Differential structure of the intronic promoter of the Bombyx mori A3 actin gene correlated with silkworm sensitivity/resistance to nucleopolyhedrovirus.

Previous reports demonstrated that actin is necessary for nucleocapsid transport and viral gene expression during nucleopolyhedrovirus infection of Bombyx mori. The first intron of B. mori A3 actin contains a cryptic promoter that drives expression of a rare isoform. We detected differences in the size and nucleotide composition of the first intron of the A3 actin gene from B. mori strain C24A, which is more resistant to nucleopolyhedrovirus than the M11A strain (22 and 95% lethality, respectively). We sought to determine if resistance to BmMNPV infection and the A3 actin promoter structure are correlated. Intrinsically bent DNA sites in these sequences, which determine curved structures, were analyzed by electrophoretic mobility assays and the helical parameters ENDS ratio, roll and twist. We found both fragments to have non-centralized bent DNA sites with distinct ENDS ratio values, nucleotide positions and two-dimensional structures. Additionally, a conformational-sensitive gel electrophoresis assay identified an allelic variation found in strain M11A that is absent in strain C24A. These data suggest that A3 actin intronic sequence variations impair virus propagation and are markers of BmMNPV-resistant populations.

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury.

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80% identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80 percent identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.

Molecular markers in commercial Bombyx mori (Lepidoptera: Bombycidae) hybrids susceptible to multiple nucleopolyhedrovirus.

The silkworm Bombyx mori L. is particularly susceptible to virus diseases, especially B. mori nucleopolyhedrovirus (BmNPV). Disease resistance, along with high productivity, are important selection criteria for developing commercial hybrids of B. mori. We used bioassays and molecular markers linked to susceptibility/resistance to baculovirus infection to analyze the response of commercial B. mori hybrids from two companies to a geographic isolate of B. mori multiple nucleopolyhedrovirus (BmMNPV) from Paraná state in Brazil. Both of these commercial lines were highly susceptible to BmMNPV, with death rates of 92 and 94%. A polymorphic fragment of approximately ~350 bp, associated with susceptibility, and an ~800-bp fragment, associated with resistance to BmMNPV, were detected in all specimens. An additional fragment of ~480 bp, recently identified by our research team as a BmMNPV genomic sequence, was detected in the infected silkworms and could be used as a molecular marker for the diagnosis of nucleopolyhedrovirus infection.

Allelic variability in the third intron of the fibroin light chain gene in Bombyx mori (Lepidoptera: Bombycidae).

Conformation-sensitive gel electrophoresis is a useful method for identifying allele polymorphism; it provides co-dominant molecular markers. Using this method, we identified genetic variability in the third intron of the fibroin light chain gene, fib-L, in six Bombyx mori strains. Only Chinese C21A strain did not demonstrate allelic alterations, showing only homoduplex DNA molecules. We found distinct heteroduplex profiles in the Japanese HAA, M12B and M19-2 and the Chinese C25B and C24-2 strains. Analysis with restriction endonuclease fingerprinting conformation-sensitive gel electrophoresis demonstrated the potential of this method for the identification of allelic variability in B. mori; this was confirmed by cloning and sequencing the different alleles. The main alteration was a 12-bp deletion in two alleles of the C24-2 strain and one allele of the HAA strain; this deletion results in specific heteroduplex DNA molecule profiles.

DNA barcode information for the sugar cane moth borer Diatraea saccharalis.

We reviewed the use and relevance of barcodes for insect studies and investigated the barcode sequence of Diatraea saccharalis. This sequence has a high level of homology (99%) with the barcode sequence of the Crambidae (Lepidoptera). The sequence data can be used to construct relationships between species, allowing a multidisciplinary approach for taxonomy, which includes morphological, molecular and distribution data, all of which are essential for the understanding of biodiversity. The D. saccharalis barcode is a previously undescribed sequence that could be used to analyze Lepidoptera biology.