Toxoneuron nigriceps is a koinobiont larval endoparasitoid of the tobacco budworm, Heliothis virescens. No koinobiont parasitoid (which develop in active, growing hosts) has been reared completely in vitro. However, T. nigriceps has been partially reared in vitro, from egg stage through 2nd larval instar and from 2nd to 3rd larval instar. Despite reaching its final larval instar, T. nigriceps failed to develop past the larval stage. In the following study, two aspects of T. nigriceps development, post-egression tissue feeding and cocoon formation through pupation, were examined to determine why T. nigriceps failed to develop in vitro beyond the larval stage and how in vitro rearing might be improved to rear T. nigriceps to adulthood. The importance of post-egression feeding and the possibility of developing an artificial post-egression diet were examined by manipulating the post-egression feeding behavior of T. nigriceps. The tissues of pre- and post-tissue feeding T. nigriceps larvae were analyzed to investigate the macronutritional benefit of post-egression feeding. A preliminary artificial post-egression diet was tested, and the putative quality of T. nigriceps reared to adulthood using this diet was examined. The formation of subterranean pupation chambers by healthy and parasitized H. virescens larvae was examined to investigate potential behavioral manipulation by T. nigriceps during this process. Finally, the potential use of artificial pupation chambers by T. nigriceps was investigated. Post-egression feeding was determined to be a vital part of T. nigriceps development, contributing to both parasitoid size and survival to adulthood. A preliminary artificial post-egression diet was developed, which facilitated rearing of T. nigriceps from 3rd larval instar to adulthood. The dimensions and burial depth of chambers formed by parasitized H. virescens were significantly different from those of chambers formed by healthy H. virescens, suggesting that T. nigriceps manipulated host behavior during excavation. Toxoneuron nigriceps was able to use a number of common laboratory items as substitute pupation chambers during cocoon formation, and the merits of several artificial chambers were discussed. With provision of an adequate post-egression and artificial pupation chamber, in vitro rearing of T. nigriceps from the larval stage to adulthood may be possible.

Submitted by Juliana Barroso Silva (juliana.barrososilva@yahoo.com.br) on 2018-03-22T19:35:28Z No. of bitstreams: 1 TESE_Juliana_Barroso_Silva.pdf: 1323265 bytes, checksum: cad99fe998c4e341d6385e39ea20e249 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-03-23T10:34:18Z (GMT) No. of bitstreams: 1 silva_jb_dr_jabo.pdf: 1323265 bytes, checksum: cad99fe998c4e341d6385e39ea20e249 (MD5) / Made available in DSpace on 2018-03-23T10:34:18Z (GMT). No. of bitstreams: 1 silva_jb_dr_jabo.pdf: 1323265 bytes, checksum: cad99fe998c4e341d6385e39ea20e249 (MD5) Previous issue date: 2018-02-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os parasitoides são insetos que utilizam outros insetos para se desenvolverem e completarem seu ciclo imaturo de desenvolvimento. Os parasitoides podem manipular a fisiologia e afetar o crescimento e desenvolvimento de seus hospedeiros, o que frequentemente resulta na morte de suas vítimas. Durante o parasitismo de Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) sobre Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), teratócitos são liberados após eclosão da larva do parasitoide no interior do hospedeiro e esses teratócitos produzem um transcrito que codifica uma serpina (CfSerpina). As serpinas são inibidores de serino proteases muitas vezes associadas à regulação da cascata de ativação de profenoloxidases em fenoloxidases, enzimas relacionadas com a melanização de corpos estranhos no hospedeiro. O presente estudo foi conduzido para investigar a função biológica da CfSerpina e avaliar seu potencial biotecnológico como agente de controle de D. saccharalis após ingestão. A CfSerpina foi produzida em sistema heterólogo em células de E. coli BL 21, purificada em coluna de níquel-sepharose e quantificada pelo método de Bradford para posterior detecção por Western blot. A proteína foi produzida em quantidades suficientes para a realização da avaliação biológica e biotecnológica. Após os ensaios, verificou-se que a CfSerpina não pode ser associada à inibição da ativação da cascata de profenoloxidases na hemolinfa de D. saccharalis. Além disso, a exploração biotecnológica da CfSerpina como uma proteína tóxica para D. saccharalis não causou mortalidade ou alterações no ganho de peso larval de D. saccharalis após ingestão e não resultou em inibição in vitro da atividade de proteases digestivas de D. saccharalis. Investigações sobre outras possibilidades de regulação da fisiologia de D. saccharalis por CfSerpina precisam ser conduzidos para definição do papel dessa proteína no processo de regulação hospedeira. / Parasitoids are insects which use other insects to develop and complete their immature cycle. Parasitoids can manipulate the physiology and affect the growth and development of their hosts, which often results in the death of their victims. During parasitism of Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) by the parasitoid Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) teratocytes are released after the eclosion of parasitoid larvae inside the host and these teratocytes produce a transcript that encodes a putative serpin (CfSerpin). Serpins are serine proteases inhibitors often associated with the regulation of the activation of prophenoloxidases into phenoloxidases, enzymes responsible for melanization of foreign bodies in the host. The study was conducted to investigate the biological function of CfSerpin, as well to evaluate its biotechnological potential to disrupt the development of D. saccharalis larvae after ingestion. CfSerpin was produced in a heterologous system using E. coli BL 21 cells, purified on a nickel-sepharose column, quantified by the Bradford method, and detected by Western blot. Purified CfSerpin was produced in sufficient amount for biological and biotechnological evaluation. CfSerpin could not be associated with the inhibition of activation of prophenoloxidases cascade in the host insect hemolymph. In addition, biotechnological exploitation of CfSerpin as a toxic protein after ingestion did not resulted in the disruption of the development of D. saccharalis larvae and no in vitro inhibition of digestive proteases of D. saccharalis by CfSerpin was observed. Investigations to unravel the role of CfSerpin on the regulation of D. saccharalis physiology still need to be conducted. / 141853/2016-6; 141604/2017-4; 159376/2014-9; 141283/2014-9 / 1747192; 1452546

Submitted by ONIEL JEREMIAS AGUIRRE GIL null (oaguirretm@outlook.com) on 2016-11-18T19:43:27Z No. of bitstreams: 1 Tese_Versão_definitiva_PDF.pdf: 2376464 bytes, checksum: 98c10cbf6c2dc00d45f713eb74c82f7f (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-11-24T15:13:11Z (GMT) No. of bitstreams: 1 gil_oj_dr_jabo.pdf: 2376464 bytes, checksum: 98c10cbf6c2dc00d45f713eb74c82f7f (MD5) / Made available in DSpace on 2016-11-24T15:13:11Z (GMT). No. of bitstreams: 1 gil_oj_dr_jabo.pdf: 2376464 bytes, checksum: 98c10cbf6c2dc00d45f713eb74c82f7f (MD5) Previous issue date: 2016-11-04 / Outra / Microcharops anticarsiae é um eficiente parasitoide da lagarta-dasoja Anticarsia gemmatalis que, também, parasita lagartas de outras espécies como Chrysodeixis includens e Spodoptera eridania. O objetivo do trabalho foi (1) determinar o ínstar larval de A. gemmatalis preferencialmente parasitado por M. anticarsiae a partir de lagartas coletadas em campo, (2) determinar o efeito de criações sucessivas de M. anticarsiae nos parâmetros biológicos do parasitoide e (3) determinar o efeito da soja transgênica sobre a longevidade de adultos do parasitoide. Os experimentos foram conduzidos nos anos agrícolas de 2014/15 e 2015/16 na FCAV/UNESP, Jaboticabal, SP, Brasil. Para a coleta das lagartas em campo, foram semeadas duas áreas de soja transgênica, uma tolerante ao herbicida glifosato e a outra resistente a insetos. Os resultados indicam que (1) M. anticarsiae preferiu parasitar lagartas de A. gemmatalis de primeiro e segundo ínstar em soja tolerante ao glifosato em campo, (2) não houve diferenças nos parâmetros biológicos de M. anticarsiae criado durante três gerações sucessivas em laboratório e (3) a longevidade total de M. anticarsiae e a longevidade de fêmeas foi mais longa em soja tolerante ao glifosato e a longevidade dos machos foi mais curta em ambas as sojas, tolerante ao glifosato e resistente a insetos. Finalmente, conclui-se que M. anticarsiae parasita lagartas de A. gemmatalis de primeiro e segundo ínstar em campo, que os parâmetros biológicos de M. anticarsiae não são afetados quando criados durante três gerações em laboratório e que a soja resistente a insetos afeta negativamente a longevidade dos machos de M. anticarsiae. / Microcharops anticarsiae is an efficient parasitoid of Anticarsiagemmatalis and also parasitizes larvae of other species such as Chrysodeixis includens and Spodoptera eridania. This work aimed to (1) determine the larval ínstar of A. gemmatalis preferentially parasitized by M. anticarsiae from larvae collected in the field, (2) determine the effect of successive laboratory rearings of M. anticarsiae in the biological parameters of the parasitoid, and (3) determine the effect of transgenic soybean in the longevity of adults of the parasitoid. the experiments were carried out in the seasons 2014/2015 and 2015/2016 in the FCAV/UNESP, Jaboticabal, SP, Brasil. Two areas were sown with transgenic soybean, the first one tolerant to glyphosate and the second one resistant to insects. The results indicate that (1) M. anticarsiae preferred to parasitize A. gemmatalis larvae on the first and second larval ínstar in soybean tolerant to glyphosate in the field, (2) there were not differences in the biological parameters of M. anticarsiae reared during three successive laboratory rearings, and (3) the longevity total of M. anticarsiae and longevity of females was longer in soybean tolerant to glyphosate and the longevity of males was shorter in soybean tolerant to glyphosate and resistant to insects. Finally, we conclude that M. anticarsiae parasitize first and second ínstar larvae of A. gemmatalis in the field, the biological parameters of M. anticarsiae are not affected when reared during three generations in the laboratory, and the soybean resistant to insects affects negatively the longevity of males M. anticarsiae.