Functional characterization of piggyBac-like elements from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

PiggyBac is a transposable DNA element originally discovered in the cabbage looper moth (Trichoplusia ni). The T. ni piggyBac transposon can introduce exogenous fragments into a genome, constructing a transgenic organism. Nevertheless, the comprehensive analysis of endogenous piggyBac-like elements (PLEs) is important before using piggyBac, because they may influence the genetic stability of transgenic lines. Herein, we conducted a genome-wide analysis of PLEs in the brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), and identified a total of 28 PLE sequences. All N. lugens piggyBac-like elements (NlPLEs) were present as multiple copies in the genome of BPH. Among the identified NlPLEs, NlPLE25 had the highest copy number and it was distributed on five chromosomes. The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals, as well as a single open reading frame transposase encoding 546 amino acids. Furthermore, NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision. A cross-recognition between the NlPLE25 transposon and the piggyBac transposon was also revealed in this study. These findings provide useful information for the construction of transgenic insect lines.

Genetic diversity analysis of Bemisia tabaci populations in Pakistan using RAPD markers

Background: The Bemisia tabaci is one of the most devastating pests of agricultural crops and ornamental plants worldwide. The genetic diversity and biotype status of the Bemisia tabaci in Pakistan was assessed by using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). A total 80 samples of B. tabaci collected from 14 districts of the Punjab province and 7 districts of the Sindh province were included. Results: All 10 primers screened in this study generated 151 scorable amplification products, of which 117 or 77 percent were polymorphic. Pairwise Nei and Li’s similarity had ranged from 0.25 to 0.88 among all individuals analyzed. Based on Nei and Li’s similarity coefficients Bemisia populations were grouped into 3 main clusters and clearly distinguished the non B biotype from the B biotype. Conclusion: The level of similarity among populations of same biotypes was high whereas between populations of non B and B biotypes appeared to be less closely related. This analysis showed that non B biotype is prevalent in both provinces however B biotype is restricted to few locations in Sindh. This monitoring of the spread of B. tabaci in Pakistan will assist in the establishment of appropriate management strategies.

Estrutura genética populacional de Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) utilizando marcadores microssatélites / Population genetic structure of Bemisia tabaci (Hemiptera: Aleyrodidae) utilizing microsatellite markers

We aimed to characterize the population genetic structure within and among five Bemisia tabaci (Gennadius) populations collected from different host plants and geographic regions by using microssatelites as a molecular marker. Each population was represented by 19 specimens. The host plants and geographic origins of these populations were described as follows: Pop 1: Squash Barreiras (BA); Pop 2: Cotton Barreiras (BA); Pop 3: Soybean Campinas (SP); Pop 4: Tomato Cruz das Almas (BA); and Pop 5: Soybean Rondonópolis (MT). Six polymorphic loci were observed, which discriminated 31 different alleles in the studied populations, with a mean number of alleles per population of 3.30 (2.67 - 4.00). Using Fisher's Exact test, it was observed that at least three populations were in Hardy-Weinberg equilibrium for most of the studied loci (six). The dendrogram (UPGMA) separated populations into groups mainly related to the geographic origin of the samples. Only population 5 differed from the others at a 0.15 distance (74.5 percent group consistency). The most similar populations were 1 and 2, with a 0.01 distance (65.3 percent). This is in agreement with their geographic origins and it was not consistent with host specificity. The results suggest considerable gene flow (7.3 percent) among all whitefly populations and indicate that a better understanding of the gene flow in populations of B. tabaci associated with different hosts is required for the management of this insect.

Karyotype and male pre-reductional meiosis of the sharpshooter Tapajosa rubromarginata (Hemiptera: Cicadellidae)

Cicadellidae in one of the best represented families in the Neotropical Region, and the tribe Proconiini comprises most of the xylem-feeding insects, including the majority of the known vectors of xylem-born phytopathogenic organisms. The cytogenetics of the Proconiini remains largely unexplored. We studied males of Tapajosa rubromarginata (Signoret) collected at El Manantial (Tucumán, Argentina) on native spontaneous vegetation where Sorghum halepense predominates. Conventional cytogenetic techniques were used in order to describe the karyotype and male meiosis of this sharpshooter. T. rubromarginata has a male karyological formula of 2n=21 and a sex chromosome system XO:XX (♂:♀). The chromosomes do not have a primary constriction, being holokinetic and the meiosis is pre-reductional, showing similar behavior both for autosomes and sex chromosomes during anaphase I. For this stage, chromosomes are parallel to the acromatic spindle with kinetic activities in the telomeres. They segregate reductionally in the anaphase I, and towards the equator during the second division of the meiosis. This is the first contribution to cytogenetic aspects on proconines sharpshooters, particularly on this economic relevant Auchenorrhyncha species. Rev. Biol. Trop. 59 (1): 309-314. Epub 2011 March 01.

Los Cicadellidae son una de las familias mejor representadas en la región neotropical. La tribu Proconiini incluye a muchos de los insectos que se alimentan de xilema y la mayoría de los vectores de organismos fitopatógenos asociados con dicho tejido de conducción. La citogenética de los Proconiini es prácticamente inexplorada. Por lo tanto, se utilizaron técnicas citogenéticas convencionales para describir el cariotipo y la meiosis en los machos de Tapajosa rubromarginata Signoret. Este cicadélido presenta el complemento cromosómico diploide de 2n=20A+X0 en los machos. Los cromosomas no presentan constricción primaria, son holocinéticos, y la meiosis es pre-reduccional, muestra un comportamiento similar tanto en los cromosomas sexuales como en los autosómicos durante la anafase I. En ese estado, los cromosomas se orientan de manera paralela a las fibras del huso acromático con actividad cinética en los telómeros y segregan de manera reduccional en la fase I y ecuacional en la fase II de la meiosis.

Genetic variation among species, races, forms and inbred lines of lac insects belonging to the genus Kerria (Homoptera, Tachardiidae)

The lac insects (Homoptera: Tachardiidae), belonging to the genus Kerria, are commercially exploited for the production of lac. Kerria lacca is the most commonly used species in India. RAPD markers were used for assessing genetic variation in forty-eight lines of Kerria, especially among geographic races, infrasubspecific forms, cultivated lines, inbred lines, etc., of K. lacca. In the 48 lines studied, the 26 RAPD primers generated 173 loci, showing 97.7 percent polymorphism. By using neighbor-joining, the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups. The major cluster, comprising 32 lines, included mainly cultivated lines of the rangeeni form, geographic races and inbred lines of K. lacca. The second cluster consisted of eight lines of K. lacca, seven of the kusmi form and one of the rangeeni from the southern state of Karnataka. The remaining eight lines formed a series of outgroups, this including a group of three yellow mutant lines of K. lacca and other species of the Kerria studied, among others. Color mutants always showed distinctive banding patterns compared to their wild-type counterparts from the same population. This study also adds support to the current status of kusmi and rangeeni, as infraspecific forms of K. lacca.

Caracterização molecular e divergência genética de Bemisia tabaci (Genn. ) (Hemiptera: Aleyrodidae) em diferentes culturas e locais de cultivo / Molecular characterization and genetic divergence of Bemisia tabaci (Genn. ) (Hemiptera: Aleyrodidae) on different crops and growing areas

Knowledge on the genetic variation of populations of Bemisia tabaci (Genn.) can improve the understanding of genetic diversity found in their biotypes and, consequently, offer guidelines for its management. In this study, the molecular characterization was performed and genetic diversity data were obtained for this insect from three regions of Brazil on different crops [cotton and soybean (Mato Grosso - MT); cabbage (Distrito Federal - DF); soybean and potato (São Paulo - SP)], using RAPD markers. RAPD analysis indicated 80.6 percent polymorphic loci and the average genetic similarity obtained by the Jaccard coefficient was 0.67. The whitefly populations collected on potato (SP) and soybean (MT) had higher genetic diversity values (0.75 and 0.72, respectively). Shannon's index (Ho) showed higher values for potato and soybean (SP e MT), and a smaller value for cabbage (DF). A high genetic divergence within and among the collected populations occurred, structured according to the regions of collection. Moreover, the great genetic similarity observed between potato (SP) and soybean (SP) populations suggested that both belong to the same biotype B and reinforces the polyphagous behavior of the species.

Variabilidade genética de Bemisia tabaci (Gennadius) biótipo B (Hemiptera: Aleyrodidae) em cultivos olerícolas em São Luís, MA / Genetic variability of the Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae) in vegetable crops in São Luís, State of Maranhão, Brazil

The RAPD technique is widely used to investigate the distinct genetic characteristics of the complex Bemisia tabaci (Gennadius), which is currently constituted of approximately 41 biotypes. The objective of this research was to characterize populations of whitefly collected in crops of agricultural producing areas in São Luís, MA, like okra, beans and pepper, using RAPD molecular markers. Females from nine whitefly populations were analyzed and compared with B. tabaci biotype B taken from poinsettia culture of Embrapa Genetic Resources and Biotechnology (Brasília, DF). Twelve out of the 20 primers tested produced specific band patterns suitable to confirm that the evaluated specimens belong to the biotype B of B. tabaci, despite the high percentage of detected polymorphism. The analysis of the 96 RAPD molecular markers generated indicated that the populations on okra, beans and pepper were grouped according to the host cultures, sharing 80, 76 and 45 percent of genetic similarity, respectively, when compared with the control population of B. tabaci biotype B. A lower selective pressure was observed with the population of whitefly collected on pepper and minor genetic variability in the whitefly populations collected on okra and bean, when compared with the control population.

A técnica de RAPD é amplamente empregada para investigar características genéticas distintas dentro do complexo Bemisia tabaci Gennadius, atualmente constituído de aproximadamente 41 biótipos. O objetivo desta pesquisa foi caracterizar populações de mosca-branca coletadas em culturas agrícolas do município de São Luís, MA, como quiabo, feijão e pimentão, utilizando marcadores moleculares RAPD. Fêmeas de nove populações de mosca-branca foram analisadas e comparadas com o biótipo B de B. tabaci proveniente de cultura de poinsétia da Embrapa Recursos Genéticos e Biotecnologia (Brasília, DF). Dos 20 iniciadores utilizados, 12 produziram padrões de bandas específicas, que permitiram confirmar que os espécimes avaliados pertencem ao grupo do biótipo B de B. tabaci, apesar da alta percentagem de polimorfismo detectado. Com os 96 marcadores moleculares RAPD gerados foi construído um dendrograma, que mostrou que as populações de quiabo, feijão e pimentão foram agrupadas de acordo com as culturas hospedeiras. A matriz de similaridade genética entre as populações de B. tabaci mostrou 80, 76 e 45 por cento similaridade genética entre as populações das culturas de quiabo, feijão e pimentão, respectivamente, quando comparadas com a população controle de B. tabaci biótipo B. Foi também observada menor pressão de seleção na população de mosca-branca coletada em pimentão e menor variabilidade genética nas populações de mosca-branca coletadas em quiabo e feijão, quando comparadas com a população controle.

Monitoramento da suscetibilidade a inseticidas em populações de Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) no Brasil / Monitoring the susceptibility to insecticides in Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations from Brazil

O monitoramento da suscetibilidade de pragas a praguicidas é essencial para programas de manejo da resistência. O objetivo deste trabalho foi verificar a variabilidade genética de populações de Bemisia tabaci (Gennadius), oriundas de diferentes áreas agrícolas, quanto a suscetibilidade a inseticidas no Brasil, por meio de dois testes diferentes. Quatro populações de mosca-branca foram testadas em relação a uma população suscetível de referência (SusIAC); duas oriundas de Goiás (GO-1 e GO-2) e outras duas da Bahia (BA-1 e BA-2). Uma técnica de bioensaio do tipo contato residual foi empregada para realização dos testes: 1) teste diagnóstico da resistência; e 2) teste para obtenção das linhas de suscetibilidade das populações. Os produtos utilizados foram acetamipride, imidaclopride, tiametoxam, clorpirifós e endosulfam. Utilizaram-se insetos adultos não separados por sexo e nem por idade nos testes. As avaliações foram realizadas em 24h, para o teste com endosulfam, e em 48h, para os demais produtos. Ambos os testes revelaram variabilidade genética quanto à suscetibilidade de mosca-branca aos inseticidas. No entanto, a discriminação entre as populações de B. tabaci foi mais evidente com o uso de testes diagnósticos. A população GO-2 foi significativamente menos suscetível aos produtos testados que a SusIAC, principalmente em relação aos neonicotinóides. A situação mais crítica de resistência de mosca-branca foi com tiametoxam, seguida pelo imidaclopride.

Monitoring the susceptibility of a pest population to pesticides is essential for resistance management programs. The objective of this research was to evaluate the genetic variability in pesticide susceptibility in populations of Bemisia tabaci (Gennadius) collected from different Brazilian agricultural regions through the use of two different tests. Four whitefly populations, two from Goiás state (GO-1 and GO-2) e two from Bahia state (BA-1 and BA-2), were tested against a susceptible reference one (SusIAC). A residual contact bioassay was used to evaluate the pesticide susceptibility of each population by using diagnostic concentration bioassays and by estimating the baseline susceptibility data to each one of the tested insecticides, acetamiprid, imidacloprid, thiamethoxam, chlorpyrifos and endosulfan. Adult insects of unknown age and sex were tested. Evaluations were performed after 24h for endosulfan and 48h for the other chemicals. Both procedures showed significant differences in the susceptibility to the pesticides among B. tabaci populations. However, the discrimination among B. tabaci populations was more evident with the use of diagnosis tests. The population GO-2 was significantly less susceptible to the tested pesticides than SusIAC, mainly to neonicotinoids. The most critical resistance situation of B. tabaci was detected to thiamethoxam, followed by imidacloprid.

Diversidade genética de Tibraca limbativentris Stål (Hemiptera: Pentatomidae) de Santa Catarina e do Rio Grande do Sul, usando marcadores RAPD / Genetic diversity of Tibraca limbativentris Stål (Hemiptera: Pentatomidae) of Santa Catarina and Rio Grande do Sul, Brazil, using RAPD

O trabalho objetivou testar protocolos de extração de DNA e caracterizar populações de Tibraca limbativentris, Stål, importante inseto-praga do arroz. Os insetos foram coletados em Joinville, Rio do Oeste e Turvo, em Santa Catarina, e Agudo, Uruguaiana, Pelotas e Palmares do Sul, no Rio Grande do Sul. Testaram-se seis protocolos de extração de DNA citados na literatura, e um novo protocolo adequado à espécie em questão. DNA de dez indivíduos de cada população foi extraído usando o melhor protocolo e reações de RAPD foram realizadas com dez iniciadores. O novo protocolo mostrou os melhores resultados e foi utilizado nas reações de PCR, que geraram 151 bandas polimórficas, permitindo acessar diferenças genéticas entre todas as populações; não ocorreram indivíduos de uma população agrupados com os de outra. A maior similaridade intrapopulacional foi encontrada em Uruguaiana (22 por cento), e a menor em Palmares do Sul (50 por cento), também a população mais divergente das demais. O valor Gst foi 0,5215, e de Nm 0,4588; esses valores refletem a pouca similaridade entre as populações. O menor Nm foi apresentado quando Palmares do Sul e Pelotas foram incluídos nas comparações, em consonância com a maior divergência apresentada por essas populações em relação às outras. Não se observou relação entre a distância geográfica e a similaridade genética das populações, o que refletirá o modelo de dispersão de T. limbativentris, ainda desconhecido. Estudos explorando as estratégias de dispersão da espécie poderiam ajudar no entendimento da distribuição do inseto, evidenciando qual a principal fonte de variabilidade genética.

The work was carried out to test DNA extraction protocols and to characterize populations of Tibraca limbativentris Stål, an important rice insect-pest. Insects were collected in Joinville, Rio do Oeste and Turvo, in Santa Catarina State, and Agudo, Uruguaiana, Pelotas and Palmares do Sul, in Rio Grande do Sul State, and six literature-referenced protocols, besides a new one, were tested. DNA from ten individuals of each population was extracted using the best protocol and RAPD reactions were carried out with ten initiators. The new protocol showed the best results and was used in the PCR reactions, that generated 151 polymorphic bands, allowing to access genetic differences among all the populations; no individuals from one population were clustered with individuals from another. The largest intrapopulacional similarity was found in Uruguaiana (22 percent), and the smallest in Palmares do Sul (50 percent), which was also the most divergent population in relation to the others. The Gst was 0.5215, and the Nm was 0.4588; these values reflect the low similarity between the populations. The smallest genic flow was obtained when Palmares do Sul and Pelotas were included in the comparisons, in accordance with the largest divergence of these two populations in relation to the others. There was no significant relation between geographic distance and genetic similarity, which can reflect unknown model of dispersion of T. limbativentris. New studies exploring the species dispersion strategies may help to understand the insect distribution and to unveil the main factors linked to the genetic variability within and between populations.

Expressed sequenced tags from Lygus lineolaris (Hemiptera: Miridae), the tarnished plant bug

Expressed sequenced tags (ESTs) were prepared to establish a baseline for molecular genetic studies of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). The largest class of identifiable ESTs (15.2%) was from genes involved in cellular metabolic functions, including physiological processes. Twenty-seven ESTs (9.8%) were from genes associated with transcription and translation, including ribosomal genes. One hundred and forty-two of the 276 unique ESTs were from genes not previously identified from any organism. Twelve sequences appear to be associated with feeding and digestion and may be targets for pest control studies

Genetic diversity of Costa Rican populations of the rice planthopper Tagosodes orizicolus (Homoptera: Delphacidae)

Tagosodes orizicolus (Homoptera: Delphacidae) is one of the main constraints of the rice production in the Neotropics. This planthopper produces severe damages as a phloem feeder, causes mechanical injury during oviposition and vectors the rice hoja blanca virus (RHBV). The main objective of this study was to determine the genetic diversity of T. orizicolus populations from three rice growing regions of Costa Rica, using RAPDs. Individuals from Guanacaste, Parrita, San Carlos and Cali-Colombia, as outgroup, were analyzed using the random primers. Phenetic relationships revealed that the Costa Rican populations were clearly separated from Cali-Colombia, sharing less than 25% similarity. Costa Rican populations were divided into two main branches separated at 30% similarity. The first branch included Guanacaste and San Carlos and the second displayed Parrita. In relation to similarity indexes within groups, the Guanacaste cluster showed the highest (over 50%) and Cali-Colombia was the most diverse (28%). The correspondence analysis confirmed the clusters of the phenogram and showed close interactions between the Parrita and San Carlos populations. The genetic separation observed could be the result of the geographic isolation among populations, but it could also be explained by the infection with the rickettsia Wolbachia pipientis. This bacterium causes cytoplasmic incompatibility in its host, which results in non-viable progeny when infected males mate with non-infected females, or when insects hosting different strains of Wolbachia mate. Then, a search for Wolbachia in previously described populations of T orizicolus was initiated. The presence of the bacteria was analyzed by PCR with 16S rDNA-specific primers for Wolbachia. The PCR analyses revealed infections of 86% in the population of San Carlos, 96% in Guanacaste, 37% in Parrita and 100% in Cali-Colombia. Crosses between individuals of T. orizicolus from Parrita and Guanacaste were performed for testing...

Fine-structural details of the photoreceptor membranes in the ocellus of the scale-insect parasite Centrodora sp. (Hymenoptora; Aphenelidae): a case of gene transfer between host and parasite?

Only one insect (the scale insect Eriococcus sp.) is known, in which photoreceptive lamellae appear to have replaced the usual arthropod rhabdom microvilli. We are now reporting the presence of photoreceptive membranes, which also appear to resemble lamellae rather than microvilli, but they are in the ocellus of the tiny wasp Centrodora sp., which parasitizes scale insect eggs. The apparently optically homogenous lens of the Centrodora ocellus measures approximately 10 µm in diameter and, thus, operates at the limits of diffraction. We calculated that the lens is capable of focusing a parallel bundle of rays on the retina of the ocellus.

RAPD markers linked to brown planthopper Nilaparvatha lugens resistance locus in rice.

Brown plant hopper, a major pest in rice causes "hopper burn" in the field. The resistance gene for brown planthopper was mapped by using 20 recombinant inbred lines (RIL's) derived from a cross between resistant line Oryza. officinalis derivative (IR 54742-2-21-12-17-6) and a susceptible rice cultivar ASD 16 using bulked segregant analysis. On an average of 4 loci were amplified and two RAPD primers amplified loci that co-segregated with resistance/susceptibility. The segregating RAPD loci were mapped using Mapmaker programme into 13 groups. The expected and the 95% confidence level were found to be 15.2 and 47.7 cM respectively, confirming the location of the brown planthopper resistant gene on the region of chromosome 4. These RAPD markers will accelerate breeding programme for brown planthopper resistance.