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Production, regeneration and biochemical precursors of the major
components of the defensive secretion of
Eurycotis floridana
(Dictyoptera, Polyzosteriinae)
Jean-Pierre Farine
*, Claude Everaerts, Dehbia Abed, Remy Brossut
Universite´ de Bourgogne, C.N.R.S, U.M.R. 5548, 6 Bd Gabriel, 21000 Dijon, France
Received 2 June 1999; received in revised form 15 February 2000; accepted 25 February 2000
Abstract
The defensive secretion of the cockroachEurycotis floridanacontains three main components, (E)-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid, which represented about 98% of the organic phase. The quantity of the aldehyde, alcohol, and acid present in the defensive secretion increased rapidly for 60 days from the imaginal moult. Following artificial discharge, the males were able to regenerate their initial volume of secretion over a 30 day period. To investigate the possible routes of biosynthesis of the three components, E. floridanawas injected with 14C-labeled fatty acids and acetate, and the incorporation of 14C into the three components were quantified 1, 6, and 24 h after milking. Our results revealed that oleic, linoleic, linolenic and palmitic acids, which constitute part of the fat body of the insect, were incorporated to the same degree into the three main components, but very slowly compared to acetate. Although it has not been possible to identify the exact route of aldehyde, alcohol and acid biosynthesis, our findings suggests that (E)-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid are preferentially biosynthesized de novo from acetate. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Eurycotis floridana; Dictyoptera; Exocrine glands; Pheromones; Defence; Biosynthesis
1. Introduction
When alarmed, adults of the large wingless cockroach
Eurycotis floridana emit a defensive secretion from the sternal gland, which opens medially on the intersegmen-tal membrane between the 6th and 7th abdominal ster-nites (Stay, 1957). The secretion is a mixture of an organic and aqueous phase (Roth et al., 1956; Dateo and Roth, 1967a,b; Brossut, 1983). While the aqueous phase contains gluconic acid, gluconolactone and glucose (Dateo and Roth, 1967a; Brossut, 1983), the organic phase is a mixture of about 40 constituents of which (E )-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid are the main components (Farine et al., 1997). Fifteen-day-old males contained about 20 to 30µl of secretion with 15 mg (E)-2-hexenal, 687µg (E)-2-hexenol and 31µg (E )-2-hexenoic acid, making up about 93, 5 and 0.3% of the
* Corresponding author. Tel:+33-03-80-39-62-95; fax:+ 33-03-80-39-62-89.
E-mail address:[email protected] (J.-P. Farine).
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 6 5 - 5
organic phase of the secretion, respectively (Farine et al., 1997).
Like other insects, some cockroaches are able to syn-thesize de novo all their linear fatty acids with 14, 16 or 18 carbon atoms (Cripps et al., 1986; Jurenka et al., 1987; de Renobales et al., 1987). In the fat body of males and females of E. floridana, palmitic (C16:0), oleic (C18:1) and linoleic (C18:2) acids are the most abundant fatty acids (respectively, 18/56/18%) whereas linolenic (C18:3) and stearic (C18:0) acids represent only a few percent (respectively, traces/4%) (Bade, 1964). After ingestion of [1-14C] acetate, both sexes are able to biosynthesize by condensation of C2 units all the fatty acids encountered (Bade, 1964).
In the first part of this study, the defensive secretion of males of E. floridana was investigated qualitatively and quantitatively over a two-month period. In the second part, the rate of refilling of the defensive glands after artificial discharge was quantified. Comparatively to many insect species where the sex pheromones are derived to fatty acids (Tillman et al., 1999), the possible role of the five linear fatty acids encountered in the fat body ofE. floridana, and that of acetate as possible pre-cursors of the major components of the defensive secretion, were also investigated.
2. Materials and methods
2.1. Insects
Colonies of E. floridana were obtained in 1989 (gift of M.S. Blum, University of Georgia, USA) and reared in glass aquaria (60×40×20 cm) at 28°C and 80% rela-tive humidity, under a 12:12 h dark:light photoperiodic regime. The insects had free access to dry dog food and water-soaked cotton pads. Newly-emerged adults used in this work were sexed and raised individually in plastic boxes (12×8×5 cm) to avoid agonistic behaviour and any resultant discharge of the defensive secretion.
2.2. Preparation of extracts
As the GC profiles were qualitatively and quantitat-ively similar in adults of both sexes, we used only males. The ontogenic pattern of the secretion was followed on males of various ages from the imaginal moult to 60 days.
For quantitative analyses of regenerative secretion, 15-day-old males were artificially “milked” (collection of the spray from the gland opening using a capillary tube). As a control, the volume of the secretion was noted using a Hamilton syringe and its major compounds quantified by GC as described below. To quantify these compounds in the remaining secretion, insects were cooled for a few minutes at 220°C before use. The glands were dissected out after various periods (from 1 h to 30 days) of regeneration. The adjacent tissues were
removed. The gland and its overlying cuticle were crushed in a vial containing 500µl of distilled methylene chloride (CH2Cl2) and 5µg of propanoic acid added as an internal standard. The extract was then filtered through glass-wool and stored at 220°C until use. To prevent underestimates due to a loss of secretion, insects that had the characteristic odor of (E)-2-hexenal during manipulations were not used. Each analysis was repli-cated using five insects.
2.3. Chemical analysis
GC analyses of extracts were performed using a Pack-ard 437 A fitted with a flame-ionization detector. A CP Wax 58 CB (30 m×0.25 mm i.d., 0.22µm film thickness, Chrompack) fused silica capillary column was used for analyses. One microliter of each sample was injected into a split-splitless injection system, operating with a split flow of 25 ml/min and a septum purge of 3 ml/min. The split port was closed during injection and then opened 30 s after injection. The column was held iso-thermally at 40°C for 2 min, then programmed to increase at a rate of 20°C/min for one min, and then at 2°C/min to 240°C. Hydrogen was used as carrier gas (50 cm/s velocity at room temperature). The injector and detector temperatures were 250 and 270°C, respectively. The amounts of the various compounds were calculated using a Shimadzu CR 4 A computer (Kyoto, Japan). The response factor was previously determined for each of the identified major compounds at 10, 100, 500 and 1,000 µg. These response factors were used for quanti-tative analyses.
A Girdel 30 GC apparatus fitted with a FID detector and a Carbowax 20M (3 m×3mm i.d., Spiral) was used for collection of the major labelled compounds. The tem-perature was programmed from 50° to 220°C at a rate of 2°C/min. The injector and detector temperatures were 250 and 270°C, respectively. Helium (20 ml/s) was used as carrier gas. The GC column effluent was split in a ratio of 1/8 (v/v) to the detector and a trap (glass tube, 30 cm×1 mm i.d.) cooled with liquid nitrogen. The tubes were immediatly washed using 500µl scintillation fluid and the radioactivity measured. Previous data revealed that, using this technique, recovery of chemical standard (E)-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid was 60, 75 and 40%, respectively. These percentages were taken into account for quantitative analyses.
2.4. Chemicals
)-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid] and propanoic acid were purchased from Interchim, France. Acetonitrile and methanol (Carlo Erba), of RS purity for HPLC and scintillation cocktail, were obtained from B.C.S. (Amersham).
2.5. In vivo incorporation of labelled precursors
After being anaesthetized by chilling at 25°C for about 10 min, milked 15-day-old insects were individu-ally injected with 1µCi of one of the studied precursors in 5µl dimethylsulfoxide (DMSO, Sigma) using a 10µl Hamilton syringe. The needle was inserted just under the cuticle between the 3rd and 4rd abdominal tergites. To verify whether the milking was efficient, the volume of the defensive secretion obtained by direct collection of the spray from the gland opening was recorded.
After 1, 6 or 24 h incubation in individual boxes at 28°C, males were killed by freezing at 220°C. The defensive glands were quickly pulled out through dissec-tion and the haemolymph and adjacent tissues removed, except for the small area of cuticle overlying the gland. A comparable lateral area of sternite 6 was used as a blank. Subsequently, the pieces were crushed in 200 µl of acetonitrile. Two microliters of each of the extracts were analyzed by GC for quantification of the major components. An aliquot of 20 µl served to collect the major compounds using the GC preparative technique as described above. Radioactivity has never been detected in other fractions collected by GC. Finally, 50 µl of the extract was directly assayed for radioactivity. The rest of the insect was crushed for 5 min by sonication in 10 ml methanol and allowed to soak for 24 h at room temperature. Preliminary data obtained using this simple method revealed that the percentage recovery of the radi-olabelled precursors was about 80%. After filtration onto glass-wool and centrifugation (5 min; 10,000g), an ali-quot of 1 ml was dissolved in 10 ml of scintillation cock-tail.
The radioactivity was measured using a Beckman LS 6000SC liquid scintillation counter. The counting efficiency was 95% for 14C. As a high percentage of injected radioactivity was lost as CO2 or other metab-olism derivates (75 to 95%, depending to the precursors and to the duration of incubation), the percentages of incorporation of the studied radiolabelled precursors into the defensive secretion (incorporation index, S) were estimated using the formulae S=(defensive gland2 stern-ite blank)/(remainder insect+defensive gland+sternite blank). Each experiments was replicated using five males.
3. Results
3.1. Production of major compounds after moulting (Fig. 1)
We estimated the level of gland filling in adult males by quantifying the major defensive components at inter-vals following the imaginal moult. The observation that the defensive gland was present in the last instar nymphs but do not produce secretions (Stay, 1957) was con-firmed by chemical analysis. In adults, (E)-2-hexenal was always quantified as the major component of the secretion. Thirty minutes after the imaginal moult, the gland contained 5.6±0.51 ng (E)-2-hexenal, 2.8±0.37 ng
(E)-2-hexenol, and only traces of the corresponding acid. One hour after the moult, 273±8.19 ng of aldehyde, 244.8±16.88 ng of alcohol, and 19.2±0.86 ng of acid were detected. The amount of the three major com-pounds increased rapidly in the same way during the first three days and then somewhat more slowly to the end of the experiment. For example, 496.84±7.24 µg of (E )-2-hexenal were encountered in one-day-old insects, 8.64±0.17 mg at 10 days, 24.22±0.18 mg at 20 days, and 45.06±0.55 mg at 60 days.
3.2. Regeneration of the defensive components (Fig. 2)
The ability of 15-day-old males to regenerate their defensive secretion was determined over a one month period after milking. The average amount of the secretion collected from each of the milked individuals was about 24.8±3.5 µl (n=30). Chemical analysis of milked males always revealed small amounts of (E )-2-hexenal and its corresponding alcohol and acid
Fig. 2. Production of the three main defensive components in 15-day-old adult males ofEurycotis floridana. The glandular content was quantified at various times after the males were milked. Insert: a subset of the same data during the first day period. Each value is an average of five samples. (64.8±1.91, 5.8±0.18 and 2.8±0.18 µg per gland, respectively), which represent, respectively, 0.43, 0.84 and 9.03% of the corresponding compounds encountered in a control male.
and 89.6±1.87 µg (versus 84.05±0.37 ng) (E )-2-hex-enoic acid.
3.3. In vivo incorporation of labelled precursors into the defensive secretion (Fig. 3)
The incorporation of various [14C]-radiolabelled pre-cursors was assessed during a 24 h period of incubation. At three time intervals, the glandular secretion was extracted and the incorporation level was determined. C18:1, C18:2, C18:3 and C16:0 radiolabelled acids were incorporated to a similar degree. About 0.5% of the reco-vered radioactivity was detected in the secretion after 1 h of incubation and 1.5% after 6 h. From this time onwards, the incorporation of labelled precursors was quite linear and stabilized near 2% at 24 h. The incorpor-ation of C18:0 acid was very slow: 0.11% of the label was found into the secretion after 1 h of incubation, and only 0.9% of the injected radioactivity was recovered after one day. By comparison, [1-14C] acetate was quickly incorporated: 1.49% of the recovered radioac-tivity was detected in the secretion after 1 h incubation, 6.27% after 6 h and 20.99% after 24 h.
Fig. 3. Incorporation of various [1-14C] substrates into the defensive secretion of adult males ofEurycotis floridana according to time after injection. Each data point represents the mean among five insects. Acetate, sodium acetate; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C16:0, palmitic acid; C18:0, stearic acid.
3.4. In vivo incorporation of the labelled precursors into the major components of the defensive secretion (Fig. 4)
After various times of incubation, the three major components of the secretion were quantified and their labelling determined relative to their amounts. After 1 h incubation (Fig. 4A), large amounts of radiollabel were incorporated into (E)-2-hexenol from acetate (152.38±14.15 dpm/µg), and about 7 to 10 times less when using C18:1 (21.06±2.16 dpm/µg), C18:2 (17.94±1.55 dpm/µg), C18:3 (14.84±1.29 dpm/µg) and C16:0 (18.91±1.08 dpm/µg) acids. (E)-2-Hexenal was 10 times less labelled than its corresponding alcohol. Radi-ollabel from C18:0 acid was not detected in (E )-2-hex-enal, (E)-2-hexenol, and (E)-2-hexenoic acid, but all of the major compounds were present in the glandular secretion (258.2±8.1, 16.4±0.5 and 4.4±0.2 µg, respectively). We did not detect any radiotracer incor-poration of any of the precursors into (E)-2-hexenoic acid.
Fig. 4. Amounts of labelled (E)-2-hexenal, (E)-2-hexenol and (E )-2-hexenoic acid found in the defensive gland of adult male ofEurycotis floridana after injection of various [1-14C] labelled substrates. Each point is an average (mean±S.E.M.) of five samples. Acetate, sodium acetate; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C16:0, palmitic acid; C18:0, stearic acid.
the acid when using acetate, C18 unsaturated and C16 saturated acids. By contrast, a spectacular decrease of (E)-2-hexenol radioactivity was noted when using lab-elled acetate (25.67±2.24 versus 152.38±14.15 dpm/µg). As mentioned above, no incorporation into (E )-2-hex-enoic acid was observed when using C16 and C18 satu-rated fatty acids as precursors.
After a 24 h incubation (Fig. 4C), (E)-2-hexenoic acid was the only component which appeared well labelled, especially when using acetate as precursor (168.57±9.94 dpm/µg). (E)-2-hexenal and (E)-2-hexenol were about three times less labelled compared to the values observed at 6 h.
4. Discussion
A vast amount of information has been accumulated on the chemistry and functional role of arthropod defens-ive secretions (Blum 1981, 1987 and references therein). Comparatively, little quantitative information is avail-able on the ability of arthropods to generate and regener-ate their defensive secretions (Fescemyer and Mumma, 1983; Baldwin et al., 1990; Rossini et al., 1997) or on the biosynthesis of the defensive components (Happ and Meinwald, 1965; Hefetz and Blum, 1978a,b; Kim and Toia, 1989; Honda, 1990; Renson et al., 1994).
It is well-known that a number of insects and animals are especially very receptive to aldehydic compounds and only few nanograms of these components were able to repel predators (Eisner et al., 1959; Blum, 1981).
As in most Polyzosteriinae species (Chada et al., 1961; Blum, 1964; Wallbank and Waterhouse, 1970), (E)-2-hexenal, (E)-hexenol and (E)-2-hexenoic acid are the main components of the defensive secretion of E. floridana (Farine et al., 1997). All of these species initially produce large amounts of secretion. In E. flori-dana, our data showed that within two days of the imaginal moult, the insect had a stock of secretion that was more than enough to respond to a potential aggressor. After an attack, insects do not regenerate their defensive secretion very rapidly. So, many of them have evolved strategies to prevent its full discharge (Fescemyer and Mumma, 1983; Baldwin et al., 1990; Rossini et al., 1997) and only a fraction is generally emitted during a single attack. We determined that the remaining defensive secretion in a 15-day-old artificially milkedE. floridanais about 0.5% of the one of its corre-sponding non-milked insect. However, this low percent-age represented about 80µg of the major defensive com-pounds. One hour later, the gland contained about 2% of its initial secretion, which corresponds to 270 µg of the three components. So, this low percentage of regen-eration is largely sufficient to repel predators.
again after the following “milking”. The percentage of (E)-2-hexenoic acid is quite stable.
In E. floridana, when 50-day-old males were artifici-ally milked, we showed that the initial level of (E )-2-hexenoic acid is regenerated in less than 10 days and that the acid always appears to be the major component of the secretion. However, while the initial level of (E )-2-hexenal was reconstituted within a 15-day period, about one month was necessary for the insect to recover its full level of the alcohol.
Fatty acids are the origin of a large pheromone struc-ture and are modified in many ways to form a multitude of aliphatic compounds (Francke and Schulz, 1999, and references therein). These compounds may be formed by transformation of the final products of the fatty acid synthesis or through modifications during their biosynth-esis. Other products, mainly C6, C8, C9, and C12 com-pounds, can be formed by oxidative cleavage of unsatu-rated fatty acids, e.g., linoleic acid. Lipoxygenases oxidize fatty acids to hydroperoxy acids, which upon chain cleavage yield aldehydes, which may be further metabolized. These processes have been intensively studied in plants (Heath and Reineccius, 1986; Hatanaka, 1996), but rarely verified to take place in insects. Never-theless, similar components have been identified as pher-omones and plant volatiles.
Radiotracer studies have confirmed that a number of insects are capable of synthesizing polyunsaturated fatty acids from labelled acetate (Dwyer and Blomquist, 1981; Blomquist et al. 1982, 1991; Cripps et al. 1986, 1990; de Renobales et al. 1986, 1987; Stanley-Samuelson et al., 1986; Borgeson et al. 1990, 1991; Borgeson and Blomquist, 1993). The role of acetate as a precursor of a number of pheromonal compounds has long been dem-onstrated in various insect species (reviewed in Blum, 1987; Prestwich and Blomquist, 1987), but little is known concerning the volatile defensive components (Hefetz and Blum, 1978a,b; Kim and Toia, 1989; Attygalle et al., 1994; Renson et al., 1994; Veith et al., 1994; Leclercq et al., 1996). For example, the green veg-etable bug, Nezara viridula, is able to incorporate 0.2% of injected sodium acetate into the major components (tridecane, (E)-2-decenal and (E)-2-hexenal) of the defensive secretion after an eight-day incubation (Gordon et al., 1963). In E. floridana, our data clearly showed that acetate is rapidly incorporated into the three major components of the defensive secretion.
The intermediates of the fatty acid metabolism and catabolism form an important pool for the biosynthesis of low molecular weight semiochemicals. The flexibility with respect to the utilization of acetate and longer fatty acids is observed in many fatty acid-derived pheromone biosyntheses (Prestwich and Blomquist, 1987; Tillman et al., 1999). During the beta-oxidation cycle, [1-14C] labelled fatty acids can be chain-shortened subsequently to produce labelled acetate (Downer, 1985). Generally,
transformations retaining the carboxy carbon lead to compounds with an even number of carbon atoms along the chain whereas decarboxylative processes yield com-pounds with an odd number of carbon atoms.
InE. floridana, the major components of the defensive secretion have an even number of carbons. So, a beta-oxidative process of the fatty acids would certainly be at the origin of these compounds. This hypothesis was in accordance with our results and could explain why the incorporation of all the tested labelled fatty acids in the defensive components of E. floridana was slower, but very similar to that obtained when using the acet-ate precursor.
In this way, the five acid precursors used were first beta-oxidized to acetyl-CoA and then resynthesized into aldehyde, alcohol or acid, but did not rule out the hypothesis that the free labelled acetates could be used to synthesize [U-14C] fatty acids. After chain-shortening through partial beta-oxidation, these acids could then be incorporated into the compounds of interest as demon-strated in the biosynthesis of many lepidopteran phero-mones (Bjostad et al., 1987). However, the high level of labelled (E)-2-hexenol which is obtained after 1 h incu-bation showed that the acetate is the preferential precur-sor of (E)-2-hexenol. The alcohol could then be rapidly converted to its corresponding aldehyde with immediate conversion to the acid (Ding and Prestwich, 1986). This might explain the high level of labelled (E)-2-hexenoic acid obtained after 6 and 24 h of incubation.
At this stage of the work, although it has not been possible to identify the exact routes of aldehyde, alcohol, and acid biosynthesis in E. floridana, we have estab-lished that one of the biosynthetic route of the major defensive components proceeded from acetate. To know if it is the unique route, comparative experiments using uniformly-labelled precursor [U-14C] acids were needed. If C6 chain was produced by chain-shortening of the fatty acids, obtained radiolabel would be more important when using [U-14C] acids.
Acknowledgements
The authors thank Dr B. Smith (Lund University, Sweden) for help in editing the English paper.
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![Fig. 3.Incorporation of various [1-14C] substrates into the defensive secretion of adult males of Eurycotis floridana according to time afterinjection](https://thumb-ap.123doks.com/thumbv2/123dok/3120834.1379306/5.598.53.548.372.705/incorporation-substrates-defensive-secretion-eurycotis-oridana-according-afterinjection.webp)
![Fig. 4.Amounts of labelled (hexenoic acid found in the defensive gland of adult male offloridanaE)-2-hexenal, (E)-2-hexenol and (E)-2- Eurycotis after injection of various [1-14C] labelled substrates](https://thumb-ap.123doks.com/thumbv2/123dok/3120834.1379306/6.598.47.289.62.451/labelled-hexenoic-defensive-oforidanae-eurycotis-injection-labelled-substrates.webp)
