- Email: [email protected]
Differential effects of fatty acid chain length on the viability of two species of cactophilic Drosophila
Chmp. Bio&em. Physioi! Great Britain
Vol. 83A,
No. 4, pp. 761--764, 1986
~3~-96~9~~6 $3.00 + 0.00 fi, 1986 Pergamon Press ttd
Printed in
DIFFERENTIAL EFFECTS OF FATTY ACID CHAIN LENGTH UN THE VIABXLITY OF TWO SPECIES OF CACTOPHILIC ~~~~~~~~~A JAMES C. FOGLEMAN* *Department
and
HENRY
W.
KIRCHER~~
of
Biological Sciences, University of Denver, Denver, CO 80208, USA, Telephone: (303) 871-3561 and IDepartment of Nutrition and Food Science, University of Arizona, Tucson, AZ 8572I. USA (Received 16 August 1985)
Abstract--I. Two columnar cacti in the Sonoran Desert, agria and organpipe, contain medium chain (C,-C,,) fatty acids. 2. Necrotic tissues of these cacti serve as feeding and breeding substrates for ~r~.su~~~f~~~Q~Q~e~S~.~ but not D. ~~~~~s~j~u~~~a. 3. Results show that capric and lauric acids are the predominant fatty acids of both cacti, 4. Fatty acid chain length exhibits a differential effect on larval viability with caprylic acid (C,) having the greatest and myristic acid (C,,,) having the least effect. 5. ~r~su~~j~arn~ja~e~s~s is more tolerant of free fatty acids than D. ~~gr~~~jruc~~u,and this partiy explains the ability of L1. moja~nsis to utilize agria and organpipe cacti.
iNTRODUCTlON
The toxicity of fatty acids and their salts on insects has a long history. In fact, they may be among the oldest insecticidal agents (Shepard, 1951). Fatty acids have been reported as effective contact insecticides against aphids, mosquitoes, housefties, Japanese beetles and some forest insects (Binder et al., 1979; and references therein). Not ail fatty acid chain lengths are equally effective in causing insect mortality. Capric {C,,) and lauric (C,,) acids were the most lethal of those tested as contact insecticides against aphids (~a~ters~eld and Gimingham, 1927; Dills and Menusan, 1935). In later tests, 1% lauric acid inhibited cricket growth (McFarlane and Wenneberry, 1965) but was not very toxic to Pseudosarcophaga afinis; this insect was killed more readily by capric acid (House, 1967). Capric acid also killed all confused flour beetles in 8 weeks when 2.5% was added to a dry Aour diet (House and Graham, 1967). More recently, I-5% of C,-C,, fatty acids in the diet of hide beetles suppressed fertjlity (Cohen and Levinson, 1972) and C,-C,, fatty acid salts were most harmful to several forest insects (Puritch, 1978). While it is apparent that the medium chain fatty acids exhibit the greatest insecticidal properties, they are, in general only minor constituents of most plant lipid fractions. Fatty acids of mast higher plants have an even number of carbon atoms and chains that are 14-22 carbon atoms long; with C,, and C,, as the most abundant forms. Although the major fatty acid in coconut oil. tauric acid is only present in small quantities in many vegetable oils {Binder ef a!., 1979). Capric acid is even more rare. _
__ _.--.__- .-~___-_-~
$Died January, 1984.
_.__
Two of the giant columnar cacti of the Sonoran Desert, Stenocereus gummosus (agria) and S. thurheri (organpipe), have been reported as containing medium-chain fatty acids (C&,2) as principle constituents of the lipid fraction (Kircher, 1982). Although the fatty acids are typically esterified to neutral triterpenes and sterol diois (K&her, 1980). some free fatty acids are released during decay of the tissue. One of the four cactophilic Rrasop~i~a species endemic to the Sonoran Desert, D. rnajal~erlsis, utilizes the necrotic tissue of agria and organpipe as feeding and breeding sites (Heed, 1978). Another sympatric drosophilid, r). nigraspira~u~a, apparently cannot utilize these cacti as host plants due to extensive larval mortality (Fellows and Heed, 1972). Instead, D. nigrnspiracula uses the necrotic tissue of Carnegiea giguntea (saguaro) as feeding and breeding substrates. Recently, Fogleman et al. (1985) reported
that a mixture of free fatty acids extracted from organpipe cactus and added to homogenized saguaro tissue at a concentration of 0.5% dry weight had a drastic effect on the larval viability of L). n~gr~~s~jracufa but had no significant effect on D. nzujaz~ensjs larvae. Although higher concentrations of organpipe fatty acids ( I .Oand 2.0% dry wt) reduced the viability of both species, the tolerance of D. ~~aja~,ens~swas greater than that of !I). ~~~rosp~ra~z~~a at all concentrations. The ability of D. mojarensis to tolerate the fatty acids which are present in the necroses of agria and organpipe cacti explains, in part, why it can use these cacti as host plants and D. nigrospiracufu cannot.
The experiments reported here attempt to extend our knowledge of the toxicity of medium chain fatty acids by examining the effect of single fatty acids of different chain length on larval viability of these two species of Drosu~~i~a. The fatty acid compositions of agria and organpipe are also reported.
JAMES C. FOGLEMAN and HENRY W. KIRCHER
762 MATERIALS
AND METHODS
Both species of D~os~phjfu were obtained from the iaboratory of Dr William Heed, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ. Media for testing larva1 viability were prepared by dissolving the free fatty acids (obtained from Sigma Chemical Co.) in ether, adding the mixture to dried saguaro powder and allowing the ether to evaporate. The powder was rehydrated and added to homogenized saguaro rot. The resulting mixture was blended to insure homogeneity, divided into six portions, and placed in half-pint milk bottles. The final concentration of the free fatty acids was either 0.5 or 1.0% on a dry weight basis. Viability tests for each species and each fatty acid were set up in triplicate with 50 first instar larvae. This larval density was used because a preliminary experiment using 50. 100,2OQ and 300 larvae per test showed that densities of 50 larvae per test yielded a higher percentage adult emergence. Larvae were given approx. 30 days to eclose into adults. The number of adults in each bottle after 30 days was used as a measure of larval viability in the medium. The fatty acids that were tested include caproic acid (C,), caprylic acid (C,), capric acid (C,,), lauric acid (C,,) and myristic acid (C,,). Control bottles contained saguaro rot without fatty acids. The data were analyzed using a one-way analysis of variance with replication (Sokal and Rohlf. 1969). The fatty acid composition of agria and organpipe cacti was determined as follows: the cactus was skinned, blended with two volumes of MeOH and filtered. The residue was extracted three times with 2: I CHCI,:MeOH. All filtrates were then combined and evaporated. Lipids were removed by ether-water extraction followed by evaporation of the ether fraction to dryness. Lipids were hydrolyzed by making a mixture of lipids, 10% KOH and 95% ethanol (1: I : IO) and refiuxing this mixture overnight on a steam bath. After saponification, the mixture was filtered and subjected to a ether-water extraction. The aqueous phase was separated. made acidic with I N HCI and extracted with ether. The ether fraction was evaporated to dryness and an excess (20 . x I of 2% w/v HSO, in methanol was added to the residue. After refluxing on a steam bath for several hours. H,O was added and the fatty acid methyl esters were extracted with PE. Gas chromatography of the fatty acid methyl esters of agria and organpipe cacti was performed on a Varian 3700GC eauiooed with dual flame ionization detectors . .. linked to an Apple He computer through an anaIog~digita1 interface (AII3, Interactive Structures). A 2 m x 2 mm id. x I/4” o.d. glass column packed with 15% DEGS on 80~100 Chromosorb W AW (Supelco cat. No. l-1904) was used. Benzene was used as the solvent for the GC analysis. Peak identification was based on retention time compared to retention times of known compounds. I
_
1
RESULTS
The fatty acid compositions of agria and organpipe are given in Table I. Although both cacti contain a diverse set of fatty acid chain lengths, with some unsaturated forms, two chain lengths are predominant. capric acid (C,,) and lauric acid (C,,). These two, together with caprybc acid (C,), make up about 80% of the fatty acids in agria and over 90% of the fatty acids in organpipe. Statistical analysis indicates that there is no significant difference between the cactus species with respect to fatty acid composition. The results of the viability studies for the two different concentrations of free fatty acids are presented in Table 2. The one-way analysis of variance tests demonstrate whether there is significant
Table
1. Rekdtive
acid fraction
composition of agria
and
of the fatly organpipe
Percentage of toial’ Fatty C* 2, Cl, Cl, , C,: !. CM Cl, C 10 I Cl, ? C,, C il / C,, : C ii( 3 Others
acid
Agrla
Organpipe
2.2 7.0 34.0 38.9 1.8 6.7 0.6 1.7 0.0 0.0 0.4 2.3 1.4 0.3 2.2
04 6.7 39.3 45.2 0.5 0.8 0.X I .o 0.0 1.4 0.2 06 1.5 0.6
I .o
*Calculated as percent of total area under all peaks.
variation in viability between treatments, i.e. different fatty acids at a particular concentration. These tests do not compare the viabilities between the two Drosophila species for each treatment since comparisons of this type are not as ecologically relevant as the effect of the treatments within a species. It can be seen in Table 2 that, while all of the one-way analysis of variance tests were statistically significant at the 0.05 level, L). ~oju~~e~s~s in 0.05% tatty acids were the teast affected by the treatments and D. nigrospirucula in 1.0% fatty acids were affected the most. Student-Newman-Keuls tests of differences between average viabilities of a species in media containing fatty acids of different chain lengths at the same concentration were also performed (Sokal and Rohlf, 1969, pp. 235-242). The results of these tests are indicated by superscripts in Table 2. For D. mojuuensis larvae in media containing 0.5% dry wt fatty acids, caprylic acid was the only one that produced a significant reduction in viability. The average viabihties in media containing the other falty acids were not significantly different from the viability in the control medium. At 1.0% dry wt, both caprylic and capric acids significantly reduced the viability of D. mojauensis larvae. Average viability in medium containing caprylic acid was reduced to near zero. On the other hand, D. nigrospiraculu was greatly affected by all fatty acids except myristic acid. The addition of caprylic or capric acids to the saguaro homogenate at a concentration of 0.5% dry wt prevented any D. nigrospiraculu from eclosing. Caproic and lauric acids also significantly reduced larval viability. At 1.O% dry wt, viability was zero on caproic, capryhc and capric acids and near zero (6.0%) on lauric acid. Only myristic acid had no significant effect on viability. DISCUSSION
The fatty acid composition of agria and organpipe cacti appears highly unusual in that typical plant fatty acids are, essentially, not present. The fact that agria and organpipe contain medium chain fatty
Cactophilic Drosophilaand fatty acids
763
Table 2. Average percent viability i standard deviation on saguaro homogenate plus free fatty acids. Results of one-way ANOVA (F) and probability (P) are also given Average
Substrate (Saguaro
D. mojarensis
plus.. .) Concentration
Control C, C, C,” GIL CM F(df=S. P
12)
4.751 <0.05 Concentration
Control C, C, C,,, C,* CM F(df=S. P
= 83.3 94.0 0.7 9.3 85.3 86.7
1.0% dry wt + I S.Ot k 6.0t + I .2$ k 6.11 k l4.0t + 6.4t
61.090 <
12)
*Superscripts level.
= 0.5% dry wt 83.3 f ILOt X8.0* 13.lt 56.7 k 4.6 76.0 i_ 6.9t 64.7+6.lt 82.7 k 7.6t
relate averages which are not significantly
acids had been previously established (Kircher, 1982) but the relative composition had never been determined. The presence of medium chain fatty acids may, in fact, be a characteristic of the genus, Stenocereus. Two related species, S. alamosensis (cina) and S. hystrix also contain medium chain fatty acids (Fogleman and Kircher, unpublished), although the relative composition is somewhat different. The fatty acids in other columnar cacti, such as saguaro, have the more typical chain lengths, C,,-C,,. There are two general conclusions that can be made based on the data in Table 2. First, although both species are significantly affected by the addition of medium chain fatty acids to the larval substrate, D. mojaaensis are much less affected than D. nigrospiracula. The F-values derived from the one-way analysis of variance tests reflect this trend clearly. At either concentration, the value for D. mojauensis is considerably less than that for D. nigrospiracula. The second conclusion is that, with respect to chain length, caprylic and capric acids have the greatest effect on the larval viability of either species. The primary question here is whether the presence of medium chain fatty acids in organpipe and agria cacti is responsible for the inability of D. nigrospiracula to utilize these cacti as host plants. The total concentration of fatty acids (all chain lengths) in fresh tissue has been estimated at about 3% dry wt for organpipe and 1.6% dry wt for agria (Fogleman et al.. 1985; Kircher. unpublished). Most of the fatty acids in the fresh tissue of these cacti, however, are monoesterified to triterpenes and sterol diols. Fatty acids which are complexed in this fashion are apparently not toxic since the addition of crude lipids (organpipe) to saguaro homogenate at concentrations up to 10% dry wt had no significant effect on the larval viability of D. mojavensis or D. nigrospiracula (Fogleman et al., 1985). Free fatty acids, as well as sterol diols and triterpenes, are released during the rotting process by microbial hydrolysis.
viability* D.
nigrospiracula 86.0 * 20.0 + 0.0 * 0.0 f 33.3 f 84.7 *
10.4t 8.7f$ a.01 0.02 15.5g 10.3t
52.324 <
* f * f k i
IO.47 O.O$ 0.01 o.ot: 4.0$ 4.2t
250.991 <
The concentration of free sterol diols has been observed to increase during rotting, but free fatty acids do not increase in concentration. Possible explanations that have been offered for this lack of increase include the metabolism of free fatty acids by lipolytic yeasts which grow in the necrosis and the chemical reactivity of free fatty acids leading to their being recomplexed with other components of the lipid fraction (Fogleman et al., 1985). The concentration of free fatty acids in necrotic organpipe tissue has been measured at around 0.5% dry wt. From the discussion above and the relative fatty acid composition given in Table 1, it appears that capric acid (C,,) is the most biologically important fatty acid in this model system. Although caprylic acid has a greater effect on larval viability. its concentration is not sufficient to cause a major reduction in viability. Laurie acid is in a relatively high concentration but does not match capric acid in its effect on viability. Caproic and myristic acids are relatively minor components with little biological impact. AIthough the fatty acids used in this study differ in their individual effects on larval viability, the toxicity of necrotic agria and organpipe tissue is a phenomenon to which all fatty acids (except myristic acid) contribute to some extent. The actual physiological basis for the toxic effect of medium chain fatty acids on cactophilic Drosophila is unknown. General speculation on this subject has been reviewed by Puritch (1978). He suggested that the toxic effect may be the result of inhibition of oxidative phosphorylation. This idea is supported by a number of animal studies cited in the review. The tolerance of D. mojavensis to medium chain fatty acids, and the intolerance of D. nigrospiracula, supports the statement that these fatty acids are, in part, responsible for the ability of the former but not the latter species to utilize necrotic sections of agria and organpipe cacti as breeding substrates. The free sterol diols which are present in these cacti are also
JAMES
764
C, FUGLEMAN and HENRYW. KIRCHER
known to reduce the viability of D. nigruspiracuia but not D. ~~j~~p~s~s (Fogleman et al., 19X5). Together, they make up a set of natural plant products which are involved in determining insect-host plant reIationships in the Sonoran Desert.
Drosophila, Kn Ecologiegicaf Generics: The Inre$ace (Edited
by Brussard P. F.) pp. 109~326, Springer, New York.
Acknowledgements-The authors would like to thank Susann Duperret for technical assistance. This work was supported by a grant (BSR-8207056) from the National Science Foundation.
House H. L. fi967) The nutr~tio~~~ status and larvicidal activities of C,-C,, saturated fatty acids in Pseudosarcuphnga afink (Diptera: Sarcophagidae). Corn. Entomol. 99, 384392. House H. L. and Graham A. R. (1967) Capric acid blended into foodstuff for control of an insect pest. Tribolium co&sum (Coleoptera: Tenebrionidae). Can. Eni. 99, 994s-999. Kircher H. W. (1980) Triterpenes in organpipe cactus.
REFERENCS
Kircher H. W. (1982) Chemical composition of cacti and its relationship to Sonoran Desert Drosophih. In Ec~k~gk~l
Phytochemistry 19, 2707-2712.
Binder R. G., Chan B. G. and Ehiger C. A. (1379) Antibiotic effects of C&-C,, fatty acid esters on pink bollworm, b&worm, and tobacco budworm. Agrie. B&f. C&em. 43, 246?-247
I
Cohen E. and h&on Z. H. (1972) The effect of fatty acids on reproduction of the hide beetle t>ermestes macufuttts (Coieoptera: Dermestidae). .l$e $5, Part f1 11, 293-299. Dills L. E. and Mensuan H. Jr (1935) A study of some fatty acids as contact insecticides. Contr. Boyce-Thompson Inst. 7, 63-82. E;;ellows D. P. and Heed W. B. (1972) Factors affecting host plant selection in desert-adapted ‘cactiphilic Dros&hila. Ecology 53, 850-858.
Fogleman J. C, Duperret S. M. and Klrcher H. W. (1985) The role of phytosterofs in host plant selection by cactophi& Dr~~u~~j~u. Lipid9 (in press). Heed W. 3. (1978) Ecology and Genetics of Sonoran Desert
Genetics and Er,alution: The Cactus
Yeast - Drwophi~u
IMoriei S?;stent (Edited by Barker J. S. F. and Starmer W. T.), pp. 143-t%% Academic Press, New York, McFarlane 3. E. and Henneberry G. 0. (1965) Inhibition of the growth of an insect by fatty acids. J. § Phf:~iol. II, 1247.-1252. Puritcb G. S. (19%) Biocidai effects of fatty acid salts on various forest insect pests. In The Pharmacologiccll Efecr of Lipids(Edikd by Kabara J. J.), pp. 105-I 12. American Oil Chemists Society. Champaign, IL. Shepard H. H. (1951) The ‘Chemistry and Action of Insecticides. McGraw-Hill. New York. Sokal R. R. and Rohlf F. J. (1969) Biomerry. W. H. Freeman, San Francisco. Tattersfield F. and Gimingham C, T. (1927) Studies on contact insecticides. Part VI. The insecticidal action of the fatty acids, the methyi esters and ammonium salts. Ana. &pi. Bioi. 14, 331-358.
Vol. 83A,
No. 4, pp. 761--764, 1986
~3~-96~9~~6 $3.00 + 0.00 fi, 1986 Pergamon Press ttd
Printed in
DIFFERENTIAL EFFECTS OF FATTY ACID CHAIN LENGTH UN THE VIABXLITY OF TWO SPECIES OF CACTOPHILIC ~~~~~~~~~A JAMES C. FOGLEMAN* *Department
and
HENRY
W.
KIRCHER~~
of
Biological Sciences, University of Denver, Denver, CO 80208, USA, Telephone: (303) 871-3561 and IDepartment of Nutrition and Food Science, University of Arizona, Tucson, AZ 8572I. USA (Received 16 August 1985)
Abstract--I. Two columnar cacti in the Sonoran Desert, agria and organpipe, contain medium chain (C,-C,,) fatty acids. 2. Necrotic tissues of these cacti serve as feeding and breeding substrates for ~r~.su~~~f~~~Q~Q~e~S~.~ but not D. ~~~~~s~j~u~~~a. 3. Results show that capric and lauric acids are the predominant fatty acids of both cacti, 4. Fatty acid chain length exhibits a differential effect on larval viability with caprylic acid (C,) having the greatest and myristic acid (C,,,) having the least effect. 5. ~r~su~~j~arn~ja~e~s~s is more tolerant of free fatty acids than D. ~~gr~~~jruc~~u,and this partiy explains the ability of L1. moja~nsis to utilize agria and organpipe cacti.
iNTRODUCTlON
The toxicity of fatty acids and their salts on insects has a long history. In fact, they may be among the oldest insecticidal agents (Shepard, 1951). Fatty acids have been reported as effective contact insecticides against aphids, mosquitoes, housefties, Japanese beetles and some forest insects (Binder et al., 1979; and references therein). Not ail fatty acid chain lengths are equally effective in causing insect mortality. Capric {C,,) and lauric (C,,) acids were the most lethal of those tested as contact insecticides against aphids (~a~ters~eld and Gimingham, 1927; Dills and Menusan, 1935). In later tests, 1% lauric acid inhibited cricket growth (McFarlane and Wenneberry, 1965) but was not very toxic to Pseudosarcophaga afinis; this insect was killed more readily by capric acid (House, 1967). Capric acid also killed all confused flour beetles in 8 weeks when 2.5% was added to a dry Aour diet (House and Graham, 1967). More recently, I-5% of C,-C,, fatty acids in the diet of hide beetles suppressed fertjlity (Cohen and Levinson, 1972) and C,-C,, fatty acid salts were most harmful to several forest insects (Puritch, 1978). While it is apparent that the medium chain fatty acids exhibit the greatest insecticidal properties, they are, in general only minor constituents of most plant lipid fractions. Fatty acids of mast higher plants have an even number of carbon atoms and chains that are 14-22 carbon atoms long; with C,, and C,, as the most abundant forms. Although the major fatty acid in coconut oil. tauric acid is only present in small quantities in many vegetable oils {Binder ef a!., 1979). Capric acid is even more rare. _
__ _.--.__- .-~___-_-~
$Died January, 1984.
_.__
Two of the giant columnar cacti of the Sonoran Desert, Stenocereus gummosus (agria) and S. thurheri (organpipe), have been reported as containing medium-chain fatty acids (C&,2) as principle constituents of the lipid fraction (Kircher, 1982). Although the fatty acids are typically esterified to neutral triterpenes and sterol diois (K&her, 1980). some free fatty acids are released during decay of the tissue. One of the four cactophilic Rrasop~i~a species endemic to the Sonoran Desert, D. rnajal~erlsis, utilizes the necrotic tissue of agria and organpipe as feeding and breeding sites (Heed, 1978). Another sympatric drosophilid, r). nigraspira~u~a, apparently cannot utilize these cacti as host plants due to extensive larval mortality (Fellows and Heed, 1972). Instead, D. nigrnspiracula uses the necrotic tissue of Carnegiea giguntea (saguaro) as feeding and breeding substrates. Recently, Fogleman et al. (1985) reported
that a mixture of free fatty acids extracted from organpipe cactus and added to homogenized saguaro tissue at a concentration of 0.5% dry weight had a drastic effect on the larval viability of L). n~gr~~s~jracufa but had no significant effect on D. nzujaz~ensjs larvae. Although higher concentrations of organpipe fatty acids ( I .Oand 2.0% dry wt) reduced the viability of both species, the tolerance of D. ~~aja~,ens~swas greater than that of !I). ~~~rosp~ra~z~~a at all concentrations. The ability of D. mojarensis to tolerate the fatty acids which are present in the necroses of agria and organpipe cacti explains, in part, why it can use these cacti as host plants and D. nigrospiracufu cannot.
The experiments reported here attempt to extend our knowledge of the toxicity of medium chain fatty acids by examining the effect of single fatty acids of different chain length on larval viability of these two species of Drosu~~i~a. The fatty acid compositions of agria and organpipe are also reported.
JAMES C. FOGLEMAN and HENRY W. KIRCHER
762 MATERIALS
AND METHODS
Both species of D~os~phjfu were obtained from the iaboratory of Dr William Heed, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ. Media for testing larva1 viability were prepared by dissolving the free fatty acids (obtained from Sigma Chemical Co.) in ether, adding the mixture to dried saguaro powder and allowing the ether to evaporate. The powder was rehydrated and added to homogenized saguaro rot. The resulting mixture was blended to insure homogeneity, divided into six portions, and placed in half-pint milk bottles. The final concentration of the free fatty acids was either 0.5 or 1.0% on a dry weight basis. Viability tests for each species and each fatty acid were set up in triplicate with 50 first instar larvae. This larval density was used because a preliminary experiment using 50. 100,2OQ and 300 larvae per test showed that densities of 50 larvae per test yielded a higher percentage adult emergence. Larvae were given approx. 30 days to eclose into adults. The number of adults in each bottle after 30 days was used as a measure of larval viability in the medium. The fatty acids that were tested include caproic acid (C,), caprylic acid (C,), capric acid (C,,), lauric acid (C,,) and myristic acid (C,,). Control bottles contained saguaro rot without fatty acids. The data were analyzed using a one-way analysis of variance with replication (Sokal and Rohlf. 1969). The fatty acid composition of agria and organpipe cacti was determined as follows: the cactus was skinned, blended with two volumes of MeOH and filtered. The residue was extracted three times with 2: I CHCI,:MeOH. All filtrates were then combined and evaporated. Lipids were removed by ether-water extraction followed by evaporation of the ether fraction to dryness. Lipids were hydrolyzed by making a mixture of lipids, 10% KOH and 95% ethanol (1: I : IO) and refiuxing this mixture overnight on a steam bath. After saponification, the mixture was filtered and subjected to a ether-water extraction. The aqueous phase was separated. made acidic with I N HCI and extracted with ether. The ether fraction was evaporated to dryness and an excess (20 . x I of 2% w/v HSO, in methanol was added to the residue. After refluxing on a steam bath for several hours. H,O was added and the fatty acid methyl esters were extracted with PE. Gas chromatography of the fatty acid methyl esters of agria and organpipe cacti was performed on a Varian 3700GC eauiooed with dual flame ionization detectors . .. linked to an Apple He computer through an anaIog~digita1 interface (AII3, Interactive Structures). A 2 m x 2 mm id. x I/4” o.d. glass column packed with 15% DEGS on 80~100 Chromosorb W AW (Supelco cat. No. l-1904) was used. Benzene was used as the solvent for the GC analysis. Peak identification was based on retention time compared to retention times of known compounds. I
_
1
RESULTS
The fatty acid compositions of agria and organpipe are given in Table I. Although both cacti contain a diverse set of fatty acid chain lengths, with some unsaturated forms, two chain lengths are predominant. capric acid (C,,) and lauric acid (C,,). These two, together with caprybc acid (C,), make up about 80% of the fatty acids in agria and over 90% of the fatty acids in organpipe. Statistical analysis indicates that there is no significant difference between the cactus species with respect to fatty acid composition. The results of the viability studies for the two different concentrations of free fatty acids are presented in Table 2. The one-way analysis of variance tests demonstrate whether there is significant
Table
1. Rekdtive
acid fraction
composition of agria
and
of the fatly organpipe
Percentage of toial’ Fatty C* 2, Cl, Cl, , C,: !. CM Cl, C 10 I Cl, ? C,, C il / C,, : C ii( 3 Others
acid
Agrla
Organpipe
2.2 7.0 34.0 38.9 1.8 6.7 0.6 1.7 0.0 0.0 0.4 2.3 1.4 0.3 2.2
04 6.7 39.3 45.2 0.5 0.8 0.X I .o 0.0 1.4 0.2 06 1.5 0.6
I .o
*Calculated as percent of total area under all peaks.
variation in viability between treatments, i.e. different fatty acids at a particular concentration. These tests do not compare the viabilities between the two Drosophila species for each treatment since comparisons of this type are not as ecologically relevant as the effect of the treatments within a species. It can be seen in Table 2 that, while all of the one-way analysis of variance tests were statistically significant at the 0.05 level, L). ~oju~~e~s~s in 0.05% tatty acids were the teast affected by the treatments and D. nigrospirucula in 1.0% fatty acids were affected the most. Student-Newman-Keuls tests of differences between average viabilities of a species in media containing fatty acids of different chain lengths at the same concentration were also performed (Sokal and Rohlf, 1969, pp. 235-242). The results of these tests are indicated by superscripts in Table 2. For D. mojuuensis larvae in media containing 0.5% dry wt fatty acids, caprylic acid was the only one that produced a significant reduction in viability. The average viabihties in media containing the other falty acids were not significantly different from the viability in the control medium. At 1.0% dry wt, both caprylic and capric acids significantly reduced the viability of D. mojauensis larvae. Average viability in medium containing caprylic acid was reduced to near zero. On the other hand, D. nigrospiraculu was greatly affected by all fatty acids except myristic acid. The addition of caprylic or capric acids to the saguaro homogenate at a concentration of 0.5% dry wt prevented any D. nigrospiraculu from eclosing. Caproic and lauric acids also significantly reduced larval viability. At 1.O% dry wt, viability was zero on caproic, capryhc and capric acids and near zero (6.0%) on lauric acid. Only myristic acid had no significant effect on viability. DISCUSSION
The fatty acid composition of agria and organpipe cacti appears highly unusual in that typical plant fatty acids are, essentially, not present. The fact that agria and organpipe contain medium chain fatty
Cactophilic Drosophilaand fatty acids
763
Table 2. Average percent viability i standard deviation on saguaro homogenate plus free fatty acids. Results of one-way ANOVA (F) and probability (P) are also given Average
Substrate (Saguaro
D. mojarensis
plus.. .) Concentration
Control C, C, C,” GIL CM F(df=S. P
12)
4.751 <0.05 Concentration
Control C, C, C,,, C,* CM F(df=S. P
= 83.3 94.0 0.7 9.3 85.3 86.7
1.0% dry wt + I S.Ot k 6.0t + I .2$ k 6.11 k l4.0t + 6.4t
61.090 <
12)
*Superscripts level.
= 0.5% dry wt 83.3 f ILOt X8.0* 13.lt 56.7 k 4.6 76.0 i_ 6.9t 64.7+6.lt 82.7 k 7.6t
relate averages which are not significantly
acids had been previously established (Kircher, 1982) but the relative composition had never been determined. The presence of medium chain fatty acids may, in fact, be a characteristic of the genus, Stenocereus. Two related species, S. alamosensis (cina) and S. hystrix also contain medium chain fatty acids (Fogleman and Kircher, unpublished), although the relative composition is somewhat different. The fatty acids in other columnar cacti, such as saguaro, have the more typical chain lengths, C,,-C,,. There are two general conclusions that can be made based on the data in Table 2. First, although both species are significantly affected by the addition of medium chain fatty acids to the larval substrate, D. mojaaensis are much less affected than D. nigrospiracula. The F-values derived from the one-way analysis of variance tests reflect this trend clearly. At either concentration, the value for D. mojauensis is considerably less than that for D. nigrospiracula. The second conclusion is that, with respect to chain length, caprylic and capric acids have the greatest effect on the larval viability of either species. The primary question here is whether the presence of medium chain fatty acids in organpipe and agria cacti is responsible for the inability of D. nigrospiracula to utilize these cacti as host plants. The total concentration of fatty acids (all chain lengths) in fresh tissue has been estimated at about 3% dry wt for organpipe and 1.6% dry wt for agria (Fogleman et al.. 1985; Kircher. unpublished). Most of the fatty acids in the fresh tissue of these cacti, however, are monoesterified to triterpenes and sterol diols. Fatty acids which are complexed in this fashion are apparently not toxic since the addition of crude lipids (organpipe) to saguaro homogenate at concentrations up to 10% dry wt had no significant effect on the larval viability of D. mojavensis or D. nigrospiracula (Fogleman et al., 1985). Free fatty acids, as well as sterol diols and triterpenes, are released during the rotting process by microbial hydrolysis.
viability* D.
nigrospiracula 86.0 * 20.0 + 0.0 * 0.0 f 33.3 f 84.7 *
10.4t 8.7f$ a.01 0.02 15.5g 10.3t
52.324 <
* f * f k i
IO.47 O.O$ 0.01 o.ot: 4.0$ 4.2t
250.991 <
The concentration of free sterol diols has been observed to increase during rotting, but free fatty acids do not increase in concentration. Possible explanations that have been offered for this lack of increase include the metabolism of free fatty acids by lipolytic yeasts which grow in the necrosis and the chemical reactivity of free fatty acids leading to their being recomplexed with other components of the lipid fraction (Fogleman et al., 1985). The concentration of free fatty acids in necrotic organpipe tissue has been measured at around 0.5% dry wt. From the discussion above and the relative fatty acid composition given in Table 1, it appears that capric acid (C,,) is the most biologically important fatty acid in this model system. Although caprylic acid has a greater effect on larval viability. its concentration is not sufficient to cause a major reduction in viability. Laurie acid is in a relatively high concentration but does not match capric acid in its effect on viability. Caproic and myristic acids are relatively minor components with little biological impact. AIthough the fatty acids used in this study differ in their individual effects on larval viability, the toxicity of necrotic agria and organpipe tissue is a phenomenon to which all fatty acids (except myristic acid) contribute to some extent. The actual physiological basis for the toxic effect of medium chain fatty acids on cactophilic Drosophila is unknown. General speculation on this subject has been reviewed by Puritch (1978). He suggested that the toxic effect may be the result of inhibition of oxidative phosphorylation. This idea is supported by a number of animal studies cited in the review. The tolerance of D. mojavensis to medium chain fatty acids, and the intolerance of D. nigrospiracula, supports the statement that these fatty acids are, in part, responsible for the ability of the former but not the latter species to utilize necrotic sections of agria and organpipe cacti as breeding substrates. The free sterol diols which are present in these cacti are also
JAMES
764
C, FUGLEMAN and HENRYW. KIRCHER
known to reduce the viability of D. nigruspiracuia but not D. ~~j~~p~s~s (Fogleman et al., 19X5). Together, they make up a set of natural plant products which are involved in determining insect-host plant reIationships in the Sonoran Desert.
Drosophila, Kn Ecologiegicaf Generics: The Inre$ace (Edited
by Brussard P. F.) pp. 109~326, Springer, New York.
Acknowledgements-The authors would like to thank Susann Duperret for technical assistance. This work was supported by a grant (BSR-8207056) from the National Science Foundation.
House H. L. fi967) The nutr~tio~~~ status and larvicidal activities of C,-C,, saturated fatty acids in Pseudosarcuphnga afink (Diptera: Sarcophagidae). Corn. Entomol. 99, 384392. House H. L. and Graham A. R. (1967) Capric acid blended into foodstuff for control of an insect pest. Tribolium co&sum (Coleoptera: Tenebrionidae). Can. Eni. 99, 994s-999. Kircher H. W. (1980) Triterpenes in organpipe cactus.
REFERENCS
Kircher H. W. (1982) Chemical composition of cacti and its relationship to Sonoran Desert Drosophih. In Ec~k~gk~l
Phytochemistry 19, 2707-2712.
Binder R. G., Chan B. G. and Ehiger C. A. (1379) Antibiotic effects of C&-C,, fatty acid esters on pink bollworm, b&worm, and tobacco budworm. Agrie. B&f. C&em. 43, 246?-247
I
Cohen E. and h&on Z. H. (1972) The effect of fatty acids on reproduction of the hide beetle t>ermestes macufuttts (Coieoptera: Dermestidae). .l$e $5, Part f1 11, 293-299. Dills L. E. and Mensuan H. Jr (1935) A study of some fatty acids as contact insecticides. Contr. Boyce-Thompson Inst. 7, 63-82. E;;ellows D. P. and Heed W. B. (1972) Factors affecting host plant selection in desert-adapted ‘cactiphilic Dros&hila. Ecology 53, 850-858.
Fogleman J. C, Duperret S. M. and Klrcher H. W. (1985) The role of phytosterofs in host plant selection by cactophi& Dr~~u~~j~u. Lipid9 (in press). Heed W. 3. (1978) Ecology and Genetics of Sonoran Desert
Genetics and Er,alution: The Cactus
Yeast - Drwophi~u
IMoriei S?;stent (Edited by Barker J. S. F. and Starmer W. T.), pp. 143-t%% Academic Press, New York, McFarlane 3. E. and Henneberry G. 0. (1965) Inhibition of the growth of an insect by fatty acids. J. § Phf:~iol. II, 1247.-1252. Puritcb G. S. (19%) Biocidai effects of fatty acid salts on various forest insect pests. In The Pharmacologiccll Efecr of Lipids(Edikd by Kabara J. J.), pp. 105-I 12. American Oil Chemists Society. Champaign, IL. Shepard H. H. (1951) The ‘Chemistry and Action of Insecticides. McGraw-Hill. New York. Sokal R. R. and Rohlf F. J. (1969) Biomerry. W. H. Freeman, San Francisco. Tattersfield F. and Gimingham C, T. (1927) Studies on contact insecticides. Part VI. The insecticidal action of the fatty acids, the methyi esters and ammonium salts. Ana. &pi. Bioi. 14, 331-358.