Project description:The migratory locust, Locusta migratoria, is a serious agricultural pest and important insect model to study insect digestion and feeding behavior. The gut is one of the primary interfaces between the insect and its environment. Nevertheless, knowledge on the gut transcriptome of L. migratoria is still very limited. With the development of two EST databases from L. migratoria (whole body and central nervous system (CNS)) and one EST database from Schistocerca gregaria (CNS), an abundance of transcript data was made available for locusts. In addition, the genome of Locusta was also recently published in an effort to create a better understanding of swarm formation and flight behavior (Wang et al., 2014). While the transcript composition of nervous tissue was relatively well studied after the development of the specific CNS-derived EST-databases from both L. migratoria and S. gregaria, little transcript profiling information is available for the digestive system at the moment. Locusts are, however, widely used as physiological model organisms regarding the regulation and control of feeding and digestion, and improved knowledge on the gut transcriptome could contribute significantly to a better understanding of their gut physiology. Therefore, we aimed to use the available sequence data to specifically identify gut-expressed transcripts in 5th larval locusts. By means of two independent self-self microarray hybridizations for two distinct tissues, the gut and the brain, a selection could be made of those ESTs that are present in the gut and/or the brain. Here, sequences that were found to be expressed in gut but not brain were further functionally annotated to shed new light on the complex physiology of the locust digestive system. Since the gut is the single most important organ in digestion, and both tissues are assumed to be involved in the regulation thereof, the resulting subset of sequences can also be valuable for further in-depth studies on the regulation of digestion. In addition, the method allowed us to rank the signal intensities, using them as a rough indicator to compare relative transcript abundance in the gut. Therefore, the data complement previously published transcript and genomic data, and provide a clear overview of the expressed portion of the genome in the gut. Taken together, the present data provide significant insight into locust larval gut physiology, and will be valuable for future studies on the insect gut. Self-self hybridisation of Cy5- and Cy3-labeled samples. One biological repeat per tissue type, i.e., brain and gut. Gut is a combination of foregut, midgut, gastric caeca and hindgut. Per tissue type, a pool was made from RNA from 3 pools of 5 locusts, and this for 3 different feeding conditions, resulting in samples derived from a total of 45 locust larvae. Feeding conditions were normally fed, fed with diet containing additional protease inhibitors (PIs), and starved locusts.
Project description:A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC₅₀s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.
Project description:Diapause is a state of arrested growth, which allows insects to adapt to diverse environments. Serine protease inhibitors (serpins) play an important role in various physiological processes, including blood coagulation, fibrinolysis, development, complement activation and extracellular matrix remodeling. We hypothesized that serpin may affect energy metabolism and thereby control diapause of migratory locust (Locusta migratoria) embryos by regulating protease cascades. A total of seven nonredundant serpin genes (named serpin1-serpin7) of L. migratoria were obtained through transcriptomic analysis. We further performed label-free proteomic sequencing and analysis of diapause and nondiapause eggs of L. migratoria, revealing significant differences in serpin7 expression. A significant reduction in diapause rate under the short photoperiod was observed in insects treated with serpin7 double-stranded RNA. Furthermore, knockdown of the serpin7 gene resulted in significant upregulation of the activity of polyphenol oxidase. We therefore propose that the observed serpin7 gene plays a crucial role in diapause, suggesting that control of energy metabolism may have potential as a future strategy for the reproductive control of insect pests.
Project description:Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1?, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1?, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1? and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts.
Project description:Ontogenetic locomotion research focuses on the evolution of locomotion behavior in different developmental stages of a species. Unlike vertebrates, ontogenetic locomotion in invertebrates is poorly investigated. Locusts represent an outstanding biological model to study this issue. They are hemimetabolous insects and have similar aspects and behaviors in different instars. This research is aimed at studying the jumping performance of Locusta migratoria over different developmental instars. Jumps of third instar, fourth instar, and adult L. migratoria were recorded through a high-speed camera. Data were analyzed to develop a simplified biomechanical model of the insect: the elastic joint of locust hind legs was simplified as a torsional spring located at the femur-tibiae joint as a semilunar process and based on an energetic approach involving both locomotion and geometrical data. A simplified mathematical model evaluated the performances of each tested jump. Results showed that longer hind leg length, higher elastic parameter, and longer takeoff time synergistically contribute to a greater velocity and energy storing/releasing in adult locusts, if compared to young instars; at the same time, they compensate possible decreases of the acceleration due to the mass increase. This finding also gives insights for advanced bioinspired jumping robot design.