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In 1931, Mungomery stated "whitegrubs have been for years past, and still are, the worst insect problem confronting the sugar industry". This statement remains true to this day, with canegrubs costing the Australian sugar industry A$7.22 million in lost production and in use of insecticides. The development of a sugarcane cultivar with resistance to canegrub attack would be a valuable addition to the recently implemented canegrub management program. This thesis examined the possibility that natural inhibitors derived from canegrubs could be incorporated in sugarcane to reduce or prevent its destruction by canegrubs. The research described here demonstrated that canegrub haemolymph contains inhibitors with activity against commercially purified enzymes and serine proteases found in crude midgut extracts. A cDNA encoding a potential canegrub protease inhibitor (DA10 12) belonging to the Ascaris family was cloned, but it did not have activity against the major canegrub midgut proteases. This protein does, however, still have potential for modification into a serine protease inhibitor suitable for use as a novel insect resistance transgene. The possibility of using haemolymph derived inhibitors as novel antimetabolites in a canegrub management strategy based on transgenic plants was also explored. The findings suggest that proteins with properties similar to those of DA10 12 will require the presence of a signal peptide and/or codon optimisation for successful expression in sugarcane. The research outlined in this thesis is the first investigation of protease inhibitors in the haemolymph of scarab larvae, and is the first report of an Ascaris family inhibitor that does not inhibit a serine protease.

Submitted by (edna.saturno@ufrpe.br) on 2016-08-01T15:49:41Z No. of bitstreams: 1 Andrea Barretto.pdf: 465481 bytes, checksum: 80d81c9df4236400373841c9117f86d9 (MD5) / Made available in DSpace on 2016-08-01T15:49:41Z (GMT). No. of bitstreams: 1 Andrea Barretto.pdf: 465481 bytes, checksum: 80d81c9df4236400373841c9117f86d9 (MD5) Previous issue date: 2009-02-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The study of blood cells, also known as hematology, has become for aquaculture a valuable tool in understanding the physiological changes that occur in organisms, however the shrimp, there is still much to discover about if the hemolymph. Considering the above objective of this work: to evaluate the effectiveness of the anticoagulant sodium citrate (10%) in blood tests, to check the influence of cooling on the total count of hemocytes (CTH), haematological data correlate with the presence of Vibrio in the hemolymph, to identify the influence of age and seasonality in the total count of haemocytes in shrimp Litopenaeus vannamei. The species were caught shrimp Litopenaeus vannamei from two nurseries of land (A and B) of mariculture Santa Cruz, located in the city of Goiana-PE. The collections are made weekly, for two cycles of cultivation (dry season and rainy season). It is the examination fresh (macro and microscopic) in all animals sampled. The total counts of hemocytes (cells/mm3) were performed immediately after collection and after five hours of chilling in a Neubauer chamber. Immediately after the counting, the samples intended for bacteriological analysis were transported in temperature to the laboratory of health of aquatic animals (LASAq), Universidade Federal Rural de Pernambuco. The data concerning the influence of cooling on the hemocytes were analyzed using the techniques of mathematical modeling (P <0.05), however the data for the bacteriological analysis,examining changes in the fresh and CTH, the shrimps were divided into four groups: cameroon with injury and with bacteria (CLCB), with injury and without bacteria (CLSB) without injury and with bacteria (SLCB) and without injury and without bacteria (SLSB). The results were submitted to analysis of variance (ANOVA) and when significant, the Tukey test for comparison between means, is adopting a significance level of 5%. At the end of the research concluded that the anticoagulant sodium citrate only adequately preserves the hemolymph of the shrimp Litopenaeus vannamei to CTH performed immediately after collection, and the CTH is not associated with the presence of Vibrio spp and examination with lesions detected in the fresh and therefore not a good tool for immediate recognition of changes in the health of marine shrimp Litopenaeus vannamei. / O estudo das células sanguíneas, também chamado como hematologia, vem se tornando para a aqüicultura um precioso instrumento no conhecimento das alterações fisiológicas que ocorrem nos organismos, no entanto na carcinicultura, ainda há muito que se desvendar a respeito da hemolinfa. Diante do exposto objetivou-se com este trabalho: avaliar a eficácia do anticoagulante citrato de sódio (10%) nas análises hematológicas, verificar a influência da refrigeração sobre a contagem total de hemócitos (CTH), correlacionar os dados hematológicos com a presença de vibrio na hemolinfa, identificar a influencia da idade e da sazonalidade na contagem total de hemócitos em camarões Litopenaeus vannamei. Foram capturados camarões da espécie Litopenaeus vannamei provenientes de dois viveiros de terra (A e B) da Maricultura Santa Cruz, situada no município de Goiana- PE. As coletas se deram semanalmente, durante dois ciclos de cultivo (período seco e período chuvoso). Procedeu-se o exame a fresco (macro e microscópico) em todos os animais amostrados. As contagens totais de hemócitos (células/mm3) foram realizadas imediatamente após a coleta e depois de cinco horas após a refrigeração, em câmara de Neubauer. Imediatamente após a realização das contagens, as amostras destinadas para as análises bacteriológicas foram transportadas, em temperatura ambiente, para o laboratório de Sanidade de Animais Aquáticos (LASAq) da Universidade Federal Rural de Pernambuco. Os dados referentes a influencia da refrigeração sobre os hemócitos foram analisados utilizando-se as técnicas de modelagens matemáticas (P < 0,05), no entanto os dados referentes àsanalises bacteriológicas, alterações no exame a fresco e CTH, os camarões foram distribuídos em quatro grupos: camarões com lesão e com bactéria (CLCB), com lesão e sem bactéria (CLSB), sem lesão e com bactéria (SLCB) e sem lesão e sem bactéria (SLSB). Os resultados foram submetidos à análise de variância (ANOVA) e, quando significativos, ao teste de Tukey, para comparação entre as médias, adotando-se o nível de significância de 5%. Ao término da pesquisa concluímos que o anticoagulante citrato de sódio somente preserva adequadamente a hemolinfa do camarão Litopenaeus vannamei para a CTH realizada imediatamente após a coleta, e que a CTH não está associada com a presença de Vibrio spp e com lesões detectadas no exame a fresco, e portanto, não é uma boa ferramenta para a constatação imediata de alterações na saúde dos camarões marinhos Litopenaeus vannamei.

The evaluation of the sub-chronic exposure to atrazine on crayfish The aim of this study is to evaluate the sub-chronic effect of atrazin on a behaviour, oxidative stress, antioxidant enzyme aktivities and biochemical profile of haemolymph in. These complex data should help to appraise the impact of this substance in the environment. The total test duration was 28 days and was divided into two periods. The first 14 days the crayfish were exposed to two concentrations of atrazine: 6.86 micrograms per liter (ATRenv = environmental concentration in the water in the Czech Republic) and 1.21 milligrams per liter (ATR10% = is coincident to 10% LC50). After the atrazine treatments the depuration 2 weeks phases in water without any chemicals followed. The results indicate that sub-chronic effect of atrazine influenced neither the behaviour of the crayfish nor the level of oxidative stress (measured by TBARS), whereas the changes of superoxiddismutase (SOD) were observed in all tissues (muscles, gills and hepatopancreas). The changes of enzyme activity were observed in catalase (CAT; hepatopancreas and the muscle tissue), glutathione S-transferase (hepatopancreas and the gills tissue), glutathione reductase (GR; the hepatopancreas tissue) and reduced glutathione (the muscle tissue). The influence of ATRenv on the biochemical profile of haemolymph at the following parameters was estimated only for lactate and alkaline, however phosphatase changes made by ATR10% were significant for glucose, ammonia, lactate and alkaline phosphatase measurements. The sub-chronical effect changed the activity of all antioxidant enzymes in hepatopancreas, muscles and the gills tissue of the observed crayfish. The presented results in this study are giving compact information of impact of atrazine on the crayfish and the whole water environment. The suggestion of using the crayfish for tests of toxicity looks like an ideal supplement for triazine herbicide estimations.

Insects maintain blood (haemolymph) Ca2+ concentrations within a narrow range in order to support the health of internal tissues and organs. The Malpighian (renal) tubules play a primary role in haemolymph Ca2+ homeostasis by sequestering excess Ca2+ within calcified biomineral deposits (Ca-rich granules) often located within type I (principal) tubule cells. Using the classic Ramsay assay, the scanning ion-selective microelectrode technique (SIET), and modifications of these two electrophysiological techniques, this thesis begins to unravel the sites and mechanisms of Ca2+ transport by the Malpighian tubules isolated from eight insects, representing seven orders. A segment-specific pattern of Ca2+ flux was observed along the length of the Malpighian tubules isolated from D. melanogaster, A. aegypti and A. domesticus and was uniform along the length in the remaining species. The majority (≥ 90%) of Ca2+ entering the tubule cells is sequestered within intracellular calcium stores in Ca2+-transporting segments of D. melanogaster and A. domesticus tubules, consistent with the presence of Ca-rich storage granules in these tubule segments. In addition, this thesis provides the first measurements of basolateral Ca2+ flux across single principal and secondary tubule cells of T. ni, where Ca2+ uptake occurs only across principal cells. Perhaps the most important finding of this thesis is that increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca2+ in isolated tubules of A. domesticus had opposite effects on tubule Ca2+ transport. The adenylyl cyclase-cAMP-PKA pathway promotes Ca2+ sequestration whereas both 5-hydroxytryptamine and thapsigargin inhibited sequestration. In contrast, tubules of the remaining species were generally insensitive to cAMP or thapsigargin and v rates of tubule Ca2+ transport were often very low. The presence of Ca-rich granules in the cells of the midgut in several of the species with low rates of tubule Ca2+ transport provide evidence for a putative role of the midgut in haemolymph Ca2+ homeostasis. Taken together, these results suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis through neuroendocrine regulated sequestration of excess Ca2+ during periods of high dietary calcium intake. Sequestration of dietary Ca2+ by the midgut may reduce Ca2+ entry into the haemolymph and therefore Ca2+ sequestration by the Malpighian tubules need not be so rapid. Finally, reversible tubule Ca2+ transport may allow internal reserves of Ca2+ (Ca-rich granules) to be returned to the haemolymph allowing insects to survive prolong periods of Ca2+ deficiency (i.e. overwintering). / Thesis / Doctor of Philosophy (PhD) / This thesis contributes to our understanding of how insects regulate the calcium content of their blood (haemolymph). Using electrophysiological techniques with improved spatial resolution (from millimeters to micrometers) this thesis sought to determine the sites, mechanisms and regulation of Ca2+ transport by insect Malpighian (renal) tubules in order to gain insights into the role of Ca-rich granules (similar to those identified in early stages of human kidney stone formation i.e. nephrolithiasis) within these tissues. Using eight insect species this thesis demonstrates that the Malpighian tubules act as dynamic Ca2+ stores that appear to be under neuroendocrine control: actively taking up Ca2+ through calcium entry channels, where the majority (≥ 90%) of excess haemolymph Ca2+ is sequestered within intracellular stores (Ca-rich granules) during period of excess dietary calcium and passively releasing Ca2+ back to the haemolymph during periods of metamorphosis or calcium deficiency (i.e. overwintering).