Strategies for exogenous RNA delivery in RNAi-mediated pest management / 生物工程学报

Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

DNA sequencing in the classroom: complete genome sequence of two earwig (Dermaptera; Insecta) species

BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.

Population genetics of Chrysoperla externa (Neuroptera: Chrysopidae) and implications for biological control / Genética de populações de Chrysoperla externa (Neuroptera: Chrysopidae) e implicações para o controle biológico

Abstract Green lacewings are insects with great potential to be use in the biological control of agricultural pests, but relatively few studies have attempted to understand the genetic structure of these agents, especially those of predatory insects. The purpose of this study was to characterize genetically populations of C. externa using sequences of subunit I of the cytochrome oxidase, a mitochondrial gene, and examine the population structure of this species in sampled areas in São Paulo state. The results indicate high genetic diversity but no genetic structure, detected by AMOVA analysis, and high levels of haplotype sharing in the network. These genetic patterns could be a consequence of environmental homogeneity provided by agroecosystem (citrus orchard), allowing gene flow among populations. Probably there is a unique population in the area sampled that could be used as a population (genetic) source for mass-reared and posterior release in these farms.

Resumo Crisopídeos são insetos com grande potencial para uso em controle biológico de pragas agrícolas, mas relativamente poucos estudos têm tentado compreender a estrutura genética destes agentes, especialmente no caso de insetos predadores. O objetivo deste trabalho foi caracterizar geneticamente populações de C. externa utilizando sequências da subunidade I do gene mitocondrial citocromo oxidase e avaliar a estruturação populacional desta espécie em áreas amostras no estado de São Paulo. Os resultados indicaram elevada diversidade genética e nenhuma estruturação genética, detectada pela AMOVA, além de elevado compartilhamento na rede haplotípica. Este padrão genético poderia ser uma consequência da homogeneidade ambiental favorecida pelos agroecossistemas (citricultura), permitindo fluxo gênico entre as populações. Provavelmente há uma única população, do ponto de vista genético, na área amostrada que poderia ser utilizada em criações massais e em liberações nas fazendas desta região.

Diversidad genérica de Trichoptera (Insecta) en dos microcuencas del Páramo Rabanal (Cundinamarca-Boyacá, Colombia)

Generic diversity of Trichoptera (Insecta) of Paramo Rabanal (Cundinamarca-Boyacá, Colombia). Trichopterans are considered an important and diverse biotic element in continental aquatic ecosystems. The purpose of this study was to evaluate the assemblages of the order Trichoptera in two subwatersheds with a gradient of disturbance. Four sampling events were conducted in two subwatersheds in the Eastern Mountain Range of the Colombian Andes. For the analysis we used rarefaction curves, Bray-Curtis’ Index and Partitioning Diversity and total richness and Shannon’s diversity as metrics. Although total richness was similar between both subwatersheds, abundance was always highest in streams within the conserved subwatershed. Each subwatershed was dominated by different genera, except Ochrotrichia, which was abundant at all sites. Alpha diversity was similar among streams in the conserved watershed, while a reduction in diversity potentially associated with the disturbance gradient was observed in streams of the disturbed subwatershed. Beta diversity (0D and ¹D) between subwatersheds and among conserved streams was similar, while in disturbed streams a similar gradient to that of alpha diversity was found. The similitude analysis clustered streams according to their conservation status. Differences found in trichopteran assemblages do confirm that the use of their attributes is adequate to assess the conservation status of stream ecosystems. Rev. Biol. Trop. 62 (Suppl. 2): 97-110. Epub 2014 April 01.

Los tricópteros son considerados un componente biótico importante y diverso de los ecosistemas acuáticos continentales. Este estudio tuvo como objetivo evaluar la comunidad del orden Trichoptera en dos microcuencas con diferente grado de perturbación. Se realizaron cuatro campañas de muestreo en dos microcuencas de la Cordillera Oriental de los Andes colombianos. Se realizaron análisis mediante curvas de rarefacción, el índice de Bray Curtis y partición de la diversidad, usando como medidas la riqueza y la diversidad de Shannon. Se encontró que, aunque la riqueza fue similar entre las dos microcuencas, la abundancia siempre fue mayor en los arroyos de la microcuenca conservada. En cada microcuenca dominaron géneros diferentes, excepto Ochrotrichia que fue abundante en todos los sitios. La diversidad alfa entre los arroyos de la microcuenca conservada fue similar, mientras que en la perturbada se observó una reducción de la diversidad asociada a un gradiente de perturbación de los arroyos. La diversidad beta (0D y ¹D) entre microcuencas y entre arroyos conservados fue similar, mientras que en los perturbados también mostró un gradiente similar al de la diversidad alfa. El análisis de similitud agrupó los arroyos según su estado de conservación. Las diferencias encontradas en los atributos de los comunidades de tricópteros confirman que su uso es adecuado para conocer el estado de conservación de los sistemas que habita.

Comparison of mtDNA extracting methods for common sarcosaphagous insects / 法医学杂志

OBJECTIVE@#To compare effects of three different methods for mtDNA extraction from common sarcosaphagous insects including cetyl trimethyl ammonium bromide (CTAB) method, sodium dodecyl sulfate-potassium acetate (SDS-KAc) method and sodium dodecyl sulfate-proteinase K (SDS-PK) method.@*METHODS@#Seventy-two insects from four species [Chrysomya megacephala (Fabricius, 1784), Eusilpha bicolor (Fairmaire, 1896), Paraeutrichopus pecoudi (Mateu, 1954), Vespa velutina (Lepeletier, 1836)] were collected from the corpses of the rabbits in Changsha district. The total DNA of above samples was extracted by CTAB, SDS-Kac and SDS-PK methods. The purity and concentration of DNA were examined by protein-nucleic acid spectrophotometry, and mtDNA were amplified by specific primers and PCR products were detected by agarose gel electrophoresis. Then PCR products were sequenced and subsequently up-loaded to GenBank.@*RESULTS@#mtDNA was successfully extracted with three methods from most of the samples. The SDS-PK method was better in DNA purity compared to other methods and the CTAB method was superior in extracting DNA from old samples, while SDS-KAc method showed no significant difference for extraction effects of different samples.@*CONCLUSION@#The most appropriate method should be chosen depending on different situations. SDS-PK method is expected to obtain high-quality DNA, while CTAB method is preferred in extracting obsolete samples. SDS-KAc method is low cost and can be used in various kinds of preliminary experiments.

Application of mtDNA polymorphism in species identification of sarcosaphagous insects / 法医学杂志

Species identification of sarcosaphagous insects is one of the important steps in forensic research based on the knowledge of entomology. Recent studies reveal that the application of molecular biology, especially the mtDNA sequences analysis, works well in the species identification of sarcosaphagous insects. The molecular biology characteristics, structures, polymorphism of mtDNA of sarcosaphagous insects, and the recent studies in species identification of sarcosaphagous insects are reviewed in this article.

Seven new species of Lachesilla (Psocodea: 'Psocoptera': Lachesillidae), in the group forcepeta from the Amazon Basin / Sete espécies novas de Lachesilla Westwood (Psocodea:'Psocoptera': Lachesillidae) do grupo forcepeta da Bacia Amazônica

Seven new species of Lachesilla in the group forcepeta, from the Amazon Basin in Brazil, Colombia and Peru, are here described and illustrated: L. amacayacuensis sp. n. (type locality: Colombia, Amazonas, Leticia, Amacayacú); L.bulbosiforceps sp. n. (type locality: Peru, Cuzco); L. cuzcoensis sp. n. (type locality: Peru, Cuzco); L. marabaensis sp. n. (type locality: Brasil, Pará, Marabá, Serra Norte); L. pilosiforceps sp. n. (type locality: Brasil, Pará, Oriximiná, Rio Trombetas); L. pilosipenna sp. n. (type locality: Peru, Cuzco); L. squamiforceps sp. n. (type locality: Colombia, Amazonas, Leticia). The Amazon Basin is the second most rich world area for species of Lachesilla.

São descritas e ilustradas sete espécies de Lachesilla do grupo forcepeta, provenientes da bacia amazônica do Brasil, Colômbia e Peru: L. amacayacuensis sp. n. (localidade-tipo: Colômbia, Amazonas, Leticia, Amacayacú); L. bulbosiforceps sp. n. (localidade-tipo: Peru, Cuzco); L. cuzcoensis sp. n. (localidade-tipo: Peru, Cuzco); L. marabaensis sp. n. (localidade-tipo: Brasil, Pará, Marabá, Serra Norte); L. pilosiforceps sp. n. (localidade-tipo: Brasil, Pará, Oriximiná, Rio Trombetas); L. pilosipenna sp. n. (localidade-tipo: Peru, Cuzco); L. squamiforceps sp. n. (localidade-tipo: Colômbia, Amazonas, Letícia). A região amazônica é a segunda mais rica em número de espécies de Lachesilla.

Suitability of leguminous cover crop pollens as food source for the green lacewing Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae)

Diversification of crops with species that provide suitable pollen for predators may reduce pest population on crops by enhancing predator effectiveness. In this paper we evaluated the suitability of leguminous cover crop pollens to the predatory green lacewing Chrysoperla externa (Hagen). The predator is commonly found in coffee agroecosystems and the plant species tested were pigeon pea and sunn hemp, which are used in organic coffee systems. Newly emerged females and males of C. externa were reared on diets containing pollen of pigeon pea, sunn hemp, or castor bean, used as a control. The reproductive success of C. externa was evaluated when females fed the pollen species and when honey was added to the diets, to verify the predator need for an extra carbohydrate source. Similar intrinsic growth rates were found for females fed on pigeon pea pollen and on sunn hemp pollen but these rates increased significantly when honey was added to the diets. Females fed with pigeon pea pollen plus honey and with sunn hemp pollen plus honey had higher intrinsic growth rates than those fed with castor bean pollen plus honey. Females fed on castor bean pollen only or on honey only, did not oviposit. Leguminous pollen species were equally suitable for C. externa especially when they were complemented with honey. The results suggest that to successfully enhance predator effectiveness, organic coffee plantation should be diversified with plant providing pollen in combination with plant providing nectar.

A diversificação das culturas com plantas que forneçam pólen nutricionalmente adequado para predadores pode reduzir a população de pragas pelo aumento da efetividade dos predadores. Neste trabalho, avaliou-se a viabilidade nutricional de polens de leguminosas utilizadas como adubação verde, para o crisopídeo Chrysoperla externa (Hagen), comumente encontrado em agroecossistemas cafeeiros. Os adubos verdes testados foram guandu e crotalária, leguminosas utilizadas em sistemas orgânicos de café. Adultos de C. externa recém-emergidos foram criados em dietas contendo pólen de guandu, de crotalária e de mamona (controle). O sucesso reprodutivo de C. externa foi avaliado nas dietas de pólen e quando mel foi adicionado ao pólen, para se verificar a necessidade do predador por fonte extra de carboidratos. A taxa de crescimento populacional do predador foi semelhante nas dietas de pólen de guandu e de crotalária. No entanto, a taxa aumentou significativamente quando mel foi adicionado às dietas. Fêmeas alimentadas com pólen de guandu e mel e com pólen de crotalária e mel tiveram crescimento superior ao das alimentadas com pólen de mamona e mel. Fêmeas alimentadas somente com pólen de mamona ou somente com mel não ovipositaram. Os polens de guandu e de crotalária foram igualmente adequados para C. externa, especialmente quando foram complementados com mel. Os resultados sugerem que para aumentar a efetividade dos predadores, os sistemas orgânicos de café deveriam ser diversificados com plantas que forneçam pólen em combinação com plantas que forneçam néctar.

Chromosome complement and meiosis in three species of the Neotropical bug genus Antiteuchus ( Heteroptera, Pentatomidae, Discocephalinae )

Orcein staining of spermatocytes was used to study the meiotic behavior of holocentric chromosomes in three member of the genus Antiteuchus (commonly known as stink bugs). We describe and illustrate the karyotype of Antiteuchus mixtus, A. sepulcralis and A. macraspis which were cytogenetically characterized as having a diploid number of 2n = 14 and an XY sex chromosome system showing pre-reductional meiosis for autosomes and post-reductional meiosis for sex chromosomes. These species were also shown to have a long diffuse stage during meiotic prophase I and aberrant harlequin-type meiocytes. The chiasma frequency was also analyzed for two of the three species studied.

Effectiveness of "Bacillus thuringiensis" strains against "Spodoptera frugiperda" (Lepidoptera: Noctuidae)

The fall armyworm ("Spodoptera frugiperda") is one of the most important pests of maize, causing up to 20(per cent) production losses when defoliation occurs near to flowering, or even complete destruction of plants. Among the alternatives to control this pest, the use of "Bacillus thuringiensis" ("Bt") has gained attention due to its efficiency and low impact on natural enemies. Strains "Bt dendrolimus" HD 37, "Bt aizawai" HD 68, "Bt kurstaki" HD 73, "Bt darmstadiensis" HD 146, and "Bt thuringiensis" 4412 were tested against second instar larvae in "in vivo" assays. Suspensions of "Bt aizawai" HD 68 and "Bt thuringiensis" 4412, containing 3 x 10 8 cells/ml, induced mortality of 100(per cent) and 80.4(per cent), respectively. To test virulence, cell concentrations of 8x10(5) to 3x10(8) cells/ml of strains "Bt aizawai" HD 68 and "Bt thuringiensis" 4412 were applied on the second instar larvae: LC(50) were 6.7x10(6) and 8.6x10(6) cells/ml, respectively.

Insect juvenile hormone: from "status quo" to high society

Juvenile hormone (JH) exerts pleiotropic functions during insect life cycles. The regulation of JH biosynthesis by neuropeptides and biogenic amines, as well as the transport of JH by specific binding proteins is now well understood. In contrast, comprehending its mode of action on target organs is still hampered by the difficulties in isolating specific receptors. In concert with ecdysteroids, JH orchestrates molting and metamorphosis, and its modulatory function in molting processes has gained it the attribute "status quo" hormone. Whereas the metamorphic role of JH appears to have been widely conserved, its role in reproduction has been subject to many modifications. In many species, JH stimulates vitellogenin synthesis and uptake. In mosquitoes, however, this function has been transferred to ecdysteroids, and JH primes the ecdysteroid response of developing follicles. As reproduction includes a variety of specific behaviors, including migration and diapause, JH has come to function as a master regulator in insect reproduction. The peak of pleiotropy was definitely reached in insects exhibiting facultative polymorphisms. In wing-dimorphic crickets, differential activation of JH esterase determines wing length. The evolution of sociality in Isoptera and Hymenoptera has also extensively relied on JH. In primitively social wasps and bumble bees, JH integrates dominance position with reproductive status. In highly social insects, such as the honey bee, JH has lost its gonadotropic role and now regulates division of labor in the worker caste. Its metamorphic role has been extensively explored in the morphological differentiation of queens and workers, and in the generation of worker polymorphism, such as observed in ants

Hematoxylin: a simple, multiple-use dye for chromosome analysis

A staining mixture of hematoxylin-iron alum combined with a strong hydrochloric hydrolysis was successfully applied for chromosome observation of several kinds of plants and some animals. Slightly different procedures were developed for different materials and objectives. For plant cells, the most important technical aspect was the use of 5 N HCl hydrolysis, which resulted in a very transparent cytoplasm, combined with an intense, specific hematoxylin stain. This technique is recommended for cytogenetical analysis in general, and it is especially indicated for practical classes, due to its simplicity and high reproducibility of results. Moreover, the deep contrast observed makes this technique very useful for sequential staining of cells previously analyzed with other stains, as well as for materials with fixation problems.

Over-expression and characterization of recombinant beta subunit of the human chorionic gonadotropin hormone synthesized in insect cells infected with a genetically engineered baculovirus.

A recombinant baculovirus, vAc beta hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the beta subunit of hCG was used to express beta hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 hr pi, secreted approximately 8.02 micrograms beta hCG/2 x 10(6) cells/ml. The recombinant beta hCG purified from insect cells exhibited increased mobility on SDS-PAGE as compared to authentic urinary beta hCG, a reflection on differences in glycosylation between insect and mammalian systems. The insect derived beta hCG, however, was identical to the native hormonal peptide in terms of immunoreactivity and bioactivity on association with alpha-subunit, as evident by its binding to rat testicular receptors and induction of steroidogenesis in a mouse Leydig cell bioassay system. The implications of using the baculovirus system to study the importance of carbohydrates for biological activity are also discussed.