Transcriptome responses to heat- and cold-stress in ladybirds (Cryptolaemus montrouzieri Mulasnt) analyzed by deep-sequencing

BACKGROUND: Changed temperature not only threaten agricultural production, but they also affect individual biological behavior, population and community of many insects, and consequently reduce the stability of our ecosystem. Insect's ability to respond to temperature stress evolved through a complex adaptive process, thus resulting in varied temperature tolerance among different insects. Both high and low extreme temperatures are detrimental to insect development since they constitute an important abiotic stress capable of inducing abnormal biological responses. Many studies on heat or cold tolerance of ladybirds have focused on measurements of physiological and biochemical indexes such as supercooling point, higher/lower lethal temperatures, survival rate, dry body weight, water content, and developmental duration. And studies of the molecular mechanisms of ladybird responses to heat or cold stress have focused on single genes, such as those encoding heat shock proteins, but has not been analyzed by transcriptome profiling. RESULTS: In this study, we report the use of Digital Gene Expression (DGE) tag profiling to gain insight into transcriptional events associated with heat- and cold-stress in C. montrouzieri. About 6 million tags (49 bp in length) were sequenced in a heat stress group, a cold stress group and a negative control group. We obtained 687 and 573 genes that showed significantly altered expression levels following heat and cold shock treatments, respectively. Analysis of the global gene expression pattern suggested that 42 enzyme-encoding genes mapped to many Gene Ontology terms are associated with insect's response to heat- and cold-stress. CONCLUSIONS: These results provide a global assessment of genes and molecular mechanisms involved in heat and cold tolerance.

Efeito de íons Cu2+ e Zn2+ em atividade Ca-ATPásica isolada de larvas de Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) / Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae

As ATPases, um importante alvo de inseticidas, são enzimas que hidrolisam o ATP e utilizam a energia liberada no processo para realizar algum tipo de trabalho celular. A larva de Pachymerus nucleorum (Fabricius) possui uma ATPase que apresenta alta atividade Ca-ATPásica, mas não expressa atividade Mg-ATPásica. Nesse trabalho, foi testado o efeito de íons zinco e cobre na atividade Ca-ATPásica dessa enzima. Mais de 90 por cento da atividade Ca-ATPásica foi inibida em 0,5 mM de íons cobre ou 0,25 mM de íons zinco. Na presença de EDTA, mas não na sua ausência, a inibição por zinco foi revertida pelo aumento da concentração de cálcio. A inibição por íons cobre, não foi revertida nem na presença e nem na ausência de EDTA. O tratamento da fração ATPase com cobre, previamente ao ensaio de atividade ATPásica, não inibiu a atividade Ca-ATPásica sugerindo que o íon cobre não liga diretamente a enzima. Os resultados sugerem que íons zinco e cobre formam complexo com o ATP e se ligam à enzima inibindo sua atividade Ca-ATPásica.

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90 percent of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae)

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.