Thesis (M.Sc. (Microbiology)) -- Mahidol University, 1979.

This study is concerned with two strigeid genera which utilise fish as their second intermediate host and piscivorous birds as a definitive host, i.e. Apatemon (Apatemon) Sudarikov, 1959 and Ichthyocotylurus Odening, 1969. Although the lifecycle has been ascertained for most Ichthyocotylurus spp., confusion and disagreement still exist as to the constituent species, while all of the life-stages have been described for only a single member of the subgenus Apatemon (Apatemon). In order to clarify species membership to these taxa and indeed the taxonomic position of the subgenus Apatemon (Apatemon) further information was required on the life-cycles and life-stages of these strigeids. Although, metacercariae from this family have been recorded from a variety of British fishes, confirmed records, i.e. those supported with life-cycle data, are limited to a single species. It was this lack of confidence in identifying metacercariae recovered from fishes and the lack of known good criteria for distinguishing the adults that prompted the present study. Collections of metacercariae from a variety of hosts and locations were made, from which all subsequent life-cycle stages were obtained. The project aims were to establish the identity of the forms occurring in British fishes, by applying discriminatory techniques to the experimentally reared life-stages. In addition to traditional methods, techniques with little previous application to these genera were used and included, scanning electron microscopy (SEM), chaetotaxy, principal components analysis (PCA), and karyology. Furthermore, behavioural aspects such as the release patterns of cercariae from their molluscan hosts were studied to investigate whether they would prove to be of diagnostic value. Metacercariae obtained from the sampling survey were tentatively identified, using all currently employed methods for their determination, i.e. morphology, nature of cyst, host and site specificities, as Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825), Apatemon gracilis (Rudolphi, 1819) and A. annuligerum (Nordmann, 1832). Material collected from Finland was considered to contain both Ichthyocotylurus spp. recovered in the U.K., as well as I. platycephalus (Creplin, 1825) and I. pileatus (Rudolphi, 1802). The Ichthyocotylurus spp. were found to be more host specific than A. gracilis, although A. annuligerum was considered oioxenic to perch Perea fluiatilis L. Records of I. erraticus from gwyniad Coregonus lavaretus (L.) and grayling Thymallus thymallus (L.), and A. gracilis from arctic charr Salvelinus alpinus (L.) constitute first listings from Britain. The large number of sensilla present on the body surface of these metacercariae, observed by SEM and chaetotaxy, precluded their diagnostic use. PCA was, however, found to be of value for distinguishing between species and determining morphological variation within a species. I. erraticus, I. variegatus and A. gracilis adults were successfully reared in experimental hosts using metacercariae from a variety of fish hosts, sites within a single fish host and geographical sites. The adults obtained enabled clarification of the identities assigned to the metacercariae. Those metacercariae believed to represent I. pileatus and A. annuligerum failed to establish in experimental hosts. Herring gulls and lesser black-backed gulls proved to be extremely good experimental hosts for both Ichthyocotylurus spp., with the vast majority of infections establishing and providing high yields of eggs and adults. These infections yielded information on the establishment, development, fecundity, site specificity, longevity and morphological variability of the adults. Aspects of the morphology and biology of I. variegatus adults recorded were found to support its validity as a species discrete from I. platycephalus which was in some doubt. The experimental hosts used for A. gracilis infections, domestic and mallard ducklings, were found to be less satisfactory. Challenges were performed with A. gracilis metacercariae from three sources, rainbow trout, salmon parr and stone loach. The latter source was the only one to result in egg producing adults, with specimens exhibiting normal morphology and demonstrating an increased longevity over adults raised from salmonid metacercariae. These findings suggest that the metacercarial host may affect the successful completion of the life-cycle. Eggs of known origin were collected for all three cultured strigeid species, enabling further life-cycle studies, these were incubated and miracidia successfully raised. Developmental periods were found to be temperature dependent and differed for the three species at 20°C: A. gracilis < I. erraticus < I. variegatus. Light microscopy revealed the morphology of all three species to be identical, as were the epidermal plate formulae and chaetotaxy, indicated by silver-staining. The nomenclature for the distribution of miracidial sensilla derived by Dimitrov et al. (1989) was amended to enable a full description of these species. Osmotic shock resulted in an improved deciliation of the miracidia compared to sonication and subsequent SEM observation confirmed the arrangement of body surface structures, while revealing sensilla forms. Behavioural aspects of I. variegatus miracidia were examined, with a maximum longevity (< 11 hours) recorded at the lowest temperature studied (l0 degrees C), and host finding demonstrated to occur by an increased turning response in the presence of substances emitted from the susceptible snail host, following an initial unresponsive dispersal phase. Ichthyocotylurus cercariae were found in naturally infected Valvata piscinalis which constitutes the first record in Britain of cercariae of this genus. Cercariae of I. erraticus and I. variegatus were successfully raised experimentally from miracidia of known identity and origin within naive, experimentally raised V. piscinalis hosts, while A. gracilis cercariae were obtained from laboratory reared Lymnaea peregra. Cercarial developmental periods within the molluscan host were found to be temperature dependent and markedly different for the strigeid genera investigated, as were their behaviour and morphology. The Ichthyocotylurus spp. exhibit a distinct diurnal emergence rhythm from their molluscan host, being shed during the hours of daylight, while A. gracilis cercariae demonstrate a reciprocal pattern, emerging during the hours of darkness. Behavioural contrasts were also observed in longevities, emergence strategies (route of exit) and swimming behaviour. The two Ichthyocotylurus spp. were extremely similar, the only cercarial features found to be of diagnostic use were: the presence or absence of eye-spots; their differing developmental periods from miracidium to cercaria; the number and distribution of sensilla when compared by PCA; and their differing longevities at 20 degrees C. Characters considered to be of value in differentiating between strigeid cercariae at the species level, including the armature, chaetotaxy pattern and resting posture, did not differ between these two species. SEM observations enabled descriptions of the variety and structure of sensilla present on different life-stages, while transmission electron microscopy revealed the internal structure of cercarial sensory structures. Experimentally raised cercariae were found to be infective and the life-cycle was completed for the three strigeid species. Host specificities were observed for I. erraticus and A. gracilis, being particularly stringent for the latter species, while site specificities recorded were as observed in natural infections. Metacercarial maturation periods (for encystment) were highly temperature dependent, being comparable for the two Ichthyocotylurus spp. and more rapid than for A. gracilis specimens.

Thesis (Ph. D.)--University of Wisconsin, 1950. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [71-73]).

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph.D. (Tropical Medicine))--Mahidol University, 2000.

The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications. / Ph. D.

Eimeria maxima é uma das principais espécies que causam a coccidiose aviária. Visando uma maior compreensão dos mecanismos moleculares envolvidos na esporulação de oocistos de E. maxima, o perfil da expressão gênica em oocistos não esporulados, parcialmente esporulados e esporulados foi avaliado utilizando a técnica ORESTES. As 20.149 leituras geradas foram pré-processadas, agrupadas, anotadas empregando-se a plataforma Egene e em seguida, analisadas estatisticamente. Um total de 1.207 contigs e 2091 singlets foi gerado. Cerca de 72% dos transcritos foram classificados como proteínas hipotéticas, 20% eram similares a proteínas de função conhecida e 8% eram proteínas conservadas. Após a análise estatística, 32% dos transcritos foram classificados como diferencialmente expressos, dos quais, a maioria (65%) era estágio-específica. Para validação dos perfis de expressão obtidos por ORESTES, a expressão diferencial de um pequeno conjunto de genes foi quantificada por RT-qPCR. Os resultados demonstraram boa correlação entre as duas técnicas. / Eimeria maxima is one of the most important causing agents of poultry coccidiosis. Aiming at obtaining a better understanding of the molecular mechanisms involved in oocyst sporulation of E. maxima, the gene expression profiles of unsporulated, sporulating (sporoblast phase) and sporulated oocyst were studied using the ORESTES methodology. The 20,149 reads generated were pre-processed, grouped, annotated employing the platform Egene and statistically analyzed. A total of 1207 contigs and 2091 singlets were generated. About 72% of the transcripts were classified as hypothetical proteins, 20% were similar to proteins of known function and 8% were conserved proteins. After statistical analysis, 32% of the transcripts were classified as differentially expressed of which 65% were stage-specific. For ORESTES validation, the differential expression was quantified by RT-qPCR to a small set of genes. The results demonstrated a good correlation between the two techniques studied.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv., 2006. / Härtill 5 uppsatser.

Eimeria maxima é uma das principais espécies que causam a coccidiose aviária. Visando uma maior compreensão dos mecanismos moleculares envolvidos na esporulação de oocistos de E. maxima, o perfil da expressão gênica em oocistos não esporulados, parcialmente esporulados e esporulados foi avaliado utilizando a técnica ORESTES. As 20.149 leituras geradas foram pré-processadas, agrupadas, anotadas empregando-se a plataforma Egene e em seguida, analisadas estatisticamente. Um total de 1.207 contigs e 2091 singlets foi gerado. Cerca de 72% dos transcritos foram classificados como proteínas hipotéticas, 20% eram similares a proteínas de função conhecida e 8% eram proteínas conservadas. Após a análise estatística, 32% dos transcritos foram classificados como diferencialmente expressos, dos quais, a maioria (65%) era estágio-específica. Para validação dos perfis de expressão obtidos por ORESTES, a expressão diferencial de um pequeno conjunto de genes foi quantificada por RT-qPCR. Os resultados demonstraram boa correlação entre as duas técnicas. / Eimeria maxima is one of the most important causing agents of poultry coccidiosis. Aiming at obtaining a better understanding of the molecular mechanisms involved in oocyst sporulation of E. maxima, the gene expression profiles of unsporulated, sporulating (sporoblast phase) and sporulated oocyst were studied using the ORESTES methodology. The 20,149 reads generated were pre-processed, grouped, annotated employing the platform Egene and statistically analyzed. A total of 1207 contigs and 2091 singlets were generated. About 72% of the transcripts were classified as hypothetical proteins, 20% were similar to proteins of known function and 8% were conserved proteins. After statistical analysis, 32% of the transcripts were classified as differentially expressed of which 65% were stage-specific. For ORESTES validation, the differential expression was quantified by RT-qPCR to a small set of genes. The results demonstrated a good correlation between the two techniques studied.