Larval gypsy moths, Lymantria dispar (Lepidoptera:Lymantriidae) were co-infected with the L. dispar nucleopolyhedrovirus (LdMNPV) and the Cotesia melanoscela (Hymenoptera:Braconidae) polydnavirus (CmeBV). CmeBV was given along with a parasitoid egg and calyx products in a stinging event, or in the form of an injection of calyx-derived extract. LdMNPV was delivered per os, integrated into artificial diet. Mortality from all sources was recorded over the subsequent three-week period. Neither parasitism nor injections of purified CmeBV with toxin had any effect on the amount of mortality caused by concurrent challenges with LdMNPV

D\u27Amico, V.

Fuester, R.

Podgwaite, J.D.

Taylor, P.

Zerillo, R.

English

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnUSDA Forest Service / UNL Faculty Publications U.S. Department of Agriculture: Forest Service --National Agroforestry Center2013Interactions between an injected polydnavirus andper os baculovirus in gypsy moth larvaeV. D'AmicoUSDA Forest Service, Northern Research Station, vincedamico@gmail.comJ.D. PodgwaiteUSDA Forest Service, Northern Research StationR. ZerilloUSDA Forest Service, Northern Research StationP. TaylorUSDA-ARS, BIIRLR. FuesterUSDA-ARS, BIIRLFollow this and additional works at: http://digitalcommons.unl.edu/usdafsfacpubThis Article is brought to you for free and open access by the U.S. Department of Agriculture: Forest Service -- National Agroforestry Center atDigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA Forest Service / UNL Faculty Publications by anauthorized administrator of DigitalCommons@University of Nebraska - Lincoln.D'Amico, V.; Podgwaite, J.D.; Zerillo, R.; Taylor, P.; and Fuester, R., "Interactions between an injected polydnavirus and per osbaculovirus in gypsy moth larvae" (2013). USDA Forest Service / UNL Faculty Publications. 266.http://digitalcommons.unl.edu/usdafsfacpub/266Short CommunicationInteractions between an injected polydnavirus and per os baculovirusin gypsy moth larvaeV. D’Amico a,⇑, J.D. Podgwaite b, R. Zerillo b, P. Taylor c, R. Fuester caUSDA Forest Service, Northern Research Station, Department of Entomology & Wildlife Ecology, 531 South College Ave, Newark, DE 19716, United StatesbUSDA Forest Service, Northern Research Station, 51 Mill Pond Rd., Hamden, CT 06514, United StatescUSDA-ARS, BIIRL, 501 S. Chapel St., Newark, DE 19713, United Statesa r t i c l e i n f oArticle history:Received 29 January 2013Accepted 31 July 2013Available online 8 August 2013Keywords:PolydnavirusBaculovirusLdMNPVLymantria disparInsect immunityCmeBVa b s t r a c tLarval gypsy moths, Lymantria dispar (Lepidoptera:Lymantriidae) were co-infected with the L. disparnucleopolyhedrovirus (LdMNPV) and the Cotesia melanoscela (Hymenoptera:Braconidae) polydnavirus(CmeBV). CmeBV was given along with a parasitoid egg and calyx products in a stinging event, or inthe form of an injection of calyx-derived extract. LdMNPV was delivered per os, integrated into artificialdiet. Mortality from all sources was recorded over the subsequent three-week period. Neither parasitismnor injections of purified CmeBV with toxin had any effect on the amount of mortality caused by concur-rent challenges with LdMNPV.Published by Elsevier Inc.1. IntroductionPolydnaviruses (PDVs) occur in many genera of ichneumonids(ichnoviruses) and braconids (bracoviruses) (Stoltz and Whitfield,1992; Webb and Strand, 2005) but replicate only in the ovariancalyx cells of female wasps. The primary function of PDVs is toimmunosuppress larvae parasitized by the wasp and overcomeencapsulation of parasitoid eggs (Stoltz et al., 1988). Additionally,Washburn et al. (1996) reported that parasitism of Heliothis zealarvae via Campoletis sonorensis (Hymenoptera:Ichneumonidae)led to immunosuppression that increased spread of the Autographacalifornica nucleopolyhedrosis virus (AcMNPV) in larval tissues.Washburn et al. (1996) theorized that the increased virulencewas due to the effects of the C. sonorensis polydnavirus on larvalhemocytes, which may normally clear early AcMNPV infection inH. zea by encapsulating infected cells.Parasitoids both produce and transmit PDVs, and also have arole in field dissemination of baculoviruses, especially thenucleopolyhedroviruses (NPVs) that infect lepidopteran hosts(Kurstak and Vago, 1967; reviewed by Cossentine, 2009). Our workstems from numerous observations of interactions between alepidopteran larva (gypsy moth), its baculovirus (LdMNPV), and abraconid parasitoid (Cotesia melanoscela). During ovipositionC.mel-anoscela injects the bracovirus CmeBV into larvae along with calyxsecretions and an egg (Stoltz et al., 1986). Parasitism by this waspranges from 0% to 80% in both samples collected in the field andreared until wasp emergence (Reardon and Podgwaite, 1976;D’Amico et al., 1999), and in larvae exposed to parasitism by C. mel-anoscela concurrently with LdMNPV (Woods and Elkinton, 1987).Guzo and Stoltz (1985) used CmeBV to immunosuppress Orgyialeucostigma to the extent that it became permissive for three para-sitoid species that under normal circumstances were unable tocomplete their life cycle within that host. CmeBV, however, didnot make gypsy moth larvae permissive for any of these parasit-oids. Lovallo et al. (2002) explored the effects of the C. congregataPDV on the immune responses of three species of lepidopteran lar-vae, including Lymantria dispar. One of the striking findings of theirwork was that despite relatively close phylogenetic relationshipsbetween parasitoid–host players, the immune responses of gypsymoth larvae were essentially unaffected by that PDV.We hypothesized that if CmeBV had the expected immunosup-pressive properties of other PDVs, then it would influence gypsymoth disease dynamics by increasing susceptibility to per osLdMNPV challenge. Separately both CmeBV and LdMNPV havebeen studied extensively, but their interactions have not beeninvestigated.0022-2011/$ - see front matter Published by Elsevier Inc.http://dx.doi.org/10.1016/j.jip.2013.07.009⇑ Corresponding author. Address: U. of Delaware, Dept. of Entomology andWildlife Ecology, 531 South Ave, Newark, DE 19716, United States. Fax: +1 302 8318889.E-mail address: vdamico@fs.fed.us (V. D’Amico).Journal of Invertebrate Pathology 114 (2013) 158–160Contents lists available at ScienceDirectJournal of Invertebrate Pathologyjournal homepage: www.elsevier .com/ locate / j ip2. Materials and methodsGypsy moth larvae were from a ‘‘New Jersey’’ strain (US ForestService, Hamden, CT). C. melanoscela adults were reared on gypsymoth larvae at the ARS Beneficial Insects Laboratory in Newark,DE. Larvae were used immediately after molting to the 2nd instar.Using methods similar to Washburn et al. (2000), larvae werechallenged per os with LdMNPV, and by parasitization or injectionwith PDV. After challenge they were reared on artificial diet (Bellet al., 1981) for three weeks, and dead individuals examined usinglight microscopy to verify LdMNPV as the cause of death. Mortal-ity data were compared using a two-factor analysis of variance.Paired t tests were used to compare individual points whereappropriate, with Pvalues of less than 0.05 considered statisticallysignificant.To explore the effects of C. melanoscela parasitism and CmeBVon LdMNPV virulence, a cohort of gypsy moth larvae were givenone of two treatments, either a sting from C. melanoscela 48 h priorto an LdMNPV dose; or an LdMNPV dose only, delivered at thesame time as that given to the stung larvae. Larvae in both treat-ments were further divided arbitrarily into groups of 30 larvaeand fed cubes of virus-incorporated diet containing 0 (control),or 500, 5,000, 10,000, 20,000 or 50,000 OBs/ml (Slavicek et al.,1992). Only larvae eating the entire cube were used; in any caseonly 25 larvae were used for each treatment. The effects of CmeBVon larval immunity are known to last at least through the four orfive days comprising the early larval development phase of C. mel-anoscela (Stoltz et al., 1986), so we considered our 48 h timingchoice to be reasonable. Stung larvae in the 0-dose group werethe controls for parasitism, and the unstung 0-dose group forLdMNPV contamination.We repeated the experiment above with a change in timing oftreatments. Two groups of larvae were given either a stung or uns-tung treatment but larvae fed on contaminated diet starting 48 hbefore being stung, rather than after. We added this treatment pairto see if a change in the order of parasitization by C. melanoscela orinfection by LdMNPV would have a strong effect on mortality fromeither agent.Using methods described by Beckage et al. (1994), an experi-ment was designed in which CmeBV was extracted from 150 fema-leC. melanoscela and then injected into larvae. One wasp equivalentof CmeBV preparation (crude ovarian extract in Grace’s medium toca. 1 ll per injected larva) was injected into each of 150 third instarlarvae with a Hamilton syringe and a 31 gauge needle. Anothergroup of 150 larvae were injected with 1 ll of Grace’s mediumonly, and another group of 150 larvae were not injected. Eachgroup was arbitrarily divided further into groups of 25 and dosedwith LdMNPV at rates of 0, 500, 5,000, 10,000, 20,000 and 50,000OBs/ml of diet. Statistical analysis was performed as above.3. ResultsThere was no virus-caused mortality in the controls in anyexperiment. Parasitism was approximately 80% in the controlsand was verified on emergence or through dissection of parasitizedlarvae. Controls are shown for comparison of developmental data.When parasitism occurred 48 h before dosing, it did not signifi-cantly change the amount of LdMNPV-caused mortality observedin larvae (Fig. 1A) (df = 1, F = 2.21, P = 0.188), although stung larvaeexperienced less mortality at the two highest doses of LdMNPV.LdMPNV mortality in larvae stung 48 h after LdMNPV challengewas similar to mortality in larvae stung prior to challenge. Thus,parasitism did not significantly change the amount of LdMNPV-caused mortality observed in larvae (Fig. 1B) (df = 1, F = 3.194,P = 0.124). In fact, stung larvae experienced more mortality at thefour highest doses of LdMNPV, although this difference did notproduce significant differences in overall mortality at the P = 0.05level.Fig. 1. (A) LdMNPV-caused mortality in gypsy moths stung by C. melanoscela asnewly emerged 2nd-instar larvae, and dosed with LdMNPV per os 48 h later, andunstung larvae given LdMNPV only. LdMNPV doses were administered to all larvaeat the same time. There was no LdMNPV mortality in any control larvae (notshown). There were no significant differences between the treatments. (B)LdMNPV-caused mortality in gypsy moths dosed with LdMNPV per os as newlyemerged 2nd-instar larvae, and stung by C. melanoscela 48 h after, and unstunglarvae given LdMNPV only. LdMNPV doses were administered to all larvae at thesame time. There was no LdMNPV mortality in any control larvae (not shown).There were no significant differences between the treatments.Fig. 2. LdMNPV-caused mortality in gypsy moths treated as newly emerged 3rd-instar larvae with: an extract of C. melanoscela ovarian calyx tissue in Grace’smedium and LdMNPV per os, Grace’s medium and LdMNPV per os, or LdMNPV peros. A two-factor ANOVA performed on the three possible treatment comparisonsshowed no significant differences between LdMNPV-caused mortality in the larvaetreated with CmeBV + Grace’s + LdMNPV versus those treated with LdMPV only(P = 0.114), or between larvae treated with CmeBV + Grace’s + LdMNPV versus thosegiven Grace’s + LdMNPV (P = 0.442).V. D’Amico et al. / Journal of Invertebrate Pathology 114 (2013) 158–160 159Results of our injection experiment (Fig. 2) were consistent withthose using parasitoids to inject CmeBV into larvae. The two-factorANOVA performed on the three possible treatment comparisonsrevealed no significant differences between LdMNPV-caused mor-tality in the larvae treated with CmeBV + Grace’s + LdMNPV versusthose treated with LdMPV only (df = 1, 4.063, P = 0.114), orbetween larvae treated with CmeBV + Grace’s + LdMNPV versusthose given Grace’s + LdMNPV (df = 1, F = 0.725, P = 0.442). Thetreatment pair that did not include CmeBV, LdMNPV versusGrace’s + LdMNPV, showed significant differences in LdMNPV-caused mortality (df = 1, F = 8.48, P = 0.04): mortality was higherin larvae not given an injection of Grace’s medium at all but thelowest NPV dose (Fig. 2).4. DiscussionIn our experiments we found no effects of parasitism or injec-tion of CmeBV on gypsy moth mortality either before or after peros challenge with LdMNPV. This is evidence that immunosuppres-sion produced by CmeBV or other C. melanoscela-derived ovariancalyx products does not compromise the defenses with whichgypsy moth larvae resist per os LdMNPV. While there are no di-rectly comparable studies of CmeBV and LdMNPV, one recent studyby McNeil et al. (2010a) showed that intrahemocoelic injections ofthe Glyptapanteles flavicoxis polydnavirus markedly increased lev-els of infection in gypsy moth larvae after injections of the buddedvirus form of LdMNPV at 3.750 tissue culture infectious dose(TCID50) units, or three to four budded virus particles. Reasonsfor this result are not yet well-understood.The earliest stages of the NPV infection process (dissolution ofpolyhedra, penetration of the peritrophic membrane, or bindingof ODV to midgut cells) are likely not subject to influence by con-current PDV infection. Washburn et al. (1996) strongly implicatedthe PDV-mediated abrogation of hemocytic encapsulation of in-fected cells as the reason for a rapid spread of AcMNPV in a ‘‘highlyrefractory’’ host, H. zea. Other work by McNeil et al. (2010b) doesnot lend strong support for that mechanism achieving the sameprotective role in the gypsy moth–LdMNPV system. They foundstrong evidence for some encapsulation and melanization afterLdMNPV challenge, but no obvious correlation between theseand observed mortality. It may be that the gypsy moth–CmeBV–LdMNPV system is somehow atypical, although we will have to re-peat our work, adding budded baculovirus injections and usingboth C. melanoscela and G. flavicoxis PDVs, before furtherconjecture.Questions raised by this and similar studies emphasize theusefulness of PDVs in unraveling the sequence and mechanismsof insect baculovirus resistance. The continued exploration ofinteractions between polydnaviruses and baculoviruses, in thissystem as well as others, is therefore likely to be fruitful on botha microbiological and ecological basis.AcknowledgmentsThe authors thank D.B. Stoltz and M.R. Strand for help and ad-vice during the early planning stages of this work, and J. Slavicekfor lengthy discussions, comments, and suggestions that were ofgreat help in improving the ms. We thank C. Bartlett for reviewingthe ms prior to its submission to JIP, and K. Hoover for her reviewand many helpful comments.ReferencesBeckage, N.B., Tan, F., Schleifer, K.W., Lane, R.D., Cherubin, L.L., 1994.Characterization and biological effects from Cotesia congregata polydnaviruson host larvae of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem.26, 165–195.Bell, R.A., Owens, C.D., Shapiro, M., Tardiff, J.R., 1981. Mass rearing and virusproduction. In: Doane, C.C., McManus, M.L., (Eds.) The Gypsy Moth: ResearchToward Integrated Pest Management. USDA Technical Bulletin 1584,Washington, D.C., pp. 599–655.Cossentine, J.E., 2009. The parasitoid factor in the virulence and spread oflepidopteran baculoviruses. Virol. Sinica 24, 305–314.D’Amico, V., Elkinton, J.S., Podgwaite, J.D., Slavicek, J.M., McManus, M.L., Burand, J.P.,1999. A field release of genetically engineered gypsy moth (Lymantria dispar L.)nuclear polyhedrosis virus (LdMNPV). J. Invertebr. Pathol. 73, 260–268.Guzo, D., Stoltz, D.B., 1985. Obligatory multiparasitism in the tussock moth, Orgyialeucostigma. Parasitology 90, 1–10.Kurstak, E., Vago, C., 1967. Transmission dun virus de la densonucleose par laparasitisma d’un hymenoptere. Rev. Can. Biol. 26, 311–316.Lovallo, N.C., McPherson, B.A., Cox-Foster, D.L., 2002. Effects of the polydnavirus ofCotesia congregata on the immune system and development of non-habitualhosts of the parasitoid. J. Insect Physiol. 48, 517–526.McNeil, J., Cox-Foster, D., Slavicek, J., Hoover, K., 2010a. Contributions of immuneresponses to developmental resistance in Lymantria dispar challenged withbaculovirus. J. Insect Physiol. 56, 1167–1177.McNeil, J., Cox-Foster, D., Gardner, M., Slavicek, J., Thiem, S., Hoover, K., 2010b.Pathogenesis of Lymantria disparmultiple nucleopolyhedrovirus in L. dispar andmechanisms of developmental resistance. J. Gen. Virol. 91, 1590–1600.Reardon, R.C., Podgwaite, J.D., 1976. Disease-parasitoid relationships in naturalpopulations of Lymantria dispar in the northeastern United States. Entomophaga21, 333–341.Slavicek, J.M., Podgwaite, J.P., Lanner-Herrara, C., 1992. Properties of two Lymantriadispar nuclear polyhedrosis virus isolates obtained from the microbial pesticideGypchek. J. Invertebr. Pathol. 59, 142–148.Stoltz, D.B., Whitfield, J.B., 1992. Viruses and virus-like entities in the parasitichymenoptera. J. Hymenopt. Res. 1, 125–139.Stoltz, D.B., Guzo, D., Cook, D., 1986. Studies on polydnavirus transmission. Virology155, 120–131.Stoltz, D.B., Guzo, D., Belland, E.R., Lucarotti, C.J., MacKinnon, E.A., 1988. Venompromotes uncoating in vitro and persistence in vivo of DNA from a braconidpolydnavirus. J. Gen. Virol. 69, 903–907.Washburn, J.O., Kirkpatrick, B.A., Volkman, L.E., 1996. Insect protection againstviruses. Nature 383, 767.Washburn, J.O., Haas-Stapleton, E.J., Tan, F.F., Beckage, N.E., Volkman, L.E., 2000. Co-infection of Manduca sexta larvae with polydnavirus from Cotesia congregataincreases susceptibility to fatal infection by Autographa californicanucleopolyhedrovirus. J. Insect Physiol. 46, 179–190.Webb, B.A., Strand, M.R., 2005. The biology and genomics of polydnaviruses. In:Gilbert, I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science.Elsevier, San Diego, CA, pp. 260–323.Woods, S.A., Elkinton, J.S., 1987. Bimodal patterns of mortality from nuclearpolyhedrosis virus in gypsy moth (Lymantria dispar) populations. J. Invertebr.Pathol. 50, 151–157.160 V. D’Amico et al. / Journal of Invertebrate Pathology 114 (2013) 158–160

Interactions between an injected polydnavirus and per os baculovirus
in gypsy moth larvae

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University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
USDA Forest Service / UNL Faculty Publications U.S. Department of Agriculture: Forest Service --National Agroforestry Center
2013
Interactions between an injected polydnavirus and
per os baculovirus in gypsy moth larvae
V. D'Amico
USDA Forest Service, Northern Research Station, vincedamico@gmail.com
J.D. Podgwaite
USDA Forest Service, Northern Research Station
R. Zerillo
USDA Forest Service, Northern Research Station
P. Taylor
USDA-ARS, BIIRL
R. Fuester
USDA-ARS, BIIRL
Follow this and additional works at: http://digitalcommons.unl.edu/usdafsfacpub
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Forest Service -- National Agroforestry Center at
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA Forest Service / UNL Faculty Publications by an
authorized administrator of DigitalCommons@University of Nebraska - Lincoln.
D'Amico, V.; Podgwaite, J.D.; Zerillo, R.; Taylor, P.; and Fuester, R., "Interactions between an injected polydnavirus and per os
baculovirus in gypsy moth larvae" (2013). USDA Forest Service / UNL Faculty Publications. 266.
http://digitalcommons.unl.edu/usdafsfacpub/266
Short Communication
Interactions between an injected polydnavirus and per os baculovirus
in gypsy moth larvae
V. D’Amico a,⇑, J.D. Podgwaite b, R. Zerillo b, P. Taylor c, R. Fuester c
aUSDA Forest Service, Northern Research Station, Department of Entomology & Wildlife Ecology, 531 South College Ave, Newark, DE 19716, United States
bUSDA Forest Service, Northern Research Station, 51 Mill Pond Rd., Hamden, CT 06514, United States
cUSDA-ARS, BIIRL, 501 S. Chapel St., Newark, DE 19713, United States
a r t i c l e i n f o
Article history:
Received 29 January 2013
Accepted 31 July 2013
Available online 8 August 2013
Keywords:
Polydnavirus
Baculovirus
LdMNPV
Lymantria dispar
Insect immunity
CmeBV
a b s t r a c t
Larval gypsy moths, Lymantria dispar (Lepidoptera:Lymantriidae) were co-infected with the L. dispar
nucleopolyhedrovirus (LdMNPV) and the Cotesia melanoscela (Hymenoptera:Braconidae) polydnavirus
(CmeBV). CmeBV was given along with a parasitoid egg and calyx products in a stinging event, or in
the form of an injection of calyx-derived extract. LdMNPV was delivered per os, integrated into artificial
diet. Mortality from all sources was recorded over the subsequent three-week period. Neither parasitism
nor injections of purified CmeBV with toxin had any effect on the amount of mortality caused by concur-
rent challenges with LdMNPV.
Published by Elsevier Inc.
1. Introduction
Polydnaviruses (PDVs) occur in many genera of ichneumonids
(ichnoviruses) and braconids (bracoviruses) (Stoltz and Whitfield,
1992; Webb and Strand, 2005) but replicate only in the ovarian
calyx cells of female wasps. The primary function of PDVs is to
immunosuppress larvae parasitized by the wasp and overcome
encapsulation of parasitoid eggs (Stoltz et al., 1988). Additionally,
Washburn et al. (1996) reported that parasitism of Heliothis zea
larvae via Campoletis sonorensis (Hymenoptera:Ichneumonidae)
led to immunosuppression that increased spread of the Autographa
californica nucleopolyhedrosis virus (AcMNPV) in larval tissues.
Washburn et al. (1996) theorized that the increased virulence
was due to the effects of the C. sonorensis polydnavirus on larval
hemocytes, which may normally clear early AcMNPV infection in
H. zea by encapsulating infected cells.
Parasitoids both produce and transmit PDVs, and also have a
role in field dissemination of baculoviruses, especially the
nucleopolyhedroviruses (NPVs) that infect lepidopteran hosts
(Kurstak and Vago, 1967; reviewed by Cossentine, 2009). Our work
stems from numerous observations of interactions between a
lepidopteran larva (gypsy moth), its baculovirus (LdMNPV), and a
braconid parasitoid (Cotesia melanoscela). During ovipositionC.mel-
anoscela injects the bracovirus CmeBV into larvae along with calyx
secretions and an egg (Stoltz et al., 1986). Parasitism by this wasp
ranges from 0% to 80% in both samples collected in the field and
reared until wasp emergence (Reardon and Podgwaite, 1976;
D’Amico et al., 1999), and in larvae exposed to parasitism by C. mel-
anoscela concurrently with LdMNPV (Woods and Elkinton, 1987).
Guzo and Stoltz (1985) used CmeBV to immunosuppress Orgyia
leucostigma to the extent that it became permissive for three para-
sitoid species that under normal circumstances were unable to
complete their life cycle within that host. CmeBV, however, did
not make gypsy moth larvae permissive for any of these parasit-
oids. Lovallo et al. (2002) explored the effects of the C. congregata
PDV on the immune responses of three species of lepidopteran lar-
vae, including Lymantria dispar. One of the striking findings of their
work was that despite relatively close phylogenetic relationships
between parasitoid–host players, the immune responses of gypsy
moth larvae were essentially unaffected by that PDV.
We hypothesized that if CmeBV had the expected immunosup-
pressive properties of other PDVs, then it would influence gypsy
moth disease dynamics by increasing susceptibility to per os
LdMNPV challenge. Separately both CmeBV and LdMNPV have
been studied extensively, but their interactions have not been
investigated.
0022-2011/$ - see front matter Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.jip.2013.07.009
⇑ Corresponding author. Address: U. of Delaware, Dept. of Entomology and
Wildlife Ecology, 531 South Ave, Newark, DE 19716, United States. Fax: +1 302 831
8889.
E-mail address: vdamico@fs.fed.us (V. D’Amico).
Journal of Invertebrate Pathology 114 (2013) 158–160
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology
journal homepage: www.elsevier .com/ locate / j ip
2. Materials and methods
Gypsy moth larvae were from a ‘‘New Jersey’’ strain (US Forest
Service, Hamden, CT). C. melanoscela adults were reared on gypsy
moth larvae at the ARS Beneficial Insects Laboratory in Newark,
DE. Larvae were used immediately after molting to the 2nd instar.
Using methods similar to Washburn et al. (2000), larvae were
challenged per os with LdMNPV, and by parasitization or injection
with PDV. After challenge they were reared on artificial diet (Bell
et al., 1981) for three weeks, and dead individuals examined using
light microscopy to verify LdMNPV as the cause of death. Mortal-
ity data were compared using a two-factor analysis of variance.
Paired t tests were used to compare individual points where
appropriate, with Pvalues of less than 0.05 considered statistically
significant.
To explore the effects of C. melanoscela parasitism and CmeBV
on LdMNPV virulence, a cohort of gypsy moth larvae were given
one of two treatments, either a sting from C. melanoscela 48 h prior
to an LdMNPV dose; or an LdMNPV dose only, delivered at the
same time as that given to the stung larvae. Larvae in both treat-
ments were further divided arbitrarily into groups of 30 larvae
and fed cubes of virus-incorporated diet containing 0 (control),
or 500, 5,000, 10,000, 20,000 or 50,000 OBs/ml (Slavicek et al.,
1992). Only larvae eating the entire cube were used; in any case
only 25 larvae were used for each treatment. The effects of CmeBV
on larval immunity are known to last at least through the four or
five days comprising the early larval development phase of C. mel-
anoscela (Stoltz et al., 1986), so we considered our 48 h timing
choice to be reasonable. Stung larvae in the 0-dose group were
the controls for parasitism, and the unstung 0-dose group for
LdMNPV contamination.
We repeated the experiment above with a change in timing of
treatments. Two groups of larvae were given either a stung or uns-
tung treatment but larvae fed on contaminated diet starting 48 h
before being stung, rather than after. We added this treatment pair
to see if a change in the order of parasitization by C. melanoscela or
infection by LdMNPV would have a strong effect on mortality from
either agent.
Using methods described by Beckage et al. (1994), an experi-
ment was designed in which CmeBV was extracted from 150 fema-
leC. melanoscela and then injected into larvae. One wasp equivalent
of CmeBV preparation (crude ovarian extract in Grace’s medium to
ca. 1 ll per injected larva) was injected into each of 150 third instar
larvae with a Hamilton syringe and a 31 gauge needle. Another
group of 150 larvae were injected with 1 ll of Grace’s medium
only, and another group of 150 larvae were not injected. Each
group was arbitrarily divided further into groups of 25 and dosed
with LdMNPV at rates of 0, 500, 5,000, 10,000, 20,000 and 50,000
OBs/ml of diet. Statistical analysis was performed as above.
3. Results
There was no virus-caused mortality in the controls in any
experiment. Parasitism was approximately 80% in the controls
and was verified on emergence or through dissection of parasitized
larvae. Controls are shown for comparison of developmental data.
When parasitism occurred 48 h before dosing, it did not signifi-
cantly change the amount of LdMNPV-caused mortality observed
in larvae (Fig. 1A) (df = 1, F = 2.21, P = 0.188), although stung larvae
experienced less mortality at the two highest doses of LdMNPV.
LdMPNV mortality in larvae stung 48 h after LdMNPV challenge
was similar to mortality in larvae stung prior to challenge. Thus,
parasitism did not significantly change the amount of LdMNPV-
caused mortality observed in larvae (Fig. 1B) (df = 1, F = 3.194,
P = 0.124). In fact, stung larvae experienced more mortality at the
four highest doses of LdMNPV, although this difference did not
produce significant differences in overall mortality at the P = 0.05
level.
Fig. 1. (A) LdMNPV-caused mortality in gypsy moths stung by C. melanoscela as
newly emerged 2nd-instar larvae, and dosed with LdMNPV per os 48 h later, and
unstung larvae given LdMNPV only. LdMNPV doses were administered to all larvae
at the same time. There was no LdMNPV mortality in any control larvae (not
shown). There were no significant differences between the treatments. (B)
LdMNPV-caused mortality in gypsy moths dosed with LdMNPV per os as newly
emerged 2nd-instar larvae, and stung by C. melanoscela 48 h after, and unstung
larvae given LdMNPV only. LdMNPV doses were administered to all larvae at the
same time. There was no LdMNPV mortality in any control larvae (not shown).
There were no significant differences between the treatments.
Fig. 2. LdMNPV-caused mortality in gypsy moths treated as newly emerged 3rd-
instar larvae with: an extract of C. melanoscela ovarian calyx tissue in Grace’s
medium and LdMNPV per os, Grace’s medium and LdMNPV per os, or LdMNPV per
os. A two-factor ANOVA performed on the three possible treatment comparisons
showed no significant differences between LdMNPV-caused mortality in the larvae
treated with CmeBV + Grace’s + LdMNPV versus those treated with LdMPV only
(P = 0.114), or between larvae treated with CmeBV + Grace’s + LdMNPV versus those
given Grace’s + LdMNPV (P = 0.442).
V. D’Amico et al. / Journal of Invertebrate Pathology 114 (2013) 158–160 159
Results of our injection experiment (Fig. 2) were consistent with
those using parasitoids to inject CmeBV into larvae. The two-factor
ANOVA performed on the three possible treatment comparisons
revealed no significant differences between LdMNPV-caused mor-
tality in the larvae treated with CmeBV + Grace’s + LdMNPV versus
those treated with LdMPV only (df = 1, 4.063, P = 0.114), or
between larvae treated with CmeBV + Grace’s + LdMNPV versus
those given Grace’s + LdMNPV (df = 1, F = 0.725, P = 0.442). The
treatment pair that did not include CmeBV, LdMNPV versus
Grace’s + LdMNPV, showed significant differences in LdMNPV-
caused mortality (df = 1, F = 8.48, P = 0.04): mortality was higher
in larvae not given an injection of Grace’s medium at all but the
lowest NPV dose (Fig. 2).
4. Discussion
In our experiments we found no effects of parasitism or injec-
tion of CmeBV on gypsy moth mortality either before or after per
os challenge with LdMNPV. This is evidence that immunosuppres-
sion produced by CmeBV or other C. melanoscela-derived ovarian
calyx products does not compromise the defenses with which
gypsy moth larvae resist per os LdMNPV. While there are no di-
rectly comparable studies of CmeBV and LdMNPV, one recent study
by McNeil et al. (2010a) showed that intrahemocoelic injections of
the Glyptapanteles flavicoxis polydnavirus markedly increased lev-
els of infection in gypsy moth larvae after injections of the budded
virus form of LdMNPV at 3.750 tissue culture infectious dose
(TCID50) units, or three to four budded virus particles. Reasons
for this result are not yet well-understood.
The earliest stages of the NPV infection process (dissolution of
polyhedra, penetration of the peritrophic membrane, or binding
of ODV to midgut cells) are likely not subject to influence by con-
current PDV infection. Washburn et al. (1996) strongly implicated
the PDV-mediated abrogation of hemocytic encapsulation of in-
fected cells as the reason for a rapid spread of AcMNPV in a ‘‘highly
refractory’’ host, H. zea. Other work by McNeil et al. (2010b) does
not lend strong support for that mechanism achieving the same
protective role in the gypsy moth–LdMNPV system. They found
strong evidence for some encapsulation and melanization after
LdMNPV challenge, but no obvious correlation between these
and observed mortality. It may be that the gypsy moth–CmeBV–
LdMNPV system is somehow atypical, although we will have to re-
peat our work, adding budded baculovirus injections and using
both C. melanoscela and G. flavicoxis PDVs, before further
conjecture.
Questions raised by this and similar studies emphasize the
usefulness of PDVs in unraveling the sequence and mechanisms
of insect baculovirus resistance. The continued exploration of
interactions between polydnaviruses and baculoviruses, in this
system as well as others, is therefore likely to be fruitful on both
a microbiological and ecological basis.
Acknowledgments
The authors thank D.B. Stoltz and M.R. Strand for help and ad-
vice during the early planning stages of this work, and J. Slavicek
for lengthy discussions, comments, and suggestions that were of
great help in improving the ms. We thank C. Bartlett for reviewing
the ms prior to its submission to JIP, and K. Hoover for her review
and many helpful comments.
References
Beckage, N.B., Tan, F., Schleifer, K.W., Lane, R.D., Cherubin, L.L., 1994.
Characterization and biological effects from Cotesia congregata polydnavirus
on host larvae of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem.
26, 165–195.
Bell, R.A., Owens, C.D., Shapiro, M., Tardiff, J.R., 1981. Mass rearing and virus
production. In: Doane, C.C., McManus, M.L., (Eds.) The Gypsy Moth: Research
Toward Integrated Pest Management. USDA Technical Bulletin 1584,
Washington, D.C., pp. 599–655.
Cossentine, J.E., 2009. The parasitoid factor in the virulence and spread of
lepidopteran baculoviruses. Virol. Sinica 24, 305–314.
D’Amico, V., Elkinton, J.S., Podgwaite, J.D., Slavicek, J.M., McManus, M.L., Burand, J.P.,
1999. A field release of genetically engineered gypsy moth (Lymantria dispar L.)
nuclear polyhedrosis virus (LdMNPV). J. Invertebr. Pathol. 73, 260–268.
Guzo, D., Stoltz, D.B., 1985. Obligatory multiparasitism in the tussock moth, Orgyia
leucostigma. Parasitology 90, 1–10.
Kurstak, E., Vago, C., 1967. Transmission dun virus de la densonucleose par la
parasitisma d’un hymenoptere. Rev. Can. Biol. 26, 311–316.
Lovallo, N.C., McPherson, B.A., Cox-Foster, D.L., 2002. Effects of the polydnavirus of
Cotesia congregata on the immune system and development of non-habitual
hosts of the parasitoid. J. Insect Physiol. 48, 517–526.
McNeil, J., Cox-Foster, D., Slavicek, J., Hoover, K., 2010a. Contributions of immune
responses to developmental resistance in Lymantria dispar challenged with
baculovirus. J. Insect Physiol. 56, 1167–1177.
McNeil, J., Cox-Foster, D., Gardner, M., Slavicek, J., Thiem, S., Hoover, K., 2010b.
Pathogenesis of Lymantria disparmultiple nucleopolyhedrovirus in L. dispar and
mechanisms of developmental resistance. J. Gen. Virol. 91, 1590–1600.
Reardon, R.C., Podgwaite, J.D., 1976. Disease-parasitoid relationships in natural
populations of Lymantria dispar in the northeastern United States. Entomophaga
21, 333–341.
Slavicek, J.M., Podgwaite, J.P., Lanner-Herrara, C., 1992. Properties of two Lymantria
dispar nuclear polyhedrosis virus isolates obtained from the microbial pesticide
Gypchek. J. Invertebr. Pathol. 59, 142–148.
Stoltz, D.B., Whitfield, J.B., 1992. Viruses and virus-like entities in the parasitic
hymenoptera. J. Hymenopt. Res. 1, 125–139.
Stoltz, D.B., Guzo, D., Cook, D., 1986. Studies on polydnavirus transmission. Virology
155, 120–131.
Stoltz, D.B., Guzo, D., Belland, E.R., Lucarotti, C.J., MacKinnon, E.A., 1988. Venom
promotes uncoating in vitro and persistence in vivo of DNA from a braconid
polydnavirus. J. Gen. Virol. 69, 903–907.
Washburn, J.O., Kirkpatrick, B.A., Volkman, L.E., 1996. Insect protection against
viruses. Nature 383, 767.
Washburn, J.O., Haas-Stapleton, E.J., Tan, F.F., Beckage, N.E., Volkman, L.E., 2000. Co-
infection of Manduca sexta larvae with polydnavirus from Cotesia congregata
increases susceptibility to fatal infection by Autographa californica
nucleopolyhedrovirus. J. Insect Physiol. 46, 179–190.
Webb, B.A., Strand, M.R., 2005. The biology and genomics of polydnaviruses. In:
Gilbert, I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science.
Elsevier, San Diego, CA, pp. 260–323.
Woods, S.A., Elkinton, J.S., 1987. Bimodal patterns of mortality from nuclear
polyhedrosis virus in gypsy moth (Lymantria dispar) populations. J. Invertebr.
Pathol. 50, 151–157.
160 V. D’Amico et al. / Journal of Invertebrate Pathology 114 (2013) 158–160


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Interactions between an injected polydnavirus and per os baculovirus in gypsy moth larvae

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