603 J.-P. Farine et al. Insect Biochemistry and Molecular Biology 30 2000 601–608
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 1
µ Ci of one of the studied precursors
in 5 µ
l dimethylsulfoxide DMSO, Sigma using a 10 µ
l Hamilton syringe. The needle was inserted just under the
cuticle between the 3
rd
and 4
rd
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
µ l
of acetonitrile. Two microliters of each of the extracts were analyzed by GC for quantification of the major
components. An aliquot of 20 µ
l 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
µ l 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; 10,000 g, 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
14
C. As a high percentage of injected radioactivity was lost as CO
2
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 gland2stern-
ite blankremainder 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
Fig. 1. Production of the three major components found in the
defensive glands of adult males of Eurycotis floridana. E-2-hexenal and its corresponding alcohol and acid were quantified by GC at vari-
ous intervals after the imaginal moult. Each point is an average mean
± S.E.M. of five samples.
604 J.-P. Farine et al. Insect Biochemistry and Molecular Biology 30 2000 601–608
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 µ
g 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 µ
l 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 of Eurycotis 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 µ
g per gland, respectively, which represent, respectively, 0.43, 0.84
and 9.03 of the corresponding compounds encountered in a control male.
The amount of the aldehyde and the alcohol slowly increased during the first day. Then, the percentage of
each of the compounds in the secretion became more important on and after the second day. One hundred per-
cent of the aldehyde was regenerated after 15 days whereas complete recovery of E-2-hexenol was only
observed at 30 days. Compared to the normal filling rate of the gland after the imaginal moult, the regeneration
of the acid was lower during the first two days 3.2 regenerated after 1 h and 10 after two days and
increased spectacularly
after five days 47
of regeneration. One hundred and thirty percent of the con-
trol amount of the acid was regenerated after 10 days. However, after 30 days regeneration, all the individuals
restored their original secretion, i.e. 28.08 ±
0.44 mg versus 26
± 0.14 mg in control males E-2-hexenal,
770.4 ±
19.65 µ
g versus 866.33 ±
5.58 µ
g E-2-hexenol,
605 J.-P. Farine et al. Insect Biochemistry and Molecular Biology 30 2000 601–608
and 89.6 ±
1.87 µ
g 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 [
14
C]-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-
14
C] 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-
14
C] substrates into the defensive secretion of adult males of Eurycotis 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
µ g, and about 7 to 10 times less
when using
C18:1 21.06
± 2.16
dpm µ
g, C18:2
17.94 ±
1.55 dpm µ
g, C18:3 14.84 ±
1.29 dpm µ
g and C16:0 18.91
± 1.08 dpm
µ g 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
µ g,
respectively. We did not detect any radiotracer incor- poration of any of the precursors into E-2-hexenoic
acid. After a 6 h period Fig. 4B, there were general
increases of detected radioactivity in the aldehyde and
606 J.-P. Farine et al. Insect Biochemistry and Molecular Biology 30 2000 601–608
Fig. 4. Amounts of labelled E-2-hexenal, E-2-hexenol and E-2-
hexenoic acid found in the defensive gland of adult male of Eurycotis floridana
after injection of various [1-
14
C] 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
µ g.
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 µ
g. E-2-hexenal and E-2-hexenol were about three times less labelled compared to the values observed
at 6 h.
4. Discussion
