diofenolan: a novel insect growth regulator in common citrus butterfly, papilio demoleus

Diofenolan: a novel insect growth regulator in commoncitrus butterfly, Papilio demoleus

Satya Singh & Krishna Kumar

Received: 13 August 2010 /Accepted: 1 March 2011 /Published online: 5 April 2011# Springer Science+Business Media B.V. 2011

Abstract Penultimate and last instar larvae of thecommon citrus swallowtail butterfly, Papilio demoleusL. (Papilionidae: Lepidoptera), were treated withdifferent doses of a novel juvenoid, diofenolan. Severaldeformities were observed as a result of topicaladministration of diofenolan which include delay inlarval–larval and larval–pupal ecdysis, ecdysial failure,mortality, severe reduction in pupation, deformedpupae and complete inhibition of adult emergence.The juvenoid diofenolan severely hampers the normalgrowth, development and metamorphosis of P. demo-leus and can be incorporated into integrated pestmanagement programs for successful control of thisimportant pest of citrus all over the world.

Keywords Common lime butterfly . Ecdysial failure .

Emergence inhibition . Juvenoid . Larval–larvalecdysis . Larval–pupal ecdysis . Metamorphosis

Introduction

Papilio demoleus L. (Papilionidae: Lepidoptera) is amajor pest of citrus all over the world. According to theUNConference on Trade and Development (UNCTAD)

in 2006, globally 140 countries are involved in citrusproduction. Of the world’s total citrus production,approximately 70% has been reported from thenorthern hemisphere, with Brazil being the top citrus-producing country followed by the USA. India rankseighth in world citrus production (FAOSTAT 2010).

Papilio demoleus larvae are voracious feeders andattack the young foliage of citrus plants, completelydefoliating plants in nurseries and orchards. Adultsare strong fliers and are introduced into new regionswith their host plant—native or cultivated citrus(Guerrero et al. 2004). It has been reported fromthroughout southern Asia, the Middle East to India,and the Indo-Pacific region (Vane-Wright and de Jong2003). In recent years P. demoleus has been recordedfrom New World countries like the DominicanRepublic (Guerrero et al. 2004), Jamaica and PuertoRico (Homziak and Homziak 2006).

Juvenoid diofenolan, a potent juvenile hormone(JH) mimic and molt inhibitor, is a photo stable andsafe insect growth regulator (IGR) used for thecontrol of scale insects and many lepidopteran pests(Pedigo 2002; Streibert et al. 1994). It interferes withthe endogenous hormone levels and disrupts thenormal growth, development, metamorphosis andmolting in insects, rendering them non-viable. Dio-fenolan has been used for the control of scale insects,many lepidopteran, dipteran and coleopteran insects(Ghoneim et al. 2007), but to the best of ourknowledge there has not been any previous recordon the effect of diofenolan on this economicallyimportant pest of citrus. This prompted us to study the

Phytoparasitica (2011) 39:205–213DOI 10.1007/s12600-011-0154-8

S. Singh (*) :K. KumarDepartment of Zoology, University of Allahabad,Allahabad 211002 U.P., Indiae-mail: [email protected]

K. Kumare-mail: [email protected]

effect of juvenoid diofenolan on the development andmetamorphosis of citrus butterfly, P. demoleus.

Materials and methods

Insect rearing The larvae of various stages werecollected from infested citrus plants and reared in alaboratory at 28±2°C, 70±5% r.h. and 10L:14Dphotoperiod. Larvae were provided with fresh lemon(Citrus aurantifolia) and bael (Aegle marmelos) leavesfor feeding in 6″×4″ glass troughs. After feeding thelarvae were shifted to tall glass jars for pupation. Thelarvae clung onto the walls of these jars and trans-formed into pupae. Adults emerging from the pupaewere kept in 1′×1′×1′ cages provided with netting anda cloth sleeve on one side for handling. Adults werefed 10% honey solutions, and fresh twigs of lemonwere provided for oviposition. The eggs were reared at28±2°C to obtain fresh batches of larvae.

Test compound Diofenolan (a.i. 99.9% ) was purchasedfrom Sigma–Aldrich Chemical Co. (Milwaukee, WI,USA) and the desired doses were prepared by dissolvinga known amount of diofenolan in 1 ml of acetone toobtain 7.5, 15, 30 and 60 μg μl−1 doses.

Experimental procedure Larvae of the desired age, i.e.,penultimate and last instar, were selected from the stockgroup of the same age and divided into batches of 20each. The larvae were treated topically with the desireddoses (7.5, 15, 30 and 60 μg μl−1) on the posteriorabdominal segments with the help of a Hamiltonsyringe (Sigma–Aldrich Chemical Co.). The larvaewere immobilized for a few seconds to allow easypenetration and avoid spillage of the compound due totheir movements. Control larvae were treated with 1 μlof acetone only. After treatment all the larvae werereared as mentioned above. All the experiments wererepeated thrice. Observations were recorded at regularintervals. Various deformities produced as a conse-quence of diofenolan treatment were categorized intodifferent grades depending upon the manifestation ofattributes such as dead as larvae, prepupae, or formationof larval–pupal and larval–pupal–adult chimera forms.

Data analysis All the data regarding larval–larval andlarval–pupal ecdysis duration were subjected toone-way ANOVA to determine significant differ-

ences between means. Correlation coefficient wasalso applied to follow the correlation betweendifferent deformities produced and doses appliedusing BioStat 2008 Professional 5.8.0 software(AnalystSoft, USA).

Results

Topical treatment of penultimate and last instar larvaewith different doses of diofenolan severely hamperednormal development and metamorphosis. In addition tomortality, several deformities were observed whichincluded delay in larval–larval and larval–pupal ecdysisresulting in prolonged larval duration, ecdysial stasis,inhibitory effect on pupation and complete inhibition ofadult emergence.

Effect of diofenolan on penultimate larval instar -Treatment of penultimate larval instars with differentdoses of diofenolan resulted in delay in larval–larvalecdysis or subsequent ecdysis into last instar. The larvalduration was prolonged significantly in all the treatedgroups as compared with the control group (F=30.9;P≤0.001; df=4, 295; f crit=4.74). The mean larvalduration of the control was 2.3±0.06 days whichincreased to 3.5±0.07, 3.5±0.09. 3.5±0.11 and 3.6±0.12 days at 7.5, 15, 30 and 60 μg doses, respectively(r=0.87, P≤0.05) (Table 1).

The larval—pupal ecdysis duration was also pro-longed significantly from 8.3±0.11 days in control to8.5±0.10, 8.9±0.10, 10.4±0.16 and 12.8±0.10 at 7.5,15, 30 and 60 μg, respectively (F=255.1; df=4, 295;P≤0.001; f crit=4.47), showing a dose-dependentresponse and a positive correlation coefficient, r=0.99(P≤0.05) (Table 1).

Pupation was severely affected and none of thetreated larvae produced any pupae except at 30 μg,where only 10.7±0.7% pupation was observed withan overall correlation coefficient of r=0.05 (P≤0.05)(Table 1).

Ecdysial failure of various degrees was the maindeformity produced as a result of diofenolantreatment at all doses tested. The larvae developeddouble head capsules, were unable to feed properlyand secreted watery fluids from the body, leadingto desiccation of the larvae while undergoingmolting. All treated larvae (100%) died due to

206 Phytoparasitica (2011) 39:205–213

ecdysial failure at 7.5, 15 and 60 μg doses, whereas91.7±1.6% ecdysial failure was recorded at a dose

of 30 μg with a negative correlation of r = −0.05(P≤0.05) (Table 1).

Table 1 Effect of topical treatment of diofenolan on penultimate instar larvae of Papilio demoleus (n=20; replicates = 3; data aremeans ± SE)

Age Dose (μg l−1) L–L ecdysis duration(days)

L–P ecdysis duration(days)

L–P mosaics(%)

Pupation(%)

Ecdysialfailure (%)

Adultemergence (%)

4th instar0–2 h

0 2.3±0.06 8.3±0.11 0±0 100±0 0±0 100±0

7.5 3.5±0.07* 8.5±0.10* 0±0 0±0 100±0 0±0

15 3.5±0.09* 8.9±0.10* 9.7±0.33 0±0 100±0 0±0

30 3.5±0.11* 10.4±0.16* 0±0 10.7±0.7 91.7±1.6 0±0

60 3.6±0.12* 12.8±0.10* 0±0 0±0 100±0 0±0

r=0.87 r=0.99 r =−0.59 r=0.05 r =−0.05

*Significant at P≤0.001, one-way ANOVA

L–L = larval–larval; L–P = larval–pupal

r = Coefficient of correlation

Types of deformities Percentage of deformities observed

7.5 μg 15 μg 30 μg 60 μg

Dead as larvae

Double head capsule 65.5±0.5 52.3±1.4 69.5±1.3 54.2±1.1

Cuticle melanized 32.7±1.4 37.5±1.4 70.5±0.5 65.6±0.6

Cuticle ruptured in parts 0±0 0±0 0±0 13.7±1.3

Charred cuticle 17.2±1.4 0±0 0±0 0±0

Disrupted pigmentation 15.6±0.6 0±0 0±0 0±0

Body and cuticle wrinkled 67.2±1.4 60.8±0.8 73.8 ±1.2 75.9±0.9

Extremely shrunken body 51.6±1.6 63.5±0.7 69.1 ±1.1 87.3±1.1

Leg tips black 18.9±1.0 37.5±1.4 29.5±0.5 44.8±0.2

Desiccated larvae 65.5±0.5 25.7±0.7 47.7±1.4 11.0±0.6

Old exuvia attached 0±0 0±0 0±0 12.7±1.2

Rectal prolapse 0±0 0±0 0±0 0±0

Loss of spots and pigmentation 0±0 13.3±0.8 0±0 0±0

Dead as prepupae

Pigmentation disrupted 0±0 0±0 14.7±0.2 0±0

Open in middle 0±0 0±0 10.6±0.7 0±0

Thoracic legs shrunken/swollen 0±0 0±0 14.7±0.2 0±0

Abdominal prolegs present 0±0 0±0 10.8±0.8 0±0

Support threads not formed 0±0 0±0 15.1±0.5 0±0

Dead as L–P mosaic

Body larval 0±0 13.8±0.7 0±0 0±0

Black cuticle in parts 0±0 14.5±1.0 0±0 0±0

Pigmentation disrupted 0±0 13.5±0.5 0±0 0±0

Pupal cuticle present in parts 0±0 14.2±0.8 0±0 0±0

Support threads not formed 0±0 13.5±0.8 0±0 0±0

Table 2 Types of abnor-malities produced after top-ical treatment of diofenolanat four different doses topenultimate instar larvae ofPapilio demoleus (n=20;replicates = 3; data aremeans ± SE)

• Control (0 rate)—normaldevelopment/ no abnormality

Phytoparasitica (2011) 39:205–213 207

Grades of deformities All the dead specimens werecategorized into various grades on the basis of

deformities or abnormalities observed. More thanone type of deformity was observed in the majority

4 1 3

6

5 7 8

Plate 1 Abnormalities produced as a result of topical treatmentof diofenolan on penultimate and last instar larvae of Papiliodemoleus 1. Shrunken larva with melanized cuticle 2. Shrunkenlarva with double head capsule 3. Extremely shrunken larvawith wrinkled body 4. Larva with wrinkled body and loss ofpigmentation 5. Dead prepupa open in thoracic region 6. L–Pmosaic with pupal hump (lateral view); 7. L–P mosaic showingpupal hump with larval head and body (dorsal view); 8. L–Pmosaic posterior end showing abdominal prolegs and silkthreads 9. Wrinkled larva with double head capsule andfragments of old cuticle 10. Extremely shrunken larva with

disrupted melanization 11. Larva with black charred cuticle 12.Larva showing wrinkled body 13. Larva with black cuticle andrectal prolapse 14. Abnormal prepupae with silk threads aroundbody 15. Deformed, open L–P–A mosaic 16. Open L–P–Amosaic showing larval prolegs and cuticle 17. Open L–P–Amosaic with small antennae outside the chrysalis, legs notdeveloped 18. Open L–P–A mosaic with larval prolegs. (Bar =1 mm; Red arrow pointing out the deformities. An = antennae,op = open pupa, Lex = larval exuvia, Abn L = abnormal leg,Abn mp = abnormal mouthparts, Abn W = Abnormal wings,LP = larval proleg)


of dead specimens as a result of diofenolan treatment(Table 2).

Dead as larvae Larvae extremely shrunk, showingwrinkled cuticle and body; disrupted normal pigmen-tation; black, melanized, charred cuticle; cuticleruptured in patches showing flakes of old cuticleattached to newly formed young cuticle; formation ofdouble head capsule; rectal prolapse; loss of bodyfluids, with desiccation; thoracic legs and abdominalprolegs shrunk with melanized tips (Plate 1: Figs. 1–4, 9–13).

Dead as prepupae Body did not attain usual commashape, cuticle pigmentation disrupted, open in middlein the thoracic and anterior abdominal segments onthe ventral side; thoracic legs shrunk; abdominalprolegs present; supporting silk threads not formed,unable to attach to the walls of the rearing jars(Plate 1: Figs. 5, 14).

Dead as chimera/mosaic having characters of morethan one stage Larval–pupal chimera: Body larvalwith head, mouthparts, thoracic legs and abdominalprolegs present; cuticle black, melanized at places;thorax black with a hump of pupal mass bulging out,green in color; support silk threads not formedproperly, found interwoven throughout the larvalbody (Plate 1: Figs. 6–8)

Adult emergence was totally suppressed as none ofthe pupae (10.7±0.7%) formed at the 30 μg dosewere able to undergo pupal–adult ecdysis and died aspupae (Table 1).

Effect of diofenolan on last larval instar The larval–pupal ecdysis duration was prolonged as a result ofdiofenolan treatment at all dose levels. The mean larval–pupal ecdysis duration in control was 9.31±0.08 days,which increased to 10.9±0.02, 12.1±0.10, 12.8±0.15and 13.4±0.14 days at the 7.5, 15, 30 and 60 μg doses,respectively (F=175.5; df=4, 295; P≤0.001; f crit=4.74). The larval–pupal ecdysis duration increased in adose-dependent manner with a correlation coefficientof r=0.90 (P≤0.05) (Table 3).

Pupation was severely hampered and only 5.66±0.7% and 6.0±0.57% pupation was observed at the7.5 and 30 μg doses. With the other doses, treatedlarvae did not produce pupae, giving a correlationcoefficient of r= −0.44 (P≤0.05), showing a negativecorrelation between doses applied and pupation.

Ecdysial failure was the most prominent deformityobserved after diofenolan treatment of last instarlarvae, similar to 4th instar: 95.6±0.66% larvae at7.5 μg and 95.3±0.32% larvae at 30 μg, while 100%larvae at 15 and at 60 μg could not molt into pupaeand died due to ecdysial stasis with loss of bodyfluids and desiccation, giving a positive correlationcoefficient of r=0.44, showing a dose-dependentresponse. Several deformities of different grades werealso observed similar to penultimate instar treatedlarvae apart from formation of a chimera havingcharacters of all the three stages—larva, pupa andadult (Table 4).

Dead as chimera/mosaic having characters of morethan one stage Larval–pupal–adult chimera: Pupadeformed; open in the middle with white undifferen-

Table 3 Effect of topical administration of diofenolan on last instar larvae ofPapilio demoleus (n=20; replicates = 3; data are means ± SE)

Age of larvae Dose (μg l−1) L–P ecdysis duration (days) Pupation (%) Ecdysial failure (%) Adult emergence (%)

5th instar0–2 h

0 9.31±0.08 100±0 0±0 100±0

7.5 10.9±0.02z 5.66±0.7 95.6±0.66 0±0

15 12.1±0.10z,y 0±0 100±0 0±0

30 12.8±0.15z 6.0±0.57 95.3±0.32 0±0

60 13.4±0.14z,y 0±0 100±0 0±0

r=0.90 r =−0.44 r=0.44

L–P = larval–pupalz Significant at P≤0.001, one-way ANOVAy Treated larvae did not pupate

r = Coefficient of correlation


tiated pupal mass showing through the opening; headpupal; adult mouthparts present, proboscis bifurcatedand twisted with pupal cuticle present on the upperside and a bulging mass present on the ventral side;antennae short, a few segmented, present outside thechrysalis; wings pupal, constricted; parts of legs in theform of white / translucent mass without anydifferentiation; abdomen pupal; larval prolegs present;larval exuviae present attached to the mid-part or atthe tip (Plate 1: Figs. 15–18).

Adult emergence was totally suppressed due todiofenolan treatment at all the doses and adults did

not emerge from a very small number of pupae(5.66±0.7 and 6.0±0.57%) formed at the 7.5 and30 μg doses (Table 3).

Discussion

Topical treatment of penultimate and last instarlarvae of P. demoleus with different doses of novelIGR diofenolan produced an array of developmentalabnormalities. These include delay in larval–larval

Types of deformities Percentage of deformities observed

7.5 μg 15 μg 30 μg 60 μg

Dead as larvae

Double head capsule 11.3±0.7 0±0 29.5±0.5 0±0

Cuticle melanized 34.4±0.6 0±0 30.2±0.9 84.4±0.6

Cuticle ruptured in parts 18.2±0.9 0±0 14.7±0.3 17.5±0.4

Charred cuticle 17.2±0.4 70.5±0.5 0±0 17.2±0.4

Disrupted pigmentation 17.5±0.4 29.5±0.5 0±0 0±0

Body and cuticle wrinkled 65.6±0.6 99.0±0.6 84.6±1.4 65.6±0.6

Extremely shrunken body 65.9±0.5 98.3±0.9 56.0±1.0 66.6±0.3

Leg tips black 34.4±0.6 0±0 0±0 34.4±0.6

Rectal prolapse 17.6±0.4 14.7±0.3 14.7±0.3 0±0

Loss of spots and pigmentation 0±0 0±0 0±0 0±0

No change in pigmentation 0±0 29.5±0.5 0±0 17.2±0.4

Dead as prepupae

Pigmentation disrupted 0±0 0±0 0±0 17.5±0.4

Open in middle 0±0 0±0 0±0 34.4±0.6

Thoracic legs shrunk/ swollen 0±0 0±0 0±0 35.1±1.0

Abdominal prolegs present 0±0 0±0 0±0 51.6±0.8

Support threads not formed 0±0 0±0 0±0 51.0±1.0

Body charred 0±0 0±0 0±0 17.6±0.4

Black cuticle formed in patches 0±0 0±0 0±0 34.4±0.6

Dead as L–P–A mosaic

Deformed open pupa 18.5±0.8 0±0 14.7±0.2 0±0

Undifferentiated pupal mass present 17.5±0.4 0±0 14.1±0.6 0±0

Mouthparts larval, head capsule present 17.2±0.4 0±0 0±0 0±0

Mouthparts adult, bifurcated/ twisted 16.6±0.3 0±0 14.7±0.3 0±0

Antennae short, outside the chrysalis 0±0 0±0 8.5±0.8 0±0

Wings constricted, pupal type 16.9±0.1 0±0 15.3±0.3 0±0

Legs undifferentiated, translucent mass 0±0 0±0 13.5±0.8 0±0

Abdomen pupal, no differentiation 98.7±0.7 0±0 96.6±1.6 0±0

Larval prolegs present 99.7±0.3 0±0 98.7±0.7 0±0

Larval exuvia attached 99.3±0.7 0±0 0±0 0±0

Table 4 Types of abnormal-ities produced after topicaltreatment of diofenolan atfour different doses to lastinstar larvae of Papiliodemoleus (n=20; replicates =3; data are means ± SE)

• Control (0 rate)—normaldevelopment/ noabnormality


as well as larval–pupal ecdysis resulting in pro-longed larval life, severe reduction in pupation andformation of chimera / mosaic forms having charac-ters of more than one developmental stage, mortality,ecdysial failure and complete inhibition of adultemergence.

Delay in larval–larval and larval–pupal ecdysis andsubsequent prolongation of larval life as a result ofdiofenolan treatment as found in the present studyshows its role as a JH mimic as well as a moltinhibitor. Exogenous application of diofenolan, apotent juvenile hormone analog (JHA) and chitinsynthesis inhibitor, may overload the insect system,interfering with the normal JH levels and activity ofthe prothoracic gland and inducing inhibitory actionon the synthesis, release and action of moltinghormone ecdysone (Hirano et al. 1998).

The normal JH level in untreated holometabolousinsects varies during its life cycle. In the penultimateinstar, the JH level is high, declines gradually and atthe end of the larval instar there is a JH-sensitiveperiod for larval vs pupal commitment. In the finallarval instar, the high JH level during the early phaseof the last instar drops to a low level during mid-stageexcept for a surge showing a small peak at the end ofthe final instar. There are two JH-sensitive periods inthe last larval instar which determine the larval vspupal or adult characters. The presence of JH duringthe JH-sensitive period is responsible for maintainingthe current state of development, whereas the absenceof JH triggers the switch in developmental pathways(Nijhout 1994).

Exogenous application of diofenolan to penulti-mate and last instar larvae in the present study couldhave flooded the insect system with JH analog/mimicat an inappropriate time, resulting in suppression ofthe developmental switch in the larvae, with the insecttending to remain in that stage for a longer duration toeliminate all the JHA before undergoing the nextmolt. Moreover, JH also has an inhibitory effect onsecretion of prothoracicotropic hormone (PTTH) inthe final larval instar and an indirect inhibitory actionon the secretion of ecdysteroids (Nijhout 1994). Theeffect of diofenolan in delaying the molts andprolonging larval life can be explained in the lightof these facts, where it mimics the action of JH.

Several other workers have shown prolongation ofimmature stages due to the action of JHAs / chitinsynthesis inhibitors in different insect species such as

Ctenocephalides felis (Meola et al. 2000); Chryso-perla rufilabris (Chen and Liu 2002); Aedes aegypti(Braga et al. 2005; Harburguer et al. 2009).

Ghoneim et al. (2007) studied the effects oflufenuron and diofenolan on the survival, growth anddevelopment of the red palm weevil (Rhynchophorusferrugineus) and found that both the IGRs hastened thedevelopment of pupae. Contrary to the present study, inwhich larval duration was prolonged in diofenolan-treated larvae, diofenolan exerted an antagonisticaction on the treated prepupae and pupae of Rh.ferrugineus, reducing the normal pupal duration. Thevariation in developmental effects as a result of JH/JHA/chitin synthesis inhibitors may be due to largespecies variation regarding the potency of thesecompounds as well as different mechanisms ofecdysteroid metabolism in different insects (Whisentonet al. 1989).

Tomkins et al. (1994) studied the effects ofdiofenolan on scale insects and found that itdisrupted the molting between instars and interferedwith the crawler production in greedy scale, Hemi-berlesia rapax. Streibert et al. (1994) studied theeffects of diofenolan on scale insects and lepidop-teran pests of deciduous fruits and citrus and foundthat diofenolan disrupted the insect-specific trans-formations from the crawler to the sessile scaleinsect or from egg to larva or larva to pupa inlepidopterans. Similar intermolt inhibitory effectswere observed in the present study where latenteffects of diofenolan were manifested during larval–larval and larval–pupal molts when penultimate andlast larval instars were treated. Suppression ofpupation was also observed by Ghoneim et al.(2007) by diofenolan when prepupae of Rh. ferrugi-neus were treated. They classified diofenolan as achitin inhibitor. Diofenolan also inhibited pupationin Coccinella septempunctata and Chrysoperla car-nea (Sechser et al. 1994).

During normal development, the intrinsic JH levelsremain negligible during pupation and this low levelof JH facilitates the pupal–adult transformation ofimaginal discs during the sensitive phase (Nijhout1994). The treatment of penultimate and last larvalinstars with diofenolan results in latent effects onpupation as the JHA could not have been metabolizedin the insect system and may persist for a longerduration, inhibiting the morphogenic changes frompupal to adult type. This results in ecdysial stasis with


several deformities in larval–pupal as well as pupal–adult transformation. Also, because of the highintrinsic JH level as a result of the presence ofendogenous JHA, diofenolan may inhibit the releaseand/or action of the PTTH and ecdysteroids (Tunazand Uygun 2004), which ultimately prevents themolting and the insect dies while undergoing larval–pupal or pupal–adult molts having characters of boththe stages in mosaic or chimera forms.

In the present study, edcysial failure / stasis was themost prominent effect, causing mortality in themajority of treated insects. These insects had varioustypes of deformities like a melanized cuticle—black,wrinkled bodies, ruptured cuticle in parts with flakesof old cuticle attached to a new one. The mortalitydue to edcysial failure could be due to directinhibition of chitin synthesis within the integumentor to the rupture of the newly formed one. Apart fromthis, mortality also results from bleeding and desic-cation, imperfect exuviations and failure of vitalhomeostatic mechanisms (Smagghe and Degheele1994), as observed in the present study.

Several authors have reported mortality due to theeffect of JH/JHA/chitin synthesis inhibitors on variousinsect species (Ghoneim et al. 2003; Hazarika et al.2009; Navrozidis et al. 1999). Some species respondby exhibiting the effects of acute toxicity, while othersdo not and this may be attributed to innate differencesin absorption, degradation and excretion of the JHAsby different insects (Granett et al. 1980); it alsodepends on the presence of JHA esterase in differentspecies (Nijhout 1994).

Complete inhibition of adult emergence was observedas a result of diofenolan treatment at all the doses in boththe penultimate and last larval instar treated larvae. It issuggestive of the fact that the JHA diofenolan persists inthe insect system for a long time and produces stronginhibitory effects in the later parts of the life cycle. Thepossible explanation for this might be that the JHAs havea strong affinity for the JH-binding proteins found in theinsect hemolymph (Nijhout 1994) These proteins pre-vent the degradation of JHs by forming a JH–proteincomplex. When JHAs are exogenously applied, theycompete for the JH binding proteins, bind to themforming a JHA–protein complex and avoid the naturaldegrading mechanisms of the system, producing long-term effects (Edwards et al. 1993). Diofenolan hasproduced excellent results for control of lepidopteransand scale insects by inhibiting their emergence, as

evident from the studies of Kerns and Tellez (1998),Pedigo (2002) and Ghoneim et al. (2003).

In the present study, treatment with all the testeddoses of diofenolan resulted in complete inhibitionof emergence, showing that diofenolan is effectiveeven at low doses. The presence of the JHAdiofenolan might have delayed or reduced orblocked the synthesis / release of eclosion hormonewhich is responsible for all the processes associatedwith molting (Nijhout 1994). Ghoneim et al. (2007)has postulated that diofenolan suppresses the chitinsynthesis and prevents normal deposition of newcuticle during apolysis, resulting in molting abnor-malities during larval–pupal or pupal–adult trans-formations. These effects are evident in the presentstudy where deformities in larval and pupal stageswere observed as a result of diofenolan treatment.

The present findings demonstrate that the JHAdiofenolan can effectively disrupt the development ofthe citrus pest P. demoleus under laboratory condi-tions. Considering its efficacy and selective mode ofaction against lepidopteran pests, apart from beingrelatively harmless to beneficial and non-targetorganisms, it may be incorporated into IPM programsalong with other bio-rational approaches for thecontrol of P. demoleus.

Acknowledgment We deeply thank the University GrantsCommission for providing financial assistance in the form of ameritorious research scholarship to S.S., the correspondingauthor.

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diofenolan: a novel insect growth regulator in common citrus butterfly, papilio demoleus

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