Isopropylesters as wetting agents from the defensive secretion of the rove beetle Coprophilus striatulus F. (Coleoptera, Staphylinidae)

Insect Biochem. Vol. 14, No. 4, pp. 383-390, 1984 0020-1790/84 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1984 Pergamon Press Ltd

ISOPROPYLESTERS AS WETTING AGENTS FROM THE DEFENSIVE SECRETION OF THE ROVE BEETLE

COPROPHILUS STRIATULUS F. (COLEOPTERA, STAPHYLINIDAE)

KONRAD DETTNER Institute for Biology II (Zoology), RWTH Aachen, Kopernikusstrasse 16, D 5100 Aachen, FRG

(Received 29 September 1983)

Abstract--The defensive gland secretion of the rove beetle Coprophilus striatulus F. (Staphylinidae, Oxytelinae) has been shown to contain p-toluquinone, p-toluhydroquinone and a series of long chained saturated and unsaturated isopropylesters which were demonstrated by gas chromatography and mass spectrometry. The co-occurrence of at least five free fatty acids was confirmed by gas chromatography. The defensive glands of C. striatulus represent the first reported source of/sopropylesters from defensive glands of insects. By contact angle measurements synthetic quinoid isopropylester mixtures have been shown to possess excellent wetting properties on chitin surfaces. Moreover the quinoid isopropylester mixture was highly effective against immersed Calliphora erythrocephala larvae. As a primitive member of the Oxytelinae C. striatulus differs distinctly from derived species of the same subfamily both with respect to the defensive gland morphology and the chemistry of the secretion.

Key Word Index: Isopropylesters, quinones, Coprophilus striatulus, Staphylinidae, defensive secretion, glands, wettability, taxonomy

INTRODUCTION MATERIALS AND METHODS

Within Coleoptera, rove beetles (Staphylinidae) are unique in having evolved different types of abdominal defensive gland systems where an astonishing variety of defensive molecules are produced. In rove beetles these defensive glands are found to be homologous at the subfamily level (Araujo, 1978; Dettner, 1983) whereas defensive gland systems of other beetle groups are usually present within a wide assemblage of several beetle families. Based on this chemical and morphological diversity rove beetles are excellent candidates for assessing taxonomic and ecological influences on the chemical composit ion of the defensive secretions.

Within the Oxytelinae subfamily it was possible to analyze the chemistry of the paired abdominal defensive glands of several highly derived species (Wheeler et al., 1972; Dettner and Schwinger, 1982). These beetles have been shown to produce an uncom- mon but similar group of defensive molecules although their life histories are quite different. When molested members of this subfamily bend their abdominal tips dorsally to emit a secretion from their abdominal defensive glands.

This study reports on the exceptional morphology and chemistry of the abdominal defensive glands of Coprophilus striatulus F., a species which holds an isolated taxonomic position as a primitive member of the Oxytelinae (Herman, 1970; Newton, 1982). Ex- perimental results and comments on the biological significance of the constituents found in the gland are also given.

Biological material and gland preparation

Thirty specimens of C. striatulus F. were collected both from horse excrement (natural habitat) and plastered house walls (where the beetles often land) in the vicinity of Aachen (Rhineland). Carefully transported beetles were immediately frozen on reaching the laboratory. Gland reservoirs were excised from frozen beetles on an ice bath and deposited in the tiny groove of a cooled wire plunger which is movable within the needle (0.1/zl Mini Injector; Precision Sampling Corporation). This method allowed injection and analysis of gland reservoirs of single beetles by capillary gas chromatography without using any solvent.

Gas chromatography (GC)-mass spectometry (MS)

Gas chromatographic analyses were performed with a Carlo Erba Fractovap 2900 capillary gas chromatograph equipped with a FID detector (cartier gas: helium; 1 ml/min) and a Spectra Physics computer integrator (System 1). Also used was a 8 m CW 20 M glass capillary column temperature programmed from 65°C (2min isothermal) to 225°C (heating rate: 5°C/min). Capillary gas chromatography-mass spectrometry (GC-MS) was performed on a Varian 3700 capillary gas chromatograph coupled to a MAT 44 quadrupole mass spectrometer which operated at 80 eV and was connected with a Varian SS 200 computer system. For GC-MS a 8 m CW 20 M glass capillary column (60-230°C; heating rate: 10°C/min) and a 20 m SE 30 glass capillary column (60°C; 3 min isothermal; 60-240°C; heating rate: 12°C/min) were used.

Esterification and bromination of the gland constituents Six gland reservoirs of C. striatulus were mixed with 2 pl

diethyl ether in a micro glass tube. After centrifugation the upper phase was sucked off and mixed with 30/11 of ethereal

383

384 KONRAD DETTNER

diazomethane solution which was generated from 100mg MNNG (l-methyl-3-nitro-l-nitroso-guanidine (N-W-N) and 0.5ml 5 M aqueous potassium hydroxide solution. After part evaporation 0.2#1 of the remaining (1#1) esterified solution was analyzed by capillary gas chromatography.

Micro-scale bromination of the glandular components was performed by adding 0.2#1 of a 1:1 (v/v) solution of bromine in tetrachloromethane to two crushed gland reservoirs deposited in the groove of the Mini injector (injector of gas chromatograph: 220°C) described above. Also four gland reservoirs were mixed with 2/~1 of a 1:1 (v/v) solution of bromine in tetrachloromethane in a micro glass tube which was closed and heated at 150°C (30 min). One tenth of this solution was analyzed by gas chromatography.

Reference compounds Methyl-, propyl- and isopropylesters from CI0 to C19

straight chain carboxylic acids (Sigma) were prepared by refluxing 200mg of the corresponding acids with 0.2 ml MeOH/BF3-MeOH complex respectively with n-propanol or isopropanol and by addition of a drop of concentrated sulphuric acid. Isopropylesters of palmitoleic acid (Sigma) and myristoleic acid (Sigma) were prepared likewise, however concentrated hydrochloric acid was used instead of sulphuric acid. Further reference compounds were p- toluquinone (Fluka) and p-toluhydroquinone (Ega).

Test solutions Test solutions were prepared according to the known and

available main gland constituents of C. striatulus (CM: Coprophilus mixture) and Bledius spectabilis (BM: Bledius mixture; Dettner and Schwinger, 1982) by dissolving 0.5 g p-toluquinone either in 4 ml isopropyldodecanoate and 1 ml isopropyltetradecanoate (CM) or in 2 ml dodecane-4-olide, 2 ml 1-undecene and 1 ml citral (BM).

Contact angle measurements Test solution (0.1/11CM, 0.1 #1BM) was placed on the

forewing surface of Locusta migratoria specimens which were mounted horizontally in a constant temperature cell (Kriiss, Hamburg; 20°C). Initial and the decreasing contact angles of these droplets on the chitin surface were measured for 2.5 to 5 min by using a contact angle microscope (Erma, Tokyo).

Biological tests Last stage Calliphora erythrocephala larvae were im-

mersed for 1 sec in 5 ml Coprophilus- or Bledius-mixture (30 specimens per experiment). Dead larvae were counted 1 hr after immersion, non-pupated specimens were registered 5 days afterwards.

RESULTS

Defensive behaviour and gland morphology

When molested by ants or other arthropods C. striatulus bends the tip of its abdomen dorsally and emits small droplets of a reddish secretion at the attacking animal. In addition the abdominal tip of C. striatulus is used as brush to ensure that attacking small arthropods are smeared repeatedly with the repellent secretion. The defensive material is produced within two complex gland systems situated within the abdominal tips and consisting of a gland tube, an efferent duct and a gland reservoir (Fig. 1). The lightly musculated gland reservoir (res) opens (op) at the anterior border of the subdivided ninth tergit (IX). Both gland reservoirs of C. striatulus show a characteristic sac like evagination (sac res) orginating close by the opening (op) of the reservoir.

Defensive secretion is produced within a lengthened gland tube (gl) which also shows a lengthened cuticular central cavity (Fig. I, right gland). Several cuticular ductules originate from the central cavity of the gland tube and lead to the secretory cells. The central cavity of the gland tube is linked by a short cuticular efferent duct (ed) to the gland reservoir.

Chemistry of the secretion

Twenty one constituents of the GC separated C. striatulus secretion could be identified (Fig. 2). They represent between 84 and 92% of the total peak area found in the GLC analysis of the beetle secretion (Table 1). The identity of most components could be confirmed by EI mass spectral data and by their retention times on a 8 m CW 20 M capillary column when compared with available authentic compounds.

p- Toluquinone (1)

p-Toluquinone represents the main constituent and toxic principle of the C. striatulus secretion (Fig. 2; peak 1). Dependent on age and previous molestation of the rove beetle 5-50/~ g p- to luquinone are seques- tered per individual. This widespread defensive mole- cule (Blum, 1981) showed a strong molecular ion at m/z 122 and co-chromatographed with authentic 2-methyl-1,4-benzoquinone. EI-mass spectral data of compound 1 are indistinguishable from authentic p- toluquinone.

p- Toluhydroquinone (21)

The mass spectrum of compound 21 revealed a M +-peak at m/z 124 and distinct further fragments at m/z 123, 107, 95, 77, 67, 55 and 41. This fragmentation pattern is identical with recorded mass spectral data of authentic p- toluhydroquinone, a compound which has been found previously within defensive glands of other Oxytelinae beetles (Dettner and Schwinger, 1982).

Saturated isopropylesters (2~1, 6, 8, 12, 13, 16, 17, 20)

EI-mass spectral data for compounds 2, 3, 4, 6, 8 and 13 altogether showed base peaks at m/z 43 and

Fig. 1. Dorsal view of an abdominal tip of Coprophilus striatulus (gl: gland tube, ed: efferent duct, res: reservoir, sac res: sac like reservoir evagination, op: opening, numbers indicate tergites). Within the right gland tube (gl) cuticular

ductules and central cavity are outlined.

Defensive secretion of Coprophilus 385

17

19 i

-11 13

e sq 12

14

'0 ' I I I

l~lin 30 20 10

1_ Fig. 2. Capillary gas chromatogram of two defensive gland reservoirs from Coprophilus striatulus by using a 8 m CW 20 M glass capillary. Analytical conditions are given in the text. The numbers of the peaks represent: 1: p-toluquinone; 21: p-toluhydroquinone; 2-4,6, 8, 12, 13, 16, 17,20: saturated isopropylesters; 5, 7, 9-11, 14, 15, 18, 19: unsaturated isopropylesters (for further explanation see Table 1).

distinct fragments at m/z 60 and 102 (Table 2; Fig. 3). The periodic succession of retention times of the six gland components on a 8 m CW 20 M capillary column together with their molecular masses of 214 (2), 228 (3), 242 (4), 256 (6), 270 (8) and 298 (13) were in favour of a homologous series of gland constituents. Characteristically the molecular ion in each instance loses 41, 42 and 59 a.m.u., indicating that there are several propyl- or isopropylesters present in the beetle secretion. Fragments at M-41 and M-42 result f rom the loss of the propylresidue from the ester moiety together with transfer o f two hydrogen atoms to produce the carboxylic acid ion respectively the pro tona ted carboxylic acid ion. The fragment at M-59 constitutes an acylium ion. The above men- tioned fragment at m/z 102 is due to a McLafferty rearrangement of the propylester with fl-cleavage and transfer o f a y-hydrogen atom.

E l -mass spectral data from a series of authentic n-propyl- and isopropylesters showed that C. striatulus secretes only isopropylesters. As compared with

n-propylesters (Fig. 3C) isopropylesters are characterized by a weak fragment at m/z 61, a distinct peak at M-42 and often a minute fragment at M-15 (Fig. 3 A and B). Finally compounds 2, 3, 4, 6, 8 and 13 co-chromatographed with authentic isopropyldecanoate (2), isopropylundecanoate (3), isopropyldodecanoate (4), isopropyltridecanoate (6), isopropyl tetradecanoate (8) and isopropylhexadecano- ate (13). The isopropylesters from the beetle remained unaffected after t reatment with bromine and showed distinctly shorter retention times on a 8 M CW 20 M capillary column as compared with authentic n-propylesters (e.g. isopropylundecanoate from C. striatulus R t: 927 sec, authentic n- propylundecanoate R,: 1056 sec; isopropyldodecanoate from C. striatulus Rt: 1075sec, authentic n-propyldodecanoate Rt: 1194 sec; 65-225°C, heating rate 5°C/min, 4 min isothermically at 65°C). Apart from p- to luquinone isopropyldodecanoate (4) represents the second main compound of the Coprophilus secretion (Fig. 2;

Table 1. Defensive gland composition of Coprophilus striatulus together with the analytical evidence for the assignments

Mean ~ peak Range of values area per beetle found

Compound ( + SD, n = 5) (~ peak area) Evidence

1. p-Toluquinone 47.7 + 9.2 38.5-56.9 2. Isopropyldecanoate 0.2 _+ 0.2 0~.5 3. lsopropylundecanoate 0.1 + 0.1 0.5-0.2 4. Isopropyldodecanoate 21.4+ 10.3 10.5-31.3 5. Isopropyldodecenoate 0.5 + 0.3 0.08-0.8 6. lsopropyltridecanoate 0.5 + 0.6 0.2-1.3 7. Isopropyltridecenoate 0.4 + 0.4 0-0.9 8. Isopropyltetradecanoate 0.6 + 0.1 0.6-0.7 9. Iso propyltetradecenoate 0.2 + 0.1 0.17-0.34

10. lsopropyltetradecenoate 0.02 + 0.02 0-0.05 11. Isopropyltetradecadienoate 5.0 + 2.5 4.~7.8 12. lsopropylpentadecanoate 0.7 -t- 0.05 0.64).7 13. lsopropylhexadecanoate 1.1 + 0.5 0.5-1.5 14. Isopropylhexadecenoate 0.17 + 0.05 0.1-0.23 15. Iso propylhexadecadienoate 0.1 _+ 0.1 0~.25 16. lsopropylheptadecanoate 0.1 + 0.1 0.05~.2 17. lsopropyloctadecanoate 6.9 + 4.4 0.3-9.8 18. lsopropyloctadecenoate 0.5 + 0.6 0-1.4 19. Isopropyloctadecadienoate 0.5 + 0.4 0-1.0 20. lsopropylnonadecanoate 0.2 + 0.3 0~.6 21. p-Toluhydroquinone 1.0 + 1.5 (~3.2

GC, MS, Br + GC, MS, Br - GC, MS, Br - GC, MS, Br - GC, MS, Br + GC, MS, Br - GC, MS, Br + GC, MS, Br - GC MS, Br + GC MS, Br + GC MS, Br + GC Br- GC MS, Br - GC MS, Br + GC MS, Br + GC Br- GC Br- GC MS, Br + GC MS, Br + GC Br- GC MS, Br -

GC = compound has similar retention time on a 8 m CW 20 M capillary column to the authentic compound. MS = identity confirmed from mass spectrum. Br + = component removed from GC profile on treatment with bromine. Br - = component unaffected on treatment with bromine.

386 KONRAD DETTNER

Table 2. EI-MS data from saturated isopropylesters recorded from Coprophilus striatulus

Molecular mass Fragments Compound (relative intensity) (relative intensity)

2. Isopropyldecanoate 214 (0.25) 173 (6), 172 (9), 155 (8), 129 (10), 102 (18), 73 (24), 61 (20), 60 (65), 43 (100), 41 (60)

3. Isopropylundecanoate 228 (0.5) 187 (7.5), 186 (8), 169 (10), 129 (5), 102 (27), 73 (18), 61 (18), 60 (63), 43 (100), 41 (53)

4. Isopropyldodecanoate 242 Fig. 3

6. lsopropyltridecanoate 256 (0.5) 215 (8), 214 (11), 197 (8), 129 (8), 102 (32), 73 (20), 61 (21), 60 (50), 43 (100), 41 (60)

8. Iso propyltetradecanoate 270 (0.5) 229 (6), 228 (8), 21 l (6), 129 (8), 102 (30), 73 (21), 61 (23), 60 (52), 43 (100), 41 (50)

13. lsopropylhexadecanoate 298 (0.2) 299 (0.2), 257 (2.5), 256 (4), 239 (2), 129 (3), 102 (22), 73 (12), 61 (13), 60 (40), 43 (100), 41 (47)

rel. I [% 100

A

5 0 -

4O 5O

rel I (%1 100

so 0

102

? 3 1115 ~ IL I L 1 0 3 200 ~L. 11~ ,2g 143 i,s? 171 ,, ,

. . L . . . ,

100 150 200

227 242

250 m/z

B 43

5 0 -

6O

102

n .1= . ~ , I ,, . L ~.~ I. , t,0 50 100 150 200

rel. I[%] 100 T¢

J 4 3

50.

0

,oo ,so

242

2~o

Fig. 3. E l -mass spectrum of authentic isopropyldodecanoate (A), co mp o u n d 4 f rom the defensive secretion of Coprophilus striatulus (B) and authentic n-propyldodecanoate (C).

227 242

250 m/z


Table 1): concentrations per beetle ranged between 0.5 and 10#g. Because of quantitative differences between the gland contents of different specimens for compounds 12, 16, 17 and 20 no mass spectra could be recorded. Nevertheless these constituents co- chromatographed with authentic /so propylpenta- decanoate (12), isopropylheptadecanoate (16), iso- propyloctadecanoate (17) and isopropylnona- decanoate (20) on a 8 m CW 20 M capillary column and remained unaffected after treatment with bromine.

Unsaturated isopropylesters (5, 7, 9-11, 14, 15, 18, 19)

Based on El -mass spectral evidence C. striatulus compounds 5, 7, 9, 10, 14 and 18 were suggested to be identical with mono-unsaturated isopropylesters whereas compounds 11, 15 and 19 have been found to contain a second double bond in the carboxylic acid chain of the ester (Table 3). The nine constituents were quantitatively removed from the GC-profile on treatment with bromine, when the gland-bromine mixture was heated previously (Table 1). Retention times of all unsaturated isopropylesters of C. striat-

ulus (Fig. 2) showed the expected approximate succession on a polar capillay column (CW 20 M: saturated, mono-unsaturated, di-unsaturated esters) with reverse retention times on a SE 30 capillary column.

As compared With the saturated esters E l -mass spectral data from the unsaturated iso propylesters of C. striatulus gave a series of strong hydrocarbon backbone fragments at m/z 41, 43, 55 (compounds 5, 7, 9, 10, 14, 18; CnH2n_l: m/z 69, 83, 97 etc.; compounds 11, 15, 19; CnH2n_3: m/z 67, 81, 95 etc.) with m/z 43 or 41 being the base peak (Table 3). The molecular ion in each instance loses 43, 59 and 60a.m.u., in most cases additional M-41, M-42, M-44, M-58 and M-61 fragments were observed. Toward higher molecular masses there appeared distinct M + 1 peaks (Table 3). The fragmentation pattern of compound 10 was very similar to authentic isopropylmyristoleate moreover the mass spectrum of compound 14 was indistinguishable from the spectrum of authentic isopropylpalmitoleate. Be- cause of the scarcity of beetle material which was available for this study it was not possible to deter- mine position and configuration of the double bonds.

Table 3. El-MS data from unsaturated isopropylesters recorded from Coprophilus striatulus Molecular mass Fragments

Compounds (relative intensity) (relative intensity) 5. lsopropyldodecenoate 240 (0.5) 198 (0.5), 197 (0.5), 196 (0.5),

7. Isopropyltridecenoate

9. Isopropyltetradecenoate

10. Isopropyltetradecenoate

11. lso propyltetradecadienoate

14. lsopropylhexadecenoate

15. Isopropylhexadecadienoate

18. Iso propyloctadecenoate

19. Isopropyloctadecadienoate

182 (0.2), 181 (0.8), 180 (2), 179 (1), 60 (17.5), 55 (65), 43 (100), 41 (97)

254 (0.25) 213 (0.5), 212 (1), 211 (0.5), 196 (0.2), 195 (2), 194 (2), 193 (0.8), 60 (14), 55 (80), 43 (85), 41 (97)

268 (1) 269 (0.3), 227 (0.5), 226 (1), 225 (0.6), 224 (0.3), 210 (0.6), 209 (4), 208 (6), 207 (1.8), 60 (20), 55 (70), 43 (100), 41 (85)

268 (0.5) 269 (0.2), 227 (0.4), 226 (0.7), 225 (0.4), 224 (0.2), 210 (0.7), 209 (3), 208 (2.5), 207 (1), 60 (17), 55 (80), 43 (100), 41 (90)

266 (0.5) 267 (0.2), 225 (0.3), 224 (2), 223 (3.5), 222 (0.1), 207 (1), 206 (2), 205 (1), 60 (11), 55 (59), 43 (81), 41 (100)

296 (0.6) 297 (0.5), 255 (0.5), 254 (1), 253 (1), 252 (0.5), 238 (1.7), 237 (6), 236 (4.2), 235 (1.8), 60 (15), 55 (86), 43 (100), 41 (86)

294 (0.2) 295 (0.5), 252 (1), 251 (2), 235 (1.5), 234 (0.6), 233 (0.2), 60 (4), 55 (70), 43 (82), 41 (100)

324 (0.1) 325 (0.2), 281 (0.4), 267 (0.4), 266 (1), 265 (1), 264 (1), 263 (0.4), 60 (7), 55 (79), 43 (100), 41 (86)

322 (0.1) 323 (0.2), 281 (0.2), 280 (1), 279 (0.5), 264 (0.6), 263 (0.5), 262 (0.2), 60 (3), 55 (60), 43 (100), 41 (90)

388 KONRAD DETTNER

20

o 15

o

~ ~o

BM

×

..41 I 1 2 3 4

Min

Fig. 4. Decreasing contact angles (°) of single droplets (0.1 # 1) of synthetic quinoid mixtures (BM: Bledius mixture, solvents: dodecane-4-olide, 1-undecene, citral; CM: Co- prophilus mixture, solvents: isopropyldodecanoate, isopropyltetradecanoate) after placing on forewing surfaces of Locusta rnigratoria (20°C). Each line shows decreasing contact angles of single droplet. Single determinations only.

From mass spectral data it seems evident that unsaturated isopropylesters with the following molecular masses are present: 240 (isopropyldodecenoate; 5), 254 (isopropyltridecenoate; 7), 268 (isopropyl- tetradecenoates; 9, 10), 266 (isopropyltetradecadienoate; 11; third main component of the secretion), 296 (isopropylhexadecenoate; 14), 294 (isopropylhexadecadienoate; 15), 324 (isopropyloctadecenoate; 18) and 322 (isopropyloctadecadienoate; 19).

Free ]atty acids When pure defensive secretion free from tissue was

treated with ethereal diazomethane the presence of free fatty acids in the glandular reservoir was seen. By co-chromatography with authentic fatty acid methylesters the C. striatulus secretion has been found to contain at least five free acids from dodecanoic to hexadecanoic acid with the C14 carboxylic acid as main constituent. These results largely agree with the gland chemistry of other Oxytelinae species where mass spectra were recorded from these fatty acid methylesters which are present as free acids within the defensive secretions (Dettner, in preparation).

Properties of synthetic beetle mL~tures One of the most important demands for arthropod

defensive secretions aimed against other arthropods is a good wettability on an arthropod cuticle (Duffey, 1976; Eisner, 1970). Due to the unavailability of sufficient natural beetle secretion contact angle measurements of a synthetic quinoid-/so propylester mixture (as found in the primitive species C. striatulus) were compared with contact angles of a quinoid-lactone-alkene-citral mixture found in the secretion of the derived rove beetle genera Bledius or Platystethus. As demonstrated in Fig. 4 both initial and succeeding contact angles of the quinoid-isopropylester mixture (CM) are distincly lowered as compared with the quinoid Bledius mixture (BM). Some minutes after placing the low volatile quinoid-isopropylester droplets on the L. mi-

gratoria surface there was still no contact angle measurable. Moreover when droplets of the synthetic C. striatulus mixture were placed on other cuticles (e.g. elytra of Tenebrio molitor, Tribolium confusum ) some minutes later it was observed that they had moistened a great surface area of the elytra when compared with the distinct droplet of the quinoid-lactone-alkene-citral mixture which is still confined to a small surface area of the cuticle some minutes afterwards.

Immersion experiments of both quinoid mixtures by using C. erythrocephal larvae showed an elevated mortality when compared with untreated larvae (Fig. 5) which is obvious both one hour (hatched area of columns) and one week after larval immersion (bro- ken lines included). Unexpectedly the isopropylester mixture (CM) was less effective when compared with the lactonic blend (BM) although reverse trends have been shown with respect to the cuticular wettability of these mixtures.

DISCUSSION

With respect to the morphology and chemistry of the defensive glands the primitive species C. striatulus shows distinct differences when compared with taxonomically more derived genera such as Bledius or Platystethus (Dettner and Schwinger, 1982). In C. striatulus the volume of the glandular reservoir is very small when compared with the body volume. There- fore only a few successive depletions of the gland reservoir may be possible when C. striatulus is repeatedly molested. Further differences consist in the extremely shortened efferent duct and the lengthened gland tube. Those differences are still evident when the fine structure of the secretory cells is compared with similar structures of the derived genera. For example apical parts of the glandular efferent ductules are considerably lengthened in the derived species (Happ and Happ, 1973) but short and wide within the primitive species C. striatulus (Dettner, Wunderle and Schwinger, in preparation).

Within Oxytelinae only p-toluquinone and p- toluhydroquinone are present as common main constituents in all defensive glands investigated. C. striat-

30 -

,

Io

CM BM C

Fig. 5. M o r t a l i t y o f Calliphora erythrocephala larvae ] h r (hatched area o f columns) and 5 days (b roken lines o f columns included) after immersion within synthetic quinoid mixtures (CM: Coprophilus mixture; BM: Bledius mixture; C: control; n = 30 larvae per experiment). Single deter-

minations only.


ulus is so far unique in its ability to sequester a series of saturated and unsaturated isopropylesters which now have been recorded for the first time from defensive glands of insects. Isopropylmyristate was found as minor constituent from head extracts and abdominal extracts of the argentine ant Iridomyrmex humilis (Cavill and Houghton, 1974). Moreover several saturated and mono-unsaturated isopropylesters from C12, C14 and C16 carboxylic acids were identified as pheromones from male specimens of Dermestes maculatus (Francke et al., 1979). Usually esters from arthropod defensive secretions either show methyl, ethyl or butyl and higher, often branched carbon skeletons as alcoholic moieties (Blum, 1981). Isopropylesters are well known as powerful wetting agents and are frequently used in cosmetic and topical medicinal preparations where good absorption through the skin is desired. There- fore isopropylesters of C14, C16 and C18 fatty acids are especially used for skin creams, lipsticks or suntan oils. Obviously in the beetle C. striatulus these esters also function as carriers but for the toxic p- toluquinone which must penetrate the cuticle of an attacking arthropod. Biological tests with defensive secretions are problematic, for target organisms of the defensive blends are seldom known and natural secretions often show considerable quantitative vari- ations (Duffey, 1976). Therefore the immersion of C. erythrocephala larvae only seems a rough method for comparing the efficiency of chemically different defensive constituents (Remold, 1963) in default of a suitable repellency test. Nevertheless different kinds and stages of dipteran larvae represent the most important organisms occurring with C. striatulus and other Oxytelinae beetles in horse- or cow-excrement. A smaller body area may be in contact with the defensive secretion depending on the attackers body volume. The elevated larval mortality found and caused by the Bledius mixture (BM) as compared with the synthetic Coprophilus mixture (CM) probably is due to the presence of 1-alkenes in the defensive secretions of taxonomically derived Oxytelinae rove beetles.

By preliminary tests it has been shown that pure quinoid-l-alkene mixtures possess excellent wetting properties on chitin surfaces still superior to the quinoid-isopropylester mixtures. Therefore smaller cuticle surface areas may be sufficient for quinoid-1- alkene-lactone (where the lactone drastically reduces the wettability) mixtures to ensure a penetration of considerable amounts of the toxic p-toluquinone by effective 1-alkenes through this barrier. Finally a lot of minor and trace constituents of the natural C. striatulus secretion have not been available for testing and it may be that these minor compounds may too drastically influence the wettability and other phys- icochemical properties of the natural secretion (Duffey, 1976).

As found previously (Dettner and Schwinger, 1982) the qualitative defensive chemistry of Oxy- telinae beetles is not correlated with the life history or habitat of the beetles but seems mainly dependent from the phylogenetic position of the beetle taxon investigated. Preliminary tests support this suggestion revealing that isopropylesters of fatty acids are present within the primitive species Deleaster dichrous

(Grav.) also (Dettner, Wunderle and Schwinger, in preparation). Therefore an evolutionary trend in replacing isopropylesters by the more effective (Cal- liphora test) 1-alkenes seems obvious towards derived Oxytelinae species. Moreover synthetic quinoid-1- alkene mixtures were characterized by lower melting points as compared with quinoid-isopropylester mixtures. During cold seasons C. striatulus probably prevents the hardening of its defensive mixture within the gland reservoir to a certain extent by sequestering additional unsaturated isopropylesters. Nevertheless it may probably be advantageous to replace the high melting isopropylesters by solvents characterized by lower melting points.

With respect to the biogenesis of the defensive compounds p-toluhydroquinone may represent the precursor of the toxic p-toluquinone as proposed for tenebrionid beetles (Happ, 1968). Furthermore fatty acid moieties of the isopropylesters might be derived from the co-occurring free fatty acids. The biogenetic origin of the isopropyl alcoholic moiety however seems an interesting question worthy of further in- vestigation.

Acknowledgements--Mr G. Schwinger (University of Ho- henheim) kindly recorded mass spectra. Mr P. Wunderle (RWTH Aachen) assisted in collecting beetle specimens. This study has been financially supported by the Deutsche Forschungsgemeinschaft (De 258/4-4).

REFERENCES

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Isopropylesters as wetting agents from the defensive secretion of the rove beetle Coprophilus striatulus F. (Coleoptera, Staphylinidae)