Doctor of Philosophy / Department of Biology / Stephen Keith Chapes / Ehrlichia chaffeensis is an obligate, intracellular bacterium that causes human monocytic ehrlichiosis (HME). The bacteria are vectored by the Lone Star tick (Amblyomma americanum), which is found primarily in the Midwestern and Southeastern United States E. chaffeensis was first reported in 1986 and HME was designated a nationally reportable disease by the United States Centers for Disease Control in 1999. Ehrlichia grows in several mammalian cell lines, but NO consensus model for pathogenesis exists for arthropods or vertebrates. Moreover, the host genes required for intracellular growth of this bacteria are unknown. We first established that the bacteria could infect and replicate both in vitro and in vivo in Drosophila melanogaster S2 cells and adult flies, respectively. We performed microarrays on S2 cells, comparing host gene expression between permissive or non-permissive conditions for E. chaffeensis growth. A total of 210 permissive, exclusive and 83 non-permissive, exclusive genes were up-regulated greater than 1.5-fold above uninfected cells. We screened flies mutant for genes identified in our microarrays for their ability to support Ehrlichia replication. Five mutant stocks were resistant to infection with Ehrlichia (genes CG6479, separation anxiety, CG3044, CG6364, and CG6543). qRT-PCR confirmed that bacterial load was decreased in mutant flies compared to wild-type controls. In particular, gene CG6364 is predicted to have uridine kinase activity. Thus, the in vivo mutation of this gene putatively disrupts the nucleotide salvage pathway, causing a decrease in bacterial replication. To further test the function of gene CG6364 in bacterial replication, we obtained cyclopentenyl cytosine (CPEC) from the National Cancer Institute. CPEC is a cytidine triphosphate (CTP) inhibitor known to deplete CTP pools in various cancers and to exhibit antiviral activity. Consequently, it inhibits de novo nucleotide synthesis, but doesn’t affect the nucleotide salvage pathway. When S2 cells were treated with CPEC and infected with Ehrlichia, an increase in bacterial replication was confirmed by qRT-PCR. Furthermore, addition of cytosine to S2 cells also resulted in increased bacterial replication. Therefore the nucleotide salvage pathway through cytidine appears necessary for bacterial replication. Our approach has successfully identified host genes that contribute to the pathogenicity of E. chaffeensis in Drosophila.

Hemocytes are an important part of a crayfish’s immune system in helping tackling both virus and bacterial infections. Dscam is a protein that can be found in hemocytes, as well as many other tissues like the brain. In the brain, Dscam is thought to be important in the establishment of neuronal connections. Previous studies have found that the neurons in the crayfish brain do not replenish themselves, but instead are replenished by hemocytes that enter through a vascular cavity that pass through the neurogenic niche. There might be a specific type of hemocyte that is drawn to the niche and because of the link between Dscam and establishment of neuronal connections, Dscam have been chosen as a potential factor for this attraction. Dscam could be upregulated at many places along the way from the HPT to the brain. In this study, antibodies have been used to view BrdU and Dscam presence in hemocytes from crayfish P. leniusculus to find out where Dscam is upregulated and in what cells they are located. It was found that Dscam is not present on newly synthesized cells but rather on more differentiated cells, suggesting that Dscam is upregulated in older HPT cells or in circulation. It was found that LPS injections are an efficient way to upregulate Dscam in hemocytes and that expression of extracellular Dscam is peaking 24 hours post LPS injection.

Drosophila melanogaster has a tubular heart called the dorsal vessel, which is composed of contractile cardiomyocytes and hemolymph filtering pericardial cells. During larval development the dorsal vessel (heart) grows in size, and the luminal space inside the heart expands, however it has not been clear which cells are responsible for laying the extracellular matrix (ECM) during this expansion. Hemocytes (white blood cells), pericardial cells and cells of the fat body are candidate cell types that may secrete ECM for assembly during the growth of the heart lumen. With gene knock-down techniques we are exploring whether hemocytes participate in assembly of the heart ECM at this location. Additionally, studies of fluorescently tagged hemocytes in intact larvae reveal that hemocytes aggregate around pericardial cells of the dorsal vessel in 3rd instars. Confocal studies of dissected larval hearts indicate that hemocytes aggregate within infoldings of basement membrane associated with pericardial cells. Hemocyte-pericardial cell association could indicate that hemocytes take up proteins that are produced by pericardial cells and deliver them to other locations or that there might be a previously unidentified hematopoietic site at the Drosophila larval heart. / Thesis / Master of Science (MSc)

The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.

Endocrine disrupting chemicals (EDCs) are compounds that can interfere with hormone signaling pathways and are now recognized as pervasive in estuarine and marine waters. One prevalent EDC in California’s coastal waters is the xenoestrogen 4-nonylphenol (4-NP), which has been shown to impair reproduction, development, growth, and in some cases immune function of marine invertebrates. To further investigate effects of 4-NP on marine invertebrate immune function we measured total hemocyte counts (THC), relative transcript abundance of immune-relevant genes, and lysozyme activity in Pacific oysters (Crassostrea gigas) following bacterial infection. To quantify these effects we exposed oysters to dissolved phase 4-NP at high (100 μg l-1), low (2 μg l-1), or control (100 μl ethanol) concentrations for 7 days, and then experimentally infected (via injection into the adductor muscle) the oysters with the marine bacterium Vibrio campbellii. 4-NP significantly altered the effects of bacterial infection had on THC. Oysters exposed to both high and low 4-NP did not experience a bacteria-induced increase in THC, as seen in control oysters. We also determined that V. campbellii infection induced differential expression of a subset of immune-related genes tested (Cg-bigdef2, Cg-bpi1, Cg-lys1, Cg-timp) in some, but not all, tissues; 4-NP exposure altered expression patterns in two of these genes (Cg-bpi1 and Cg-tgase). Exposure to 4-NP alone also caused differential expression in some genes (Cg-bpi1, Cg-galectin1, Cg-clec2). Lastly, low levels of 4-NP significantly increased lysozyme activity 24 h post-infection. These results suggest that exposure to 4-NP can alter both cellular and humoral immune responses to bacterial infection in C. gigas.

No description available.

Le travail de thèse s’inscrit dans la problématique très actuelle des mortalités massives de naissain et de juvéniles d’huîtres creuses, C. gigas, et des questionnements autour de l’implication des pesticides dans ce phénomène. La première partie de la thèse a été consacrée à l’étude des effets de pesticides sur les capacités hémocytaires de l’huître creuse (in vitro et in vivo). L’effet immuno-modulateur des xénobiotiques sélectionnés (seul ou en mélange) a été exploré principalement au travers de la cytométrie en flux. La deuxième partie de la thèse a concerné l’étude des effets d’un mélange de pesticides sélectionnés sur le virus OsHV-1 et l’infection qu’il induit chez l’huître creuse. Les effets des pesticides sur le virus lui-même ont été évalués. Les expériences réalisées n'ont pas permis de mettre en évidence, dans les conditions testées, de dégradation des particules virales en présence des polluants. D’autre part, leurs effets sur les huîtres elles-mêmes ont été explorés après traitement des animaux avec un mélange de pesticides lors d’essais de pathologie expérimentale. Il a ainsi pu être montré que des huîtres préalablement contaminées par un mélange de pesticides à des concentrations retrouvées dans l’environnement semblaient être plus sensibles à l’infection virale. La troisième partie de la thèse a concerné l’étude de l’autophagie chez l’huître creuse, C. gigas. La publication du génome complet de cette espèce en 2012 a ouvert de nouvelles possibilités pour étudier l'immunité innée. L’étude de l’autophagie réalisée pour la première fois chez l’huître creuse a consisté lors d’une première étape en la recherche in silico de gènes impliqués dans cette voie et des protéines correspondantes par western blotting. Puis, le rôle de ce processus important dans l’immunité innée a été exploré au travers d’essais de reproduction d’infections en présence ou non de modulateurs de l’autophagie. Les résultats obtenus semblent montrer que l’autophagie soit un processus important pouvant être impliqué dans les capacités de défense de l’huître creuse vis-à-vis d’infections virales et bactériennes. / The thesis work is part of the very current issue on mass mortality outbreaks affecting Pacific oyster, C. gigas, spat and juveniles and questions about the involvement of pesticides in this phenomenon. The first part of this thesis was devoted to study the effects of pesticides on hemocyte parameters in the Pacific oyster (in vitro and in vivo). The immunomodulator effect of selected pesticides (alone or in mixture) has been explored principally through flow cytometry. The second part concerned the study of the effects of a pesticides mixture on OsHV-1 itself. No direct effects have been reported on the viral particles in presence of the pollutants. Moreover, pesticide effects on Pacific oysters were also explored through experimental pathology assays after treatment of animals with a polluant mixture. Results showed that pesticide treated oysters appeared more susceptible to the viral infection in experimental conditions. The third part concerned the study of autophagy in the Pacific oyster, C. gigas. The publication of the complete genome in 2012 has opened up new possibilities for the study of innate immunity in this species. The study of autophagy for the first time in oysters consisted in a first step in the in silico search for genes involved in this pathway and the corresponding proteins by Western blotting. Then, the role of this important process in innate immunity has been explored through reproduction infections tests with or without modulators of autophagy. Results showed that autophagy appeared as involved in defence mechanisms against viral and bacterial infection in Pacific oysters.

Doctor of Philosophy / Department of Entomology / Michael R. Kanost / James R. Nechols / Cellular encapsulation is the immune response in which insects protect themselves from multicellular parasites such as nematodes or parasitoids. During an encapsulation episode, certain insect hemocytes become attracted to a foreign invader and aggregate on its surface. In short order, the invading entity will become entrapped within a capsule comprised of thousands of hemocytes, thus rendering the parasite harmless to the insect host. Although the process of cellular encapsulation has been known for a great many years, very little knowledge yet exists regarding the biochemistry underlying capsule formation. It would seem likely that cell surface adhesion proteins mediate this immune response. In a series of in vivo encapsulation assays in the tobacco hornworm, Manduca sexta, a collection of anti-hemocyte monoclonal antibodies (mAbs) was screened for their ability to inhibit cellular encapsulation. Two of the mAbs that inhibited this immune response and incidentally specifically bind plasmatocytes, MS13 and MS34, were used to isolate a ≈ 90 kDa protein. Several short peptide sequences contained within this protein were acquired via Edman degradation. Degenerate primers based on two of these peptide sequences and total RNA from M. sexta hemocytes were used to perform RT-PCR and 5´ and 3´ RACE. This resulted in a full-length cDNA sequence of 2426 bp. A 2301 bp open reading frame within this cDNA sequence codes for a protein of 767 residues. This protein, denominated [Beta]Ms1, exhibits significant sequence homology to the [Beta]-subunits of integrins, which are a family of transmembrane, heterodimeric glycoproteins that possess adhesive properties. Analysis of recombinant segments of [Beta]Ms1 showed that the protein produced from the PCR product is the antigen to MS13 and MS34 and that these mAbs bind to the region of the integrin that contains the extracellular binding site. Northern blot analysis of various M. sexta tissues together with immunofluorescence labeling with MS13 and MS34 shows that [Beta]Ms1 is solely expressed in plasmatocytes. The totality of these experiments demonstrates that integrins are essential for the cellular immune response of encapsulation.

Molluscs as well as all other invertebrates rely on innate immune response only. Their internal defense system is capable of destroying most pathogens. However, there are some exceptions, e.g. some snails serve as intermediate hosts for some trematodes. Trematodes are able to develop inside these snails due to intervention in the snail internal defense system. The submitted thesis describes hemocyte activities of two lymnaeid snails, Lymnaea stagnalis a Radix lagotis, and the influence of Trichobilharzia regenti infection on R. lagotis hemocytes. Hemocytes of both species exposed to various chemicals produced different amounts of H2O2 and NO. The response varied between both lymnaeid species. The amount of circulating hemocytes was elevated in R. lagotis snails due to T. regenti infection. However, the infenction attenuated hemocyte activities monitored by us. Hemocyte basal NO production was decreased as well as phagocytosis of bacteria, cell adherence and pseudopodia formation. Toxicity of L. stagnalis plasma against T. regenti miracidia was also described. Mechanisms used by trematodes to interact with the snail internal defense system will help us to understand why one species is suitable for the develepment of the trematode whereas another closely related species kills it. Powered by TCPDF (www.tcpdf.org)

Made available in DSpace on 2014-12-17T14:10:18Z (GMT). No. of bitstreams: 1 LigiaGR.pdf: 427991 bytes, checksum: fd90b864e24611c634c5ec114b00d8f7 (MD5) Previous issue date: 2008-08-29 / The main problem faced by the shrimp industry are the infectious diseases. The hypodermal and hematopoietic necrosis infection (IHHN) is one of the major cause of disease in the cultured shrimp, Litopenaeus vannamei. Environmental changes involving water quality, oxygen concentration, salinity, temperature, stocking density, presence of pathogens, among others, triggering a stressing condition for the cultured shrimp, weakening them and allowing the outbreak of diseases. The stress on the animal leads to a change in the molecules immune response components, which can be used as indicators of shrimp health. Thus, the objective of the present study was to evaluate the effect of salinity, stocking density and IHHNV infection on the L. vannamei shrimp. The immune parameters used to check the shrimp health were the total hemocytes counts (THC), the agglutinating activity (AA) and the clotting time (CT) of the serum of shrimp. These parameters were analyzed in healthy and IHHNV-infected shrimp, grown in low (0-0.5 ), medium (19-24 ) and high (> 38 ) salinity, and extensive (7-12 cam.m-2), semi-intensive (15-25 cam.m-2) and intensive (33-45 cam.m -2) stocking density. The IHHNV infection rate was significantly higher in low salinity (P<0.005) and intensive density (P<0.005), both stressful conditions for L. vannamei. Low salinity significantly increased THC (P<0.05) and decreased and CT (P<0.05) in healthy and infected shrimp, but AA (P<0.05) significantly decreased in healthy shrimp at medium salinity. Culture intensification did not affect the THC, AA and CT of healthy and infected shrimp (P>0.05). The IHHNV infection did not affect any immune parameters of shrimp cultured at different salinities and stocking densities. It is necessary to emphasize that this study was conducted in shrimp grown in ponds, where several environmental factors are acting simultaneously. Thus, further studies are needed about the influence of other environmental factors on the immune parameters of shrimp cultured in pond / O principal problema enfrentado pela ind?stria do camar?o s?o as enfermidades de origem infecciosa. A Infec??o Hipodermal e Necrose Hematopoi?tica (IHHN) ? uma das principais causas de doen?as no camar?o de cultivo Litopenaeus vannamei. Altera??es do ambiente de cultivo envolvendo qualidade da ?gua, concentra??o de oxig?nio, salinidade, temperatura, densidade de estocagem, presen?a de pat?genos, entre outros, desencadeiam uma situa??o de estresse nos camar?es cultivados, debilitando-os e permitindo a instala??o de enfermidades. O estresse desencadeia no animal a altera??o de mol?culas componentes da resposta imune, que podem ser usadas como indicadores de sa?de do camar?o. Desta forma, o objetivo do presente trabalho foi avaliar o efeito da salinidade, densidade de estocagem e infec??o pelo IHHNV na imunidade do camar?o de cultivo L. vannamei. Os par?metros imunes utilizados para monitorar as condi??es de sa?de dos camar?es foram o n?mero total de hem?citos (THC), a atividade aglutinante (AA) e o tempo de coagula??o (TC) dos soros dos camar?es. Estes par?metros foram analisados em camar?es saud?veis e infectados pelo IHHNV, cultivados em baixa (0-0,5 ), m?dia (19-24 ) e alta (>38 ) salinidades e em extensiva (7-12 cam.m-2 ), semi-intensiva (15-25 cam.m-2) e intensiva (33-45 cam.m-2) densidades de estocagem. A taxa de infec??o pelo IHHNV foi significativamente maior em baixa salinidade (P<0,005) e na densidade intensiva (P<0,005), ambas condi??es estressantes para o L. vannamei. Baixa salinidade significativamente aumentou THC (P<0,05) e diminuiu TC (P<0,05) de camar?es saud?veis e infectados, mas AA (P<0,05) de camar?es saud?veis diminui significativamente na m?dia salinidade. A intensifica??o do cultivo n?o afetou THC, AA e TC de camar?es saud?veis e infectados (P>0,05). A infec??o pelo IHHNV n?o afetou nenhum dos par?metros imunes dos camar?es cultivados nas diferentes salinidades e densidades de estocagem. ? necess?rio enfatizar que o presente estudo foi realizado em camar?es cultivados em viveiros, onde v?rios fatores ambientais est?o atuando simultaneamente. Desta forma, s?o necess?rios estudos adicionais sobre a influ?ncia de outros fatores ambientais nos par?metros imunes de camar?es nas condi??es de cultivo