Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.
BackgroundThe expression of intermediate filaments (IFs) is a hallmark feature of metazoan cells. IFs play a central role in cell organization and function, acting mainly as structural stress-absorbing elements. There is growing evidence to suggest that these cytoskeletal elements are also involved in the integration of signalling networks. According to their fundamental functions, IFs show a widespread phylogenetic expression, from simple diblastic animals up to mammals, and their constituent proteins share the same molecular organization in all species so far analysed. Arthropods represent a major exception in this scenario. Only lamins, the nuclear IF proteins, have so far been identified in the model organisms analysed; on this basis, it has been considered that arthropods do not express cytoplasmic IFs.ResultsHere, we report the first evidence for the expression of a cytoplasmic IF protein in an arthropod - the basal hexapod Isotomurus maculatus. This new protein, we named it isomin, is a component of the intestinal terminal web and shares with IFs typical biochemical properties, molecular features and reassembly capability. Sequence analysis indicates that isomin is mostly related to the Intermediate Filament protein C (IFC) subfamily of Caenorhabditis elegans IF proteins, which are molecular constituents of the nematode intestinal terminal web. This finding is coherent with, and provides further support to, the most recent phylogenetic views of arthropod ancestry. Interestingly, the coil 1a domain of isomin appears to have been influenced by a substantial molecular drift and only the aminoterminal part of this domain, containing the so-called helix initiation motif, has been conserved.ConclusionsOur results set a new basis for the analysis of IF protein evolution during arthropod phylogeny. In the light of this new information, the statement that the arthropod phylum lacks cytoplasmic IFs is no longer tenable.See commentary article: http://www.biomedcentral.com/1741-7007-9-16.
Farnesoic acid O-methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative-FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real-Time PCR in medfly pre-imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre-imaginal putative-FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well-known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control.
A gene potentially involved in juvenile hormone (JH) biosynthesis was previously identified in Ceratitis capitata as the putative-farnesoic acid O-methyltransferase (FAMeT). Since JH is involved in insect reproduction, we silenced the putative-FAMeT expression by RNA interference in Ceratitis capitata to evaluate its implication in egg production. FAMeT gene expression was knocked down in females and males after eclosion and in 1- and 2-day-old females. Treated specimens were left to mate with each other or with untreated partners to evaluate the extent of each sex influencing egg production. Gene silencing was investigated by Real-Time PCR. Results unambiguously showed that FAMeT has a measurable role on the fertility of both medfly sexes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.