602 J.-P. Farine et al. Insect Biochemistry and Molecular Biology 30 2000 601–608
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, 185618 whereas linolenic C18:3 and stearic C18:0 acids represent only a few
percent respectively, traces4 Bade, 1964. After ingestion of [1-
14
C] acetate, both sexes are able to biosynthesize by condensation of C
2
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 of E. 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 500
µ l of distilled methylene
chloride CH
2
Cl
2
and 5 µ
g 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., 0.22
µ m 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 mlmin and a septum purge of 3 mlmin.
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 °
Cmin for one min, and then at 2
° Cmin to 240
° C. Hydrogen was used as carrier gas
50 cms 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
µ g. 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
° Cmin. The injector and detector temperatures were
250 and 270 °
C, respectively. Helium 20 mls was used as carrier gas. The GC column effluent was split in a
ratio of 18 vv to the detector and a trap glass tube, 30 cm
× 1 mm i.d. cooled with liquid nitrogen. The tubes
were immediatly washed using 500 µ
l 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 Sodium [1-
14
C] acetate 56.8 mCimmol, [1-
14
C] pal- mitic acid 57 mCimmol, [1-
14
C] stearic acid 58 mCimmol, [1-
14
C] oleic acid 56 mCimmol, [1-
14
C] linoleic acid 53 mCimmol and [1-
14
C] linolenic acid 52 mCimmol were obtained from NEN Research Pro-
ducts. The major components of the secretion [E-2-
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
