Identification of Halloween Genes and RNA Interference-Mediated Functional Characterization of a Halloween Gene shadow in Plutella xylostella
KeywordEcdysteroids – Genetic Aspects
Ecdysteroids – Analysis
RNA – Genetic Aspects
Genetic Research – Genetic Aspects
Genetic Research – Analysis
Genes – Genetic Aspects
Genes – Analysis
RNA – Analysis
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AbstractEcdysteroids play an essential role in controlling insect development and reproduction. Their pathway is regulated by a group of enzymes called Halloween gene proteins. The relationship between the Halloween genes and ecdysteroid synthesis has yet to be clearly understood in diamondback moth, Plutella xylostella (L.), a worldwide Lepidoptera pest attacking cruciferous crops and wild plants. In this study, complete sequences for six Halloween genes, neverland ( nvd ), shroud ( sro ), spook ( spo ), phantom ( phm ), disembodied ( dib ), shadow ( sad ), and shade ( shd ), were identified. Phylogenetic analysis revealed a strong conservation in insects, including Halloween genes of P. xylostella that was clustered with all other Lepidoptera species. Three Halloween genes, dib , sad , and shd were highly expressed in the adult stage, while nvd and spo were highly expressed in the egg and pupal stages, respectively. Five Halloween genes were highly expressed specifically in the prothorax, which is the major site of ecdysone production. However, shd was expressed predominantly in the fat body to convert ecdysone into 20-hydroxyecdysone. RNAi-based knockdown of sad , which is involved in the last step of ecdysone biosynthesis, significantly reduced the 20E titer and resulted in a longer developmental duration and lower pupation of fourth-instar larvae, as well as caused shorter ovarioles and fewer fully developed eggs of P. xylostella . Furthermore, after the knockdown of sad , the expression levels of Vg and VgR genes were significantly decreased by 77.1 and 53.0%. Meanwhile, the number of eggs laid after 3 days was significantly reduced in sad knockdown females. These results suggest that Halloween genes may play a critical role in the biosynthesis of ecdysteroids and be involved in the development and reproduction of P. xylostella . Our work provides a solid basis for understanding the functional importance of these genes, which will help to screening potential genes for pest management of P. xylostella.
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Mechanism and consequences for avoidance of superparasitism in the solitary parasitoid Cotesia vestalisChen, Wen-Bin; Vasseur, Liette; Zhang, Shuai-Qi; Zhang, Han-Fang; Mao, Jun; Liu, Tian-Sheng; Zhou, Xian-Yong; Wang, Xin; Zhang, Jing; You, Min-Sheng; et al. (Nature Research, 2020)A parasitoid's decision to reject or accept a potential host is fundamental to its fitness. Superparasitism, in which more than one egg of a given parasitoid species can deposit in a single host, is usually considered sub-optimal in systems where the host is able to support the development of only a single parasitoid. It follows that selection pressure may drive the capacity for parasitoids to recognize parasitized hosts, especially if there is a fitness cost of superparasitism. Here, we used microsatellite studies of two distinct populations of Cotesia vestalis to demonstrate that an egg laid into a diamondback moth (Plutella xylostella) larva that was parasitized by a conspecific parasitoid 10 min, 2 or 6 h previously was as likely to develop and emerge successfully as was the first-laid egg. Consistent with this, a naive parasitoid encountering its first host was equally likely to accept a healthy larva as one parasitized 10 min prior, though handling time of parasitized hosts was extended. For second and third host encounters, parasitized hosts were less readily accepted than healthy larvae. If 12 h elapsed between parasitism events, the second-laid egg was much less likely to develop. Discrimination between parasitized and healthy hosts was evident when females were allowed physical contact with hosts, and healthy hosts were rendered less acceptable by manual injection of parasitoid venom into their hemolymph. Collectively, these results show a limited capacity to discriminate parasitized from healthy larvae despite a viability cost associated with failing to avoid superparasitism.
Non-photorealistic Rendering with Cartesian Genetic Programming using Graphic Processing UnitsBakurov, Illya; Department of Computer ScienceNon-photorealistic rendering (NPR) is concerned with the algorithm generation of images having unrealistic characteristics, for example, oil paintings or watercolour. Using genetic programming to evolve aesthetically pleasing NPR images is a relatively new approach in the art field, and in the majority of cases it takes a lot of time to generate results. With use of Cartesian genetic programming (CGP) and graphic processing units (GPUs), we can improve the performance of NPR image evolution. Evolutionary NPR can render images with interesting, and often unexpected, graphic effects. CGP provides a means to eliminate large, inefficient rendering expressions, while GPU acceleration parallelizes the calculations, which minimizes the time needed to get results. By using these tools, we can speed up the image generation process. Experiments revealed that CGP expressions are more concise, and search is more exploratory, than in tree-based approaches. Implementation of the system with GPUs showed significant speed-up.
The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cellsCentre for Biotechnology (Brock University, 2005-05-28)Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.