Biological response of Anticarsia gemmatalis (Lepidoptera: Noctuidae) to Bacillus thuringiensis berliner var. kurstaki at sublethal concentrations

Rafael Coelho Ribeiro ¹ , Hany Armed Fouad ² , Evaldo Morais da Silva³ , Mariana Casari Parreira⁴ , Jefferson dos Santos Martins ⁵ , Sinara Dias Silva ⁶ , Paula Emanuelly Pantoja Lobato ⁷ , Ana Laura Pinheiro Ruivo Monteiro⁸ , Cleia Gomes Vieira e Silva Medeiros ⁹ , , Elonha Rodrigues dos Santos¹⁰

¹Universidade Federal do Pará – UFPA, Tv padre Antônio franco s/n-Brazil

²Sohag University, Faculty of Agriculture, Plant Protection Department, Egypt

³Universidade Federal do Pará, Tv padre Antônio franco s/n, Brazil

⁴Universidade dos Açores, Instituto de Investigação e Tecnologias Agrárias e do Ambiente (IITAA), Rua Capitão João D'Ávila, São Pedro, 9700- 042, Angra do Heroísmo, Açores, Portugal

⁵Universidade Rural da Amazônia- UFRA, aveninda presidente trancredo neves, n° 2501, bairro terra irme, cep: 66077830, Belém-Pa., Brazil

⁶ Instituto Agro lorestal de Assessoria Técnica da Amazônia, Rua 24 de outubro, 236 - São Benedito, CEP: 68400-000 - Cametá-Pa, Brazil

⁷Universidad Federal do Pará ;Endereço Completo da Instituição: Tv Padre Antônio Franco, Cametá/PA, Brazil

⁸Universidade Federal do Pará. Campus Guamá/Belém, Av. Augusto Corrêa, no 1, Guamá, Belém (PA) - CEP: 66.075-110, Brazil

⁹Instituto Federal de Roraima, Campus Novo Paraíso, Vila de Novo Paraíso, Km 512, BR 174, no município de Caracaraí, Roraima, Brazil

¹⁰Instituto Multidisciplinar de Rondônia - MULTIRON Faculdade Santo André – FASA, Rua Aníbal Ribeiro Batista; número:4078; Residencial Orleans; CEP: 76985-784 - Vilhena – RO, Brazil

Corresponding Author Email: evaldomorais@ufpa.br

DOI : https://doi.org/10.51470/ABF.2024.4.2.39

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INTRODUÇÃO

Soybean [Glycine max (L.) Merr.] has great social and economic value as one of the largest vegetable oil sources with low cost and high protein content for human and animal consumption [3]. Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae), a key pest of the soybean crops in Brazil and other American countries, causes extreme defoliation with a caterpillar consuming about 110 cm² of soybean leaves, This makes necessary applications of chemical insecticides in areas with A. gemmatalis occurrence, but alternative methods are necessary to manage this pest. The biology of A. gemmatalis was studied in the field [7] and this this pest has, normally, five to six instars, with some individuals reaching eight instars [5].

Synthetic insecticides have ecological and toxicological risks, besides high costs, while alternative methods of pest control can be cheaper [14]. Microorganisms used in biological control programs of insect pests, especially from the eighties, reduced conventional insecticide use, lowered environmental contamination, and could increase cultivation revenues [10]. Bacillus thuringiensis (Bt), a soil bacteria that produces crystals (cry), has high toxicity to insect pests and is globally considered an effective biopesticide. The toxicity of Bt to insects is due to its proteins of molecular mass ranging from 25 kDa to 140 kDa [4].

The objectives are to study mortality, and development of immature and biological parameters of the soybean caterpillar with B. thuringiensis var. kurstak (Dipel WP^®) at sub-lethal concentrations.

MATERIAL E MÉTODOS

Soybean caterpillars were obtained from mass rearing of the Laboratory of Biological Control of the Universidade Federal de Viçosa (UFV) in Viçosa, Minas Gerais state, Brazil and maintained under laboratory conditions at 25,0 ± 4°C, relative humidity of 70 ± 4%, and photoperiod of 12 hours.

Soybean plants were grown in plastic pots in a greenhouse of the UFV without pesticide use. Fresh leaves of soybean were collected and transferred to laboratory. Disks (10,75 cm²) from leaves of these plants were prepared one hour before the experiment.

The experiment was carried out in the Laboratory of Biological Control of the Institute of Applied Biotechnology (BIOAGRO) of the UFV. The Bacillus thuringiensis var. kurstaki (Dipel WP^® 16,000 ITU.mg^-1) activity was studied with newly emerged A. gemmatalis caterpillars in plastic containers (500 mL) with artificial diet until the third instar, when they were individualized in Petri dish (9 cm diameter) for 24 hours without food. Five concentrations (0,5; 1; 2; 4; 6; 8 μg mL) of B. thuringiensis were tested, and the control had distilled water. Twenty disks (10,75 cm²) of soybean leaves per B. thuringiensis concentration were dipped for ten seconds and left for thirty minutes to dry under laboratory conditions. After this period, the leaves were transferred to Petri dishes (9,0 x 1,5 cm), each with one A. gemmatalis larva. Four replications were used with one larva of this pest each one with soybean leaf disks treated with B. thuringiensis according to the treatment or in the control. After this, larvae were transferred to an artificial diet to complete their life cycle.

The LC₅₀ and LC₉₀ after 24 h and at last instar, development period (third instar to end of pupa period), larva weight gain, pupa weight, and dry weight of food ingested and feces were evaluated to obtain the B. thuringiensis effect on A. gemmatalis development and biology. The weight was obtained with an analytical precision scale (± 0,001 g) in milligrams.

Mortality (%) of A. gemmatalis was corrected with Abbott’s formula [1]. The effective B. thuringiensis concentration was analyzed using probit. Data from the biology experiment was subjected to variance analysis (ANOVA). Differences between treatments were determined by Tukey’s significant difference test (P ≤ 0,05).

RESULTADOS E DISCUSSÃO

The B. thuringiensis effects on third instar A. gemmatalis larvae showed that this insect is more susceptible at early than at later instars. The mortality of this pest was higher after 4 μg.mL^-1 than with other concentrations lower (Figure 1). The a toxicity de LC₉₀ of B. thuringiensis in after larva period was folding higher than the LC₉₀ for the 24 h after treatment.

The development of caterpillars neonates at end of pupa period (days), weight gain, pupa weight, and dry weight of food ingested and faeces on larva of A. gemmatalis was not modified after treatment with B. thuringiensis at all

Means followed by the same letter (s), per row, do not differ at 5% probability according to Tukey test. Statistical analyzes was not made with 6 and 8 μg/mL concentrations due to their number of replications lower than three of larvae completing this stage. * From 3^rd instar larvae at end of pupal period.

The survival of A. gemmatalis third instar caterpillars varied with the concentrations B. thuringiensis (0,5; 6 and 8 μg.mL^-1 ). The percentage of adult emergence of this pest decreased as the B. thuringiensis concentrations

The Cry proteins of the bacterium B. thuringiensis kill Lepidoptera insect pests [6]  with lower environmental impact than most chemical insecticides [8]. Differences in A. gemmatalis susceptibility to the B. thuringiensis concentrations may be due to the protein Cry 1Ba levels from the gene Cry1B [12].

The increasing mortality (%) of A. gemmatalis third instar larvae after 24 h, starting at the concentration of 4 μg.mL^-1 in a dose-dependent manner, will reduce adult emergence at higher B. thuringiensis. These results are similar to those of other studies with this bacteria on Apis mellifera Linnaeus (Hymenoptera: Apidae), Ostrinia nubilalis (Hiibner) (Lepidoptera: Crambidae, Diatraea saccharalis Fabr. (Lepidoptera: Crambidae) and storage pests [7-9]. Bacillus thuringiensis crystals are solubilized and activated by proteases in the insect midgut. The δ-endotoxins (Cry toxins) bind to midgut epithelial cells; and form pores, causing cytoplasm vacuolization and increasing cellular volume cell lysis which eventually leads to insect death [10].

Lower values of the LC₅₀ and LC₉₀ for A. gemmatalis at the larva period end compared to those after 24 hours may be due to the action mode of B. thuringiensis needing 24 h to express its toxicity. Besides the impact of this bacterium reduces the energy (i.e., total protein, glycogen, and lipids) by lowering food digestion due to its toxic effect reducing digestive enzymes as a-amilase e proteases. Besides, B. thuringiensis toxins bind to cell receptors of the epithelial of the medium intestine resulting in its perforation [15].

The lack of impact on development period, last instar larvae, and pupa weight with B. thuringiensis concentrations lower than 4 μg.mL^-1 will allow the larva recovering after healing its medium intestine. This is due to low B. thuringiensis toxins binding to medium intestin receptors or to insecticide activation by insect proteases [2]. Similar effects were reported on larva and pre-pupa (days), pupa weight (g), sex ratio, survival (%) and larvae-adult period of Spodoptera cosmioides (Walker, 1858) (Lepidoptera: Noctuidae) submitted to this protein Cry1Ac, even being specific to Lepidoptera [13]. The mortality of third instar A. gemmatalis was higher with B. thuringiensis at the concentration of 4 μg.mL^-1 or higher in a dose-dependent manner, without effect in the biological parameters studied.

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Figure 1 Concentration–mortality responses of Anticarsia gemmatalis after 24 hours feeding on soybean leaf treated with Bacillus thuringiensis sub-lethal concentrations. Means followed by the same letter (s) do not differ at 5% according to Tukey test.

Figure 2 Anticarsia gemmatalis adult emergence (%) with different concentrations of Bacillus thuringiensis. Means followed by the same letter (s) do not differ at 5% according to Tukey test.