Genome Analysis of Cytochrome P450s and Their Expression Profiles in Insecticide Resistant Mosquitoes, Culex quinquefasciatus

Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab) and resistant mosquito populations, two different field populations of mosquitoes (HAmCq and MAmCq), and field parental mosquitoes (HAmCq G0 and MAmCqG0) and their permethrin selected offspring (HAmCq G8 and MAmCqG6). While the majority of the P450 genes were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental strains and the susceptible S-Lab strain, HAmCqG8 and MAmCqG6 were found to up-regulate 11 and 6% of total P450 genes in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults. Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or down-regulated in the HAmCqG8 and MAmCqG6 mosquito populations in response to insecticide selection. Several genes were identified as being up- or down-regulated in either the larvae or adults for both HAmCqG8 and MAmCqG6; of these, CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450 genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of mosquitoes to changes in cellular environment

Padrão do acúmulo do fitoecdisteróide 20-hidroxiecdisona em diferentes órgãos de Pfaffia glomerata

Os ecdisteróides são hormônios esteróides encontrados em insetos e plantas. Em insetos, vários processos que ocorrem durante o seu desenvolvimento são iniciados ou controlados por pulsos específicos do zooecdisteróide 20-hidroxiecdisona (20E). Em plantas, a biossíntese, a regulação e a função precisa do fitoecdisteróide 20E permanecem desconhecidas e três hipóteses sobre suas funções potenciais têm sido propostas: i) compostos fitohormonais; ii) compostos protetores contra insetos fitófagos não adaptados; e iii) fonte de fitoesteróides polihidroxilados requeridos no crescimento e proliferação celular. A 20E foi detectada nas raízes de Pfaffia glomerata, que foi selecionada para a condução de outros experimentos. Entre os 71 acessos de P. glomerata analisados, o maior acúmulo de 20E (1,48 g) foi encontrado nas raízes do acesso 13, que foi escolhido para a realização das análises de altura, massa seca, detecção, concentração e acúmulo de 20E. Durante o desenvolvimento do acesso 13, foram identificadas três fases distintas. Na primeira fase (0 60 dias), ocorreu intenso aumento da altura em combinação com um baixo acúmulo de matéria seca. Em contraste, durante a segunda fase (60 120 dias), ocorreu intenso acúmulo de matéria seca e um pequeno aumento da altura. Durante a terceira fase (120 210 dias), ocorreu acúmulo de matéria seca menos intenso em combinação com um pequeno aumento da altura. A 20E foi constantemente detectada em todos os órgãos analisados, mas sua concentração variou durante o desenvolvimento de P. glomerata. As maiores concentrações de 20E foram seqüencialmente encontradas em flores (0.8221%), raízes (0,6658%), folhas (0,6042%), e caules (0,2445%). Em contraste, as menores concentrações de 20E foram consecutivamente encontradas em caules (0,1379%), folhas (0,2118%), raízes (0,4224%), e flores (0,4731%). Enquanto os caules apresentaram as menores variações nas concentrações de 20E (0,1379% - 0,2445%), seguidos pelas raízes (0,4224% - 0,6658%) e flores (0,4731% - 0,8221%), as folhas apresentaram as maiores variações nas concentrações de 20E (0,2118% - 0,6042%). Apesar da existência de variação na porcentagem de 20E em cada órgão analisado, o seu acúmulo apresentou um padrão específico. O padrão do acúmulo de 20E revelou que este foi predominantemente acumulado nas raízes (após 60 dias), enquanto nas folhas (após 60 dias) e nos caules (após 120 dias) o conteúdo total de 20E foi mantido constante, apresentando pequena variação.Universidade Federal Rural da AmazôniaThe ecdysteroids are steroid hormones found in insects and plants. In insects, several developmental processes are controlled or elicited by specific pulses of the zooecdysteroid 20-hydroxyecdysone (20E). In plants, the biosynthesis, regulation, and precise functions of the phytoecdysteroid 20E remain unknown and three major hypotheses for their potential roles have been proposed: i) phytohormonal compounds; ii) protective compounds against unadapted phytophagous insects; and iii) supply of polyhydroxylated phytosterols required for growth and cell proliferation. The 20E was detected in the roots of P. glomerata that was chosen to carry out further investigations. Among the 71 analyzed accessions of P. glomerata, the highest root 20E accumulation (1.48 g) was found in the accession 13, which was selected to perform analysis of height, dry matter, 20E occurrence, concentration and accumulation. During the development of the accession 13, it was identified three distinct phases. In the first (0 60 days), an intense increase of height occurred in combination with a lower dry matter accumulation. In contrast, during the second (60 120 days), an intense dry matter accumulation occurred with a lower increase of height. During the third (120 210 days), a less intense dry matter accumulation occurred in combination with a lower increase of height. The 20E was constantly detected in all analyzed organs, but its concentration was variable throughout the development of P. glomerata. The highest 20E concentration was sequentially found in flowers (0.8221%), roots (0.6658%), leaves (0.6042%), and stems (0.2445%). In contrast, the lowest 20E concentration was consecutively found in stems (0.1379%), leaves (0.2118%), roots (0.4224%), and flowers (0.4731%). While stems showed the less variable 20E concentration (0.1379% - 0.2445%), followed by roots (0.4224% - 0.6658%), and flowers (0.4731% - 0.8221%), leaves showed the more variable 20E concentration (0.2118% - 0.6042%). There is a variation of 20E percentage in each analyzed organ, but its accumulation followed a specific pattern. The accumulation pattern of 20E revealed that 20E was predominantly accumulated in roots (after 60 days) whereas in leaves (after 60 days) and stems (after 120 days), the 20E total content was kept constant, showing a little variation

Structure, organization, and functions of cellulose synthase complexes in higher plants

Annually, plants produce about 180 billion tons of cellulose making it the largest reservoir of organic carbon on Earth. Cellulose is a linear homopolymer of β(1-4)-linked glucose residues. The coordinated synthesis of glucose chains is orchestrated by specific plasma membrane-bound cellulose synthase complexes (CelS). The CelS is postulated to be composed of approximately 36 cellulose synthase (CESA) subunits. The CelS synthesizes 36 glucose chains in close proximity before they are further organized into microfibrils that are further associated with other cell wall polymers. The 36 glucose chains in a microfibril are stabilized by intra- and inter-hydrogen bonding which confer great stability on microfibrils. Several elementary microfibrils come together to form macrofibrils. Many CESA isoforms appear to be involved in the cellulose biosynthetic process and at least three types of CESA isoforms appear to be necessary for the functional organization of CelS in higher plants

Azadirachtin biosynthesis induction in Azadirachta indica A. Juss cotyledonary calli with elicitor agents

The use of cell and plant tissues culture techniques to produce economically important active metabolites has been growing. Among these substances, azadirachtin (AZA), produced by the neem tree (Azadirachta indica), has received considerable attention due to its bioinsecticide action. The main goal of this work was to analyze the AZA levels in neem cotyledonary calli. The calli were grown in agitated Woody Plant Medium (WPM) liquid medium, supplemented with glucose (Gl), hydrolyzed casein (HC) and methyl jasmonate (MeJ) as elicitor agent. An interaction was observed between these substances, depending on in vitro cultivation time with orbital agitation. The highest concentrations (average of 0.2470 µg g-1) of AZA were produced in the first and second weeks of culture when the cell mass was grown in a medium with 2% Gl v/v, 500 mg L-1 HC and 100 µM of MeJ. This corresponded to approximately 57% of the AZA content stored in the donor plants seeds, used as a source of explants to induce in vitro callus formation. It was concluded that the nutrition, as well as the concentration of MeJ as signal transduction of secondary metabolism in neem cells, might influence the AZA content produced in vitro

Effects of flask sealing and growth regulators on in vitro propagation of neem (Azadirachta indica A. Juss.)

In vitro propagation of neem (Azadirachta indica A. Juss.) may offer an efficient alternative to seed propagation of this species. For optimization of in vitro propagation, different basal salt formulations, growth regulators, and culture container sealants (polytetrafluoroethylene hydrophobic membranes [PTFE]) were evaluated. Nodal segments cultured on Murashige and Skoog (MS) medium showed the highest shoot formation per explant (1.67). Explants cultured in flasks containing MS medium with 0.5 mg L−1 benzyladenine, 0.5 mg L−1 kinetin, and 0.05 mg L−1 naphthaleneacetic acid, and sealed with two PTFE membranes, produced the highest number of shoots (4.04). In contrast, explants cultured in flasks without membranes showed leaf chlorosis and senescence. For plant recovery, regenerants were acclimatized in a substrate of coconut fiber and eucalyptus bark (1:1) and showed 80% survival. Our results indicated that the use of flasks with vents was beneficial for in vitro propagation of this important plant

Biochemical and morpho-anatomical analyses of strawberry vitroplants hyperhydric tissues affected by BA and gelling agents

In vitro propagation has become an effective practice for large-scale production of strawberry plants. The objective of this study was to evaluate the hyperhydricity and the multiplication capacity of two strawberry varieties (Fragaria x ananassa Duch. 'Dover' and 'Burkley') propagated in vitro. Plants maintained in MS medium supplemented with 1.0 mg L-1 BA were individualized and transferred to the same medium solidified with Agar (6.5 g L-1) or Phytagel® (2.5 g L-1) and BA at different concentrations (0; 0.5; 1.0; 2.0 and 3.0 mg L-1). Biochemical and anatomical analyses were carried out, as well as the analysis of the morphological hyperhydricity characteristics. The analysis of data showed: a) the increase in cytokinin concentration increased hyperhydricity frequency in both varieties; b) at concentrations up to 2.0 mg L-1 BA, the replacement of Agar by Phytagel® induced a higher formation of hyperhydric shoots; and c) the addition of BA induced oxidative stress, which is characterized by increased antioxidant activity and lipid peroxidation, as well as alterations at the cellular level, such as malformation of stomata and epidermal cells. In conclusion, the culture medium containing 0.5 mg L-1 BA solidified with Agar provided lower hyperhydricity percentages in association with higher rates of shoot proliferation in strawberry.A propagação in vitro tem se destacado como uma técnica efetiva na produção em larga escala de plantas sadias de morangueiro. Neste trabalho estudamos a hiperhidricidade associado a capacidade de multiplicação in vitro na propagação de duas variedades de morangueiro (Fragaria x ananassa Duch. "Dover" e "Burkley"). Plantas mantidas em meio de cultura MS, suplementadas com 1.0 mg L-1 de BA fora individualizadas e transferidas para o mesmo meio com Ágar (6.5 g L-1) ou Phytagel® (2,5 g L-1) e BA em diferentes concentrações (0; 0,5; 1,0; 2,0 e 3,0 mg L-1). Foram realizadas análises bioquímica e anatômicas, além da caracterização morfológica do material hiper-hídrico. A análise dos dados mostrou: a) o incremento na concentração de citocinina aumentou a freqüência de hiper-hidricidade para ambas as variedades; b) concentrações maiores que 2,0 mg L-1 de BA, com a substituição do Ágar pelo Phytagel® induziu a maior formação de ramos hiper-hídricos; e c) a adição de BA induziu o estresse oxidativo, caracterizado pelo incremento da atividade antioxidante e peroxidação de lipídeos, bem como alterações a nível celular, como má formação dos estômatos e células epidérmicas. Concluindo, o meio de cultura contendo 0,5 mg L-1 de BA solidificado com Ágar promoveu menor porcentagem de hiper-hidricidade associado com maiores taxas de proliferação de gemas em morangueiro