Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) / Identificação molecular, caracterização e análise de expressão de um gene inibidor de serina protease da lagarta Helicoverpa armigera (Lepidoptera: noctuidae) na cultura de algodão

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341-N350), the serpin signature, (F367-F375) and a predicted P1-P1′ cleavage site (L357-S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Resumo Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.

Genetic characterization of silkworm (Bombyx mori L. ) strains (Lepidoptera: Bombicidae) with different geographical originon the basis of isozyme markers

The aim of the present study was to assess genetics variations within and among strains with different geographical origin, belonging to Bulgarian germplasm bank of mulberry silkworm (Bombyx moriL.)and to establish their relationships using isoenzyme markers. Polyacrilamide gel electrophoresis (PAGE) was used to study the isoenzymes of nonspecific esterases (EST), malate dehydrogenase (MDH) and acid phosphatase (ACP) from haemolymph, phosphoglucomutase (PGM) and hexokinase (HK) from silk glands and alkaline phosphatase (ALP) from midgut of mulberry silkworm (Bombyx moriL.). Variability was evident in all of these enzyme systems among twelve strains from Bulgarian germplasm resources of B. mori.Total of nine lociwere detected. All of them (100%) were polymorphic. “Null” alleles in four lociwere determined. Intra-and inter-strain polymorphism was obtained. The degree of polymorphism ranged from 0% to 77.80%. Low levels of observed heterozygosity in comparison with the expected one have been calculated in almost all of strainsas well as deviations from Hardy-Weinberg equilibrium in almost all analyzed loci, resultingof excess of homozygotes. The value of FSTwas 0.4903. The dendrogram constructed with the values of genetic distance manifests that Romanian strain Cislau Tokay is formed one main clade while the rest strains studied (from Bulgaria, Japan, China, Vietnam, Spain, Syria and Egypt) are formed the other clade withsubclades. The genetic data of the tested strains from different geographical regions, would be used for identifying suitable parents for breeding programs with a view to yield improvement in this species with economic significance.

Molecular characterization of brinjal shoot and fruit borer, Leucinodes orbonalis (Guenée) (Lepidoptera: Crambidae) based on mitochondrial marker cytochrome oxidase I and their phylogenetic relationship.

Shoot and fruit borer, Leucinodes orbonalis is an important insect pest infesting brinjal or eggplant in India. Molecular characterization of nine different populations belonging to various brinjal growing regions was done using Cytochorome C Oxidase I (COI) gene. Nucleotide analysis of genetic diversity and phylogenetic analysis of the COI indicate that the L. orbonalis from different geographical regions are homogenous. The results showed less nucleotide diversity (π = 0.007895) and overall mean distance (0.008±0.003). Topologies of neighbour-joining (NJ) trees indicate all the populations belong to single major clade. Therefore, it is inferred that there was no significant molecular diversity within L. orbonalis of different geographical locations of India with respect to COI.

Exploitation of mitochondrial nad6 as a complementary marker for studying population variability in Lepidoptera

The applicability of mitochondrial nad6 sequences to studies of DNA and population variability in Lepidoptera was tested in four species of economically important moths and one of wild butterflies. The genetic information so obtained was compared to that of cox1 sequences for two species of Lepidoptera. nad6 primers appropriately amplified all the tested DNA targets, the generated data proving to be as informative and suitable in recovering population structures as that of cox1. The proposal is that, to obtain more robust results, this mitochondrial region can be complementarily used with other molecular sequences in studies of low level phylogeny and population genetics in Lepidoptera.

Genetic diversity of the sunflower caterpillar (Chlosyne lacinia saundersii Doubleday and Hewitson) (Lepidoptera: Nymphalidae) populations determined by molecular RAPD markers

Chlosyne lacinia saundersii is one of the most important pests of sunflower and it is the main target of insecticides applications. Larvae were collected in Londrina (PR), Santa Maria (RS), Dourados (MS), Ribeirão Preto (SP), Brasília (DF), Barreiras (BA), Uberaba (MG) and Vilhena (RO). Genomic DNA was extracted and amplified with ten-mer primers, which produced 101 loci. The size of the RAPD amplicons ranged from 180 to 2564 bp. Polymorphism among populations ranged from 31 percent to 67 percent, with the highest polymorphisms of 57 percent and 67 percent being detected in Uberaba and Vilhena populations, respectively. Populations with the highest similarity determined with Dice coefficient were from Ribeirão Preto and Barreiras, while insects from Londrina showed the highest similarity among them. Gene flow of C. lacinia saundersii 1.1 was lower than those previously observed for the noctuid Anticarsia gemmatalis Hübner, suggesting that C. lacinia saundersii populations are more isolated than the ones of this noctuid. Through the Analysis of Molecular Variance (AMOVA), RAPD variance was 33.64 percent among geographical populations and 66.36 percent within populations. These results suggest that populations of C. lacinia saundersii are genetically structured.

Chlosyne lacinia saundersii é uma das mais importantes pragas da cultura do girassol e o principal alvo das aplicações de inseticidas. As larvas foram coletadas em Londrina (PR), Santa Maria (RS), Dourados (MS), Ribeirão Preto (SP), Brasília (DF), Barreiras (BA), Uberaba (MG) e Vilhena (RO). O DNA genômico foi extraído e amplificado com dez primers, que produziram 101 locos. O tamanho das amplificações de RAPD variou de 180 a 2564 pb. O polimorfismo entre as populações variou de 31 por cento a 67 por cento, com maior polimorfismo 57 por cento e 67 por cento, detectado em populações de Uberaba e Vilhena, respectivamente. As populações com maior similaridade determinada com o coeficiente de Dice foram de Ribeirão Preto e Barreiras, enquanto os insetos coletados em Londrina apresentaram maior similaridade entre eles. O fluxo gênico de C. lacinia saundersii de 1,1 foi menor que o observado para a Anticarsia gemmatalis Hübner Noctuidae, sugerindo que as populações de C. lacinia saundersii estão mais isoladas do que estes noctuideos. Através da análise de variância molecular (AMOVA), RAPD a variação foi de 33,64 por cento entre as populações geográficas e 66,36 por cento dentro das populações. Estes resultados sugere que as populações de C. lacinia saundersii são geneticamente estruturadas.

Estandarización de un bioensayo y evaluación preliminar de tres formulaciones comerciales de bacillus thuringiensis sobre tuta absoluta (meyrick) (lepidoptera: gelechiidae) / Standardising a bioassay for the preliminary evaluation of threecommercial bacillus thuringiensis formulations against tutaabsoluta (meyrick) (lepidoptera: gelechiidae)

La polilla del tomate (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) es una de las plagas más devastadorasdel tomate en Colombia y países suramericanos, produciendo pérdidas de hasta el 100% en cultivos sin protección.En 2009, T. absoluta se detectó en España, Portugal y países del mediterráneo, además de Inglaterra,Bulgaria y Alemania. Para su control se utilizan insecticidas químicos que generan resistencia e impactoambiental y de salud. La alternativa de utilizar biopesticidas contra esta plaga es de importancia creciente. Eneste estudio se evaluaron cinco métodos de bioensayo para medir adecuadamente la toxicidad sobre larvasde T. absoluta de tres productos comerciales: Dipel®, XenTary® y Turilav®, formulaciones a base de Bacillusthuringiensis (Bt). El método de Inmersión del folíolo, con el producto Dipel®, causó el 100% de mortalidadde larvas y 96% de supervivencia del testigo; este método presentó diferencias significativas al segundo(F=0,025, p>0,05) y cuarto (F=0,0018, p>0,05) día después de la aplicación (DDA). El método de Aspersiónfoliar por aerógrafo produjo 100% de mortalidad de larvas con Dipel® al segundo DDA (F=7,94x10-10,p> 0,05), y produjo diferencias significativas también al cuarto DDA (F=3,45x10-6, p>0,05). Los métodosFoliolos sumergidos y Medio de cultivo provocaron una alta mortalidad en el control por lo que fueron rechazados.El uso de Dipel®, XenTari® y Turilav® en concentración de 1,25 g/L causó entre 80-100% demortalidad entre el segundo y octavo DDA en tres métodos evaluados válidos (1, 2, 3), además corrobora laactividad biológica de B. thuringiensis sobre este insecto plaga.

The tomato moth (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) is one of the most devastating tomatopests in Colombia and South-American countries, producing losses of up to 100% in unprotected crops. T.absoluta was detected in Spain, Portugal and Mediterranean countries in 2009, as well as England, Bulgaria andGermany. Chemical insecticides are used for controlling it; however, they produce resistance and an environmentaland human health impact. Finding an alternative to using biopesticides against this pest is becomingincreasingly important. This study evaluated five bioassay methods measuring three commercial products’toxicity on T. absoluta larvae: Dipel, XenTary and Turilav Bacillus thuringiensis (Bt) -based formulations. The leafdipping bioassay method caused 100% larvae mortality with Dipel, the control group having 95% survivalrate. The other products showed significant differences on the 2nd (F=0.025, p>0.05) and 4th (F=0.0018,p>0.05) days after application (DAA). The leaf spray airbrush method produced 100% larvae mortality withDipel on the 2nd DAA, having significant differences from the other products tested on 2nd (DAA F=7.94 x10-10, p>0.05 ), 4th (F=3.45x10-6, p>0.05 ) and 8th (F=1.07x10-5, p>0.05 ) DAA. Submerged leaflet and culturemedium methods caused high mortality in controls and were thus rejected. A variation of the leaflet immersionmethod was standardised. The three commercial products produced high mortality in Lab conditionsregarding T. absolute larvae control at 1.25 g/L concentration, thereby corroborating the biological activity ofB. thuringiensis against this insect pest.

Diferenciación morfológica y molecular de Oleria makrena (Hewitson) y Oleria fumata (Haensch) (Lepidoptera: Ithomiinae) / Molecular and morphological differentiation of Oleria makrena (Hewitson) and Oleria fumata (Haensch) (Lepidoptera: Ithomiinae)

Oleria spp. (tribu Olerinii) es un género de gran interés a nivel ecológico y evolutivo entre los Ithomiinae. La identificación de sus especies con base en caracteres morfológicos puede ser compleja debido a que la mayoría de ellas están involucradas en anillos miméticos y poseen grandes similitudes. En muestreos de mariposas ithomiinae realizados en el Suroeste Antioqueño, Colombia, se encontraron especímenes con patrones alares que dificultaron el registro de una o dos especies en la zona de estudio. Los especímenes se examinaron en detalle incluyendo observaciones de los genitales de machos y hembras y obtención de secuencias nucleotídicas además de los caracteres tradicionales de venación y coloración alar. Se verificó la presencia de Oleria makrena (Hewitson) y Oleria fumata (Haensch) y se precisó la utilidad de cada uno de los caracteres en la diferenciación de las dos especies. En los machos, la separación de ambas especies pudo realizarse con base en la morfología alar y coincidió con la realizada mediante los genitales y las secuencias de ADN. Sin embargo, en el caso de las hembras los patrones de venación alar fueron confusos y se requirió el uso de los genitales. Las secuencias de la región corta de ADN (gen ND4) usada, permitieron realizar una diferenciación rápida y acertada de las especies que coincidió con la obtenida por los genitales tanto femeninos como masculinos. El análisis del fragmento de 579 pb señaló la existencia de 46 diferencias nucleotídicas entre las dos especies.

Oleria (Olerinii tribe) is a genus of great ecological and evolutionary importance among the Ithomiinae (Nymphalidae). Species identification is very complex since most of them are involved in mimicry rings and share large morphological similarities. Ithomiinae specimens collected in the Antioquia Southwest - Colombia, showed confuse wing color pattern generating diffi culties to define the presence of one or two species at the sampled region. Collected specimens were studied in detail using male and female genitalia and nucleotide sequences besides traditional character of wing color and venation. Oleria makrena (Hewitson) and Oleria fumata (Haensch) were registered and the relative utility of each character for species identification was defi ned. For males, wing color pattern was enough to differentiate both species, and the separation of specimens by this character was congruent with the separation obtained by using genitalia and DNA sequences. However, for females, wing patterns were not enough for the adequate separation or species and genitalia-based characters were necessary. DNA sequences of a short fragment of the mitochondrial gene ND4 allowed for the rapid and precise differentiation of both species, coinciding with the separation using genitalia for both males and females. The analysis of 579 base pairs (bp) indicated 46 nucleotide differences between both species.

Barcoding lepidoptera: current situation and perspectives on the usefulness of a contentious technique / Barcoding lepidoptera: situação atual e perspectivas sobre a utilidade de uma técnica controversa

Faced by a growing need of identification and delimitation of new and established cryptic species that are being lost at an increasing rate, taxonomists can now more than ever take advantage of an enormous variety of new molecular and computational tools. At this moment they should be open to all new available technologies in the so called "technology-driven revolution" in systematics. The use of the "DNA barcode" has been discussed by those applying successfully this approach to identify and diagnose species and by those who believe that the flaws in the use of this molecular marker are as many as to negate the worth of its employment. For insects of the order Lepidoptera neither side seems totally correct orwrong, and although many groups of lepidopterans have been taxonomically resolved by using exclusively or additionally this marker for diagnoses, for others the "barcode" helped little to resolve taxonomic issues. Here we briefly present some pros and cons of using DNA barcode as a tool in taxonomic studies, with special attention to studies with groups of Lepidoptera developed in the last few years.

A necessida de crescente de identificação e delimitação de novas espécies, ou de espécies crípticas já estabelecidas, que estão sendo perdidas a uma taxa também crescente, tem levado diversos especialistas a utilizar em uma variedade de ferramentas moleculares e computacionais. Neste momento, taxonomistas devem estar atentos a toda nova tecnologia disponível na chamada "revolução dirigida pela tecnologia"na sistemática, que tem entre as novas ferramentas moleculares a utilização de"DNA barcodes". O uso de "DNA barcode" tem sido amplamente discutido por aqueles que aplicam essa abordagem com sucesso para identificar e diagnosticar espécies, e por aqueles que acreditam que são tantos os problemas no uso desse marcador molecular que não se justifica seu emprego. Para insetos da ordem Lepidoptera nenhum lado parece estar totalmente certo ou errado e, embora alguns grupos de lepidópteros tenham sido resolvidos taxonomicamente pelo uso exclusivo ou adicional desse marcador, para outros o "barcode" ajudou pouco a resolver problemas taxonômicos. Aqui nós apresentamos brevemente prós e contras do uso de "DNA barcode" como ferramenta em estudos taxonômicos, com atenção especial para estudos com grupos de Lepidoptera desenvolvidos nos últimos anos.

Morphology and biology of Porphyrosela minuta Clarke 1953 (Lepidoptera: Gracillariidae, Lithocolletinae) in Uruguay

Porphyrosela minuta Clarke is a gracillariid that causes damage on white clover (Trifolium repens) in Uruguay. In this article, the eggs, all the larval instars, the pupa and the external morphological characteristics of the adult are described. Information about the insect's biology under laboratory and field conditions is presented. The eggs are laid singly on the upper surface of the leaflets. The larvae make extensive mines that appear as white blotches. The larvae develop through five instars and do not remain exposed during their development. At 25°C the life cycle lasted 16.2 days, made up of 3.0 for eggs, 8.8 for larvae and 4.4 for pupae. Females began laying on the first night, and during an oviposition period that varied between five and 12 days deposited an average of 71.8 eggs. It is a multivoltine species and seven to nine generations occur between November and the end of March.

Porphyrosela minuta Clarke es un gracilárido que causa daños sobre trébol blanco (Trifolium repens) en Uruguay. En este artículo, son descriptos huevo, todos los instares larvarios y pupa. Características morfológicas externas del adulto son igualmente incluidas. Se aporta información sobre la biología en condiciones de laboratorio y campo. Los huevos son depositados simples sobre la epidermis superior de los foliolos. Las larvas forman grandes minas a modo de manchas blancas. La larva pasa por cinco estadios sin permanecer expuesta durante su desarrollo. A 25°C el ciclo de vida requirió de 16,2 días en completarse, correspondiendo 3,0 a huevos, 8,8 a larva y 4,4 a pupa. Las hembras iniciaron la puesta la primera noche y durante un periodo de oviposición que osciló entre cinco y 12 días depositaron en promedio 71,8 huevos. Es una especie multivoltina que desarrolla de siete a nueve generaciones de noviembre a fines de marzo.

Detection of genetic polymorphism in the populations of brinjal shoot and fruit borer, Leucinodes orbonalis (Guenee).

In the present study six different populations of L. orbonalis were collected and subjected to analysis of genetic variability in terms of carboxylesterase isozyme pattern and DNA polymorphism using RAPD-PCR. Pattern of carboxylesterase revealed a similar isozyme cluster in the populations namely, sivaganga (population-3), dindigal (population-4), virudhunagar (population-5) and coimbatore (population-6). Similarly, the populations of L. orbonalis recorded 3 distinct randomly amplified polymorphic DNA markers in all populations grouped above. This pattern of genetic variability in the populations was also supported by the analysis of the similarity indices and UPGMA dendrogram.

An attempt to establish cell lines from Dione Juno (Cramer, 1779) and Methona themisto (Hubner)

Susceptible insect cell lines to specific pathogens of agricultural insect pests could be directly applied to special programs of their biological control; or this could be also used in the propagation of vectors of expression, such as baculovirus for genetic enginnering. In this paper, several attempts of establishing cell lines from different tissues of two Lepidoptera, Dione juno, an injurious pest of the fruit-bearing plants Passifloraceae and Methona themisto, a pest of an urban ornamental tree, Brunfelsia sp., are described. Two cell cultures could be obtained from D. juno: One, Dj-0387, from macerated neonate larvae and the other, Dj3-0687, from minced ovaries of 5th instar larvae. Their morphological characteristics, viability and populational growth rate were similar. Approximately after 60 passages in vitro for Dj-0387 and 40 passages for Dj3-0687, these cell cultures became inviable. Apparently, these numbers of passages were their critical age limit to establish in vitro. Otherwise, at the 30th passage, mainly in the case of Dj-0387, the origin of the "larval beings"was observed. This phenomenon could be explained by the presence of the imaginal discs of the newborn larvae, that could resume development in vitro.