Recognition behavior of the cassava mealybug Phenacoccus manihoti Matile-Ferrero (Homoptera: Pseudococcidae) at the leaf surface of different host plants

Journal of Insect Behavior, Vol. 11, No. 3, 1998

Recognition Behavior of the Cassava MealybugPhenacoccus manihoti Matile-Ferrero (Homoptera:Pseudococcidae) at the Leaf Surface of DifferentHost Plants

Sophie Renard,1,5 Paul-Andre Calatayud,2 Jean-Sebastien Pierre,3 andBruno Le Ru4

Accepted November 15, 1997; revised December 17, 1997

The testing behavior and test probing which are a part of host-plant acceptancebehavior were studied in P. manihoti. Attention was focused on the testingbehavior of three plants of the Manihot genus (Incoza, M'Pembe, and Faux-caoutchouc) and a weed of the cassava fields (Talinum). This enabled us notonly to characterize the associated behavior but also to show that the mealybugis able to distinguish between different host plants when walking on the leaf.The video description of the test probing of first- and fourth-instar larvae onM'Pembe and on the Faux-caoutchouc shows that the succession of the phasesis similar. After a first phase characterized by the repeated intervention of themealybug sensorial organs, a second phase, more mechanical, with up-and-down head movements, is observed. At this time, the stylets pass through theepidermic and inner tissues. Finally, a third phase, during which the mealybugbecomes more agitated, is observed: it stands up using its rear legs and pushesthe upper part of its body against the plant. The stylets continue their progres-sion, which is principally intercellular, until they reach the phloem. Longer andlonger immobility periods are observed over a period of time. We used thecoupled videocamera and electrical penetration graph technique to see the rela-tions existing between outer and inner plant events, i. e., between the behavioralitems and the electrical signals characteristic of the stylets pathway in the plant.

1 UER de Zoologie Generale et Appliquee, Faculte des sciences agronomiques, 2 Passage desDeportes. B-5030 Gembloux, Belgique.

2LIN-BPG, Pare Scientifique Agropolis 2. Bat. B5-B6, 34297 Montpellier Cedex 5, France.3Laboratoire de Zoologie, ENSAR, 65 Route de Saint-Brieuc, F-35042 Rennes Cedex, France.4Laboratoire d'Entomologie Fondamentale et Appliquee de l'Universite de Rennes I, Avenue du

General Leclerc, 35042 Rennes Cedex, France.5To whom correspondence should be addressed. (32) 81 62 23 12. e-mail: [email protected].

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0892-7553/98/0500-0429$15.00/0 C 1988 Plenum Publishing Corporation

KEY WORDS: Phenacoccus manhoti; Pseudococcidae; cassava; testing behavior; test probing.

INTRODUCTION

The mealybug P. manihoti, a native of South America, has been one of themain pests of cassava (Manihot esculenta Crantz; Euphorbiaceae) in tropicalAfrica since the beginning of the seventies (Silvestre, 1973; Herren, 1987).Research has focused on biological control. As far back as 1982, Epidinocarsislopezi (Hymen. Encyrtidae), a native of South America, was successfully intro-duced and allowed the mealybug populations to be controlled in numerous eco-logical situations (Herren and Neuenschwander, 1991). Nevertheless,considerable damage is still observed in cassava fields cultivated on poor soil(Neuenschwander et al., 1990; Le Ru et al., 1991).

A plant of integrated control has been developed better to regulate themealybug populations under these particular ecological conditions. Biologicalcontrol can be improved by using appropriate cultural techniques and by apply-ing varietal control. The latter method, which consists in using resistant vari-eties, supposes a thorough knowledge of interactions between the mealybug andthe plant. Research is already in progress and findings indicate that cassavaresistance is polygenically expressed according to the three components definedby Painter (1951), that is, antixenosis, antibiosis, and tolerance (Tertuliano etal., 1993; Le Ru and Tertuliano, 1993). At present we are especially interestedin antixenosis, i.e., the understanding of plant selection mechanisms of themealybug.

According to a number of authors, there are two steps in plant selectionby a phytophagous insect; localization and plant acceptance. Distance local-ization, which involves the senses of smell and sight, depends on physical andchemical factors such as odor, shape, and plant color. When the insect is onthe plant, it uses sight, touch, and its senses of smell and taste, as well asphysical (color, hairiness) and chemical stimuli (olfactive and gustative) (Ber-nays and Chapman, 1994). These stimuli, which differ from one plant to anotherand from one genotype to another, are known to influence acceptance of theplant by the insect (Stadler, 1986). It is therefore necessary to understand fullythis behavior and its possible variations according to the plant resistance mech-anisms to a pest (Bernays and Chapman, 1994). The numerous studies on plantacceptance by Hemiptera indicate that it is very similar from one species to thenext (Backus, 1988). For Homoptera Sternorrhyncha, which are fairly similarto mealybugs, acceptance is the result of a succession of behavioral steps: testing

We observed that the stylet progression in the plant was more difficult in theresistant hybrid Faux-caoutchouc than in the sensitive M 'Pembe variety.

Renard, Calatayud, Pierre, and Le Ru430

of the plant surface and external tissues, penetration, and the phloem test(Klingauf, 1987). During the first step, the insect shows typical behavior ofwalking and probing, during which it moves its antennae, drums, and rubs theplant surface with its antennae, legs, and labium according to a given behavioralsequence (Ibbotson and Kennedy, 1959). Only the cuticule, or at most the leafepidermis, is probably explored by the labium and the stylets; nonhost plantsare usually rejected during this first step (Klingauf, 1987). During the secondstep, the insect generally puts its stylets in the plant tissues to reach the phloemcells or, occasionally, xylem cells. The penetration, which is relatively long(from 15 min to hours), is in general intercellular except for the Lachnidae(Aphididae), in which it is intracellular. The final acceptance of the plant bythe aphid depends on the qualitative and quantitative properties of the phloem.

The description of the stylet penetration and phloem testing by P. manihoti(Calatayud et al., 1994) confirmed the similarity of these two acceptance behav-ior steps of plants by mealybugs, aphids, and white flies. The testing behaviorof the surface and of the outer tissues of the plant have not yet been describedfor the mealybug. This description is important because the rare studies of itindicate that phytophagous insects "exhibit behavior patterns that can be inter-preted as an examination of the quality of the surface and acceptance or rejectionmay follow without further testing" (Chapman and Bernays, 1989). It alsoreveals how the mealybug, whose labium and antennae tips have diversifiedchemical sensorial equipment (La Ru et al., 1995a, b), proceeds to detect byolfaction and contact the chemical substances at the leaf surface.

This paper describes this behavior in P. manihoti and determines how it ismodified when the plants have different antixenotic resistance levels to the pest.The behavior description concerns, first, selection of the penetration site (testingbehavior) on the basis of visual observations to establish the characteristicethograms of walks and probes which lead to the first acceptance of the plantand, then, test probing using a videocamera to describe the different behavioralitems when the insect stops on the plant. The videocamera technique and theelectrical penetration graph (EPG) technique are used simultaneously to specifythe stylet position in the plant during the second behavioral step.

MATERIALS AND METHODS

Plants

We used four plants: two cassava varieties (Incoza and M'Pembe, Manihotesculenta Crantz, Euphorbiaceae), a hybrid of M. esculenta and M. glazioviiMull arg. (Faux-caoutchouc), and another species called Talinum (Talinumtriangularae Jack, Portulacaceae). The three plants of the Manihot genus were

431Recognition Behavior of the Cassava Mealybug at the Leaf Surface

chosen on the basis of their antixenotic resistance level (Painter, 1951) to P.manihoti, which was determined at the time of field varietal screenings. Faux-caoutchouc is the least antixenotic plant, followed by M'Pembe, Incoza, and,finally, Talinum, a common weed, which is a good, though not the preferred,host of the cassava mealybug in the field (Neuenschwander and Madojemu,1986). It is a highly antixenotic plant to the mealybug, even if it is an excellentsubstitution plant in the laboratory (Tertuliano et al., 1993). Plants were obtainedfrom 20-cm cuttings planted vertically at two-thirds of their height in soil placedin plastic bags (30 x 22 cm). Cuttings were placed in a glasshouse until the 9-to 10-leaf stage was reached (9-10 weeks; approximate height, 60 cm). Plantswere watered twice a week and then transferred to the controlled insect rearingroom: temperature, 23 + 5°C; relative humidity, 70 + 5%; and photoperiod,12 L:12D.

Insects

The mealybug strain used in this study came from the Congo (Pool region).Phenacoccus manihoti reproduces by thelytokous parthenogenesis. We used fourdistinct clones maintained isolated and for several generations on the four hostplants studied to limit the possible incidence of parental trophic nutrition. Insectswere maintained in a controlled insect rearing room at 25 + 2°C and 70 + 5%RH.

Experiment 1: Testing Behavior

This study was performed with same-aged recently hatched neonates because(L1) they are responsible for plant colonization in the field. Observations beginin the morning when one larva is deposited on a leaf situated near the plantapex (rank 3 to 6),6 which is the preferential fixation site. For Talinum, a stemtip (four to five leaves) is considered. In the two cases, a honey ring on thestem prevents the mealybug from leaving the experimental unit. The observationduration (90 minutes), which is sufficient for mealybug fixation, was determinedby preliminary tests on M'Pembe. Twenty mealybugs were observed for eachplant.

Results were represented by ethograms, which allow a succession of insectmovements (called "items") to be visualised over a period of time. Accordingto Klingauf's model on aphids, the following items were observed: walking onthe upper or lower leaf side and test probing (the insect stops and puts its styletsinto the plant tissues).

6The rank is determined from the tip of the plant. The first leaf fully expanded is called number 1.

432 Renard, Calatayud, Pierre, and Le Ru

Experiment 2: Test Probing (Video)

This experiment was carried out on M'Pembe and Faux-caoutchouc (FC)petioles coming from rank 3 to 6 leaves. They were placed horizontally toobserve the insect in profile and each of its movements, including that of itslabium.

We worked with neonate larvae (L1) but also with fourth instar larvae (L4)larvae because the latter are easier to observe and fix more rapidly on the patiole.To accelerate the fixation, L1 were starved for 3 to 4 days and L4 for 2 h.7

Insects were brought up specifically on each variety for several generations.After being placed on the petiole, the mealybug was filmed for 22 min

once it had stopped. We have determined that a period of 22 min is sufficientfor each mealybug to end its "test probing." Twenty-one L4 were observed onM'Pembe and FC, while only 10 and 5 L1 were considered on M'Pembe andFC, respectively, because of their difficulty of fixing on that support.

Results were analyzed statistically according to the Pierre and Kaspermethod (1990), which leads to the production of flowcharts on factorial maps.The mealybugs movements can be examined without taking into account theirduration and the moment at which they were produced.

Succession and transition matrices were established and then analyzed bycorrespondence analysis (van der Heiden, 1987). However, to avoid disturbancedue to diagonal elements (autosuccessions) whose values were either zero,unknown, or undetermined (Escofier, 1984; van der Heiden, 1987, cited by vanBaaren et al., 1993), some modifications according to the Pierre and Kaspermethod (1990) were made. This method has a double aim: to obtain a descriptionof the sequential structure of behavioral patterns (from the interpretation offactorial axes) and to place the patterns relative to one another in space, theirdistance being inversely related to the frequency of succession. In the flowchartobtained, two patterns succeeding each other frequently are both close and linkedby thick arrows. Conversely, two patterns which rarely follow each other willbe far apart and linked by thin arrows. This method has already been used tocompare behavioral sequences of insects under other experimental conditions(van Baaren et al., 1995; Caillaud et al., 1995).

A total of 80 items was observed. Because of the proximity of many ofthem on the flowchart, a clustering according to the average linkage methodbased on Euclidean distances was realized with SYSTAT software. Nine itemgroups were obtained (Table I).

7Starvation does not influence the results of the experiment because we did not test the acceptabilityof the plant or the rapidity of probing, but just the steps of the penetration.

Recognition Behavior of the Cassava Mealybug at the Leaf Surface 433


Experiment 3: Test Probing (Video Coupled with EPG)

The video system was described for experiment 2. Stylet penetration wasmonitored in the plant by a single-channel DC EPG system (Tjallingii, 1998)with a 10°-O input resistance (Model EPG 86 summer course, Van de Pers Inc.,The Netherlands). A gold wire (2-3 cm; P, 25 um) was then fixed on thedorsum of the insect moving [according to the Montlor and Tjallingii (1989)method, cited by van Helden and Tjallingii (1993)] with a water-based silverpaint.

The mealybug was starved during 2 h with the gold wire already fixed(connection with the amplifier) to minimize the manipulation stress just beforethe beginning of the experiment. The experiment took place in a Faraday cage.Fourth-instar larvae were used because their size facilitates manipulation andobservation compared to neonate larvae. We used a standard EPG chart recorder(0-75 Hz; Mini-writer, Ankersmit, GER). Time 0 corresponds to the start ofpenetration of the insect (Fig. 3).

Items were reported on EPG recordings and summarized for each plant ina two-way contingency table in which the total number of associations (on 15mealybugs) between an item and an electrical signal was noted. A test of inde-

Table I. Items Associated with the Host Acceptance Behavior by P. manihoti and AbbreviationsUsed in the Text and Figures

TlaFL

P-A

P-FTF

P-A-T

AcPS

P1AcPS

P1Ac

Antennal and labial drumming of the plant surface, leg rubbing of the surfaceMaintenance of labium against the plant surface, sometimes accompanied by

foreleg rubbing (P1), by antennal drumming, and by up-and-downmovements of the head, like a hammer (T)

Antennal movements accompanied by foreleg (P1 A) or intermediate-leg (P2A)rubbing of the plant surface

Antennal vibration and foreleg (P1F) or rear-leg (P2F) rubbingUp-and-down head movements accompanied by leg rubbing and by antennal

movements from front to backUp-and-down movements accompanied by fore- and intermediate-leg rubbing

and sometimes antennal drumming and antennal movements from front toback

Movements indicating the start of a more accentuated activity: the mealybugstands up using its rear and intermediate legs together (P23S) or separately(P2S) (P3S), first suddenly (Ac) and then for a long time (PS). Othermovements involving antennae or forelegs (P123S) are observedsimultaneously. Activity periods alternate with immobility periods (1), whichbecome longer and longer when approaching 22 min.

Foreleg rubbing of the leaf surface (P1) often followed by sudden (Ac) andthen prolonged movements (P3S), standing up using the legs, sometimesaccompanied by antennal movements (A) or intermediate- and rear-legrubbing

The same as P1AcPS, but sudden movements are not followed by prolongedmovements

pendence (Dagnelie, 1975) and a correspondence analysis (Figs. 4 and 5) (Palm,1993) were performed to study these associations. To compare the two plantsin terms of items—electrical signal associations, electrical signal duration, andnumber of potential drops, a t test (Student-Fisher) was employed (Table IV).

RESULTS

Experiment 1: Testing Behavior

When selecting the fixation site, a succession of walks and stops is observed.When walking on the leaf, the mealybug maintains its antennae up and forwardand its labium drums the plant surface slowly. When stopping, the ventral partof its last antennal segment and the labium tip drum the plant surface intensivelywhile its legs rub the surface (especially forelegs).

Nine ethograms (Types 1 to 9) representative of the plant selection behaviorof P. manihoti were established. Each plant is characterized by a relative pro-portion of each ethogram which is specific to it (Fig. 1).

In Table II, we can distinguish between a short fixation (less than 15 min)and a long fixation (more than 15 min). Indeed, a duration of 15 min is sufficientto allow the mealybug stylets not only to pass through the epidermial tissuesbut also to begin their progression in the parenchyma. After that the probabilityof leaving the nutrition site is very slight.

All the mealybugs (100%) made a long probe on Incoza, compared to 85%on M'Pembe, 65% on FC, and 55% on Talinum. Among these, 80% prefer thelower leaf side of Incoza, versus 45% on M'Pembe, 20% on FC, and 0% onTalinum.

Type 1 is the most encountered behavioral type on Incoza. In other words,the mealybugs quickly went under the leaf (2.7 min), where they rapidly madea long probe (after 11.9 min).

On M'Pembe, the behavior observed is especially of type 4. Mealybugsremained on the upper leaf side, where they made a probe relatively quickly(after 20.2 min). However, 20% of the individuals (type 5) tried to leave theleaf by going on the petiole. M'Pembe seems to be less favorable than Incozafor insect penetration.

On Fc, the penetration is represented by types 4, 5, and 6. Sixty percentof the mealybugs remained on the upper leaf side, where 35% made a longprobe and 25% made short and numerous probes. Twenty-five percent of theindividuals moved for a long time (68.3 min), changing sides frequently.

On Talinum, type 8 is the most important behavior observed. Mealybugswalked nearly all the time (76.2 min) whatever the face of the leaf, and theytried to leave the leaf by climbing on the petiole (6.2). The nonhost character


Renard, Calatayud, Pierre, and Le Ru

Fig. 1. Characteristic ethograms of the mealybug testing behavior observed during 90 min onleaves of the four plants.

436


of Talinum is more accentuated than for FC since long walks are associatedwith attempts to escape.

Experiment 2: Test Probing (Video)

Observation of 21 L4 on M'Pembe and FC

Correspondence Analysis of the Modified Matrix. Axis 1 is associated, onthe negative side, with the behavioral group AcPS observed at the end of thesequence. This group corresponds to sudden movements of the mealybug, whichstands up using both its rear and its intermediate legs, suggesting a mainlymechanical activity.

Axis 2 is associated, on the positive side, with TLaF, L, P-A, and P-Fbehavioral items observed in the beginning of the sequence and characterizedby leaf surface drumming and rubbing with the antennae, labium, and legs,suggesting the use of sensorial equipment. On the negative side, it is associatedwith TF and P-A-T behavioral items observed in the middle of the sequence,which correspond to up-and-down movements of the mealybug head (like ahammer), suggesting mechanical activity but less intensive than for axis 1. Inthe case of FC, the AcPS group is subdivided into Ac and PS. Only PS isassociated, on the negative side, with axis 1, while the Ac group is associated,on the positive side, with axis 2.

Finally, the axes distinguish three behavioral item groups: those associatedwith plant surface recognition (axis 2, positive side); those suggesting a begin-ning of test probing, with possible stylet penetration in the more external tissuesof the plant (axis 2, negative side); and finally, those suggesting a strong pen-etration into the deeper tissues of the leaf (axis 1, negative side). In the case ofFC, the groups are less individualized on account of the more numerous itemswhich are nearly observed.

Flowchart (Fig. 2). The flowchart shows the sequence of a standard testprobing, which can be divided into three stages (Figs. 2a and b). The first onecorresponds to the recognition of a fixation site by the insect. During this stage,the mealybug drums the leaf surface with the ventral tip of its antennae and itslabium while it rubs it with its forelegs (TLaF), and afterward, it maintains itslabium against the surface (L). This stage is followed by the rubbing of theplant surface with the forelegs, accompanied either by a simple antennal vibra-tion (P-F) or by other antennal movements (P-A). The second stage correspondsto the beginning of the test probing, with possible stylet penetration in theepidermic plant tissues. The mealybug moves its head up and down frequentlyand rubs the leaf surface with its legs (P-A-T) or moves its antennae (vibration).The third stage corresponds to sudden movements, which become prolongedand consist, for the mealybug, in standing up using its intermediate and rearlegs (AcPS). They are associated with other movements such as those of anten-


Fig. 2. Flowcharts of the test probing of fourth-instar (L4) mealybugs on M'Pembe and Faux-caoutchouc and of first-instar (L1) mealybugs on the same plants.

nae. This stage suggests stylet penetration into the deeper tissues of the leaf.Sometimes, the third stage is observed directly after the first one.

Observation of 10 L1 on M'Pembe and 5 L1 on FC

Because of the difficulty in filming L1, the video sequences began laterthan for the L4. The transition frequencies from one item group to another(arrows) and the observation frequencies of the items groups (circles) weremultiplied by 2.1 in M'Pembe and 4.2 in FC to allow their comparison withthe L4 (21 individuals).

Correspondence Analysis of the Modified Matrix (Figs. 2c and d). Axis 2is associated, on the positive side, with the P-A-T behavioral group observedat the beginning of the sequence. It corresponds to up-and-down movements ofthe head suggesting mechanical stylet penetration into the plant. Axis 3 is asso-ciated, on the negative side, with the P1AcPS behavioral group observed duringthe whole sequence. This group is characterized by frequent contact of theforelegs with the leaf surface, followed by more intensive mechanical activity.It consists for the mealybug in standing up, first suddenly and afterward moreslowly, using its intermediate and rear legs.

In the case of FC, the P1AcPS group is subdivided into P1Ac and P3S,P23S, and P123S. The latter two are associated with axis 3, on the negativeside, while the others are associated with axis 2, respectively, on the positiveand negative side. Like L4, this behavioral sequence seems to correspond todeep stylet penetration into the leaf tissues. It is more difficult to conclude herebecause the AcPS group is representative of activities distributed over the entirefixation sequence.

Flowchart (Figs. 2c and d). The flowchart shows the following sequence.The first stage corresponds to the beginning of the test probing. At first, themealybug drums the leaf surface with its forelegs (P1 in AcPS and P1 Ac) whileit may move its head up and down (T in P-A-T). Stylets are thought to bepresent in the superficial tissues of the leaf. After that, the mealybug stands upsuddenly, using its intermediate and rear legs (Ac in AcPS and P1 Ac). Thesemovements can become slower and longer (P3S, P23S, and P123S in AcPS).Stylets probably penetrate deeper into the leaf tissues.

Experiment 3: EPG Coupled with Video Recording

In this experiment, A, B, C, and pdc electrical signals and 12 behavioralitems previously defined in Table I were taken into account. Items were notgrouped because the notion of time (duration) is present when searching forcorrespondences with electrical signals. Electrical signals are representative ofevents occurring within the plant during the insect-plant relationships and weredescribed by Calatayud et al. (1994). The A signal corresponds to the estab-


lishment of the electrical contact between the buccal cavity of the mealybug andthe plant tissues. The B signal corresponds to the first salivary secretions whichare implicated in plant recognition and the cellular layer penetration in thestylets. The C signal is associated with salivation and with an intercellularprogression of the stylets, intersected by intracellular punctures (pdc signal orpotential drops). During the latter, sudden drops in the voltage level and a fastreturn to the original level are observed. Figure 3 shows 5 min of an EPGrecording. Table III shows the results for the two varieties.

Because of the difficulty of interpreting the results from a two-way contin-gency table, we first performed an test of independence between the behavioralitems. The null hypothesis (independence) was rejected and a correspondenceanalysis (Figs. 4a and 4b) was then conducted. We can conclude that axis 1opposes, for the two plants, the A and B signals of the beginning of the sequency(labium contact with the plant, passage of the epidermic layers) to the C andpdc signals of the end of the sequence (pathway in the parenchyma and puncturein the cells). In the same way, it opposes the Ac, A, PIA, P1 (P2 for the FC),and I items representative of increased sensorial activity and of the beginningof an activity phase, to the P2S, P23S, P3S, and P13S items corresponding tomore intensive mechanical activity. In the case of Incoza, the I item is associatedwith the A and B signals, while P23S, P3S, and P13S are associated with pdc.

Fig. 3. EPG recording of the stylet pathway of a fourth-instar mealybug larva in a petiole of theIncoza variety during 5 min. A, B, C, and pdc are the electrical signals, while the other abbre-viations are the items observed.



Fig. 4. Results of the factorial analysis showing the relation between the elec-trical signals and the behavioral items for Incoza and Faux-caoutchouc. A, B,C, and pdc are the electrical signals, while the other abbreviations are the itemsobserved.

Recognition Behavior of the Cassava Mealybug at the Leaf Surface

Table III. Total Frequency of the Items (on 15 Mealybugs) for the Four Electrical SignalsMonitored on Incoza and Faux-caoutchouc

Items

IncozaIAP1P1AAcP3SP23SP2P13SCTL

Faux-caoutchoucIAP1P1AAcP2SP3SP23SP2P13S

A

15451300000

11583200020

Electrical signal

B

32594

1451301

211113101802350

C

10134551536724623

74

942841392112

117422010

pdc

15483

111817023

1479866

3615

12

In the case of FC, the Ac item is associated with the B signal, while P3S isassociated with pdc.

This analysis allows us to conclude that the principal difference betweenthe two varieties is situated in the A and B signals. Indeed, the Ac item isproportionally more frequent in the B than in the A signal, whatever the variety,but the difference is far more accentuated for the FC. Moreover, the frequencyof the Ac item is nearly the same for the A signal in Incoza as in the FC, whileit is more frequent for the B signal in FC than in Incoza. This suggests that theepidermic structures of the FC are harder than those of Incoza. Indeed, the Aand B signals are representative of the beginning of the puncture and of sali-vation, and the mechanical activity (Ac) is more accentuated in the case of FC.

Comparing the association frequencies of electrical signals and the behav-ioral items for the two plants studied by a t test (Student-Fisher), we observea significative difference. Indeed, the P3S item is more frequently encounteredduring the C signal and the potential drops more in the FC than in the Incoza.As the P3S signal corresponds to the mealybug standing up using its rear legsand its movement seems to respond to the need of helping the stylet pathway

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Table IV. Results (Means) of the t Test (Student-Fisher)

Parameters

Number of potential dropsDuration of the A signalDuration of the B signal*Duration of the C signal (of 22 min)Mean duration of a pddc*

Incoza

1.31.2 min3.2 min

17.6 min17.5 min

Faux-caoutchouc

1.31.2 min2.2 min

18.6 min15.9 s

* Significant mean difference at 95%.

between and in the parenchyma cells, the stylets should experience more diffi-culties to progress inter- and intracellulary in the Fc than in the Incoza.

Comparing the number of potential drops and the duration of electricalsignals by a t test (Student-Fisher) (Table IV), we see that differences exist.Indeed, the B signal, which corresponds to salivations on first plant recognition,and the potential drops, which should intervene in the plant testing, are onaverage longer both in the sensitive Incoza variety than in the FC.

DISCUSSION AND CONCLUSION

The selection behavior of the fixation site by P. manihoti described in ourstudy is similar to that of other Homopteres (Backus, 1988), in particular, tothat of Aphids (Klingauf, 1969, 1973). Like aphids, during the selection of thefixation site, the mealybug alternates walks and stops. According to Klingauf'scriteria (1973), the behavior observed on Incoza, that is, a quick passage on thelower leaf side followed by a nearly immediate fixation, corresponds to a pre-ferred host plant (only slightly antixenotic). On the contrary, that observed onTalinum, that is, long walks, especially on the upper leaf side, accompanied bynumerous side changes and by attempts to escape, corresponds to a nonpreferredhost plant (highly antixenotic). Behaviors encountered on M'Pembe and FC areintermediate: an increase in the duration of walks and side changes from theleast (M'Pembe) to the most antixenotic (FC). These results indicate that simplewalk on the leaf surface allows the insect to differentiate between the two plants.The following classification was obtained: the antixenosis increases from Incozato M'Pembe, from M'Pembe to FC, and from FC to Talinum. For the plant ofthe Manihot genus, this classification differs from that previously mentioned,which was an increase in the antixenosis from FC to M'Pembe and fromM'Pembe to Incoza. But it was obtained from field observations which integratedthe two steps of the plant selection by the insect, that is, the distance localizationand the acceptance when the insect is on the plant. This difference can be related


to a modification of the plant's antixenosis expression because of the experi-mental condition differences. It can also indicate that the antixenosis expressedby the cassava for the mealybug is found not only directly in contact with theplant by means of the substances of the phylloplane and of the deeper tissues,but also in the odors emitted by the plant. The antixenosis level should differwhen expressed at a distance or on plant contact. Indeed, Storer and van Emden(1995) showed, on the Aphis gossypii Glover (Homoptera, Aphididae)/Chry-santhemums model, the existence of an antixenotic level difference accordingto whether the aphid is or is not in contact with the plant. As underlined bythese authors, "The degree of antixenosis which can [be] exhibit[ed] will there-fore reflect the overall processed response made by the insect acting on infor-mation received from the numerous sensory sources." During this selection stepof the fixation site, we have mentioned that the mealybug raises its antennaeand vibrates them while it drums the plant surface with its antennae and labiumtip. The main part of the chemical sensorial equipment being located in theseorgans (Le Ru et al., 1995a, b), it is probable that the chemical characteristicsof the plant phylloplane play a prominent part in this step.

When the penetration site is chosen, whatever the instar (L1 or L4) or theplant (Incoza or FC), we can distinguish three phases in the behavior of P.manihoti. During the first one, the insect repeatedly drums and rubs the plantsurface with the ventral part of its antennae, labium, and leg tips and its antennaevibrate. At the end of the phase, the labium is maintained in contact with thephylloplane without stylet penetration into the plant tissues. According to Stadler(1986), the insect probably evaluates the nutritional quality of the plant duringthis phase, the recognition having already taken place during the fixation siteselection. The repeated contacts of the olfactory and gustatory sensorial organs,located principally on the antennae and labium tip of P. manihoti (Le Ru et al.,1995a, b), indicate that the mealybug uses its sensorial equipment particularlyand that it is able, like aphids (Klingauf, 1971; Greenway et al., 1978), toperceive the odors present in the thin air layer of the leaf surface (called theboundary layer) just as the surface waves and chemical compounds in it. Theserepeated contacts of the sensorial organs with the plant should increase theamount of sensory input to the central nervous system (Blaney and Duckett,1975). In addition, they should allow better evaluation of the chemical char-acteristics of the phylloplane to the extent where the wax composition variesfrom one place to another and in the function of the physiological state on asame plant (Eglinton and Hamilton, 1967) and where the most polar compoundsare heterogeneously distributed on the leaf surface. During the second phase,the mealybug quickly moves its head up and down and its antennae forward andbackward while it rubs the plant surface with its legs and inserts its stylets intothe epidermic tissues and probably into the layers just under it. The first sali-


vations are monitored. The third phase is indicative of great activity of themealybug. The insect strongly presses the front part of its body on the plantwith insistence while it raises its abdomen with its rear and intermediate legsand the stylets penetrate into the more inner tissues of the leaf. Longer andlonger immobility periods are observed during this time. The latter two phasescorrespond to the stylet probing in the tissue-feeders that Sogawa subdividedinto two steps: test probing (our second phase) and exploration probing (ourthird phase). In this study, we described principally the test probing and thebeginning of exploration probing. Indeed, our observations were made during22 min, while the necessary time to reach the phloem is more than 90 min inP. manihoti (Calatayud et al., 1994). The test probing has been described bynumerous authors (Sogawa, 1973; Klingauf, 1973, 1987; Backus, 1988) withoutdetermination of the exact moment of stylet penetration in the plant. To ourknowledge, only Hardie et al. (1992) have studied the exact relation betweenAphid fabae Scopoli (Homoptera, Aphididae) behavior at the leaf surface andstylet penetration into the plant tissues. These authors showed that penetrationtimes were closely correlated with the cessation of antennation and body move-ment; nevertheless they did not distinguish between test probing and explorationprobing.

Antixenosis globally induces a greater number of behavioral items whateverthe behavioral phase: those implicating the mobilization of the sensorial organslocated on the antennae, the labium, and the legs as well as those involvinghead and abdomen movements. This results in more confused general behaviorof the mealybug on the FC (Fig. 3). Indeed, it is frequent that poorer adaptationto a situation leads to less regular and less stereotyped sequences (turning back,hesitations, probes, and numerous mistakes). On several occasions the followingobservations were made on insects under experimental conditions: choice ofdifferent hosts in parasitoids (van Baaren et al., 1993), and choice of plants witha different degree of resistance to aphids (Caillaud et al., 1995). The fact wasestablished by several classical ethologists (Tinbergen, 1953). During the firstphase, the antixenotic resistance induces more antennae vibrations and phyllo-plane rubbing by the legs. To the extent that repeated contacts increase theamount of sensory input to the central nervous system (Blaney and Duckett,1975), this behavior suggests that stimulating compounds are less abundant onFC than on Incoza and that the resistance expressed by the plant is of a chemicaltype at this phase of acceptance behavior. During test probing and exploratoryprobing, the greater number of sudden raisings of the rear end by pushing upon its hindlegs on the FC shows that the mealybug has more difficulties goingthrough epidermic layers and parenchyma, suggesting that the plant resistanceis probably of a rather mechanical type during this phase. A more difficult styletpathway in the plant tissues implies a longer duration of A, B, and C signals


as demonstrated by Montlor and Tjallingii (1989) in lettuce aphids. Since theC signal was not observed until the end, we cannot compare our results withthose of these authors. The difference in puncture duration pdc between theIncoza and the FC suggests that the resistance could also be expressed duringthe exploratory probing by the chemical characteristics of the plant. The shortestduration of the pdc on resistant plants was also demonstrated in P. manihoti byCalatayud et al. (1994).

Plant recognition and acceptance by a phytophagous insect are determinedeither by the presence of a mixture of different chemical compounds on the plantsurface, which are attractive and stimulating compounds or which are repellentand deterrent compounds, or by specific chemical compounds of the plant play-ing a determining role (Bernays and Chapman, 1994). Both behavior (this study)and sensory equipment (Le Ru et al., 1995a, b) show that the recognition andthe first acceptance phase at the plant by P. manihoti are determined by chemicalcharacteristics of the plant surface. Many Hemipteroidea with a restricted host-plant range are known to be stimulated to feed by specific host-associated chem-icals (Chapman, 1982). For instance, sinigrin in Brevicoryne brassicae L. (vanEmben, 1972), spartiene in Acyrthosipum spartei Koch (Smith, 1996), andphlorizine in Aphis pomi De Greer (Klingauf, 1971) stimulate probing. By anal-ogy, the number of contact chemical receptors being reduced on the antennaand the mealybug being specific to the genus Manihot, we can wonder if thesecondary substances of cassava that convey the main chemical informationabout plant quality are not implicated in that recognition. Besides, it was dem-onstrated that cyanogenic glycosides should be implicated in the plant selectionmechanisms by P. manihoti (Calatayud, 1993). In that context, it will be desir-able to screen the cassava leaf surface substances, in particular, the secondarysubstances, and then to determine how they vary with the antixenosis expressedby the cassava and, finally, to try to determine by electrophysiology if the contactchemical receptors of the antenna and labium are sensitive to those substances.During stylet probing, it is accepted in the phloem-feeding Homoptera such asP. manihoti that stylet penetration is probably determined by a combination ofphysical and chemical characteristics of the plant (Dreyer and Campbell, 1987).Calatayud et al. (1994) showed that the prephloemien interactions play a veryimportant role in plant acceptance by the cassava mealybug. These results sug-gest, among others, that phenolic acids and cyanogenic compounds could beimplicated in the insect fixation. Our study suggests that these chemical char-acteristics probably intervene particularly during the exploratory probing in rela-tion with intracellular punctures. During test probing, which correspondsessentially to the stylet passing through the epidermic layers of the plant, themealybug behavior showed a strong penetration, this penetration being moredifficult on the most antixenotic plant. The physical characteristics of its epi-


dermic layers certainly play a determining role. Therefore, an ultrastructuralstudy of its tissues and their modifications during the stylet progression onvarieties expressing different antixenosis levels should be conducted.

ACKNOWLEDGMENTS

This research was financed by the FRIA (Fond pour la formation a laRecherche dans l'Industrie et dans l'Agriculture). I thank Madam Bock for herhelp with the English translation.

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Documents

Recognition behavior of the cassava mealybug Phenacoccus manihoti Matile-Ferrero (Homoptera: Pseudococcidae) at the leaf surface of different host plants