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The beetle Anthonomus grandis Boheman, 1843, is the main cotton pest, causing enormous losses in cotton. The breeding of genetically modified plants with A. grandis resistance is seen as an important control strategy. However, the identification of molecules with high toxicity to this insect remains a challenge. The susceptibility of A. grandis larvae to proteins (Cry1Ba, Cry7Ab, and Mpp23Aa/Xpp37Aa) from Bacillus thuringiensis Berliner, 1915, with toxicity reported against Coleopteran, has been evaluated. The ingestion of different protein concentrations (which were incorporated into an artificial diet) by the larvae was tested in the laboratory, and mortality was evaluated after one week. All Cry proteins tested exhibited higher toxicity than that the untreated artificial diet. These Cry proteins showed similar results to the control Cry1Ac, with low toxicity to A. grandis, since it killed less than 50% of larvae, even at the highest concentration applied (100 µg·g-1). Mpp/Xpp proteins provided the highest toxicity with a 0.18 µg·g-1 value for the 50% lethal concentration. Importantly, this parameter is the lowest ever reported for this insect species tested with B. thuringiensis proteins. This result highlights the potential of Mpp23Aa/Xpp37Aa for the development of a biotechnological tool aiming at the field control of A. grandis.

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KEY MESSAGE: pGhERF105 and pGhNc-HARBI1 promoters are highly responsive to CBW infestation and exhibit strong activity in vegetative and reproductive tissues, increasing their potential application in GM crop plants for pest control. The main challenge to cotton (Gossypium hirsutum) crop productivity is the constant attack of several pests, including the cotton boll weevil (CBW, Anthonomus grandis), which uses cotton floral buds for feeding and egg-laying. The endophytic nature of the early developmental stages of CBW makes conventional pesticide-based control poorly efficient. Most biotechnological assets used for pest control are based on Bacillus thurigiensis insecticidal Cry toxins or the silencing of insect-pest essential genes using RNA-interference technology. However, suitable plant promoter sequences are required to efficiently drive insecticidal molecules to the target plant tissue. This study selected the Ethylene Responsive Factor 105 (GhERF105) and Harbinger transposase-derived nuclease (GhNc-HARBI1) genes based on available transcriptome-wide data from cotton plants infested by CBW larvae. The GhERF105 and GhNc-HARBI1 genes showed induction kinetics from 2 to 96 h under CBW's infestation in cotton floral buds, uncovering the potential application of their promoters. Therefore, the promoter regions (1,500 base pairs) were assessed and characterized using Arabidopsis thaliana transgenic plants. The pGhERF105 and pGhNc-HARBI1 promoters showed strong activity in plant vegetative (leaves and roots) and reproductive (flowers and fruits) tissues, encompassing higher GUS transcriptional activity than the viral-constitutive Cauliflower Mosaic Virus 35S promoter (pCaMV35S). Notably, pGhERF105 and pGhNc-HARBI1 promoters demonstrated more efficiency in driving reporter genes in flowers than other previously characterized cotton flower-specific promoters. Overall, the present study provides a new set of cotton promoters suitable for biotechnological application in cotton plants for pest resistance.

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BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

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The cotton boll weevil Anthonomus grandis Boheman (Coleoptera: Curculionidae) is the main pest in cotton crop around the world, directly affecting cotton production. In order to establish a sequential sampling plan, it is crucial to understand the spatial distribution of the pest population and the damage it causes to the crop through the different developmental stages of cotton plants. Therefore, this study aimed to investigate the spatial distribution of adults in the cultivation area and their oviposition and feeding behavior throughout the development of the cotton plants. The experiment was conducted in Maracaju, Mato Grosso do Sul, Brazil, in the 2012/2013 and 2013/2014 growing seasons, in an area of 10,000 m2, planted with the cotton cultivar FM 993. The experimental area was divided into 100 plots of 100 m2 (10 × 10 m) each, and five plants per plot were sampled weekly throughout the crop cycle. The number of flower buds with feeding and oviposition punctures and of adult A. grandis was recorded throughout the crop cycle in five plants per plot. After determining the aggregation indices (variance/mean ratio, Morisita's index, exponent k of the negative binomial distribution, and Green's coefficient) and adjusting the frequencies observed in the field to the distribution of frequencies (Poisson, negative binomial, and positive binomial) using the chi-squared test, it was observed that flower buds with punctures derived from feeding, oviposition, and feeding + oviposition showed an aggregated distribution in the cultivation area until 85 days after emergence and a random distribution after this stage. The adults of A. grandis presented a random distribution in the cultivation area.

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Foram estudados, em câmara climatizada, a 30°C, umidade relativa de 60 ± 10 por cento e fotofase de 14h, os efeitos dos hospedeiros alternativos Euscepes postfasciatus (Fairmaire) e Callosobruchus maculatus (Fabricius) na reprodução e ataque de Catolaccus grandis (Burks), parasitóide do bicudo-do-algodoeiro. Os hospedeiros alternativos C. maculatus e E. postfasciatus, proporcionaram ao parasitóide, taxas de desenvolvimento superiores às apresentadas por A. grandis. As taxas de parasitismo variaram de 44,3 por cento no hospedeiro alternativo E. postfasciatus a 39,9 por cento em A. grandis. O hospedeiro natural (bicudo) foi o que originou a maior porcentagem de pupas fêmeas (73,7 por cento). C. grandis apresentou a mesma aceitação para oviposição em ambos hospedeiros alternativos, porém a maior produção de ovos foi obtida sobre E. postfasciatus. A oogênese do parasitóide foi estimulada de forma semelhante na presença dos hospedeiros A. grandis e E. postfasciatus, e ambos superaram o hospedeiro alternativo C. maculatus em número de ovos depositados nos cinco primeiros dias. A qualidade do hospedeiro afetou o período de oviposição e a longevidade do parasitóide, sendo o hospedeiro A. grandis responsável por menor expectativa de vida e menor período de oviposição. Larvas de E. postfasciatus ou C. maculatus podem ser usadas como hospedeiros alternativos de C. grandis e estimulam a oogênese em fêmeas de C. grandis.

The effects of the factitious hosts Euscepes postfasciatus (Fairmaire) and Callosobruchus maculatus (Fabricius) on the reproduction and attack rates of Catolaccus grandis (Burks), parasitoid of the cotton boll weevil, were studied in bioclimate chambers, at 30°C, relative humidity of 60 ± 10 percent, and a 14L:10D photoperiod. The factitious hosts C. maculatus and E. postfasciatus provided higher development rates than A. grandis. The parasitism rates ranged from 44.3 percent in E. postfasciatus to 39.9 percent in A. grandis. The natural host (cotton boll weevil) originated the highest female pupae percentage (73.7 percent). C. grandis showed the same acceptance for oviposition in both factitious hosts; however, the highest production of eggs was obtained on E. postfasciatus. The parasitoid oogenesis was stimulated in a similar way in the presence of the host A. grandis and factitious host E. postfasciatus, and both overcame the factitious host C. maculatus in number of eggs deposited in the first five days. The host quality affected the oviposition period and the longevity of the parasitoid, being the host A. grandis responsible for smallest life expectation and oviposition period. Larvae of E. postfasciatus and C. maculatus might be used as factitious hosts of C. grandis. These factitious hosts serve as hosts for parasitoid females of C. grandis to stimulate oogenesis.

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The evaluation of the life expectancy for adults of Bracon sp. was carried out at 26 ± 2°C temperature, RH of 75 ± 5% and photophase of 12 h. The life expectancy (ex) was the highest at the beginning of life, with values of 7.46 days. The results show that the realease age of Bracon sp. aiming the biological control of the cotton boll weevil, is from the adult emergence to the 15 th day, when survival was maximum.

A esperança de vida para adultos de Bracon sp foi avaliada em câmara climatizada regulada a temperatura de 26 ± 2°C, UR de 75 ± 5% e fotofase de 12 h. Foi registrada maior esperança de vida (ex) no início da vida, que foi de 7,46 dias. Os resultados evidenciaram que a liberação de Bracon sp. para o controle biológico do bicudo, deve ser a partir da emergência do adulto até o 15o dia, quando a sobrevivência foi máxima.

RESUMO

The evaluation of the life expectancy for adults of Bracon sp. was carried out at 26 ± 2°C temperature, RH of 75 ± 5% and photophase of 12 h. The life expectancy (ex) was the highest at the beginning of life, with values of 7.46 days. The results show that the realease age of Bracon sp. aiming the biological control of the cotton boll weevil, is from the adult emergence to the 15 th day, when survival was maximum.

A esperança de vida para adultos de Bracon sp foi avaliada em câmara climatizada regulada a temperatura de 26 ± 2°C, UR de 75 ± 5% e fotofase de 12 h. Foi registrada maior esperança de vida (ex) no início da vida, que foi de 7,46 dias. Os resultados evidenciaram que a liberação de Bracon sp. para o controle biológico do bicudo, deve ser a partir da emergência do adulto até o 15o dia, quando a sobrevivência foi máxima.