Three series of studies were conducted to quantify the effectiveness of dietary incorporation of b-1,3-glucan (BG) from Schizophyllum commune in enhancing the immunity and resistance of grass prawn Penaeus monodon to vibriosis and white spot syndrome virus (WSSV) infections. In the first series of studies, three experiments were conducted to evaluate the effectiveness of dietary b-1,3-glucan on shrimp growth and resistance to vibriosis. Weight gain, survival and feed efficiency of juvenile shrimp (0.5 ¡Ó 0.1 g) were not significantly different (P>0.05) after being fed the diets containing BG 0, 0.2, 2, 10 g/kg diet for 18 weeks. Subadult shrimp (20.4 ¡Ó 2.1 g) fed the diet containing of BG 2 g/kg diet for 10 days showed a significantly (P<0.001) enhanced resistance against vibriosis. Postlarvae fed with the BG diet (2 g/kg diet) were more resistant (P<0.001) against starvation and V. harveyi challenges than the postlarvae fed non-BG diet. Additive disease resistance was observed when polyphosphorylated L-ascorbic acid (PAA) was used together with BG. In challenge tests with V. damsela, shrimp fed with PAA (0.2 g/kg diet) + BG (2 g/kg diet) diet for 20 days had a survival rate up to 60%. In the second series of studies, two experiments were conducted to evaluate the effectiveness of dietary b-1,3-glucan on wound healing and immunity in spawners. Dietary supplement of BG reduced the chance of infections, but did not help wound healing as did the supplement of PAA. And regardless of indoor or outdoor rearing, the survival rate of brooder (135 ¡Ó 25 g) fed the BG (2 g/kg diet) diet was higher (P<0.001) than that of the non-BG group. Fed the BG brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production then the control group. Third series of studies evaluated the effectiveness against white spot syndrome virus (WSSV). Six days after being challenged with WSSV, 12.2 % of the BG-treated (2 g/kg diet for 15 days) postlarvae (PL15) and 20 % BG-treated (2 g/kg diet for 20 days) juveniles (5.5 ¡Ó 0.5 g) were still alive; while all non-BG-treated shrimp died. In order to quantify the effectiveness of BG to WSSV, juveniles (6.5 ¡Ó 0.4 g) were fed diets containing graded levels of BG. The results showed that shrimp fed the diet containing BG 10 g/kg for 20 days had the highest (P<0.001) survival rate (42 %) among all groups. Shrimp that received diets supplemented with BG at a dosage >2 g/kg recuperated 9 ~ 12 days after WSSV challenge; while the group fed diets with no or 1 g/kg BG suffered from rapid decrease in total haemocyte count, phagocytic activity, phenoloxidase, O2-, superoxide dismutase (SOD) production and subsequent high mortality. The results in this study showed that b-1,3-glucan is effective in enhancing the phagocytic activity, phenoloxidase, O2- and SOD productions and consequently the resistance of postlarval, juvenile, subadult and brooder P. monodon against vibriosis and viral infections. Since prolonged use of BG, even at optimal dietary levels, decreased the immunity of the shrimp, care therefore must be taken to maximize its effectiveness. A cycle of dietary BG supplement of 2 ~ 10 g/kg diet for 20 days with an intermission of 10 days may serve the purposes.

No description available.

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.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-24T16:26:34Z No. of bitstreams: 1 Jennifer Guimaraes.pdf: 774518 bytes, checksum: 992bf3a690cf42445cc397d5068f8f46 (MD5) / Made available in DSpace on 2016-11-24T16:26:34Z (GMT). No. of bitstreams: 1 Jennifer Guimaraes.pdf: 774518 bytes, checksum: 992bf3a690cf42445cc397d5068f8f46 (MD5) Previous issue date: 2012-02-01 / Diatraea flavipennella (Box) is considered a major pest of economic importance to the culture of sugar cane in Northeast Brazil. Biological control with the fungus Metarhizium anisopliae (Metch.) Sorok., may be a viable alternative, as this pathogen is already used for other pests in this crop. This work aimed to evaluate the effects of this fungus at different concentrations, against the third instar larvae of D. flavipennella and to investigate the immune response, by analysing the types of hemocytes, and phenoloxidase, total protein and nitric oxide, tritation as well as the possible differences in the biochemical profile of hemocytes. It was observed the effect of M. anisopliae against larvae of D. flavipennella, sprayed at concentrations of 103, 104, 105 conidia/mL, which showed differences regarding to the larval period,which extended up to 72.0 days and 25.0 days in treatment the control, respectively The results for hemocytes revealed that the most common cells when sprayed at concentrations of 103, 105, 107 conidia/mL were prohemocytes, spherulocytes, plasma cells and granulocytes in relation to adipohemocytes and oenocytoids. The of nitric oxide titration was different between the control and the concentration 107 conidia/mL (24), while phenoloxidase activity was similar among treatments at 24h and was higher only at the concentration 107 conidia/mL after 60h. The total protein concentration in 107 conidia/ml (60h) was higher relatively to the control. In the biochemical profile of hemocytes there was an alteration in carbohydrates, lipids and total proteins contents at reating to the fungus. The results indicate that M. anisopliae may be used in Integrated Pest Management of D. flavipennella in northeastern sugar crops, because of the its pathogenicity and interfere with their development even when exposed to small concentrations. / Diatraea flavipennella (Box) é considerada uma das principais pragas de importância econômica para a cultura da cana-de-açúcar no nordeste brasileiro. O controle biológico com o fungo Metarhizium anisopliae (Metch.) Sorok., pode ser uma alternativa viável, considerando que esse entomopatógeno já é empregado para outras pragas nesta cultura. Este trabalho teve como objetivo avaliar os efeitos deste fungo, em diferentes concentrações, sobre lagartas do terceiro ínstar de D. flavipennella e investigar sua resposta imune, através da análise quantitativa dos tipos de hemócitos, da produção de fenoloxidase, proteína total e óxido nítrico, e possíveis diferenças no perfil bioquímico dos hemócitos. Foi observado o efeito de M. anisopliae sobre lagartas de D. flavipennella, pulverizado nas concentrações de 103, 104, 105 conídios/mL, no qual apresentou diferenças com relação ao período larval, prolongando até 72,0 dias nos tratamentos e 25,0 dias na testemunha. Os resultados referentes aos hemócitos revelaram que as células mais freqüentes quando pulverizado nas concentrações de 103, 105, 107 conídios/mL foram prohemócitos, esferulócitos, plasmócitos e granulócitos, em relação aos adipohemócitos e oenocitóides. O nível de oxido nítrico apresentou diferença entre a testemunha e a concentração 107 conídios/mL (24h), enquanto a atividade de fenoloxidase foi semelhante entre os tratamentos em 24h e superior na concentração 107 conídios/mL em 60 h. A proteína total na concentração 107 conídios/mL (60h) foi maior diferindo da testemunha. No perfil bioquímico dos hemócitos, houve uma alteração em carboidratos, lipídeos e proteínas em reação ao fungo. Os resultados indicam que o fungo M. anisopliae pode ser utilizado no Manejo Integrado de D. flavipennella nos canaviais nordestinos, por apresentar patogenicidade e interferir no seu desenvolvimento mesmo quando expostas a pequenas concentrações.

Trade-off between reproduction and longevity is a widely accepted fact, yet proximate mechanisms are scarcely understood. In this work I tested differences in lifespan between diapause and non-diapause males of a model insect, the linden bug Pyrrhocoris apterus. Also the role of juvenile hormone in regulation of longevity and immunity (measured by relative phenoloxidase activity) was assessed. In addition to that, I examined if juvenile hormone is the mediator of reduction in longevity induced by mating.

No description available.

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.