Physiological Entomology (2009) 34, 98–102 DOI: 10.1111/j.1365-3032.2008.00653.x

S H O R T C O M M U N I C A T I O N

© 2008 The Authors98 Journal compilation © 2008 The Royal Entomological Society

Introduction

The sweetpotato whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), a pest species complex of increasing worldwide importance, comprises over 20 biotypes, including the highly polyphagous B biotype (syn. B. argentifolii Bellows & Perring) that has a vast range of over 500 host species ( Perring, 1995 ). The high degree of polyphagy, ingestion of phloem sap, and transmission of over 60 geminiviruses all contribute to their pest status ( Byrne et al. , 1990 ). Presently, the whitefly occurs heavily on greenhouse grown vegetables in China ( Hou et al. , 2007 ) and their management relies heavily on imidacloprid and other chemicals but B. tabaci from different regions in the world have already shown resist-ance to all major types of insecticides ( Cahill et al. , 1996 ).

Azadirachtin, a steroid-like tetranortriterpenoid derived from neem trees ( Azadirachta indica ), is a strong anti-feedant, deterrent and growth regulator for insects ( Mordue Luntz, 2004 ).

The fact that azadirachtin is selective toward phytophagous insects with minimal toxicity to beneficial insects increases its potential value to pest management ( Naumann & Isman, 1996; Mitchell et al. , 2004 ) and both foliar and systemic application of azadirachtin can provide effective control against whiteflies and other piercing insect pests ( Thoeming et al. , 2003; Kumar & Poehling, 2006; Wen et al. , 2008b ). Application of azadirachtin reduces landing and oviposition of whiteflies and other piercing insect pests on host plants ( Coudriet et al. , 1985; Nisbet et al. , 1993; Prabhaker et al. , 1999; Kumar et al. , 2005; Kumar & Poehling, 2007; Wen et al. , 2007, 2008a ) but little is known regarding the effects of azadirachtin on behaviour after landing on host plants, although the behavioural responses of Bemisia to plant surfaces are central to the effects of applied azadirachtin and the ability of the insect to cause economic damage.

Morphological and chemical characteristics of the leaf sur-face provide specific cues for host evaluation but the final acceptance of a host may come only after some probing ( Walker, 1987; Isaacs et al. , 1999; Freeman et al. , 2001 ). Topically or systemically applied azadirachtin may affect host-plant evaluation behaviour of whitefly at the leaf sur-face, during movement of the stylets, or after sieve element

Influence of foliar and systemically applied azadirachtin on host-plant evaluation behaviour of the sweetpotato whitefly, Bemisia tabaci

J I - H U I W E N 1 , K E - J I A N L I N 1 , M A O - L I N H O U 1 , W E I L U 1 and J I A - W E N L I 2 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of

Agricultural Sciences, Beijing, China and 2 College of Bio-safety Sciences and Technology, Hunan Agricultural University,

Changsha, China

Abstract . The behaviour of female adult Bemisia tabaci is observed for a period of 20 min after initial contact with untreated cucumber leaves, or leaves either foliar or systemically treated with azadirachtin, to determine whether application of aza-dirachtin affects the host-evaluation behaviour and whether the behaviour on treated leaves differs between application methods. Application of azadirachtin deters set-tling of the whiteflies on host plants. The whiteflies probe for shorter duration and less frequently but spend longer and engage more frequently in labial grooming on treated leaves than on untreated leaves. Behavioural transition between probing and other behavioural elements is less common, and that between labial grooming and other behavioural elements more common, on both treated leaves than on untreated leaves. No difference is detected in the host-evaluation behaviour of B. tabaci between leaves foliar treated and systemically treated with azadirachtin.

Key words . Azadirachtin , behaviour , Bemisia tabaci , host evaluation , whitefly .

Correspondence: Dr Mao-Lin Hou, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 Yuanmingyuan W. Road, Beijing 100193, China. Tel.: +86 10 6283 3985; fax: +86 10 6283 3985; e-mail: mlhou@ippcaas.cn

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puncture ( Jiang et al. , 1999; Freeman et al. , 2001 ). The present study aims to determine whether contact with aza-dirachtin affects the host-evaluation behaviour of B. tabaci and whether the behaviour on treated leaves differs between application methods.

Materials and methods

Insects

Colonies of B. tabaci B biotype were maintained on potted cucumber plants (c.v. Zhongnong 16) in a greenhouse under a LD 16 : 8 h photoperiod at 24 – 30 °C. The animals were reared for at least five generations before being used in the tests. Female whitefly adults aged less than 2-days old were used in the observations.

Plants and azadirachtin treatments

Cucumber plants were planted in 0.5-L pots with vermicu-lite and 2.14 g compound fertilizer (N : P 2 O 5 : K 2 O � 15 : 15 : 15%, Shandong Luxi Chemicals Inc., China) in insect-proof cages in the greenhouse. The second true leaf of a 2-week-old cucumber plant was used in all observations. On the observa-tion day, required leaves cut at the base of petioles were ran-domly assigned to one of the three treatments: untreated, foliar treated, or systemically treated with azadirachtin. To obtain untreated, foliar treated and systemically treated leaves, the petiole was inserted into a 20-mL glass vial con-taining either 5 mL of tap water or a 6 mg L −1 aqueous solu-tion of 0.3% azadirachtin EU (Yunnan Zhongke Biological Industrial Inc., China) (used as systemically treated leaves). The leaves were kept overnight in the greenhouse for approx-imately 14 h. Leaves with petiole inserted in the tap water were then either dipped into a 6 mg L −1 aqueous solution of 0.3% azadirachtin EU for 10 s and left to air dry (for approx-imately 2 h) (used as foliar treated leaves), or left untreated. Each leaf was used once for one individual whitefly and then discarded.

Influence of azadirachtin treatment on host-evaluation behaviour

A petiolate cuboid arena (43 × 28 × 23 mm) constructed, as previously described ( Walker, 1987 ), from transparent plastic was used to observe the behaviour of the female whitefly adults. The box was open on the bottom surface (43 × 28 mm) and the handle was 50 mm long. Immediately before observation, a leaf piece of the size of 43 × 28 mm was cut in the center of a leaf and placed onto a moist filter paper on a piece of plastic of similar size, with abaxial side upwards. After a female whitefly cooled for 1 min at −4 °C was placed gently onto the center surface of the leaf piece, the plastic piece was fastened with rubber onto the open side of the box. The insect was observed under cool light source through a microscope until it took the normal upright stance on the leaf surface, when observations began. The behav-ioural state ( Table 1 ) of a whitefly was observed continuously for approximately 20 min, and recorded on an MP3 (F500C; Aigo, China) player. Observations were made five times for each treatment in random order in a day, until a total of 67 replicates were obtained for each treatment.

The WAV files created by the MP3 player were played back on computer so that the sequence and duration of each behavioural element of each observation could be determined.

Statistical analysis

All frequency data were analysed using a chi-squared test. Transition matrices of whitefly behaviour were created for each treatment by transferring the observational data from columns of sequences into matrices of preceding and suc-ceeding behavioural elements. Logical zeroes were entered into the diagonal where one behaviour follows the same behaviour ( Fagen & Young, 1978 ). Thereafter, each first-order transition ( Slater, 1973 ) from one behaviour to another was analysed to determine those transitions that were signifi-cantly greater than expected by chance, using the chi-squared test. This was performed only for those transitions with a

Table 1. Ethogram of behavioural elements of Bemisia tabaci observed on cucumber leaves (adapted from Isaacs et al. , 1999 ).

Behavioural element Description

Stationary Standing motionless, with the labium retracted under the head Walk Walking across the leaf surface Labial dab Insect making rapid up and down head movements, with the labium pointing

down and its tip touching the leaf surface during head movements Probe Insect not moving across the leaf surface, with the labium tip stationary on

the leaf surface Groom Rapid movements of the legs across the body surface (other than labium)

and abdominal wax pads Labium groom Rapid movements of the legs across the labium Oviposition Movement on the leaf over a restricted area, with the abdomen arched

toward the leaf surface and the abdomen tip placed on the leaf surface

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frequency greater than 1% of the total number of transitions ( Isaacs et al. , 1999 ) and, to reduce the likelihood of type II errors caused by multiple comparisons, a critical P -value of 0.001 was employed ( Sokal & Rohlf, 1995 ). Comparisons of the transition frequencies between treatments were also made with a chi-squared test. To determine the influence of aza-dirachtin and its route of contact on behaviour of B. tabaci , the frequency of each main behavioural element was com-pared between treatments. Analysis of variance was used to examine the effect of treatment method on whitefly behaviour, where appropriate ( Sokal & Rohlf, 1995 ), followed by com-parison of means. Non-normally distributed data were analysed using the Kruskal – Wallis test.

Results and Discussion

Application of azadirachtin, either foliar or systemic, deters settling of the whiteflies on host plants. On untreated leaves, the whiteflies spent 1.7% of the total observation time off the leaves, whereas, for leaves either foliar or systemically treated with azadirachtin, they stayed significantly longer (13.6% and 11.6% of the total observation time, respectively) off the leaves ( � 2 � 16.4, d.f. � 2, P < 0.001) ( Table 2 ). The present data confirm earlier findings that azadirachtin has deterrent activities against whitefly ( Kumar et al. , 2005; Kumar & Poehling, 2007; Wen et al. , 2007 ).

The acceptance of a host plant by piercing insects depends on a series of behaviours, including, among others, the prob-ing of the labium tip on the leaf surface ( Freeman et al. , 2001 ). In the present study, probing dominated the time-budget of these insects in all the three treatments, comprising 62.3%, 44.4% and 52.5% of the whole duration on leaves untreated, foliar and systemically treated with azadirachtin, respectively ( Table 2 ). These data show the same pattern as that in a study by Isaacs et al. (1999) using the same species.

In the present study, both foliar and systemic treatments show significant effects on durations and frequencies of, and frequencies of transition from and to, probing and labial grooming. Probing of the whiteflies was significantly shortened ( � 2 � 40.4, d.f. � 2, P < 0.001), whereas labial grooming

was prolonged ( � 2 � 15.5, d.f. � 2, P < 0.001), on treated leaves ( Table 2 ). The whiteflies probed less frequently ( F 2,198 � 12.4, P < 0.001) and engaged more frequently in labial grooming ( � 2 � 10.5, d.f. � 2, P � 0.005) on both treated leaves than on untreated leaves ( Table 3 ). Frequencies of transition between probing and other behavioural elements were less common ( � 2 > 10.2, d.f. � 1, P < 0.001), and those between labial grooming and other behavioural ele-ments more common ( � 2 > 13.2, d.f. � 1, P < 0.001), on both treated leaves than on untreated leaves ( Table 4 ). In an investigation of the effects of imidacloprid on after-landing behaviours of the whiteflies on cotton leaves, Isaacs et al. (1999) report a 50% reduction in the total time spent probing on systemically treated leaves and a more than three-fold increase in labial grooming activity on foliar and systemi-cally treated leaves.

The specific reasons for the reduced duration and low fre-quency in probing behaviour of the whiteflies on azadirach-tin-treated leaves are not clear. During the course of labium tip contact with leaf surface and sieve elements, the white-flies have access to mechano- and chemosensory stimuli ( Hunter et al. , 1996 ). The present results may be due to the direct detection of adverse effects of azadirachtin on cucum-ber leaf tissue and the quality of phloem sap, or the toxic effect of ingested azadirachtin. Because B. tabaci has nine chemosensilla that project into the precibarial chamber ( Hunter et al. , 1996 ), ingested phloem sap may be sampled prior to passing into the gut. If a deterrent or anti-feedant compound, such as azadirachtin, is present in the plant flu-ids, its detection at this point may trigger termination of the ongoing probing and inhibition of further probing, thus resulting in reduced probing durations and less frequent probing. Interestingly, labial grooming is increased in the whiteflies on both foliar and systemically treated leaves in the present observation, which is contrary to the probing. The present data and those of Isaacs et al. (1999) may lend support to the direct detection hypothesis. However, further detailed study is required to determine the mechanisms of host chemistry detection in whiteflies. Furthermore, stylet movement through the tissues is not monitored in the present study; further electrophysiological studies should be

Table 2. Effects of azadirachtin treatment on the durations of behavioural elements of Bemisia tabaci .

Behavioural elements Untreated Foliar Systemic Treatment effect ( P )

Probing * 735.9 ± 20.7 a 468.2 ± 28.9 b 559.4 ± 32.0 b < 0.001 Grooming * 186.4 ± 13.3 a 197.2 ± 18.3 a 187.6 ± 17.0 a NS Stationary † 82.5 ± 11.8 a 147.9 ± 17.6 b 118.3 ± 14.3 ab 0.008 Walking † 99.0 ± 10.5 a 93.5 ± 9.5 a 89.0 ± 9.7 a NS Labial grooming * 15.1 ± 3.1 a 46.9 ± 8.2 b 37.8 ± 8.8 b < 0.001 Labial dabbing * 55.2 ± 5.1 a 80.6 ± 8.9 a 66.6 ± 9.6 a NS Oviposition * 7.9 ± 2.9 a 4.5 ± 2.2 a 2.9 ± 1.5 a NS Off leaf * 20.1 ± 7.0 a 160.9 ± 31.1 b 137.6 ± 27.8 b < 0.001

Overall treatment effects are calculated using analysis of variance for stationary and walking, and with the Kruskal – Wallis test for other behavioural ele-ments due to differences in variability between treatments. Values within a row followed by different superscript letters are significantly different from each other at the corrected P -value. Data are expressed as the mean ± SE. * Mann – Whitney comparison of means; critical P -value � 0.0167. † Dunn’s comparison between means; critical P -value � 0.0167. NS, not significant.

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performed to elucidate the relationship between stylet movement and azadirachtin treatment.

In the present study, behavioural elements other than prob-ing and labial grooming are not affected by the treatment of azadirachtin, and the behaviours do not differ between the whiteflies on foliar treated and systemically treated leaves within the 20-min assay ( Tables 2 – 4 ). However, Kumar & Poehling (2006) report more stable effects of systemic appli-cation of azadirachtin against the whiteflies compared with foliar application over a 7-day post-application period.

Considering the rapid dissipation of the active ingredients of azadirachtin, it is necessary to detect the persistence effects of azadirachtin on the behaviours of the whiteflies.

Bemisia tabaci is polyphagous and is exposed to a broad array of toxic plant secondary compounds during contact with plants, either on the leaf surface, during probing, or dur-ing phloem sap ingestion ( Byrne et al. , 1990 ). The ability to detect these compounds prior to acquiring a lethal dose as well as the employment of a behavioural mechanism to avoid further contact with these compounds would confer a clear

Table 3. Effects of azadirachtin treatment on frequency of behavioural elements of Bemisia tabaci on cucumber leaves.

Behavioural elements Untreated Foliar Systemic Treatment effect ( P )

Probing * 13.0 ± 0.4 a 9.7 ± 0.4 b 11.0 ± 0.6 b < 0.001 Grooming * 7.8 ± 0.4 a 7.5 ± 0.5 a 7.2 ± 0.5 a NS Stationary * 3.5 ± 0.3 a 4.7 ± 0.4 a 4.4 ± 0.4 a NS Walking * 6.9 ± 0.5 a 6.6 ± 0.6 a 6.9 ± 0.6 a NS Labial grooming † 1.7 ± 0.2 a 2.6 ± 0.3 b 2.9 ± 0.3 b 0.005 Labial dabbing * 7.1 ± 0.4 a 7.2 ± 0.5 a 7.5 ± 0.6 a NS Oviposition † 0.36 ± 0.11 a 0.18 ± 0.07 a 0.12 ± 0.06 a NS

Overall treatment effects are calculated using the Kruskal – Wallis test for labial grooming and oviposition, due to differences in variability between treatments, and with analysis of variance for other behavioural elements. Values within a row followed by different superscript letters are significantly different from each other at the corrected P -value. Data are expressed as the mean ± SE. * Dunn’s comparison between means; critical P -value � 0.0167. † Mann – Whitney comparison of means; critical P -value � 0.0167. NS, not significant.

Table 4. Frequencies of transitions between all observed behavioural elements for adult female Bemisia tabaci on cucumber leaves that were either untreated, or treated with azadirachtin as a foliar or systemic application.

Preceding behavioural element

Succeeding behavioural element

Stationary Walk Groom Labial groom Labial dabs Probe Oviposition

Untreated leaves Stationary — 40 58 16 47 64 4 Walk 40 — 57 12 195 135 1 Groom 64 73 — 30 71 274 2 Labial groom 13 16 31 — 22 31 0 Labial dabs 12 63 37 16 — 337 3 Probe 49 255 330 40 134 — 14 Oviposition 15 2 1 0 3 2 — Foliar treated leaves Stationary — 53 89 19 59 73 6 Walk 47 — 81 20 157 96 1 Groom 105 92 — 40 88 154 2 Labial groom 36 18 32 — 38 51 0 Labial dabs 32 64 76 33 — 261 0 Probe 37 189 208 64 120 — 3 Oviposition 7 2 1 0 1 1 — Systemically treated leaves Stationary — 58 91 22 37 67 0 Walk 61 — 53 15 226 106 1 Groom 78 113 — 37 70 166 0 Labial groom 35 21 39 — 36 45 0 Labial dabs 30 74 51 35 — 320 0 Probe 38 218 226 71 137 — 8 Oviposition 1 2 2 0 5 0 —

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adaptive advantage. The present study reveals distinct behav-ioural modifications in response to azadirachtin treatment, characterized by the inhibition of probing and an increase in labial grooming.

Acknowledgements

This study was financially supported by MOST Research Program for Public Good (2004DIB4J156) and a grant from MOA (2008326005), P.R. China.

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Accepted 2 September 2008 First published online 27 October 2008