Made available in DSpace on 2015-03-26T13:30:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1603340 bytes, checksum: 135e2fd308ff1af60f963623548c94b3 (MD5) Previous issue date: 2006-02-17 / Empresa Brasileira de Pesquisa Agropecuária / The resistance of the insect-pests to fumigants has been frequently reported in stored grains and a present is one of the great obstacles to the control programs involving the use of chemical pesticides. This study was therefore carried out to detect phosphine resistance in populations of Tribolium castaneum, Rhyzopertha dominica and Oryzaephilus surinamensis and to recognize the existence of adaptative disadvantages of these insects in the absence of phosphine, by correlating the instantaneous rate of population growth (ri) with the resistance levels observed in different populations of the species under study. Twelve distinct populations of T. castaneum, ten of R. dominica, and eight of O. surinamensis collected in the states of Goiás, Mato Grosso, Minas Gerais and São Paulo were surveyed. The bioassays for the detection of phosphine resistance were carried out following the standard method recommended by the Food and Agriculture Organization of the United Nations (FAO), which it is based on the exposure of 50 non-sexed adult insects, in four replicates, to the discriminating concentration (DC) of the fumigant for a 20 hourperiod. Later, the concentration-response bioassays were carried out generating the concentration-mortality curves that were used to estimate LC50 and LC95 of the populations pertaining to those three species. The LC50 were used to discriminate the standard population for susceptibility in each species. The measurements of the body mass, CO2 production, and instantaneous rate of population increase (ri) in each population of each species were correlated to the resistance ratio at the LD50. The resistance ratio in T. castaneum ranged from 1,0 to 186,2-fold, whereas in R. dominica it ranged from 2,0 to 71,0-fold, and the lowest one from 1,9 to 32,2-fold for O. surinamensis. The results obtained in the resistance detection bioassays pointed out ten populations of T. castaneum, nine of R. dominica and seven of O. surinamensis that were resistant to phosphine. The relationship between the body mass and CO2 production was positive and significant indicating an increase in CO2 production with the individual body mass. The same effect is observed in the interaction between the instantaneous rate of population increase (ri) and body mass. In addition, the populations with lower CO2 production showed higher resistance ratio (RR) for all species, what is related to the phosphine resistance mechanism. The populations with higher ri showed lower resistance ratio (RR). This result indicates a worse reproductive performance of the resistant populations compared with the susceptible ones. Thus, the management strategies that are based on the interruption of the phosphine fumigation over determined time for the reestablishment of the susceptibility and eventual reintroduction of the fumigant shows a good perspective as a management tactic since the resistant populations under study showed adaptative disadvantage in relation to the susceptible ones in the absence of phosphine. / A resistência a inseticidas fumigantes em insetos-praga de grãos armazenados tem sido freqüentemente registrada e, atualmente, tem se tornado um dos grandes obstáculos aos programas de controle envolvendo o uso de produtos químicos. Com isso, objetivou-se neste estudo detectar populações de Tribolium castaneum, Rhyzopertha dominica e Oryzaephilus surinamensis resistentes à fosfina, além de aferir a existência de desvantagens adaptativas destas, na ausência da fosfina, correlacionando-se produção de CO2, massa corpórea e taxa instantânea de crescimento populacional (ri), com os níveis de resistência observados nas diferentes populações das três espécies em estudo. Foram utilizadas 12 populações distintas de T. castaneum, 10 de R. dominica e oito populações de O. surinamensis, coletadas nos Estados de Goiás, Mato Grosso, Minas Gerais e São Paulo. Os bioensaios para detecção de resistência foram conduzidos conforme método-padrão, recomendado pela Food and Agriculture Organization of the United Nations (FAO), que é baseado na exposição de 50 insetos adultos, em quatro repetições, por um período de 20 horas à concentração discriminante (CD) do gás fumigante. Posteriormente, foram conduzidos bioensaios de concentração-resposta gerando, assim, as curvas de concentração-mortalidade que foram utilizados para estimar as CL50 e CL95 das populações das três espécies. As CL50, por sua vez, foram utilizadas para discriminar a população padrão de suscetibilidade de cada espécie e o nível de resistência das demais populações. A mensuração da massa corpórea, produção de CO2 e taxa instantânea de crescimento (ri) de cada população, das três espécies avaliadas, foram correlacionados à razão de resistência para CL50. A razão de resistência para CL50 em T. castaneum variou de 1,0 a 186,2 vezes, enquanto em R. dominica foi de 2,0 a 71,0 vezes e em O. surinamensis observou-se a menor variação, de 1,9 a 32,2 vezes. Os resultados obtidos nos bioensaios de detecção de resistência indicaram 10 populações de T. castaneum, nove de R. dominica e sete de O. surinamensis resistentes a fosfina. A relação entre massa corpórea e produção de CO2 foi significativa, indicando aumento na produção de CO2 com o incremento da massa corpórea dos indivíduos, nas três espécies avaliadas. O mesmo efeito é observado na interação entre taxa instantânea de crescimento populacional (ri) e a massa corpórea. As populações com menor produção de CO2 apresentaram maior razão de resistência (RR) para CL50, para as três espécies avaliadas, fato que está relacionado ao mecanismo de resistência dos insetos à fosfina. As populações com maiores valores médios de ri apresentaram menor razão de resistência (RR) para CL50, nas três espécies avaliadas. Este resultado indica pior desempenho reprodutivo das populações resistentes, em relação às populações suscetíveis na ausência do fumigante. Portanto, estratégias de manejo da resistência que são baseadas na interrupção da fumigação com fosfina, por determinado tempo visando o restabelecimento da suscetibilidade e eventual reintrodução do fumigante apresentam boa perspectiva de sucesso. Isto porque, as populações resistentes avaliadas neste estudo apresentaram desvantagens adaptativas, em relação às suscetíveis, na ausência da fosfina.

Doctor of Philosophy / Department of Entomology / James F. Campbell / Phillip E. Sloderbeck / Robert "Jeff" J. Whitworth / Knowledge of factors influencing the establishment, persistence and distribution of stored-product pests aids the development of effective Integrated Pest Management (IPM) programs in food storage and processing facilities. This research focused primarily on Tribolium castaneum, which is one of the most important pests of mills. Populations of T. castaneum from different food facilities can potentially be interconnected by either their own dispersal behavior or by human transportation. Population genetic structure analyses based on microsatellites and other insertion-deletion polymorphisms (“indels”) showed that populations from different mills around the US are genetically distinct from each other, but the level of differentiation was not correlated with the geographic distance. A potential source of insect infestation within a food facility is spillage that accumulates outside or movement from bulk storage facilities on site. Results from three facilities showed that most stored-product species were captured both inside and outside buildings, but T. castaneum was rarely captured outside of the facilities. Spatial distribution of all species outside was associated with the proximity of buildings, not necessarily with areas with accumulated spillage. T. castaneum populations inside facilities are potentially exposed to frequent genetic bottlenecks resulting from structural fumigations. Changes in allele frequencies through time, based on the analysis of microsatellites and other indels in individuals collected in a mill, confirmed bottleneck effects. To understand how spatial distribution of T. castaneum within a mill could be influenced by environmental and physical factors, a range of variables were measured at each trap location. There was significant variation among trap locations regarding beetle captures and the variables measured, but increase in beetle captures correlated only with increase in temperature and spillage production. Tribolium castaneum response to visual cues could influence attraction to pheromone and kairomone olfactory cues used in traps. Results of laboratory experiments showed that adults respond to tall narrow black shapes and placing traps in front of these shapes can increase captures. This research provides new insights into factors influencing the spatial distribution of T. castaneum and could help in improving monitoring programs for this important pest of the food industry.

Doctor of Philosophy / Department of Entomology / David C. Margolies and Yoonseong Park / Organisms are capable of sensing environmental conditions through diverse mechanisms. Transient receptor potential channels (TRPs) are a cation channel family that has been found to function in diverse sensing mechanisms. In this dissertation, I identified the function of several TRPs in thermosensing and mechanosensing in the red flour beetle, Tribolium castaneum. Candidate TRPs were chosen based on homology to TRPs found and studied in Drosophila melanogaster. To identify the function of candidate TRPs in T. castaneum, I suppressed the expression of target genes by RNA interference technique and investigated the phenotype of each treated beetle. Temperature is a major limiting environmental factor for organisms. I tested the function of candidate TRPs in thermotaxis (behavior) and thermal acclimation (physiology). Using bioinformatics approaches, I identified three candidate TRPs – painless, pyrexia, and trpA1 – involved in high temperature sensing. To test thermotactic behavior, I investigated beetle movement on a temperature arena with two separate temperature zones. Thermal acclimation was tested by pre-exposing beetles to either 42 °C for 10 min. When treated with double stranded RNA of TRPA1 (dstrpA1), the thermotactic response of beetles at 39 and 42 °C was reduced when compared to control groups. With pre-exposure at 42 °C, survivorship of dstrpA1-treated beetles significantly increased after one minute exposure at 52 °C compared to beetles that were not pre-exposed. With dspainless treatment, beetles showed lower response to thermal acclimation and lower long-term survivorship. Beetles treated with dspyrexia showed lower recovery after heat treatment without pre-exposure at 42 °C. To identify the function of candidate TRPs in mechanosensing, I evaluated dsRNA treated beetles for survival, walking behavior, and tonic immobility. Treatment with dsnompC and dstrpA5 resulted in failure in eclosion, causing 93 % mortality in both treatments. Survivors in dsnompC showed defects in elytra sclerotization. In dsnanchung and dsinactive treatments, adults showed abnormal walking behavior and reduced walking speed that were likely caused by defects of mechanosensing in folding of the joint between the femur and tibia. For tonic immobility, beetles with dsnanchung, dsinactive, dswaterwitch and dsick2 (insect cytokine 2) treatments showed increased sensitivity to mechanical stimulation leading to tonic immobility.

Master of Science / Department of Grain Science and Industry / Subramanyam Bhadriraju / During heat treatment, the ambient temperature of grain-processing facilities is raised to 50-60°C for at least 24 hours to manage stored-product insects. Young larvae (first instars) of the red flour beetle, Tribolium castaneum (Herbst), are the most heat tolerant stage at 50-60°C. A thermal death kinetic (TDK) model predicted survival of T. castaneum young larvae exposed to six constant elevated temperatures between 42 and 60ºC. The model is based on logarithmic survival of T. castaneum as a function of time and logarithmic reduction in larval survival as a function of temperature. The model was validated with 12 independent temperature datasets collected during heat treatments of pilot-scale and commercial grain-processing facilities. Young larval survival in plastic boxes/vials with flour was used to validate model predictions. The heating rate to 50°C from the ambient among the 12 datasets ranged from 0.9-7.8°C/h. Mean absolute deviations between observed and predicted larval survival for 10 of the 12 datasets ranged from 2.1-11.4%; it was 16.2 and 18.3% for two other datasets. The TDK model can be used to predict survival of young larvae of T. castaneum based on time-dependent temperature profile obtained at any given location during heat treatment of grain-processing facilities. In three commercial grain-processing facilities heat treatments were conducted for 24-27.7 hours using forced-air gas heaters. Temperatures attained and survival of 20 eggs, 20 young larvae, and 20 adults of T. castaneum in bioassay vials at various locations were determined. Across all three facilities, 5 out of 2720 adults in 136 vials, 1 out of 960 young larvae in 48 vials, and 0 out of 1760 eggs in 88 vials were alive at the end of the heat treatment. In each facility, the time in hours for 1% predicted survival of T. castaneum young larvae was positively related to how quickly temperatures reached 50°C, and negatively related to rate of heating to 50°C from the ambient, time above 50°C in hours, and the maximum temperature. Bioassays with T. castaneum life stages and the TDK model can be used to gauge effectiveness of facility heat treatments.

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Doctor of Philosophy / Department of Entomology / James F. Campbell / Kun Yan Zhu / Aerosol applications of reduced risk insecticides such as synergized pyrethrin and insect growth regulators (IGR) are part of food industry integrated pest management programs. Since aerosols cannot penetrate into hidden areas exploited by pests such as the red flour beetle, Tribolium castaneum, the potential for these insecticides to effect beetle populations was evaluated. Because IGRs do not cause immediate mortality, the potential of horizontal transfer for an IGR from treated to untreated individuals was also examined. Results showed that when untreated T. castaneum, larvae or pupae, were added to flour containing methoprene, IGR, treated larvae, pupae or adults, the untreated individuals exhibited evidence of methoprene exposure (external deformities and reduced survival). Evaluation of the different mechanisms of transfer indicated that contact with methoprene treated individuals or flour that had been in contact with treated individuals may be the primary method of methoprene transfer. Since aerosols are often applied as a combination of IGR and pyrethrin with a carrier, the effect of these components was evaluated. Applications of synergized pyrethrin caused knockdown of adults but affected adults recovered and progeny production was not effected. Exposure of eggs to these insecticides reduced egg hatch. Food material accumulations inside food facilities can potentially increase or reduce insecticide efficacy. Evaluation of different flour residue levels, representing different sanitation levels, revealed that sanitation alone reduced immature development. As flour residue depths increased more individuals developed into adults but very few developed in the insecticide treatments. Food facilities that use aerosol insecticides apply them at regular intervals, so the cumulative effects of these treatments were considered. Experiments evaluating repeated insecticide exposures indicated that the direct morality from synergized pyrethrin not the horizontal transfer of methoprene was the primary factor in population reduction. Overall findings suggested that methoprene is highly mobile between different surfaces. Exposure of untreated individual beetle larvae to treated larvae or pupae or to flour that has been in contact with exposed beetles can have detrimental effects on development or survival, but these effects may be highly variable and even in cumulative exposures the overall level of population suppression is limited.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Hulya Dogan / Larvae of the Indian meal moth, Plodia interpunctella (Hübner), have the ability to invade or penetrate packaging materials and infest the food within. Energy bars with three polypropylene packaging types were challenged with eggs (for first instars), third, and fifth instars of P. interpunctella to determine package resistance to larvae at 28°C and 65% r.h. to determine which provided the greatest protection against P. interpunctella larval penetration. Third and fifth instars showed a higher propensity to infest all packaging variations. First instars showed a reduction in package pentration abiltiy compared to third and fifth instars. Methoprene is an insect growth regulator labeled in the USA for use as an aerosol spray, a residual surface treatment, and as a grain protectant, and recently has been impregnated into a polymer-based packing film to prevent insect infestations of packaged products. The objectives of these studies were 1) determine the effect of short term exposure time and temperature on four week old larvae, continual exposure on egg-to-adult emergence of beetles, and sub-lethal effect on adults of the red flour beetle, Tribolium castaneum (Herbst) and warehouse beetle, Trogoderma variabile Ballion, on the inside and outside surfaces of methoprene-treated woven packaging material at 27 and 32°C at 60% r.h.; 2) evaluate fecundity, egg hatch, and egg-to-adult emergence of T. castaneum and T. variabile, when exposed to two methoprene-impregnated polymer packaging on the inside and outside surfaces at 27 and 32°C at 60% r.h.; and 3) determine the effect of methoprene-treated foil packaging on larval emergence, penetration, and invasion ability of T. variabile and P. interpunctella at 27°C and 60% r.h. Short term exposure results indicated that adult emergence from larvae of T. castaneum and T. variabile decreased with increasing exposure time. The number of eggs laid per female of T. castaneum and T. variabile did not vary from their controls. Continual exposure demonstrated 100% supression of T. castaneum adult emergence, irrespective of exposure to outside or inside surfaces. T. variabile exposed to inside surfaces were unaffected and normal adult emergence was reduced in those exposed to outside surfaces.. The number of T. variabile eggs laid per female was not significantly different among polymer packaging types. The methoprene-treated polyethylene terephthalate to polyethylene packaging, PET-PE reduced the number of T. castaneum eggs laid per female. Both polymer packaging reduced the percent hatch of both species. No T. castaneum adults emerged on the inside surface of PET-PE and both sides of the polyethlyene to polyethylene (PE-PE). Egg-to-adult emergence of T. variabile was arrested at the pupal stage on the outside surface of PE-PE packaging. The PET-PE packaging greatly reduced the number of normal adults by 87 to 97% when exposed to inside surfaces at both temperatures. The foil packaging had no significant effect on hatch of either species. T. variabile were unable to penetrate/invade any foil packages. P. interpunctella invaded all packaging containing pinholes. Therefore, continual exposure of T. castaneum and T. variabile to methoprene impregnated packaging could be a vaiable tool to protect food packages.

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Intraspecific genetic diversity is an important attribute of natural populations and is deemed critical for their adaptive potential and persistence. However, we have limited empirical understanding of the impact of genetic diversity on population performance under different conditions. For my dissertation, I conducted long-term laboratory experiments with populations of the flour beetle Tribolium castaneum to test the consequences of genetic variation for population dynamic stability and niche evolution. In Chapter 1, I show that genetic variation prevented population extinction in a novel habitat. In addition, genetically diverse populations were more stable, both in a novel heterogeneous habitat and in their ancestral habitat. In the ancestral habitat, alleles from a single founding lineage dominated the dynamics, leading to increased stability of genetically diverse populations. However, such as selective effect was not observed in the novel heterogeneous habitat. Therefore, while genetic variation within populations increased their stability and persistence, the magnitude of the impact and its mechanism depended on the selective habitat. In Chapter 2, I ask whether genetic variation also facilitates resource niche expansion, i.e., use of a novel resource. Using stable carbon isotopes, I analyzed diets of beetles sampled from the above experiment and quantified the rate of change in resource use. Contrary to theoretical predictions, I found that genetic variation for resource use had no effect on the rate of niche evolution. Furthermore, behavioral niche expansion accounted for most of the adaptation to the novel resource, and the behavioral change hindered subsequent evolutionary change in resource use. It is thus apparent that in the short term, behavioral plasticity in niche use may impose far greater constraints on niche evolution than the amount of standing genetic variation. Mathematical models predict that intraspecific competition generates selection for niche evolution, and that genetic variation increases the response to selection. Therefore, I hypothesized that the impact of genetic variation on resource niche evolution may depend on the degree of intraspecific competition. In the final chapter of this thesis, I describe results of an experiment to test this hypothesis. I found that genetic variation and competition indeed interacted to increase the rate of niche expansion in T. castaneum, but that their impacts were temporally variable. Furthermore, the two factors acted on different components of niche evolution: while competition only affected the degree of niche expansion, genetic variation also promoted maintenance of individual variation in resource use. In summary, my thesis describes experiments to test for the ecological and evolutionary impacts of intraspecific genetic variation; and its interaction with behavioral plasticity, intraspecific competition, and resource availability. Genetic diversity and behavioral plasticity are common features of living organisms, and therefore it is vital to understand their combined consequences for population ecological and evolutionary dynamics. In addition, natural populations often face intense competition for limited resources. Hence the experimental results presented here can help us to better understand how populations overcome these resource constraints, given their specific genetic composition. Biologists are increasingly aware that the intricate connection between ecological and evolutionary dynamics is important to gain a more complete understanding of population biology. The work described here represents one of the few experiments providing such detailed mechanistic understanding of the interactions between- and consequences of - key ecological and evolutionary parameters. Finally, the results have important implications for conservation biology, because they show that the effects of genetic diversity can vary greatly depending on a number of population and environmental parameters. / text