043986

043986
 1338 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 61, No. 5
reported another culicine gynandromorph in which the Table 1Melting points of carbonyl compounds pro-
specimen was also anteriorly female and posteriorly male. duced by C. leclularius.
No observations were reported on internal reproductive
structures. Melting point in "C
Oliver and Delfin (1967) treated the ventral surface
Compound Known Unknown Mixed
of 21 S with a weak solution of "pholate-acetone,"
and reported the probable experimental production of 2
gynandromorphs in a Pacific Coast tick, Dermacenter
Ethanal 167-168.5 166.5-168.5 167 -168
2-butanonc 116-117 115 -117 115.5-117
occidenfalis Marx. In these ticks tlie mosaicism was A^-K-hexenal 147-148 146 -148 146.5-147
somewhat complicated. The individuals appeared as bi- A^it-octenal 127-128.5 127 -128 127 -128
lateral gynandromorphs from the dorsal aspect, but ven-
trally they were essentially female in appearance. Both
specimens had female-appearing genital regions. Both filtered through cheesecloth to remove tissue debris. The
individuals were mated to males. One produced eggs, a aqueous suspension containing the odorous material was
normal percent of which hatched, and the young ticks poured into a 2-liter boiling flask and steam distilled.
had regular larval and nymphal stages and matured into The steam distillate (about 500 ml) was then extracted
normal adults. Oliver and Delfin expressed no opinion with ether in a liquid-liquid extractor for 24 hr. A sat-
as to the mode of origin for their 2 specimens but gave urated solution of sodium bisulfite was added to the ether
a summary of reported ways through which gynandro- extract to remove carbonyl compounds. Following this
morphs might be assumed to originate. step, the mixture was shaken for 4 hr on a rotary shaker
and the organic and aqueous phases were then separated
REFERENCES CITED with a separatory funnel.
Lee, V. H. 1967. Gynandromorphism in the sabethine The ether extract containing any alcohols or esters was
Trichoprosopon digitatwn (Rondani). Mosquito dried over anhydrous magnesium sulfatc, filtered, and
News 27: 420-7. concentrated to a small volume, and then set aside for
Oliver, J. H., Jr., and E. D. Delfin. 1967. Gynandro- future analysis.
morphism in Dermacentor occidenfalis (Acari: Ixod- The sodium bisulfite solution was treated with an ex-
idae) Ann. Entoniol. Soc. Amer. 60(5) : 1119-21. cess amount of sodium carbonate and steam distilled, and
Smith, T. L. 1938. Gcnetical studies on the wax moth tlie liberated cnrhonyls were trapped in a receiver con-
Calleria melloneUd L. Genetics 23: 115-37. taining 2,4-dinitrophenylhydrazine (.0.2% in 2NHC1).
1965. External morphology of tlic larva, pupa, and
adult of the wax moth, Galleria melloiiella L. J. The solution containing the 2,4-dinitroplicnylhydra,iones
Kans. Entomol. Soc. 38: 287-310. (2,4-DNPH’s) was allowed to stand overnight at room
Taylor, D., K. Meadows, and N. Branch, 1966. Gynan- temperature. The resulting 2,4-DNPH precipitate was
dromorphism in Culex (Linnaeus) mosquitoes col- then filtered from the aqueous mixture, washed with
lected in the Tampa Bay area 1962 through 1964. 2NHC1, and then with water and dried. This 2,4-DNPH
Mosquito News 26: 8-10. precipitate was dissolved in a small amount of benzene:
heptane (1:1) and chromatographed by thin-layer chro-
matography. The filtrate remaining after the removal
of the 2,4-DNPH’s was extracted with 100 ml of petrol-
Carbonyl. Compounds Produced by the Bed eum ether and after separation the solution was again

/.Bug, Cimex lectularius1’2 extracted witli 100 ml of benzene. The petroleum ether
*.
Q Q_
and benzene extracts were then evaporated to dryness
under reduced pressure. The residue from each extract
R. P. COLLINS was dissolved in benzene ;heptane (1:1) and chroma-
Division of BiologyRegulatory Biology Section tographed by thin-layer chromatography. Preliminary
University of Connecticut, Storrs 06268 investigation revealed that the same compounds were
present in the 2,4-DNPH precipitate, the petroleum ether
The bed bug, dmex lectularius L., emits an odor extract, and the benzene extract; therefore, all the frac-
which was described by Kemper (1926) as an "obnoxious tions were combined.
sweetness." The scent apparatus, in adults, is situated in The unknown compounds were separated into classes
the metathorax at the base of the abdomen, against the following the method of Schwartz and Parks (1963).
inner face of the ventral wall. The scent apparatus con- Thin-layer plates (20 X 20 cm) were coated with mag-
sists of glands and a reservoir. nesia, spotted, and developed in chloroform :hexane
The analytical data presented by Schildknecht (1964) (85:15). In this procedure saturated aldehydes appear
suggest that C. lectularivs produces A^-it-hexenal and tan, 2-enats rust-red, 2,4-dicnals lavender, and methyl
A^n-octenal. In the present study tlic odorous constitu-
ents produced by C. lectularius were studied and the Table 2.Retention times of 2,4-DNPH derivatives
work of Schildknecht (1964) has been confirmed and from C. leclularins.
extended.
The bugs were cultured, using essentially the same Retentii>n time in
procedures described by Davis (1956). Several thou- miri;sec
sand mature insects were collected over a 12-month pe- Compound
riod and stored at 15C until used. The insects were derivative Known Unknown
ground in a chilled mortar containing 1000 ml of distilled
water and the resulting homogenate (?H 6.3) was Ethanal 3:12 3:12
2-butanone 4:10 4:12
1
Heniiptera: Cimicidae. A^t-hexenal 12:48 12:48
a Endorsed and communicated by Norman T. Davis. Accepted A^n-octenal 23:24 23:26
for publication February 26, 1968.
September 1968:1 SCIENTIFIC NOTES 1339
cedure described by Soukup et al. (1964). Identification
was made by comparing the retention times of the un-
known compounds with those of known compounds. The
instrument used was an F and M 5750. A flame ioniza-
00 00 tion detector was used and the column was 6-ft, ^-in.
00 00 stainless steel. The liquid phase was a 10% silicone UC
00 00 W98 coated on 80-100-mesh acid-washed, silanized
chromosorb W. Operating conditions were; column
temp 250C, injector temp 280C, detector temp 300C,
00 00 helium flow rate 30 ml/min, hydrogen flow rate
90 ml/min, air flow rate 500 ml/min, inlet pressure
50 psi, outlet pressure atmosphere, and sample size 2 or
3 iiiliter. Table 2 shows the results.
7 8
The 2,4-DNPH carbonyl derivatives were identified also
by comparing the Ri values of the unknown 2,4-DNPH
derivatives with the Rt values of known 2,4-DNPH
derivatives using thin-layer chromatographic techniques.
Fig. I.Thin-layer chromatogram of known 2,4- Fig. 1 shows these results.
DNPH derivatives and 2,4-DNPH derivatives obtained As final proof, the infrared spectrum of each of the
from C. lectularius. 1, Suspected ethanal; 2, known unknown 2,4-DNPH derivatives was compared with
ethanal; 3, suspected 2-butanone; 4, known 2-butanone; the infrared spectrum of. a corresponding authentic
5, suspected A^-hexenal; 6, known A^M-hexenal; sample, and the infrared spectra confirmed the identity
7, suspected A^i-octenal; 8, known A^-K-octenal; of ethanal, 2-butanone, A^n-hexenal, and A^-octenal.
9, mixture of unknown 2,4-DNPH’s; 10, mixture of
known 2,4-DNPH derivatives; (a = ethanal, b == 2- Each 2,4-DNPH derivative (10-40 nig) was mixed with
butanone, c = A^w-hexenal, d = A^K-octenal). Rela- 250 mg of anhydrous KBr; a few drops of spectrograde
tive R( values of a, b, c, and d are 0.30, 0.56, 0.65, and chloroform were added and the mixture ground to a fine
0.76, respectively. Solvent system petroleum ether :ethyl powder using a mullite mortar and pestle. The ground
acetate .-ether (90:5:5). Adsorbent silica gel G, layer mixture was then pressed into a pellet under pressure
0.5 mm thick. (20,000 psi for 5 min) with ,a 20-ton Loomis press.
Each KBr pellet was scanned (12-min scan) on a Perkin-
ketones gray. Thin-layer chromatography of the un- Elmer Infracord Model 137 B Spectrophotometer.
known mixture, on magnesia-coated plates, revealed 4 It has been shown in the present study that C. lectul-
spots. The color reactions indicated that 1 compound arius produces, in addition to A^ra-hexenal and A2-^-
was a saturated aldehyde, 2 were unsaturated aldehydes octenal, both ethanal and 2-butanone. The major con-
with 1 double bond, and 1 was a methyl ketone. stituent was A^it-hexenal and it undoubtedly contributes
The R( of unknown aldehydes and ketone 2,4-DNPH’s to the characteristic odor associated with this insect. The
were compared with the R< of known aldehyde and ke- other carbonyls also contribute to the overall flavor
tone 2,4-DNPH’s, to obtain a tentative identification of essence, and preliminary gas-chromatographic analyses
the compounds. Thin-layer chromatographic analyses re- suggest very strongly that components in the neutral
vealed 4 spots with Rt similar to ethanal, 2-butanone, alcohol and. ester fractions also contribute to the odor
A^-w-hexenal, and A^-octenal. produced by C, lectularius.
Preparative thin-layer plates coated with silica gel
G (0.5 mm thick) were then run to obtain enough ma- ACKNOWLEDGMENTS
terial for further analytical work. The solvent system
employed was petroleum ether :ethyl acetate :ethyl ether I thank Dr. Norman T. Davis of the Biology Depart-
(90:5:5), and the solution containing the unknown corn-, ment for help in rearing the insects and I thank also
pounds was applied to the plates by a Morgan applica- Mr. K. Kalnins and Miss Lisa Helmboldt for technical
tor (1962). The resulting bands were scraped from the assistance.
plates and eluted with hot ethanol. The silica gel was
-emoved from the solution by nitration and the nitrate REFERENCES CITED
was concentrated. Each compound was rechromato-
graphed separately on silica gel G plates to be certain Davis, N. T. 1956. The morphology and functional
that no impurities were present. The rechromatographed anatomy of the male and female reproductive systems
of Cimex lectularius L. (Heteroptera, Cimicidae).
bands were scraped from the plates and eluted with Ann. Entomol. Soc. America 49: 466-93.
hot ethanol. Water was added to the ethanol solution
until it became turbid and the solution was heated until Keniper, H. 1936. Die Bettwonze und ihre Bekampfung.
clear, then filtered and cooled. The solution was allowed Z. Kleintierk. Peiztierk. 12: 1-107. 18 abb. (Band
4 der Schr. Hgg. Zool.)
to stand overnight at room temperature; the resulting
crystals were collected by filtration. To obtain sharp Morgan, M. E. 1962. A device for simultaneous appli-
melting points it was necessary to recrystallize a 2nd cation of multiple spots on thin-layer chromatographic
time from hot ethanol, and in some cases it was neces- plates. J. Chromatogr. 9: 379-51.
sary to sublime the material to obtain sharp melting Morgan, M. E., D, A. Forss, and S. Patton. 1957. Vola-
points. The microsublimations were made on a Kofler tile carbonyl compounds produced in skim milk by
micro-hot state apparatus following the procedure de- high heat treatment. J. Dairy Sci. 40: 571-8.
scribed by Morgan et al. (1957). All melting points Schildknecht, H. 1964. Defensive substances of the
were corrected and obtained with a "RCH" Kofier arthropods, their isolation and identification. Angew.
micro-hot stage apparatus. Table 1 shows these results. Chem. Int. Ed. Engl. 3: 73-82.
The 2,4-DNPH’s of the unknown carbonyls also were Schwarrz, D. P., and 0. W. Parks. 1963. Thin-layer
separated and identified by the gas chromatographic pro- chromatography of carbonyl compounds; separation
 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 61, No. 5
1340
of aliphatic carbonyl 2,4-dinitrophenylhydrazones :nto
classes. Microchem. J. 7: 403-6.
Soukup, R. J., R. J. ScarpelUno, and E_Damelczik. W.
Gas chromatographic separation of 2,4-dimtrophenyl-
hydrazone derivatives of carbonyl compounds. Anal.
Chem. 36: 225-6.

Serial Sections of Pseudopityophthorus

spp.1.2
i i

3987 4 CHARLES 0. REXRODE AND

CHARLES R. KRAUSE3
are of
Oak bark beetles, Pseudopityophthorus spp.,
little importance as forest pests since they attack small
dead and dying branches of oak. However, they -iaye
the past 12
been the object of much investigation during
years as possible vectors of the oak wilt fungus, Cerato-
Berry
cystis fagacearum (Bretz) Hunt (Rexrode 1968).
ot
and Bretz (1966) and others found that when adulis
(Zimmermann) emerge from a disease
P minutissimus
fungus on
source they carry some form of the oak wilt
or within their bodies. But we do
not know wher-; on
their bodies and how the beetles carry the pathogen
In our studies on the internal anatomy of oak bark
would
beetles we needed a sectioning technique that
have any
make it possible to find whether the adults
special structures for carrying the fungus.
Because
larvae and adults are small (1-2 mm long) and the
not
adult integument is very chitinous, dissection was
practical. . , .

.
Kennedy (1949) gave several methods for studying the
internal anatomy of soft-bodied insects. However, _no
chitm
method has been found for softening sclerotized
without injuring the softer tissues to some extent.
Daven-
port (1960) stated that Diaphanol (dihydrochloracefcic FIG. 1 (top).Sagittal view through proventnculus of
dam-
acid) will soften exoskeleton sufficiently with little Pseudopityophthorus pruwosiis, x50.
no long;r on
age to soft tissues. However, this reagent is FIG. 2 (bottom).Frontal view of a proventncular plate
the market. Gray (1954) recommended 10% scdmm oiP.prwnosis, X500.
hydroxide, 6% sodium hypochlorite, and an equal-parts
mixture of chloral hydrate and phenol to soften the exo-
Bouin’s* fixative (Guyer 1953) at 55C for 8-15 min.
skelton, but he states that this mixture does not work Place, in a vacuum oven at 55C. and infiltrate 3 times,
well on all insects. Storch and Chadwick (1964) pre- releasing the vacuum gradually from 25 in. of mercury to
pared serial sections of whole insects using a modification atmospheric pressure in approximately 10 min.6
of the double-embedding method, but they did not name 2. Remove beetles from Bouin’s fixative and wash 3
the insect used in their study.
times in 70% ethyl alcohol. Allow 30 min/wash in 25"C
Carlisle (1960) successfully softened with chitmase the mercury."
vacuum oven at 24 in of
integument of the copepod Calanus, the prawn Pala’mon,
3. Place beetles in 95%, then in 100% ethyl alcohol, and
’
and the insect Locusta. Chitinase is an extraction of the
then in ethyl ether" (1 min in each). Blot on filter paper
common commercial mushroom in a 35% saline solution.
and quickly return to 100% ethyl alcohol.
It acts cnzymatically on the chitin of the integument and 4. Pass beetles through 100, 95, 70, and 35% ethyl
not on the protein constituents (Carlisle 1960). We used
alcohol and distilled water (1 min in each). Then place
chitinase as a softening agent, and have developed a 37C.
in buffered chitinase7 solution for 48 hr at
technique for producing serial sections of oak bark beetle
5 Remove insects from chitinase solution and wash in
larvae and adults. The technique may be adaptable to distilled water. Pass through 35, 70,5 85, 95, and 100%
other scolytids. ethyl alcohol (1 hr in each).
The following procedure will produce good sections
(Fig. 1, 2). Timing of each step in the technique .s im- ^Alcoholic Bouin’s fixative (Smith’s modification): Picric acid
portant. (saturated aqueous solution) 45 parts, ethyl alcohol (95%) acid 45

1. Kill and fix beetle larvae and adults in hot alcoholic parts, Formaline (reagent grade) 5 parts, glacial acetic
S parts. .. , ,
-
.
.
-
6 Beetles can remain in this solution overnight.
0 The ether helps dissolve certain lipids that are structural

Coleoptera: Scolytidae.
1 constituents of the chitinous integument. .
., . ,,, ,
1 Chitinase: 100 g mushrooms steeped overnight in 100 ml
Accepted for publication February 28, 196S.
2
the residue, store
35% w/v sodium chloride solution; centrifugewith
.
Research Entomologist and Biological Laboratory techni-
3
extract at 42C. Dilute when using at 1:5 acetate butter.
cian respectively. Forest Insect and Disease Research Labora-
tory; Forest Service, U.S. Department of Agriculture, Delaware, Acetate buffer: 7 ml of 0.2 M sodium acetate 13.01 g/500 ml
water + 3 ml of 0.2 M glacial acetic acid 6.005 g/500 ml water.
Ohio.