Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions

<p>Abstract</p> <p>Background</p> <p>To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, <it>Schistocerca gregaria </it>and two different software programs (geNorm and Normfinder) were applied to assess the stability of thesegenes.</p> <p>Results</p> <p>We have identified seven orthologs of commonly used housekeeping genes in the desert locust. The selected genes were the orthologs of actin, <it>EF1a, GAPDH, RP49, TubA1, Ubi</it>, and <it>CG13220</it>. By employing real time RT-PCR we have analysed the expression of these housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated <it>Sg-EF1a</it>, <it>Sg-GAPDH </it>and <it>Sg-RP49 </it>as most stable genes, while Normfinder ranked <it>Sg-RP49</it>, <it>Sg-EF1a </it>and <it>Sg-ACT </it>as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were <it>Sg-EF1a, Sg-GAPDH </it>and <it>Sg-Ubi </it>according to geNorm, while Normfinder determined <it>Sg-GAPDH</it>, <it>Sg-Ubi </it>and <it>Sg-ACT </it>as the most stable housekeeping genes.</p> <p>Conclusion</p> <p>To perform transcript profiling studies on brains of the desert locust, the use of <it>Sg-RP49</it>, <it>Sg-EF1a </it>and <it>Sg-ACT </it>as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were <it>Sg-GAPDH</it>, <it>Sg-Ubi </it>and <it>Sg-EF1a</it>. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.</p

An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype

Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis.status: publishe

Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster

Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage.publisher: Elsevier articletitle: Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster journaltitle: Peptides articlelink: http://dx.doi.org/10.1016/j.peptides.2014.07.014 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

Molecular characterization of a short neuropeptide F signaling system in the tsetse fly, Glossina morsitans morsitans

Neuropeptides of the short neuropeptide F (sNPF) family are widespread among arthropods and found in every sequenced insect genome so far. Functional studies have mainly focused on the regulatory role of sNPF in feeding behavior, although this neuropeptide family has pleiotropic effects including in the control of locomotion, osmotic homeostasis, sleep, learning and memory. Here, we set out to characterize and determine possible roles of sNPF signaling in the haematophagous tsetse fly Glossina morsitans morsitans, a vector of African Trypanosoma parasites causing human and animal African trypanosomiasis. We cloned the G. m. morsitans cDNA sequences of an sNPF-like receptor (Glomo-sNPFR) and precursor protein encoding four Glomo-sNPF neuropeptides. All four Glomo-sNPF peptides concentration-dependently activated Glomo-sNPFR in a cell-based calcium mobilization assay, with EC50 values in the nanomolar range. Gene expression profiles in adult female tsetse flies indicate that the Glomo-sNPF system is mainly restricted to the nervous system. Glomo-snpfr transcripts were also detected in the hindgut of adult females. In contrast to the Drosophila sNPF system, tsetse larvae lack expression of Glomo-snpf and Glomo-snpfr genes. While Glomo-snpf transcript levels are upregulated in pupae, the onset of Glomo-snpfr expression is delayed to adulthood. Expression profiles in adult tissues are similar to those in other insects suggesting that the tsetse sNPF system may have similar functions such as a regulatory role in feeding behavior, together with a possible involvement of sNPFR signaling in osmotic homeostasis. Our molecular data will enable further investigations into the functions of sNPF signaling in tsetse flies.publisher: Elsevier articletitle: Molecular characterization of a short neuropeptide F signaling system in the tsetse fly, Glossina morsitans morsitans journaltitle: General and Comparative Endocrinology articlelink: http://dx.doi.org/10.1016/j.ygcen.2016.06.005 content_type: article copyright: © 2016 Published by Elsevier Inc.status: publishe

Neuropeptide Receptors as Possible Targets for Development of Insect Pest Control Agents

Vaious insect species have a severe impact on human welfare and environment and thus force us to continuously develop novel agents for pest control. Neuropeptides constitute a very versatile class of bioactive messenger molecules that initiate and/or regulate a wide array of vital biological processes in insects by acting on their respective receptors in the plasmamembrane of target cells. These receptors belong to two distinct categories of signal transducing proteins, i.e., heptahelical or G protein-coupled receptors (7TM, GPCR) and single transmembrane containing receptors. An increasing amount ofevidence indicates that insect neuropeptide-receptor couples play crucial roles in processes as diverse as development, metabolism, ecdysis and reproduction. As such, they gain growing interest as promising candidate targets for the development of a new generation of species- and receptor-specific insect control agents that may generate fewer side effects. In this chapter, we will present some examples of insect neuropeptide receptors and aim to demonstrate their fundamental importance in insect biology.Advances in Experimental Medicine and Biologystatus: publishe

Evolutionary conservation of bursicon in the animal kingdom.

Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The ovicidal, larvacidal and adulticidal properties of 5,5'-Dimethyl-2,2'-Bipyridyl against Drosophila melanogaster

Insecticide resistance has limited the number of available chemical options for insect pest control. Hence there is a need for new chemistries with novel modes of action. Here we investigate the mode of action for an insecticide that has not yet been released for commercial use. The ovicidal, larvacidal and adulticidal effects of 5,5′-dimethyl -2, 2′-dipyridyl (termed Ha44), which is being developed as a treatment for head lice, were evaluated in the Drosophila melanogaster model system. Ha44 demonstrated significant activity against embryos and was capable of arresting development at a number of stages of embryogenesis. The effects of Ha44 on embryos was shown to be reversible following the addition of the metal ions Fe(II) and Fe(III), Cu and Zn. When larvae were exposed to Ha44, lethality was recorded at similar concentrations to those observed for embryos. Using an eYFP reporter system it was shown that Ha44 was able to reduce the levels of both copper and zinc in the digestive tract, confirming the binding of Ha44 to these metals in vivo. Ha44 has further been shown to inhibit a zinc containing metalloproteinase in vitro. Exposure of adult flies to Ha44 resulted in lethality, but at higher concentrations than those observed for embryos and larvae. The median lethal dose in adult flies was shown to be associated with the type of exposure, with an LD-50 of 1.57 mM being recorded following the direct contact of flies with Ha44, while an LD-50 of 12.29 mM was recorded following the ingestion of the compound. The capacity of Ha44 to act on all stages of the life-cycle and potentially via a range of targets suggests that target site resistance is unlikely to evolve

Comparative genomics of leucine-rich repeats containing G protein-coupled receptors and their ligands.

Leucine-rich repeats containing G protein-coupled receptors (LGRs) constitute a unique cluster of transmembrane proteins sharing a large leucine-rich extracellular domain for hormone binding. In mammals, LGRs steer important developmental, metabolic and reproductive processes as receptors for glycoprotein hormones and insulin/relaxin-related proteins. In insects, a receptor structurally related to human LGRs mediates the activity of the neurohormone bursicon thereby regulating wing expansion behaviour and remodelling of the newly synthesized exoskeleton. In the past decade, novel insights into the molecular evolution of LGR encoding genes accumulated rapidly due to comparative genome analyses indicating that the endocrine LGR signalling system likely emerged before the radiation of metazoan phyla and expanded throughout evolution. Here, we present a short survey on the evolution of LGRs and the hormones they interact with.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: sh.jinfo:eu-repo/semantics/publishe