- Email: [email protected]
Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes
Journal of Invertebrate Pathology 104 (2010) 239–241
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes Songsak Roekring, Duncan R. Smith * Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, Thailand
a r t i c l e
i n f o
Article history: Received 13 January 2010 Accepted 2 April 2010 Available online 9 April 2010 Keywords: Aedes Apoptosis Brevidensovirus Caspase Densovirus
a b s t r a c t The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Mosquitoes of the Aedes genus are vectors of some of the most significant human viral pathogens (Tolle, 2009). In particular, A. aegypti or the yellow fever mosquito is a vector for yellow fever, dengue and chikungunya, while A. albopictus, or the Asian Tiger mosquito is a vector for dengue, chikungunya and West Nile virus (Tolle, 2009). Collectively, viruses transmitted by these mosquitoes are responsible for tens to hundreds of millions of infections of humans each year, and thus impose a significant burden on human health and health resources worldwide. In the current absence of effective vaccines (Pierson et al., 2008) or specific antivirals (Perera et al., 2008) for the majority of these diseases, effective amelioration of this burden has fallen primarily on mosquito control programs (Lounibos, 2002). While classic control strategies have relied upon the use of chemical insecticides, increasing resistance to these chemicals in mosquito populations (Roberts and Andre, 1994), as well as increasing concern as to the unregulated usage of these chemicals in the environment has limited their scope and application. Alternate approaches to vector control include the use of larvicidal entomopathogenic bacteria such as Bacillus spp (Federici et al., 2007), predatory fish (Willems et al., 2005) and copepods such as Mesocyclops aspericornis (Russell et al., 1996). Densonucleosis viruses or densoviruses are invertebrate viruses belonging to the subfamily Densovirinae within the family parvoviridae, genus Brevidensovirus (Murphy et al., 1995). Denosviruses are non-enveloped, icosahedral particles, with a diameter of 18– 22 nm, with an approximately 4 kb linear single stranded DNA
* Corresponding author. Fax: +66 662 441 9906. E-mail address: [email protected] (D.R. Smith). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.04.002
genome which contains several inverted repeats (Bando et al., 1990). To date several mosquito densoviruses have been characterized, including the Aedes densovirus designated AeDNV isolated from an A. aegypti mosquito colony (Lebedeva et al., 1973), an A. albopictus densonucleosis virus designated AalDNV, isolated from a persistently infected subclone of C6/36 cells (Jousset et al., 1993) and a densovirus isolated from adult A. albopictus mosquitoes in Thailand designated AThDNV (Kittayapong et al., 1999). AeDNV, AalDNV and AThDNV have all been shown to be highly pathogenic against A. aegypti larvae (Barreau et al., 1996; Hirunkanokpun et al., 2008; Jousset et al., 1993). However, experiments have shown that challenge of successive generations of A. aegypti with AThDNV results in an increasing proportion of surviving mosquitoes (Roekring et al., 2006), suggesting that some form of adaptation to the densovirus is occurring in these mosquitoes. This study sought to provide evidence as to the mechanism of mortality in denosovirus infected A. aegypti mosquitoes. 2. Materials and methods 2.1. Mosquitoes and densovirus challenge Captive bred A. aegypti mosquitoes were raised and challenged with AThDNV exactly as described elsewhere (Roekring et al., 2006). Briefly, larvae were raised in 2 l water bowls and fed on mouse feeding powder, while adults were reared in an insectarium at 27 °C, humidity 65–70% and were fed on 10% sugar water. For egg maturation, female mosquitoes were allowed to feed on Wistar rats. For AThDNV challenge, larvae at 4 h post hatching were washed twice in sterile distilled water before immersion in sterile distilled water to which the densovirus stock was added for 48 h.
240
S. Roekring, D.R. Smith / Journal of Invertebrate Pathology 104 (2010) 239–241
Following this, larvae were transferred to 2 l water bowls where they were fed as normal until hatched. Dead larvae, pupae and adults were collected daily. Survivors were those larvae that reached the adult stage. 2.2. TUNEL assay Mosquito samples used for TUNEL analysis were fixed in Davison’s AFA for 2–4 h before being dehydrated and embedded in paraffin using standard histological methods. Sections (4 lm thick) were cut and mounted on Superfrost Plus Slides (Fisher Scientific, PA, USA). Prior to hybridization, slides with sections were incubated on their sides at 45 °C overnight, deparaffined with xylene and then rehydrated through an ethanol series to water. Tissue sections were then pre-treated with 20 mg/L proteinase K for 30 min, and analyzed with an In Situ Cell Death Detection Kit, POD (Roche Applied Science, Germany) according to the manufacture’s guidelines. Controls included the TUNEL reaction mixture without terminal transferase (negative control) and samples treated with DNaseI (positive control). Sections were visualized by light microscopy. 2.3. Fluorescent in situ hybridization (FISH) FISH analysis was undertaken as described previously (Roekring et al., 2006). Briefly, a fluorescein-labeled DNA probe was prepared by PCR using a plasmid template containing an AThDNV fragment from open reading frame 3 (O’Neill et al., 1995) to amplify a 350 bp DNA fragment. Labeling was carried out using fluorescein-12-dUTP (Roche Molecular Biochemicals, Germany) according to the manufacturers protocol. Mosquito samples were processed as for TUNEL analysis (above) to the rehydration step. The in situ hybridization protocol followed that described by Lightner (Lightner, 1996) and sections were counterstained with propidium iodide. 2.4. Caspase 3-like activity assay Caspase 3-like activity was measured using the ApoAlertTM caspase colorimetric assay kit (Clontech laboratories, Inc. Mountain View, CA) according to the manufacturer’s protocol. Briefly, pools of seven mosquitoes were homogenized in 350 ll of the manufacturers provided chilled lysis buffer. Whole cell lysates were centrifuged at maximum speed in an eppendorf centrifuge at 4 °C for
10 min. 50 ll of each supernatant were aliquoted into new microcentrifuge tubes and placed on ice and subsequently, 50 ll of 2 reaction buffer containing 10 mM DTT was added. As appropriate, either 1 ll chilled DMSO or 1 ll of inhibitor (1 mM DEVD-fmk) were added and samples were incubated at 4 °C for 30 min, followed by the addition of 5 ll caspase 3 substrate (1 mM DEVDpNA) or 5 ll of chilled DMSO to the appropriate tubes. Absorbance was read with a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA) at 405 nm and the activity unit were determined according to the instruction provide with the kit. Samples from experiments were assayed in duplicate and experiments were undertaken as three independent replicates. All values are expressed as the mean ± standard deviation (SD).
3. Results and discussion While densoviruses are known to be highly pathogeneic towards Aedes mosquito larvae (Barreau et al., 1996; Hirunkanokpun et al., 2008; Jousset et al., 1993), studies have suggested that repeated challenge of successive generations of mosquitoes surviving prior densovirus challenge results in significantly reduced mortality (Roekring et al., 2006). Therefore, understanding both the mechanism of mortality in densovirus infected mosquitoes, as well as the basis for decreased susceptibility to successive generation challenge are important for developing denosoviruses as anti-mosquito biological control agents. Using a previously established challenge protocol (Roekring et al., 2006) two generations of A. aegypti mosquitoes were challenged with AThDNV. Specimens (both surviving and moribund) from the 2nd generation challenge were examined for the presence of apoptotic cells using the TUNEL assay to detect 30 -DNA hydroxyl groups, a hall mark of apoptosis induced DNA fragmentation (Gavrieli et al., 1992), as well as for the presence of AThDNV by fluorescent in situ hybridization (FISH) using a primer specific to the AThDNV genome (O’Neill et al., 1995). Results (Fig. 1) showed that while apoptotic cells were detected by TUNEL staining following densovirus challenge, relatively little TUNEL staining was observed in control (non-challenged) mosquitoes. These results were consistent in all tissues examined and no specific tissue distribution was observed. Interestingly mosquitoes surviving the challenge showed some degree of reactivity to the TUNEL reagent (Fig. 1). Positive control samples (DNase I treated) showed extensive staining in all nuclei examined, while and negative control sections (no terminal transferase in the reaction mix)
Fig. 1. Apoptosis in Aedes aegypti mosquitoes after densovirus challenge. Abdominal sections of surviving and moribund mosquitoes from a 2nd generation densovirus challenge together with sections from control (naïve) mosquitoes were examined by TUNEL to detect apoptotic cells (dark brown stain) and by FISH with a AThDNV specific probe (orange signal) and counter staining with propidium iodide (red signal). Leftmost FISH panel shows a section through an ovary of a surviving adult female.
S. Roekring, D.R. Smith / Journal of Invertebrate Pathology 104 (2010) 239–241 Table 1 Caspase 3-like activity in lysates of whole mosquitoes.
*
Specimens assayed
Unit caspase 3 activity ± SD
AThDNV infected mosquitoes + substrate (DEVD-pNA) Uninfected (na) mosquitoes + substrate (DEVD-pNA) AThDNV infected mosquitoes + substrate (DEVDpNA) + caspase inhibitor (DEVD-fmk) AThDNV infected mosquitoes (no substrate)
6.97 ± 0.88* 3.35 ± 0.11 1.85 ± 0.26 1.89 ± 0.47
P < 0.05 (Student’s T-test), as compared individually to the three other groups.
showed no evidence of staining (data not shown). The presence of AThDNV was clearly seen in both surviving and moribund mosquitoes upon FISH analysis (Fig. 1). To confirm that the increased numbers of TUNEL positive cells in AThDNV infected mosquitoes were the results of the induction of apoptosis, the levels of activated caspase 3-like activity, an executioner caspase (Shi, 2002) were investigated in 2nd generation AThDNV challenged mosquitoes, in comparison with unchallenged (naïve) mosquitoes. Controls included the inclusion of a caspase 3 inhibitor in the reaction mix, as well as the omission of the substrate. Results (Table 1) showed a greater level of specific caspase 3-like activation in moribund challenged mosquitoes as compared to unchallenged mosquitoes. Together, these results suggest that AThDEN induces apoptosis in infected mosquitoes. Acknowledgments SR was supported by a Mahidol University Postdoctoral Fellowship. DRS is supported by Mahidol University and the Thailand Research Fund. References Bando, H. et al., 1990. Terminal structure of a densovirus implies a hairpin transfer replication which is similar to the model for AAV. Virology 179, 57–63.
241
Barreau, C. et al., 1996. Pathogenicity of the Aedes albopictus parvovirus (AaPV), a denso-like virus, for Aedes aegypti mosquitoes. J Invertebr Pathol 68, 299–309. Federici, B.A. et al., 2007. Developing recombinant bacteria for control of mosquito larvae. J. Am. Mosq. Contr. Assoc. 23, 164–175. Gavrieli, Y. et al., 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119, 493–501. Hirunkanokpun, S. et al., 2008. Evaluation of mosquito densoviruses for controlling Aedes aegypti (Diptera: Culicidae): variation in efficiency due to virus strain and geographic origin of mosquitoes. Am. J. Trop. Med. Hyg. 78, 784–790. Jousset, F.X. et al., 1993. A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae. Virus Res. 29, 99–114. Kittayapong, P. et al., 1999. A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand. Am. J. Trop. Med. Hyg. 61, 612–617. Lebedeva, O.P. et al., 1973. Investigation of a virus disease of the densonucleosis type in a laboratory culture of Aedes aegypti. Acta Virol. 17, 253–256. Lightner, D.V., 1996. A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. Special publication of the world aquaculture society. Lounibos, L.P., 2002. Invasions by insect vectors of human disease. Annu. Rev. Entomol. 47, 233–266. Murphy, F.A., et al., 1995. Virus taxonomy. Sixth report of the international committee on taxonomy Oj viruses. Arch Virol. (Suppl. 10), 208–239. O’Neill, S.L. et al., 1995. Insect densoviruses may be widespread in mosquito cell lines. J. Gen. Virol. 76 (Pt 8), 2067–2074. Perera, R. et al., 2008. Closing the door on flaviviruses: entry as a target for antiviral drug design. Antivir. Res. 80, 11–22. Pierson, T.C. et al., 2008. Structural insights into the mechanisms of antibodymediated neutralization of flavivirus infection: implications for vaccine development. Cell Host Microbe. 4, 229–238. Roberts, D.R., Andre, R.G., 1994. Insecticide resistance issues in vector-borne disease control. Am. J. Trop. Med. Hyg. 50, 21–34. Roekring, S. et al., 2006. Challenging successive mosquito generations with a densonucleosis virus yields progressive survival improvement but persistent, innocuous infections. Dev. Comp. Immunol. 30, 878–892. Russell, B.M. et al., 1996. Surveillance of the mosquito Aedes aegypti and its biocontrol with the copepod Mesocyclops aspericornis in Australian wells and gold mines. Med. Vet. Entomol. 10, 155–160. Shi, Y., 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 9, 459–470. Tolle, M.A., 2009. Mosquito-borne diseases. Curr. Probl. Pediatr. Adolesc Health Care. 39, 97–140. Willems, K.J. et al., 2005. A comparison of mosquito predation by the fish Pseudomugil signifier Kner and Gambusia holbrooki (Girard) in laboratory trials. J. Vector Ecol. 30, 87–90.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes Songsak Roekring, Duncan R. Smith * Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, Thailand
a r t i c l e
i n f o
Article history: Received 13 January 2010 Accepted 2 April 2010 Available online 9 April 2010 Keywords: Aedes Apoptosis Brevidensovirus Caspase Densovirus
a b s t r a c t The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Mosquitoes of the Aedes genus are vectors of some of the most significant human viral pathogens (Tolle, 2009). In particular, A. aegypti or the yellow fever mosquito is a vector for yellow fever, dengue and chikungunya, while A. albopictus, or the Asian Tiger mosquito is a vector for dengue, chikungunya and West Nile virus (Tolle, 2009). Collectively, viruses transmitted by these mosquitoes are responsible for tens to hundreds of millions of infections of humans each year, and thus impose a significant burden on human health and health resources worldwide. In the current absence of effective vaccines (Pierson et al., 2008) or specific antivirals (Perera et al., 2008) for the majority of these diseases, effective amelioration of this burden has fallen primarily on mosquito control programs (Lounibos, 2002). While classic control strategies have relied upon the use of chemical insecticides, increasing resistance to these chemicals in mosquito populations (Roberts and Andre, 1994), as well as increasing concern as to the unregulated usage of these chemicals in the environment has limited their scope and application. Alternate approaches to vector control include the use of larvicidal entomopathogenic bacteria such as Bacillus spp (Federici et al., 2007), predatory fish (Willems et al., 2005) and copepods such as Mesocyclops aspericornis (Russell et al., 1996). Densonucleosis viruses or densoviruses are invertebrate viruses belonging to the subfamily Densovirinae within the family parvoviridae, genus Brevidensovirus (Murphy et al., 1995). Denosviruses are non-enveloped, icosahedral particles, with a diameter of 18– 22 nm, with an approximately 4 kb linear single stranded DNA
* Corresponding author. Fax: +66 662 441 9906. E-mail address: [email protected] (D.R. Smith). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.04.002
genome which contains several inverted repeats (Bando et al., 1990). To date several mosquito densoviruses have been characterized, including the Aedes densovirus designated AeDNV isolated from an A. aegypti mosquito colony (Lebedeva et al., 1973), an A. albopictus densonucleosis virus designated AalDNV, isolated from a persistently infected subclone of C6/36 cells (Jousset et al., 1993) and a densovirus isolated from adult A. albopictus mosquitoes in Thailand designated AThDNV (Kittayapong et al., 1999). AeDNV, AalDNV and AThDNV have all been shown to be highly pathogenic against A. aegypti larvae (Barreau et al., 1996; Hirunkanokpun et al., 2008; Jousset et al., 1993). However, experiments have shown that challenge of successive generations of A. aegypti with AThDNV results in an increasing proportion of surviving mosquitoes (Roekring et al., 2006), suggesting that some form of adaptation to the densovirus is occurring in these mosquitoes. This study sought to provide evidence as to the mechanism of mortality in denosovirus infected A. aegypti mosquitoes. 2. Materials and methods 2.1. Mosquitoes and densovirus challenge Captive bred A. aegypti mosquitoes were raised and challenged with AThDNV exactly as described elsewhere (Roekring et al., 2006). Briefly, larvae were raised in 2 l water bowls and fed on mouse feeding powder, while adults were reared in an insectarium at 27 °C, humidity 65–70% and were fed on 10% sugar water. For egg maturation, female mosquitoes were allowed to feed on Wistar rats. For AThDNV challenge, larvae at 4 h post hatching were washed twice in sterile distilled water before immersion in sterile distilled water to which the densovirus stock was added for 48 h.
240
S. Roekring, D.R. Smith / Journal of Invertebrate Pathology 104 (2010) 239–241
Following this, larvae were transferred to 2 l water bowls where they were fed as normal until hatched. Dead larvae, pupae and adults were collected daily. Survivors were those larvae that reached the adult stage. 2.2. TUNEL assay Mosquito samples used for TUNEL analysis were fixed in Davison’s AFA for 2–4 h before being dehydrated and embedded in paraffin using standard histological methods. Sections (4 lm thick) were cut and mounted on Superfrost Plus Slides (Fisher Scientific, PA, USA). Prior to hybridization, slides with sections were incubated on their sides at 45 °C overnight, deparaffined with xylene and then rehydrated through an ethanol series to water. Tissue sections were then pre-treated with 20 mg/L proteinase K for 30 min, and analyzed with an In Situ Cell Death Detection Kit, POD (Roche Applied Science, Germany) according to the manufacture’s guidelines. Controls included the TUNEL reaction mixture without terminal transferase (negative control) and samples treated with DNaseI (positive control). Sections were visualized by light microscopy. 2.3. Fluorescent in situ hybridization (FISH) FISH analysis was undertaken as described previously (Roekring et al., 2006). Briefly, a fluorescein-labeled DNA probe was prepared by PCR using a plasmid template containing an AThDNV fragment from open reading frame 3 (O’Neill et al., 1995) to amplify a 350 bp DNA fragment. Labeling was carried out using fluorescein-12-dUTP (Roche Molecular Biochemicals, Germany) according to the manufacturers protocol. Mosquito samples were processed as for TUNEL analysis (above) to the rehydration step. The in situ hybridization protocol followed that described by Lightner (Lightner, 1996) and sections were counterstained with propidium iodide. 2.4. Caspase 3-like activity assay Caspase 3-like activity was measured using the ApoAlertTM caspase colorimetric assay kit (Clontech laboratories, Inc. Mountain View, CA) according to the manufacturer’s protocol. Briefly, pools of seven mosquitoes were homogenized in 350 ll of the manufacturers provided chilled lysis buffer. Whole cell lysates were centrifuged at maximum speed in an eppendorf centrifuge at 4 °C for
10 min. 50 ll of each supernatant were aliquoted into new microcentrifuge tubes and placed on ice and subsequently, 50 ll of 2 reaction buffer containing 10 mM DTT was added. As appropriate, either 1 ll chilled DMSO or 1 ll of inhibitor (1 mM DEVD-fmk) were added and samples were incubated at 4 °C for 30 min, followed by the addition of 5 ll caspase 3 substrate (1 mM DEVDpNA) or 5 ll of chilled DMSO to the appropriate tubes. Absorbance was read with a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA) at 405 nm and the activity unit were determined according to the instruction provide with the kit. Samples from experiments were assayed in duplicate and experiments were undertaken as three independent replicates. All values are expressed as the mean ± standard deviation (SD).
3. Results and discussion While densoviruses are known to be highly pathogeneic towards Aedes mosquito larvae (Barreau et al., 1996; Hirunkanokpun et al., 2008; Jousset et al., 1993), studies have suggested that repeated challenge of successive generations of mosquitoes surviving prior densovirus challenge results in significantly reduced mortality (Roekring et al., 2006). Therefore, understanding both the mechanism of mortality in densovirus infected mosquitoes, as well as the basis for decreased susceptibility to successive generation challenge are important for developing denosoviruses as anti-mosquito biological control agents. Using a previously established challenge protocol (Roekring et al., 2006) two generations of A. aegypti mosquitoes were challenged with AThDNV. Specimens (both surviving and moribund) from the 2nd generation challenge were examined for the presence of apoptotic cells using the TUNEL assay to detect 30 -DNA hydroxyl groups, a hall mark of apoptosis induced DNA fragmentation (Gavrieli et al., 1992), as well as for the presence of AThDNV by fluorescent in situ hybridization (FISH) using a primer specific to the AThDNV genome (O’Neill et al., 1995). Results (Fig. 1) showed that while apoptotic cells were detected by TUNEL staining following densovirus challenge, relatively little TUNEL staining was observed in control (non-challenged) mosquitoes. These results were consistent in all tissues examined and no specific tissue distribution was observed. Interestingly mosquitoes surviving the challenge showed some degree of reactivity to the TUNEL reagent (Fig. 1). Positive control samples (DNase I treated) showed extensive staining in all nuclei examined, while and negative control sections (no terminal transferase in the reaction mix)
Fig. 1. Apoptosis in Aedes aegypti mosquitoes after densovirus challenge. Abdominal sections of surviving and moribund mosquitoes from a 2nd generation densovirus challenge together with sections from control (naïve) mosquitoes were examined by TUNEL to detect apoptotic cells (dark brown stain) and by FISH with a AThDNV specific probe (orange signal) and counter staining with propidium iodide (red signal). Leftmost FISH panel shows a section through an ovary of a surviving adult female.
S. Roekring, D.R. Smith / Journal of Invertebrate Pathology 104 (2010) 239–241 Table 1 Caspase 3-like activity in lysates of whole mosquitoes.
*
Specimens assayed
Unit caspase 3 activity ± SD
AThDNV infected mosquitoes + substrate (DEVD-pNA) Uninfected (na) mosquitoes + substrate (DEVD-pNA) AThDNV infected mosquitoes + substrate (DEVDpNA) + caspase inhibitor (DEVD-fmk) AThDNV infected mosquitoes (no substrate)
6.97 ± 0.88* 3.35 ± 0.11 1.85 ± 0.26 1.89 ± 0.47
P < 0.05 (Student’s T-test), as compared individually to the three other groups.
showed no evidence of staining (data not shown). The presence of AThDNV was clearly seen in both surviving and moribund mosquitoes upon FISH analysis (Fig. 1). To confirm that the increased numbers of TUNEL positive cells in AThDNV infected mosquitoes were the results of the induction of apoptosis, the levels of activated caspase 3-like activity, an executioner caspase (Shi, 2002) were investigated in 2nd generation AThDNV challenged mosquitoes, in comparison with unchallenged (naïve) mosquitoes. Controls included the inclusion of a caspase 3 inhibitor in the reaction mix, as well as the omission of the substrate. Results (Table 1) showed a greater level of specific caspase 3-like activation in moribund challenged mosquitoes as compared to unchallenged mosquitoes. Together, these results suggest that AThDEN induces apoptosis in infected mosquitoes. Acknowledgments SR was supported by a Mahidol University Postdoctoral Fellowship. DRS is supported by Mahidol University and the Thailand Research Fund. References Bando, H. et al., 1990. Terminal structure of a densovirus implies a hairpin transfer replication which is similar to the model for AAV. Virology 179, 57–63.
241
Barreau, C. et al., 1996. Pathogenicity of the Aedes albopictus parvovirus (AaPV), a denso-like virus, for Aedes aegypti mosquitoes. J Invertebr Pathol 68, 299–309. Federici, B.A. et al., 2007. Developing recombinant bacteria for control of mosquito larvae. J. Am. Mosq. Contr. Assoc. 23, 164–175. Gavrieli, Y. et al., 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119, 493–501. Hirunkanokpun, S. et al., 2008. Evaluation of mosquito densoviruses for controlling Aedes aegypti (Diptera: Culicidae): variation in efficiency due to virus strain and geographic origin of mosquitoes. Am. J. Trop. Med. Hyg. 78, 784–790. Jousset, F.X. et al., 1993. A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae. Virus Res. 29, 99–114. Kittayapong, P. et al., 1999. A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand. Am. J. Trop. Med. Hyg. 61, 612–617. Lebedeva, O.P. et al., 1973. Investigation of a virus disease of the densonucleosis type in a laboratory culture of Aedes aegypti. Acta Virol. 17, 253–256. Lightner, D.V., 1996. A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. Special publication of the world aquaculture society. Lounibos, L.P., 2002. Invasions by insect vectors of human disease. Annu. Rev. Entomol. 47, 233–266. Murphy, F.A., et al., 1995. Virus taxonomy. Sixth report of the international committee on taxonomy Oj viruses. Arch Virol. (Suppl. 10), 208–239. O’Neill, S.L. et al., 1995. Insect densoviruses may be widespread in mosquito cell lines. J. Gen. Virol. 76 (Pt 8), 2067–2074. Perera, R. et al., 2008. Closing the door on flaviviruses: entry as a target for antiviral drug design. Antivir. Res. 80, 11–22. Pierson, T.C. et al., 2008. Structural insights into the mechanisms of antibodymediated neutralization of flavivirus infection: implications for vaccine development. Cell Host Microbe. 4, 229–238. Roberts, D.R., Andre, R.G., 1994. Insecticide resistance issues in vector-borne disease control. Am. J. Trop. Med. Hyg. 50, 21–34. Roekring, S. et al., 2006. Challenging successive mosquito generations with a densonucleosis virus yields progressive survival improvement but persistent, innocuous infections. Dev. Comp. Immunol. 30, 878–892. Russell, B.M. et al., 1996. Surveillance of the mosquito Aedes aegypti and its biocontrol with the copepod Mesocyclops aspericornis in Australian wells and gold mines. Med. Vet. Entomol. 10, 155–160. Shi, Y., 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 9, 459–470. Tolle, M.A., 2009. Mosquito-borne diseases. Curr. Probl. Pediatr. Adolesc Health Care. 39, 97–140. Willems, K.J. et al., 2005. A comparison of mosquito predation by the fish Pseudomugil signifier Kner and Gambusia holbrooki (Girard) in laboratory trials. J. Vector Ecol. 30, 87–90.