An Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) mutant, vApAg, induces apoptosis in a cell culture derived from Anticarsia gemmatalis (UFL-AG-286), reducing viral progeny. We have investigated apoptosis induction in vivo by vApAg in A. gemmatalis larvae and its correlation to infectivity reduction. LC50, LD50, LT50 and the mean time to death of larvae were determined for vApAg and AgMNPV. Apoptosis was accessed for hemocytes of infected larvae using light and transmission electron microscopy. All types of hemocytes can be infected by vApAg. After 12 h post-infection (h p.i.), typical cellular modifications associated to nucleopolyhedrovirus infection were observed. Apoptosis becomes evident after 24 h p.i., and massive after 72 h p.i. Necrosis of infected cells was also observed. Despite cell death, hemocytes produced budded viruses and polyhedra. Pl and gh1-type hemocytes presented phagocytic activity. Agarose gel electrophoresis revealed fragmentation of hemocytes DNA at late times post-infection. The LC50 and LD50 were between five- and six-fold higher for vApAg. The LT50 and the mean time to death were higher for vApAg in a same treatment or for a similar mortality induced by AgMNPV. These results show correlation of apoptosis and the reduced infectivity of vApAg in A. gemmatalis larvae. © 2007 Elsevier B.V. All rights reserved.13001/02/15182192Aljanabi, S.M., Martinez, I., Universal and rapid salt extraction of high quality genomic DNA for PCR-based techniques (1997) Nucleic Acids Res., 25, pp. 4692-4693Ashe, P.C., Berry, M.D., Apoptotic signaling cascates (2003) Prog. NeuroPsychopharmacol. Biol. Psychiatry, 27, pp. 199-204Carpes, M.P., de Castro, M.E., Soares, E.F., Villela, A.G., Pinedo, F.J., Ribeiro, B.M., The inhibitor of apoptosis gene (iap-3) of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) encodes a functional IAP (2005) Arch. Virol., 150, pp. 1549-1562Castro, M.E.B., Ribeiro, B.M., Production of viral progeny in insect cells undergoing apoptosis induced by a mutant Anticarsia gemmatalis nucleopolyhedrovirus (2001) Microbiol. Res., 156, pp. 369-376Clarke, T.E., Clem, R.J., Lack of involvement of haemocytes in the establishment and spread of infection in Spodoptera frugiperda larvae infected with the baculovirus Autographa californica M nucleopolyhedrovirus by intrahaemocoelic injection (2002) J. Gen. Virol., 83, pp. 1565-1572Clarke, T.E., Clem, R.J., Insect defenses against virus infection: the role of apoptosis (2003) Int. Rev. Immunol., 22, pp. 401-424Clarke, T.E., Clem, R.J., In vivo induction of apoptosis correlating with reduced infectivity during baculovirus infection (2003) J. Virol., 77, pp. 2227-2232Clem, R.J., Baculoviruses and apoptosis: the good, the bad and the ugly (2001) Cell Death Differ., 8, pp. 137-143Clem, R.J., Miller, L.K., Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus (1993) J. Virol., 67, pp. 3730-3738Clem, R.J., Fechheimer, M., Miller, L.K., Prevention of apoptosis by a baculovirus gene during infection of insect cells (1991) Science, 254, pp. 1388-1390Clem, R.J., Robson, M., Miller, L.K., Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94 (1994) J. Virol., 68, pp. 6759-6762Dai, X., Shi, X., Pang, Y., Su, D., Prevention of baculovirus-induced apoptosis of BTI-Tn-5B1-4 (Hi5) cells by the p35 gene of Trichoplusia ni multicapsid nucleopolyhedrovirus (1999) J. Gen. Virol., 80, pp. 1841-1845Finney, D.J., (1971) Probit Analysis. third ed., , Cambridge University Press, New YorkGranados, R.R., Infectivity and mode of action of baculoviruses (1980) Biotechnol. Bioeng., 22, pp. 1377-1405Granados, R.R., Guoxun, L., Derksen, A.C.G., McKennna, K.A., A new insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus (1994) J. Invertebr. Pathol., 64, pp. 260-266Grasela, J.J., McIntosh, A.H., In vitro and in vivo host range of Anticarsia gemmatalis multiple nuclear polyhedrosis virus (1998) In Vitro Cell. Dev. Biol., 34, pp. 79-83Greene, G.L., Leppla, N.C., Dickerson, W.A., Velvetbean caterpillar: a rearing procedure and artificial medium (1976) J. Econ. Entomol., 69, pp. 487-548Haas-Stapleton, E.J., Washburn, J.O., Volkman, L.E., Pathogenesis of Autographa californica M nucleopolyhedrovirus in fifth instar Spodoptera frugiperda (2003) J. Gen. Virol., 84, pp. 2033-2040Hoover, K., Washburn, J.O., Volkman, L.E., Midgut-based resistance of Heliothis virescens to baculovirus infection mediated by phytochemicals in cotton (2000) J. Insect Physiol., 46, pp. 999-1007Johnson, D.W., Maruniak, J.E., Physical map of Anticarsia gemmatalis Nuclear Polyhedrosis Virus (AgMNPV-2) DNA (1989) J. Gen. Virol., 70, pp. 1877-1883Kerr, J.F.R., Wyllie, A.H., Currie, A.R., Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics (1972) Br. J. Cancer, 26, pp. 239-257Matos, T.G.T., Giugliano, L.G., Ribeiro, B.M., Báo, S.N., Structural and ultrastructural studies of Anticarsia gemmatalis midgut cells infected with the baculovirus A. gemmatalis nucleopolyhedrovirus (1999) Int. J. Insect Morphol. Embriol., 28, pp. 195-201Mead, G.P., Ratcliffe, N.A., Renwrantz, L.R., The separation of insect hemocyte types on Percoll gradients: methodology and problems (1986) J. Insect Physiol., 32, pp. 167-177Means, J.C., Penabaz, T., Clem, R.J., Identification and functional characterization of AMVp33, a novel homolog of the baculovirus caspase inhibitor p35 found in Amsacta moorei entomopoxvirus (2007) Virology, 358, pp. 436-447Morales, L., Moscardi, F., Sosa-Gomez, D.R., Paro, F.E., Soldorio, I.L., Fluorescent brighteners improve Anticarsia gemmatalis (Lepidoptera: Noctuidae) nucleopolyhedrovirus (AgMNPV) activity on AgMNPV susceptible and resistant strains of the insect (2001) Biol. Control., 20, pp. 247-253Moscardi, F., Assessment of the application of baculoviruses for control of Lepidoptera (1999) Ann. Rev. Entomol., 44, pp. 257-289Oliveira, J.V.C., Wolff, J.L.C., Garcia-Maruniak, A., Ribeiro, B.M., Castro, M.E.B., Souza, M.L., Moscardi, F., Zanotto, P.M.A., Genome of the most widely used viral biopesticide: Anticarsia gemmatalis multiple nucleopolyhedrovirus (2006) J. Gen. Virol., 87, pp. 3233-3250O'Reilly, D.R., Miller, L.K., Luckow, V.A., (1992) Baculovirus Expression Vectors. A Laboratory Manual. first ed., , W.H. Freeman and Company, New YorkPombo, V., Velloso, L.M., Ribeiro, B.M., Báo, S.N., Structural and ultrastructural changes during the infection of UFL-AG-286 cells with the baculovirus AgMNPV (1998) J. Invertebr. Pathol., 72, pp. 239-245Ribeiro, B.M., Souza, M.L., Kitajima, E.W., Taxonomia, caracterização molecular e bioquímica de vírus de insetos (1998) Controle Microbiano de Insetos, pp. 481-507. , Alves S.B. (Ed), FEALQ, PiracicabaSieburth, P.J., Maruniak, J.E., Growth characteristics of a continuous line from the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) (1988) In Vitro Cell. Dev. Biol., 24, pp. 195-198Sieburth, P.J., Maruniak, J.E., Susceptibility of an established cell line of Anticarsia gemmatalis (Lepidoptera: Noctuidae) to three nuclear polyhedrosis viruses (1988) J. Invertebr. Pathol., 52, pp. 453-458Silveira, E.B., Ribeiro, B.M., Báo, S.N., Morphological studies of apoptosis in insect cells infected with vApAg, an Anticarsia gemmatalis nucleopolyhedrovirus mutant (1999) J. Submicrosc. Cytol. Pathol., 31, pp. 543-554Silveira, E.B., Ribeiro, B.M., Báo, S.N., Characterization of larval haemocytes from the velvetbean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) (2003) J. Submicrosc. Cytol. Pathol., 35, pp. 129-139Silveira, E.B., Cordeiro, B.A., Ribeiro, B.M., Báo, S.N., Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvetbean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) (2004) Tissue Cell, 36, pp. 171-180Silveira, E.B., Cordeiro, B.A., Ribeiro, B.M., Báo, S.N., In vivo apoptosis induction and reduction of infectivity by an Autographa californica multiple nucleopolyhedrovirus p35- recombinant in hemocytes from the velvet bean caterpillar Anticarsia gemmatalis (Hubner) (Lepidoptera: Noctuidae) (2005) Res. Microbiol., 156, pp. 1014-1025Trudeau, D., Washburn, J.O., Volkman, L.E., Central role of haemocytes in Autographa californica M nucleopolyhedrosis pathogenesis in Heliothis virescens and Helicoverpa zea (2001) J. Virol., 75, pp. 996-1003Washburn, J.O., Kirkpatrick, B.A., Volkman, L.E., Insect protection against viruses (1996) Nature, 383, p. 767Washburn, J.O., Haas-Stapleton, E.J., Tan, F.F., Beckage, N.E., Volkman, L.E., Co-infection of Manduca sexta larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa californica M nucleopolyhedrovirus (2000) J. Insect Physiol., 46, pp. 179-190Zhang, P., Yang, K., Daí, X., Pang, Y., Su, D., Infection of wild-type Autographa californica multicapsid nucleopolyhedrovirus induces in vivo apoptosis of Spodoptera litura larvae (2002) J. Gen. Virol., 83, pp. 3003-3011