Fernando Hercos Valicente

Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm ( Diabrotica virgifera virgifera )

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

Analysis of the genome of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV-19) and of the high genomic heterogeneity in group II nucleopolyhedroviruses

The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-19), was completely sequenced and shown to comprise 132,565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-19 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-19 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-19 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes and rearrangements. Synthenic maps for the genomes of group II NPVs revealed that gene collinearity was observed only within certain clusters. Analysis of the dynamics of gene gain and loss along the phylogenetic tree of the NPVs showed that group II had only five defining genes and supported the hypothesis that these viruses form ten highly divergent ancient lineages. Crucially, more than 60 % of the gene gain events followed a power-law relation to genetic distance among baculoviruses, indicative of temporal organization in the gene accretion process.

Plasmid patterns of efficient and inefficient strains of Bacillus thuringiensis against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

Bacillus thuringiensis harbors genes encoding Cry proteins found in chromosomes or plasmids of different sizes (4-150 Mb). Although the smaller plasmids are more abundant in B. thuringiensis, their specific function is unknown. As for the megaplasmids, their main recognized function is to harbor cry genes, although the sequencing of some of these plasmids indicates the occurrence of other important genes. This work used a new protocol for practical and rapid extraction of plasmid DNA in order to characterize the plasmid patterns of Brazilian strains belonging to Embrapa Milho e Sorgo research center B. thuringiensis bank. We tried to further assess the relationship of plasmid patterns with strains belonging to the same serovars and strains causing 100% and no mortality to Spodoptera frugiperda (J.E. Smith) larvae. It was possible to characterize 59 strains based on the migration of bands in agarose gel. Strains belonging to the same serovars showed different plasmid sizes (from 1,636 bp to 23,200 bp), with the exception of two strains belonging to serovar galleriae. The strain T09 Bt tolworthi showed a plasmid migration pattern identical to strains belonging to serovar galleriae. Plasmid patterns differed for 46 strains, confirming that this is a useful tool to discriminate specific strains. However, it was not possible to associate the plasmid pattern or the occurrence of particular plasmids with the pathogenicity of a given species towards S. frugiperda larvae.

Molecular characterization of Bacillus thuringiensis using rep-PCR

The genetic divergence of 65 strains of Bacillus thuringiensis (Bt) was determined using Rep-PCR. Based on the repetitive sequences the BOX primer was the most informative with 26 fragments, followed by ERIC (19) and REP (10), generating a total of 55 fragments. The dendogram shows that ten groups were formed when 45% was the average distance of the population: group 1 with 41,5% of the isolates, 33,8% of the isolates were distributed in other groups and 24,6% did not formed distinct group. 53,2% of the isolates from Embrapa are in the group 1, and 29,8% of the isolates are distributed in other groups. Bt strains from USDA and Institute Pasteur showed more variability.

Nomuraea rileyi (Hypocreales: Clavicipitaceae) in Helicoverpa armigera (Lepidoptera: Noctuidae) Larvae in Brazil

Summary Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is one of the most important polyphagous pests globally. It was reported in Brazil at the end of the 2012/2013 crop season. The aim of this study was to report the occurrence of an entomopathogenic fungus on H. armigera larvae in Brazil. We collected 589 larvae from cotton plantations in Bahia State, Brazil, and transported them to the laboratory of biological control of Embrapa Milho e Sorgo in Sete Lagoas, Minas Gerais State, Brazil. Of the 320 dead larvae, 106 were infected by Nomuraea rileyi (Farl.) Samson (Hypocreales: Clavicipitaceae), causing 33.1% of the total mortality. This is the first report on the natural occurrence of the fungus N. rileyi infecting H. armigera larvae in Brazil.

fAFLP analysis of Brazilian Bacillus thuringiensis isolates

A total of 65 Bacillus thuringiensis (Bt) isolates were subjected to analysis of genetic relationship using fAFLP (fluorescent Fragment Length Polymorphism), in order to determine the genetic diversity within a group of Bt strains. 26 strains from different subspecies were identified as it follows: 9 kindly provided by the USDA (United States Department of Agriculture), 9 kindly provided by the Institute Pasteur and eight from Embrapa Maize and Sorghum Bt Collection, and 39 strains with no subspecies information also from Embrapa’s Bt Collection. DNA sample was double digested with restriction enzymes Eco RI and Mse I, and the fragments were linked to adapters. Selective amplification reactions were performed using five primer combinations and the amplified fragments were separated by gel electrophoresis on an ABI377 sequencer. Genetic distances were obtained by the complement of the Jaccard coefficient and the groups were performed by the UPGMA method. Five primer combinations generated 495 scorable fragments and 483 were found to be polymorphic. Out of 26 subspecies, strains 344 and T09 (B. thuringiensis subsp. tolworthi) showed the highest similarity (15%), while isolates HD3 B. thuringiensis subsp finitimus and T24 B. thuringiensis subsp neoleonensis were the most genetically distant (92%). B. thuringiensis isolates with no subspecies identification, found in samples from Goiás State showed higher similarity forming a group with an average distance of 6%, and the closest subspecies to this group was B. thuringiensis subsp thuringiensis (HD2) with 52% of similarity. This similarity may be due to the fact that these organism exchange genetic material by conjugation, and it is relatively common to have evolutionary characteristics of their ancestors.

Characterization of Bacillus thuringiensis Using Plasmid Patterns, AFLP and Rep-PCR

Molecular characterization of Bacillus thuringiensis (Bt) strains may be used to characterize DNA, protein, and genetic variability among Bt isolates. Different techniques have been used to discriminate between different isolates with different properties such as genetic profile, genetic variability, and proteins. The most common technique used for characterization of genetic profile and toxicity prediction is the polymerase chain reaction (PCR); for genetic diversity it is the repetitive element polymorphism (Rep-PCR) using ERIC, REP, and BOX primers. Amplified fragment length polymorphism (AFLP) is used to understand genetic variability and detect molecular markers, and plasmid characterization is important to detect the number and plasmid patterns. B. thuringiensis proteins can be characterized according to their size in SDS PAGE. In general, for most techniques the advantage is that it is not necessary to know about the genome of the species to be studied, it is not so expensive, and the results are reproducible.