Identification of Bacillus thuringiensis Strains for the Management of Lepidopteran Pests.

Bacillus thuringiensis (Bt)-based bioinsecticides and transgenic plants expressing proteins with insecticidal activity (Cry and Vip) have been successfully used in several integrated pest management programs worldwide. Lepidoptera comprise some of the most economically important insect pests of the major agricultural crops. In this study, the toxicity of 150 Bt strains was evaluated against Helicoverpa armigera (Hübner) larvae. Eight strains (426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658) showed high insecticide activity against H. armigera and were therefore tested against Anticarsia gemmatalis (Hübner), Spodoptera cosmioides (Walker), Chrysodeixis includens (Walker), and Diatraea saccharalis (Fabricius) larvae. Our results showed that most of the Bt strains were also toxic to these lepidopteran species. The biochemical and molecular analyses of these strains revealed that they had a similar protein profile; however, their cry and vip gene contents were variable. In addition, the median lethal concentration (LC50) of the selected strains indicated that the strains 1636, 1641, and 1658 were the most effective against H. armigera, showing LC50 values of 185.02, 159.44, and 192.98 ng/cm2, respectively. Our results suggest that the selected Bt strains have great potential to control the lepidopteran pests H. armigera, A. gemmatalis, D. saccharalis, S. cosmioides, and C. includes.

RNAi-Mediated Suppression of Laccase2 Impairs Cuticle Tanning and Molting in the Cotton Boll Weevil (Anthonomus grandis).

The cotton boll weevil, Anthonomus grandis, is the most economically important pest of cotton in Brazil. Pest management programs focused on A. grandis are based mostly on the use of chemical insecticides, which may cause serious ecological impacts. Furthermore, A. grandis has developed resistance to some insecticides after their long-term use. Therefore, alternative control approaches that are more sustainable and have reduced environmental impacts are highly desirable to protect cotton crops from this destructive pest. RNA interference (RNAi) is a valuable reverse genetics tool for the investigation of gene function and has been explored for the development of strategies to control agricultural insect pests. This study aimed to evaluate the biological role of the Laccase2 (AgraLac2) gene in A. grandis and its potential as an RNAi target for the control of this insect pest. We found that AgraLac2 is expressed throughout the development of A. grandis with significantly higher expression in pupal and adult developmental stages. In addition, the immunolocalization of the AgraLac2 protein in third-instar larvae using specific antibodies revealed that AgraLac2 is distributed throughout the epithelial tissue, the cuticle and the tracheal system. We also verified that the knockdown of AgraLac2 in A. grandis resulted in an altered cuticle tanning process, molting defects and arrested development. Remarkably, insects injected with dsAgraLac2 exhibited defects in cuticle hardening and pigmentation. As a consequence, the development of dsAgraLac2-treated insects was compromised, and in cases of severe phenotypic defects, the insects subsequently died. On the contrary, insects subjected to control treatments did not show any visible phenotypic defects in cuticle formation and successfully molted to the pupal and adult stages. Taken together, our data indicate that AgraLac2 is involved in the cuticle tanning process in A. grandis and may be a promising target for the development of RNAi-based technologies.

Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae).

The relative quantification of gene expression is mainly realized through reverse transcription-quantitative PCR (RT-qPCR). However, the accuracy of this technique is deeply influenced by the expression stability of the reference genes used for data normalization. Therefore, the selection of suitable reference genes for a given experimental condition is a prerequisite in gene expression studies. Dichelops melacanthus (Hemiptera: Pentatomidae) is an important phloem sap-sucking insect pest of soybean, wheat, and maize in Brazil. Most of the genetic and molecular biology studies require gene expression analysis. Nevertheless, there are no reports about reference genes for RT-qPCR data normalization in D. melacanthus. In this study, we evaluated the expression stability of nine candidate reference genes (nadh, sdhb, gapdh, fau, ef1a, rpl9, ube4a, gus and rps23) in different developmental stages, body parts, sex, starvation-induced stress and dsRNA exposure by RefFinder software that integrates the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt method. Our results showed that ef1a and nadh are the most stable reference genes for developmental stages, fau and rps23 for sex, ube4a and rps23 for body parts, rpl9 and fau for starvation stress, and nadh and sdhb for dsRNA exposure treatment. The reference genes selected in this work will be useful for further RT-qPCR analyses on D. melacanthus, facilitating future gene expression studies that can provide a better understanding of the developmental, physiological, and molecular processes of this important insect pest. Moreover, the knowledge gained from these studies can be helpful to design effective and sustainable pest management strategies.

Requirement of simultaneous assessment of crystal- and supernatant-related entomotoxic activities of Bacillus thuringiensis strains for biocontrol-product development.

Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (ß-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting ß-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 10(7) endospores mL(-1) caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted ß-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.