Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution

Silencing RNAs expressed from W-linked PxyMasc “retrocopies” target that gene during female sex determination in Plutella xylostella

The Lepidoptera are an insect order of cultural, economic, and environmental importance, representing ∼10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori), the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (Plutella xylostella), a globally important, highly invasive, and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in P. xylostella—PxyMasc, a homolog of B. mori Masculinizer—which, although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly Piwi-interacting RNAs [piRNAs]) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly arranged, multicopy arrays found exclusively on the W (female-specific) chromosome, which we term “Pxyfem”. Analysis of the Pxyfem sequences showed that they are partial complementary DNAs (cDNAs) of PxyMasc messenger RNA (mRNA) transcripts, likely integrated into transposable element graveyards by the noncanonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the “shortcuts” that retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects’ biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment.Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos.Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control

Genetics and cytogenetics of sex determination in Diachasmimorpha longicaudata (Hymenoptera, Braconidae)

El parasitoide bracónido Diachasmimorpha longicaudata es ampliamente utilizado como controlador biológico de moscas de los frutos; sin embargo, es escaso el conocimiento disponible sobre su genética. A fin de aportar información y mejorar su cría masiva, se estudió su sistema de determinación del sexo mediante la realización de cruzamientos de alta y baja endogamia, utilizando herramientas de citogenética para determinar el nivel de ploidía de la descendencia. Asimismo, se analizó su espermatogénesis y su cariotipo en detalle, realizando aportes a la Teoría de Interacción Mínima, desarrollada para el estudio de la evolución del cariotipo en Hymenoptera. En este parasitoide el sexo se determina por haplodiploidía (machos haploides, hembras diploides), pero en condiciones de alta endogamia ocurre un sesgo significativo de la proporción de sexos hacia machos, debido a la generación de machos diploides. El sexo estaría determinado por el estado de un promedio de tres loci no ligados. Los individuos homocigotas para los loci sexuales se desarrollan en machos diploides. Esta información permitirá mejorar los protocolos de cría masiva de la especie, tratando de minimizar el nivel de endogamia a fin de evitar un incremento en la producción de machos. El estudio citogenético permitió corroborar el número cromosómico descripto previamente. Se analizó la cantidad, composición y distribución de la heterocromatina constitutiva, se mapearon los “clusters” de genes ribosomales y se determinó la cantidad de “clusters” activos. La información obtenida permitió aportar más evidencias a favor de la Teoría de Interacción Mínima.Diachasmimorpha longicaudata is a braconid parasitoid widely used as biological control agent against fruit flies. In spite of its importance, little is known about its genetics. Different aspects were studied in order to provide more information and to optimize its massive production. Its sex determination system was analyzed using inbred and outbred crosses, and cytogenetic tools to analyze the ploidy level of the descendants. Also its spermatogenesis and karyotype were analyzed in detail, contributing to the Minimum Interaction Theory proposed for the study of karyotype evolution in Hymenoptera. Sex is determined by haplodiploidy (males are haploid, females are diploid), but under inbreeding a significant bias towards male progeny is observed due to the generation of diploid males. Sex is presumably determined by a mean of three independent loci. Homozigotes for the sex loci develop into diploid males. In order to avoid an increment in male production, this information would be useful to improve the protocols of massive production of this species, regarding to minimizing the inbreeding levels. The cytogenetic study allowed the corroboration of the chromosome number of the species. The amount, composition and distribution of constitutive heterochromatin were analyzed. The ribosomal gene clusters were located and the quantity of active sites was determined. The obtained information gave more evidence in favor of the Minimum Interaction Theory.Fil:Carabajal Paladino, Leonela Z.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution. Introductio

Intron-derived small RNAs for silencing viral RNAs in mosquito cells.

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3' UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes

AePUb promoter length modulates gene expression in Aedes aegypti

Abstract Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects

AePUb promoter length modulates gene expression in Aedes aegypti

Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects

X-ray doses to safely release the parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) reared on Anastrepha fraterculus larvae (Diptera: Tephritidae)

Diachasmimorpha longicaudata is a koinobiont larval parasitoid that is currently used to control fruit flies of the genera Anastrepha, Ceratitis and Bactrocera. In the rearing process, a fraction of the host larvae that are exposed to parasitoids escape from parasitism and develop into viable and fertile flies. This creates the need to eliminate emerging flies before the parasitoids are shipped for release, increasing costs due to additional handling steps. Exposure of fly eggs or larvae to gamma-irradiation before they are parasitised has been used to reproductively sterilise hosts, or even inhibit their emergence. Our aim was to determine whether X-ray radiation applied to Anastrepha fraterculus third instar larvae before they are exposed to parasitoids, inhibits fly emergence in non-parasitised larvae without affecting the performance of the parasitoids that emerge from parasitised larvae. Three X-ray doses: 6250.2 R, 8333.6 R and 10417 R (equivalent to 60, 80 and 100 Gy, respectively) and one γ-ray dose (100 Gy) were tested. Fly emergence decreased with increasing doses of radiation, showing null values for the higher X-ray dose and the dose of 100 Gy. Irradiation showed either no impact or a positive effect on parasitism rate and fecundity. Sex rate was biased towards females in almost every dose. We conclude that the two types of radiation evaluated here were equally effective in suppressing fly emergence with no detrimental effects on the biological quality of the produced parasitoids. X-rays offer an alternative method of irradiation than the conventional radiation source, i.e. γ-rays. These results represent a significant improvement in the development of a biological control programme against A. fraterculus.12 page(s