Submitted by (edna.saturno@ufrpe.br) on 2017-02-16T13:06:03Z No. of bitstreams: 1 Marcos Antonio Cerqueira.pdf: 307415 bytes, checksum: a0c882402c940a3ff2dd4c74fe826638 (MD5) / Made available in DSpace on 2017-02-16T13:06:03Z (GMT). No. of bitstreams: 1 Marcos Antonio Cerqueira.pdf: 307415 bytes, checksum: a0c882402c940a3ff2dd4c74fe826638 (MD5) Previous issue date: 2003-02-17 / The Tambaqui fish Colossoma macropomum (Cuvier, 1816) , is a typical species from the amazon state, which the cultive to find oneself in ascendance, mainly with the increase of the post-larval production. This work has as objetive to determine the effect of the colors in the post-larval survival to the tambaqui fish during the incubation phases. The experiment was realized with the support a hatchery with the 60L of capacity, in the pisciculture station of Balbina, Amazon State, Brazil. The experiment was formed with four conditions and five replies to each. In the three conditions the hatcheries were envolved with cellophane sheets with differents colors and the last conditions the hatchery wasn’t envolved. Each condition was represented by a color : A- blue; M- brown; P- black and T- translucent (environmental light). To the experiments was obtained any effect of the colors on the fertilization (F(3;16) = 0,53, p > 0,05). However, the larval eclosion was influenceated by the colors: the “T” and “A” conditions presented the better performance (F(3;16) = 4,65, p < 0,05). The rate of the post-larval prodution was influenced by the colors too: the “T” and “M” conditons presente better income (F(3;16) = 4,76, p < 0,05). Thus is possible to suggest that in some phases of the fish reprodution was influenceated by the colors. / O tambaqui Colossoma macropomum (Cuvier, 1816) é uma espécie nativa de região Amazônica, cuja criação encontra-se em ascendência principalmente com o aumento da produção de pós-larvas, o que justifica o desenvolvimento de pesquisa visando otimizar a produção deste insumo. Assim, o objetivo deste trabalho foi determinar o efeito da cor na sobrevivência de pós-larvas de tambaqui durante a incubação. O experimento foi realizado em incubadoras de 60L na estação de piscicultura de Balbina e constou de quatro condições, com cinco réplicas cada. Em três delas, as incubadoras foram envoltas com papel celofane de diferentes cores e na quarta condição (controle) as incubadoras não foram cobertas por papel. Cada uma delas representou uma condição: A - azul; M - marrom, P - preta e T - translúcida (cor natural). Não ocorreu efeito da cor sobre fertilização dos ovos (F(3;16) = 0,53, p > 0,05) cujas taxas variaram de 46,95%, na condição P a 56,95%, na condição T. No entanto, houve influência da cor na eclosão de larvas: as condições T, com taxa de 43,76% e A, com taxa de 30%, apresentaram melhor sobrevivência que as demais (F(3;16) = 4,65, p < 0,05). A taxa de produção de pós-larvas também foi influenciada pelas cores: as condições T, com taxa de 18,26% e M com taxa de 10,36%, apresentaram melhor sobrevivência (F(3;16) = 4,76, p < 0,05) do que as demais. Assim, é possível sugerir que as cores podem influenciar algumas fases da reprodução de peixes.

Le moustique Aedes aegypti représente un problème de santé publique majeur, car il est le principal vecteur des virus de la Fièvre Jaune, de la Dengue, du Chikungunya et du Zika à l’échelle mondiale. Dans la région des Amériques, ce moustique a été introduit, depuis le continent africain au cours du XVIIème siècle. À la Martinique, entre 2013 et 2016, cette espèce a transmis de façon active les virus de le Dengue, du Chikungunya et du Zika, plaçant l’île en situation épidémique. Sur l’île, la Dengue est devenue endémique depuis près de 20 ans, avec 7 grandes épidémies (1995, 1997, 2001, 2005, 2007, 2010, 2013) ; celle de 2010 a touché près de 40 000 personnes et causé 18 décès. Le Chikungunya est apparue en Décembre 2013 et l’épidémie qui a duré jusqu’en Janvier 2015 a touché 72 520 personnes (dont 83 décès). Le Zika est apparu en Décembre 2015 et l’épidémie a duré toute l’année 2016 (36 000 cas estimés au 30 Septembre). C’est dans ce contexte de circulation active des arbovirus, et dans la volonté d’améliorer les connaissances actuelles sur la bioécologie du vecteur Ae. aegypti, qu’a été initié ce travail sur le thème « Aedes aegypti à la Martinique : écologie et transmission des arbovirus ». La détection puis la caractérisation des virus dengue et chikungunya dans les populations naturelles du moustique ont confirmé le rôle vectoriel d’Ae. aegypti. Ces résultats permettent d’envisager la mise en place d’une veille entomo-virologique dans le cadre de la surveillance des virus circulant sur l’île ; cette veille entomo-virologique pouvant avoir une application opérationnelle (contrôle de foyers émergents). Ensuite, des études portant sur l’écologie larvaire du moustique ont été initiées. L’étude physicochimique des eaux des gîtes larvaires, les retombées de deltaméthrine suite à un traitement spatial et leurs impacts sur le développement larvaire et les traits de vie du moustique ont permis de confirmé que le phénomène de résistance aux insecticides des populations locales d’Ae. aegypti est un frein dans la stratégie de lutte contre le vecteur, dans la mesure où le développement de ce dernier n’était pas affecté. À l’échelle locale, ces études combinées, visent à compléter les données et connaissances sur le moustique, en vue d’une gestion plus efficace de ce dernier et des risques sanitaires et épidémiologiques qui lui sont associés. / Aedes aegypti mosquito is a major public health problem because it is the main vector of Yellow Fever, Dengue, Chikungunya and Zika viruses worldwide. In the Americas, the mosquito was introduced from Africa during the seventeenth century. In Martinique, between 2013 and 2016, the species has actively transmitted Dengue, Chikungunya and Zika viruses, placing the island in an epidemic situation. On the island, Dengue has become endemic in nearly 20 years, with 7 major epidemics (1995, 1997, 2001, 2005, 2007, 2010, 2013); the 2010 epidemic affected almost 40,000 people and caused 18 deaths. Chikungunya virus was introduced in December 2013 and the epidemic lasted until January 2015 and affected 72,520 people (including 83 deaths). Zika virus was introduced in December 2015 and the epidemic lasted throughout 2016 (36,000 cases estimated up to September 30th). In this context of active circulation of arboviruses, and the will to improve the current knowledge on the bioecology of the vector Ae. aegypti, we initiated the work on "Aedes aegypti in Martinique : ecology and transmission of arboviruses."Detection and characterization of Dengue and Chikungunya viruses in natural populations of the mosquito have first confirmed the role of Ae. aegypti as the main vector of these arboviruses on the island. These results allow to consider the establishment of a virological monitoring tool for surveillance of viruses circulating on the island; this can have an operational application such as the control of emerging households.Then, studies of larval mosquito ecology have been initiated. Physicochemical studies of breeding sites waters, impact of deltamethrin after spatial spray and their impacts on larval development and mosquito life traits showed no effect on the general reserves of emerging adults confirmed that the phenomenon of insecticide resistance of local populations of Ae. aegypti is an obstacle in the strategy against the vector. Locally, these studies are intended to supplement data and knowledge about the mosquito, for a more efficient management of the sanitary and epidemiological risks associated.

The butterfly family Lycaenidae (including the Riodinidae) contains an estimated 30% of all butterfly species and exhibits a diverse array of life history strategies. The early stages of most lycaenids associate with ants to varying degrees, ranging from casual facultative coexistence through to obligate association where the long-term survival of the butterfly is dependent on the presence of its attendant ants. Attendant ants guard the butterflies against predators and parasites during their vulnerable period of larval growth and pupation. The caterpillars, in return, reward the ants by providing attractive secretions from specialized glands in their cuticle. The prevalence of caterpillar-ant associations in the species rich Lycaenidae is in contrast with other Lepidoptera, where ant association appears only as isolated cases in otherwise non ant-associated lineages. This has led to the proposal that ant association may have influenced lycaenid diversification or even enhanced the rates of speciation in the group. In contrast, facultative ant-associated butterflies exhibit high levels of host plant integrity, so it is reasonable to assume that host plants may have played a significant role in their diversification. Since the influence of ants (or plants) on diversification is independent of geographic speciation modes such as vicariance or peripheral isolates, there is an underlying inference of sympatric speciation. Certain prerequisites thought to be important for sympatric speciation, such as mating on the host plant (or in the presence of the appropriate ant) as well as ant dependent oviposition preferences are characteristic of many obligate myrmecophiles. Not surprisingly, it has been suggested that evidence for sympatric speciation is more likely to be found in the Insecta since this additional mode of diversification could account for the large numbers of insect species. This thesis tested the diversification processes in obligate and facultative ant associated lycaenids using comparative methodologies in hierarchical molecular phylogenetic analyses. First, several hypotheses relating to the influence of ants on diversification in obligately ant associated lycaenid butterflies were tested in a phylogeographic analysis of the Australian endemic Jalmenus evagoras. The phylogeographic analysis revealed that regional isolation of butterfly subpopulations coincident with locally adapted ant taxa could generate a phylogenetic pattern in which related lycaenids would be seen to associate with related or ecologically similar ants. Likewise, ecological shifts in habitat preferences by lycaenids could lead to co-diversification with habitat specialist ants, even though in both cases, the ants may play only an incidental role in the diversification process. A comparative methodology was then applied in a molecular phylogenetic analysis of the genus Jalmenus to test for a signal of diversification consistent with shifts in ant partners, and to infer the processes by which ants could influence speciation. Several other specific hypotheses relating to monophyly and taxonomy were also examined. Comparative analysis of the Jalmenus phylogeny found that attendant ant shifts coincided with high levels of sympatry among sister species. This pattern could be explained by sympatric speciation; however, data suggested it was more likely that ant shifts occurred during butterfly population expansions as a result of vegetation and climate changes in the Pleistocene. Fragmentation of populations associating with novel ants could promote rapid ecological and behavioural changes and this could result in reproductive isolation of conspecifics when in secondary contact. Diversification would then continue in sympatry. In contrast, secondary contact of populations associating with the same ant species would result in homogenisation of the two lycaenid lineages or the extinction of one. A phylogeographic analysis of the facultative myrmecophiles, Theclinesthes albocincta/T. hesperia, was then undertaken to infer the evolutionary processes (such as the effects of host plant shifts) that could result in extant demographics. Species-specific questions of taxonomy, relative population ages and dispersal routes in arid Australia were also addressed. Results from the analysis suggested the two taxa were conspecific and had diversified in the late Pleistocene as a consequence of isolation in refugia in and around the arid areas of mainland Australia. However, as was the case in the J. evagoras population analysis in which attendant ant shifts were not detected, host plant shifts were not detected in the population analysis of T. albocincta/hesperia. Host plant or attendant ant shifts manifest more frequently at the species level, thus it was necessary to test the influence of host plant shifts at this higher level. The comparative methodology was then applied to a molecular phylogenetic analysis of the facultative ant-associated section Theclinesthes (comprising Theclinesthes, Sahulana and Neolucia) to test for modes of diversification consistent with host plant shifts. The relative importance of other influences on diversification was also assessed. Akin to the Jalmenus analysis, the prediction that sister species ranges should broadly overlap when a shift in host plants had taken place was upheld in the comparative analysis. Species in the genera Jalmenus and Theclinesthes were found to have diversified in the Pleistocene so were subject to the same climatic oscillations that influenced patterns of vegetation expansion and contraction across much of Australia. Thus, the similarity and predictability of relationships in the comparative analyses based on biological data suggested that host plant shifts have influenced diversification in facultative myrmecophiles by inhibiting gene flow in secondary contact in similar fashion to that of attendant ant shifts identified in the Jalmenus phylogeny. Interpretation of data in these analyses suggested that allopatric diversification was the most common mode of speciation. Isolation was inferred to be the result of fragmentation following long distance dispersal across wide expanses of marginal habitat, or vicariance following the closing of biogeographical barriers. However, attendant-ant and host-plant shifts clearly played an important role in the diversification process, and in the maintenance of species integrity among lycaenid butterflies. Furthermore, exceptions to the predicted patterns of range overlap and ecological shifts provided clues to additional modes of diversification including shifts in habitat preferences and an unusual temporal shift following changes in specific host plant phenology resulting in allochronic diversification. Inferring modes of diversification using comparative methods based on range overlap and biological traits in a phylogenetic context is not new; however, the interpretation presented in this thesis is in contrast with contemporary methods. It is clear that the patterns of species range overlap and the ecological preferences of sister taxa are intimately related among lycaenid species that diversified during the Pleistocene. As a result, different influences on diversification can be highlighted in phylogenies when applying existing comparative methodologies but without necessarily drawing the same conclusions about modes of diversification. A more inclusive explanation for patterns of range overlap among sister taxa is detailed, a consequence of which is a method for estimating rates of extinction in a phylogeny where comprehensive distributional, biological and taxonomic data are available. These patterns and predictions may be applicable to a range of taxa, especially those that have diversified in the Pleistocene. Plans for future studies are outlined.

Discovery of a new host-parasite relationship, <i>Parelaphostrongylus odocoilei</i> in Dalls sheep (<i>Ovis dalli dalli</i>) in the Canadian North, prompted the first investigation of the geographic distribution, pathogenesis, ecology and epidemiology of this parasite, as well as the related protostrongylid <i>Protostrongylus stilesi</i>, at Subarctic latitudes (60-65ºN). All protostrongylid parasites have an indirect life-cycle, where first-stage larvae are shed in the feces of a mammalian definitive host, penetrate the foot of a gastropod intermediate host, and develop to infective third-stage larvae. <p> Protostrongylid larvae were recovered from over 2000 fecal samples from thinhorn sheep (<i>Ovis dalli</i>) and other hosts for <i>P. odocoilei</i> and <i>P. stilesi</i> across northwestern North America (38-69 ºN). Through novel application of molecular techniques to identify morphologically indistinguishable first-stage larvae, new records for <i>P. odocoilei</i> were established at 20 locations. This provided insight into the historical origins and biogeography of this new host-parasite relationship, and greatly expanded the known geographic range of both protostrongylids. <p> Clinical effects, including a neurological syndrome, were described in five thinhorn sheep experimentally infected with <i>P. odocoilei</i>. Neural and respiratory pathology in these five sheep were compared with over 50 wild Dalls sheep from a population naturally infected with <i>P. odocoilei</i> and <i>P. stilesi</i>. In the end stages, diffuse verminous interstitial pneumonia associated with P. odocoilei led to respiratory failure, and may have acted as a predisposing factor for bacterial pneumonia, which caused sporadic mortalities in this wild population. <p> At Subarctic latitudes, seasonal patterns in host and parasite availability, including larval shedding by Dalls sheep and larval development in experimentally infected gastropods, suggested that lambs become infected with <i>P. odocoilei</i> in a narrow seasonal window in their first fall on winter range. In combination with laboratory experiments, a degree day model for temperature-dependent larval development was developed, validated, and applied to describe and predict the effects of climate warming on protostrongylid parasites of thinhorn sheep in northern North America. In a future of climate warming, the narrow seasonal window for parasite development and transmission would be significantly extended, leading to amplification of populations of <i>P. odocoilei</i> and <i>P. stilesi</i> in endemic regions, and possibly range expansion of <i>P. odocoilei</i>. This may have consequences for the health of thinhorn sheep, as well as other wildlife that are important resources in the Canadian North.

Discovery of a new host-parasite relationship, <i>Parelaphostrongylus odocoilei</i> in Dalls sheep (<i>Ovis dalli dalli</i>) in the Canadian North, prompted the first investigation of the geographic distribution, pathogenesis, ecology and epidemiology of this parasite, as well as the related protostrongylid <i>Protostrongylus stilesi</i>, at Subarctic latitudes (60-65ºN). All protostrongylid parasites have an indirect life-cycle, where first-stage larvae are shed in the feces of a mammalian definitive host, penetrate the foot of a gastropod intermediate host, and develop to infective third-stage larvae. <p> Protostrongylid larvae were recovered from over 2000 fecal samples from thinhorn sheep (<i>Ovis dalli</i>) and other hosts for <i>P. odocoilei</i> and <i>P. stilesi</i> across northwestern North America (38-69 ºN). Through novel application of molecular techniques to identify morphologically indistinguishable first-stage larvae, new records for <i>P. odocoilei</i> were established at 20 locations. This provided insight into the historical origins and biogeography of this new host-parasite relationship, and greatly expanded the known geographic range of both protostrongylids. <p> Clinical effects, including a neurological syndrome, were described in five thinhorn sheep experimentally infected with <i>P. odocoilei</i>. Neural and respiratory pathology in these five sheep were compared with over 50 wild Dalls sheep from a population naturally infected with <i>P. odocoilei</i> and <i>P. stilesi</i>. In the end stages, diffuse verminous interstitial pneumonia associated with P. odocoilei led to respiratory failure, and may have acted as a predisposing factor for bacterial pneumonia, which caused sporadic mortalities in this wild population. <p> At Subarctic latitudes, seasonal patterns in host and parasite availability, including larval shedding by Dalls sheep and larval development in experimentally infected gastropods, suggested that lambs become infected with <i>P. odocoilei</i> in a narrow seasonal window in their first fall on winter range. In combination with laboratory experiments, a degree day model for temperature-dependent larval development was developed, validated, and applied to describe and predict the effects of climate warming on protostrongylid parasites of thinhorn sheep in northern North America. In a future of climate warming, the narrow seasonal window for parasite development and transmission would be significantly extended, leading to amplification of populations of <i>P. odocoilei</i> and <i>P. stilesi</i> in endemic regions, and possibly range expansion of <i>P. odocoilei</i>. This may have consequences for the health of thinhorn sheep, as well as other wildlife that are important resources in the Canadian North.

Regulation of transcription is an important molecular mechanism through which organisms can respond to environmental change. Environmentally-related transcriptional variation can play a significant role in evolution, potentially acting as a mechanism for the formation of new adaptive phenotypes. Organisms are most sensitive to the influence of external environmental variation during development, yet very few studies have explored environmentally-related transcriptional variation in early life history stages. Marine invertebrate metamorphosis, where intimate larval-environment interactions trigger settlement onto the benthos and a drastic morphological shift from larval to adult form, exemplifies the influence of environment on development. Variation in both the timing of metamorphosis and the form of specific metamorphic inductive cues, even at an intraspecific level, suggests that larvae have molecular mechanisms for surviving settlement and metamorphosis in a range of environmental conditions. The extent of transcriptional variation at metamorphosis remains largely unknown due to limited information on both the natural inductive cues and the molecular mechanisms directing metamorphosis in marine invertebrates. Contributing to current understanding of the interplay between genes, environment and phenotype during development, I explored molecular and ecological aspects of metamorphosis in a marine invertebrate, the tropical abalone Haliotis asinina. First, I employed cDNA microarray methods to identify candidate genes and document widespread transcriptional changes occurring in Haliotis asinina larvae during larval development and metamorphosis. Microarray results reveal that as the abalone veliger larva matures, it requires coordinated regulation of temporally different gene batteries involved in a wide range of physiological and developmental processes associated with the transition to a new, benthic habitat. All candidate genes showed changes in expression following exposure of larvae to coralline algae, an external inductive cue, demonstrating the remarkable effect of environment on transcription during marine invertebrate metamorphosis. 144 genes, ~40% of which are novel, were identified as candidates for a role in H. asinina metamorphosis. This high proportion of novel genes indicates that the conserved signaling pathways operating in marine invertebrate metamorphosis likely regulate the expression of taxon-specific genes. The relationship between abalone larvae and their natural inductive cue, coralline algae, is species-specific. To characterize the metamorphic cue preferences of Haliotis asinina larvae from Heron Island Reef, Australia, I documented larval induction response to a number of different coralline algae species commonly found in adult H. asinina habitat. H. asinina larvae exhibit highly specific responses to induction of metamorphosis by different coralline algae species, with 0 – 100% metamorphosis by 48 hours post induction depending on algae species. Unlike any other abalone species studied, the most effective inducers of Heron Island Reef H. asinina are articulated corallines of the genus Amphiroa. Comparing the response of different larval families to select species of coralline algae indicated that coralline algae community composition is likely to significantly impact H. asinina population structure. Additionally, I compared larval response to dead and live coralline algae to show that induction specificity is driven by chemical, not physical, properties. Characterization of the surface cell biomarkers of three different coralline algae species indicates that algal biomolecular composition relates to variations in H. asinina induction response. To explore the influence of variation in larval induction environment, I compared transcription patterns of 17 metamorphosis-related genes in Haliotis asinina larvae induced by three different species of coralline algae. H. asinina post-induction gene expression profiles vary according to the species of coralline algae inducer. This transcriptional variation occurs in genes with diverse functions and spatial expression patterns, highlighting the global nature of the impact of benthic microhabitat on gene expression. The environment-specific modulation of gene expression in H. asinina post-induction may be a means for marine invertebrates to cope with changes in their settlement environment at metamorphosis. Genes expressed in the larval sensory structures acting to detect external metamorphic cues may be particularly good candidates for studying environmentally-related transcriptional variation. I identified three novel genes expressed in putative sensory structures of Haliotis asinina larvae just prior to metamorphosis. The spatial and temporal expression patterns of these genes correlate with changes in larval ciliation patterns throughout metamorphosis, strongly suggestive of a role in metamorphic initiation. The three genes exhibit significant overlap in spatial expression profiles, indicative of genetic crosstalk between different sensory systems at metamorphosis. Transcriptional variation in gastropod sensory system genes may have assisted the evolution of different metamorphic inductive cues for different species. The results presented here establish an important role for transcriptional variation during marine invertebrate metamorphosis. Transcriptional variation underlies the morphological change from larval to adult body plan and also appears to assist larval recruitment in variable benthic habitats. Modulation of gene expression at metamorphosis in response to the environment may ultimately influence marine invertebrate species biogeography and evolution.

The principle aim of the current research was to modify the larval development assay (LDA) for use with Cooperia from cattle. A series of experiments were conducted in order to modify the LDA protocol to determine the most appropriate culture media and incubation temperature. These initial experiments concluded that, of the protocols examined, a culture medium of 1/8th the concentration of E. coli (EC) + 1/4th the concentration of yeast extract (YE) as generally used to culture sheep nematodes, at a culture temperature of 18ºC, resulted in the optimum number of Cooperia larvae developed to the third larval stage (L3). However, the number of eggs that developed to L3 was still generally low. A comparison was then made using isolates from a farm with a history of resistance in Cooperia to ivermectin (IV) and benzimidazoles (BZ) and two farms with a history of no resistance in this parasite. These experiments were undertaken using 1/8EC + 1/4YE media protocol and 1/2EC + 1/2YE concentration of the standard culture media for sheep nematodes. These three isolates were cultured at temperature of 18ºC and 25 ºC in the commercially available DrenchRite® 96-well microtitre assay plates which contained BZ, levamisole (LV) and IV in doubling dilutions within an agar matrix. The LD50 values were determined from a dose response curve. The resulting LD50 values were very variable, especially for the IV analogues. There was no obvious difference between the resistant and susceptible farms for the LD50 values of BZ or IV. A secondary aim of this research was to investigate the potential usefulness of the larval feeding inhibition assay (LFIA). This was adopted as published and it was determined it could be used to distinguish between susceptible and resistant Teladorsagia circumcincta with a resistance ratio of at least six. This research concluded that further research is required to fully optimise the LDA for Cooperia in cattle but adequate dose response curves were determined to indicate it struggles to distinguish BZ and IV resistance. The LFIA deserves to be further investigated as it offers some scope to detect ivermectin resistance in cattle nematodes as the dose response curves demonstrated a good repeatability for T. circumcincta from sheep. Comparing LDA and LFIA, both assays seemed to be useful but the latter was considered to have greater potential.

O carrapato bovino Boophilus microplus está presente em áreas tropicais e subtropicais no mundo e é um ectoparasito hematófago que causa inúmeras perdas à bovinocultura, através da espoliação ao bovino ou das doenças que transmite como vetor. Atualmente, o principal método de controle empregado baseia-se em produtos químicos, que são onerosos e contaminam o ambiente. Estudos a respeito da ecologia, comportamento e fisiologia de B. microplus são importantes para o desenvolvimento de novos métodos de controle do carrapato. A resposta ao estresse oxidadivo e a morte celular programada, examinadas neste estudo, permitem compreender melhor os mecanismos fisiológicos usados pelo carrapato adulto e seus ovos e larvas para sobreviver no ambiente. A resposta a estresse oxidativo foi analisada em ovos e larvas. A atividade de GST e outras moléculas envolvidas em mecanismos de proteção contra estresse oxidativo variaram dependendo do tempo transcorrido após a postura e eclosão. A cinética do consumo de oxigênio apresentou correlação positiva com o aumento na atividade de GST durante a embriogênese. Um alto conteúdo de substâncias reativas ao ácido tiobarbitúrico foi observado em extratos de ovos e larvas, indicando que carrapatos apresentam alto estresse oxidativo durante a embriogênese e desenvolvimento larval. Além disso, a atividade de GST apresentou forte correlação com peroxidação de lipídeos, uma indicação de seu papel na defesa antioxidante em ovos. Com o objetivo de melhor caracterizar o processo de morte celular, que elimina tecidos não mais necessários e leva à degeneração de glândulas salivares, ovários e singânglios, foi feita uma investigação usando ensaios de visualização de fragmentação de DNA em gel de agarose, teste cometa e TUNEL e a ativação da via apoptótica foi analisada usando ensaio de caspase. A fragmentação de DNA e a atividade enzimática de caspase-3 foram observadas em glândulas salivares e ovários 48 e 72 h após a remoção do carrapato do hospedeiro; em singânglios estes parâmetros foram mantidos em baixos níveis após 48 h. Estes resultados obtidos sugerem que há um controle refinado de manutenção de tecido através de apoptose. / The cattle tick Boophilus microplus is present in tropical and subtropical areas in the world and it is a haematophagous ectoparasite that causes several losses to cattle breeding, through the bovine exploiting and diseases transmitted. Currently, the main control method is based on chemicals, which are expensive and contaminate the environment. Studies about ecology, behavior and physiology of B. microplus are important to develop new methods for the tick control. The data examined in this study allow understanding the physiologic mechanisms used by the tick, eggs and larvae to survive in the environment. For this, the oxidative stress response in eggs and larvae and the cell death in different tissues of adult tick females during the pre-oviposition period were analyzed. The oxidative stress response was analyzed in eggs and larvae and the results showed that the GST activity and other molecules involved in mechanism of protection against oxidative stress varied depending on the time elapsed after oviposition and eclosion. A positive correlation was observed between the oxygen consumption kinetics and the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In addition, GST activity presented strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs. In order to better characterize the cell death process that eliminates unnecessary tissues, the degeneration of salivary glands, ovaries and synganglia was investigated using DNA fragmentation in agarose gel, comet and TUNEL assays, and apoptosis activation pathway by the caspase assay. DNA fragmentation and enzymatic activity of caspase-3 were observed in salivary glands and ovaries at 48 and 72 h after tick removal from the host; in synganglia these parameters were maintained at low levels upon 48 h. These results obtained suggest that there is a refined control of tissue maintenance through apoptosis.

O carrapato bovino Boophilus microplus está presente em áreas tropicais e subtropicais no mundo e é um ectoparasito hematófago que causa inúmeras perdas à bovinocultura, através da espoliação ao bovino ou das doenças que transmite como vetor. Atualmente, o principal método de controle empregado baseia-se em produtos químicos, que são onerosos e contaminam o ambiente. Estudos a respeito da ecologia, comportamento e fisiologia de B. microplus são importantes para o desenvolvimento de novos métodos de controle do carrapato. A resposta ao estresse oxidadivo e a morte celular programada, examinadas neste estudo, permitem compreender melhor os mecanismos fisiológicos usados pelo carrapato adulto e seus ovos e larvas para sobreviver no ambiente. A resposta a estresse oxidativo foi analisada em ovos e larvas. A atividade de GST e outras moléculas envolvidas em mecanismos de proteção contra estresse oxidativo variaram dependendo do tempo transcorrido após a postura e eclosão. A cinética do consumo de oxigênio apresentou correlação positiva com o aumento na atividade de GST durante a embriogênese. Um alto conteúdo de substâncias reativas ao ácido tiobarbitúrico foi observado em extratos de ovos e larvas, indicando que carrapatos apresentam alto estresse oxidativo durante a embriogênese e desenvolvimento larval. Além disso, a atividade de GST apresentou forte correlação com peroxidação de lipídeos, uma indicação de seu papel na defesa antioxidante em ovos. Com o objetivo de melhor caracterizar o processo de morte celular, que elimina tecidos não mais necessários e leva à degeneração de glândulas salivares, ovários e singânglios, foi feita uma investigação usando ensaios de visualização de fragmentação de DNA em gel de agarose, teste cometa e TUNEL e a ativação da via apoptótica foi analisada usando ensaio de caspase. A fragmentação de DNA e a atividade enzimática de caspase-3 foram observadas em glândulas salivares e ovários 48 e 72 h após a remoção do carrapato do hospedeiro; em singânglios estes parâmetros foram mantidos em baixos níveis após 48 h. Estes resultados obtidos sugerem que há um controle refinado de manutenção de tecido através de apoptose. / The cattle tick Boophilus microplus is present in tropical and subtropical areas in the world and it is a haematophagous ectoparasite that causes several losses to cattle breeding, through the bovine exploiting and diseases transmitted. Currently, the main control method is based on chemicals, which are expensive and contaminate the environment. Studies about ecology, behavior and physiology of B. microplus are important to develop new methods for the tick control. The data examined in this study allow understanding the physiologic mechanisms used by the tick, eggs and larvae to survive in the environment. For this, the oxidative stress response in eggs and larvae and the cell death in different tissues of adult tick females during the pre-oviposition period were analyzed. The oxidative stress response was analyzed in eggs and larvae and the results showed that the GST activity and other molecules involved in mechanism of protection against oxidative stress varied depending on the time elapsed after oviposition and eclosion. A positive correlation was observed between the oxygen consumption kinetics and the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In addition, GST activity presented strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs. In order to better characterize the cell death process that eliminates unnecessary tissues, the degeneration of salivary glands, ovaries and synganglia was investigated using DNA fragmentation in agarose gel, comet and TUNEL assays, and apoptosis activation pathway by the caspase assay. DNA fragmentation and enzymatic activity of caspase-3 were observed in salivary glands and ovaries at 48 and 72 h after tick removal from the host; in synganglia these parameters were maintained at low levels upon 48 h. These results obtained suggest that there is a refined control of tissue maintenance through apoptosis.

Entre os meses de junho e agosto 2006 dois surtos de intoxicação pelas larvas de P. flavipes ocorreram, um em ovinos (surto 1) e outro em bovinos (surto 2). O surto nos ovinos ocorreu nos meses de junho e julho no município de Encruzilhada do Sul RS e morreram 25 ovinos de um rebanho de 175 ovinos e 11 bovinos. Os ovinos eram mantidos em uma área de 40 hectares de pasto nativo. O surto nos bovinos ocorreu no mês de agosto no município de Sombrio SC e morreram 17 animais que estavam num lote de 77 bovinos, o rebanho total da propriedade era de 280 animais. O lote de bovinos estava em uma área de 90 hectares onde dois animais morreram devido à ingestão das larvas de P. flavipes. O lote foi transferido para outra área onde morreram mais 15 animais em um período de 5 dias. Seis ovinos do surto 1 e seis bovinos do surto 2 foram necropsiados. Grande quantidade de agrupamentos de larvas de P. flavipes foram encontradas no campo e no rúmen dos animais de ambos os surtos. Larvas de P. flavipes foram coletadas em ambos os surtos e foram armazenadas congeladas a -20º. Larvas frescas e larvas congeladas foram administradas para 6 ovinos, por meio de uma seringa com a ponta cortada. A menor dose letal foi de 7,5 g/kg em administração única. Animais que receberam doses sub-letais de 5 g/kg e posteriormente doses letais de 10 g/kg e 15 g/kg em intervalos de 15 e 30 dias, mostraram menor sensibilidade à intoxicação, um animal não adoeceu e outro adoeceu apenas depois de receber uma dose de 15 g/kg. Os animais intoxicados experimentalmente mostraram sinais de doença cerca de 48 horas e morreram cerca de 54 horas após a intoxicação. O exame bioquímico realizado em intervalos de 12 horas revelou alterações apenas nos animais que adoeceram. Os níveis séricos de GGT apresentavam-se elevados depois de 24 horas da intoxicação e continuaram a se elevar até a morte, AST apresentou aumento significativo cerca de 30 horas após a dosificação e posteriormente decaindo até a morte, os níveis séricos de glicose sofreram queda próximo à morte dos animais. As lesões de necropsia observadas nos casos espontâneos e experimentais foram semelhantes e mais consistentes no fígado, que se apresentava com acentuação do padrão lobular e com áreas de coloração amarelada com petéquias subcapsulares. Foram observados também edemas cavitários, edema da parede da vesícula biliar, perirrenal e na região inicial do duodeno, pâncreas e abomaso. Além das hemorragias no fígado havia hemorragias no tecido subcutâneo, coração, e mucosas e serosas da cavidade abdominal. O principal achado histopatológico era caracterizado por necrose coagulativa centrolobular ou massiva associada à hemorragia e congestão centrolobular e degeneração e tumefação hepática na região periportal. Observou-se ainda, depleção e necrose linfóide nos centros germinativos de linfonodos, nas placas de Peyer e na polpa branca do baço. A microscopia eletrônica demonstrou lesão hepática com hepatócitos necróticos, e dilatação dos espaços de Disse e endotélio vascular. Foi observada também intensa proliferação de reticulo endoplasmático liso no animal que recebeu doses graduais das larvas. As larvas descongeladas e dessecadas a 100ºC por 24 horas foram administradas a um coelho e mostraram-se tóxicas. Outros dois coelhos receberam frações sólidas e liquidas das larvas respectivamente e apenas o coelho que recebeu a fração sólida morreu. / Between June and August 2006, two outbreaks of P. flavipes larvae poisoning were observed. In outbreak 1 occurred in a farm located at the county of Encruzilhada do Sul, Rio Grande do Sul State, Brazil, 25 out of 175 sheep were affected and died. Although there were 11 cattle in the same paddock, none of them was affected. Animals were kept in a 40 hectares paddock of native pastures. In outbreak 2, occurred in a farm located at county of Sombrio, Santa Catarina State, 11 out of 77 cattle were affected and died. In total, 6 sheep and 6 cattle from respective outbreaks were necropsied. High numbers of compact masses containing up to 150 larvae were scattered on the paddocks in which animals were grazing. Larvae were collected and frozen at –20ºC. Perreyia flavipes larval body fragments and heads were found in the forestomach contents of all necropsied animals. Fresh and thawed larvae were administered to six sheep by a tip-cut plastic syringe. The lower single lethal dosis was 7,5 g/kg. Animals which received an initial sublethal doses of 5 g/kg and subsequently were dosed with lethal 10 to 15 g/kg at 15 and 30 days intervals showed lower susceptibility to the intoxication. Animals that were experimentally poisoned showed signs of disease about 48 h after dosing and died in approximately 54 h. Biochemical tests performed at 12 h intervals showed changes only in diseased animals. Serum levels of GGT started being higher 24 h after intoxication and kept enhancement until death. AST serum levels were significatively enhanced about 30 h after dosing and then, decreased to death. Glucose serum levels decreased close to the death. Necropsy lesions were similar in both, spontaneous and experimental cases and were most prominent in the livers, which had enhanced lobular pattern and yellowish areas with subcapsular pinpoint hemorrhages. Edema in body cavities, gallbladder wall, perirenal tissues, initial portion of duodenum, pancreas, and abomasum was also seen. Hemorrhages were also present in subcutaneous tissues, heart, and in mucosal and serosal membranes of the abdominal cavity. The principal histological finding was centrolobular to massive coagulative necrosis associated with centrolobular hemorrhage and congestion and periportal degeneration and tumefaction. There also were lymphoid depletion and necrosis in the germinative centres of lymph nodes, Peyer’s patches, and white pulp of the spleen. Transmission electron microscopy showed necrotic hepatocytes and dilatation in the space of Disse and vascular endothelium. Severe proliferation of smooth endoplasmic reticulum was also seen in animal receiving more than one dosis of larvae. Thawed larvae were desiccated at 100ºC for 24 h and administered to one rabbit that also became ill. Additional two rabbit were dosed with solid or liquid fractions of larvae and only which received the solid fraction died.