Calatayud, Paul-André

Nardon, C.

Rahbé, Y.

P. A. Calatayud

C. Nardon

Y. Rahbé

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Entomologia Experimentalis et Applicata

A new technique to immobilize an aphid or a mealybug on plants using a high‐frequency microcautery unit

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1- . . L 
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Eiitomol(igi,6‘ici Experi/rieiitnlis et Applictrttr SO: 239-241, 1996. 
@ 1996 Klicwer Auideitiic Publishers. Printed iii Belgiiini. 
239 
A new technique to immobilize an aphid or a mealybug on plants using a 
high-frequency microcautery unit 
P. A,. Calatayud’, C. Nardon2 & Y. Rahbé2 ’ ORSTOM, Laboratoire d’Entomologie, Parc ScientiJque Agropolis II, 34397 Montpellier Cedex 5, Frame 
21NRA-INSA, Laboratoire de Biologie Appliquée 406, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, 
France 
Accepted: November 7, 1995 
Key words: Macrosiplzuni albifrons, Phenacoccus manihoti, electrical penetration graphs, stylet pathways, stylec- 
tomy, phloem sap 
Introduction 
Light microscopy is the oldest tool used to describe 
stylet pathways of homopterous insects within their 
host plant. Since 1960, electrical recording of pene- 
tration behaviour by aphids has been introduced by 
McLean & Kinsey (1 967) and modified from an AC 
to a DC device called the electrical penetration graph 
technique (EPG) by Tjallingii (1978). EPG, especially 
when combined with microscopy, is unique in describ- 
ing the details of penetration activity, and in studying 
the feeding behaviour of phloem- or xylem-feeding 
insects, 
Difficulties in linking wave-forms obtained by EPG 
and localisation of the stylets in plant tissues by 
microscopy are mainly due to problems in immobi- 
lizing the insect. We describe a simple technique with 
a modified use of a high-frequency (HF) microcautery 
unit that seems useful both for stylet immobility and, 
in some cases, for phloem sap collection. 
Materials and methods 
AS in EPG experiments, a gold wire (2-3 cm, 
017.5 pm) was fixed on the dorsum of the insect with a 
water-based silver paint. Then, the gold wire was glued 
to a copper wire which was plugged into the amplifier 
‘probe’ of the EPG-device (for an eventual recording of 
an EPG trace), or was directly connected to the ‘injec- 
tion needle’ of the microcautery ‘probe’ (connected to 
the HF microcautery unit (Syntech), see Figure la). 
After a few hours (time necessary for reaching and 
feeding on phloem, especially for the mealybug) the 
HF pulse was completed. 
We applied this technique to Macrosiphum alb- 
ifvons (Aphididae) on lupine Lupirius albus cv. Lublanc 
(Fabaceae) and Phenacoccus manihoti (Pseudococci- 
dae) on cassava Manihot esculenta CY. Fetonegbodgi 
(Euphorbiaceae). 
Light microscopy was used to reveal alterations in 
tissue after administering the HF pulse. In the case of 
stylet amputation after the HF pulse, exudates were 
collected for chemical analysis (with a microcapil- 
lary), and volumes were estimated by weighing (1 pg  
accuracy). As for previous stylectomy analyses (Rahbé 
et al., 1990), sugars were identified and measured by 
a gas chromatographic method, and amino acids were 
assayed by ion-exchange and ninhydrin detection. 
Results and discussion 
With an appropriate power adjustment, depending on 
the insect species (i.e. 15 W and 12 W during 0.6 s 
for M. albifrons and I! manihoti respectively), the HF 
pulse caused the death of the insect and resulted in the 
complete immobility of the stylets. Light microscopy 
observations revealed no visible change in plant tissues 
following the electrical pulse but transmission electron 
microscopy was not carried out to confirm this phe- 
nomenon on a smaller scale. An interesting aspect of 
this procedure is the very simple wiring system, allow- 
ing recording of an EPG trace, and shortly after the 
immobilization of the stylets for microscopy. It should 
be easier to use than the standard stylectomy general- 
-- 
240 
a 
Insect with gold wire 
fixed on the dorsum 
I 
EPG amplifier 
HF microcautery unit 
"Injection needle" 
HF pulse results 
b C 
Labium# 
Slyleis Exudate 
Leaf Leaì 
Figure I. Schematic setup used for EPG and subsequent HF microcautery pulse. Insect wiring (a) and HF pulse results with Mucrosiphum 
ulb@rms feeding on lupine: paaid stylet amputation (b) or total stylet amputation (c). 
ly employed for EPGhistology correlation (Tjallingii 
& Hogen Esch, 1993), especially for insects where 
stylectomy is extremely difficult due to their anatomy. 
Interestingly and surprisingly, this technique 
allowed the exudation of phloem sap through the labi- 
umlstylet stump of the aphid (Figures l b  and IC): 26 
'shots' were performed on M. albifrons adults feeding 
on lupine leaves, resulting in a success rate of exudation 
of 69%, which is a better result than for standard stylec- 
tomy (i.e. 44% (Rahbé et al., 1990)). The mean sap 
volume collected was 91 nl, less than in the standard 
technique (121 nl on leaves), with a mean flow rate of 
7 nl min-'. Analysis of sugars and amino acids showed 
that the exudate obtained was pure phloem sap (mean 
values: 1890 mh4 of sucrose and 338 mM of amino 
acids; no fructoselglucose contamination detected, and 
high amide content, both reflecting the authenticity of 
phloem sap sampling). According to standard analysis 
for lupine (Pate et al., 1979), asparagine was the main 
N-transporter (50%, Figure 2). The continuous exu- 
dation of phloem sap obtained after electrical shock 
confirmed the maintenance of the phloem's integrity, 
as observed by light microscopy. Nevertheless, precau- 
tions should be taken: the stylet amputation shown in 
Figure l b  was interpreted as a partial disruption of the 
mouth parts of the insect. Analyses of sugars revealed 
trehalose traces in such exudates, indicating contam- 
ination by haemolymph. In contrast, stylet amputa- 
tions as shown in Figure IC, resulted in uncontaminat- 
ed samples (no trehalose detected). Usually, phloem 
exudation occurred as presented in Figures l b  and IC. 
Nevertheless, we observed two cases where only the 
stylets were amputated, and where the labium was sev- 
ered but still connected to the rest of the dead aphid. 
In these cases, as in standard stylectomy, the HF pulse 
resulted in the ejection of the aphid, leaving only the 
% 
6o 1 
50 - 
do - 
30 - 
t o  - 
10 - 
0 -  
'-\SF Thr Ser Asn Glu Gln Pro Ala Vnl Ileu Leu Tyr Phe Lys Hist 
Figure 2. Typical amino acid profile of exudates obtained after HF 
pulse on lupine's aphid. 
stylet stump, and no contamination with haemolymph 
occurred. These results led us to adapt the HF cautery 
device for better stylet amputations by changing HF 
frequency and power. 
In contrast to our results with M. albifrons, 
stylectomy was never observed with our target 
insect Pheriacoccus manihoti. Whereas immobiliza- 
tion occurred invariably, stylet amputation seemed to 
be dependent on the insect species, and may not have 
resulted only from the electrical shock. In M, albifons, 
stylet amputation took place relatively close to the leaf 
surface, and the insect twitched, excreting at the same 
time honeydew. Therefore, a physical reaction coup- 
led with the HF pulse, involving some kind of vio- 
24 1 
lent reflex movement of the aphid head, may result 
in  stylectomy. It is surprising that phloem flow was 
not prevented, indicating that no overheating and no 
withdrawal movement occurred. The observed species- 
specific differences in response may be traced to dif- 
ferences in anatomy and behavioural reactions of the 
insects to the shock. 
I 
Acknowledgments 
We thank B. Boher for his help with the histological 
techniques used for the mealybug, and J. Guillaud and 
P. Jourand for their technical assistance. S. Manguin is 
thanked for her English corrections. 
References 
McLean, D. L. & M. G. Kinsey, 1967. Probing behaviour of pea 
aphid. 1. Annals of the Entomological Society of America 60: 
400-406. 
Pate, J. S., C. A. Atkins, D. L. Mc Neil & D. B. Layzell, 1979. 
Transport of organic solutes in phloem and xylem of a nodulated 
Legume. Plant Physiology 63: 1082-1088. 
Rahbé, Y., B. Delobel, P. A. Calatayud & G. Febvay, 1990. Phloem 
sap composition of lupine by aphid stylectomy: Methodology, 
variation in major constituants and detection of minor solutes. In: 
D. C. Peters, J. A. Webster & C. S. Chlouber (eds), Proceedings of 
Aphid-Plant Interactions: Populations to Molecules. Oklahoma 
State University, Stillwater, Oklahoma, p 307. 
Tjallingii, W. E, 1978. Electronic recording of penetration behaviour 
by aphids. Entomologia Experimentalis et Applicata 24: 721- 
730. 
Tjallingii, W. E & Th. Hogen Esch, 1993. Fine structure of aphid 
stylet routes in plant tissues in correlation with EPG signals. 
Physiological Entomology 18: 317-328. 



A new technique to immobilize an aphid or a mealybug on plants using a high-frequency microcautery unit

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A new technique to immobilize an aphid or a mealybug on plants using a high-frequency microcautery unit

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