Cuticular extracts inducing aggregation in the German cockroach, Blattella germanica (L.)

Journal of Insect Physiology 44 (1998) 909–918

Cuticular extracts inducing aggregation in the German cockroach, Blattella germanica (L.) C. Rivault b

a,*

, A. Cloarec a, L. Sreng

b

a CNRS UMR 6552, Universite´ de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France CNRS UPR 9024, LNB, Laboratoire de Neurobiologie, Communication Chimique, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

Received 17 November 1997; received in revised form 3 March 1998; accepted 31 March 1998

Abstract German cockroaches Blattella germanica (L.) are gregarious insects. An aggregation pheromone contributes to the maintenance of aggregates. Choice experiments checked the efficiency of different solvents, i.e. dichloromethane, methanol and pentane, in extracting attractive substances and compared the attractiveness of extracts of different parts of the body. Dichloromethane and pentane were the most efficient solvents tested for extracting the attractive substances. Methanol whole body extracts appeared inefficient to induce aggregation. The proportion of larvae attracted to conditioned papers decreased in relation to the size of cuticular surface washed, from whole body to half-body and again to a section of the body cut in three. Attractive substances appear to be present on all parts of the body. Chemical analysis by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) showed that the active extracts contained only cuticular hydrocarbons. In addition to behavioural tests, differences between the composition of methanol extracts and that of the extracts for the other two solvents were revealed by GC. These results indicated that the cuticular hydrocarbons operate as an aggregation pheromone in Blattella germanica.  1998 Elsevier Science Ltd. All rights reserved. Keywords: Cuticular extracts; Aggregation pheromone; German cockroach

1. Introduction Aggregation pheromones act as a recognition signal allowing proximity tolerance between individuals of the same group (Jaffe, 1987). Cockroaches have proved to be good biological material for studies on gregariousness. Aggregate cohesion based on interindividual attraction in Blattella germanica (L.) (Dictyoptera: Blattellidae) was reported for the first time by Ledoux (1945). Contrary to long-distance attraction by sexual pheromones, cockroach social attraction based on olfaction occurs only at short distances and the stimulus is perceived by the antennae. Choice tests with groups of Blattella germanica larvae showed that these larvae were attracted onto papers conditioned by the odour of their conspecifics. Our behavioural data confirmed that the attractive odour was produced and perceived by larvae

* Corresponding author. Fax: 33-(0)2-99-28-69-27; E-mail: [email protected] 0022–1910/98/$19.00  1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 6 2 - 6

at all developmental stages. This attractive odour was an aggregation pheromone and strains from different locations present variations of the specific odour which are detected by the larvae (Rivault and Cloarec, 1998). There are many discrepancies in the literature concerning the chemical nature of Blattella aggregation pheromone and its origin. Ishii and Kuwahara (1967, 1968) showed that cockroach faeces contain an attractive substance produced by rectal pad cells and that it is also adsorbed on to the body surface. They considered this substance to be an aggregation pheromone. Attempts by Ritter and Persoons (1975) to isolate the aggregation pheromone from faeces indicated that the mixture of active factors included free fatty acids. MacFarlane and Alli (1986) identified lactic acid in extracts of filter paper conditioned by Blattella germanica larvae as the attractive factor. Sakuma and Fukami (1990, 1991) identified alkylamines as the aggregation pheromone and obtained maximum arrestant activity with extracts of the posterior end of the abdomen. They isolated two compounds labelled blattellastanoside A and B which have steroid skel-

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etons and chlorine atoms and are secreted by the centre part of the supra-anal plate surrounding the anus (Sakuma and Fukami, 1993). However, no exocrine glandular system capable of secreting these substances has yet been found. These discrepancies between results concerning attractive substances considered to act as aggregation pheromones in Blattella germanica led us to reconsider the problem of the chemical identity of the aggregation pheromone and to try to localize the possible site(s) of secretion of this active substance(s). Behavioural tests (Rivault and Cloarec, 1998) were performed to check the efficiency of different solvents in extracting attractive substances and to compare the attractiveness of extracts of different parts of the body of Blattella germanica (L.) larvae in choice tests.

2. Material and methods 2.1. Behavioural tests Our experiments were conducted with Blattella germanica. Our test situation was similar to that described by Ishii and Kuwahara (1967). A group of 20 first instar larvae, placed in a Petri dish (140 mm in diameter and 20 mm high), was presented a choice between two filter papers (60 × 15 mm, folded in four to form a ‘W’). The choice was always between a control paper and a conditioned paper. Tests lasted 24 h and were carried out under an L : D 12 : 12 light–dark cycle at 25°C. Tests were prepared during the rest phase which occurs during the light period of the cockroach photoperiod. Larvae aggregate when resting, therefore 24 h later, during the following rest period and after one active period during the dark phase allowing for exploration of the dish, the positions of the larvae in the Petri dish were recorded. During a test, larvae were deprived of food and water. 2.1.1. Test larvae Larvae present an activity peak during the middle of an instar (Dabouineau and Rivault, 1988). As the first instar lasts 7 days, only 4-day-old larvae were tested. Until they were required for testing, groups of larvae having hatched the same day were kept in boxes with food and water ad lib. 2.1.2. Extraction of the attractive substances Three different solvents were used: dichloromethane, methanol and pentane (all purified for trace analyses). Extractions were made either over short (2 min) or long (60 min) periods. Attractive substances were extracted by dipping all or part of the body of 15 sixth instar larvae into 1.5 ml of

the different solvents. Seven different types of extracts were prepared. 1. whole bodies. Two other types of extracts were obtained by dipping half the body into the solvent: 2. anterior portion of the body, including head and thorax; 3. posterior portion of the body, comprising all the abdomen. Finally, bodies were cut simultaneously into three parts: 4. heads, cut off at the neck with scissors; 5. final abdominal segments, cut off between segments 6 and 7 with scissors; 6. remaining parts of the bodies (without head and final abdominal segments used in (4) and (5). 7. To increase concentration of active components in extracts, 50 sixth instar larvae were used in 4 ml of solvent. 2.1.3. Filter papers Conditioned papers were prepared, corresponding to the six different types of extracts. When an extraction was finished, conditioned papers, at a ratio of 10 papers for 1.5 ml of extract, were impregnated and left until the solvent had completely evaporated. These filter papers, which were the conditioned papers, were used the day they were prepared. Control papers were prepared by impregnating 10 clean papers with 1.5 ml of pure solvent and left until the solvent had completely evaporated. 2.1.4. Behavioural test analysis There were three possible positions for the larvae in the dish: on the control paper, on conditioned paper or elsewhere in the dish. We considered that larvae that did not go onto the papers exhibited an interesting behavioural response which was important to take into account because filter papers were a more attractive substrate than the plastic dish (Rivault and Cloarec, 1998). For each series of experiments, we calculated the mean number of animals per test observed in each of the three different positions. 2.2. Chemical analysis 2.2.1. Gas chromatography (GC) For each extract, 15 sixth instar larvae were anaesthetized with CO2 and immersed in 1.5 ml of pentane, of methanol or of dichloromethane for 2 min, then the insects were removed from solvents. Samples were concentrated under nitrogen flow (50 ␮l) prior to the injection. Samples of 2 ␮l were analyzed by gas chromatography (GC) on a Delsi 200 DN coupled to an Enica 31 integrator and equipped with a flame ionization detector (FID) and a CPsil 5 column (25 m × 0.25 mm ID) with

C. Rivault et al. / Journal of Insect Physiology 44 (1998) 909–918

a split–splitless injector. The temperature programme started at 90°C for 3 min, then increased gradually to 230°C at 15°C/min and then to 320°C at 5°C/min (final time 10 min). The carrier gas was helium. 2.2.2. Gas chromatography–mass spectrometry (GC– MS) analysis GC–MS analysis was carried out using a Hewlett Packard 5890 gas chromatograph coupled to a 5989 A high-resolution mass spectrometer. The whole analysis system was controlled by an HPUX MS Chemstation. The analyses were first performed in an electronic impact mode (EI; 70 eV). Positive chemical ionization (CI; 150 eV/CH4+) was used over the mass range m/z 100 to m/z 650. During chemical ionization, methane was used at a pressure of 1.5 bars. The gas chromatograph, containing a CPsil 5 column (25 m × 0.25 mm ID), was programmed from 100°C for 3 min, then programmed to 230°C at 15°C/min and then to 320°C at 5°C/min (final time 10 min). 2.2.3. Silica gel microcolumn purification procedure In order to verify the attractiveness of cuticular hydrocarbons in the whole body extracts, a purification procedure was used. To separate cuticular hydrocarbons from other components in the different extracts, we used a silica gel microcolumn procedure. Extracts from either 15 or 50 sixth instar larvae in 4 ml of the different solvents were concentrated under nitrogen flow into a small volume (about 0.2 ml). Samples were purified by chromatography on a 4 cm microcolumn in a Pasteur pipette (0.5 × 7 cm) packed with 0.5 g of silica gel (silice chromagel, 230–400 mesh, SDS, France), activated overnight at 120°C in a dry oven, and eluted with 2 ml of pentane (or dichloromethane). Purified samples were used for behavioural tests as described above. Samples from each extract were compared with respect to GC pattern before and after passing through a silica gel microcolumn.

3. Results These experiments aimed, first, to find the most efficient solvent for extracting the substances which induce cockroaches to aggregate, and second, to localize the production site(s) of the aggregation pheromone and then to identify it. 3.1. Efficiency of solvents in extracting the attractive substances Extracts of the whole body were obtained with three solvents, dichloromethane, methanol and pentane, at two different extraction durations (Table 1). Dichloromethane. The results of choice tests showed that larvae were significantly attracted to conditioned

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papers when dichloromethane was used for extraction (ttest, p ⬍ 0.001). Very few larvae were attracted to control papers or remained in the dish. Extractions lasted either 2 or 60 min. The fact that the cockroach bodies remained for longer (60 min) in the solvent did not increase significantly the proportion of larvae attracted to conditioned papers (t-test, p > 0.05). Pentane. Larvae were significantly attracted to conditioned papers when pentane was used for extraction (ttest, p ⬍ 0.001). Longer pentane extractions increased the attractiveness of conditioned papers (t-test, p ⬍ 0.001). Methanol. Larvae were not significantly attracted to papers conditioned with methanol extracts (t-test, p > 0.05). Methanol whole body extracts, compared to extracts with dichloromethane or pentane, appeared inefficient to extract substances inducing aggregation behaviour. Increased extraction duration increased the attractiveness of methanol extracts even though their level of attractiveness remained non-significant as many larvae did not settle on either paper. The attractiveness of methanol extracts remained significantly lower than that of dichloromethane- or pentane-conditioned papers. Two solvents, dichloromethane and pentane, were efficient in extracting active substances able to induce aggregation behaviour. Methanol was not efficient and was therefore discarded from further experiments. Pentane is an apolar solvent which extracts cuticular hydrocarbons; dichloromethane extracts polar molecules and some apolar molecules including cuticular hydrocarbons; methanol extracts polar molecules. Our results showed that the two solvents that extract apolar molecules like cuticular hydrocarbons are the most efficient for extracting the attractive substances. 3.2. Extract efficiency of different body portions As whole body dichloromethane and pentane extracts were attractive, we repeated choice tests with papers conditioned with extracts of either the anterior or the posterior part of the body in order to localize the emission of the attractive substances (Table 2). Conditioned papers were always more attractive than control papers whatever the extracting solvent (t-test, p ⬍ 0.001). Therefore both the anterior and the posterior parts of the body secrete the attractive substances. Extracts of the anterior part of the body were not significantly more attractive than those of the posterior part (t-test, p > 0.05) for both extracting solvents. However, when dichloromethane was used, these papers were not as attractive as papers conditioned with whole body extracts (t-test, p ⬍ 0.001). When pentane was used, the same tendency was observed: 60 min whole body extracts were significantly more attractive than part extracts (t-test, p ⬍ 0.001). Extracts of the anterior and posterior parts of the body

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Table 1 Whole body extracts Solvent Methanol

Dichloromethane

Pentane

Extraction conditions

Control paper

Conditioned paper

Dish

60 min 15 L6

Mean S.E. N

3.76 1.03 33

7.48 1.44 33

8.76 1.47 33

2 min 15 L6

Mean S.E. N

3.49 1.19 39

1.44 0.75 39

15.10 1.32 39

Silica 15 L6

Mean S.E. N

1.5 1.5 10

4.2 2.64 10

14.3 2.79 10

60 min 15 L6

Mean S.E. N

0.10 0.09 10

15.10 2.48 10

4.80 2.49 10

2 min 15 L6

Mean S.E. N

0.34 0.24 29

15.45 1.16 29

4.21 1.11 29

60 min 50 L6

Mean S.E. N

0.5 0.33 20

19.45 0.34 20

0.05 0.05 20

Silica 15 L6

Mean S.E. N

2.2 1.23 20

6.7 2.02 20

11.1 2.21 20

Silica 50 L6

Mean S.E. N

3.53 1.33 30

2.4 1.11 30

18.03 4.23 30

60 min 15 L6

Mean S.E. N

0 0 10

18.5 1.32 10

1.5 1.32 10

2 min 15 L6

Mean S.E. N

2.15 1.09 20

13.4 1.72 20

4.3 1.56 20

60 min 50 L6

Mean S.E. N

0 0 10

17.9 1.99 10

2.1 1.99 10

Silica 15 L6

Mean S.E. N

1.77 0.84 30

2.7 1.16 30

15.53 1.40 30

Silica 50 L6

Mean S.E. N

2.4 1.22 20

11.45 1.89 20

6.15 1.8 20

Results of choice tests expressed as mean number ( ± S.E.) of larvae on control papers, conditioned papers and in dish for extracts of whole bodies. N: number of tests; L6: sixth instar larvae. Extracts using three different solvents (pentane, dichloromethane and methanol), at two different extraction durations (2 min and 60 min) and two different concentrations (15 or 50 L6) were tested. In addition, papers were conditioned either directly with solvent extracts or with extracts after a silica gel procedure (Silica).

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Table 2 Half-body extracts Solvent Dichloromethane

Pentane

Control paper

Conditioned paper

Dish

Anterior part

Mean S.E. N

1.68 0.81 40

8.95 1.47 40

9.38 1.49 40

Posterior part

Mean S.E. N

2.65 0.92 40

10.98 1.35 40

6.45 1.29 40

Anterior part

Mean S.E. N

3.55 1.06 40

12.22 1.39 40

4.22 1.19 40

Posterior part

Mean S.E N

5.37 1.28 40

11.87 1.41 40

2.75 1.03 40

Results of choice tests expressed as mean number ( ± S.E.) of larvae on control papers, conditioned papers and in dish for extracts of anterior or posterior half of the body. N: number of tests. Two different solvents were used. Extractions lasted 2 min.

attracted similar proportions of larvae. This could mean that the emission of the attractive substances is not limited to one of these parts. These results indicate that the glandular systems of the head or the abdominal extremity were not involved in the secretion of the attractive substances. In order to localize more precisely the site of production of the attractive substances, we prepared conditioned papers with three separate parts of the body (head, posterior abdominal segments and rest of body) (Table 3). Papers conditioned with one of these three parts were not significantly attractive. The proportions of larvae on the control paper and remaining in the dish were greater than on the conditioned paper (t-test, p > 0.05 in all cases). Extracts of one part were not significantly more attractive than extracts of either of the other parts (t-test, p > 0.05), and this was true for extracts with either of the two solvents. Papers conditioned with one third of the body were significantly less attractive than the conditioned papers in the previous experiments (ttest, p > 0.001); attractive substances appeared to be present on all three parts of the body although at levels too low to attract the larvae significantly. With the same number of sixth instar larvae, the attractiveness of conditioned papers increased when the body surface used to prepare the extracts increased. The proportion of larvae attracted to conditioned papers increased in relation to the size of cuticular surface washed, from third-body to half-body and again to whole body extracts (Tables 1 and 2). Papers with whole body extracts were as attractive as pheromone-conditioned papers used in a previous study (t-test, p > 0.05) (Rivault and Cloarec, 1998). These results showed that the attractive substance was

not concentrated on a particular part of the body but rather spread over the whole body surface. These results lead to the conclusion that secretion of aggregation pheromone is not due to any particular glandular system located in the head or in the abdominal tip. 3.3. Gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) analyses Whole body extracts of 15 Blattella germanica larvae were analysed by GC and GC–MS. All the components of these extracts were identified as cuticular hydrocarbons and the extracts contained only cuticular hydrocarbons (Table 4). The hydrocarbons we identified were similar to those previously reported for Blattella germanica (Augustynowicz et al., 1987; Carlson and Brenner, 1988; Jurenka et al., 1989). GC profiles for pentane and dichloromethane extracts gave identical results. The methanol extracts chromatogram differed from the two previous chromatograms. Comparisons between gas chromatograms of pentane (or dichloromethane) and methanol extracts (Fig. 1) show that some hydrocarbons such as n-heptacosane (1), n-octacosane (7) and n-nonacosane (12) have almost disappeared, 5-methylheptacosane (3), 3-methylheptacosane (5), 5-methylnonacosane (15) and 3-methylnonacosane (17) are extracted in low proportions with methanol whereas other compounds like 9,11- and 13-methylheptacosane (2), 5,9- and 5,11dimethylheptacosane (6) and 11,15- and 13,17-dimethylnonacosane (16) are extracted in high proportions with this solvent. In addition to differences revealed by GC between the composition of methanol extracts and that of the extracts

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Table 3 Body sections Solvent

Extraction duration

Dichloromethane

2 min

Pentane

Dichloromethane

Pentane

2 min

60 min

60 min

Control paper

Conditioned paper

Dish

Head

Mean S.E. N

1.17 0.47 30

6.03 1.37 30

12.80 1.40 30

Abdominal extremity

Mean

2.04

7.57

10.39

S.E. N

1.09 28

1.68 28

1.72 28

Rest of body

Mean S.E. N

1.81 0.63 31

7.90 1.43 31

10.29 1.43 31

Head

Mean S.E. N

3.4 1.35 20

7.95 1.71 20

8.65 1.90 20

Abdominal extremity

Mean

3.21

8.84

7.94

S.E. N

1.34 19

1.87 19

1.85 19

Rest of body

Mean S.E. N

3.7 1.34 20

5.75 1.58 20

10.55 1.93 20

Head

Mean S.E. N

2.00 1.90 10

5.90 2.85 10

12.10 3.06 10

Abdominal extremity

Mean

3.60

5.90

10.50

S.E. N

1.55 10

2.30 10

2.29 10

Rest of body

Mean S.E. N

1.00 0.75 10

3.50 2.12 10

15.50 2.15 10

Head

Mean S.E. N

11.00 2.85 10

8.10 2.96 10

1.00 0.65 10

Abdominal extremity

Mean

6.00

9.50

4.40

S.E. N

2.54 10

2.74 10

2.40 10

Mean S.E. N

12.5 2.85 10

5.30 2.48 10

2.2 1.88 10

Rest of body

Results of choice tests expressed as mean number ( ± S.E.) of larvae on control papers, conditioned papers and in dish for extracts of three different body sections. N: number of tests. Two different solvents (dichloromethane and pentane) and two different extraction durations (2 min and 60 min) were used.

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Table 4 Cuticular hydrocarbons of Blattella germanica Peak

Compounds—Fragments 1 2

3 4 5 6

7 8

9

10 11 12 13

14 15 16

17 18

19

20

21

n-Heptacosane 9,11- and 13Methylheptacosane 140/141, 280/281, 168/169, 252/253,196/197, 224/225 5-Methylheptacosane 308, 337 11,15-Dimethyheptacosane 168/169, 196/197, 239, 267 3-Methylheptacosane 365 5,9- and 5,11Dimethyheptacosane 155, 280/281, 351, 379, 183, 252/253, 351, 379 n-Octacosane 3,11- and 3,9Dimethyheptacosane 155, 280/281, 379, 183, 252/253, 379 12- and 14Methyloctacosane 182/183, 252/253, 210/211, 224/225 2-Methyloctacosane 365 4-Methyloctacosane 365, 336/337 n-Nonacosane 9,11-, 13- and 15Methylnonacosane 140/141, 308/309, 168/169, 280/281, 196/197, 252/253 7-Methylnonacosane 112/113, 336/337, 407 5-Methylnonacosane 336, 337, 365 11,15- and 13,17Dimethylnonacosane 168/169, 224/225, 239, 295, 196/197, 267 3-Methylnonacosane 364, 365, 393 5,9- and 5,11Dimethylnonacosane 155, 308/309, 351, 379, 183, 280/281, 351, 379, 211, 252/253, 351, 379 3,7-, 3,9- and 3,11Dimethylnonacosane 127, 336/337, 379, 407, 155, 308/309, 379, 407, 183, 280/281, 379, 407 11-, 13- and 15Methyltriacontane 168/169, 294/295, 196/197, 266/267, 224/225, 238/239 4,8- and 4,10Dimethyltriacontane 141, 169, 308/309, 336/337

MW

TC

CI (CH4)

380 394

27 28

379 393

394

28

393

408

29

407

394

28

393

408

29

407

394 408

28 29

393 407

408

29

407

408 408

29 29

407 407

408 422

29 30

407 421

422

30

421

422

30

421

436

31

435

422

30

421

436

31

435

436

31

435

436

31

435

450

32

449

[continued overleaf

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Table 4 Continued] Cuticular hydrocarbons of Blattella germanica 22

23

24

25

11-, 13- and 15Methylhentriacontane 168/169, 308/309, 196/197, 280/281, 224/225, 252/253 13,17- and 11,15Dimethylhentriacontane 168/169, 239, 252/253, 323, 196/197, 224, 267, 295 5,9- and 5,11Dimethylhentriacontane 84/85, 155, 336/337, 407, 435, 183, 308/309, 407, 435 10 and 12Methyldotriacontane 154/155, 182/183, 308/309, 336/337

450

32

449

464

33

463

464

33

463

464

33

463

MW: molecular weight; TC: total carbon; CI(CH4): chemical ionization.

for the other two solvents, the behavioural tests revealed a lack of attractiveness of methanol extracts (Table 1). 3.4. Extract efficiency of cuticular hydrocarbons A silica gel microcolumn procedure was applied to obtain purified cuticular hydrocarbons from our extracts. GC profiles of extracts before and after passing through a silica gel microcolumn gave the same peak patterns (Fig. 1). No qualitative loss was detected. Conditioned papers with silica gel-purified extracts obtained with only 15 larvae were not attractive in behavioural tests (Table 1). The great majority of test larvae remained in the dish and avoided both control and conditioned papers whatever the solvent. Compared to results presented above (Table 1) conditioned papers had completely lost their attractiveness. The presence of solvent traces on papers induced avoidance. As the concentration of active components in the extracts after the silica gel microcolumn procedure was reduced, these behavioural tests were repeated with extracts obtained with 50 larvae. In this case, papers conditioned with purified pentane extracts obtained with 50 larvae were attractive (Table 1), although they were slightly less attractive than non-purified extracts. This supports our hypothesis of a loss of concentration during the silica gel procedure. However, papers conditioned with purified dichloromethane extracts obtained with 50 larvae were still not attractive, although they had the same chromatogram profiles. This appears to be simply a quantitative problem. These results allow us to conclude that pure cuticular hydrocarbon extracts induced aggregation in behavioural tests.

4. Discussion Results of behavioural tests showed that extracts in pentane or dichloromethane contained an attractive substance whereas it did not occur in methanol extracts. This attractive substance can be identified as the aggregation pheromone. Aggregation behaviour induced by papers impregnated with whole body pentane or dichloromethane solvent extracts was similar to that induced by papers conditioned by body contact (Rivault and Cloarec, 1998) (t-test, p > 0.05 in all cases). Our results show clearly that the attractive substance was not concentrated on a particular part of the body but rather spread over the whole body surface. The amounts of attractive substance obtained following solvent extractions were proportional to the body surface extracted. In ants, the analysis of recognition, estimated by aggressive responses instead of attraction, showed that similar levels of aggressive responses were induced by the whole body and by different parts of the body of foreign workers introduced into a colony. This indicated that sufficient amounts of the recognition odour were not carried by a single part of the body in ants (Bonavita-Cougourdan et al., 1987). In contrast to previous claims (Ritter and Persoons, 1975; Sakuma and Fukami, 1993), our results show clearly that neither the glandular systems of the head nor that of the last abdominal segments appear to be particularly involved in the secretion of the attractive substances in Blattella germanica. In Blaberus craniifer (Brossut et al., 1974) and in Eublaberus distanti (Brossut, 1979) the aggregation pheromones identified are secreted by the mandibular glandular system. Although Blattella germanica possess mandibular glands, these glands do not appear to be particularly involved in the production of attractive substances. Our

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data seem to contradict the results of Ishii and Kuwahara (1967), MacFarlane and Alli (1986), and Sakuma and Fukami (1990, 1991), who indicated that the active substances were mainly localized in the abdominal part of the body, in the rectal pad cells. Faeces, passing by these rectal pads, collect the attractive substances and diffuse them. Chemical analyses by GC and GC–MS of extracts made with the three solvents revealed that all the components were cuticular hydrocarbons. All these components have already been identified as Blattella germanica cuticular hydrocarbons by previous authors (Augustynowicz et al., 1987; Carlson and Brenner, 1988; Jurenka et al., 1989). For the first time, we have shown that cuticular hydrocarbons are involved in aggregation behaviour in Blattella germanica. The facts that a few of these components were either not extracted or extracted in lower proportions with methanol (Fig. 1) and that methanol extracts were not attractive in behavioural tests suggest that the absent components which are apolar molecules play a part in inducing aggregation behaviour. In termites the apolar fraction of the cuticular extract containing hydrocarbons has the greatest effect but the polar fraction accounted for the chemical signature to a lesser degree (Cle´ment and Bagne`res, 1998). However, this information needs to be supplemented with further experimentation because correlational studies establishing that some compounds vary among chemical analyses in association with discrimination behaviour are not sufficient proof (Breed, 1998). There is growing evidence that cuticular hydrocarbons play an important role in intra- and inter-colony recognition mechanisms in many social insects (Nowbahari et al., 1990; Bonavita-Cougourdan and Cle´ment, 1994; Lorenzi et al., 1995; Vauchot et al., 1996). Cuticular hydrocarbons eliciting aggregation behaviour may play a similar role in sub-social insects like cockroaches, as Blattella germanica are able to distinguish between strain odours (Rivault and Cloarec, 1998). Fig. 1. Comparisons between gas chromatograms of cuticular hydrocarbons from extracts of 15 sixth instar larvae (a) in pentane and (b) in methanol. As chromatograms for pentane and dichloromethane extracts were exactly the same, only that for pentane is given. As the silica gel procedure did not modify the chromatograms for any of the solvent extracts, only chromatograms before this procedure are given. Samples of 2 ␮l of each extract were analysed by gas chromatography (GC) on a Delsi 200 DN coupled to an Enica 31 integrator and equipped with a flame ionization detector (FID). Conditions: Cpsil 5 capillary column (25 m × 0.25 mm ID) with a split–splitless injector; temperature programme started at 90°C for 3 min, then increased gradually to 230°C at 15°C/min and then to 320°C at 5°C/min (final time 10 min); carrier gas: helium at a flow rate 2 ml/min. Peaks are numbered according to the components identified by GC–MS in Table 4. Peaks 13, 18, 19 and 20 present about the same intensities in pentane (a) and in methanol (b) extracts, peaks 1, 7, and 12 have almost disappeared in (b), and peaks 3, 5, 15 and 17 are less intensive in (b) than in (a) and other peaks (2, 6 and 16) are more intensive in (b) than in (a).

Acknowledgements We thank G. Dusticier for help in monitoring mass spectrometry data.

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