Molecular insights on the historical dispersion of Piezodorus guildinii (Hemiptera: Pentatomidae) in Brazil.

Piezodorus guildinii (Westwood, 1837) (Hemiptera: Pentatomidae) is an important arthropod pest of soybean (Glycine max (L.) Merr.) throughout American continents. However, the historical events associated with its dispersion are poorly understood. In this study, we employed a phylogeographic approach to investigate the origin and demographic history of P. guildinii in Brazil. We analyzed the cytochrome c oxidase subunit I and Cytb gene sequences of P. guildinii individuals collected in Brazil's 5 soybean production macro-regions and cross-referenced this information with sequences available in public databases. Our findings support an older Caribbean basin establishment for the current genealogical strains of P. guildinii, with subsequent dispersion to Brazil around 0.97 Mya. No secondary dispersion of this species from the Caribbean region to soybean areas in Brazil was identified. The Brazilian populations of P. guildinii are genetically structured across the country's soybean macro-regions and show strong signals of continuous demographic and spatial expansion in Brazil, which may be accelerated by the soybean cropping landscape in the country. The populations from the northern region (MR5) are older than the Central and South populations. The signs of demographic expansion indicate that P. guildinii populations are increasing their effective size in soybean regions, which could reflect its importance as a soybean pest in the coming years.

Influence of adjuvants added to teflubenzuron spray on resistant and susceptible strains of the soybean looper Chrysodeixis includens (Lepidoptera: Noctuidae) / Influência da adição de adjuvantes à calda de pulverização de teflubenzuron em linhagens resistentes e suscetíveis da lagarta falsa-medideira Chrysodeixis includens em soja (Lepidoptera: Noctuidae)

ABSTRACT: The soybean looper (SBL), Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), is a soybean and cotton pest in South America countries. Field-evolved resistance of SBL to inhibitors of chitin biosynthesis has been reported in Brazil; however, this mode of action is still widely used against SBL. On this basis, we conducted laboratory bioassays to investigate if adjuvants (Nimbus®, TA 35®, Break-Thru® S 240, and Rizospray Extremo®) added to the teflubenzuron spray increase the mortality of SBL strains (resistant, heterozygous, and susceptible to chitin biosynthesis inhibitors). Using chromatography analysis, we also evaluated the amount of teflubenzuron on soybean leaves when applied alone or in combination with adjuvants. In laboratory bioassays, the biological activity of teflubenzuron increased against the susceptible SBL strain when adjuvants were added. In contrast, no relevant effects of adjuvants added to the teflubenzuron spray against heterozygous and resistant SBL larvae were detected. In leaf bioassays, even leaves from the upper third part of the plants containing a significantly higher amount of teflubenzuron (3.4 mg/kg vs 1.7 and 0.6 mg/kg); the mortality of SBL strains was similar when teflubenzuron was applied alone or in mixture with adjuvants. Our findings indicated that adjuvants added to teflubenzuron spray do not provide a substantial increase in the mortality of SBL strains resistant to chitin biosynthesis inhibitors. Therefore, it is necessary to reduce the use of this mode-of-action insecticide against SBL and to give preference to other insecticides or control tactic.

RESUMO: A lagarta falsa-medideira, Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), é uma praga da soja e do algodão nos países da América do Sul. A resistência de C. includens a inibidores da biossíntese de quitina tem sido relatada no Brasil. Entretanto, esse modo de ação ainda é amplamente utilizado para controle de C. includens. Com base nisso, conduzimos bioensaios em laboratório para investigar se adjuvantes (Nimbus®, TA 35®, Break-Thru® S 240 e Rizospray Extremo®) adicionados à calda inseticida de teflubenzuron aumentam a mortalidade de linhagens de C. includens (resistentes, heterozigotos e suscetíveis a inibidores da biossíntese de quitina). Usando análise cromatográfica, também avaliamos a quantidade de teflubenzuron em folhas de soja quando aplicado isolado ou em combinação com adjuvantes. Em bioensaios de laboratório, a atividade biológica do teflubenzuron aumentou para a linhagem suscetível quando os adjuvantes foram adicionados à calda inseticida. Em contraste, nenhum efeito relevante de adjuvantes adicionados ao teflubenzuron foi detectado para os heterozigotos e resistentes. Em bioensaios de folhas, mesmo naquelas do terço superior das plantas, as quais apresentaram uma maior deposição de teflubenzuron (3,4 mg/kg vs 1,7 e 0,6 mg/kg); a mortalidade das linhagens de C. includens foi semelhante quando o teflubenzuron foi aplicado isolado ou com adjuvantes. Nossos resultados indicam que os adjuvantes adicionados ao teflubenzuron não fornecem um aumento substancial na mortalidade de linhagens de C. includens resistentes aos inibidores da biossíntese de quitina. Portanto, é necessário reduzir o uso desse modo de ação para o manejo de C. includens e dar preferência a outros inseticidas ou tática de controle.

Influence of adjuvants added to teflubenzuron spray on resistant and susceptible strains of the soybean looper Chrysodeixis includens (Lepidoptera: Noctuidae) / Influência da adição de adjuvantes à calda de pulverização de teflubenzuron em linhagens resistentes e suscetíveis da lagarta falsa-medideira Chrysodeixis includens em soja (Lepidoptera: Noctuidae)

The soybean looper (SBL), Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), is a soybean and cotton pest in South America countries. Field-evolved resistance of SBL to inhibitors of chitin biosynthesis has been reported in Brazil; however, this mode of action is still widely used against SBL. On this basis, we conducted laboratory bioassays to investigate if adjuvants (Nimbus®, TA 35®, Break-Thru® S 240, and Rizospray Extremo®) added to the teflubenzuron spray increase the mortality of SBL strains (resistant, heterozygous, and susceptible to chitin biosynthesis inhibitors). Using chromatography analysis, we also evaluated the amount of teflubenzuron on soybean leaves when applied alone or in combination with adjuvants. In laboratory bioassays, the biological activity of teflubenzuron increased against the susceptible SBL strain when adjuvants were added. In contrast, no relevant effects of adjuvants added to the teflubenzuron spray against heterozygous and resistant SBL larvae were detected. In leaf bioassays, even leaves from the upper third part of the plants containing a significantly higher amount of teflubenzuron (3.4 mg/kg vs 1.7 and 0.6 mg/kg); the mortality of SBL strains was similar when teflubenzuron was applied alone or in mixture with adjuvants. Our findings indicated that adjuvants added to teflubenzuron spray do not provide a substantial increase in the mortality of SBL strains resistant to chitin biosynthesis inhibitors. Therefore, it is necessary to reduce the use of this mode-ofaction insecticide against SBL and to give preference to other insecticides or control tactic.

A lagarta falsa-medideira, Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), é uma praga da soja e do algodão nos países da América do Sul. A resistência de C. includens a inibidores da biossíntese de quitina tem sido relatada no Brasil. Entretanto, esse modo de ação ainda é amplamente utilizado para controle de C. includens. Com base nisso, conduzimos bioensaios em laboratório para investigar se adjuvantes (Nimbus®, TA 35®, Break-Thru® S 240 e Rizospray Extremo®) adicionados à calda inseticida de teflubenzuron aumentam a mortalidade de linhagens de C. includens (resistentes, heterozigotos e suscetíveis a inibidores da biossíntese de quitina). Usando análise cromatográfica, também avaliamos a quantidade de teflubenzuron em folhas de soja quando aplicado isolado ou em combinação com adjuvantes. Em bioensaios de laboratório, a atividade biológica do teflubenzuron aumentou para a linhagem suscetível quando os adjuvantes foram adicionados à calda inseticida. Em contraste, nenhum efeito relevante de adjuvantes adicionados ao teflubenzuron foi detectado para os heterozigotos e resistentes. Em bioensaios de folhas, mesmo naquelas do terço superior das plantas, as quais apresentaram uma maior deposição de teflubenzuron (3,4 mg/kg vs 1,7 e 0,6 mg/kg); a mortalidade das linhagens de C. includens foi semelhante quando o teflubenzuron foi aplicado isolado ou com adjuvantes. Nossos resultados indicam que os adjuvantes adicionados ao teflubenzuron não fornecem um aumento substancial na mortalidade de linhagens de C. includens resistentes aos inibidores da biossíntese de quitina. Portanto, é necessário reduzir o uso desse modo de ação para o manejo de C. includens e dar preferência a outros inseticidas ou tática de controle.

Effects of intraguild interactions on Anticarsia gemmatalis and Chrysodeixis includens larval fitness and behavior in soybean.

BACKGROUND: Crop pest management requires an understanding of the complex interactions among species that potentially impact crop yield. In soybean, the velvetbean caterpillar, Anticarsia gemmatalis (Hübner), and the soybean looper, Chrysodeixis includens (Walker), are described as key pests, sharing the same feeding guild. We assessed the intraguild interactions of these species under laboratory conditions. Fitness cost study was conducted to examine the influence of competition on insect development. A video tracking system was used to evaluate behavioral parameters during larval interactions in scenarios with and without food availability. RESULTS: In the fitness cost assay, pupal weight was not significantly affected, regardless of sex. However, larval and pupal survival were influenced by the competition, especially in third versus fifth instar scenarios. We detected 40.00% cannibalism and 46.67% predation when A. gemmatalis and C. includens third instars competed with A. gemmatalis fifth instar, respectively. Distance moved, distance between larvae, body contact (food available) and frequency in food of C. includens larvae were negatively affected by interactions. Anticarsia gemmatalis larvae showed highly active behavior, moving twice or more the distance compared to C. includens larvae, and A. gemmatalis spent more time in body contact with food. CONCLUSION: Our results suggest that A. gemmatalis has a competitive advantage over C. includens. This study provides important information regarding lepidopteran behavior in soybean. We recommended that additional studies are necessary to understand the effects of interactions, especially in field conditions. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Field-evolved resistance to chitin synthesis inhibitor insecticides by soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae), in Brazil.

Soybean looper (SBL), Chrysodeixis includens (Walker), is an economically important soybean and cotton pest in Brazil. Here, we selected an SBL strain resistant to teflubenzuron using F2 screening, estimated the resistance allele frequency, characterized the inheritance of resistance, investigated fitness costs, evaluated patterns of cross-resistance, and determined the magnitude of resistance. The teflubenzuron-resistant strain (Teflu-R) was selected from field-collected populations with an estimated allele frequency of 0.1700. Estimated LC50 values were 0.010 and 363.61 µg a.i. cm-2 for the susceptible (Sus) and Teflu-R strains, respectively, representing a 36,361-fold resistance ratio (RR). The LC50 values of reciprocal crosses were 1.02 and 0.59 µg a.i. cm-2, suggesting that resistance is autosomally inherited. The low survival of reciprocal crosses (16 and 20%) on teflubenzuron-sprayed leaves indicates incomplete recessive resistance. The number of segregations influencing resistance was 2.72, suggesting a polygenic effect. The Teflu-R strain showed longer development periods as well as lower survival and population growth than the Sus strain, revealing fitness costs. The Teflu-R strain also showed high cross-resistancesto other chitin inhibitor insecticides, such as novaluron (RR = 6147-fold) and lufenuron (RR = 953-fold), but low cross-resistance to methoxyfenozide, flubendiamide, and indoxacarb (RR < 3.45-fold). On discriminatory concentrations of teflubenzuron and novaluron, populations of SBL showed survival rates from 15 to 52%, indicating field resistance to these insecticides. Our findings indicated that resistance to teflubenzuron in SBL is autosomal, recessive, polygenic, and associated with fitness cost. We also found a high cross-resistance to other benzoylphenylureas and a high frequency of resistance to this mode-of-action in SBL in Brazil.

Population Genomics of the Neotropical Brown Stink Bug, Euschistus heros: The Most Important Emerging Insect Pest to Soybean in Brazil.

Recent changes in soybean management like the adoption of transgenic crops and no-till farming, in addition to the expansion of cultivated areas into new virgin frontiers, are some of the hypotheses that can explain the rise of secondary pests, such as the Neotropical brown stink bug, Euschistus heros, in Brazil. To better access the risk of increased pests like E. heros and to determine probabilities for insecticide resistance spreading, it is necessary first to access the levels of the genetic diversity, how the genetic diversity is distributed, and how natural selection is acting upon the natural variation. Using the genotyping by sequencing (GBS) technique, we generated ~60,000 single-nucleotide polymorphisms (SNPs) distributed across the E. heros genome to answer some of those questions. The SNP data was used to investigate the pattern of genetic structure, hybridization and natural selection of this emerging pest. We found that E. heros populations presented similar levels of genetic diversity with slightly higher values at several central locations in Brazil. Our results also showed strong genetic structure separating northern and southern Brazilian regions (FST = 0.22; p-value = 0.000) with a very distinct hybrid zone at the central region. The analyses also suggest the possibility that GABA channels and odorant receptors might play a role in the process of natural selection. At least one marker was associated with soybean and beans crops, but no association between allele frequency and cotton was found. We discuss the implications of these findings in the management of emerging pests in agriculture, particularly in the context of large areas of monoculture such as soybean and cotton.

The Role of Rainfall in Sternechus subsignatus (Coleoptera: Curculionidae) Adult Emergence From the Soil After Its Winter Dormant Period.

In this work, we report the effect of rainfall on Sternechus subsignatus Boheman, 1836, adult emergence after winter dormancy. This weevil is a univoltine soybean pest found in northwestern Argentina, a subtropical region with dry winters and rainy summers. Before harvest, fully grown larvae burrow into the soil where they overwinter. In the spring, they emerge as adults and recolonize the crop during its planting and early vegetative stages. Our study examines the seasonal timing of adult emergence with the aim of improving chemical control strategies and avoiding unnecessary pesticide applications. To do so, we developed a regression model to predict adult emergence onset as a function of cumulated rainfall after 1st November. The regression with the highest coefficient of determination between cumulated rainfall and adult emergence onset was Emergence onset (Julian day) = -7.98 Ln(cumulative rainfall) + 65.7. The negative relationship showed that adults emerged earlier in wet years than in dry years. Also it was observed that adults emerged from late November to mid-March, in pulses following periods of rainfall. Males were more abundant than females at first, but then the reverse was true toward the end of the period. In most cases, there was a suggestion of relationship (though not significantly) between peaks of adult emergence with peaks of rainfall 15 d before adult emergence. These results reveal that rainfall has a significant impact on the beginning and dynamics of adult emergence from the soil.

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) / Purificação de uma enzima "tipo tripsina" não-solúvel do intestino da lagarta da soja (Anticarsia gemmatalis Hübner)

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.

Comprometer a digestão de proteínas dos insetos pelo uso de inibidores específicos de endoproteases tem sido amplamente estudado como um método de controle de pragas alternativo ao uso dos inseticidas convencionais. No processo de otimização desta estratégia, o conhecimento da diversidade das endoproteases do inseto alvo torna-se crucial. Neste sentido, uma enzima "tipo-tripsina" ligada à membrana obtida do intestino de larvas do 5° instar de A. gemmatalis foi purificada. A fração insolúvel do extrato do intestino foi solubilizada com 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) e submetida à uma cromatografia de afinidade em uma coluna de p-aminobenzamidina, seguida por uma cromatografia de troca-aniônica. O rendimento da enzima purificada foi de 11% com fator de purificação de 143 e uma atividade específica final de 18.6 µM min.-1 mg-1 de proteína usando N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) como substrato. Após a separação da amostra purificada por SDS-PAGE e incubação subsequente com caseína, uma única banda ativa com massa molecular aparente de 25 kDa foi observada. A massa molecular determinada por espectrometria de massa (MALDI-TOF) foi de 28,632 ± 26 Da. O baixo rendimento e as dificuldades em purificar grandes quantidades da enzima limitaram caracterização complementar. A observação desta enzima, no entanto, é mais uma etapa no processo de conhecer as endoproteases presentes no intestino de A. gemmatalis, alvos potenciais de inibidores de proteases naturais ou especificamente projetados.

Stimulatory male volatiles for the Neotropical brown stink bug, Euschistus heros (F. ) (Heteroptera: Pentatomidae) / Semioquímicos produzidos pelo macho do percevejo Neotropical Euschistus heros (Fabr. ) (Heteroptera: Pentatomidae)

Volatiles compounds collected from the male Neotropical brown stink bug, Euschistus heros (F.), a serious Central and South American soybean pest, have been reevaluated. The proportion of three methyl esters is found to be quite different from previous study. A new blend is proposed as the male-produced stimulatory volatiles based on gas chromatographic-electroantennographic detection (GC-EAD) techniques. The three GC-EAD-active components reproducibly found in volatiles collected from males are methyl (2E,4Z)-decadienoate (53 percent), methyl 2,6,10-trimethyldodecanoate (3 percent), and methyl 2,6,10-trimethyltridecanoate (44 percent) respectively. Males of this hemipteran species needed to reach an age of ~15 days to produce enough male-specific compounds to be detected by capillary column GC equipped with flame ionization detector (FID). The amounts of these three stimulatory volatiles increased with age and appeared to reach a maximum at ~35 days old, with the release rate of ~2.5 mug/day/male and the ratio of these three components seemed not to be affected by aging.

Compostos voláteis obtidos de machos do percevejo marrom Neotropical Euschistus heros (F.), uma das principais pragas da cultura da soja na região da América Central e Sul, foram reavaliados. A proporção dos três ésteres já identificados mostrou-se bem diferente da determinada em estudos anteriores. Uma nova combinação de ésteres metílicos é proposta como voláteis estimulatórios produzidos pelos machos, baseando-se nos resultados obtidos das análises por detecção eletroantenográfica acoplada ao cromatógrafo gasoso (GC-EAD). Os três componentes ativos detectados por CG-EAD nos voláteis coletados da aeração de machos foram os (2E,4Z)-decadienoato de metila (53 por cento), 2,6,10-trimetildodecanoato de metila (3 por cento), e 2,6,10-trimetiltridecanoato de metila (44 por cento). A liberação dos voláteis pelos machos adultos alcança níveis detectáveis para análise por cromatografia gasosa capilar com detector de ionização de chamas quando os insetos estão com aproximadamente 15 dias na fase adulta. A quantidade dos três compostos estimulatórios aumentou com a idade dos percevejos e atingiu um nível máximo de produção com aproximadamente 35 dias de fase adulta. A taxa de liberação desses compostos é aproximadamente de 2,5 mig/macho/dia e a proporção desses três componentes parecem não ser afetada com a idade após a maturidade sexual.

Desenvolvimento de uma técnica de criação para Cerotoma arcuatus Olivier (Coleoptera: Chrysomelidae) em laboratório / Development of a rearing technique for Cerotoma arcuatus Olivier (Coleoptera: Chrysomelidae) in laboratory

Studies were carried out aiming to develop a laboratory rearing technique for Cerotoma arcuatus Olivier. For larvae rearing, soybean and bean seedlings grown on vermiculite substrate were used. Viability and duration of larva-adult period were evaluated. In the adult phase, the egg-laying capacity under temperatures of 20°C, 25°C and 30°C as well as the feeding preference on soybean, bean, cowpea and pumpkin leaves were evaluated through free choice, double choice and no choice tests. Soybean seedlins grown on vermiculIte was the most adequate substrate for rearing C. arcuatus. Insects reared on bean seedlings had the viability of the larva-adult period reduced. Egg laying capacity was not affected by rearing substrate or temperature. As the temperature increased a higher percentage of females that did not oviposit as well as a more concentrated oviposition rhythm were observed. C. arcuatus adults preferred soybean, bean and pumpkin leaves as compared to cowpea leaves. The use of soybean seedlings grown on vermiculite for the immature phases and soybean leaves for the adults made possible the continuous rearing of C. arcuatus in the laboratory.

Realizaram-se estudos com o objetivo de desenvolver um método de criação de Cerotoma arcuatus Olivier em laboratório. Para a criação das larvas, foram testadas plântulas de soja e feijoeiro, utilizando-se vermiculita como substrato, avaliando-se a viabilidade e a duração do período larva-adulto. Em adultos avaliou-se a capacidade de postura nas temperaturas de 20, 25 e 30°C, bem como a preferência alimentar em soja, feijoeiro, caupi e abóbora, através de testes de livre escolha, dupla escolha e sem chance de escolha. O substrato mais adequado para criação de C. arcuatus foi plântulas de soja em vermiculita, pois os insetos criados em feijoeiro tiveram a viabilidade do período larva-adulto reduzida. A capacidade de postura não foi afetada pelo substrato de criação e nem pelas temperaturas, sendo que à medida que se aumentou a temperatura, observou-se maior percentual de fêmeas que não realizaram postura e um ritmo de postura mais concentrado. Adultos de C. arcuatus têm preferência por folhas de soja, feijoeiro e abóbora em relação a caupi. Utilizando-se o substrato alimentar soja em vermiculita para as fases imaturas, e, folhas de soja para adultos, foi possível criar C. arcuatus, continuamente em laboratório.

Cinética da esporulação e viabilidade de conídios de Nomuraea rileyi (Farlow) samson sobre cadáveres da lagarta-da-soja, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), em condições de campo / Sporulation kinetics and conidial viability of Nomuraea rileyi (Farlow) samson on cadavers of the velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lep: Noctuidae) in the field

The entomopathogenic fungus Nomuraea rileyi (Farlow) Samson produces epizootics on populations of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, resulting in its natural control. However, sometimes this phenomenon does not occur soon enough to prevent pest population outbreaks and damage to the crop. Observations of N. rileyi conidial production on the surface of A. gemmatalis cadavers in the field showed that high relative humidity and precipitation determine the sporulation dynamics. In fact, initiation of sporulation is delayed and conidia production is reduced when A. gemmatalis cadavers infected with N. rileyi are exposed to water stress (relative humidity 25-30%) in the laboratory. Field observations also demonstrated that N. rileyi conidial viability remain high (>75% germination) until 10 days on A. gemmatalis cadaver surface, acting as an inoculum source for this period.

O fungo entomopatogênico Nomuraea rileyi (Farlow) Samson pode produzir epizootias em populações de lagarta-da-soja, Anticarsia gemmatalis Hübner, controlando essa praga naturalmente. No entanto, o fenômeno nem sempre ocorre a tempo de evitar que populações de A. gemmatalis atinjam o nível de dano econômico na cultura. Observações sobre a produção de conídios em cadáveres de A. gemmatalis em condições de campo mostraram que alta umidade relativa e precipitação são fatores determinantes na cinética da esporulação do fungo in vivo. O início da esporulação foi retardado e a produção de conídios diminuída quando cadáveres de A. gemmatalis infectados com N. rileyi foram submetidos a períodos longos de estresse hídrico (umidade relativa de 25-30%). Observações no campo também demonstraram que a viabilidade de conídios de N. rileyi pode permanecer elevada (>75% germinação) por períodos de até 10 dias na superfície de cadáveres, servindo como fonte de inóculo durante esse período.