Juvenile hormone analog technology: effects on larval cannibalism and the production of Spodoptera exigua (Lepidoptera: Noctuidae) nucleopolyhedrovirus.

Abstract

The production of a multiple nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), has been markedly increased by using juvenile hormone analog (JHA) technology to generate a supernumerary sixth instar in the species. In the current study we compared the incidence of cannibalism in S. exigua fifth and sixth instars reared at low (two larvae per dish) and a high density (10 larvae per dish). The incidence of cannibalism was significantly higher in fifth instars compared with sixth instars and increased with rearing density on both instars. Infected larvae were more prone to become victims of cannibalism than healthy individuals in mixed groups comprising 50% healthy + 50% infected larvae in both instars reared at high density. Instar had a marked effect on occlusion body (OB) production because JHA-treated insects produced between 4.8- and 5.6-fold increase in OB production per dish compared with fifth instars at high and low densities, respectively. The insecticidal characteristics of OBs produced in JHA-treated insects, as indicated by LD50 values, were similar to those produced in untreated fourth or fifth instars. Because JHA technology did not increase the prevalence of cannibalism and had no adverse effect on the insecticidal properties of SeMNPV OBs, we conclude that the use of JHAs to generate a supernumerary instar is likely to be compatible with mass production systems that involve gregarious rearing of infected insects.

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BIOLOGICAL AND MICROBIAL CONTROL
Juvenile Hormone Analog Technology: Effects on Larval Cannibalism
and the Production of Spodoptera exigua (Lepidoptera: Noctuidae)
Nucleopolyhedrovirus
SONIA ELVIRA,1TREVOR WILLIAMS,2AND PRIMITIVO CABALLERO1,3,4
J. Econ. Entomol. 103(3): 577Ð582 (2010); DOI: 10.1603/EC09325
Theproductionofamultiplenucleopolyhedrovirus(SeMNPV)ofthebeetarmyworm,
Spodoptera exigua (Hu ¨bner) (Lepidoptera: Noctuidae), has been markedly increased by using ju-
venile hormone analog (JHA) technology to generate a supernumerary sixth instar in the species. In
the current study we compared the incidence of cannibalism in S. exigua Þfth and sixth instars reared
at low (two larvae per dish) and a high density (10 larvae per dish). The incidence of cannibalism
was signiÞcantly higher in Þfth instars compared with sixth instars and increased with rearing density
on both instars. Infected larvae were more prone to become victims of cannibalism than healthy
individuals in mixed groups comprising 50% healthy ? 50% infected larvae in both instars reared at
high density. Instar had a marked effect on occlusion body (OB) production because JHA-treated
insects produced between 4.8- and 5.6-fold increase in OB production per dish compared with Þfth
instars at high and low densities, respectively. The insecticidal characteristics of OBs produced in
JHA-treated insects, as indicated by LD50values, were similar to those produced in untreated fourth
or Þfth instars. Because JHA technology did not increase the prevalence of cannibalism and had no
adverse effect on the insecticidal properties of SeMNPV OBs, we conclude that the use of JHAs to
generate a supernumerary instar is likely to be compatible with mass production systems that involve
gregarious rearing of infected insects.
ABSTRACT
KEYWORDS
Spodopteraexigua,cannibalism,juvenilehormone,nucleopolyhedrovirusproduction
Cannibalism, or intraspeciÞc predation, is a frequent
behavior in many species of Lepidoptera during the
larval stage (Pierce 1995, Chapman et al. 2000). Al-
though cannibalism can confer direct Þtness beneÞts
in the form of increased development rate, fecundity,
or the removal of potential competitors (Joyner and
Gould 1985, Church and Sherratt 1996), it also can
haveprofoundconsequencesoninsectpopulationdy-
namics(Reedetal.1996),theriskofacquiringdisease
(Rudolf and Antonovics 2007), and the inclusive Þt-
ness of cannibals that consume kin (Fox 1975, Polis
1981).
The prevalence of cannibalistic behavior is often
density-dependent, with an increasingly higher inci-
dence at higher densities, even when food is not lim-
iting (Polis 1981). Cannibalism may also be stage de-
pendent in certain species of Lepidoptera with later
instars often showing a greater tendency to engage in
intraspeciÞc predation, compared with their younger
and smaller conspeciÞcs, especially when larvae of
differentstagesareenclosedtogether(Chapmanetal.
1999a,b).
Baculoviruses form the basis for many biological in-
secticideswithprovencommercialpotential(Moscardi
1999). However, one factor that limits their commer-
cial development is the need to produce these viruses
in living insects, which requires the maintenance of
largeinsectcoloniesinmassrearingfacilities(Shapiro
1986). Recently, we applied juvenile hormone analog
(JHA) technology to the production of a nucleopoly-
hedrovirus (SeMNPV) of the beet armyworm, Spo-
doptera exigua (Hu ¨bner) (Lepidoptera: Noctuidae).
TheapplicationofJHAcompoundsresultsinmultiple
changes in the developing insect that have been re-
viewed in detail (Gilbert et al. 2000, Wilson 2004).
Treating Þfth instars with methoprene or fenoxycarb
resulted in the generation of a supernumerary sixth
instar, that when infected by feeding on viral occlu-
sion bodies (OBs), yielded an almost three-fold in-
crease in the number of OBs produced in each larva,
with no signiÞcant reduction in OB potency, com-
pared with OBs produced in infected Þfth instar con-
speciÞcs (Lasa et al. 2007).
As SeMNPV is now being produced commercially
under the name of Spod-X (Certis USA, Columbia,
MD),Vir-ex(Biocolor,Almeria,Spain),andasvarious
products produced by local companies in southern
1Departamento de Produccio ´n Agraria, Universidad Pu ´blica de
Navarra, Pamplona, 31006, Spain.
2Instituto de Ecologõ ´a AC, Xalapa 91070, Veracruz, Mexico.
3Microbial Bioinsecticides, Instituto de Agrobiotecnologõ ´a, CSIC-
Universidad Pu ´blica de Navarra-Gobierno de Navarra, Mutilva Baja
31192, Spain.
4Corresponding author, e-mail: pcm@unavarra.es.
0022-0493/10/0577Ð0582$04.00/0 ? 2010 Entomological Society of America

Page 2

and southeast Asia, we examined the inßuence of
applying JHA technology to the prevalence of canni-
balism of groups of S. exigua supernumerary instars in
the laboratory and the impact that cannibalistic be-
haviorhasontheproductionandinsecticidalpotency
of SeMNPV OBs.
Materials and Methods
Insect Colony, JHA, and Virus Strain. S. exigua lar-
vae were obtained from a laboratory colony main-
tained at 25 ? 2?C, 70 ? 5% humidity, and a photo-
period of 16:8 (L:D) h in the Universidad Pu ´blica de
Navarra, Pamplona, Spain. An artiÞcial diet adapted
from Hoffman and Lawson (1964) was used to feed
larvae.Fifthinstarswereobtainedfromthecolonyand
were individually treated topically with 3 ?l of a com-
mercialfenoxycarbproduct(Zambu25W,AgroArte ´s,
Castello ´n, Spain) that had been diluted to a concen-
tration of 2.5 ?g active ingredient (AI)/ml in distilled
water. Treated larvae were incubated individually for
5 d on artiÞcial diet until they had molted to the
supernumerary sixth instar.
OBs of a Spanish nucleopolyhedrovirus isolate
SeMNPV-SP2 (Caballero et al. 1992) were produced
byorallyinoculatingS.exiguafourthinstars.OBswere
collected from insects that subsequently died of poly-
hedrosisdisease,puriÞedbycentrifugation(Mun ˜ozet
al. 1997), quantiÞed by counting in triplicate in a
Neubauer chamber (Hawsksley, Lancing, United
Kingdom), and stored at 4?C until required.
Supernumerary Instar and Rearing Density Effects
onCannibalism.GroupsofÞfthandsixthinstarswere
allowed to feed on droplets of 10% (wt:vol) sucrose,
0.001% (vol:vol) ßuorella blue food dye, and a con-
centration of OBs of 4.0 ? 105or 1.0 ? 106OBs/ml,
which had been established previously to result in
?90% infection in Þfth and sixth instars, respectively.
Larvae that drank the suspension within 10 min were
selected for inclusion in the experiment and were
placed in petri dishes at densities of either two or 10
larvae per dish. Larvae were assigned to one of six
treatments at each density on a random basis accord-
ing to their condition. These were 1) healthy Þfth
instars, 2) infected Þfth instars, 3) 50% healthy ? 50%
infected Þfth instars, 4) healthy sixth instars, 5) in-
fectedsixthinstars,and6)50%healthy?50%infected
sixth instars. Healthy and infected larvae in mixed
treatments were differentiated by marking dorsally
with indelible inks that had been found not to affect
survivalinpreliminarytests.Eachpetridishcontained
a piece of semisynthetic diet that represented excess
food for the period of the experiment. Dishes were
incubated at 28 ? 2?C for 6 d, and the number of
surviving larvae was noted daily. We considered that
larvae have been cannibalized when observed to be
dead and partially or totally consumed. The experi-
mentwasperformed20timesforeverytreatmentand
density.Thenumberofinsectsthatwerecannibalized
and the total number of insects tested (n) were used
to determine the proportion of cannibalism (p) by
ÞttinggeneralizedlinearmodelsusingGLIMfourwith
a binomial error structure speciÞed (Numerical Al-
gorithms Group 1993). In the absence of overdisper-
sion, the results of such analyses closely approximate
tothechi-squaredistribution.Theerrorsofbinomially
distributed means become increasingly asymmetrical
astheyapproachthelimitsofthedistribution(1or0).
Resultsareshownaspercentagesofcannibalisminthe
corresponding Þgures.
Supernumerary Instar and Rearing Density Effects
onOBProductionandPotency.Aftera6-dincubation
period at 28 ? 2?C, the number of dead larvae was
noted, and the disappearance of infected and healthy
insects in mixed treatments was similarly recorded.
Petri dishes were placed at ?20?C until completely
frozen, which facilitated collection of infected larvae
thattendedtobecomeliqueÞedatroomtemperature.
Healthy larvae that may have acquired an infection in
the mixed treatments were not collected. Frozen
cadavers of the infected larvae were individually
weighedandplacedin50-mltubescontaining30mlof
sterile distilled water. The suspension was Þltered
through Þne steel gauze to remove insect debris. The
total number of OBs produced in each dish was de-
termined in triplicate using a Neubauer improved
countingchamberunderphasecontrastmicroscopyat
400?magniÞcation.Theprocedurewasperformed20
times at the lower density and 10 times at the higher
density. OB production and weights of frozen larvae
were normalized by log transformation and were sub-
jected to analysis of variance (ANOVA) in SPSS, ver-
sion 15.0 (SPSS Inc., Chicago, IL).
The insecticidal activity of OBs collected was com-
pared with the original inoculum produced in fourth-
instar individualized larvae. Pathogenicity was deter-
mined in S. exigua second instars from the laboratory
colonybyusingamodiÞeddropletbioassaytechnique
(Hughes and Wood 1996). Late Þrst instars were
starved for 12 h at 25 ? 2?C and allowed to molt to
thenextinstaroverperiodof10h.Groupsof30larvae
were allowed to feed on droplets of 10% sucrose,
0.001% ßuorella blue food dye and one of Þve con-
centrationsofOBsintherangeof3.03?103to2.45?
105OBs/ml, calculated previously to result in mortal-
ities of between 10 and 90%. Control larvae were
treatedidenticallybutfedonasolutionofsucroseand
fooddyealone.Foreachconcentration,25larvaethat
had ingested the OB suspension within 10 min were
placedindividuallyinthecellsofatissuecultureplate
containing diet and incubated at 25 ? 2?C. Mortality
was noted at 5 d postinoculation. The bioassay was
performedthreetimes.Resultsweresubjectedtologit
regression in GLIM with a binomial error distribution
speciÞed (Numerical Algorithms Group 1993).
Results
Supernumerary Instar and Rearing Density Effects
on Cannibalism. The prevalence of cannibalism (Fig.
1AandB)wassigniÞcantlyhigheramonglarvaeinthe
high density treatments compared with the low den-
sity treatments (?2? 40.5, df ? 1, P ? 0.001). At a
density of two larvae per dish (Fig. 1A) losses due to
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cannibalism not differ signiÞcantly in any treatment
(?2? 5.9, df ? 5, P ? 0.31). In contrast, at a density
of 10 larvae per dish (Fig. 1B) cannibalism was sig-
niÞcantly higher in the Þfth-instar treatments involv-
ing virus infected and a mixture of healthy ? infected
larvae,comparedwithgroupscomprisinghealthyÞfth
instars alone (?2? 25.3, df ? 5, P ? 0.001), whereas
all treatments involving sixth instars were intermedi-
ateforcannibalism(Fig.1B).Novirusinfectionswere
observed in any of the healthy larvae of either instar
at both densities.
A closer examination of the patterns of cannibalism
in the mixed 50% healthy ? 50% infected treatments
(Fig. 2) revealed that at high density, the percentage
of insects that fell victim to cannibalism was signiÞ-
cantly higher among infected larvae of both instars
(?2? 12.40, df ? 1, P ? 0.001). Host instar also had a
signiÞcant effect with sixth instars showing a reduced
prevalence of cannibalism compared with Þfth instars
(?2? 5.84, df ? 1, P ? 0.016). In contrast, the prev-
alence of cannibalism was too low in the low density
treatments to permit a reliable analysis; in total two
infected larvae fell victim to cannibalism in the Þfth-
instar treatment, whereas one infected larva was can-
nibalized in the sixth instar treatment.
Supernumerary Instar and Rearing Density Effects
on OB Production and Potency. The Þnal weight of
sixth instars (Fig. 3A) was between 3- and 3.5-fold
greaterthanthatofÞfthinstarsatbothdensities(?2?
342.7, df ? 7, P ? 0.001). As a result, the total yield of
OBs per larva (Fig. 3B) was 4.8Ð5.6-fold higher in
infected sixth instars from the 100% infected treat-
ment and the 50% infected ? 50% healthy treatment
(inwhichonlyOBsfrominitiallyinfectedinsectswere
quantiÞed) at densities of two larvae per dish (?2?
58.7, df ? 3, P ? 0.001) and 10 larvae per dish (?2?
29.6, df ? 3, P ? 0.001). OB yield per mg of body
weight(?SE)rangedbetween8.56?106?1.47?106
and 1.07 ? 107? 1.18 ? 106but did not differ signif-
icantly between instars at a density of two larvae per
dish (F ? 0.97; df ? 3, 79; P ? 0.41) or 10 larvae per
dish (F ? 1.25; df ? 3, 39; P ? 0.31), irrespective of
whethergroupsoflarvaewereallinfectedorwhether
groups initially comprised mixtures of healthy ? in-
fectedlarvae.Asexpectedgiventhenumberofinsects
present, the total production of OBs per dish was
highest in high density treatments (?2? 103.4, df ? 7,
P?0.001),withthehighestmean?SEvalueof3.43?
1010? 1.96 ? 109OBs per dish in dishes containing
solely infected sixth instars (Fig. 3C). This repre-
sented a 4.8-fold increase per dish compared with the
OBproductioninÞfthinstarsataninitialdensityof10
larvae per dish. Similarly, at an initial density of two
larvae per dish, overall OB production was 5.8-fold
greater in sixth instars compared with Þfth instars.
The potencies of OBs produced in individualized
fourthinstarsrepresentedacontroltreatment(Table1).
There was no signiÞcant interaction between dose and
treatment(?2?2.25,df?2,P?0.32)suchthatregres-
sions could be Þtted in parallel with a common slope
(?SE) of 0.666 ? 0.048. The estimated LD50values did
Fig. 1.
the Þfth and sixth instar reared at densities of two larvae per
dish(A)and10larvaeperdish(B).Verticalbarsindicatethe
binomially-distributed 95% conÞdence interval (CI). Col-
umnslabeledwithidenticallettersdidnotdiffersigniÞcantly
for comparisons between treatments (P ? 0.05; Generalized
Linear Model).
Percentage of cannibalism of S. exigua larvae in
Fig. 2.
and infected Þfth and sixth instars reared at a density of 10
larvae per dish in groups consisting of 50% healthy ? 50%
infected. Vertical bars indicate SE. Asterisks next to hori-
zontal bars placed above columns indicate statistical differ-
ences between the corresponding treatments as determined
by Þtting generalized linear models with a binomial error
structure (*, P ? 0.05; ***, P ? 0.001).
Percentage of cannibalism observed in healthy
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not differ signiÞcantly between instars with values of
10.0,8.6,and12.9forthefourthinstarcontrols,Þfth,and
sixthinstars,respectively.Noevidenceofoverdispersion
was observed in data sets, and no mortality by virus
infection was registered in control insects.
Discussion
Cannibalismhasbeenreportedwidelyinmanyspe-
cies of lepidopterous larvae. This study demonstrates
that cannibalism was a frequent behavior in a labora-
torypopulationofS.exiguaalthoughtoalesserextent
than observed in species such as Heliothis virescens
(F.) (Gould et al. 1980), Spodoptera frugiperda (J.E.
Smith) (Chapman et al. 1999a, b), or Helicoverpa zea
(Boddie) (Joyner and Gould 1985). As expected, the
frequency of cannibalism increased with increasing
density. Many other studies have demonstrated the
relationship between the incidence of cannibalism
and the degree of crowding (Chapman et al. 1999b).
In fact, higher frequency of cannibalism occurs when
hostsareexposedtoovercrowdingornutritionalstress
(Vasconcelos 1996), which could have an effect on
pathogen transmission. High-density morphs differ
from conspeciÞcs reared at low-densities in a variety
of features including color, behavior, and develop-
mental time (Tojo et al. 1985, Pener 1991). These
changes are generally assumed to have evolved in
response to increased intraspeciÞc competition for
foodordensity-dependentpredationpressures.More-
over, larvae develop more quickly but attain smaller
size at pupation and are more susceptible to disease
when reared at high than at low densities (Goulson
and Cory 1995).
The frequency of cannibalism was also affected by
the stage of the individuals involved; cannibalism was
higher in Þfth instars compared with supernumerary
sixth instars generated by JHA treatment. JHAs tend
to cause severe disruptions in the organization of the
central nervous system, salivary glands and muscula-
tureinadose-dependentmanner(RestifoandWilson
1998). In consequence, marked changes occur in the
physiological and developmental behavior of the in-
dividuals treated. We observed that sixth instars were
noticeably less agile and apparently less willing to
engageinaggressiveinteractionsthantheirÞfth-instar
conspeciÞcs.
Cannibalism has been reported as a viable route of
horizontal transmission of several viruses in natural
and laboratory populations of Lepidoptera, including
nucleopolyhedroviruses in
(Hu ¨bner) (Dhandapani et al. 1993), Mamestra bras-
sicae L. (Vasconcelos 1996), S. frugiperda (Chapman
et al. 1999b), granulovirus in Plodia interpunctella
(Hu ¨bner) (Boots 1998), and an iridescent virus of S.
frugiperda (Williams and Herna ´ndez 2006). In the
current study, cannibalism rates increased when
healthy and infected larvae were enclosed together,
compared with those reared in separate groups. In-
Helicoverpa armigera
Fig. 3.
(milligrams) at death (6 d postinfection; A), OB production
per larva (B), and total OB production per petri dish (C).
Vertical bars indicate SE. In all cases columns labeled with
identical letters did not differ signiÞcantly for comparisons
between treatments (ANOVA; P ? 0.05).
Density and host instar effects on larval weight
Table 1.
in fifth instars or supernumerary sixth instars compared with con-
trol OBs produced in fourth instars
Logit regression analysis of SeMNPV OBsaproduced
Source of OBs
(host stage)
Intercept
? SE
LD50b
(OBs per larvae)
95% CI
Upper Low
Fourth instar (control) 1.53 ? 0.16
Fifth instar
Sixth instar
10.0a
8.6a
12.9a
12.5
10.6
16.1
8.0
6.8
10.3
1.42 ? 0.17
1.70 ? 0.17
aOBs were bioassayed by feeding to S. exigua second instars.
bLD50values followed by identical letters did not differ signiÞ-
cantly as determined by pairwise t-tests (P ? 0.05).
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deed, the highest incidence of cannibalism was ob-
served in the high-density treatment involving 50%
healthy and 50% infected Þfth instars.In line with our
Þndings, the patterns of transmission of lethal NPV
diseaseinsmallboxescontainingamajorityofhealthy
M.brassicaelarvaedependedonthestagespresentand
to a lesser extent on host density (Vasconcelos et al.
2002).
Viral infection seems to alter the risk of injury in
interactionswithconspeciÞcs,becauseinfectedlarvae
may become less vigorous and therefore less capable
of defending themselves than uninfected larvae, and
hence more likely to become the victims of cannibals
(Poprawski and Yule 1990, Boots 1998). The sluggish
responsesofinfectedinsectsprobablyresultfromma-
jor changes in the metabolic activity of infected in-
sects(ThompsonandSikorowski1981).Larvaldensity
also has been observed to have a signiÞcant inßuence
on susceptibility to virus infection in Spodoptera ex-
empta (Walker); insects reared at a high density were
considerablymoreresistanttoNPVdiseasethanthose
reared in isolation (Reeson et al. 1998).
ThisstudyfurtherunderlinestheÞndingsofLasaet
al. (2007) who reported approximately three-fold in-
creases in the production of OBs per larva in individ-
ually reared S. exigua sixth instars that had been
treated with methoprene or fenoxycarb in the previ-
ous instar. In the current study, we observed that
rearing density had a signiÞcant effect on OB produc-
tion in fenoxycarb treated insects with increases of
between 4.8- and 5.6-fold over Þfth instars reared at
high and low densities, respectively. This means that
JHA technology could potentially be applied to bac-
ulovirus production in this and many other lepidop-
teran species. In the case of S. exigua, this technology
hadnoadverseeffectsontheinsecticidalpropertiesof
SeMNPV OBs or the prevalence of cannibalism. JHA
technology is therefore likely to be compatible with
massproductionsystemsthatemploypracticesinvolv-
ing gregarious rearing of infected insects.
Acknowledgments
We thank Noelia Gorria for insect rearing. The study
receivedÞnancialsupportfromComisio ´nInterministerialde
CienciayTecnologicaprojectsPET2005-0130andAGL2008-
05456-CO3-01. S.E. received a studentship from the Univer-
sidad Pu ´blica de Navarra.
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