μg/ml (CQ-r), while chloroquine IC₅₀ were 81.5 μg/ml (CQ-s) and 86.5 μg/ml (CQ-r). EW–AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW–AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW–AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW–AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies.     

Potential of Lariophagus distinguendus (Förster) (Hymenoptera: Pteromalidae) to suppress the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) in bagged and bulk stored maize

Grains are often stored in jute bags in developing countries, especially in Africa, as well as in small quantities in bulk. Parasitoids suitable for biological control of stored-product pests should be able to find their hosts in bulk grain or in jute bags over a certain distance in a warehouse containing stacks of bagged grain. The potential of using Lariophagus distinguendus for the biological control of Sitophilus zeamais was assessed in maize stored in jute bags and bulk grain. The ability of the parasitoid to penetrate the jute cloth and the grain mass and parasitize its host was studied under controlled conditions of 25 ± 1 °C and 65 ± 5% RH. Experiments were carried out in small 5-kg jute bags containing 28 d old S. zeamais larvae within infested maize kernels, and in cylinders filled with maize grains and containing caged hosts at different depths. L. distinguendus parasitized S. zeamais in the jute bags and in the storage cylinders at various depths. In the jute bag experiment, out of the 60 L. distinguendus adults released, a mean ± SD of 7.03 ± 1.78% and 6.34 ± 1.01% of the 40 females and of the 20 males released, respectively, entered the jute bags. Significantly, no differences were found between the female and male L. distinguendus that entered the bags. Mean reduction of S. zeamais in the jute bags by parasitoids was 81%. The parasitic wasps also significantly reduced the emergence of S. zeamais in bulk maize. At depths of 20–45 cm from the grain surface, mean reduction of S. zeamais was 74%, while from 95 to 100 cm, mean reduction was 34%. When results from depths lower than 50 cm were pooled and compared with pooled data from depths higher than 90 cm, there was a significant reduction in parasitism at depths of more than 90 cm. For depths below 50 cm a mean of 5.3 L. distinguendus adult offspring per cage emerged compared with a mean of 2.6 at depths of more than 90 cm. These results support the approach to utilize L. distinguendus as a component in the integrated control of S. zeamais in bagged or bulk stored maize.     

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ～ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca^+ 2, Mn^+ 2 and Mg^+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.