Aproximación a la filogenia de Spodoptera (Lepidoptera: Noctuidae) con el uso de un fragmento del gen de la citocromo oxidasa I (COI) / Approach to Spodoptera (Lepidoptera: Noctuidae) phylogeny based on the sequence of the cytocrhome oxydase I (COI) mitochondrial gene

The genus Spodoptera includes 30 species of moths considered important pests worldwide, with a great representation in the Western Hemisphere. In general, Noctuidae species have morphological similarities that have caused some difficulties for assertive species identification by conventional methods. The purpose of this work was to generate an approach to the genus phylogeny from several species of the genus Spodoptera and the species Bombyx mori as an out group, with the use of molecular tools. For this, a total of 102 S. frugiperda larvae were obtained at random in corn, cotton, rice, grass and sorghum, during late 2006 and early 2009, from Colombia. We took ADN samples from the larval posterior part and we analyzed a fragment of 451 base pairs of the mitochondrial gene cytochrome oxydase I (COI), to produce a maximum likelihood (ML) tree by using 62 sequences (29 Colombian haplotypes were used). Our results showed a great genetic differentiation (K2 distances) amongst S. frugiperda haplotypes from Colombia and the United States, condition supported by the estimators obtained for haplotype diversity and polymorphism. The obtained ML tree clustered most of the species with bootstrapping values from 73-99% in the interior branches; with low values also observed in some of the branches. In addition, this tree clustered two species of the Eastern hemisphere (S. littoralis and S. litura) and eight species of the Western hemisphere (S. androgea, S. dolichos, S. eridania, S. exigua, S. frugiperda, S. latifascia, S. ornithogalli and S. pulchella). In Colombia, S. frugiperda, S. ornithogalli and S. albula represent a group of species referred as “the Spodoptera complex” of cotton crops, and our work demonstrated that sequencing a fragment of the COI gene, allows researchers to differentiate the first two species, and thus it can be used as an alternative method to taxonomic keys based on morphology. Finally, the ML tree did not cluster S. frugiperda with S. ornithogalli, suggesting that both species do not share the same recent ancestral even though they coexist in cotton. We suggest sequencing other genes (mitochondrial and nuclear) to increase our understanding of this genus evolution.

En este trabajo se secuenció un fragmento de 451pb del gen mitocondrial de la citocromo oxidasa I (COI) en 62 secuencias del género Spodoptera y una secuencia de Bombix mori (grupo externo). Los resultados mostraron gran diferenciación genética (distancia K2) entre los haplotipos de Spodoptera frugiperda de Colombia y Estados Unidos, según los estimadores de diversidad haplotípica, diversidad y polimorfismo nucleotídicos calculados. Un árbol de ML agrupó las especies con valores de bootstrap entre 73-99% en las ramas internas. No obstante algunas ramas presentaron bajos valores de bootstrap. Este árbol formó un grupo constituido por las especies del hemisferio oriental (S. littoralis y S. litura) y también agrupó las especies localizadas en el hemisferio occidental (S. androgea, S. dolichos, S. eridania, S. exigua, S. frugiperda, S. latifascia, S. ornithogalli y S. pulchella). Esto demuestra que el árbol agrupó las especies con base en su origen geográfico. Contrariamente, el árbol no agrupó a S. frugiperda con S. ornithogalli, demostrando que a pesar de que ambas coexisten en el cultivo de algodón, no comparten un ancestro común reciente. En Colombia, estas especies forman parte del “complejo Spodoptera” del algodón, y nuestros resultados demuestran que la secuenciación de este gen permite diferenciarlas sin necesidad del uso de claves taxonómicas de sus estadios larvales. Este trabajo es una aproximación a la filogenia de este género, por lo cual la inclusión de más genes (mitocondriales y nucleares) son necesarios para futuros trabajos.

Zygomycetes from herbivore dung in the ecological reserve of Dois Irmãos, Northeast Brazil

Thirty-eight taxa of Zygomycetes distributed in 15 genera were recorded from tapir (Tapirus terrestris), camel (Camelus bactrianus), horse (Equus caballus), deer (Cervus elaphus), agouti (Dasyprocta aguti), donkey (Equus asinus), llama (Llama glama) and waterbuck (Kobus ellipsiprymnus) dung collected at the Reserva Ecológica de Dois Irmãos located in Recife, State of Pernambuco, Northeast Brazil. The samples were collected on a monthly basis from June 2005 to May 2006, taken to the laboratory and incubated in moist chambers. Higher number of taxa was observed in the excrements of tapir, followed by deer and donkey. The highest number of species was detected for Mucor, followed by Pilobolus. Statistical analyses showed significant differences in richness of Zygomycetes taxa between the herbivore dung types. Differences of species composition, however, were weak. Seasonality influenced the Zygomycetes species composition but not its richness. Variations in taxa composition between ruminants and non-ruminants dung were non significant.

Characterization and comparison of Serratia marcescens isolated from edible cactus and from silkworm for virulence potential and chitosan susceptibility

Representative strains of Serratia marcescens from an edible cactus plant and silkworms were characterized and a comparison based on their cellular fatty acid composition, 16S rRNA and groE gene sequence analysis as well as silkworm virulence and chitosan susceptibility was carried out. Results from this study indicate that there are no significant differences between the phenotypic and molecular characterization, virulence and chitosan susceptibility of the S. marcescens strains from the cactus plant and silkworms. Silkworms inoculated with S. marcescens from either plant or silkworm resulted in nearly 100 percent mortality. Chitosan solution exhibited strong antibacterial activity against S. marcescens. This activity increased with the increase of chitosan concentration and incubation time regardless of the strain source. Also, the results indicate that the plant associated S. marcescens maybe plays a possible role in the contamination of humans and animals, in particular silkworms, while chitosan showed a potential to control the contamination caused by S. marcescens.

Genetic analysis of scattered populations of the Indian eri silkworm, Samia cynthia ricini Donovan: differentiation of subpopulations

Deforestation and exploitation has led to the fragmentation of habitats and scattering of populations of the economically important eri silkworm, Samia cynthia ricini, in north-east India. Genetic analysis of 15 eri populations, using ISSR markers, showed 98 percent inter-population, and 23 percent to 58 percent intra-population polymorphism. Nei's genetic distance between populations increased significantly with altitude (R² = 0.71) and geographic distance (R² = 0.78). On the dendrogram, the lower and upper Assam populations were clustered separately, with intermediate grouping of those from Barpathar and Chuchuyimlang, consistent with geographical distribution. The Nei's gene diversity index was 0.350 in total populations and 0.121 in subpopulations. The genetic differentiation estimate (Gst) was 0.276 among scattered populations. Neutrality tests showed deviation of 118 loci from Hardy-Weinberg equilibrium. The number of loci that deviated from neutrality increased with altitude (R² = 0.63). Test of linkage disequilibrium showed greater contribution of variance among eri subpopulations to total variance. D'2IS exceeded D'2ST, showed significant contribution of random genetic drift to the increase in variance of disequilibrium in subpopulations. In the Lakhimpur population, the peripheral part was separated from the core by a genetic distance of 0.260. Patchy habitats promoted low genetic variability, high linkage disequilibrium and colonization by new subpopulations. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original S. c. ricini gene pool.

Comparative genetic diversity and genetic structure of three chinese silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini donovan (Lepidoptera: Saturniidae)

The genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L., Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan were comparatively assessed based on RAPD markers. At the species level, A. pernyi and B. mori showed high levels of genetic diversity, whereas S. cynthia ricini showed low level of genetic diversity. However, at the strain level, A. pernyi had relatively highest genetic diversity and B. mori had lowest genetic diversity. Analysis of molecular variance (AMOVA) suggested that 60 percent and 72 percent of genetic variation resided within strains in A. pernyi and S. cynthia ricini, respectively, whereas only 16 percent of genetic variation occurred within strains in B. mori. In UPGMA dendrogram, individuals of A. pernyi and B. mori formed the strain-specific genetic clades, whereas those of S. cynthia ricini were distributed in a mixed way. The implications of these results for the conservation and utilization in breeding programs of three silkworm species are discussed.

Comparison of Phenotypic and Genetic Performance of Local Silkworm Groups and Two Commercial Lines

Five Iranian native silkworm groups: Baghdad, Khorasan Orange, Guilan Orange, Khorasan Pink, Khorasan Lemon, and 107 and 110 commercial lines (12 families from each breed) were randomly selected and reared during 2003-2005 (five generations in spring and autumn). In each family, 30 male and 30 female cocoons were individually recorded for weight, shell weight and shell ratio. From among the native groups, the highest average in all three traits belonged to Baghdad and Khorasan Pink, and the lowest to Khorasan Orange and Khorasan Lemon. From among the commercial lines, the highest average in all three traits belonged to 107. In comparing heritabihty for cocoon weight in native groups, the highest heritabihty belonged to Guilan Orange (0.5147) and Khorasan Orange (0.5036) and the lowest heritabihty belonged to Khorasan Pink (0.0967). In the two other traits, the highest heritabihty belonged to Khorasan Orange and Baghdad and the lowest to Khorasan Pink. In the commercial lines, linellO had higher heritabihty than linel07 for cocoon weight and cocoon shell weight. In all the groups, genetic correlations between cocoon weight and cocoon shell weight were high, expect for the Baghdad group. There was médium or low genetic correlation among cocoon weight, cocoon shell weight and cocoon shell ratio.

Amplificação por PCR da região controle do DNA mitocondrial de raças de bombyx mori / Bombyx mori mitocondrial DNA control region amplification by PCR / Amplificación por pcr de la región control del dna mitocondrial de razas de Bombyx mori

A sericicultura é uma importante atividade agroindustrial no Brasil, sendo o Estado do Paraná responsável por aproximadamente 90% de toda a produção nacional. A localização de marcadores genéticos para o bicho-da-seda (Bombyx mori L.) é importante para a diferenciação intra e interespecífica e para o uso em melhoramento genético da espécie, sendo o DNA mitocondrial (DNAmt) um dos marcadores genéticos mais utilizados no estudo de insetos. O objetivo deste trabalho foi amplificar a região controle do DNA mitocondrial de quatro raças de Bombyx mori, pela técnica de Polymerase Chain Reaction (PCR). Obteve-se a amplificação de um fragmento de aproximadamente 750 pb referente à região controle, demonstrando a eficiência da metodologia empregada para a amplificação desta região específica do DNAmt de Bombyx mori.

Sericulture is an important agro industrial activity in Brazil, mainly in Paraná State, that is responsible for 90% of the national production. For the genetic improvement of silkworm (Bombyx mori L.), it is necessary to develop techniques to amplify molecular markers in order to use it on genetic analysis. Mitochondrial DNA (mtDNA) has been widely used as a molecular marker for insects and provides suitable markers for studies on genetic variability and molecular characterization. The control region is the major non-coding region of animal mtDNA and has been responsible for providing important evolutionary data about many insect groups. The aim of this paper was to amplify the mtDNA control region of four Bombyx mori strains by polymerase chain reaction (PCR). A 750 bp fragment including the control region was amplified showing the efficiency of the methodology to amplify specific regions of the Bombyx mori mtDNA.

La sericicultura es una importante actividad agroindustrial en Brasil, siendo el Estado del Paraná responsable por aproximadamente 90% de toda la producción nacional. La localización de marcadores genéticos para el gusano de seda (Bombyx mori L.) es importante para la diferenciación intra e interespecífica y para el uso en mejoramiento genético de la especie, siendo el DNA mitocondrial (DNAmt) uno de los marcadores genéticos más utilizados en el estudio de insectos. El objetivo de esta investigación fue amplificar la región control del DNA mitocondrial de cuatro razas de Bombyx mori, por la técnica de Polymerase Chain Reaction (PCR). Se obtuvo la amplificación de un fragmento de aproximadamente 750 pb referente a la región control, demostrando la eficiencia de la metodología empleada para la amplificación de esta región específica del DNAmt de Bombyx mori.

Macromolecular array patterns of silk gland secretion in social Hymenoptera larvae

The cocoon, produced by most holometabolous insects, is built with silk that is usually produced by the larval salivary gland. Although this silk has been widely studied in the Lepidoptera, its composition and macromolecular arrangement remains unknown in the Hymenoptera. The macromolecular array patterns of the silk in the larval salivary gland of some meliponids, wasps, and ants were analyzed with polarized-light microscopy, and they were compared with those of Bombyx mori (Lepidoptera). There is a birefringent secretion in the glandular lumen of all larvae, due to filamentous structural proteins that display anisotropy. The silk in the distal, middle and proximal regions of the secretory portion of Formicidae and Vespidae glands presented a lattice optical pattern. We found a different pattern in the middle secretory portion of the Meliponini, with a zigzag rather than a lattice pattern. This indicates that the biopolymer fibers begin their macromolecular reorganization at this glandular region, different from the Formicidae and the Vespidae, in which the zigzag optical pattern was only found at the lateral duct. Probably, the mechanism of silk production in the Hymenoptera is a characteristic inherited from a common ancestor of Vespoidea and Sphecoidea; the alterations in the pattern observed in the Meliponini could be a derived characteristic in the Hymenoptera. We found no similarity in the macromolecular reorganization patterns of the silk between the Hymenoptera species and the silkworm.

The expression of GFP under the control of fibroin promotor in primary ovarian cells of Antheraea pernyi.

The fibroin promoter can stably express foreign gene in lepidopteran cells. Total RNA was extracted from the gland of silkworm, Antheraea pernyi and the transcription initiation site of fibroin gene of A. pernyi was identified by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector (pGFP-N2/Fib) was constructed by use of replacing the CMV promoter with the fibroin promoter. The results of visual screening under a fluorescent inverted microscope and Western blot analysis indicated that the GFP gene was expressed in the primary cells of ovary origins from A. pernyi.

A comparative phylogenetic analysis of full-length mariner elements isolated from the Indian tasar silkmoth, Antheraea mylitta (Lepidoptera: saturniidae).

Mariner like elements (MLEs) are widely distributed type II transposons with an open reading frame (ORF) for transposase. We studied comparative phylogenetic evolution and inverted terminal repeat (ITR) conservation of MLEs from Indian saturniid silkmoth, Antheraea mylitta with other full length MLEs submitted in the database. Full length elements from A. mylitta were inactive with multiple mutations. Many conserved amino acid blocks were identified after aligning transposase sequences. Mariner signature sequence, DD(34)D was almost inva ri able although a few new class of elements had different signatures. A. mylitta MLEs (Anmmar) get phylogene ti cally classified under cecropia subfamily and cluster closely with the elements from other Bombycoidea superfamily members implying vertical transmission from a common ancestor. ITR analysis showed a conserved sequence of AGGT(2-8N)ATAAGT for forward repeat and AGGT(2-8N)ATGAAAT for reverse repeat. These results and additional work may help us to understand the dynamics of MLE distribution in A. mylitta and construction of appropriate vectors for mariner mediated transgenics.

Accelerated molecular evolution of insect orthologues of ERG28/C14orf1: a link with ecdysteroid metabolism?

We have analysed the evolution of ERG28/C14orf1, a gene coding for a protein involved in sterol biosynthesis. While primary sequence of the protein is well conserved in all organisms able to synthesize sterols de novo, strong divergence is noticed in insects, which are cholesterol auxotrophs. In spite of this virtual acceleration, our analysis suggests that the insect orthologues are evolving today at rates similar to those of the remaining members of the family. A plausible way to explain this acceleration and subsequent stabilization is that Erg28 plays a role in at least two different pathways. Discontinuation of the cholesterogenesis pathway in insects allowed the protein to evolve as much as the function in the other pathway was not compromised.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally.

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.