A Possible Working Hypothesis of Host Translation Inhibitory Factor, CpBV15, Encoded in the Genome of Cotesia plutellae Bracovirus

( Nalini Madanagopal ),( Yong Gyun Kim ) 한국응용곤충학회 2006 한국응용곤충학회 학술대회논문집 Vol.2006 No.9

Biochemical Characterization of C-type Lectin in the Diamondback Moth, Plutella xylostella (Yponomeutidae: Lepidoptera)

Madanagopal, Nalini,Kim, Yong-Gyun Korean Society of Applied Entomology 2007 Journal of Asia-Pacific Entomology Vol.10 No.3

Lectins due to their affinity to carbohydrate moiety are involved in diverse functions like cell attachment in embryogenesis, organogenesis and cellular trafficking as well as nonself recognition in immune responses. Agglutinating activity was detectable in Plutella xylostella (Yponomeutidae: Lepidoptera) against 14 different species including bacterial and yeast cells, among which the whole body homogenate of P. xylostella agglutinated Providencia vermicola, Flavobacterium sp., and Saccharomyces cerevisiae with high titers. On analysis of physico-chemical properties, this putative agglutinating factor (s) was specifically dependent on the presence of $Ca^{++}$ for its activity and was reversibly sensitive to EDTA. The agglutinating activity was stable at pH 6-8, but was heat-labile. The agglutinating factor (s) was proteinaceous in nature as it was completely precipitable by ammonium sulphate. Its carbohydrate binding activity was demonstrated by inhibition assay, which revealed that methyl ${\alpha}$-D-mannopyranoside inhibited agglutination against P. vermicola. In contrast, P. xylostella parasitized by an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera), also showed the agglutination properties with somewhat higher activity than the nonparasitized. Carbohydrate inhibition assay with methyl ${\alpha}$-D-mannopyranoside was detectable at one-fold higher concentration in the homogenate of the parasitized larvae, suggesting that the agglutinating factor (s) is inducible or due to de novo parasitism-specific synthesis. These results suggest the presence of calcium-dependent lectin in P. xylostella and an alteration in the agglutinating property by C. plutellae parasitization.

Biochemical Characterization of C-type Lectin in the Diamondback Moth, Plutella xylostella (Yponomeutidae: Lepidoptera)

Nalini Madanagopal,김용균 한국응용곤충학회 2007 Journal of Asia-Pacific Entomology Vol.10 No.3

Lectins due to their affinity to carbohydrate moiety are involved in diverse functions like cell attachment in embryogenesis, organogenesis and cellular trafficking as well as nonself recognition in immune responses. Agglutinating activity was detectable in Plutella xylostella (Yponomeutidae: Lepidoptera) against 14 different species including bacterial and yeast cells, among which the whole body homogenate of P. xylostella agglutinated Providencia vermicola, Flavobacterium sp., and Saccharomyces cerevisiae with high titers. On analysis of physico-chemical properties, this putative agglutinating factor (s) was specifically dependent on the presence of Ca++ for its activity and was reversibly sensitive to EDTA. The agglutinating activity was stable at pH 6-8, but was heat-labile. The agglutinating factor (s) was proteinaceous in nature as it was completely precipitable by ammonium sulphate. Its carbohydrate binding activity was demonstrated by inhibition assay, which revealed that methyl α-D-mannopyranoside inhibited agglutination against P. vermicola. In contrast, P. xylostella parasitized by an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera), also showed the agglutination properties with somewhat higher activity than the nonparasitized. Carbohydrate inhibition assay with methyl α-D-mannopyranoside was detectable at one-fold higher concentration in the homogenate of the parasitized larvae, suggesting that the agglutinating factor (s) is inducible or due to de novo parasitism-specific synthesis. These results suggest the presence of calcium-dependent lectin in P. xylostella and an alteration in the agglutinating property by C. plutellae parasitization.

Parasitism by Cotesia glomerata Induces Immunosuppression of Pieris rapae: Effects of Ovarian Protein and Polydnavirus

Nalini Madanagopal,Yonggyun Kim 한국응용곤충학회 2006 Journal of Asia-Pacific Entomology Vol.9 No.4

A gregarious endoparasitoid wasp, Cotesia glomerata, parasitizes the cabbage butterfly, Pieris rapae. During wandering larval stage for pupal metamorphosis, the parasitoid larvae egress from the parasitized host to form cocoons thus eventually leading to death of the host. This study focused on the effect of C. glomerata parasitization on cellular immune response of P. rapae. For this purpose, an ideal anticoagulant buffer was formulated to procure the hemocytes in native form with morphological, behavioral, and functional characteristics. The hemocytes selectively encapsulated only DEAE beads under in vitro conditions and a quantitative study revealed about 70% of the beads being encapsulated. On the other hand, calyx fluid from C. glomerata injected to P. rapae markedly inhibited the spreading ability of the hemocytes in a dose-dependent manner and also attenuated the in vitro encapsulation response of the hemocytes against the cationic bead. The calyx fluid contained polydnavirus as well as ovarian proteins. The isolated polydnavirus genome consisted of variously sized-segments with their unequal amounts. The P. rapae injected with the calyx fluid expressed several polydnaviral genes within 2 h. These results suggest that the immunosuppression of the parasitized P. rapae may be induced by the polydnaviral gene products as well as ovarian proteins.

Pyriproxyfen Inhibits Hemocytic Phagocytosis of the Beet Armyworm, Spodoptera exigua

Nalini Madanagopal,Yongjoon Lee(이용준),Yonggyun Kim(김용균) 한국농약과학회 2007 농약과학회지 Vol.11 No.3

An Evidence for Host Translation Inhibitory Factor Encoded in a Polydnavirus, Cotesia glomerata Bracovirus, Genome and Its Expression in Parasitized Cabbage White Butterfly, Pieris rapae

Nalini Madanagopal,김용균 한국응용곤충학회 2007 Journal of Asia-Pacific Entomology Vol.10 No.4

Polydnavirus is a DNA virus symbiotic to some endoparasitic wasps and plays a critical role in accomplishing successful parasitic life cycle of host wasps. Host translation inhibitory factor (HTIF) has been found in some polydnaviral genomes and performs parasitic functions leading to host immunosuppression and redirecting host nutrient usage to wasp development. The cabbage white butterfly, Pieris rapae, parasitized by a gregarious endoparasitoid, Cotesia glomerata, undergoes several physiological alterations including immune malfunctioning and failure of pupal metamorphosis. C. glomerata possesses its own symbiotic polydnavirus, C. glomerata bracovirus (CgBV). Its genome consisted of at least 12 segments in unequal amounts. Parasitized P. rapae hemolymph contained HTIF-like protein, which was determined through an immunoblotting assay using HTIF antibody of C. plutellae bracovirus (CpBV). RT-PCR using HTIF primers of CpBV produced an HTIF-like gene in P. rapae larvae parasitized by C. glomerata. Also, this HTIF-like gene was encoded in CgBV genome and its partial sequence of CgBV showed highly homology (98.5%) to amino acid sequence of an HTIF of CpBV, called CpBV15α. These results suggest that a common HTIF- like moiety may be shared among Cotesia-associated bracovirus.

An Evidence for Host Translation Inhibitory Factor Encoded in a Polydnavirus, Cotesia glomerata Bracovirus, Genome and Its Expression in Parasitized Cabbage White Butterfly, Pieris rapae

Madanagopal, Nalini,Kim, Yong-Gyun Korean Society of Applied Entomology 2007 Journal of Asia-Pacific Entomology Vol.10 No.4

Polydnavirus is a DNA virus symbiotic to some endoparasitic wasps and plays a critical role in accomplishing successful parasitic life cycle of host wasps. Host translation inhibitory factor (HTIF) has been found in some polydnaviral genomes and performs parasitic functions leading to host immunosuppression and redirecting host nutrient usage to wasp development. The cabbage white butterfly, Pieris rapae, parasitized by a gregarious endoparasitoid, Cotesia glomerata, undergoes several physiological alterations including immune malfunctioning and failure of pupal metamorphosis. C. glomerata possesses its own symbiotic polydnavirus, C. glomerata bracovirus (CgBV). Its genome consisted of at least 12 segments in unequal amounts. Parasitized P. rapae hemolymph contained HTIF-like protein, which was determined through an immunoblotting assay using HTIF antibody of C. plutellae bracovirus (CpBV). RT-PCR using HTIF primers of CpBV produced an HTIF-like gene in P. rapae larvae parasitized by C. glomerata. Also, this HTIF-like gene was encoded in CgBV genome and its partial sequence of CgBV showed highly homology (98.5%) to amino acid sequence of an HTIF of CpBV, called $CpBV15{\alpha}$. These results suggest that a common HTIF-like moiety may be shared among Cotesia-associated bracovirus.

Parasitism by Cotesia glomerata Induces Immunosuppression of Pieris rapae: Effects of Ovarian Protein and Polydnavirus

Madanagopal, Nalini,Kim, Yong-Gyun Korean Society of Applied Entomology 2006 Journal of Asia-Pacific Entomology Vol.9 No.4

A gregarious endoparasitoid wasp, Cotesia glomerata, parasitizes the cabbage butterfly, Pieris rapae. During wandering larval stage for pupal metamorphosis, the parasitoid larvae egress from the parasitized host to form cocoons thus eventually leading to death of the host. This study focused on the effect of C. glomerata parasitization on cellular immune response of P. rapae. For this purpose, an ideal anticoagulant buffer was formulated to procure the hemocytes in native form with morphological, behavioral, and functional characteristics. The hemocytes selectively encapsulated only DEAE beads under in vitro conditions and a quantitative study revealed about 70% of the beads being encapsulated. On the other hand, calyx fluid from C. glomerata injected to P. rapae markedly inhibited the spreading ability of the hemocytes in a dose-dependent manner and also attenuated the in vitro encapsulation response of the hemocytes against the cationic bead. The calyx fluid contained polydnavirus as well as ovarian proteins. The isolated polydnavirus genome consisted of variously sized-segments with their unequal amounts. The P. rapae injected with the calyx fluid expressed several polydnaviral genes within 2 h. These results suggest that the immunosuppression of the parasitized P. rapae may be induced by the polydnaviral gene products as well as ovarian proteins.

Transient expression of parasitism specific-protein synthesis inhibitor, CpBV15β during the early larval stage of Plutella xylostella

Madanagopal Nalini,Yonggyun Kim 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.05

The diamondback moth, Plutella xylostella parasitized by its endoparasitoid Cotesia plutellae undergoes various physiological alterations which involves immunosuppression and developmental arrest. Its symbiotic virus, C. plutellae bracovirus (CpBV) is highly essential for their successful parasitization which possesses more than 136 genes encoded. CpBV15βunique in CpBV genome is expressed at low levels in early and at higher levels during late parasitization period. This gene product alters the hemocyte-spreading behavior through inhibition of protein synthesis under in vitro conditions. In the current study, we investigated its specific involvement in physiological functions in the host by transient expression and RNA interference techniques. The open reading frame of CpBV15βwas cloned into a eukaryotic expression vector and this recombinant CpBV15β was transfected into healthy non-parasitized 3rd instar P. xylostella by microinjection. CpBV15βwas expressed as early as 24 h and was consistent up to 72 h. Due to the expression of this gene, the hemolymph storage protein levels were significantly reduced and the ability of the hemocytes to adhere and spread on extracellular matrix was altered or reduced, wherein CpBV15βwas detectable in the cytoplasm of hemocytes based on indirect immunofluorescence assay. To confirm the role of CpBV15β, its double stranded RNA could efficiently recover the functional efficacy of hemocytes towards non-self and synthesis of storage proteins. Thus these results clearly demonstrate the role of CpBV15βin altering the host physiology by involving in cellular immune response and host protein synthesis.

Hemolin: a stage-specific and inducible gene involved in humoral and cellular immune responses in the diamondback moth, Plutella xylostella

Madanagopal Nalini,Yonggyun Kim 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.05

Hemolin is a member of the immunoglobulin (Ig) superfamily and contains four Ig domainsthat are similar to neural cell adhesion molecules. It has been regarded as a recognition molecule at immune challenge in insects. This study showed that hemolin of Plutella xylostella was expressed during pupal and adult stages but absent in all larval instars without any immune challenge. It is, however, strongly induced by the injection of Escherichia coli or its lipopolysaccharide in hemocytes, fat body and gut. A double-stranded RNA (dsRNA) interference experiment revealedits role in activation of prophenoloxidase (PPO) in the hemolymph during bacterial infection. Also its involvement in cellular defense was investigated in its mediation of the adherence of hemocytes to rat blood erythrocytes which was knocked down by its dsRNA. Finally, its physiological significance in pupal stage was confirmed by using dsRNA, which significantly prevented adult development. Therefore, it is concluded that hemolin plays roles in both immune and adult development in P. xylostella.