Dissertations / Theses on the topic 'Invertebrate immunity'

Invertebrates show enhanced immunity and even specific primed immunity in response to repeat infections, analogous to vertebrate adaptive immunity. Little is known of the mechanism for this phenomenon, or which molecules are involved. A candidate gene for the underlying mechanism for a pathogen-specific response in invertebrate immunity is Down syndrome cell adhesion molecule (Dscam). Dscam can produce thousands of different protein isoforms through the mutually exclusive splicing of many exon variants contained within variable regions of the gene. It is an important receptor of the invertebrate nervous system but has been implicated in having a role in immunity. Dscam has been shown to be involved in phagocytosis across members of the Pancrustacea, and it has been reported to respond in a pathogen-specific manner in mosquitoes and crayfish. In this thesis, I have investigated the splicing of Dscam in response to diverse pathogens in different host species. In the Anopheles mosquito, I cloned and sequenced a fragment of Dscam spanning across two of its variable exon regions to enable me to detect mutually exclusively splice variants and their associations in different treatments (Chapter 2). I discovered that the expression diversity of the hypervariable Dscam is higher in parasite-exposed mosquitoes. In Chapter 3, I extended the study to the more experimentally amenable Drosophila fruit fly. A new Illumina-based sequencing assay was developed and implemented to examine more closely Dscam expression in response to diverse pathogens. The new method successfully quantified non-random expression of Dscam variable exons 4 and 6. I also describe a small but detectable effect of pathogen-exposure on the expression of Dscam exon 4 variants. In Chapter 4, I expanded the work of Chapter 3 to study tissue-specific Dscam expression in response to well-characterised immune elicitors of Drosophila. I describe how exon 4 variants were expressed in a tissue-specific manner, but not exon 6 variants. I also found a small exon 4-by-tissue-by-pathogen effect, which although detectable, did not dominate over the tissue effects. Finally, in Chapter 5, I turned to the crustacean, Daphnia, to study Dscam expression in a natural host-parasite interaction and in a clonal organism. I describe the non-random expression of exons 4 and 6, and another small effect of pathogen-exposure on the expression of Dscam exon 4. My work aimed to further investigate the putative pathogen-specific alternative splicing of the hypervariable Dscam receptor. The data presented quantified the constitutive expression of Dscam exons 4 and 6 in different pancrustacean species. The data also suggest that infection-responsive splicing of Dscam may occur but that effects are small, and may be diluted within the background of the highly important Dscam expression of the nervous system if they exist at all. The study supports the high-throughput sequencing method for future studies of alternative splicing and Dscam expression.

The innate immune system is a critical first line of defense against invading pathogens. Innate immunity directly detects pathogens, sets up an appropriate adaptive response, and can directly kill pathogens. Drosophila may lack an adaptive immune response, but have a robust innate immune system with a variety of defense effector mechanisms. While the responses to bacteria, fungi, and RNA viruses have been well characterized, not much is known about the response to DNA viruses. My studies have set out to characterize the Drosophila immune response to a DNA virus, utilizing the large dsDNA virus, Invertebrate Iridescent Virus 6 (IIV-6). IIV-6 infection causes shortened lifespan, and in later stages of infection, flies present with abdominal swelling and iridescent blue color. Our objectives were to identify pathways flies use to protect themselves from IIV-6 infection, determine how this protection is mediated, and to identify any immune inhibitors that IIV-6 uses to suppress innate immune signaling. I have found that IIV-6 strongly up-regulates a class of stress proteins with unknown function, termed Turandots, after infection in vivo or in vitro. This induction is dependent upon viral replication, requires JAK-STAT activation, and activation of p38b MAPK. In addition, the unpaireds, which function as JAK-STAT ligands, are upregulated after IIV-6 infection in a p38b-dependent manner. Together, this data suggests that p38b activation leads to production of unpaired cytokines and activation of JAK-STAT signaling to induce Turandots. I have also found that IIV-6 infected cells secrete protective factors. This response is induced within 12 hours of IIV-6 infection, exosome-mediated, and provides robust protection to naive cells challenged with an mCherry-expressing strain of IIV-6. Additionally, IIV-6 inhibits two major immune responses in Drosophila, the IMD and Toll pathways. Stimulation of IIV-6 infected Drosophila S2* cells with either IMD or Toll stimulators results in very poor antimicrobial peptide responses. Yet, IMD and Relish are still cleaved upon stimulation in IIV-6 infected cells, indicating that the block is downstream. In support of this finding, IIV-6 infected flies respond very poorly to infection with the enterobacteria Erwinia carotovora carotovora compared to mock-injected flies.

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.

Les populations de bivalves exploités subissent régulièrement des épizooties qui affaiblissent voire déciment les stocks, et qui peuvent avoir des conséquences majeures pour l’aquaculture. Ces maladies, dues à des virus, bactéries, ou parasites, se développent particulièrement au printemps et en été. Ces périodes de l’année offrent également des conditions propices aux efflorescences de micro-algues toxiques, dont des dinoflagellés du genre Alexandrium. Ainsi, le risque de co-occurrence d’efflorescences d’Alexandrium sp. et de maladies infectieuses chez les bivalves est élevé. Or, ces micro-algues synthétisent et excrètent des neurotoxines et des composés cytotoxiques responsables d’altérations physiologiques chez les bivalves. L’objectif de cette thèse est d’évaluer les effets combinés d’une exposition à Alexandrium sp. et d’une infection par des agents pathogènes sur la physiologie des bivalves, à travers l’étude de différentes interactions tripartites bivalve – pathogène – Alexandrium sp. Les résultats de ce travail indiquent que différents profils de réponse existent en fonction des espèces impliquées dans ces interactions. Ainsi, une exposition à Alexandrium sp. peut augmenter le taux d’infection par des agents pathogènes chez des bivalves ou au contraire le diminuer. Les réponses hémocytaires associées peuvent traduire l’implication des défenses immunitaires dans ces modulations hôte-pathogène. De plus, l’exposition à des agents pathogènes peut interférer avec le processus d’accumulation de toxines algales dans les tissus des bivalves, illustrant la complexité de ces interactions. Ces résultats, associés à l’observation de lésions tissulaires chez les bivalves peuvent traduire l’altération des activités de nutrition (filtration, digestion…). Ce travail de thèse apporte une meilleure compréhension de l’implication des efflorescences toxiques dans le développement des maladies touchant les bivalves d’intérêt commercial, mais également de l’implication de l’environnement biotique des bivalves sur l’accumulation de phycotoxines réglementées
Bivalve populations undergo regular epidemics that weaken or decimate exploited stocks and thus limit aquaculture. These diseases are caused mainly by viruses, bacteria or parasites, and occur primarily during spring and summer. This period of the year also provides favorable conditions for toxic dinoflagellate blooms, including species of the genus Alexandrium. Thus, the risk of Alexandrium sp. blooms and infectious diseases co-occurring in bivalves is high. However, these micro-algae synthesize and excrete toxins and cytotoxic compounds responsible for physiological changes in bivalves and could lead to an immuno-compromised status.The objective of this thesis is to evaluate the combined effects on bivalve physiology of exposure to the toxic dinoflagellate, Alexandrium sp., and infection by pathogens, through the study of different bivalve - pathogen - Alexandrium sp. tripartite interactions. The results of this work highlight the species-specific nature of these impacts.Thus, exposure to Alexandrium catenella reduces the herpesviruses infection in oyster Crassostrea gigas, whereas the dinoflagellate A. fundyense increases the susceptibility of C. virginica oyster to the parasite Perkinsus marinus, probably via immuno-suppression, as suggested by the partial inhibition of hemocyte responses. Additionally, the effect of a toxic algal bloom on oyster susceptibility to opportunistic diseases when exposed to a new microbial environment (simulating a transfer) was evaluated. Hemocyte responses to a changing microbial environment were suppressed by exposure to A. catenella, although no new bacterial infection was detected.Finally, exposure to pathogens or to a new microbial environment interferes with the processes by which oysters exposed to A. catenella accumulate algal toxins, illustrating the complexity of these interactions. These results provide a better understanding of the involvement of toxic algal blooms in the development of diseases affecting commercial bivalve species, but also of the involvement of the bivalve biotic environment in the accumulation of regulated toxins

Les LBP/BPIs sont des protéines importantes de la réponse antimicrobienne des mammifères, encore mal caractérisées chez les invertébrés. L'objectif de ce projet était d'élucider le rôle des LBP/BPIs dans la réponse immunitaire du gastéropode B. glabrata. Nous avons montré que l'une des LBP/BPI (BgLBP/BPI1) était une protéine majeure de la glande albumène et des masses d'oeufs et possédait les activités attendues des BPIs telle que la capacité de perméabilisation des bactéries.De plus, nous avons découvert une nouvelle activité biocide (anti-oomycète) inconnue jusqu'alors chez les LBP/BPI, et démontré que BgLBP/BPI1 influence à la fois la production d'oeufs et leur protection contre les infections à oomycètes. Nous avons ensuite examiné la diversité et l'évolution des BgLBP/BPIs et montré qu'au moins 5 LBP/BPIs, regroupées en 3 clades phylogénétiques, étaient exprimées chez B. glabrata. Une étude de l'expression de représentants de ces 3 clades a montré qu'ils étaient exprimés dans différents tissus, appuyant l'hypothèse de l'acquisition de spécificités fonctionnelles par les membres de cette famille multigénique
LBP/BPIs are important immune factors of the mammalian antimicrobial response,poorly characterized in invertebrates. The aim of this work was to elucidate the role of LBP/BPIs in the immune response of the fresh-water snail B. glabrata. Firstly, we showed that one member, BgLBP/BPI1, was highly abundant in the albumen gland and the egg masses. Importantly, in addition to the expected activities of BPIs, such as the induction of bacterial permeability, we discovered a novel biocidal (antioomycete) activity that was unsuspected so far. We demonstrated that BgLBP/BPI1 is a major fitness-related protein, acting on both egg production and offspring protection against oomycete infections. Then, we investigated the sequence diversity and evolution of this LBP/BPI protein family and showed that at least 5 LBP/BPIs were expressed in B. glabrata, belonging to three distinct phylogenetic clades. Expression studies of representatives of the three clades showed that they are expressed in different tissues, differently regulated, and therefore supported the hypothesis of the acquisition of functional specificities by the members of this multigenic family

Ce travail a contribué à la compréhension des bases moléculaires de l'immunité de l'huître creuse par la caractérisation la diversité de trois effecteurs antimicrobiens de C. gigas et par l'appréhension du rôle de cette diversité dans les mécanismes de défense. Des analyses phylogénétiques de deux peptides antimicrobiens (AMPs), Cg-Défensines (Cg-Defs) et Cg-Proline rich peptide (Cg-Prp), et d'une protéine de type Bactericidal Permeability Increasing protein, Cg-BPI, nous a permis montrer la grande diversité pour les 2 AMPs, qui est générée par plusieurs mécanismes génétiques et par des pressions de sélection directionnelles, suggèrant une diversité fonctionnelle des variants. L'importance biologique de cette diversité a été étudiée pour trois variants de Cg-Defs. Une forte activité antimicrobienne a été mise en évidence contre les bactéries à Gram positive, mais celle-ci diffère selon les variants. De plus, nous avons démontré que le mécanisme d'action des Cg-Defs contre S. aureus repose sur l'inhibition de la biosynthèse du peptidoglycane par le piegeage de son précurseur, le lipide II. Finalement, l'expression des transcrits et la localisation de ces effecteurs en réponse à une infection par un Vibrio pathogène ont montré un réseau complexe des profils d'expression des différents antimicrobiens, au niveau des populations hémocytaires et des tissus d'huître, suggérant une interaction entre les antimicrobiens du fait de leur colocalization. La combinaison entre les familles ou entre les variants d'une même famille produit de fortes activités synergiques qui élargissent les spectres d'activité. Ainsi, la diversité produit par la coévolution entre hôte et pathogènes pourrait améliorer l'activité des AMPs d'huître, lui conférant une plus grande protection contre les pathogènes de son environnement
This work contributed to the knowledge of the molecular bases of oyster immunity by the characterization of the diversity of three antimicrobials of C. gigas and the understanding of the role played by their diversity in the oyster antimicrobial response. Phylogenetic analyses of two antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline rich peptide (Cg-Prp), and one Bactericidal Permeability Increasing protein, Cg-BPI, led us to the identification of a high diversity for both AMPs. Further analyses showed that this diversity is generated by gene duplication, allelic recombination and directional selection pressures, suggesting their functional diversification. The biological meaning of AMP diversity was investigated for the three major variants of Cg-Defs, which revealed a strong but variable potency against Gram-positive bacteria. We evidenced that oyster defensins kill S. aureus through binding to the cell wall precursor lipid II, resulting in the inhibition of peptidoglycan biosynthesis. Finally, transcript expression and localization of oyster antimicrobials after a pathogenic infection evidenced a complex network in their expression profiles in hemocyte populations and oyster tissues, suggesting a potential interplay between antimicrobials as a result of their colocalization. Indeed, the combination of oyster antimicrobials produced strong synergistic activities that enlarged their antimicrobial spectra. Thus, the diversity of oyster antimicrobials may provide significant means in acquiring functional divergence, probably concerned in the evolutionary arms race between hosts and their pathogens.From our data, it would provide oysters with a higher protection against the potential pathogens from their environment

The host-parasite relationship of the hookworm Ancylostoma ceylanicum was explored in a hamster model system, focusing on intestinal mucosal responses to infection. Primary infection induced a rapid reduction in villous height culminating in excess of 75% reduction by day 35. Crypts of Lieberkuhn increased in depth achieving maximum depth by day 35. Mitotic figures in crypts and mast cells increased until day 28. Goblet cells increased continuously from background levels of 50 cell/mm² to exceed 300 cells/mm² by day 42. Paneth cell numbers declined in infected animals. Termination of infection by anthelmintic restored background values of intestinal architecture and goblet cell numbers within 7 days, but mast cells took longer and Paneth cell numbers increased beyond values in naïve controls. Mucosal changes are therefore dependent on the presence of worms, intensity of infection and change dramatically with time. Mucosal changes were studied in hamsters experiencing secondary infections following anthelmintic abbreviation of the immunizing infection, superimposed challenge infection and trickle infections. The kinetics of the responses were compared to animals experiencing primary infections and naive controls. Among the findings were: 1) continuous reduction in villous height and a marked increase in crypt depth from day 10 after challenge in abbreviated primary-challenged hamsters compared to little change in hamsters given superimposed challenge. 2) marked mast, goblet, and Paneth cell and eosinophil responses. 3) less intense mast cell responses in abbreviated primary-challenged compared to superimposed challenge animals 4) after a superimposed challenge poor goblet cell responses because levels were already high at the time of challenge, little change in Paneth cells but intense eosinophil responses 5) slower changes in mucosal architecture and mast cell responses in trickle-infected animals eventually exceeding those in primary infected animals. 6) less marked goblet and Paneth cell responses in trickle-infected groups but more intense, persistent increases in eosinophils Cyclosporin A's (CsA) usefulness as an immunosuppressive therapy for blocking T cell control of immunity was explored. However, CsA turned out to have marked anthelmintic properties and reductions in worm burden confounded the interpretation of mucosal changes.

Les abeilles sont confrontées à la menace mondiale du syndrome d'effondrement des colonies (CCD), entraînant des décès de colonies et une diminution de leur nombre, affectant leur contribution environnementale et agronomique dans la pollinisation des plantes et des cultures commerciales, en plus de la production de miel. L'exposition aux pesticides peut être l'une des principales causes conduisant au CCD en affaiblissant le système immunitaire des abeilles et en altérant leurs réponses immunitaires. Les maladies de la nosémose causées par Nosema spp. peuvent avoir une contribution significative au CCD lorsque les abeilles sont exposées à différents pesticides simultanément. Dans cette étude, plusieurs facteurs de risque sont évalués, y compris les néonicotinoïdes les plus utilisés dans le monde, l'imidaclopride et l'amitraz qui est le pesticide utilisé directement en contact avec les abeilles pour traiter l'infection par les acariens. L'effet de ces pesticides est évalué au niveau de la stimulation immunitaire par zymosan A pour imiter l'infection par Nosema. L'effet des pesticides sur les produits cellulaires antimicrobiens, les réponses cellulaires et l'expression de gènes connexes est démontré
Honeybee are facing the global threat of colony collapse disorder (CCD) leading colony deaths and decline in their numbers affecting their environmental and agronomic contribution in pollination of plants and commercial crops in addition to honey production. Pesticide exposure may be of the main causes leading to CCD by weakening the immune system of honeybees and impairing their immune responses. Nosemosis diseases caused by Nosema spp. may have a significant contribution to CCD when bees are exposed to different pesticides simultaneously. Multiple risk factors are assessed in this study including the most used neonicotinoids worldwide, imidacloprid and amitraz which is the pesticide used directly in contact with honeybees to treat mite infection. Th effect of these pesticides is evaluated at the level of immune stimulation by zymosan A to mimic Nosema infection. The effect of pesticides on antimicrobial cells products, cellular responses and related genes' expression are demonstrated

Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-22Bitstream added on 2014-06-13T18:09:00Z : No. of bitstreams: 1 perez_dg_me_rcla.pdf: 821493 bytes, checksum: 938fad7997660774f8c919574b04e6e2 (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Embora os invertebrados sejam conhecidos pela grande facilidade de acúmulo de poluentes presentes em seu ambiente, e muitos serem utilizados como espécies sentinelas em estudos de biomonitoramento, pouco ainda é conhecido sobre o impacto de toxicantes sobre o sistema imune desses animais. Nesse sentido, os hemócitos desempenham um papel fundamental: estas células circulam livremente através da hemolinfa dos invertebrados e atuam no reconhecimento de materiais estranhos ao organismo, mediando e efetuando reações de defesa celular. Diferentes tipos morfológicos podem ser reconhecidos, mas ainda há controvérsia entre os pesquisadores sobre a exata classificação dos hemócitos, devido à diversidade de técnicas para preservação e observação dessas células. A classificação mais aceita atualmente agrupa os hemócitos em sete tipos principais: pró-hemócitos, plasmatócitos, granulócitos, esferulócitos, adipohemócitos, oenocitóides e coagulócitos. Por meio da utilização de técnicas histológica, histoquímica e ultra-estrutural, o presente trabalho objetivou caracterizar morfofisiologicamente os hemócitos circulantes na hemolinfa do diplópodo Rhinocricus padbergi, bem como aqueles encontrados por entre as células da camada de corpo gorduroso no intestino médio de animais expostos a substratos contendo diferentes amostras de lodo de esgoto, resíduo gerado nas Estações de Tratamento de Esgoto (ETEs). Este resíduo tem sido cogitado como um bom condicionador de solo em áreas degradadas e como um potencial fertilizante agrícola, apesar do risco de estar contaminado com patógenos e/ou metais pesados. A partir das análises realizadas, foram identificados três tipos morfológicos distintos de hemócitos circulantes na hemolinfa dessa espécie: pró-hemócitos, plasmatócitos e granulócitos (subtipos I e II). Também foram observadas células com características...
Although invertebrates are known for ease accumulation of pollutants present in their environment and several are used as sentinel species in biomonitoring studies, little is known about the impact of toxicants on the immune system of these animals. In this sense, hemocytes play an important role: these cells circulate freely through the hemolymph of invertebrates and act in the recognition of foreign materials to the organism, mediating and performing cellular defence reactions. Different morphological types are recognized, but there is still controversy among the researchers about the exact classification of the hemocytes due to the diversity of techniques for preservation and observation of these cells. Currently, the most accepted classification groups the hemocytes into seven main types: pro-hemocytes, plasmatocytes, granulocytes, spherulocytes, adipohemocytes, oenocytoids and coagulocytes. By histological, histochemical and ultra-structural techniques, the present study aimed to characterize morpho-physiologically the hemocytes circulating in the hemolymph of the diplopod Rhinocricus padbergi, as well as those found among the cells of the fat body layer of the midgut of animals exposed to substrates containing samples of sewage sludge, residue generated in the Sewage Treatment Plants (STPs). This residue has been considered as a good soil conditioner on degraded areas and as a potential agricultural fertilizer, despite the risk of being contaminated with pathogens and/or heavy metals. From the analyses carried out, it was identified three distinct morphological types of hemocytes circulating in the hemolymph of this species: pro-hemocytes, plasmatocytes and granulocytes (subtypes I and II). It was also observed cells with intermediate characteristics between pro-hemocytes and plasmatocytes, suggesting a probable cellular differentiation in the hemolymph... (Complete abstract click electronic access below)

Orientador: Carmem Silvia Fontanetti Christofoletti
Banca: Elaine Cristina Mathias da Silva Zacarin
Banca: Carminda da Cruz Landim
Resumo: Embora os invertebrados sejam conhecidos pela grande facilidade de acúmulo de poluentes presentes em seu ambiente, e muitos serem utilizados como espécies sentinelas em estudos de biomonitoramento, pouco ainda é conhecido sobre o impacto de toxicantes sobre o sistema imune desses animais. Nesse sentido, os hemócitos desempenham um papel fundamental: estas células circulam livremente através da hemolinfa dos invertebrados e atuam no reconhecimento de materiais estranhos ao organismo, mediando e efetuando reações de defesa celular. Diferentes tipos morfológicos podem ser reconhecidos, mas ainda há controvérsia entre os pesquisadores sobre a exata classificação dos hemócitos, devido à diversidade de técnicas para preservação e observação dessas células. A classificação mais aceita atualmente agrupa os hemócitos em sete tipos principais: pró-hemócitos, plasmatócitos, granulócitos, esferulócitos, adipohemócitos, oenocitóides e coagulócitos. Por meio da utilização de técnicas histológica, histoquímica e ultra-estrutural, o presente trabalho objetivou caracterizar morfofisiologicamente os hemócitos circulantes na hemolinfa do diplópodo Rhinocricus padbergi, bem como aqueles encontrados por entre as células da camada de corpo gorduroso no intestino médio de animais expostos a substratos contendo diferentes amostras de lodo de esgoto, resíduo gerado nas Estações de Tratamento de Esgoto (ETEs). Este resíduo tem sido cogitado como um bom condicionador de solo em áreas degradadas e como um potencial fertilizante agrícola, apesar do risco de estar contaminado com patógenos e/ou metais pesados. A partir das análises realizadas, foram identificados três tipos morfológicos distintos de hemócitos circulantes na hemolinfa dessa espécie: pró-hemócitos, plasmatócitos e granulócitos (subtipos I e II). Também foram observadas células com características... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Although invertebrates are known for ease accumulation of pollutants present in their environment and several are used as sentinel species in biomonitoring studies, little is known about the impact of toxicants on the immune system of these animals. In this sense, hemocytes play an important role: these cells circulate freely through the hemolymph of invertebrates and act in the recognition of foreign materials to the organism, mediating and performing cellular defence reactions. Different morphological types are recognized, but there is still controversy among the researchers about the exact classification of the hemocytes due to the diversity of techniques for preservation and observation of these cells. Currently, the most accepted classification groups the hemocytes into seven main types: pro-hemocytes, plasmatocytes, granulocytes, spherulocytes, adipohemocytes, oenocytoids and coagulocytes. By histological, histochemical and ultra-structural techniques, the present study aimed to characterize morpho-physiologically the hemocytes circulating in the hemolymph of the diplopod Rhinocricus padbergi, as well as those found among the cells of the fat body layer of the midgut of animals exposed to substrates containing samples of sewage sludge, residue generated in the Sewage Treatment Plants (STPs). This residue has been considered as a good soil conditioner on degraded areas and as a potential agricultural fertilizer, despite the risk of being contaminated with pathogens and/or heavy metals. From the analyses carried out, it was identified three distinct morphological types of hemocytes circulating in the hemolymph of this species: pro-hemocytes, plasmatocytes and granulocytes (subtypes I and II). It was also observed cells with intermediate characteristics between pro-hemocytes and plasmatocytes, suggesting a probable cellular differentiation in the hemolymph... (Complete abstract click electronic access below)
Mestre

Isolement, à partir de l'hémolymphe immune de phormia terrae-novae, deux nouveaux peptides sélectivement actifs contre les bactéries gram positives. Ces molécules sont chargées positivement et comportent 40 résidus avec 3 ponts disulfures. Le nom de phormicine est proposé pour ces nouveaux peptides antibiotiques d'insectes.

Etude des antigenes de surface de nippostrongylus brasiliensis susceptibles d'induire une reaction immunitaire. L'immunisation de souris par ces antigenes induit une immunite protectrice. Deux proteines p43, p14 sont plus particulierement impliquees dans l'immunisation. Un protocole de vaccination identique a ete transpose avec succes chez la souris avec des antigenes de eimeria falciformis

With the progress in sequencing of the honey bee genome new data become available which allows the search and identification of genes coding for homologous proteins found in other organism. Two genes coding for c-type lysozymes were identified in the genome of A. mellifera through an online-based BLAST search. Expression of both intron-less genes seems not to be under the regulatory control of either of the two pathways involved in humoral insect immunity, i.e. Toll and Imd, since no NF-κB transcription factor binding sites are found upstream of the genes. The encoded Lys-1 and Lys-2 are 157 and 143 amino acid long, respectively, and share a sequence similarity of 90%. Further in silico analysis revealed a signal peptidase cleavage site at the N-terminus of each amino acid sequence, strongly suggesting a secretion of the enzymes into the surrounding environment of the producing cells. Sequence alignments of both amino acid sequences with other c-type lysozymes identified the highly conserved active site glutamic acid (Glu32) as well as eight highly conserved cysteine residues. However, an important aspartic acid (Asp50) in the active site that helps to stabilize a substrate intermediate during catalysis is replaced by a serine residue in the lysozymes of A. mellifera. The replacement of the active site aspartic acid in the honey bee lysozymes suggests a different catalytic mechanism and/or a different substrate-specificity in respect to other c-type lysozymes. Furthermore, 3D-models of Lys-1 and Lys-2 were generated based on the sequence similarity of A. mellifera lysozymes with other c-type lysozymes. The published 3D structure of the lysozyme from the silkmoth Bombyx mori, which shares the highest sequence similarity of all available structures with A. mellifera lysozymes, was used as template for the construction of the 3D-models. The models of Lys-1 and Lys-2 suggest that both enzymes resemble, in large part, the structure of B. mori lysozyme. In order to identify the set of AMPs in the hemolymph of A. mellifera, hemolymph of immunized bees was analyzed. Applying SDS-polyacrylamide gel electrophoresis and mass spectrometry on hemolymph from immunized bees, three out of the four peptides were identified, i.e. abaecin, defensin 1 and hymenoptaecin. Furthermore, Lys-2 was identified in the hemolymph by mass spectrometry, conclusively demonstrating the presence of a lysozyme in the hemolymph of A. mellifera for the first time. However, the protein levels of Lys-2 were not affected by bacterial injection, suggesting that the gene expression of the putative antibacterial protein is not under the regulatory control of the Imd and/or Toll pathway. Besides the abovementioned antimicrobial peptides, the 76 kDa large transferrin was also identified. Transferrin is an iron-binding protein that has been implicated in innate immunity in the honey bee. Furthermore, the effect of pathogenic dose, the timeline of peptide induction and the age-related accumulation of the aforementioned AMPs were studied. The intensity of expression of the antimicrobial peptides, abaecin, defensin 1, and hymenoptaecin as well as transferrin increased proportionally with the amount of bacteria injected into the hemocoel. No such effect was observed for the protein levels of Lys-2. Furthermore, up-regulation of the three antibacterial peptides and transferrin was observed within the first 24 h following infection with E. coli (gram-). Infection with the gram+ bacterium Micrococcus flavus resulted in high and moderate protein levels for transferrin and abaecin, respectively, whereas hardly any accumulation of hymenoptaecin was observed, indicating that the gene expression of abaecin and transferrin is somehow positively correlated, and would suggest a shared regulatory pathway that differs from that of hymenoptaecin. Although bacterial infections didn’t seem to stimulate the production of Lys-2, different concentrations in the hemolymph were observed in bees of different ages, suggesting a correlation between the expression of Lys-2 and the age-related division of labor of adult worker honey bees, also known as age polyethism. The results further allow a proposed causal connection between the age-dependent accumulation of Lys-2 and the hemolymph titer of the gonotrophic hormone juvenile hormone, which is the “behavioral pacemaker” in adult honey bees
Mit der fortschreitenden Sequenzierung des Bienengenoms werden staendig neue Daten zur Verfuegung gestellt, die eine gezielte Suche und Identifizierung von homologen Protein in der Honigbiene ermoeglichen. Mittels einer web-basierenden BLAST-Suche konnten im Genom zwei Gene identifiziert werden, welche fuer Lysozyme des C-typs kodieren. Genauere Untersuchung der Genloci konnten zeigten, dass beide Geneabschnitte keine Bindestellen fuer Transkriptionsfaktoren der NF-κB-Familie aufweisen, und daher davon auszugehen ist, dass die Lysozyme-Gene nicht unter der Kontrolle der beiden regulatroischen Pathways Toll bzw. Imd, von denen man annimmt, dass sie die humorale Immunantwort regulieren, stehen. Die beiden Gene lyz1 und lyz2 kodieren ein 157 bzw. ein 143 Aminosaeure langes Protein, welche zu 90% sequenzielle Aehnlichkeiten aufweisen. Durch weitere in silico Analyse der Protein konnten an den N-termini Erkennungssignale der Signalpeptidase gefunden werden, welche darauf schliessen lassen, dass beide Lysozyme in die Zellumgebung sezerniert werden. Mittels Sequenzvergleiche beider A. mellifera Lysozyme mit anderen C-typ Lysozymen konnte die hochkonservierte und fuer den katalytische Aktivitaet essentielle Aminosaeure Glutamat 32 (Glu32), sowie acht konservierte Cysteine identifiziert werden. Erstaunlicherweise fehlt das fuer den katalytischen Mechanismus essentielle Aspartat 50 (Asp50), welches fuer die Stabilizierung von Intermediaerprodukten wichtig ist. In A. mellifera Lysozymen ist dieses Aspartat durch ein Serin substituiert, was darauf schliessen laesst, dass die beiden Enzyme einen anderen Mechanism und/oder eine andere Substratspezifitaet aufweissen als dies fuer die C-typ Familie der Fall ist. In diesem Zusammenhang wurde ein evolutionaerer Pathway vorgesschlagen, der eine moegliche Transition von Serin zu Aspartat erklaert. Schliesslich konnten aufgrund der Sequenzhomologien zu anderen C-typ Lysozyme und anhand von bestehende Strukturen, 3D-Strukturmodelle der beiden Lysozyme erstellt werde. Die Modelle haben gezeigt, dass die Struktur der beide Enzyme groessenteils den Strukturen andere C-typ Lysozyme gleicht. Zur Identifiezung vom AMPs in A. mellifera, wurde die Hemoplymphe immunisierte Bienen elektrophoretisch und massenspektrometrisch analysisiert. Die drei AMPs Abaecin, Defensin 1 und Hymenoptaecin konnten dabei verifiziert werden. Darueber hinaus wurde zum ersten Mal das Lys-2 in der Hemolymphe nachgewiessen. Anders als bei den drei Oben erwaehnten AMPs, wurde das Lys-2 jedoch nicht durch bakterielle Infektion induziert. Die konstitutive Expression des Lys-2 laesst darauf schliessen, dass es nicht, wie bereits erwaehnt, unter der Kontrolle der beiden immunspezifischen, regulatorischen Pathways Toll und Imd steht. Zusaetlich zu den AMPs wurde das 76 kDa grosse Transferrin, ein Eisen-bindendes Protein, identifiziert, welches eine Rolle in der angeborenen Immunitaet zugesprochen wird. Anhand der genauen Bestimmung der Peptide wurde desweiteren der Effekt der bakteriellen Dosis, der Zeitverlauf der Induktion und die alterabhaengige Induktion naeher untersucht. Die Intensitaet der Expression der AMPs Abaecin, Defensin 1 und Hymenoptaecin sowie Transferrin nahm proportional mit der Menge an injezierten Bakterien zu. Die Akkumulation von Lys-2 wurde dagegen nicht beeinflusst. Desweiteren konnte eine Hochregulierung der Expressionslevel der drei AMPs und Transferrin, nach erfolgter Infektion mit E. coli, innerhalb der ersten 24 Stunden beobachtet werden. Infektion mit dem gram+ Bakterium Micrococcus flavus resultiert dagegen in hoch bzw. moderate Expression von Transferin und Abaecin, jedoch war kaum ein Anstieg der Expression von Hymenoptaecin zu verzeichnen. Dies laesst daraus schliessen, dass die Expression von Abaecin und Transferrin positiv korreliert sind und spricht fuer einen gemeinsamen regulatorischen Pathway, der sich von dem des Hymenoptaecin unterscheidet. Trotz der Beobachtung, dass die Expression von Lys-2 nicht durch bakterielle Infection induziert wird, konnten unterschiedliche Expressionsmuster in Bienen unterschiedlichen Alters beobachtet werden, welche auf eine Korrelation zwischen der Expression des Lys-2 und der altersabhaengigen Arbeitsteilung adulter Honigbienen, dem sogenennten Alterspolythismus, schliessen lassen. Aufgrund dessen, koennte man einen kausalen Zusammenhang zwischen der alterabhaengigen Akkumulation des Lys-2 und dem Hemolymphtiter des gonotrophen Juvenilhormons, welches fuer die Verhaltensaenderung adulter Bienen verantwortlich ist, sehen