Sphenophorus levis (Coleoptera: Curculionidae) é uma das principais pragas de importância econômica na cana-de-açúcar. Seu ataque pode causar a morte da planta, promovendo falhas na rebrota e dependendo do nível de infestação, inviabilizar novos cortes. Além disso, é considerado um inseto de difícil manejo. Dentre as razões, destacam-se o hábito críptico de suas larvas, que se alimentam no interior do colmo da cana, e dos adultos, que permanecem boa parte de seu tempo abaixo da palhada sobre o solo. Por esta razão, os métodos tradicionais de controle, como os agroquímicos têm sido pouco eficientes. Neste sentido, a busca por novos conhecimentos e novas estratégias para o manejo desta praga tem se tornado frequente. Assim, o objetivo principal deste trabalho foi o de reinvestigar o feromônio de S. levis, visando a possível existência de novos compostos químicos na sua composição. Além disso, foram estudados ainda a morfologia e morfometria de S. levis, visando uma ágil e fácil separação por sexo nesta espécie, e uma amostragem populacional das formas biológicas de S. levis em campo, ao longo de uma safra, tendo em vista o manejo desta praga. As fêmeas apresentaram maior tamanho corporal que os machos, porém, houve sobreposição entre o tamanho mínimo da fêmea e máximo do macho, comprometendo uma distinção pelo tamanho. A separação dos sexos em S. levis pode ser feita observando-se a parte ventral do abdome, com os machos apresentando toda a região pilosa e as fêmeas somente com pelos na região apical. Uma maior concavidade presente no final do abdome dos machos, também pode ser utilizada como um parâmetro auxiliar nesta separação. Observando-se a flutuação populacional das diferentes fases biológicas de S. levis, por meio de trincheiras, verificou-se que as larvas ocorrem praticamente durante todos os meses do ano, enquanto que pupas e adultos recém emergidos, em menos da metade das amostras. O pico populacional de larvas, pupas e adultos recém emergidos ocorreu em outubro. Para os adultos, a amostragem por meio de iscas indicou que o pico de maior intensidade foi em dezembro. Na etapa de reinvestigação do feromônio de agregação de S. levis, foram obtidas respostas positivas nas antenas de machos e fêmeas para três compostos químicos. Um destes compostos foi o álcool 2-metil-4-octanol, já identificado anteriormente por Zarbin et al. (2003). Os outros dois, são novos compostos denominados 2,3-butanodiol diacetil (2,3 diacetilbutano) e meso 2,3 butanodiol diacetil (meso 2,3 diacetilbutano). Os três compostos sintéticos foram testados em GC-EAG, porém ainda sem uma resposta conclusiva. Novos testes necessitam ainda ser conduzidos para elucidar a composição química do feromônio de agregação de S. levis. / Sphenophorus levis (Coleoptera: Curculionidae) is one of the main pests of economic importance in sugarcane in Brazil. The attack can result plant death promoting the regrowth and failures depending on the level of infestation, derail further cuts. S. levis is a hard management pest. Among the reasons, it highlights the cryptic habits of larva, which feed inside the sugarcane stalk, and adults live in the soil surface, above of straw. Due to this, the traditional control methods such as agrochemicals have been inefficient. The search for new knowledge and new strategies for the management of this pest has become frequent. Thus, the main objective of this work was to reinvestigate the S. levis pheromone, aiming the possible existence of new chemical compounds in its composition. Beside that, this study aimed to morphometric and morphological comparison between males and females, seeking sex separation in a fast and effective manner. Moreover, population sample of S. levis in field was performed, aiming at an improvement in planning of the management of this pest. Females of S. levis have a greater body size than males; however this parameter cannot be used as a sex indicator, because there was overlap between the minimum and maximum body sizes of females and males respectively. S. levis sex separation can be carried observing abdomen ventral side, where males present hairy venter and females just a tuft in the apical region, and by a greater concavity of the end of the abdomen in males. The fluctuation of S. levis biological stages through trenches, it was found that the larvae occurs during almost all months of the year, while pupae and recently emerged adults in less than half of the samples. The population peak for larvae, pupae and recently emerged adults was in October. For adults, the sampling through baits indicated that the peak intensity was higher in December. Then, in the reinvestigate the S. levis pheromone, natural extracts from males showed responses of antennas of males and females of the species for three chemical compounds. One of these compounds was the alcohol 2-methyl-4-octanol, previously identified by Zarbin et al. (2003). The other two are new compounds, called 2.3-butanediol diacetil (2.3 diacetilbutano) and meso 2.3 butanediol diacetyl (meso 2.3 diacetilbutano). The three synthetic compounds were tested in GC-EAG, but there is still no conclusive answer. New tests still need to be conducted to elucidate the chemical composition of S. levis aggregation pheromone.

<p>In insect pest management, the plant volatiles and pheromones associated with host-plant location can be used to manipulate insect pest behavior by attracting or “pulling” insects from a valuable resource. Conversely, deterrents can be used to prevent behaviors or “push” insects away from a resource. If combined, attractants and deterrents can have powerful synergistic effects that promote greater response than the individual components. This dissertation explores the use of attractants and deterrents of the specialist herbivore and challenging agricultural pest, the striped cucumber beetle, <em>Acalymma vittatum</em>, to ultimately develop a push-pull management system. </p> <p><br></p> <p>In first chapter, we examine the combination of two striped cucumber beetle attractants in attract-and-kill mass trapping: live striped cucumber beetles as a proxy for aggregation pheromone, and cucurbit floral volatiles. In the second chapter, we examine natural products—essential oils, pawpaw extract, squash bugs, and kaolin clay—as a means for repelling or deterring beetles from cucurbit crops. Lastly, we combine the findings of previous chapters as way of using both attractive and deterrents to further modify striped cucumber beetle behavior and to observe potential synergies in removing these pests from cucurbit crops. In this, we use the aggregation pheromone and floral lures in attract-and-kill trapping with the deterrent kaolin. </p> <p><br></p> <p>We found that while aggregation pheromones and floral lures were useful in trapping striped cucumber beetles, floral lures may potentially distract pollinators. Striped cucumber beetle response to floral lures varied across the season and were most attractive in the late growing season, when plants were in bloom. The tested natural products did not successfully prevent beetles from colonizing plants, but instead deterred the specialist herbivore from feeding. While the attractant and deterrent did not have a synergistic effect, they remained complementary in that aggregation pheromones were useful in reducing pest populations, while kaolin clay deterred feeding. Thus, pest management systems should be flexible in timing and type of management used, and should look toward other metrics, such as feeding damage, rather than population density thresholds to measure management success. </p>

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19377号 / 農博第2147号 / 新制||農||1037(附属図書館) / 学位論文||H28||N4957(農学部図書室) / 32391 / 新制||農||1037 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 吉村 剛, 教授 藤井 義久, 准教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Pheromone lures and trap crops are appealing pest management tools that use insect and/or plant volatiles to reduce pest populations on crops of interest. Generating pheromone-emitting trap plants may allow for a continuing and highly-specific attraction of insect pests without repeated and costly application of synthetic pheromones. These trap plants may also be used to develop area-wide pest management strategies. As a proof-of-principle study we tested the possibility of producing the pheromone of the crucifer flea beetle Phyllotreta cruciferae in transgenic plants. P. cruciferae is an important pest of Brassica crops. In the presence of a host plant, males emit an aggregation pheromone, which attracts both males and females. Himachaladiene, a sesquiterpene, has been identified as a key component of the aggregation pheromone of P. cruciferae. In a close relative, Phyllotreta striolata, the compound is synthesized by a two-step pathway with an isoprenyl diphosphate synthase (PsIDS3) making (Z,E)-farnesyl diphosphate (FPP), which is converted by a terpene synthase (PsTPS1) to himachaladiene. Transient transformation of N. benthamiana with PsIDS3-TPS1 co-localized to the plastid resulted in the emission of himachaladiene and other known PsTPS1 products. Daily emissions of himachaladiene were approximately 1 µg per plant, which is six-fold higher than emissions from individual male flea beetles. Stable transformation of Arabidopsis thaliana with the same vector construct resulted in transgenic plants that expressed PsTPS1 and PsIDS3 transcripts, but no himachaladiene or other PsTPS1 products were present in volatile collections or leaf extracts of these plants. Moreover, no PsTPS1 enzyme activity was observed, indicating that post-transcriptional/translational effects prevent proper expression or targeting of functional PsIDS3 and/or PsTPS1 proteins in A. thaliana. Overall, this study demonstrates that the key component of the P. cruciferae aggregation pheromone, himachaladiene, can be transiently produced and emitted in a plant system at rates that are biologically relevant for insect attraction. However, further work is required for the stable production of the pheromone in plants. In addition, preliminary results are presented for the development of simple two-choice arenas that may allow for assessment of the movement of beetles toward host plant leaf tissue. This work can inform future efforts in developing methods for the economic production of himachaladiene in a plant system or the establishment of transgenic plants for the production and deployment of himachaladiene in a field setting. / Master of Science / The crucifer flea beetle is an important pest of vegetable and oilseed Brassica crops such as broccoli, cabbage and canola. Feeding by beetles has its greatest impact on crop health and yield in the early spring, when adult beetles emerge from overwintering sites and feed on newly- emerging Brassica seedlings. Currently these insects are controlled using broad spectrum insecticides. A general awareness of the negative aspects of insecticides drives the search for alternative pest management strategies that could diversify our management strategies and reduce reliance on insecticides. Previous work has found that the crucifer flea beetle navigates to its host plants, in part, through plant-emitted volatiles. After locating the plant host, males emit a volatile aggregation pheromone that when blended with host plant volatiles increases attraction. Here work towards the development of a specialized trap crop is presented. Plants were engineered to emit a key component of the crucifer flea beetle aggregation pheromone. In an engineered non-host plant, Nicotiana benthamiana, transient production of the aggregation pheromone was established. However, in an engineered Brassica plant, Arabidopsis thaliana, no aggregation pheromone was detected despite evidence of the presence and expression of the required biosynthetic genes for its production. A discussion on alternative engineering strategies for A. thaliana is presented. In addition, preliminary results are presented for the development of a simple behavior assay to assess the attraction of beetles toward different smells. This work can inform future efforts aimed at developing methods for the economic production of the aggregation pheromone in a plant system or the establishment of plants for the production and deployment of the aggregation pheromone in a field setting.

Harlequin bugs are orange and black aggregation pheromone emitting stink bug pests, specifically of cole crops such as kale, broccoli and collards. This nearly loyal crop preference makes an interesting challenge for trapping them and helping farmers predict pest severity. Harlequin bugs can be found in much of North America, and are a serious problem in the southeastern United States. Presumably their persistence into northern regions is limited by extreme winters. In 2014 and 2015 the arctic polar vortex extended into mid-latitudes bringing a blanket of sustained sub-freezing temperatures to much of the United States. We used these events to determine effects of extreme winter weather on harlequin bug survival. In both years we observed nearly identical low temperatures of -15oC and this linked to high (80-96%) harlequin bug mortality. In the lab we measured exact lethal freezing temperatures in harlequin bugs (i.e. supercooling points) to see if a physiological metric could be used to predict overwinter survival. Harlequin bug adults froze and died at -10.4oC, and similarly, their larger juvenile stages freeze at -11.0oC. Freshly hatched harlequin bugs and unhatched eggs froze at considerably lower temperatures with eggs forming ice crystals at -23.2oC and recent hatches at -21.6oC. Now with an understanding of how harlequin bugs likely survive winter extreme, we can then work on developing a trap to tally their populations in the spring and predict summer and fall pest severity. In the lab and field, harlequin bug adults and large nymphs were more likely found on green and black colors, and statistically less frequently on yellow, white, purple or red colors with the exception of adult females, which were most attracted to red and green in the lab, but green and black in the field. To increase harlequin bug attraction to and termination at traps square corrugated plastic panels were wrapped with an insecticide netting and baited with harlequin bug aggregation pheromone, murgantiol. Bugs were effectively drawn to the panels, with green panels having significantly more dead harlequin bugs and fewer dead beneficial lady beetles (Coleoptera: Coccinellidae) at their base than yellow panels. Thus, green was chosen as the ideal trap color to use for another field experiment that evaluated three trap types -- a corrugated plastic square panel, pyramidal trap, and ramp trap -- each with three lure treatments, murgantiol alone or murgantiol plus a low or high rate of mustard oil. More bugs were killed with the pyramidal trap than with the panel trap or the ramp trap, and more bugs were killed at traps containing murgantiol combined with benzyl isothiocyanate than at those with murgantiol alone. This research demonstrated that with the proper visual elements and odors, harlequin bugs can be drawn to traps and effectively killed after contact with insecticide-incorporated netting. / MSLFS / Harlequin bugs are orange and black aggregation pheromone emitting stink bug pests, specifically of cole crops such as kale, broccoli and collards. This nearly loyal crop preference makes an interesting challenge for trapping them and helping farmers predict pest severity. Harlequin bugs can be found in much of North America, and are a serious problem in the southeastern United States. Presumably their persistence into northern regions is limited by extreme winters. In 2014 and 2015 the arctic polar vortex extended into mid-latitudes bringing a blanket of sustained sub-freezing temperatures to much of the United States. We used these events to determine effects of extreme winter weather on harlequin bug survival. In both years we observed nearly identical low temperatures of -15℃ and this linked to high (80-96%) harlequin bug mortality. In the lab we measured exact lethal freezing temperatures in harlequin bugs (i.e. supercooling points) to see if a physiological metric could be used to predict overwinter survival. Harlequin bug adults froze and died at -10.4℃, and similarly, their larger juvenile stages freeze at -11.0℃. Freshly hatched harlequin bugs and unhatched eggs froze at considerably lower temperatures with eggs forming ice crystals at -23.2℃ and recent hatches at -21.6℃. Now with an understanding of how harlequin bugs likely survive winter extreme, we can then work on developing a trap to tally their populations in the spring and predict summer and fall pest severity. In the lab and field, harlequin bug adults and large nymphs were more likely found on green and black colors, and statistically less frequently on yellow, white, purple or red colors with the exception of adult females, which were most attracted to red and green in the lab, but green and black in the field. To increase harlequin bug attraction to and termination at traps square corrugated plastic panels were wrapped with an insecticide netting and baited with harlequin bug aggregation pheromone, murgantiol. Bugs were effectively drawn to the panels, with green panels having significantly more dead harlequin bugs and fewer dead beneficial lady beetles (Coleoptera: Coccinellidae) at their base than yellow panels. Thus, green was chosen as the ideal trap color to use for another field experiment that evaluated three trap types – a corrugated plastic square panel, pyramidal trap, and ramp trap – each with three lure treatments, murgantiol alone or murgantiol plus a low or high rate of mustard oil. More bugs were killed with the pyramidal trap than with the panel trap or the ramp trap, and more bugs were killed at traps containing murgantiol combined with benzyl isothiocyanate than at those with murgantiol alone. This research demonstrated that with the proper visual elements and odors, harlequin bugs can be drawn to traps and effectively killed after contact with insecticide-incorporated netting.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Knowledge about biology and animal behavior is critical to understanding how a particular organism interacts with its environment. Identifying the sex of individuals is an essential step in conducting behavioral studies. An integral part of animal behavior is communication, which can be intermediated by pheromones. Within different groups, insects are the animals use pheromones the most in order to carry out their fundamental activities. Cratosomus flavofasciatus, a Coleoptera of the Curculionidae family, popularly called the Orange-tree-borer, is considered one of the main citrus pests, occurring in Sergipan orchards. The objective of this work was to evaluate the morphological characteristics that allow the sexual differentiation of C. flavosfaciatus and to evaluate the attractiveness of C. flavosfaciatus to the synthetic compounds that make up the pheromone of this species. To verify the sexual dimorphism of C. flavofasciatus, a geometric morphometry analysis was first made using 80 adult individuals of each sex. There were 27 landmarks in the ventral region in each individual. The anatomical landmarks 2, 9, 10, 11, 18, 21 were the ones that contributed most to the distinction between males and females, which represent the region of the rostrum, the final portion of the abdomen and the end of the thorax. For sex confirmation, a withdrawal of the genitalia of both sexes was also performed. In females it was possible to recognize the following structures: sternite VIII and bursa copulatrix. In males, it was possible to observe the gastric spicule and the aedeagus with a pair of parammero. The Procrustes Anova showed significant differences between the sexes (P<0.001) and the size (centroid) in relation to sex (P <0.001), but there was no difference in shape in relation to size (P = 0.229) and the form in relation to sex in conjunction with the size (P = 0.707). Principal component analysis demonstrated an evident differentiation between the genders based on the ventral region of the species. The first two main components explained 51.909% (PC1 + PC2: 39.563% + 12.346%) of the shape variation. The discriminant analysis indicated a significant difference (permutation test = 10,000 permutations; P < 0.001) for the sexual dimorphism of the ventral region. The determination of the adult sex of C. flavofasciatus through the last abdominal sternum is as accurate as the dissection of the genitalia. The behavioral response of C. flavofasciatus to synthetic pheromonal compounds (a mixture of 2-((1R, 2S)-1-methyl-2-(prop-1-en-2-yl)cyclobutil)ethanol (grandisol), (E)-2-(3,3-dimethyl-cyclohexylidene)ethanol (alcohol E) and (Z)-2-(3,3-dimethyl-cyclohexylidene)ethanol (alcohol Z)) and for their host plants Cordia curassavica (Maria-Preta) and Citrus sinensis (Orange Tree) were evaluated by olfactory tests performed on a Y-tube olfactometer. Both sexes were significantly more attracted to C. sinensis stalks (P <0.05) than to filtered air. Tests with C. curassavica stalk attracted only males (P = 0.02). When the insects had the opportunity to choose between C. curassavica stalks and C. sinensis stalks, males preferred those from C. curassavica (P = 0.01), whereas females showed no significant preference (P> 0.05). The combination of the C. curassavica and C. sinensis volatiles associated with the synthetic pheromone at 100ppm concentration was more attractive for both sexes (P <0.05) than for the control (hexane together with the host plant). On the other hand, at the concentration of 10ppm, only the association with C. curassavica attracted males (P = 0.009). These results can be used as a basis for field testing, contributing to the development of an environmentally safe and effective method for monitoring orange tree borer. / O conhecimento sobre a biologia e o comportamento animal é fundamental para compreensão de como determinado organismo interage com o seu ambiente. A identificação do sexo dos indivíduos é um passo essencial na execução de estudos comportamentais. Um ponto integrante do comportamento animal é a comunicação, a qual pode ser intermediada por feromônios. Dentre os vários grupos, os insetos são os animais que mais utilizam os feromônios para desempenhar suas atividades fundamentais. Cratosomus flavofasciatus, um coleóptero da família Curculionidae, popularmente chamado de broca-da-laranjeira, é considerado uma das principais pragas do citros, apresentando grande ocorrência em pomares sergipanos. O presente trabalho teve como objetivo avaliar características morfológicas que permitam a diferenciação sexual de C. flavosfaciatus e avaliar a atratividade de C. flavosfaciatus aos compostos sintéticos que compõem o feromônio de agregação dessa espécie e à suas plantas hospedeiras. Para verificar o dimorfismo sexual de C. flavofasciatus, primeiramente foi feito uma análise de morfometria geométrica utilizando 80 indivíduos adultos de cada sexo. Foram feitos 27 marcos anatômicos (landmarks) na região ventral em cada indivíduo. Os marcos anatômicos 2, 9, 10, 11, 18 e 21 foram os que mais contribuíram para a distinção entre machos e fêmeas, os quais representam a região do rostro, a porção final do abdômen e o final do tórax. Para confirmação do sexo, foi realizada também a retirada da genitália de ambos os sexos. Nas fêmeas foi possível o reconhecimento das seguintes estruturas: esternito VIII e bursa copulatrix. Já nos machos foi possível observar a espícula gastral e o edeago com par de parâmeros. A Anova de Procrustes demonstrou diferença significativa da forma (P < 0,001) e do tamanho (centróide) (P < 0,001) entre os sexos, porém não houve diferença da forma em relação ao tamanho (P = 0,229) bem como da forma em relação ao sexo em conjunto com o tamanho (P = 0,707). A análise de componentes principais demonstrou uma evidente diferenciação entre os sexos com base na região ventral da espécie. Os dois primeiros componentes principais explicaram 51,91% (PC1 + PC2: 39,56% + 12,35%) da variação da forma. A análise de discriminantes indicou diferença significativa (teste de permutação = 10000 permutações; P < 0,001) quanto ao dimorfismo sexual da forma da região ventral. A determinação do sexo em adultos de C. flavofasciatus através do último esternito abdominal é tão precisa quanto à dissecação da genitália. A resposta comportamental de C. flavofasciatus aos compostos feromonais sintéticos (mistura de 2-((1R, 2S)-1-metil-2-(prop-1-en-2-il)ciclobutil)etanol (grandisol), (E)-2-(3,3-dimetilciclohexilideno)etanol (álcool E) e (Z)-2-(3,3-dimetilciclohexilideno)etanol (álcool Z)) e para suas plantas hospedeiras Cordia curassavica (Maria-Preta) e Citrus sinensis (Laranjeira) foi avaliada por meio de testes olfativos realizados em um olfatômetro em “Y”. Ambos os sexos foram significativamente mais atraídos para os caules de C. sinensis (P < 0,05) que para o ar filtrado. Já os testes com caules de C. curassavica atraíram significativamente somente os machos (P = 0,02). Quando os insetos tiveram a oportunidade de escolher entre caules de C. curassavica e caules de C. sinensis, os machos preferiram os de C. curassavica (P = 0,01), já as fêmeas não apresentaram preferência significativa (P > 0,05). A combinação dos voláteis de C. curassavica e C. sinensis associadas ao feromônio sintético na concentração de 100ppm foi mais atrativa para ambos os sexos (P < 0,05) que para o controle (hexano juntamente com a planta hospedeira). Por outro lado, na concentração de 10ppm, somente a associação com C. curassavica atraiu apenas os machos (P = 0,009). Tais resultados podem ser utilizados como base para testes de campo, contribuindo para o desenvolvimento de um método ambientalmente seguro e eficaz para o monitoramento da broca-da-laranjeira.