Genetic diversity among eight <Emphasis Type="Italic">Dendrolimus</Emphasis> species in Eurasia (Lepidoptera: Lasiocampidae) inferred from mitochondrial COI and COII,and nuclear ITS2 markers

Background

Moths of genus Dendrolimus (Lepidoptera: Lasiocampidae) are among the major pests of coniferous forests worldwide. Taxonomy and nomenclature of this genus are not entirely established, and there are many species with a controversial taxonomic position. We present a comparative evolutionary analysis of the most economically important Dendrolimus species in Eurasia.

Results

Our analysis was based on the nucleotide sequences of COI and COII mitochondrial genes and ITS2 spacer of nuclear ribosomal genes. All known sequences were extracted from GenBank. Additional 112 new sequences were identified for 28 specimens of D. sibiricus, D. pini, and D. superans from five regions of Siberia and the Russian Far East to be able to compare the disparate data from all previous studies. In total, 528 sequences were used in phylogenetic analysis. Two clusters of closely related species in Dendrolimus were found. The first cluster includes D. pini, D. sibiricus, and D. superans; and the second, D. spectabilis, D. punctatus, and D. tabulaeformis. Species D. houi and D. kikuchii appear to be the most basal in the genus.

Conclusion

Genetic difference among the second cluster species is very low in contrast to the first cluster species. Phylogenetic position D. tabulaeformis as a subspecies was supported. It was found that D. sibiricus recently separated from D. superans. Integration of D. sibiricus mitochondrial DNA sequences and the spread of this species to the west of Eurasia have been established as the cause of the unjustified allocation of a new species: D. kilmez. Our study further clarifies taxonomic problems in the genus and gives more complete information on the genetic structure of D. pini, D. sibiricus, and D. superans.

     

Cytochalasans and PMA induce IL-2 receptors on CD8+ lymphocytes.

The cytochalasans, fungal metabolites that interact with actin, can affect lymphocyte proliferation; high concentrations inhibit lectin-induced proliferation and low concentrations augment it. The phorbol ester tumor promoter, PMA, alone is not mitogenic for primary lymphocytes but enhances the activity of mitogenic lectins. Because the cytochalasans have been reported to increase intracellular Ca2+ and because PMA activates protein kinase C, lymphocytes were treated with PMA and cytochalasin B (CyB) to determine if this combination would induce DNA synthesis. While this treatment by itself did not cause proliferation, lymphocytes cultured with PMA and CyB overnight, washed, and recultured with IL-2 proliferated to the same degree as lymphocytes stimulated with Con A. Three different cytochalasans, cytochalasin B, cytochalasin D, and chaetoglobosin C, all of which bind to cellular actin with different affinities and only one of which affects glucose transport, induced IL-2 receptors in combination with PMA. Flow cytometric analysis with an antibody to the IL-2 receptor alpha subunit confirmed the induction of receptors on CD8+ cells. However, no IL-2 was produced after the exposure of lymphocytes to the combination of cytochalasans and PMA. Therefore, there was sufficient signal to induce IL-2 receptor expression but not to induce IL-2.     

Comparative efficacy of two controlled-release gypsy moth mating disruption formulations

The effects of aerial applications of the gypsy moth sex pheromone, disparlure, on mating disruption and suppression of growth of populations of the gypsy moth, Lymantria dispar (L.), were investigated. Two formulations of disparlure, plastic laminate flakes applied in a single application and polymethacrylate beads applied in two applications, were compared in two separate tests conducted in 1993 and 1994. The beads were applied in two applications spaced 2 weeks apart because preliminary tests had indicated that they released pheromone too rapidly to maintain adequate emission rates throughout the period of male flight. In 1993, the flakes were applied at a rate of 50 g a.i./ha, and the beads were applied at a rate of 15 g a.i./ha for each application. In 1994, the flakes were applied at a rate of 75 g a.i./ha and the beads were applied at rates of 32.5 and 42.5 g a.i./ha for the two applications. Beads with larger average particle size were used in 1994 to prolong disparlure release. The treatments applied in 1993 resulted in >97% reduction in mating and >82% suppression of population growth in the following year. Because of a 1995 collapse of gypsy moth populations in the vicinity of the tests, reliable population growth data were not available for the treatments applied in 1994, but significant mating disruption did occur under both treatments. Based on measurements of residual disparlure after field aging, the flakes released 32 and 48% of their disparlure content during the 6 weeks of male moth flight in 1993 and 1994, respectively. The smaller beads used in 1993 released 75% of their disparlure content, and the larger beads used in 1994 released 52% of their disparlure content, during the 6 weeks of male flight. The biological efficacy data suggest that the bead and flake formulations, as applied in these tests, have similar effects on gypsy moth mating disruption and subsequent population growth. Based on the observed release rates from both 1993 and 1994, a single application of the beads would provide emission rates equal to or greater than those provided by the flakes when applied at an equal dose.     

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

[3H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12-O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [³H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [³H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [³H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.     

Cytokines secreted by bone-metastatic breast cancer cells alter the expression pattern of f-actin and reduce focal adhesion plaques in osteoblasts through PI3K

Breast cancer frequently metastasizes to bone, resulting in osteolytic lesions. These lesions, formed by activated osteoclasts, cause pain, an increased susceptibility to fractures, and hypercalcemia. It has been shown that breast cancer cells communicate with osteoblasts and subsequently stimulate osteoclast activity; however, little research has focused on understanding the interaction between breast cancer cells and osteoblasts. We recently reported that conditioned medium from MDA-MB-231 breast cancer cells inhibited the differentiation of MC3T3-E1 osteoblasts through the secretion of transforming growth factor beta (TGFbeta). In addition, the breast cancer conditioned medium altered MC3T3-E1 morphology, the pattern of actin stress fibers, and reduced focal adhesion plaques. In the current study, we identified the mechanism used by MDA-MB-231 cells to cause these effects. When MC3T3-E1 osteoblasts were cultured with MDA-MB-231 conditioned medium preincubated with neutralizing antibodies to platelet derived growth factor (PDGF), insulin-like growth factorII (IGFII), and TGFbeta, focal adhesion plaques and actin stress fiber formation were restored. These cytokines were further found to signal through PI3Kinase and Rac. In conclusion, TGFbeta, PDGF, and IGFII might be good therapeutic targets for treating breast cancer-induced osteolytic lesions.     

Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users

In this study, we describe two cases of human immunodeficiency virus type 1 (HIV-1) intersubtype superinfection with CRF01_AE and subtype B strains, which occurred in two injection drug users participating in a prospective cohort study in Bangkok, Thailand. In both cases, the superinfecting strain was detected by molecular and serologic analyses several weeks after complete seroconversion to the primary infection with a strain belonging to a different subtype. Superinfection occurred despite specific T-cell and humoral antibody responses to the primary virus. In both cases, cross-subtype immune responses were limited or absent prior to the second infection. These data show that, in some individuals, the quality and quantity of the immune response elicited by primary HIV-1 infection may not protect against superinfection. This finding has important implications for vaccine design. HIV-1 vaccines, at a minimum, will need to include potent, broadly protective, conserved immunogens derived from several group M subtypes.     

Stimulatory beetle volatiles for the Asian longhorned beetle,Anoplophora glabripennis (Motschulsky)

Two male-specific beetle volatiles were found that elicited strong gas chromatographic-electroantennographic responses from both sexes of Asian longhorned beetle adults, Anoplophora glabripennis. The secretion consisted of a approximately 1:1 (v/v) blend of functionalized dialkyl ethers, 4-(n-heptyloxy)butanal and 4-(n-heptyloxy)butan-1-ol. These compounds are chemically unusual natural products that are previously unknown from insects. Laboratory olfactometer studies showed that a blend of 10 microg of each synthetic compound on a filter paper strip was significantly attractive to ALB adults.