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On the chemical structure of the apical droplet from eggs of Culex pipiens
r. Insect Physiol., 1973, Vol. 19, pp. 861 to 875. Pmgamon Press. Printed in Great Britain
ON THE CHEMICAL STRUCTURE OF THE APICAL DROPLET FROM EGGS OF CULEX PIPIENS ARNON
AHARONI”
and GUNTER
ZWEIGt
University of California, Davis, California, U.S.A., and Life Science Division, Syracuse University Research Corporation, Syracuse, N.Y. (Received
19 June 1972)
Abstract-The chemical nature of the apical droplet from eggs of Culex pip&as was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12/%hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14p-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with P-OH myristic acid. Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive 32P0,-3 and 35S04-2’ showed no significant incorporation of phosphorus, but a sulphur-containing aniohic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as monoand diglycerides. The structural evidence explains in part the surfactant properties of the apical drop. INTRODUCTION
EGGS of C&x and Culiseta mosquitoes exude microscopic droplets of a clear liquid at their posterior end above the water surface on which the eggs float as rafts composed of several hundred of eggs. These droplets were first described by GOELDI (1905) and were later shown by ILTIS and ZWEIG- (1962) to possess extremely high surfactant properties. These latter authors also tentatively, identified the droplet as a non-phosphorous glyceride which on acid hydrolysis yielded several long-chain fatty acids. The present report is a detailed study of the chemical composition of the apical droplet from eggs of Culex pipiens, including an investigation of the homogeneity and elemental composition of the droplet. MATERIALS
AND
METHODS
Rearing of the mosquitoes The mosquitoes were reared by a modified method of NEEDHAM et al. (1959), starting a colony from the eggs of an autogenous strain of Culex pipiens pipiens (L.). * Deceased. t Requests for reprints should be addressed to Gunter Zweig at Syracuse, New York. 861
862
AFCNONAHARONI ANDGUNTERZWEXG
Larvae were grown in small Pyrex glass dishes (15 x 18.5 in.) filled with tap water which was continuously aerated. A total amount of 3 g of high-energy, proteinrich food was supplied. After pupation, about 400 pupae were transferred to a glass dish containing about 250 ml of tap water and placed in a screen-covered wooden cage. The adults were reared at 23°C and 75% r.h. Sugar was supplied by the method of ELIASON (1963) by depositing the sugar containing a red food colour on a microscope slide. In some experiments H,35S0, (4 mc) was incorporated into the sugar. For 32P-studies, 3 ml of 10% (w/v) sucrose solution containing 20 PC of Hs32P0, was poured into a 6 ml. test tube stoppered with a cotton plug and placed in a cage. Blood meals were given once every 4-5 days by placing a featherless chicken (kindly supplied by Dr. Ursula Abbott, Poultry Department, University of California, Davis) into the cage for a period of 8 hr.
Collection of the apical droplets Newly laid egg rafts were transferred daily to Petri dishes lined with Whatman No. 1 filter paper saturated with water. The dishes remained covered for 24-30 hr. The droplets which formed during this period were collected under the dissecting microscope by the method of ILTIS and ZWEIG (1962) by touching the apical droplets with the tip of a small glass rod. The droplets which adhered to the glass tip by surface tension were washed into a 15 ml centrifuge tube with several drops of chloroform and stored in solution until future use. If material was needed for analyses, the chloroform was evaporated under an air stream at 45 to 50°C. The average yield of apical droplets was about 861 pg per hundred egg rafts.
Preparation of methyl esters from droplet hydrolysate The methanol-HCI-method of STOFFEL et al. workers (1959) was employed. About 1 mg of apical droplets was dissolved in 4 ml of O-1 N HCl in anhydrous methanol prepared according to VOGEL (1959). The solution was refluxed for 1 hr at 70” to 80°C in a glycerol bath and concentrated to 1.5 ml under a stream of nitrogen. Two millilitres of distilled water were added and the methyl esters extracted with three 7 ml portions of petroleum ether (30” to 60°C). The petroleum ether fraction was concentrated to about O-1 ml in a stream of nitrogen, and aliquots were analysed by gas-liquid chromatography (see below).
Gas-liquid chromatography (GLC) For the separation and identification of the methyl esters of the fatty acids the following equipment and conditions were used: Aerograph Model A-600B gas chromatograph (Varian Company) ; hydrogen flame ionization detector; 1 mV Leeds and Northrup recorder; 5 ft stainless steel column, + in. o. d., packed with 20% (w/w) glycol adipate polymer (LAC 446) on 30/60 mesh Chromosorb; nitrogen gas flow, 34 ml/min; hydrogen flow through detector, 25 ml/min. One microlitre of the hydrolysate in petroleum ether, corresponding to 10 pg of apical drop material, was chromatographed isothermally at 190 _F1°C. Under these experimental conditions a mixture of known methyl esters of fatty acids could
THEAPICAL DROPLET FROMEGGSOFCULEX
PIPIENS
863
be resolved consisting of saturated Crs, C14, Crs, C,, /?-OH-C,,, and /&OH-C!,, fatty acids. Preliminary identification of unknown methyl esters was made on the basis of retention times. Relative amounts of fatty acids were made by measuring peak areas with a planimeter. Absolute quantitative determination of /Lmyristic acid was made by comparing peak area with that of standard amounts of authentic material.
Thin-layer chromatography This technique was employed for testing the homogeneity of the original apical drops and for a confirmatory test of P-myristic acid. Chromatostrips, 14 x 1.3 x O-2 cm, were coated with silica gel G (Research Specialties Company) and dried for 30 min at 120°C. The following solvents were used to develop apical drops: (a) chloroform-methanol-water, 60 : 30 : 4, v/v; (b) benzene-acetone, 70 : 30, v/v. For methyl esters of fatty acids, ethyl acetate was used as solvent (TSCHESCHEet al., 1961). For the detection of apical drops, 2’,7’-dichlorofluorescein (O*21o/ow/v in methanol) or iodine vapours were employed (MANGOLDand MALINS, 1960). To detect the methyl esters of fatty acids a spray of 0.04% (w/v) bromthymol blue in 0.01 N NaOH was used (JATZKEWITZand MEHL, 1960).
PwiJication of hydrolysate The aqueous phase after the extraction of the methyl esters of fatty acids from the hydrolysate was passed through a 25 x 1 cm column containing about 25 g wet wt. of Dowex 1-4X, 50 to 100 mesh, hydroxyl form. Collection was started when the pH of the eluate became basic and was continued with repeated water washes until it returned to neutral pH. The combined eluate was continuously stirred with a sufficient amount of Dowex 50-4X-H+ until the pH had dropped to 3. (The final solution did not contain any H&SO, as tested with saturated BaCI,.) The water was removed by lyophylization, and the residue dissolved in about 1 ml of absolute methanol. Aliquots of this solution were chromatographed on paper as described below.
Identification of glycuol by paper chromatography Ascending paper chromatography using Whatman No. 1 paper and 1-butanol saturated with water were employed (BLOCK et al., 1958). Reducing compounds such as glycerol were detected by dipping the developed chromatogram into the silver nitrate-acetone reagent (BLOCK, et al., 1958) followed by spraying with O-5 N NaOH in 95% (v /v ) eth anol. The brown background of the paper could be cleared up by washing the chromatogram with a photographic fixer solution.
Separation of hydroxy-fatty
acids
A modification of the method for the separation of methyl and hydroxy-fatty acids by KANESHIROand MARR (1963) was graphic column (8.75 x l-5 cm) fitted with a 100 ml reservoir of silicic acid, 325-mesh (Bio Rad Lab.) and washed with about
esters of fatty acids used. A chromatowas filled with 5 g 150 ml of petroleum
864
AFCYON AHARONIAND GWNTERZWEIG
ether (30 to 60°C). The solution to be resolved was dissolved in 4 ml of petroleum ether and poured onto the column. The methyl esters of the fatty acids were eluted with 38 ml of petroleum ether-dðyl ether,’ 96 : 4, v/v. The methyl esters of the hydrox-fatty acids were taken off the column with 30 m of diethyl ether. Hydrogenation of methyl esters of unsaturated fatty acids The catalytic hydrogenation was based on the method by FARQUHARet al. (1959). About 1 mg of methyl esters from the apical drop hydrolysate in 5 ml of petroleum ether and 12 mg of platinum oxide (Adam’s catalysts; Matheson, Coleman, and Bell) were placed into a 10 ml beaker within a 1 litre suction flask. The suspension was continuously stirred with a magnetic stirrer. A partial vacuum of 250 mm Hg was applied to the suction flask and hydrogen admitted to 1 atm. This step was repeated after 5 min and stirring continued for 3.5 hr. The suspension was filtered through Whatman No, 1 paper (caution! PtO, catalysts are a fire hazard when allowed to dry on paper). The paper was washed with 7 ml of petroleum ether and the filtrate evaporated to about O-1 ml under nitrogen. Aliquots of this concentrate were analysed by gas-liquid chromatography (see above). RADIOCHEMICAL
TECHNIQUES
Liquid scintillation countilzg 32P- and 35S-Samples were counted with a Packard Tri-carb semi-automatic liquid scintillation spectrometer at a voltage of 900 V. Two counting solutions were used: non-aqueous (HAYES, 1962)-0.5% (w/v) 2,5-diphenyloxazol (PPO) and 0.03% (w/v) 2,2-p-phenylene-bis-(5-phenyloxazole) (POPOP) in redistilled toluene; aqueous (Y. Vadya, personal communication)-15 g of PPO, O-4 g of POPOP, 150 g of naphthalene dissolved in dioxane-toluene-ethanol(ll40 : 1140 : 720, v/v). Autoradiographic
technique
The developed chromatostrips were mounted into a cut-out section of a larger piece of cardboard, so that the glass and cardboard surfaces were flush. This was then loaded with Kodak Medical X-ray film in a light-tight holder, and after 2 to 3 weeks’ exposure, the film was processed by normal darkroom procedures. RESULTS Table 1 and Figs. 1 and .2 demonstrate that by esterification with methanolhydrochloric acid the main components of the apical drop among the fatty acids are P-OH myristic acid (72%) and P-OH 1auric acid (13.34%). It should be noted that there is a small amount of unsaturated fatty acids (see starred peaks in Figs. 1 and 2). A method in which the hydrolysate is esterified with diazomethane yielded much smaller quantities of B-OH lauric acid (8*6o/o) and P-OH myristic acid (13.8%). Three unsaturated fatty acids were found when this method was used with retention times between those of lauric and palmitic acids which are probably
X65
THE APICAL DROPLET FROM EGGS OF. CULEX PIPIENS
C,, and CT,, unsaturated fatty acids. It is possible that the change in the relative amounts of P-OH and unsaturated fatty acids following esterification with diazomethane was due to dehydration (VORBECK et al., 1961). TABLE
1. FATTY ACID COMPOSITION
OF APICAL DROP
Fatty acid
%
Laurie Myristic Palmitic Stearic B-OH Laurie B-OH Myristic Unsaturated C,, and Cl4 Unknown
0.16 0.23 8.93 4.75 13.34 72.00 0.39 0.26
TIME IN MINUTES
FIG. 1. GLC hydrochloric
showing acid,
fatty acids in apical drop material and
those
of
a standard
0.
(b) esterified
* Unsaturated
by methanol-
C,,
and C,,.
Hydrogenation of the fatty acids The two starred peaks in Figs. 1 and 2 were believed to be unsaturated fatty acids. Hydrogenation of standard methyl linoleate to stearic was achieved by the technique described in Materials and Methods, and this method was employed to determine if these unknown peaks actually corresponded to unsaturated fatty acids. Following methylation, the solutions were hydrogenated. Fig. 3 demonstrated
ARNON AHARONIAND
866
GUNTER~WEIG
Mixture of standard with .pmI Column remp: 183°C Gas f&v: 25 ml/nlin. Attenuation: 8-X
I 9
I 12
15
TIME
drops material
I 9
I 3
IN MINUTES
FIG. 2. GLC of mixture of fatty acids in apical drop esterified by methanolhydrochloric acid and fatty acids of a standard; chart paper run simultaneously. * Unsaturated C,, and C,,.
C&OH
Sample: Column. temg: Gas flow: Attenuation:
-S.mple: Conditions:
Apical drop material 183°C 28 ml/r&. 8-X
4-S&M
Apical drop materiot after hydrogenation Sme as obore CM
TIME
Cl4
I
I
b
3
0
IN MINUTES
FIG. 3. GLC showing hydrogenation of fatty acids in the apical drop material esterified by methanol-hydrochloric acid. * Unsaturated C,, and CT,,. ? Probably impurities of the solvent.
THE
APICAL
DROPLET
FROM
EGGS
OF
CULEX
PIPIENS
867
that these components in fact were converted into C,, and C,, saturated fatty acids indicating that unsaturated fatty acids were present in the original mixture. IDENTIFICATION
OF HYDROXY
FATTY
ACIDS
The total quantity of suspected hydroxy fatty acids was estimated to be about 85 per cent of the total fatty acids (see Fig. 1 and Table 1). Further tests were run to confirm the identity of the two hydroxy fatty acids. Identification
by column chromatography
When a standard mixture of aliphatic and hydroxy fatty acids was placed on a silicic acid column, the aliphatic acids were eluted with a solvent composed of petroleum ether and diethyl ether (96 : 4, v/v) and the more polar hydroxy fatty acids were eluted with diethyl ether alone. The chromatogram in Fig. 4 demonstrates that P-OH lauric and 85 per cent of /?-OH myristic acid were recovered almost quantitatively from the apical drop material by this technique.
TIME IN MINUTES
4. GLC chromatogram showing fractionation of apical drop material esterified by methanol hydrochloric acid by column chromatography. (A) Petroleum ether-diethyl ether (96 : 4, v/v) elution. (B) Diethyl ether elution. (C) Total fatty acids. FIG.
IdentiJication. of B-OH my&tic
acid by thin-layer
The methyl ester of standard P-OH
chromatography
myristic
acid together
with the methyl The chromatostrips were developed with ethyl acetate (TSCHESCHE, 1961) and sprayed with bromothymol blue. The R, value of the standard P-OH myristic acid was found esters of the fatty acids of the apical drops were chromatographed.
27
ARNON AHARONIAND GUNTERZWEIG
868
to be identical with that of the largest component of the fatty acids of the apical drops. (R, = O-65.) Thin-layer chromatogram is not reproduced. Quantitative
estimation
of hydroxy
myristic
acid
A 1% aliquot of the fatty acid fraction from 1.00 mg of apical material (collected from 115 rafts) was hydrolysed, esterified, and injected into the gas-liquid chromatography column. The peak area was equivalent to an area of a peak formed by 3.46 pg of standard B-OH myristic acid. Thus, the total P-OH myristic acid content in 1.00 mg. of apical drop material was estimated to be 35 per cent.
ANALYSIS
Preparation
OF GLYCEROL
of the apical drop material for glycerol analysis
Apical drop material (about 3 mg) collected from 300 rafts was esterified using diazomethane following reffuxing with sodium methylate in methanol Extraction of the fatty acid methyl esters with petroleum ether would leave the neutral water-soluble fraction in the aqueous phase. Sodium bisulphate which was added during the esterification interfered in subsequent chromatography and had to be removed by ion exchange. The aqueous solution which eluted from an anion exchange column (Dowex 1) was collected when the pH became basic. Since sodium hydroxide was eluted from the column simultaneously with glycerol, the rise in pH indicated the elution of NaOH and, consequently, the emergence of the glycerol from the column. Following the removal of sodium ions by mixing with a cation exchanger (Dowex 50), the aqueous solution was lyophilized, and the suspected glycerol residue redissolved in absolute ethanol for paper chromatography. Analysis
of the glycerol by paper chromatography
The alcoholic solution containing non-polar simultaneously with a standard of glycerol on chromatograms such as the one shown in Fig. that the colour density of the spot from the apical drop was approximateIy twice as intense an estimated concentration of 30%. Fractionation
of apical drop material
by thin-layer
substances was chromatographed Whatman No. 1 filter paper and 5 were obtained. It was observed ethanolic fraction of about 3 mg as that of a 50 pg standard, giving
chromatography
Chromatostrips of the apical drop material were prepared using two different solvent systems. Fig. 6(a) shows fractionation of the apical drops into four major components with benzene-acetone as a solvent system, while with the chloroformmethanol-water solvent, the apical drop material was fractioned into three components (Fig. 6b). It may be seen that fractions I + II -I- III are much slower in the benzene-acetone solvent than in the chloroform-methanol-water solvent. The R, of fraction IV in the benzene-acetone solvent was about the same as that of fraction III in the chloroform-methanol-water solvent.
THEAPICALDROPLETFROM
EGGS OF
CULEXPIPIENS
869
In order to obtain information on the chemical nature of the components of the apical drops, each component was sprayed with three different colour reagents. Positive reactions to each of the colour reagents by each of the components was observed ; bromothymol blue may detect O-1 to l-0 pg of most lipids (JATZKEWITZ and MEHL, 1960). 2’,7’-Dichloroflurescein was used to detect the presence of non-polar lipids (MANGOLD and MALINS, 1960). The spots were also exposed to iodine vapours (MANGOLD and MALINS, 1960). The positive reaction of the last test indicated that these fractions contained unsaturated lipids (MANGOLD, 1961). (Q) Jl
Sol*ent
Origin
front
J
III
II
I
Y
FIG. 6. Thin-layer chromatostrips of the apical drop material. (a) Chromatostrip run with benzene-acetone (70 : 30, v/v). (b) Chromatostrip run with chloroform-methanol-water (60 : 30 : 4, v/v). Shaded areas represent spots appearing on autoradiogram, from 35S-label. ANALYSIS
FOR PHOSPHORUS
AND
SULPHUR
Analysis of phosphorus ILTIS and ZWEIG (1962) reported several tests for the analysis of phosphorus in the apical drops. Mosquitoes were fed either with 10% sucrose solution containing 10 PC H,s2PO,/ml or on a chicken which had previously been injected intravenously with 2.5 mc of Na,ssPO,. The apical drops collected from these mosquitoes failed to show any significant radioactivity. To confirm these results, mosquitoes in these studies were fed with 10% sucrose solution containing 7 PC H332P0,/ml. The eggs and the apical drops were counted separately in a liquid scintillation spectrometer. Table 2 indicates that even though the eggs showed significant radioactivity, no significant counts were detected in the apical drop material.
Analysis of sulphur ILTIS and %vEIG (1962) proposed that the apical drop may contain a sulpholipid. The above assumption was proposed on the basis of a comparison of the
infrared absorption spectrum of the apical drop with that of 6-sulpho-6-deoxy-dn-gluco-pyranosyl diglyceride which has been isolated from photosynthetic algae. High surfactant properties have been ascribed to this compound by BENSON, et al. (1959). Radioactive tests and thin-layer chromatography were carried out in order to establish the presence and the behaviour of sulphur in the apical drop material.
ARNONAHARONIANDGUNTERZWEIG
870
TABLE 2. RADIOACTIVE ANALYSISFOR 35SAND32P*
Sample
Isotope
Background of the counting solution (c.p.m.)
Sample activity (c.p.m.)
32P 32P 3% 3%
20 24 22 35
2217 27 3236 217
9 egg rafts Apical drops from 15 rafts 9 egg rafts Apical drops from 1.5 rafts
* These results represent average of two trials.
Mosquitoes were fed on a dry sugar containing H,35S0,. The apical drops which were dissolved in chloroform were counted in a non-aqueous counting solution in a liquid scintillation spectrometer. It may be seen from Table 2 that both the eggs and the apical drops showed counts well over the background of the counting solution. These data indicated that some sulphur-containing compound was present in the apical drops. Autoradiography was carried out (CHASE, 1958) in order to locate the a5S component by fractionation of the apical drops. Fig. 6(b) shows that when chloroform-methanol-water (60 : 30 : 4) was used, the 35S component partially covered spot I on the chromatostrip. In order to determine if the sulphur-containing material actually was identical with spot I, the apical drop material was chromatographed with a second solvent consisting of benzeneacetone (70 : 30, v/v). With this solvent (Fig. 6a) the sulphur-containing compound remained at the origin. Further investigations of the sulphur-containing fraction were carried out. Sixty rafts were collected from mosquitoes which were fed on sucrose containing
z f0 .-
2
““‘I
4
6
8
IO
12
14
v’+,y--1;1.2
4
6
8
Wavelength
IO Microns
FIG. 7.
12
14
I
871
FIG. 5. Glycerol analysis by paper chromatography. (1) Ethanolic solution containing glycerol from hydrolyzed apical drop from 300 egg rafts. (2) 5 pg of standard glycerol. (3) 10 pg of standard glycerol. (4) 15 pg of standard glycerol. (5) 50 ,ug of standard glycerol.
THE APICALDROPLETFROM EGGSOF CULEX
873
PIPIENS
Hz5S0,. The apical drop material was collected and refluxed with methanol and 0.5 N sodium methylate. The aqueous solution after extraction of the fatty acids by petroleum ether was passed through an anion exchange column (Dowex-1) followed by mixing the elute with a cation exchanger (Dowex-50). Both the saltfree aqueous solution and the petroleum ether extract were analysed for 35S. No counts above the background of the counting solution were observed in any of the factions. The anion exchange column was then washed with 10% KOH, and this solution showed 202 counts while the background showed 95 counts. INFRARED
SPECTRUM
OF THE
APICAL
DROP
Table 3 shpws the infrared spectrum of the apical drop material and that of an SZq/, pure sample of glyceryl monoricinoleate. The 5.75~ band corresponds to an ester linkage and coincides with the absorption band found by ILTB and ZWEIG (1962) in their investigation of the apical drop. The bands at 2.95, 6.92, and TABLE 3. INFRARED ABSORPTION SPECTRA OF APICAL DROP MATERIAL AND OF GLYCERYLMONORICINOLEATR
Conditions of operation Origin Purity
Prism Resolution Response Gain Speed Suppression Scale
Apical drop material Davis, California Natural product film from solution on 1.5 mm KBr NaCl 927 x 2 11.00 2.0 16 8 lx
Glyceryl monoricinoleate Calbiochem 82% (Grade C). 1.5 mm KBr
Solid on
NaCl 980 11.00 4.0 8 All lx
846 TVmay indicate secondary alcohol groups of the hydroxy fatty acids. The absorption peaks at 6.35, 6.81, and 10.55 ,u indicate the presence of unsaturated fats in the apical drops. ,Although the exact nature of the primary alcohol group (9.5 or 9.7’2 p) is unknown, it is possible that these peaks may be due to mono- or diglycerides. HYPOTHESIS
ON CORRELATION BETWEEN CHEMICAL STRUCTURE PHYSICAL PROPERTIES OF APICAL DROP
AND
An interesting physical phenomenon noted by ILTIS and ZWEIG (1962) is the effect of the apical drops on the surface tension of an aqueous solution. Their measurements showed that the apical drops from eggs of C. pi@r~ lowered the surface tension of water from an initial value of 70.05 dynes/cm at 21°C to 32.1
874
ARNON
AHARONIAND GUNTERZWEIG
dynes/cm. The surface tension then rose within 90 min to a constant value of 66 dynes/cm. It is of interest to correlate the chemical structure of the apical drop with its surfactant properties. The interfacial tensions between long-chain aliphatic alcohols and acids on the one hand and water on the other are about 10 to 15 dynes/ cm (GLASSTONE, 1958). Thus, it is likely that the hydroxyl and the carbonyl groups which are abundant in the apical drop material give rise to this surfactant property. The sulphur-containing compound in the apical drop material may also play an important r81e in lowering the surface tension, as some sulphur-containing compounds have surfactant properties. The subsequent increase of the surface tension of the water from a minimum of 32.1 dynes/cm to a constant value of 66 dynes/cm indicates that the surface-active ingredient of the apical drop is soluble in water. The overall relationship between the physical, chemical, and physiological characteristics of the apical drop should lead to a further understanding of the biological nature of this material. It is possible that the property of the apical drop is important biologically in protecting the mosquito egg from adverse environmental conditions such as inversion during stormy weather. It is also possible as JLTIS (personal communication) indicated that the apical drop serves to protect the egg from microbial or fungal contamination. HINTON (1968) demonstrated that the apical droplet contained an ant-repellant substance and ascribes its formation to a defence mechanism against possible predators. Acknowledgements-This paper is taken from the thesis by Arnon Aharoni, University of California, 1964. We thank the American Cyanamid Company for a grant-in-aid which made this research possible. We also want to express thanks to the following people for technical advice and assistance during the course of this research: Professor F. N. STRONG, Messrs. FRED ILTIS, PAUL ALLEN, BASIL BOWERS,and DAVID L. GUTNICK,all at the University of California, Davis, where this work was completed. This paper was submitted at the urging of Professor MURRAY S. BLUM at the University of Georgia, although the work was completed in 1964. REFERENCES BENSONA. A., DANIELH., and WISERE. (1959) A sulfolipid in plants. Proc. nut. Acad. Sci. U.S.A. 45, 1582-1587. BLOCKR. J., DURRUME. L., and ZWEIG G. (1958) A Mum& of Paper Ckromatograpky and Paper Electrophoresis, pp. 178, 180, 202. Academic Press, New York. CHASEG. D. (1959) P?%ziples of Radioisotope Methodology, pp. 216-218. Burgess, New York. ELIASOND. A. (1963). Feeding adult mosquitoes on solid sugar. Nature, Lond. ZOO, 289. FARQUHAR J. W., INSULLW., ROSENP., STOFFELW., and AHRENSE. H. (1959) The analysis of fatty acid mixtures by gas-liquid chromatography. Nutrition Rev. (suppl.) 17, 29-30. GLASSTONES. (1958) The Elements of Physical Chemistry, p. 151. Van Nostrand, New Jersey. GOELDIE. A. (1905) OS mosquitos no para. Mem. Mus. Goeldi (Mus. Paraense) de Hist. Nat. e Ethnographia, IV. HAYES F. N. (1962) Solutes and solvents for liquid scintillation counting. Packard Tech. Bull. 1, 5. HINTON H. E. (1968) Structure and protective devices of the-egg of the mosquito Culex pipiens. J. Insect Physiol. 14, 145-161.
THE APICALDROPLETFRO&fEGGS OF CULEX
PIPIENS
875
ILTIS W. G. and %&ZIG G. (1962) Surfactant in apical drop of eggs of some culicine mosquitoes. Ann. ent. Sot. Am. 55, 409-415. JATZKEWITZH. and MEHL E. (1960) Thin layer chromatography of brain lipides-hydrolytic and breakdown products. Hoppe-Seyler’s 2. physiol. Chem. 320, 251. KANESHIROT. and MARR A. G. (1963) Hydroxy fatty acids of Azotobacter agilis. Biochim. Biophys. Acta 70, 271-277. MANGOLDH. K. and MALINS D. C. (1960) Fractionation of fats, oils, and waxes on thin layers of silicic acid. y. Am. Oil them. Sot. 37, 383. NEEDHAMJ. G., LUTZ F. E., WELCH P. C., and GALTSOFF P. S. (1959) Culture Methodsfor Invertebrate Animals, pp. 386-388. Dover, New York. STOFFEL W., CHU F., and AHRRNSE. H. (1959) Analysis of long-chain fatty acids by gasliquid chromatography. Analyt. Chem. 31, 307-308. TSCHESCHER., LAMPERT F., and SNATZKEG. (1961) Dtinnschicht- und Ionenaustauscherpapier-Chromatographie von Triterpenoiden. J. Chromatography. 5, 217. VOGEL A. I. (1959) A textbook of Practical Organic Chemistry, p. 79. Longmans, Green & Co., London. VORBECKM. C., LEONARDM. R., LEE F. A. and PEDER~ONC. S. (1961) Preparation of methyl esters of fatty acids for gas liquid chromatography. Analyt. Chem. 33, 1512.
ON THE CHEMICAL STRUCTURE OF THE APICAL DROPLET FROM EGGS OF CULEX PIPIENS ARNON
AHARONI”
and GUNTER
ZWEIGt
University of California, Davis, California, U.S.A., and Life Science Division, Syracuse University Research Corporation, Syracuse, N.Y. (Received
19 June 1972)
Abstract-The chemical nature of the apical droplet from eggs of Culex pip&as was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12/%hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14p-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with P-OH myristic acid. Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive 32P0,-3 and 35S04-2’ showed no significant incorporation of phosphorus, but a sulphur-containing aniohic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as monoand diglycerides. The structural evidence explains in part the surfactant properties of the apical drop. INTRODUCTION
EGGS of C&x and Culiseta mosquitoes exude microscopic droplets of a clear liquid at their posterior end above the water surface on which the eggs float as rafts composed of several hundred of eggs. These droplets were first described by GOELDI (1905) and were later shown by ILTIS and ZWEIG- (1962) to possess extremely high surfactant properties. These latter authors also tentatively, identified the droplet as a non-phosphorous glyceride which on acid hydrolysis yielded several long-chain fatty acids. The present report is a detailed study of the chemical composition of the apical droplet from eggs of Culex pipiens, including an investigation of the homogeneity and elemental composition of the droplet. MATERIALS
AND
METHODS
Rearing of the mosquitoes The mosquitoes were reared by a modified method of NEEDHAM et al. (1959), starting a colony from the eggs of an autogenous strain of Culex pipiens pipiens (L.). * Deceased. t Requests for reprints should be addressed to Gunter Zweig at Syracuse, New York. 861
862
AFCNONAHARONI ANDGUNTERZWEXG
Larvae were grown in small Pyrex glass dishes (15 x 18.5 in.) filled with tap water which was continuously aerated. A total amount of 3 g of high-energy, proteinrich food was supplied. After pupation, about 400 pupae were transferred to a glass dish containing about 250 ml of tap water and placed in a screen-covered wooden cage. The adults were reared at 23°C and 75% r.h. Sugar was supplied by the method of ELIASON (1963) by depositing the sugar containing a red food colour on a microscope slide. In some experiments H,35S0, (4 mc) was incorporated into the sugar. For 32P-studies, 3 ml of 10% (w/v) sucrose solution containing 20 PC of Hs32P0, was poured into a 6 ml. test tube stoppered with a cotton plug and placed in a cage. Blood meals were given once every 4-5 days by placing a featherless chicken (kindly supplied by Dr. Ursula Abbott, Poultry Department, University of California, Davis) into the cage for a period of 8 hr.
Collection of the apical droplets Newly laid egg rafts were transferred daily to Petri dishes lined with Whatman No. 1 filter paper saturated with water. The dishes remained covered for 24-30 hr. The droplets which formed during this period were collected under the dissecting microscope by the method of ILTIS and ZWEIG (1962) by touching the apical droplets with the tip of a small glass rod. The droplets which adhered to the glass tip by surface tension were washed into a 15 ml centrifuge tube with several drops of chloroform and stored in solution until future use. If material was needed for analyses, the chloroform was evaporated under an air stream at 45 to 50°C. The average yield of apical droplets was about 861 pg per hundred egg rafts.
Preparation of methyl esters from droplet hydrolysate The methanol-HCI-method of STOFFEL et al. workers (1959) was employed. About 1 mg of apical droplets was dissolved in 4 ml of O-1 N HCl in anhydrous methanol prepared according to VOGEL (1959). The solution was refluxed for 1 hr at 70” to 80°C in a glycerol bath and concentrated to 1.5 ml under a stream of nitrogen. Two millilitres of distilled water were added and the methyl esters extracted with three 7 ml portions of petroleum ether (30” to 60°C). The petroleum ether fraction was concentrated to about O-1 ml in a stream of nitrogen, and aliquots were analysed by gas-liquid chromatography (see below).
Gas-liquid chromatography (GLC) For the separation and identification of the methyl esters of the fatty acids the following equipment and conditions were used: Aerograph Model A-600B gas chromatograph (Varian Company) ; hydrogen flame ionization detector; 1 mV Leeds and Northrup recorder; 5 ft stainless steel column, + in. o. d., packed with 20% (w/w) glycol adipate polymer (LAC 446) on 30/60 mesh Chromosorb; nitrogen gas flow, 34 ml/min; hydrogen flow through detector, 25 ml/min. One microlitre of the hydrolysate in petroleum ether, corresponding to 10 pg of apical drop material, was chromatographed isothermally at 190 _F1°C. Under these experimental conditions a mixture of known methyl esters of fatty acids could
THEAPICAL DROPLET FROMEGGSOFCULEX
PIPIENS
863
be resolved consisting of saturated Crs, C14, Crs, C,, /?-OH-C,,, and /&OH-C!,, fatty acids. Preliminary identification of unknown methyl esters was made on the basis of retention times. Relative amounts of fatty acids were made by measuring peak areas with a planimeter. Absolute quantitative determination of /Lmyristic acid was made by comparing peak area with that of standard amounts of authentic material.
Thin-layer chromatography This technique was employed for testing the homogeneity of the original apical drops and for a confirmatory test of P-myristic acid. Chromatostrips, 14 x 1.3 x O-2 cm, were coated with silica gel G (Research Specialties Company) and dried for 30 min at 120°C. The following solvents were used to develop apical drops: (a) chloroform-methanol-water, 60 : 30 : 4, v/v; (b) benzene-acetone, 70 : 30, v/v. For methyl esters of fatty acids, ethyl acetate was used as solvent (TSCHESCHEet al., 1961). For the detection of apical drops, 2’,7’-dichlorofluorescein (O*21o/ow/v in methanol) or iodine vapours were employed (MANGOLDand MALINS, 1960). To detect the methyl esters of fatty acids a spray of 0.04% (w/v) bromthymol blue in 0.01 N NaOH was used (JATZKEWITZand MEHL, 1960).
PwiJication of hydrolysate The aqueous phase after the extraction of the methyl esters of fatty acids from the hydrolysate was passed through a 25 x 1 cm column containing about 25 g wet wt. of Dowex 1-4X, 50 to 100 mesh, hydroxyl form. Collection was started when the pH of the eluate became basic and was continued with repeated water washes until it returned to neutral pH. The combined eluate was continuously stirred with a sufficient amount of Dowex 50-4X-H+ until the pH had dropped to 3. (The final solution did not contain any H&SO, as tested with saturated BaCI,.) The water was removed by lyophylization, and the residue dissolved in about 1 ml of absolute methanol. Aliquots of this solution were chromatographed on paper as described below.
Identification of glycuol by paper chromatography Ascending paper chromatography using Whatman No. 1 paper and 1-butanol saturated with water were employed (BLOCK et al., 1958). Reducing compounds such as glycerol were detected by dipping the developed chromatogram into the silver nitrate-acetone reagent (BLOCK, et al., 1958) followed by spraying with O-5 N NaOH in 95% (v /v ) eth anol. The brown background of the paper could be cleared up by washing the chromatogram with a photographic fixer solution.
Separation of hydroxy-fatty
acids
A modification of the method for the separation of methyl and hydroxy-fatty acids by KANESHIROand MARR (1963) was graphic column (8.75 x l-5 cm) fitted with a 100 ml reservoir of silicic acid, 325-mesh (Bio Rad Lab.) and washed with about
esters of fatty acids used. A chromatowas filled with 5 g 150 ml of petroleum
864
AFCYON AHARONIAND GWNTERZWEIG
ether (30 to 60°C). The solution to be resolved was dissolved in 4 ml of petroleum ether and poured onto the column. The methyl esters of the fatty acids were eluted with 38 ml of petroleum ether-dðyl ether,’ 96 : 4, v/v. The methyl esters of the hydrox-fatty acids were taken off the column with 30 m of diethyl ether. Hydrogenation of methyl esters of unsaturated fatty acids The catalytic hydrogenation was based on the method by FARQUHARet al. (1959). About 1 mg of methyl esters from the apical drop hydrolysate in 5 ml of petroleum ether and 12 mg of platinum oxide (Adam’s catalysts; Matheson, Coleman, and Bell) were placed into a 10 ml beaker within a 1 litre suction flask. The suspension was continuously stirred with a magnetic stirrer. A partial vacuum of 250 mm Hg was applied to the suction flask and hydrogen admitted to 1 atm. This step was repeated after 5 min and stirring continued for 3.5 hr. The suspension was filtered through Whatman No, 1 paper (caution! PtO, catalysts are a fire hazard when allowed to dry on paper). The paper was washed with 7 ml of petroleum ether and the filtrate evaporated to about O-1 ml under nitrogen. Aliquots of this concentrate were analysed by gas-liquid chromatography (see above). RADIOCHEMICAL
TECHNIQUES
Liquid scintillation countilzg 32P- and 35S-Samples were counted with a Packard Tri-carb semi-automatic liquid scintillation spectrometer at a voltage of 900 V. Two counting solutions were used: non-aqueous (HAYES, 1962)-0.5% (w/v) 2,5-diphenyloxazol (PPO) and 0.03% (w/v) 2,2-p-phenylene-bis-(5-phenyloxazole) (POPOP) in redistilled toluene; aqueous (Y. Vadya, personal communication)-15 g of PPO, O-4 g of POPOP, 150 g of naphthalene dissolved in dioxane-toluene-ethanol(ll40 : 1140 : 720, v/v). Autoradiographic
technique
The developed chromatostrips were mounted into a cut-out section of a larger piece of cardboard, so that the glass and cardboard surfaces were flush. This was then loaded with Kodak Medical X-ray film in a light-tight holder, and after 2 to 3 weeks’ exposure, the film was processed by normal darkroom procedures. RESULTS Table 1 and Figs. 1 and .2 demonstrate that by esterification with methanolhydrochloric acid the main components of the apical drop among the fatty acids are P-OH myristic acid (72%) and P-OH 1auric acid (13.34%). It should be noted that there is a small amount of unsaturated fatty acids (see starred peaks in Figs. 1 and 2). A method in which the hydrolysate is esterified with diazomethane yielded much smaller quantities of B-OH lauric acid (8*6o/o) and P-OH myristic acid (13.8%). Three unsaturated fatty acids were found when this method was used with retention times between those of lauric and palmitic acids which are probably
X65
THE APICAL DROPLET FROM EGGS OF. CULEX PIPIENS
C,, and CT,, unsaturated fatty acids. It is possible that the change in the relative amounts of P-OH and unsaturated fatty acids following esterification with diazomethane was due to dehydration (VORBECK et al., 1961). TABLE
1. FATTY ACID COMPOSITION
OF APICAL DROP
Fatty acid
%
Laurie Myristic Palmitic Stearic B-OH Laurie B-OH Myristic Unsaturated C,, and Cl4 Unknown
0.16 0.23 8.93 4.75 13.34 72.00 0.39 0.26
TIME IN MINUTES
FIG. 1. GLC hydrochloric
showing acid,
fatty acids in apical drop material and
those
of
a standard
0.
(b) esterified
* Unsaturated
by methanol-
C,,
and C,,.
Hydrogenation of the fatty acids The two starred peaks in Figs. 1 and 2 were believed to be unsaturated fatty acids. Hydrogenation of standard methyl linoleate to stearic was achieved by the technique described in Materials and Methods, and this method was employed to determine if these unknown peaks actually corresponded to unsaturated fatty acids. Following methylation, the solutions were hydrogenated. Fig. 3 demonstrated
ARNON AHARONIAND
866
GUNTER~WEIG
Mixture of standard with .pmI Column remp: 183°C Gas f&v: 25 ml/nlin. Attenuation: 8-X
I 9
I 12
15
TIME
drops material
I 9
I 3
IN MINUTES
FIG. 2. GLC of mixture of fatty acids in apical drop esterified by methanolhydrochloric acid and fatty acids of a standard; chart paper run simultaneously. * Unsaturated C,, and C,,.
C&OH
Sample: Column. temg: Gas flow: Attenuation:
-S.mple: Conditions:
Apical drop material 183°C 28 ml/r&. 8-X
4-S&M
Apical drop materiot after hydrogenation Sme as obore CM
TIME
Cl4
I
I
b
3
0
IN MINUTES
FIG. 3. GLC showing hydrogenation of fatty acids in the apical drop material esterified by methanol-hydrochloric acid. * Unsaturated C,, and CT,,. ? Probably impurities of the solvent.
THE
APICAL
DROPLET
FROM
EGGS
OF
CULEX
PIPIENS
867
that these components in fact were converted into C,, and C,, saturated fatty acids indicating that unsaturated fatty acids were present in the original mixture. IDENTIFICATION
OF HYDROXY
FATTY
ACIDS
The total quantity of suspected hydroxy fatty acids was estimated to be about 85 per cent of the total fatty acids (see Fig. 1 and Table 1). Further tests were run to confirm the identity of the two hydroxy fatty acids. Identification
by column chromatography
When a standard mixture of aliphatic and hydroxy fatty acids was placed on a silicic acid column, the aliphatic acids were eluted with a solvent composed of petroleum ether and diethyl ether (96 : 4, v/v) and the more polar hydroxy fatty acids were eluted with diethyl ether alone. The chromatogram in Fig. 4 demonstrates that P-OH lauric and 85 per cent of /?-OH myristic acid were recovered almost quantitatively from the apical drop material by this technique.
TIME IN MINUTES
4. GLC chromatogram showing fractionation of apical drop material esterified by methanol hydrochloric acid by column chromatography. (A) Petroleum ether-diethyl ether (96 : 4, v/v) elution. (B) Diethyl ether elution. (C) Total fatty acids. FIG.
IdentiJication. of B-OH my&tic
acid by thin-layer
The methyl ester of standard P-OH
chromatography
myristic
acid together
with the methyl The chromatostrips were developed with ethyl acetate (TSCHESCHE, 1961) and sprayed with bromothymol blue. The R, value of the standard P-OH myristic acid was found esters of the fatty acids of the apical drops were chromatographed.
27
ARNON AHARONIAND GUNTERZWEIG
868
to be identical with that of the largest component of the fatty acids of the apical drops. (R, = O-65.) Thin-layer chromatogram is not reproduced. Quantitative
estimation
of hydroxy
myristic
acid
A 1% aliquot of the fatty acid fraction from 1.00 mg of apical material (collected from 115 rafts) was hydrolysed, esterified, and injected into the gas-liquid chromatography column. The peak area was equivalent to an area of a peak formed by 3.46 pg of standard B-OH myristic acid. Thus, the total P-OH myristic acid content in 1.00 mg. of apical drop material was estimated to be 35 per cent.
ANALYSIS
Preparation
OF GLYCEROL
of the apical drop material for glycerol analysis
Apical drop material (about 3 mg) collected from 300 rafts was esterified using diazomethane following reffuxing with sodium methylate in methanol Extraction of the fatty acid methyl esters with petroleum ether would leave the neutral water-soluble fraction in the aqueous phase. Sodium bisulphate which was added during the esterification interfered in subsequent chromatography and had to be removed by ion exchange. The aqueous solution which eluted from an anion exchange column (Dowex 1) was collected when the pH became basic. Since sodium hydroxide was eluted from the column simultaneously with glycerol, the rise in pH indicated the elution of NaOH and, consequently, the emergence of the glycerol from the column. Following the removal of sodium ions by mixing with a cation exchanger (Dowex 50), the aqueous solution was lyophilized, and the suspected glycerol residue redissolved in absolute ethanol for paper chromatography. Analysis
of the glycerol by paper chromatography
The alcoholic solution containing non-polar simultaneously with a standard of glycerol on chromatograms such as the one shown in Fig. that the colour density of the spot from the apical drop was approximateIy twice as intense an estimated concentration of 30%. Fractionation
of apical drop material
by thin-layer
substances was chromatographed Whatman No. 1 filter paper and 5 were obtained. It was observed ethanolic fraction of about 3 mg as that of a 50 pg standard, giving
chromatography
Chromatostrips of the apical drop material were prepared using two different solvent systems. Fig. 6(a) shows fractionation of the apical drops into four major components with benzene-acetone as a solvent system, while with the chloroformmethanol-water solvent, the apical drop material was fractioned into three components (Fig. 6b). It may be seen that fractions I + II -I- III are much slower in the benzene-acetone solvent than in the chloroform-methanol-water solvent. The R, of fraction IV in the benzene-acetone solvent was about the same as that of fraction III in the chloroform-methanol-water solvent.
THEAPICALDROPLETFROM
EGGS OF
CULEXPIPIENS
869
In order to obtain information on the chemical nature of the components of the apical drops, each component was sprayed with three different colour reagents. Positive reactions to each of the colour reagents by each of the components was observed ; bromothymol blue may detect O-1 to l-0 pg of most lipids (JATZKEWITZ and MEHL, 1960). 2’,7’-Dichloroflurescein was used to detect the presence of non-polar lipids (MANGOLD and MALINS, 1960). The spots were also exposed to iodine vapours (MANGOLD and MALINS, 1960). The positive reaction of the last test indicated that these fractions contained unsaturated lipids (MANGOLD, 1961). (Q) Jl
Sol*ent
Origin
front
J
III
II
I
Y
FIG. 6. Thin-layer chromatostrips of the apical drop material. (a) Chromatostrip run with benzene-acetone (70 : 30, v/v). (b) Chromatostrip run with chloroform-methanol-water (60 : 30 : 4, v/v). Shaded areas represent spots appearing on autoradiogram, from 35S-label. ANALYSIS
FOR PHOSPHORUS
AND
SULPHUR
Analysis of phosphorus ILTIS and ZWEIG (1962) reported several tests for the analysis of phosphorus in the apical drops. Mosquitoes were fed either with 10% sucrose solution containing 10 PC H,s2PO,/ml or on a chicken which had previously been injected intravenously with 2.5 mc of Na,ssPO,. The apical drops collected from these mosquitoes failed to show any significant radioactivity. To confirm these results, mosquitoes in these studies were fed with 10% sucrose solution containing 7 PC H332P0,/ml. The eggs and the apical drops were counted separately in a liquid scintillation spectrometer. Table 2 indicates that even though the eggs showed significant radioactivity, no significant counts were detected in the apical drop material.
Analysis of sulphur ILTIS and %vEIG (1962) proposed that the apical drop may contain a sulpholipid. The above assumption was proposed on the basis of a comparison of the
infrared absorption spectrum of the apical drop with that of 6-sulpho-6-deoxy-dn-gluco-pyranosyl diglyceride which has been isolated from photosynthetic algae. High surfactant properties have been ascribed to this compound by BENSON, et al. (1959). Radioactive tests and thin-layer chromatography were carried out in order to establish the presence and the behaviour of sulphur in the apical drop material.
ARNONAHARONIANDGUNTERZWEIG
870
TABLE 2. RADIOACTIVE ANALYSISFOR 35SAND32P*
Sample
Isotope
Background of the counting solution (c.p.m.)
Sample activity (c.p.m.)
32P 32P 3% 3%
20 24 22 35
2217 27 3236 217
9 egg rafts Apical drops from 15 rafts 9 egg rafts Apical drops from 1.5 rafts
* These results represent average of two trials.
Mosquitoes were fed on a dry sugar containing H,35S0,. The apical drops which were dissolved in chloroform were counted in a non-aqueous counting solution in a liquid scintillation spectrometer. It may be seen from Table 2 that both the eggs and the apical drops showed counts well over the background of the counting solution. These data indicated that some sulphur-containing compound was present in the apical drops. Autoradiography was carried out (CHASE, 1958) in order to locate the a5S component by fractionation of the apical drops. Fig. 6(b) shows that when chloroform-methanol-water (60 : 30 : 4) was used, the 35S component partially covered spot I on the chromatostrip. In order to determine if the sulphur-containing material actually was identical with spot I, the apical drop material was chromatographed with a second solvent consisting of benzeneacetone (70 : 30, v/v). With this solvent (Fig. 6a) the sulphur-containing compound remained at the origin. Further investigations of the sulphur-containing fraction were carried out. Sixty rafts were collected from mosquitoes which were fed on sucrose containing
z f0 .-
2
““‘I
4
6
8
IO
12
14
v’+,y--1;1.2
4
6
8
Wavelength
IO Microns
FIG. 7.
12
14
I
871
FIG. 5. Glycerol analysis by paper chromatography. (1) Ethanolic solution containing glycerol from hydrolyzed apical drop from 300 egg rafts. (2) 5 pg of standard glycerol. (3) 10 pg of standard glycerol. (4) 15 pg of standard glycerol. (5) 50 ,ug of standard glycerol.
THE APICALDROPLETFROM EGGSOF CULEX
873
PIPIENS
Hz5S0,. The apical drop material was collected and refluxed with methanol and 0.5 N sodium methylate. The aqueous solution after extraction of the fatty acids by petroleum ether was passed through an anion exchange column (Dowex-1) followed by mixing the elute with a cation exchanger (Dowex-50). Both the saltfree aqueous solution and the petroleum ether extract were analysed for 35S. No counts above the background of the counting solution were observed in any of the factions. The anion exchange column was then washed with 10% KOH, and this solution showed 202 counts while the background showed 95 counts. INFRARED
SPECTRUM
OF THE
APICAL
DROP
Table 3 shpws the infrared spectrum of the apical drop material and that of an SZq/, pure sample of glyceryl monoricinoleate. The 5.75~ band corresponds to an ester linkage and coincides with the absorption band found by ILTB and ZWEIG (1962) in their investigation of the apical drop. The bands at 2.95, 6.92, and TABLE 3. INFRARED ABSORPTION SPECTRA OF APICAL DROP MATERIAL AND OF GLYCERYLMONORICINOLEATR
Conditions of operation Origin Purity
Prism Resolution Response Gain Speed Suppression Scale
Apical drop material Davis, California Natural product film from solution on 1.5 mm KBr NaCl 927 x 2 11.00 2.0 16 8 lx
Glyceryl monoricinoleate Calbiochem 82% (Grade C). 1.5 mm KBr
Solid on
NaCl 980 11.00 4.0 8 All lx
846 TVmay indicate secondary alcohol groups of the hydroxy fatty acids. The absorption peaks at 6.35, 6.81, and 10.55 ,u indicate the presence of unsaturated fats in the apical drops. ,Although the exact nature of the primary alcohol group (9.5 or 9.7’2 p) is unknown, it is possible that these peaks may be due to mono- or diglycerides. HYPOTHESIS
ON CORRELATION BETWEEN CHEMICAL STRUCTURE PHYSICAL PROPERTIES OF APICAL DROP
AND
An interesting physical phenomenon noted by ILTIS and ZWEIG (1962) is the effect of the apical drops on the surface tension of an aqueous solution. Their measurements showed that the apical drops from eggs of C. pi@r~ lowered the surface tension of water from an initial value of 70.05 dynes/cm at 21°C to 32.1
874
ARNON
AHARONIAND GUNTERZWEIG
dynes/cm. The surface tension then rose within 90 min to a constant value of 66 dynes/cm. It is of interest to correlate the chemical structure of the apical drop with its surfactant properties. The interfacial tensions between long-chain aliphatic alcohols and acids on the one hand and water on the other are about 10 to 15 dynes/ cm (GLASSTONE, 1958). Thus, it is likely that the hydroxyl and the carbonyl groups which are abundant in the apical drop material give rise to this surfactant property. The sulphur-containing compound in the apical drop material may also play an important r81e in lowering the surface tension, as some sulphur-containing compounds have surfactant properties. The subsequent increase of the surface tension of the water from a minimum of 32.1 dynes/cm to a constant value of 66 dynes/cm indicates that the surface-active ingredient of the apical drop is soluble in water. The overall relationship between the physical, chemical, and physiological characteristics of the apical drop should lead to a further understanding of the biological nature of this material. It is possible that the property of the apical drop is important biologically in protecting the mosquito egg from adverse environmental conditions such as inversion during stormy weather. It is also possible as JLTIS (personal communication) indicated that the apical drop serves to protect the egg from microbial or fungal contamination. HINTON (1968) demonstrated that the apical droplet contained an ant-repellant substance and ascribes its formation to a defence mechanism against possible predators. Acknowledgements-This paper is taken from the thesis by Arnon Aharoni, University of California, 1964. We thank the American Cyanamid Company for a grant-in-aid which made this research possible. We also want to express thanks to the following people for technical advice and assistance during the course of this research: Professor F. N. STRONG, Messrs. FRED ILTIS, PAUL ALLEN, BASIL BOWERS,and DAVID L. GUTNICK,all at the University of California, Davis, where this work was completed. This paper was submitted at the urging of Professor MURRAY S. BLUM at the University of Georgia, although the work was completed in 1964. REFERENCES BENSONA. A., DANIELH., and WISERE. (1959) A sulfolipid in plants. Proc. nut. Acad. Sci. U.S.A. 45, 1582-1587. BLOCKR. J., DURRUME. L., and ZWEIG G. (1958) A Mum& of Paper Ckromatograpky and Paper Electrophoresis, pp. 178, 180, 202. Academic Press, New York. CHASEG. D. (1959) P?%ziples of Radioisotope Methodology, pp. 216-218. Burgess, New York. ELIASOND. A. (1963). Feeding adult mosquitoes on solid sugar. Nature, Lond. ZOO, 289. FARQUHAR J. W., INSULLW., ROSENP., STOFFELW., and AHRENSE. H. (1959) The analysis of fatty acid mixtures by gas-liquid chromatography. Nutrition Rev. (suppl.) 17, 29-30. GLASSTONES. (1958) The Elements of Physical Chemistry, p. 151. Van Nostrand, New Jersey. GOELDIE. A. (1905) OS mosquitos no para. Mem. Mus. Goeldi (Mus. Paraense) de Hist. Nat. e Ethnographia, IV. HAYES F. N. (1962) Solutes and solvents for liquid scintillation counting. Packard Tech. Bull. 1, 5. HINTON H. E. (1968) Structure and protective devices of the-egg of the mosquito Culex pipiens. J. Insect Physiol. 14, 145-161.
THE APICALDROPLETFRO&fEGGS OF CULEX
PIPIENS
875
ILTIS W. G. and %&ZIG G. (1962) Surfactant in apical drop of eggs of some culicine mosquitoes. Ann. ent. Sot. Am. 55, 409-415. JATZKEWITZH. and MEHL E. (1960) Thin layer chromatography of brain lipides-hydrolytic and breakdown products. Hoppe-Seyler’s 2. physiol. Chem. 320, 251. KANESHIROT. and MARR A. G. (1963) Hydroxy fatty acids of Azotobacter agilis. Biochim. Biophys. Acta 70, 271-277. MANGOLDH. K. and MALINS D. C. (1960) Fractionation of fats, oils, and waxes on thin layers of silicic acid. y. Am. Oil them. Sot. 37, 383. NEEDHAMJ. G., LUTZ F. E., WELCH P. C., and GALTSOFF P. S. (1959) Culture Methodsfor Invertebrate Animals, pp. 386-388. Dover, New York. STOFFEL W., CHU F., and AHRRNSE. H. (1959) Analysis of long-chain fatty acids by gasliquid chromatography. Analyt. Chem. 31, 307-308. TSCHESCHER., LAMPERT F., and SNATZKEG. (1961) Dtinnschicht- und Ionenaustauscherpapier-Chromatographie von Triterpenoiden. J. Chromatography. 5, 217. VOGEL A. I. (1959) A textbook of Practical Organic Chemistry, p. 79. Longmans, Green & Co., London. VORBECKM. C., LEONARDM. R., LEE F. A. and PEDER~ONC. S. (1961) Preparation of methyl esters of fatty acids for gas liquid chromatography. Analyt. Chem. 33, 1512.