Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-07T20:11:26Z No. of bitstreams: 1 fernandakarinadejesus.pdf: 3610115 bytes, checksum: e48129ad65c4e1db0c972d14804b52df (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-11T15:49:57Z (GMT) No. of bitstreams: 1 fernandakarinadejesus.pdf: 3610115 bytes, checksum: e48129ad65c4e1db0c972d14804b52df (MD5) / Made available in DSpace on 2016-10-11T15:49:57Z (GMT). No. of bitstreams: 1 fernandakarinadejesus.pdf: 3610115 bytes, checksum: e48129ad65c4e1db0c972d14804b52df (MD5) Previous issue date: 2009-02-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O encantamento e o interesse pela biologia da fase juvenil de vida livre de nematóides parasitos motivaram a realização desses estudos. Nematóides entomopatogênicos (NEPs) possuem em seu ciclo de vida tal fase juvenil, semelhante à existente em outros grupos de nematóides parasitos e que difere desses principalmente por possuir simbiose com bactérias. Esses juvenis (JIs) possuem capacidade infectante e de sua sobrevivência, encontro e infecção de novos hospedeiros no ambiente depende a continuidade do ciclo de vida. Porém, há pouca compreensão acerca da interação entre os JIs e as variações nas características do solo, dentre as quais a umidade se destaca pela importância na biologia desses animais. Nesse contexto, objetivou-se estudar a influência da umidade do substrato na sobrevivência e manutenção da infectividade de JIs de Heterorhabditis amazonensis Andaló, Nguyen & Moino Jr, 2006, espécie nativa do Brasil. Tal influência foi avaliada em dois estudos independentes. No primeiro, comparou-se, ao longo de 30 dias, a quantidade de JIs vivos recuperados em amostras de terra em dois grupos, um exposto à dessecação através da perda de água por evaporação natural, partindo-se da umidade inicial de 80%, “grupo evaporação” e outro, com umidade constante de 80% ao longo de todo experimento, “grupo controle”. Dentre o observado, destacam-se (1) a conservação da infectividade em todos os JIs recuperados; (2) a acentuada mortalidade nos três primeiros dias em ambos os tratamentos; (3) o fato dessa maior mortalidade ter ocorrido em condições úmidas de substrato e (4) a conservação da umidade no grupo controle ao longo do experimento não refletiu em vantagem significativa para a sobrevivência dos juvenis desse grupo. No segundo estudo, foi realizada uma comparação da quantidade de JIs recuperados ao longo de 30 dias entre três grupos com condições constantes diferentes de umidade do substrato, 100, 75 e 30%. Destacam-se entre os resultados (1) a conservação da infectividade em todos os JIs recuperados, (2) a rápida mortalidade nos três primeiros dias de observação e (3) o fato da condição mais seca do substrato, umidade 30%, ter sido mais favorável à sobrevivência dos JIs. Em ambos os estudos, as umidades utilizadas referem-se a percentagens em relação à umidade de saturação; o substrato utilizado foi previamente esterilizado e completamente seco; as amostras de terra foram mantidas a 25°C, UR90%; testou-se a infectividade dos JIs em larvas do besouro Tenebrio molitor Linnaeus, 1785 e, com base na literatura, foi desenvolvida a técnica utilizada para recuperação dos juvenis. Os resultados encontrados permitiram inferir que condições secas em substrato de granulometria fina são favoráveis à sobrevivência dos JIs, a       acentuada mortalidade nos primeiros três dias parece ser uma característica da espécie e independente da umidade, a compreensão da interferência da umidade sobre a biologia de JIs depende do estudo de sua interação com os demais fatores ambientais presentes no solo e é variável de acordo com a condição resultante dessa interação, de tal forma que a umidade favorável à biologia desses animais varie na razão com que influencie e seja influenciada pelas demais características ambientais disponíveis. / The present study was motivated by the fascination and interest regarding the biology of the juvenile phase of parasitic nematodes. The juvenile stages of entomopathogenic nematodes (EPNs) are very similar to those of other groups of parasitic nematodes, except for the occurrence of symbiosis between their larvae and bacteria. These juveniles have infective capacity and the continuity of their life cycle depends on surviving at the external environment, finding and infecting new hosts. However, little is known about the interaction of infective juveniles (IJs) of these nematodes and the soil characteristics, among which moisture content stands out for its strong influence on the biology of these animals. Therefore, the aim of this work was to investigate the influence of soil moisture on the survival and persistence of infectivity of IJs of Heterorhabditis amazonensis Andaló, Nguyen & Moino Jr, 2006, a native Brazilian specie. This influence was evaluated in two independent studies. The first experiment aimed to compare the number of living IJs recovered from soil samples of two experimental groups. The evaporation group was subjected to natural evaporation after an initial moisture content of 80% and the control group was maintained at constant moisture level of 80%. Among the results were: (1) the continuing infectivity of all the IJs recovered; (2) the high mortality in the first three days in both groups; (3) this high mortality occurred under moist substrate conditions and (4) the fact that the conservation of moisture in the control group during the experiment was not reflected in a significant advantage for the survival of the juveniles in this group. In the second study, the same comparison was made during 30 days for IJs in constant substrate moisture conditions of 100, 75 and 30%. The most notable results were: (1) the persistence of infectivity in all the IJs; (2) the rapid mortality in the first three days of observation, of 88, 79 and 59%, respectively; and (3) the fact that the driest soil condition, of 30%, was most favorable for the survival of the IJs, with significantly higher numbers until the 30th day of observation. In both studies, the humidity levels refer to the percentages in relation to the saturation point, and the substrate used was previously sterilized and completely dried and the soil samples were maintained at 25 °C, 90% RH. The infectivity of the larvae recovered at both experiments was tested in larvae of the beetle Tenebrio molitor Linnaeus, 1785. The technique to recover the juveniles was developed based on the literature. The results permit inferring that dry conditions and fine granulometry of soil       are favorable to the survival of IJs of this specie of EPN and the high mortality in the first three days appears to be a characteristic of this specie and does not depend on the substrate’s moisture content. Finally, several scenarios may come out from the dynamic interaction between the moisture and other soil conditions. The relative importance of moisture to the biology of these animals possibly will vary at each resulting scenario.

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Thesis (MScAgric (Conservation Ecology and Entomology)--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The obscure mealybug, Pseudococcus viburni (Signoret) (Pseudococcidae), is one of the common and serious pests of apples and pears in South Africa. The management of this pest in South Africa is dominated by the use of insecticides, while research into using natural enemies for biological control of mealybugs is still ongoing. Increasing concern over the environmental impact, pesticide residues in fruits, resistance, and expense associated with frequent use of insecticides make it necessary to investigate alternative biological control methods, such as the use of entomopathogenic nematodes, for the control of mealybugs. Entomopathogenic nematodes have proven comparable or even superior to chemicals in controlling certain insect pests, without residue problems or a harmful effect on the environment. An important aspect of using endemic nematodes includes the identification of species of nematodes and their symbiotic bacterial cells. A study was carried out to describe a new species of Steinernema, which was recovered during a previous survey in citrus orchards in three provinces of South Africa. Morphometrics, morphology, crossbreeding, drawings, light microscopy and Scanning Electron Microscopy (SEM) photographs were used to describe the new species. A cryopreservation method has been simplified and optimised for the long-term storage of Steinernema khoisanae (SF87) and Heterorhabditis zealandica (J34). Different cryoprotectants used included 15% glycerol, 8% ethylene glycol and 8% dimethyl sulfoxide (DMSO), in which S. khoisanae was incubated at room temperature for periods of two, three, four and five days, followed by a methanol wash. An optimum survival rate of 69% was obtained for S. khoisanae after a four-day incubation period in 15% glycerol. This technique has been used for the cryopreservation of H. zealandica, with a 78% survival rate. The thawed nematodes of both species were able to infect Galleria mellonella larvae after 42 days of cryopreservation (-196ºC) and were able to complete their life cycles. / AFRIKAANSE OPSOMMING: Die ligrooswitluis, Pseudococcus viburni (Signoret) (Pseudococcidae), is een van die algemene en ernstige peste van appels en pere in Suid-Afrika. Die bestuur van hierdie pes word tans in Suid-Afrika deur die gebruik van insekdoders gedomineer terwyl navorsing oor die gebruik van natuurlike vyande vir die beheer van P. viburni nog aan die gang is. Die verhoogde kommer oor die omgewing, residue in vrugte, weerstand, en die koste verbonde aan die gereelde gebruik van chemiese middels maak dit nodig om alternatiewe biologiese metodes van beheer, soos die gebruik van entomopatogeniese nematodes vir die beheer van witluis, te ondersoek. In ander lande is reeds aangetoon dat entomopatogeniese nematodes onder sekere omstandighede en vir sekere insekte gelykwaardige of selfs beter beheer kan gee as chemiese middels. ʼn Belangrike aspek van die gebruik van endemiese nematodes vir die beheer van insekte sluit die korrekte identifikasie van die spesies met hul geassosieerde bakteriese simbionte in. ʼn Nuwe spesie van Steinernema is uit ʼn vorige opname van entomopatogeniese nematodes in sitrusboorde in drie provinsies van Suid-Afrika geïsoleer. Morfometrie, morfologie, kruisteling, ligmikroskoop en SEM fotografie is gebruik om ʼn nuwe spesies te beskryf. ʼn Kriopreserveringsmetode is ontwikkel en ge-optimaliseer vir die langtermyn bewaring van Steinernema khoisanae (SF87) en Heterorhabditis zealandica (J34). Verskillende kriobeskermingsmiddels insluitend 15% gliserol, 8% dimetiel sulfokied (DMSO) en 8% etileen glikol, waarin S. khoisanae vir periodes van twee, drie, vier, en vyf dae geïnkubeer is, is teen kamertemperatuur, getoets, gevolg deur ʼn metanolbad. Optimum oorlewing van 69% is verkry vir S. khoisanae nadat die infektiewe larwes (IJ) vir vier dae in 15% gliserol gehou is. Hierdie tegniek is ook toegepas op H. zealandica, met 78% oorlewing van die IJ. Die ontvriesde nematodes van beide spesies was in staat om Galleria mellonella larwes suksesvol te infekteer en hulle lewensiklus te voltooi nadat hulle vir 45 dae onder kriopreservering gehou is teen -196ºC.

Thesis (MScAgric (Conservation Ecology and Entomology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The banded fruit weevil, Phlyctinus callosus (Schönherr), is a key pest of apples, nectarines and grapevines in the southern areas of the Western Cape. The control of P. callosus is not satisfactory and the insecticides used to control this insect have not proved to be effective since the development of tolerance to pyrethroids and acephate. A control method that can be used, despite it being very labour-intensive, is that of tree trunk barriers. The use of such a method will prevent the weevils from reaching the fruit, as they are unable to fly. Alternative control options, such as the use of entomopathogenic nematodes, are urgently needed for the control of P. callosus. Entomopathogenic nematodes belonging to the Steinernematidae and Heterorhabditidae are ideal biocontrol agents for incorporation into an integrated pest management programme. In order to develop control strategies for P. callosus, large numbers and a predictable quantity of different weevil stages are needed. Especially large numbers of larvae are needed, as this is the stage that will be targeted with nematodes. One of the aims of the current study was to assess various artificial diets for rearing larvae of P. callosus. Though adult weevils were easily collected from orchards, it was very difficult to obtain large numbers of larvae. Modified versions of an agar diet, as well as different carrot based diets, were tested at 21°C. The highest percentage survival obtained for the agar diet was 50% and 60% for one type of carrot diet. A better rearing method proved to be that of planting full-grown carrots in pots, kept at 25°C, resulting in the attainment of the highest percentage survival rate of 90%. A study was undertaken to assess how long, and at what temperature, P. callosus eggs could be stored. A mean percentage hatch of 45.7% was obtained when eggs were stored at 4°C for 70 days. Eggs started hatching after 47 days and 10 days, when stored at temperatures of 11°C and 14°C, respectively. If the aim of the employment of such a method is only to delay egg hatching, the two temperatures (11°C and 14°C) will be suitable. For the following part of the study, several entomopathogenic nematode isolates were evaluated for their potential use as biological control agents against P. callosus. The susceptibility of P. callosus larvae and adults to nematode infection was assessed in the laboratory by screening for their mortality, using different nematode isolates. Larvae were found to be more susceptible to nematode infection than adults. Heterorhabditis isolates were found to cause higher levels of mortality than the Steinernema isolates during screening, when a concentration of 400 infective juveniles (IJ) per insect V was used. Biological characteristics, such as the effect of different temperatures on nematode activity and the minimum concentration of nematodes needed to obtain acceptable levels of control for P. callosus, were also investigated. The percentage mortality ranged from no infection to 75% after four days for the larvae, and the SF41 isolate of Heterohabditis zealandica was selected as the most promising isolate for further laboratory experiments. The vertical movement of nematodes in sand, compared with such movement in sandy loam soil, and the biology of H. zealandica in P. callosus larvae was also investigated in laboratory bioassays. After four days, the LD50 and LD90 values were 96 IJ/50 μl and 278 IJ/50 μl, respectively. Nematodes were found to be inactive at 11°C, with the highest mortality rate of P. callosus resulting from nematode infection being recorded at 25°C. A higher percentage mortality rate was obtained with the sandy loam soil (95.2%) than with the sand (77.5%). Heterorhabditis zealandica could successfully complete its life cycle in 6th instar P. callosus larvae. The study showed that P. callosus larvae are suitable hosts for H. zealandica, and that the control of P. callosus in the field by the selected isolate holds promise. The persistence of the SF41 isolate of H. zealandica at different concentrations was investigated in the last part of the study. The experiment took place in a blueberry orchard, subject to a high rate of infestation by P. callosus. Concentrations of 0, 20, 30 and 45 IJ/cm2 were topically applied, with persistence being evaluated for days 1, 35 and 84. Percentage persistence for 30 IJ/cm2 was calculated as 87.5% for days 35 and 84. The persistence of soil samples taken on day one, and kept in plastic containers at room temperature, was again evaluated on day 128, with the finding that both 30 IJ/cm2 and 45 IJ/cm2 caused 100% mortality of Tenebrio molitor (L.). Results indicated good persistence of H. zealandica after 84 days in field conditions, with a high maintenance of P. callosus populations. The study indicated the potential use of H. zealandica for the control of P. callosus, with the possibility of persistence for at least three months. Future research into the control of P. callosus with nematodes should aim to investigate the technical aspects of field application. The current study shows that entomopathogenic nematodes have potential for controlling the soil stages of P. callosus. The capacity to rear large numbers of P. callosus larvae in the laboratory, for later use in laboratory and field trials, is of key importance. / AFRIKAANSE OPSOMMING: Die gebande vrugtekalander, Phlyctinus callosus (Schönherr), is ʼn groot plaag in appel- en nektarienboorde sowel as wingerde in die suidelike gebiede van die Wes-Kaap. Phlyctinus callosus word nie voldoende beheer nie, en plaagdoders wat voorheen gebruik is om dié insek in toom te hou, het doeltreffendheid ingeboet weens weerstandontwikkeling teen piretroϊede en asefaat. ʼn Alternatiewe beheermetode is stamsperbande. Omdat die kalanders nie kan vlieg nie, moet hulle teen stamme uitklim om die vrugte te bereik. Stamsperbande versper dus die insekte se toegang tot die vrugte, maar is baie arbeidsintensief. Meer haalbare metodes vir die beheer van P. callosus is daarom dringend nodig, en die gebruik van entomopatogeniese nematodes blyk ʼn besliste moontlikheid te wees. Entomopatogeniese nematodes, wat tot die Steinernematidae en Heterorhabditidae behoort, is uitstekende biobeheermiddels vir insluiting by geϊntegreerde plaagbeheerprogramme. Om doeltreffende beheerstrategieë vir P. callosus te bedink, is groot en voorspelbare hoeveelhede kalanders nodig veral groot hoeveelhede larwes, aangesien nematodes op hierdie ontwikkelingstadium gemik sal wees. Die eerste doel met die studie was dus om ʼn kunsmatige dieet vir die teling van P. callosus larwes te ontwikkel. Volwasse kalanders kon maklik in vrugteboorde ingesamel word, maar groot hoeveelhede larwes was moeiliker bekombaar. Aangepaste weergawes van ʼn agardieet sowel as verskillende worteldiëte is by 21°C beproef. Die hoogste persentasie larwale groei en -oorlewing op die agardieet was 50%, en 60% op een bepaalde soort worteldieet. Die beste teelmetode blyk egter volgroeide wortels te wees wat in potte geplant is en by 25°C gehou word. Dié metode het ʼn oorlewingspersentasie van 90% opgelewer. ʼn Studie is onderneem om te bepaal hoe lank en by watter temperature P. callosus eiers vir toekomstige gebruik geberg kan word. ʼn Gemiddelde uitbroeipersentasie van 45.7% is verkry toe eiers vir 70 dae by 4°C geberg is. Eiers wat onderskeidelik by 11°C en 14°C geberg is, het ná 47 en 10 dae onderskeidelik begin uitbroei. Indien die doel is om die eiers slegs stadiger te laat uitbroei, sal hierdie twee temperature dus geskik wees. VII Hierna is verskeie entomopatogeniese nematode-isolate vir moontlike gebruik as biologiese beheermiddels vir P. callosus beoordeel. Phlyctinus callosus larwes en volwassenes se vatbaarheid vir nematode infeksie is in die laboratorium bepaal deur dit met behulp van verskillende nematodeisolate vir mortaliteit te toets. Dié toetse het getoon dat larwes meer vatbaar is vir nematode infeksie as volwassenes. In die proefnemings het die Heterorhabditis-isolate hoër mortaliteit as die Steinernema-isolate veroorsaak teen ʼn konsentrasie van 400 infektiewe larwes (IJ) per insek. Biologiese eienskappe, soos die uitwerking van verskillende temperature op nematode aktiwiteit, sowel as die minimum konsentrasie nematodes om aanvaarbare vlakke van beheer uit te oefen, is ondersoek. Die persentasie mortaliteit vir die larwes het ná vier dae tussen 0% en 75% gewissel, en die SF41-isolaat van Heterohabditis zealandica is as die belowendste isolaat vir die res van die proefnemings gekies. Die vertikale beweging van nematodes in sand teenoor leemgrond, sowel as die biologie van H. zealandica in P. callosus larwes, is ook bestudeer. Ná vier dae was die LD50- en LD90-waardes onderskeidelik 96 en 278 IJ/50 μl. Wat temperatuur betref, is daar bevind dat nematodes onaktief is by 15°C, terwyl die hoogste mortaliteit van P. callosus larwes as gevolg van nematode infeksie by 25°C aangeteken is. Die mortaliteit was hoër in die leemgrond (95.2%) as in die sandgrond (77.5%). Heterorhabditis zealandica kon sy lewensiklus suksesvol in 6de instar P. callosus larwes voltooi. Die studie het derhalwe getoon dat P. callosus larwes geskikte gashere is vir H. zealandica, en dat hierdie isolaat dus in die praktyk ʼn doeltreffende beheermiddel vir P. callosus kan wees. Die oorlewing van verskillende konsentrasies H. zealandica is ten slotte bestudeer. Die proefneming is in ʼn bloubessieboord met ʼn groot populasie P. callosus uitgevoer. Konsentrasies van 0, 20, 30 en 45 IJ/cm2 is op die grond (uitwendig) toegedien, en oorlewing is op dag 1, 35 en 84 gemeet. Die persentasie oorlewing vir die 30 IJ/ cm2 konsentrasie was 87.5% op sowel dag 35 as 84. Oorlewing in grondmonsters wat op dag een ingesamel en by kamertemperatuur in plastiekhouers geberg is, is weer op dag 128 beoordeel. Daar is bevind dat sowel die 30 IJ/cm2 as die 45 IJ/cm2 konsentrasie 100% mortaliteit by T. molitor veroorsaak het. Heterorhabditis zealandica blyk ʼn goeie oorlewing te hê ná 84 dae in veld kondisies wat erg met P. callosus besmet is, en is dus ʼn moontlike beheermiddel vir P. callosus, met potensiële oorlewing vir minstens drie maande.