Intestinal proteases profiling from Anticarsia gemmatalis and their binding to inhibitors.

Although the importance of intestinal hydrolases is recognized, there is little information on the intestinal proteome of lepidopterans such as Anticarsia gemmatalis. Thus, we carried out the proteomic analysis of the A. gemmatalis intestine to characterize the proteases by LC/MS. We examined the interactions of proteins identified with protease inhibitors (PI) using molecular docking. We found 54 expressed antigens for intestinal protease, suggesting multiple important isoforms. The hydrolytic arsenal featured allows for a more comprehensive understanding of insect feeding. The docking analysis showed that the soybean PI (SKTI) could bind efficiently with the trypsin sequences and, therefore, insect resistance does not seem to involve changing the sequences of the PI binding site. In addition, a SERPIN was identified and the interaction analysis showed the inhibitor binding site is in contact with the catalytic site of trypsin, possibly acting as a regulator. In addition, this SERPIN and the identified PI sequences can be targets for the control of proteolytic activity in the caterpillar intestine and serve as a support for the rational design of a molecule with greater stability, less prone to cleavage by proteases and viable for the control of insect pests such as A. gemmatalis.

Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

Protease inhibitory, insecticidal and deterrent effects of the trypsin-inhibitor benzamidine on the velvetbean caterpillar in soybean.

The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

Bottom-Up Mechanisms Generate the Same Temporal Pattern of Attack by a Specialist and a Generalist Caterpillar on Short-Lived Plants.

The local population dynamics of insect herbivores in ephemeral patches of short-lived plants are poorly known. We investigated whether a specialist and a generalist caterpillar exhibit contrasting temporal patterns of attack during plant development and also assessed bottom-up forces related to plant ontogeny that govern such population trends. Immature stages of the polyphagous Trichoplusia ni (Hübner) and the oligophagous Plutella xylostella (L.) were sampled throughout the development of cabbage (Brassica oleracea L. var. capitata L.) crops. We measured protein and glucosinolate contents and insect performance with regard to plant age and leaf strata. The populations of both caterpillar species changed in close parallel throughout plant development, and a nonlinear temporal pattern of egg laying was reproduced in sequential population patterns of the larval stages until pupation. Reduced protein availability and insect performance coincided with a decline in egg laying and subsequent larval abundance in mature plants. By standardizing the plant size, we found that young and nutritious plants support proportionately more insects than large and mature plants. In our models of the population oscillations, the interaction between plant size and quality provided a strong causal explanation for the densities of both oligophagous and polyphagous caterpillars. Patches of fast-growing herbaceous plants are very common worldwide in the form of crop fields, and a generalized temporal pattern of attack may be widespread among caterpillars, regardless of their feeding specialization. Our results highlight the role of bottom-up forces in shaping the population dynamics of caterpillars in such systems.

Does cypermethrin affect enzyme activity, respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

Insecticides cause a range of sub-lethal effects on targeted insects, which are frequently detrimental to them. However, targeted insects are able to cope with insecticides within sub-lethal ranges, which vary with their susceptibility. Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) to sub-lethal exposure to the pyrethoid insecticide cypermethrin. We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure. Lower respiration rate and lower activity were also expected in insecticide-resistant insects since these traits are also likely to favor survivorship under insecticide exposure. Curiously though, cypermethrin did not affect activity of digestive and energy metabolism enzymes, and even reduced the activity of some enzymes (particularly for cellulase and cysteine-proteinase activity in this case). There was strain variation in response, which may be (partially) related to insecticide resistance in some strains. Sub-lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects, which is likely to impair their fitness. However, such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced). Walking activity varies with strain and may minimize insecticide exposure, which should be a concern, particularly if associated with (physiological) insecticide resistance.

Biochemical responses of Anticarsia gemmatalis (Lepidoptera: Noctuidae) in soybean cultivars sprayed with the protease inhibitor berenil.

The damage caused by Anticarsia gemmatalis motivates this study on the adaptive mechanisms of the insect to soybean. The lipoxygenase pathway produces and releases jasmonic acid, involved in the regulation of the plant defense genes, which encodes protease inhibitor (PI) production. Three soybean cultivars IAC-18, IAC-24, and Foscarin-31 were sprayed with water and berenil, a synthetic inhibitor, at 0.60 and 1.0% (w/v) and then infested with A. gemmatalis larvae. The lipoxygenase (LOX) activity increased in the leaves of Foscarin-31, IAC-18, and IAC-24 by 87, 81, and 78%, respectively, after 24 h of A. gemmatalis damage. IAC-18 revealed the lowest increase in PI when compared to the other cultivars. Protease, amidase, and esterase activities in soybean larvae dropped drastically after berenil application. PIs may be included in the control strategies of A. gemmatalis in soybean by lowering the digestive enzyme activity in the larval midgut, thus affecting insect growth and development.