Role on serine protease inhibitors in the pathogenisis of Steinernema carpocapsae

Tese de Doutoramento, Biologia (Biologia Celular e Molecular), 18 de Novembro de 2013, Universidade dos Açores.

População de Pratylenchus brachyurus no cultivo de soja e sua influência na produtividade de grãos.

As limitações legais para o avanço da cultura da soja em novas áreas, pelo valor pouco atrativo do milho segunda safra e pelo baixo vigor das sementes recebidas, o interesse do produtor em cultivar soja na segunda safra tem sido crescente no Estado de Mato Grosso. Entretanto, o cultivo de soja na segunda safra possui entraves que limitam a produtividade, como o menor fotoperíodo e o maior risco de ocorrência de seca no período reprodutivo da cultura. Outra preocupação é a multiplicação e a manutenção dos nematoides das lesões radiculares (P. brachyurus ) em altas populações no solo, quando se cultivam plantas hospedeiras durante longo período do ano. Há relatos de perdas de até 50% na produtividade de grãos de soja, em lavouras comerciais da região Centro - Oeste infestadas com P. brachyurus (FRANCHINI et al., 2014). Além da ação espoliadora e mecânica, esse nematóide causa ferimentos nas raízes o s quais favorecem a entrada de fungos e bactérias, potencializando os danos e levando à diminuição da produtividade (FONSECA, 2012). A rotação com culturas não hospedeiras é a principal forma de controle desses parasitas, visto que não existem fontes de resistência varietal. O objetivo do trabalho foi o de avaliar a influência de sucessões de cultura, principalmente de soja sobre soja 2ª safra, na população do nematoide das lesões radiculares e na produtividade de grãos na cultura da soja cultivada na safra.

Isolation and characterization of the female sex pheromone of the sugar beet cyst nematode, heterodera schachtii, and an analysis of male precopulatory behaviour /

The sugar beet cyst nematode, Heterodera schachtii, is a major agricultural pest. The disruption of the mating behaviour of this plant parasite in the field may provide a means of biological control, and a subsequent increase in crop yield. The H. schachtii female sex pheromone, which attracts homospecific males, was collected in an aqueous medium and isolated using high performance liquid chromatography. Characterization of the male-attractive material revealed that it was heat stable and water soluble. The aqueous medium conditioned by female H. schachtii was found to be biologically active and stimulated male behaviour in a concentration dependent manner. The activity of the crude pheromone was specific to males of H. schachtii and did not attract second stage juveniles. Results indicated that vanillic acid, a putative nematode pheromone, is not an active component of the H. schachtii sex pheromone. Male H. schachtii exhibited stylet thrusting, a poorly understood behaviour of the male, upon exposure to the female sex pheromone. This behaviour appeared to be associated with mate-finding and was used as a novel indicator of biological activity in bioassays. Serotonin, thought to be involved in the neural control of copulatory behaviour in nematodes, stimulated stylet thrusting. However, the relationship between stylet thrusting induced by the sex pheromone and stylet thrusting induced by serotonin is not clear. Extracellular electrical activity was recorded fi-om the anterior region of H. schachtii males during stylet thrusting, and appeared to be associated with this behaviour. The isolation of the female sex pheromone of H. schachtii may, ultimately, lead to the structural identification and synthesis of the active substance for use in a novel biological control strategy.

Mermithid (nematoa: mermithidae) infections of black flies (Diptera: Simuliidae) : seasonal variation and developmental characteristics /

Mermithid nematodes (Nematoda: Mermithidae) parasitize larval, pupal and adult black flies (Diptera: Simuliidae), oftentimes resulting in partial or complete host feminization. This study was designed to characterize parasite-host seasonal variation and to estabUsh the developmental life stage at which feminization is initiated. Data indicate that the total adult population of black flies collected from Algonquin Provincial Park throughout the spring of 2004 was comprised of 31.8% female, 67.8% male and 0.4% intersex individuals. Of the total population, 0.6% was infected by mermithid nematodes (69.0% female, 3.5% male and 27.6% intersex). Seasonal infection trends established over a 12-month period revealed that black flies with different life histories host the same mermithid subfamilies, while black flies with similar life histories host mermithids from different subfamilies. If a simuliid species simultaneously hosts two mermithid species, these parasites are from different subfamilies. Molecular mermithid identification revealed two mermithid subfamilies, Me.somermithinae and Gastromermithinae, present in the simuliid hosts. Mermithid colour variation was not found to be a reliable species indicator. The developmental stage at which feminization is initiated was determined by examining gonad morphology and meiotic chromosomal condition. Results indicate that mermithid-infected black flies exhibit feminization prior to larval histoblast formation. Larvae can be morphologically male (testes present) or female (ovaries present), with morphological males exhibiting either male (achiasmate) or female (chiasmate) meiotic chromosomes; morphological females were only genetically female. Additionally, mermithid infection inhibits simuliid gonad development.

Meloidogne incognita (nematode) parasitism of Lycopersicon esculentum (tomato) plants : Ethylene action in susceptible and resistant host responses

Involvement of ethylene in the etiology of tomato plants (Lycopersicon esculentum) infected with the root-knot nematode (Meloidogyne incognita) was investigated. Endogenous root concentrations of ethylene were not significantly different in uninfected resistant var. Anahu and susceptible var. Vendor plants. Exposure of resistant plants to high doses of infectious nematode larvae did not affect root ethylene concentrations during the subsequent 30 day period. The possibility that ethylene may be involved in the mechanism of resistance is therefore not supported by these experiments. In no experiments did ethylene concentrations in roots of susceptible plants increase significantly subsequent to ~ incognita infestation. This result is not consistent with the hypothesis in the literature which suggests that increased ethylene production accompanies gall formation. Growth of susceptible tomato plants was affected by ~ incognita infestation such that root weights increased (due to galling), stem heights decreased and top weights increased. The possibility that alterations in stem growth resulted from increased production of 'stress' ethylene is discussed. Growth of resistant plants was unaffected by exposure to high doses of ~ incognita and galls were never detected on the roots of these plants. Root ethane concentrations generally varied in parallel with root ethylene concentrations although ethane concentrations were without exception greater. In 4 of 6 experiments conducted ethane/ethylene ratios increased significantly with time. These results are discussed in the light of published data on the relationship between ethane and ethylene synthesis. The term infested is used throughout this thesis in reference to plants whose root systems had been exposed to nematodes and does not distinguish between the susceptible and resistant response.

Studies on oxamyl : analytical method development and investigation of fate in peach seedlings and corn seeds

A high performance liquid chromatographic method employing two columns connected in series and separated~y·a.switching valve has been developed for the analysis of the insecticide/ nematicide oxamyl (methyl-N' ,N'-dimethyl-N-[(methylcarbamoyl) oxy]-l-thiooxarnimidate) and two of its metabolites. A variation of this method involving two reverse phase columns was employed to monitor the persistence and translocation of oxamyl in treated peach seedlings. It was possible to simultaneously analyse for oxamyl and its corresponding oxime (methyl-N',N'-dimethyl-N-hydroxy-l-thiooxamimidate}, a major metabolite of oxamyl in plants, without prior cleanup of the samples. The method allowed detection of 0.058 pg oxamyl and 0.035 p.g oxime. On treated peach leaves oxamyl was found to dissipate rapidly during the first two-week period, followed by a period of slow decomposition. Movement of oxamyl or its oxime did not occur in detectable quantities to untreated leaves or to the root or soil. A second variation of the method which employed a size exclusion column as·the first column and a reverse phase column as the second was used to monitor the degradation of oxamyl in treated, planted corn seeds and was suitable for simultaneous analysis of oxamyl, its oxime and dimethylcyanoformamide (DMCF), a metabolite of oxamyl. The method allowed detection of 0.02 pg oxamyl, 0.02 p.g oxime and 0.005 pg DMCF. Oxamyl was found to persist for a period of 5 - 6 weeks, which is long enough to permit oxamyl seedtreatment to be considered as a potential means of protecting young corn plants from nematode attack. Decomposition was found to be more rapid in unsterilized soil than in sterililized soil. DMCF was found to have a nematostatic effect at high concentrations ( 2,OOOpprn), but at lower concentrations no effect on nematode mobility was observed. Oxamyl, on the other hand, was found to reduce the mobility of nematodes at concentrations down to 4 ppm.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Suppression of long-branch attraction artefacts in the animal phylogeny using a site-heterogeneous model

Affiliation: Département de Biochimie, Université de Montréal

Le Baylisascaris procyonis dans le sud du Québec : prévalence chez le raton laveur et étude de contamination des latrines

Le raton laveur (Procyon lotor) est porteur de nombreux parasites dont certains sont des agents de zoonose. Il nous est apparu opportun d’étudier la faune parasitaire intestinale du raton laveur québécois, particulièrement l’espèce Baylisascaris procyonis. L’étude a donc porté sur l’examen de matières fécales (n = 301) et de contenus intestinaux (n = 203) de 351 ratons laveurs provenant de deux programmes gouvernementaux sur la rage du raton laveur et d’excréments (n = 409) provenant de latrines dans des parcs nationaux du Québec. Des oeufs de B. procyonis étaient excrétés par 23,3 % des ratons laveurs, alors que 29,1 % étaient porteurs de vers. La charge parasitaire moyenne des vers B. procyonis de tout stade variait de 8,5 chez les adultes à 27,1 chez les juvéniles. Chez ces derniers, la charge parasitaire était 11,4 fois supérieure à celle des ratons adultes (p < 0,0001) dont 23,7 fois plus de vers immatures (p < 0,0001) et 9,1 fois plus de vers matures (p = 0,01). Le nombre d’oeufs était 24,7 fois plus élevé chez les ratons juvéniles (p = 0,02) et 50,0 fois moins élevé chez la femelle allaitante. La probabilité du raton d’excréter des oeufs l’automne était de 11,1 (IC95 %: 1,21-101,60) par rapport au printemps et de 21,1 (IC95 %: 2,91-153,18) par rapport à l’été (p < 0,01). La sensibilité (Se) et la spécificité (Sp) de la coproscopie comparée à la nécropsie (norme étalon) ont été calculées en se basant sur l’observation des vers matures (Se : 81,8 %; Sp : 97,7 %) et des vers de tout stade (Se : 53,9 %; Sp : 97,0 %). De plus, la recherche des parasites intestinaux nous a permis de découvrir des ookystes de Giardia (6/159), espèce parasitaire nouvellement rapportée chez le raton. En 2007, nous avons trouvé, par échantillonnage unique, des oeufs de B. procyonis dans 15,8 % des latrines (n = 165) identifiées dans des parcs nationaux du sud du Québec et en 2008, par échantillonnage multiple, dans 89,7 % des latrines (n = 26) actives situées dans des secteurs accessibles à l’homme dans trois parcs nationaux de la Montérégie. Le potentiel zoonotique de B. procyonis est un problème de santé publique qui pourrait devenir sérieux étant donné le pourcentage élevé de ratons laveurs qui excrètent le parasite et qui contaminent des secteurs accessibles par les humains.

Propriétés électrophysiologiques des canaux ioniques formés par la toxine nématicide Cry5Ba du bacille de Thuringe dans les bicouches lipidiques planes

Les toxines Cry sont des protéines synthétisées sous forme de cristaux par la bactérie bacille de Thuringe pendant la sporulation. Elles sont largement utilisées comme agents de lutte biologique, car elles sont toxiques envers plusieurs espèces d’invertébrées, y compris les nématodes. Les toxines Cry5B sont actives contre certaines espèces de nématodes parasites, y compris Ankylostoma ceylanicum un parasite qui infeste le système gastro-intestinal des humains. Jusqu’au présent, le mode d’action des toxines Cry nématicides reste grandement inconnu, sauf que leurs récepteurs spécifiques sont des glycolipides et qu’elles causent des dommages importants aux cellules intestinales. Dans cette étude, on démontre pour la première fois que la toxine nématicide Cry5Ba, membre de la famille des toxines à trois domaines et produite par la bactérie bacille de Thuringe, forme des pores dans les bicouches lipidiques planes en absence de récepteurs. Les pores formés par cette toxine sont de sélectivité cationique, à pH acide ou alcalin. Les conductances des pores formés sous conditions symétriques de 150 mM de KCl varient entre 17 et 330 pS, à pH 6.0 et 9.0. Les niveaux des conductances les plus fréquemment observés diffèrent les uns des autres par environ 17 à 18 pS, ce qui est compatible avec l’existence d’arrangement d’un nombre différent de pores élémentaires similaires, activés de façon synchronisée, ou avec la présence d’oligomères de tailles variables et de différents diamètres de pores.

Meiobenthos of Cochin backwaters in relation to macrobenthos and environmental parameters

Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology

Parasite distribution and histopathological studies on certain commercially important fishes of cochin area

In order to check the damage caused by the parasites, even though it is difficult in open waters, a proper understanding of the seasonal variation in the distribution of the parasite and other factors like age of the host, sex of the host, which affect distribution of parasite is a must. Although several workers have carried out investigations on the taxonomy of metazoan parasites of marine and brackish water Fishes of India, very little attempt is made to correlate such investigations with the host and the environment. In this thesis such an attempt is made by the researcher. In chapter one the literature related to the prevalence, mean intensity of infection, and histopathological changes caused by the metazoan parasites, in particular by helminths, copepods and isopods, was reviewed. Chapter two contains observations on the distribution pattern of parasites in relation to the season, sex, and size of the host. It was found that the prevalence rate of radiorynchus_indicus infecting the alimentary canal of Tachysurugs mgacuglatus, Ergasilus sp. infecting the gills of T maculatus, and Lernaeeniicus ramosus found on the body surface of Qeimgipteirfugs jagonicus was higher during monsoon season. But agarna malayi found in the opercular chamber and Ehilometra cephalus infecting the gonads of valamugil speiglari showed a higher prevalence rate during the postmonsoon season. This was discussed on the basis of the hydrographical characteristics prevailing in the study area during the three different seasons. It was also observed that the sex of the host did not influence significantly the distribution pattern of the parasites. The reasons for this were also discussed. Invariably, the size of the host was found to influence the parasite distribution pattern. It was observed that the prevalence rate showed an increase with increase in size of the fish. This was discussed on the basis of food habits of the host, along with other aspects An attempt was made in chapter three to study the histopathological effects of‘ the various parasites on their respective sites of attachments on host Fishes. It was found that except Rhadinorhyhchugs indicus, all other parasites produced damages of varying intensity, in the form of hypertrophy, rhyperplasia, haemorrhage, tissue disruption and ulcers. Interestingly, E. indicus, an acantho— cephalid with a powerful proboscis for attachment was found not to cause any serious damage to the intestine of the host Fish. All these aspects are included in the third and final chapter of the thesis.

Effect of industrial pollution on the benthic communities of a tropical estuary

Cochin backwaters, a tropical barbuilt estuary is well known for its prawn, molluscan and demersal fisheries. Also it formed the dumping area for sewage,235 retting of husks and discharge of effluents from industries located on either side of it. As a result the fishery is being gradually dwindled year after year due to the lowering of the water quality. The effect of industrial polution on the benthic community of this tropical estuary was worked out. An area extending over 21 km from the mouth of the estuary to upstream of industrial belt was selected. Temporal and spatial variations of 16 environmental parameters at 9 stations along the area were monitored monthly during 1981. Benthic fauna of these 9 stations consisted of amphipods , polychaetes, isopods, tanaidaceans, molluscs and other crustaceans (Decapods, Acetes, Alpheids, Balanus, insect larvae, chironomid larvae, cumacea and some fresh water forms ). Apart from these, sea anemone, flat worms, nematodes, sipunculoids, echinoderms and fishes were also encountered. 75 species belonging to 31 faunal groups were identified. Of these 31 groups, amphipods, polychaedes, isopods, tanaidaceans and molluscs were numerically abundant. Rest of the 26 groups (including 13 riverine forms) were found less significant due to their rare occurrence/low numerical abundance. Polychaetes and molluscs were the only major groups present at all the stations.

Macrobenthos And Its Relation To Ecosystem Dynamics In The Cochin Estuary

Cochin estuary is a shallow brackish water body situated on the south west coast of India. It is a tropical positive estuary extending between 90 40’ and 100 12’ N and 760 10’and 760 30’ E with its northern boundary at Azhikode and southern boundary at Thannermukkom bund.The abundance of benthic fauna in an ecosystem shows the close relationship to its environment and reflects the characteristics of an ecological niche. Seasonal and monthly variations in the distribution of macrobenthos in relation to sediment characteristics were conducted in Cochin estuary from 2009-10 periods. Oxidation-reduction potential showed reducing trends that affected the distribution and diversity of fauna. Seasonal variations in water quality and river discharge pattern affected the faunal composition in the different stations. Sewage mixing was the principal source of organic pollution in the Cochin estuary. The sediment pH was generally on the alkaline side ranging from 4.99 at St.9 and 8.33 at St.1.The Eh ranged from -11mV at St.3 to -625mV at St.2.The temperature varied from 260C to 320C in the estuary. The moisture content ranged from 1.63 to 12.155%, that of organic carbon from 0 09 at St. 6 to 4.29% at St.9 and that of organic matter from 0.16 to 7.39%. Seasonally, the average of Eh was highest during the monsoon (156.22 mV) and in the pre monsoon (140.94 mV). The average pH for the 9 study stations was 7.68 during monsoon period and 7.08 during post monsoon. Based on group wise seasonal analysis, the average mean abundance was maximum for polychaetes (43.47) followed by nematodes (33.62), crustaceans (21.62), molluscs (11.94) and Pisces (0.05) in the estuary. Monsoon season was most favourable for benthic faunal abundance followed by the post monsoon period in the study. The series of human interventions like dredging, discharge of industrial effluents, urbanisation and related aspects had a strong influence on the distribution, abundance of benthic macrofauna in the wetland.

Cereal/legume rotations affect chemical properties and biological activities in two West African soils

A more widespread use of cereal/legume rotations has been suggested as a means to sustainably meet increasing food demands in sub-Saharan West Africa. Enhanced cereal yields following legumes have been attributed to chemical and biological factors such as higher levels of mineral nitrogen (Nmin) and arbuscular mycorrhizae (AM) but also to lower amounts of plant parasitic nematodes. This study was conducted under controlled conditions to examine the relative contribution of AM, plant parasitic nematodes and increased nitrogen (N) and phosphorus (P) availability to cereal/legume rotation effects on two West African soils. Sample soils were taken from field experiments at Gaya (Niger) and Fada (Burkina Faso) supporting continuous cereal and cereal/legume rotation systems and analysed for chemical and biological parameters. Average increases in cereal shoot dry matter (DM) of rotation cereals compared with continuous cereals were 490% at Gaya and 550% at Fada. Shoot P concentration of rotation millet was significantly higher than in continuous millet and P uptake in rotation cereals was on average 62.5-fold higher than in continuous cereals. Rotation rhizosphere soils also had higher pH at both sites. For the Fada soil, large increases in Bray1-P and organic P were observed in bulk and rhizosphere soils. Plant parasitic nematodes in roots of continuous cereals were 60–80-fold higher than in those of rotation cereals. In both cropping systems mycorrhizal infection rates were similar at 37 days after sowing (DAS) but at 57 DAS AM infection was 10–15% higher in rotation sorghum than in continuous sorghum. This study provides strong evidence that cereal/legume rotations can enhance P nutrition of cereals through improved soil chemical P availability and microbiologically increased P uptake.