Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis

The Sphenophorus levis (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC (Vmax = 1017.60 ± 135.55, Km = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (Ki = 38.52 ± 1.20 μM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.

An Assessment of Steinernema rarum as a Biocontrol Agent in Sugarcane with Focus on Sphenophorus levis, Host-Finding Ability, Compatibility with Vinasse and Field Efficacy

In Brazil, countless insect species attack and damage sugarcane (Saccharum officinarum L.), which is an extremely important crop since it is planted on more than 10 million hectares. Among these insects, the sugarcane billbug, Sphenophorus levis (Coleoptera: Curculionidae), is of great importance as the larvae open tunnels in the rhizome of the plant, causing high damage and losses. This insect is attracted mainly to vinasse, which is the liquid fraction generated from the alcohol production and discarded onto the sugarcane fields for fertigation. Toward a novel control method for S. levis, the native entomopathogenic nematode Steinernema rarum (Pam 25) was compared with S. carpocapsae (IL 1) and Heterorhabditis bacteriophora (HBEN01) in respect to their ability to search for larvae of two insect hosts (Galleria mellonella and S. levis) within the cane rhizome. The selected nematode S. rarum was also assessed for rate effects, its survival in vinasse and field efficacy to control sugarcane pests S. levis, Hyponeuma taltula (Lepidoptera, Erebidae) and Leucothyreus alvarengai (Coleoptera: Melolonthidae). Steinernema rarum exhibited superior virulence to G. mellonella and S. levis larvae inserted into the cane rhizomes (75–78% mortality) compared to S. carpocapsae (30–53%) and H. bacteriophora (18–28%). Vinasse affected S. rarum when infective juveniles were suspended in the liquid compost for more than 6 h but did not affect the nematode when kept on the straw and soil treated with the compost. Steinernema rarum tested at 1–3 × 108 infective juveniles/ha on the sugarcane field caused 74.1, 56.3 and 50.6% control of S. levis, L. alvarengai and H. taltula, respectively.

Report of the mite Lasioseius prorsoperitrematus Abo-Shnaf, Sánchez & Moraes, 2016 (Acari: Blattisociidae) in Brazil associated with the insect Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae)

The sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae), is a pest of sugarcane in Brazil. Lasioseius prorsoperitrematus Abo-Shnaf, Sánchez & Moraes, 2016 (Acari: Blattisociidae), described from the Dominican Republic, is reported from Brazil in association with S. levis in Iracemápolis, São Paulo state. The actual role of each organism in this association needs to be verified. A key to species of Lasioseius Berlese, 1916 recorded from Brazil associated to Coleoptera is provided.

Entomopathogenic fungi on the control of Sphenophorus levis in sugarcane crop

Brazil is the largest producer of sugarcane crop in the world,but its production can be highly affected by the pest Sphenophorus levis. The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana can be used for the control of this pest, but are not always effective in the field, lacking information on application methods, doses and formulations. We evaluate the efficacy of M. anisopliae IBCB 425 and B.bassiana IBCB 66 in controlling S. levis and other soil pests at sugarcane crop. The bioassay was conducted in a split plot design, where each of the 4 treatments were repeated 5 times and distributed over an area of 5.000 m2. The treatments for both fungi consisted of: Wettable powder (WP) formulation 225 g conidia ha-1, WP formulation 450 g conidia ha-1, granule (GR) formulation 10 Kg ha-1 and Control (no fungal application). WP fungi formulations were applied to the ratoon with a tractor using a ratoon cutter and an pesticide applicator. The GR fungi formulation was manually applied on the ratoons. The evaluations were performed after 21, 60 and 100 days of fungi application. B. Bassiana fungus reduced the pest population but did not differ from the control, however for M. Anisopliae fungus there were significant differences between treatments on damaged stems. The fungus M. anisopliae was effective in controlling S. levis larvae and adults in the field and WP formulation had faster action than the GR, the dose of 225 g conidia ha-1 was enought to reduce S. levis population.

Steinernema rarum para o controle de Sphenophorus levis, Hyponeuma taltula e Leucothyreus sp. na cultura da cana-de-açucar e sua compatibilidade com vinhaça

O Brasil é o maior produtor mundial de cana-de-açúcar. Dentre as pragas que atacam a cultura, o bicudo-da-cana, Sphenophorus levis (Coleoptera: Curculionidae), tem ganhado destaque nos últimos anos devido à sua crescente disseminação e os grandes prejuízos causados ao setor sucroalcoleiro. Os métodos atuais recomendados para o controle desse inseto, tanto culturais, como baseados na aplicação de produtos químicos, não têm gerado resultados satisfatórios, principalmente em virtude do comportamento do inseto. Uma alternativa para o manejo populacional dessa praga é a utilização de agentes de controle biológico, como os nematoides entomopatogênicos (NEPs), porém até o momento não há disponível no mercado um produto comercial a base desses entomopatógenos. Com este estudo foi possível compreender os aspectos relacionados à formulação, aplicação e eficiência de um produto a base de NEPs, que seja eficiente para o controle de pragas de solo em cana-de-açúcar e em outras culturas/ambientes.

Application of signal correction for Sphenophorus levis control and higher quality production in mechanized harvesting of sugarcane ratoon

Higher quality mechanized agricultural operations can be achieved with the use of the Global Navigation Satellite System (GNSS) signal positioning tools (correction signals), allowing a higher accuracy, which is extremely important to reduce operating costs and waste of inputs, in addition to allowing a more effective pest control. Thus, the aim of this study was to evaluate the behavior of the mean execution error of the positioning and pass-to-pass design in the operation of sugarcane ratoon cutting and insecticide application. Furthermore, the efficiency of controlling Sphenophorus levis through non-automatic steering (NS) and use of autopilot (RTX and RTK correction signals) in a sugarcane production plot of an experimental area located in the city of Motuca, SP, Brazil were evaluated for a total of 150 points by means of the statistical process control, analysis of variance, and descriptive statistics. Fipronil was the insecticide used for S. levis control. The evaluations consisted of the measurement of the mean execution error of the project during tractor operation in ten strides and five replications, in addition to the pass-to-pass (parallelism error) error between strides of the tractor-ratoon cutter assembly. In all strides, the mean execution error and mean error of the tractor-seeder assembly were within both the acceptable limit and the stipulated by the signal manufacturer, with values lower than 3.8 cm. The control charts were efficient to evaluate the behavior of RTX signal quality, facilitating the visualization within the limits of the project execution errors and pass-to-pass, in addition to contributing with an S. levis control 27.16% higher than the conventional control in the cutting operation of sugarcane ratoon.