Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants

Odorant-binding proteins (OBPs) were discovered almost three decades ago, but there is still considerable debate regarding their role(s) in insect olfaction, particularly due to our inability to knockdown OBPs and demonstrate their direct phenotypic effects. By using RNA interference (RNAi), we reduced transcription of a major OBP gene, CquiOBP1, in the antennae of the Southern house mosquito, Culex quinquefasciatus. Previously, we had demonstrated that the mosquito oviposition pheromone (MOP) binds to CquiOBP1, which is expressed in MOP-sensitive sensilla. Antennae of RNAi-treated mosquitoes showed significantly lower electrophysiological responses to known mosquito oviposition attractants than the antennae of water-injected, control mosquitoes. While electroantennogram (EAG) responses to MOP, skatole, and indole were reduced in the knockdowns, there was no significant difference in the EAG responses from RNAi-treated and water-injected mosquito antennae to nonanal at all doses tested. These data suggest that CquiOBP1 is involved in the reception of some oviposition attractants, and that high levels of OBPs expression are essential for the sensitivity of the insect’s olfactory system

ANTIMYCOBACTERIAL, ANTIMICROBIAL AND ANTIFUNGAL ACTIVITIES OF GERANIUM OIL-LOADED NANO CAPSULES

Objective: The aim of this study was to perform the first ever investigation of the effect of activities in the nano capsules containing Geranium oil (NC1) against different species of pathogens such as Mycobacterium genus (both fast growing and slow growing), bacterial, and yeasts.Methods: The GO was analyzed by GC and GC/MS. Nano capsule suspensions (NC) were prepared by interfacial deposition of a preformed polymer method and the MICs were determined for the antimycobacterial, antimicrobial, and antifungal activities.Results: GO-loaded nano capsules (NC1) presented nano metric mean diameters (188 nm), polydispersity indices below 0.149, pH (5.5), and zeta potentials (about-10.8 mV). The MICs were determined for the antimycobacterial, antimicrobial, and antifungal activities. The NC1 was effective to Mycobacterium smegmatis (149.7 µg ml-1), M. abscessos (35.9 µg ml-1), M. massiliense (35.9 µg ml-1), M. avium (71.8 µg ml-1), Enterococcus faecalis, Streptococcus sp. (149.7 µg ml-1) and Listeria monocytogenes (35.9 µg ml-1). The NC1 was able to significantly reduce the number of cells of C. albicans (by approximately 5 log), 4 log the number of cells of C. dublinensis, C. glabrata, and C. krusei, and 2 log the number of cells of C. parapsilosis compared to the control group.Conclusion: Our study showed that the geranium oil-loaded nano capsules have antimycobacterial activities similar to free oil. The GO was effective in inhibiting the formation of germ tubes of Candida albicans, yet the nano capsule containing GO failed to inhibit the formation of this important virulence factor.Â

Interações multitróficas de Aphis (Toxoptera) e seus simbiontes secundários

Insect-symbiont interactions have many bioecological consequences to the host. Their relationships expand through a complex network that includes other microorganisms, interactions with the environment and other trophic levels. An extensive literature has been produced on bacterial symbionts and aphids, especially for polyphagous aphids from North America and Europe, such as Acyrthosiphon pisum (Harris). They indicated symbionts influence host nutrition, heat tolerance, defense against natural enemies, virus transmission, host plant exploitation, among others. However, the outcome from host-symbiont interactions is context-dependent, with the expressed phenotype depending on intra and interspecific variations, symbiont strain, and biotic/abiotic stimuli. We explored the interaction between aphids and associated symbionts, aiming to contribute to this field by exploring new aphid systems, and focused on Aphis (Toxoptera) citricidus (Kirkaldy) and associated microbiota. We investigated the influence of the feeding habits on symbiont diversity in an oligophaogus, A. citricida, as compared to a polyphagous species, Aphis (Toxoptera) aurantii Boyer de Fonscolombe. We employed several approaches (biological, metagenomics, genomic, and proteomic) to investigate i) the impact of host plants on fitness traits and primary symbiont abundance in the oligophagous and polyphagous species, ii) differences in the draft genome of the primary aphid symbiont between A. citricidus and A. aurantii, iii) the influence of host plant in secondary symbiont distribution in A. citricidus, iv) secondary symbiont richness and abundance in both aphids, and investigate the effects of Spiroplasma infections v) in the fitness traits, vi) transcriptome and ii) proteome of A. citricidus when reared on two host plants (sweet orange and orange jasmine). Our data indicated that sweet orange is a better host plant than orange jasmine for both aphids, and that A. citricidus was more negatively affected by lower-quality host than A. aurantii. A. citricidus and A. aurantii had different strategies regarding Buchnera growth and the use of food in different stages of development. We observed differences in the draft genome of Buchnera associated to A. citricidus and A. aurantii. Host plant affected secondary symbiont abundance, but Spiroplasma was the most abundant symbiont in both aphids. Spiroplasma had neutral effects on A. citricidus biology, but affected host transcriptome and proteome. The host plant affected gene expression of A. citricidus, but the effect was dependent on Spiroplasma infection. Transcriptome analysis indicated Spiroplasma down-regulated aphid immune response genes on sweet orange, while regulating an entire different set of genes on orange jasmine, mainly chaperonins. Gene transcription of A. citricidus was strongly influenced by the host plant. But while a large number of transcripts were up-regulated in uninfected aphids in sweet orange as compared to orange jasmine, the same set of genes had an opposite pattern of expression in Spiroplasma-infected aphids. Comparative proteomic analysis of Spiroplasma-infected and uninfected aphids on sweet orange and orange jasmine demonstrated regulation of a larger number proteins on orange jasmine than on sweet orange. Spiroplasma down-regulated the immune response of aphids and up-regulated proteins related to nutritional processes when developing on a low-quality host plant, orange jasmine, while no such trend was observed on sweet orange.A associação insetos - bactérias simbiontes resulta em várias implicações bioecológicas para o hospedeiro e se estende em uma rede de interações que inclue outros microrganismos, o ambiente e outros níveis tróficos. A interação bactérias simbiontes e pulgões tem sido amplamente estudada, especialmente em espécies polífagas do hemisfério norte, tal como o pulgão modelo Acyrthosiphon pisum (Harris). Essas pesquisas indicam que simbiontes influenciam vários aspectos ecológicos do hospedeiro, tais como: nutrição, resistência ao estresse térmico e inimigos naturais, capacidade de transmissão de fitovírus, uso de plantas hospedeiras, entre outros. Entretanto, o resultado da interação simbionte-pulgão é contexto dependente, no qual o fenótipo depende da variabilidade intra- e interespecífica, da linhagem do simbionte e de diversos fatores bióticos e abióticos. Assim, este trabalho aborda as interações do pulgão Aphis (Toxoptera) citricidus (Kirkaldy) e seus simbiontes, com o objetivo de contribuir com um sistema ecológico diferente. Foi investigada a influência da estratégia alimentar na diversidade da microbiota, comparando-se uma espécie oligófaga, A. citricidus, a outra polífaga, Aphis (Toxoptera) aurantii Boyer de Fonscolombe. Foram utilizadas diversas abordagens experimentais (biológica, metagenômica, genômica, transcritômica e proteômica) para atingir os objetivos de i) investigar o impacto das plantas hospedeiras na aptidão biológica de A. citricidus e A. aurantii e sua consequência ao simbionte primário, ii) explorar as diferenças no genoma do simbionte primário de A. citricidus e A. aurantii, iii) investigar a influência da planta hospedeira na distribuição de simbiontes secundários em A. citricidus, iv) pesquisar a riqueza e abundância de simbiontes secundários nas duas espécies de pulgão e indagar sobre a influência de Spiroplasma v) na aptidão biológica, vi) no transcritoma e vii) na proteômica de A. citricidus em duas plantas hospedeiras (laranja vs. murta). Os resultados indicaram ser laranja o melhor hospedeiro para ambos pulgões, sendo A. citricidus mais sensível à planta hospedeira que A. aurantii. As duas espécies de pulgão utilizam estratégias distintas para controlar o crescimento de Buchnera, assim como a utilização do alimento influenciou diferentemente cada fase do desenvolvimento dos pulgões estudados. A planta hospedeira também influenciou a abundância de simbiontes secundários; porém, Spiroplasma foi o simbionte secundário mais abundante em ambas espécies. Spiroplasma não afetou a biologia de A. citricidus, mas causou alterações no transcritoma e no proteoma do hospedeiro. A planta hospedeira também exerceu forte regulação na transcrição gênica de A. citricidus, mas esse efeito foi dependente da infecção do pulgão por Spiroplasma. Análises de transcritoma em pulgões infectados indicaram a regulação de transcritos relacionados à resposta imunológica quando em laranja, mas de chaperoninas em murta. A regulação gênica de A. citricidus foi fortemente influenciada pela planta hospedeira, mas enquanto insetos livres de Spiroplasma apresentaram superexpressão gênica em laranja quando comparada a murta, pulgões infectados com esse simbionte apresentaram padrão de expressão oposto para o mesmo conjunto de transcritos. Análises comparativas do proteoma de pulgões infectados ou não por Spiroplasma nas plantas hospedeiras estudadas indicaram diminuição dos mecanismos de defesa em favor de um aumento de proteínas ligadas à nutrição em insetos infectados quando se alimentando em murta

Genetic diversity of Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae) and molecular characterization of the associate strains of Wolbachia

Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae) é um inseto exótico no Brasil, registrado desde a década de 40. Apesar dos danos diretos que pode causar, só recebeu atenção como praga em 2004, quando foi confirmada a doença Huanglonbing (HLB) em citros, a qual foi posteriormente relacionada a D. citri como inseto-vetor. Atualmente, não há cura para plantas doentes. Portanto, o manejo do HLB é baseado na retirada de árvores infectadas e no controle dos psilídeos, o qual se fundamenta no controle biológico clássico, ação natural de predadores e no controle químico. Porém, o uso contínuo e intenso de agrotóxicos é sempre problemático devido ao desequilíbrio que pode causar nos agroecossistemas. Portanto, investigar as populações de D. citri no Brasil e discutir possíveis medidas alternativas para o controle ou manejo do vetor/doença são desejáveis. Assim, este trabalho apresenta como objetivos i) avaliar a diversidade genética intra e interpopulacional de D. citri e verificar a existência de estruturação populacional no Brasil, especialmente entre as populações que se distribuem na principal região citrícola do país; ii) determinar a ocorrência e frequência de infecção por Wolbachia em diferentes populações desse psilídeo, além de caracterizar a diversidade desse simbionte e definir a relação entre os haplótipos do hospedeiro e as linhagens de Wolbachia,visando fornecer subsídios para o controle alternativo deste inseto-vetor. A análise de diversidade genética utilizou o gene da citocromo oxidase I (COI) como marcador molecular. Os cálculos dos índices de fixação F indicaram a presença de estrutura genética e os cálculos dos índices de diversidade molecular mostraram um quadro de expansão populacional recente. Já a caracterização de Wolbachia via tipagem pela análise de sequências de multilocos (multilocus sequencing typing - MLST) indicou a ocorrência de cinco STs (173, 174, 175, 225 e 236), enquanto que análises das sequências do wsp indicaram quatro tipos de Wolbachia. O ST-173 foi predominante, estando presente em todas as populações, enquanto que os demais foram populações-específicos. Análises da relação hospedeiro-simbionte não revelou a existência de relações específicas entre os haplótipos do hospedeiro e os STs de Wolbachia, nem a diminuição da diversidade nucleotídica de D. citri frente à infecção por mais de um ST do simbionte. A relação entre os STs de Wolbachia e as consequências desta diversidade na bioecologia do hospedeiro ainda permanecem desconhecidas, mas a constatação de que Wolbachia ocorre em todas as populações e de que há um ST com distribuição abrangente abre caminhos para a utilização desse simbionte no desenvolvimento de estratégias alternativas de controle de D. citri.Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae) is an exotic insect in Brazil, first recorded in the 40s. Although it causes direct damage, D. citri only received attention as a pest in 2004, when the \"Huanglonbing\" (HLB) disease was confirmed infecting citrus in Brazil, and subsequently related to D. citri as vector. Currently, there is no cure for diseased plants, and the management of HLB is based on the removal of infected trees and psyllid control, which is based on classical biological control, natural predation and chemical control. However, the continuous use of pesticides is always problematic due to the imbalance it can cause in agrosystems. Therefore, investigations on the populations structure of D. citri in Brazil and possible alternative measures for vector/disease control or management are highly desired. Thus, this study was aimed to i) evaluate the intra and interpopulation genetic diversity of D. citri and verify the existence of population structuring in Brazil, especially in the main citrus producing region of the country, ii) determine the natural rate of infection and characterize the Wolbachia strains associated with different populations of D. citri and iii) investigate the relationship among host haplotypes and the strains of Wolbachia, in order to provide data for the alternative control of this vector. The analysis of genetic diversity was done using the cytochrome oxidase I (COI) gene as a molecular marker. The fixation rates F calculated indicated the presence of genetic structure, while the molecular diversity indices pointed to a recent population expansion of D. citri in Brazil. The multilocus sequence typing analysis (\"multilocus sequencing typing - MLST\") for Wolbachia demonstrated the presence of five new sequence-types (STs = 173, 174, 175, 225 and 236), while only four were identified when using the wsp sequences as markers. The ST-173 was the predominant ST associated with D. citri, being present in all populations, while others were specific to certain populations. Analysis of the hostsymbiont relationship did not reveal the existence of specific interactions between host haplotypes and the STs of Wolbachia, or the decrease of nucleotide diversity of D. citri in populations infected by more than one ST. The effects of Wolbachia diversity on the host bioecology are still unknown, but the fact that Wolbachia is fixed in all populations and that there is a ST broadly distributed throughout the D. citri populations highlights the use of Wolbachia for the development of alternative strategies to control D. citri

Spatio-temporal variation of aquatic macrophyte cover in a reservoir using landsat images and google earth engine

Orientador: Prof. Dr. Tobias BleningerCoorientadora: Dra.Bruna Arcie PolliDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Recursos Hídricos e Ambiental. Defesa : Curitiba, 12/08/2022Inclui referências: p. 59-66Área de concentração:Resumo: A presença de grandes quantidades de macrófitas aquáticas em reservatórios pode desencadear diversos impactos no ecossistema local e o uso de metodologias convencionais para seu monitoramento, como coletas in loco, retorna apenas informações do momento presente. Com isso em vista, este estudo teve como objetivo mapear a variação espaço-temporal da cobertura de macrófitas utilizando imagens Landsat 5, 7 e 8 para o período de 1984-2021 no reservatório Jupiá. Também foram verificadas como algumas variáveis ambientais que impactam no crescimento de macrófitas (como variação do uso do solo, temperatura, precipitação e vazões de entrada no reservatório) se comportaram ao longo dos anos. Além disso, também foram construídas curvas e mapas da permanência de macrófitas nesse reservatório para serem usadas como base de comparação de novos mapeamentos. Para realizar isto, foi necessário determinar o melhor conjunto de índice espectral e intervalo de classificação das macrófitas. Como principais resultados, foram observadas variações interanuais na disposição espacial das macrófitas e tendência de crescimento da área, sendo este último possivelmente causado pela implantação de um reservatório a montante do reservatório Jupiá. Foi observado que além da magnitude da vazão, a forma como há incremento nas vazões também é relevante em termos de deslocamento de macrófitas dentro do reservatório. Apesar da quantidade de imagens sem interferências ter sido uma limitação, a construção de uma série histórica de ocupação de macrófitas se mostrou satisfatória e poderá auxiliar na tomada de decisões de manejo desses organismos.Abstract: The presence of large amounts of aquatic macrophytes in reservoirs can trigger several impacts on the local ecosystem and the use of conventional methodologies for their monitoring, such as in loco activities, only returns information from the present moment. With that in mind, this study aimed to map the spatiotemporal variation of macrophyte cover using Landsat 5, 7 and 8 images for the period 1984-2021 at Jupiá reservoir, in southeast of Brazil. It was also verified how some environmental variables that impact the growth of macrophytes (such as variation in land use, temperature, precipitation and reservoir inflows) behaved over the years. In addition, curves and maps of the permanence of macrophytes in this reservoir were also constructed to be used as a basis for comparing new mappings. To accomplish this, it was necessary to determine the best set of spectral index and classification range of macrophytes. As main results, interannual variations were observed in the spatial arrangement of macrophytes and the area's growth trend, the latter possibly caused by the implementation of a reservoir upstream of the Jupiá reservoir. It was observed that in addition to the magnitude of the flow, the way in which there is an increase in the flows is also relevant in terms of the displacement of macrophytes within the reservoir. Although the number of images without interference was a limitation, the construction of a historical series of macrophyte occupation proved to be satisfactory and could help in decision-making for the management of these organisms

The genetic structure of an invasive pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid Diaphorina citri is currently the major threat to the citrus industry as it is the vector of Candidatus Liberibacter, the causal agent of huanglongbing disease (HLB). D. citri is native to Asia and now colonizes the Americas. Although it has been known in some countries for a long time, invasion routes remain undetermined. There are no efficient control methods for the HLB despite the intensive management tools currently in use. We investigated the genetic variability and structure of populations of D. citri to aid in the decision making processes toward sustainable management of this species/disease. We employed different methods to quantify and compare the genetic diversity and structure of D. citri populations among 36 localities in Brazil, using an almost complete sequence of the cytochrome oxidase I (COI) gene. Our analyses led to the identification of two geographically and genetically structured groups. The indices of molecular diversity pointed to a recent population expansion, and we discuss the role of multiple invasion events in this scenario. We also argue that such genetic diversity and population structure may have implications for the best management strategies to be adopted for controlling this psyllid and/or the disease it vectors in Brazil

Chitin-degrading enzymes from an actinomycete ectosymbiont of Acromyrmex subterraneus brunneus (Hymenoptera: Formicidae)

Microbes have many mechanisms to exert their inhibitory activity against target pests. One such mechanism involves the production and secretion of hydrolytic enzymes, such as chitinases, which are produced naturally by plants in response to attack by insect herbivores and phytopathogens and have been sought as an additional factor to enhance pest management. Thus, our main aim was to screen the diverse actinomycete community associated with the integument of Acromyrmex subterraneus brunneus for a chitinase-producing strain and to characterize its chitinases. We identified isolate ENT-21-a Streptomyces sp.-as a chitinase-producer and our data indicate that this isolate produces a chitinolytic complex that contains a chitinase and a high-molecular-weight beta-N-acetylglucosaminidase (> 100 kDa) when cultured in Chitin-Czapek broth. The presence of chitinases in the genome of this isolate was checked by diagnostic PCR, and two chitinase genes belonging to family 18 group A and family 19 were verified. The chitinolytic activity of the crude extract was observed at pH values ranging from 3.8 to 11.0, with the highest chitinase activities recorded at pH 9.0 and 9.5, whereas optimum beta-N-acetylglucosaminidase activity was observed over a narrow pH range, between pH 4.7 and 5.1. We describe some biochemical and molecular properties of the chitinase and beta-N-acetylglucosaminidase produced by ENT-21, and discuss the potential for exploitation of these enzymes for pest control.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP