On species delimitation, hybridization and population structure of cassava whitefly in Africa.

The Bemisia cassava whitefly complex includes species that cause severe crop damage through vectoring cassava viruses in eastern Africa. Currently, this whitefly complex is divided into species and subgroups (SG) based on very limited molecular markers that do not allow clear definition of species and population structure. Based on 14,358 genome-wide SNPs from 62 Bemisia cassava whitefly individuals belonging to sub-Saharan African species (SSA1, SSA2 and SSA4), and using a well-curated mtCOI gene database, we show clear incongruities in previous taxonomic approaches underpinned by effects from pseudogenes. We show that the SSA4 species is nested within SSA2, and that populations of the SSA1 species comprise well-defined south-eastern (Madagascar, Tanzania) and north-western (Nigeria, Democratic Republic of Congo, Burundi) putative sub-species. Signatures of allopatric incipient speciation, and the presence of a 'hybrid zone' separating the two putative sub-species were also detected. These findings provide insights into the evolution and molecular ecology of a highly cryptic hemipteran insect complex in African, and allow the systematic use of genomic data to be incorporated in the development of management strategies for this cassava pest

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for tw

Structural evolution and flip-flop recombination of chloroplast DNA in the fern genus Osmunda

The evolution and recombination of chloroplast genome structure in the fern genus Osmunda were studied by comparative restriction site mapping and filter hybridization of chloroplast DNAs (cpDNAs) from three species — 0. cinnamomea, 0. claytoniana and 0. regalis . The three 144 kb circular genomes were found to be colinear in organization, indicating that no major inversions or transpositions had occurred during the approximately 70 million years since their radiation from a common ancestor. Although overall size and sequence arrangement are highly conserved in the three genomes, they differ by an extensive series of small deletions and insertions, ranging in size from 50 bp to 350 by and scattered more or less at random throughout the circular chromosomes. All three chloroplast genomes contain a large inverted repeat of approximately 10 kb in size. However, hybridizations using cloned fragments from the 0. cinnamomea and 0. regalis genomes revealed the absence of any dispersed repeats in at least 50% of the genome. Analysis with restriction enzymes that fail to cleave the 10 kb inverted repeat indicated that each of the three fern chloroplast genomes exists as an equimolar population of two isomeric circles differing only in the relative orientation of their two single copy regions. These two inversion isomers are inferred to result from high frequency intramolecular recombination between paired inverted repeat segments. In all aspects of their general organization, recombinational heterogeneity, and extent of structural rearrangement and length mutation, these fern chloroplast genomes resemble very closely the chloroplast genomes of most angiosperms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46956/1/294_2004_Article_BF00418530.pd

Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins

Global FAW population genomic signature supports complex introduction events across the 1 Old World 2 3

16 Accurate genomic knowledge can elucidate the global spread patterns of invasive pests. The high-17 profile invasive agricultural pest Spodoptera frugiperda (fall armyworm; FAW) is a case in point. Native to the 18 Americas, the FAW was first reported in West Africa in 2016 and has rapidly spread to over 64 countries across 19 the Old World, resulting in significant economic losses. The chronological order of reported detections has led 20 to the hypothesis that the FAW moved eastwards across Africa and then Asia, however genomic evidence 21 remains lacking to test this hypothesis and to identify the potential origin of invasive populations. Using a whole 22 genome sequencing approach, we explored the population genomic signatures of FAW populations from the 23 Americas and the Old World. Analyses of complete mitochondrial DNA genomes identified 12 maternal lineages 24 across the invasive range, while genomic signatures from 870 high-quality nuclear genome-wide single 25 nucleotide polymorphic (SNP) DNA markers identified five distinct New World populations that broadly reflected 26 their native geographical ranges and absence of host-plant preference status. Unique FAW populations in the 27 Old World were also identified that suggested multiple introductions underpinned their rapid global spread. We 28 identified in Asian FAW individuals, genomes lacking evidence of admixture; while analysis of identified complex 29 substructure revealed significant directional geneflow from Asia into East Africa, in contrast to a simple east-to-30 west spread. Our study highlights the need for population genomics approaches in analysing complex pest 31 invasions, and the importance of international partnership to address global biosecurity challenges presented 32 by emerging high priority insect pests. 33 34 35 3

First Study of Different Insect Cells to Triatoma Virus Infection

The use of viruses for biological control is a new option to be considered. The family Dicistroviridae, which affects only invertebrates, is one of the families that have been proposed for this purpose. The Triatoma virus (TrV), a member of this family, affects triatomine transmitters of Chagas disease, which is endemic in Latin America but also expanding its worldwide distribution. To this end, we attempted virus replication in Diptera, Aedes albopictus (clone C6/36) and Lepidoptera Spodoptera frugiperda (SF9, SF21) and High Five (H5) cell lines. The methodologies used were transfection process, direct inoculation (purified virus), and inoculation of purified virus with trypsin. Results were confirmed by SDS-PAGE, Western blotting, RT-PCR, electron microscopy, and immunofluorescence. According to the results obtained, further analysis of susceptibility/infection of H5 cells to TrV required to be studied.Centro de Estudios Parasitológicos y de VectoresFacultad de Ciencias Veterinaria