206 Jurenka et al. Archives of Biochemistry and Physiology 37:206–214 (1998)

Hydrocarbon Profiles of Diapausing and Reproductive Adult Face ( autumnalis) Russell A. Jurenka,* Donald Holland, and Elliot S. Krafsur

Department of Entomology, Iowa State University, Ames, Iowa

Hydrocarbons present on the cuticle surface of adult face flies, , were identified by GC-MS and quan- tified by GC. Hydrocarbons consisted of n-, monomethyl, and dimethyl alkanes ranging in chain length from 23–29 carbons. Also present were monounsaturated alkenes with chain lengths of 23, 25, 27, and 29 carbons. Wild-caught flies were extracted and hydrocarbon profiles determined for both dia- pausing and reproductive adult males and females. Few quali- tative differences were found between males and females in the hydrocarbon profile. Differences in percent composition were found between diapausing and reproductive flies in monounsaturated alkenes, 4 and 45%, n-alkanes, 24 and 37%, monomethylalkanes, 57 and 15%, and dimethylalkanes, 15 and 2%, respectively, for females. A small difference was found in the total amount of hydrocarbon present, 7.3 ± 0.6 and 9.7 ± 1.1 mg/, between diapausing and reproductive female flies, respectively. Adult males also exhibited a similar change in hydrocarbon profile and amount between diapausing and reproductive flies. A laboratory strain of face flies originat- ing in Minnesota was also analyzed, and again similar dif- ferences were observed in both male and female flies that were kept under a reproductive or diapause condition. Arch. Insect Biochem. Physiol. 37:206–214, 1998. © 1998 Wiley-Liss, Inc.

Key words: diapause; hydrocarbons; face fly; alkenes; methylbranched al- kanes

INTRODUCTION usually cannot take in additional water. Very little work has been done on documenting the role of Cuticular hydrocarbons play important roles hydrocarbons and cuticular lipids in diapausing in the physiology of . The most important insects. Recently it has been demonstrated that roles are in chemical communication and in pro- the puparium from a diapausing of the flesh tection against water loss (Blomquist et al., 1987). fly, Sarcophaga crassipalpis, contained twice as The role of hydrocarbons as pheromones and spe- much hydrocarbon as the puparium from a non- cies recognition cues is well documented (Roelofs and Cardé, 1971; Howard and Blomquist, 1982; Blomquist et al., 1993; Howard, 1993). Hydrocar- bons, along with other cuticular lipids, also play *Correspondence to: Russell Jurenka, Department of Ento- a major role in preventing desiccation in most in- mology, 411 Science II, Iowa State University, Ames, IA 50011- 3222. Email: [email protected] sects (Hadley, 1981, 1994). Diapausing insects are especially vulnerable to water loss in that they Received 19 May 1997; accepted 8 September 1997

© 1998 Wiley-Liss, Inc. Face Fly Hydrocarbons 207 diapausing pupa (Yoder et al., 1992). However, and the hexane extract analyzed by GC and GC- no differences were found in the hydrocarbon pro- MS. Preliminary thin-layer chromatographic file in these insects (Yoder et al., 1995). analysis indicated that over 95% of the hexane Face flies, Musca autumnalis, overwinter in extract contained hydrocarbon. Therefore, the a reproductive diapause in which mating behavior hexane extract was analyzed directly by GC. A is suspended, vitellogenesis does not occur, and the Hewlett-Packard (Wilmington, DE) 5890 GC fat body becomes greatly hypertrophied (Stoffolano with a flame ionization detector and an oven and Matthysse, 1967). In early fall, diapausing flies temperature-programmed from 60°C to 300°C disappear into overwintering hibernacula and pre- at 7°C/min, with an SE-30 (30 m × 0.25 mm sumably do not have access to water or food until i.d.) capillary column (Alltech, Deerfield, IL) was the next April (Krafsur et al., 1985). Therefore, the utilized for separation and quantification of hy- hydrocarbon profile and content must be adequate drocarbons. A Hewlett-Packard 5972 mass to provide continued protection for diapausing flies selective detector was utilized to identify hy- over a 6 month interval. In the current study, we drocarbons. Components were separated using identified hydrocarbons found on reproductive and a DB-1 (30 m × 0.32 mm i.d.) capillary column diapausing face flies. We found that reproductive (J&W Scientific, Folsom, CA) temperature-pro- flies had a greater amount of alkenes and lesser grammed from 60°C to 300°C at 10°C/min. Hy- amounts of methyl-branched alkanes than diapaus- drocarbons were identified based on retention ing flies. However, both male and female flies had times to known standards and published mass similar hydrocarbon profiles. spectrometer diagnostic ions (Blomquist et al., 1987; Yoder et al., 1995). Double bond position in alkenes was deter- MATERIALS AND METHODS mined by purification through argentation chro- Insects matography followed by derivatization with Face flies were caught in the wild near Ames, dimethyldisulfide and analysis by GC-MS. Hex- Iowa, and were stored at –20° to –80°C. A recently ane extracts from a group of six (female) and established Minnesota strain of flies was provided seven (male) reproductive wild-caught adults were by Dr. R.D. Moon (University of Minnesota). They combined. Alkenes were purified by using columns were shipped as pupae and, when emerged, were of 20% (w/w) silver nitrate in silica gel as described sexed and kept separately under two different in Dillwith et al. (1981). Purified alkenes were light and temperature regimens. One group des- derivatized with dimethyl disulfide and iodine to tined to become reproductive was placed at a 16:8 produce the thiomethyl ethers as described in L:D cycle with a corresponding temperature cycle Francis and Veland (1981). GC-MS was performed of 30°:25°C, and, after 4 days, which was equal to as described above.

70 degree days above a threshold of 12°C (DD>12°C), the flies were extracted. The other group destined RESULTS AND DISCUSSION to enter diapause was placed at ambient tempera- Identification of Hydrocarbons ture and photoperiod during the months of Octo- ber and November in Ames, Iowa, and, after 51 A total ion chromatograph obtained from or 59 days, equal to 66 DD>12°C, the flies were ex- GC-MS analysis of wild caught diapause and tracted. After flies were extracted for hydrocar- reproductive flies is shown in Figure 1. The ma- bons, they were dissected, and the fat body was jor components were identified by characteris- graded according to the criteria of Read and Moon tic ion fragment patterns and comparative (1986). Male flies with hypertrophied fat body retention times to known n-alkane standards were considered to be in diapause. Females with (Blomquist et al., 1987). The n-alkanes had previtellogenic ovaries and hypertrophied fat chain lengths from 23–29 carbons, with odd body were considered to be in diapause. Only flies chain lengths predominating. The main methyl- that were reproductive or in diapause were used branched alkanes were determined by compari- in this study. son of retention times to n-alkane standards and from characteristic ion fragments (Yoder et al., Hydrocarbon Extraction and Analysis 1995). A monomethyl series with the methyl Single flies were extracted in 200 µl hex- group at positions 9, 11, and 13 was found with ane containing 1 µg heneicosane as an internal chain lengths of 23, 25, 27, and 29 carbons. The standard. After 5 min the flies were removed other major monomethylalkanes were the series 208 Jurenka et al.

Fig. 1. Total ion chromatogram fromwild-caught adult female face flies that were deter- mined to be in a reproductive or diapausing condition. Numbes refer to hydrocarbons listed in Tables 2 and 3. Face Fly Hydrocarbons 209 of 3-methylalkanes with smaller amounts of 5- cent compositions between male and female re- methylalkanes. The major dimethylalkanes had productive face flies. Some variation occurred in branching patterns of 3,x- and 5,x- with chain the pentacosenes, with position 6 isomer occur- lengths of 25 and 27 carbons where x = 9, 11, 13, ring in greater abundance in females, whereas 15. Small amounts of 5,x-dimethylnonacosane males had more of the position 12 isomer. were also found. Also present were small amounts It is interesting that the position 9 isomer of internally branched 11, 15-dimethylalkanes is present in relatively low amounts for all chain with chain lengths of 25, 27, and 29 carbons. The lengths. The position 9 alkenes are biosynthesized n-alkanes were previously identified from face fly by chain-elongating oleic acid and then decaboxy- cuticular extracts; however, the mono and lating to form the hydrocarbon, as occurs in the dimethylalkanes were not previously identified (Dillwith et al., 1981). Oleic acid is a (Uebel et al., 1975). very abundant fatty acid but apparently is not The alkenes were identified based on re- used to a great extent in the face fly for alkene tention times, and the relative high abundance biosynthesis. The more abundant alkenes are of the diagnostic ions at m/z 69 and 83 and the probably biosynthesized by chain-elongating dif- M+ was found to be two less than the corre- ferent chain length fatty acids with a position 9 sponding saturated alkane. The double bond po- double bond. For example, the major alkene for sitions of the alkenes were established by GC-MS each chain length could be biosynthesized by of dimethyldisulfide derivatives. Double bonds elongating 9-docosenoic acid by two, four, six or were found as indicated in Table 1. We found a eight carbons followed by decarboxylation to form series of positional isomers starting at position 10-tricosene, 12-pentacosene, 13-heptacosene, and 8 for all four chain length alkenes and at 6 for 13-nonacosene, respectively. The other alkenes pentacosene and heptacosene. Some of these alk- could be biosynthesized by chain-elongating 9-eico- enes had been previously identified from cuticu- senoic acid, 9-tetracosenoic acid, 9-hexacosenoic lar extracts of face flies (Uebel et al., 1975) acid, or 9-octacosenoic acid and then decarboxy- where positions of the double bonds were deter- lating to form the alkene. 6-pentacosene and 6- mined by reductive ozonolysis and analysis of heptacosene could be made by chain-shortening aldehyde fragments by GC. They found double 9-octacosenoic acid and 9-tricontanoic acid, re- bonds in the 6, 10, 11, and 12 positions of spectively, followed by decarboxylation. This pro- pentacosene, the 10, 11, 12, and 13 positions of posed biosynthetic pathway is currently being heptacosene, and the 10, 11, 12, 13, and 14 po- investigated. sitions of nonacosene. The major alkenes were (Z)-14-nonacosene, (Z)-13-nonacosene, and (Z)- Percent Composition of Hydrocarbons From 13-heptacosene (Uebel et al., 1975). Identifica- Reproductive and Diapausing Flies tion of the tricosenes and double bonds in The percent composition of hydrocarbons positions 8 and 9 in our study was aided by from wild adult male and female flies is presented analysis of derivatives by GC-MS. As shown in in Table 2. Flies were caught during various pe- Table 1, the alkenes were found in similar per- riods of the year and after extraction were dis-

TABLE 1. Position of Double Bonds in Alkenes Isolated From Wild-Caught Reproductive Male and Female Face Flies* Tricosenes Pentacosenes Heptacosenes Nonacosenes Double bond Percent Percent Percent Percent position Ions Female Male Ions Female Male Ions Female Male Ions Female Male 6 131, 313 19 3 131, 341 2 tr 8 159, 257 4 2 159, 285 4 2 159, 313 6 2 159, 341 2 1 9 173, 243 8 2 173, 271 2 1 173, 299 2 4 173, 327 2 6 10 187, 229 62 38 187, 257 20 12 187, 285 10 9 187, 313 21 18 11 201, 215 26 58 201, 243 27 28 201, 271 11 11 201, 299 7 9 12 215, 229 29 55 215, 257 15 26 215, 285 10 16 13 229, 243 54 47 229, 271 35 28 14 243, 257 22 22 *The diagnostic ions for the dimethyldisulfide derivatives are shown. Percent composition of individual alkenes as determined from the diagnostic ions is also shown. tr = trace amount, <1%. 210 Jurenka et al.

TABLE 2. Percent Composition of the Hydrocarbons From Adult Male and Female Wild-Caught Face Flies That Were Determined to Be in Diapausing or Reproductive Physiological States Female Male Diapause Reproductive Diapause Reproductive Diapause Hydrocarbona April (6)b May (5) June (6) September (5) June (7) September (4) 1. Tricosenes nd 0.4 ± 0.04 0.4 ± 0.02 nd 0.7 ± 0.1 nd 2. n-tricosane 0.1 ± 0.02 4.1 ± 0.2 2.8 ± 0.3 0.8 ± 0.1 2.8 ± 0.2 0.8 ± 0.3 3. 9-,11-,13-me-23 0.1 ± 0.01 0.7 ± 0.1 0.9 ± 0.04 1.1 ± 0.3 1.4 ± 0.03 1.0 ± 0.3 4. 3-me-23 0.2 ± 0.02 0.4 ± 0.03 0.5 ± 0.02 1.0 ± 0.1 0.7 ± 0.04 1.0 ± 0.3 5. n-tetracosane 0.3 ± 0.04 0.5 ± 0.02 0.5 ± 0.02 0.4 ± 0.1 0.5 ± 0.02 0.5 ± 0.1 6. Pentacosenes 1.1 ± 0.04 11.2 ± 1.6 12.3 ± 0.9 1.1 ± 0.1 14.8 ± 0.9 1.1 ± 0.5 7. n-pentacosane 22.9 ± 2.0 19.2 ± 0.7 15.2 ± 0.9 17.3 ± 2.1 12.3 ± 0.6 16.2 ± 2.5 8. 9-,11-,13-me-25 4.3 ± 0.3 2.1 ± 0.1 3.8 ± 0.4 23.6 ± 1.9 5.8 ± 0.4 25.8 ± 0.5 9. 5-me-25 3.6 ± 0.1 0.3 ± 0.02 0.7 ± 0.1 3.6 ± 0.3 1.3 ± 0.1 3.6 ± 0.2 10. 11,15-dime-25 0.3 ± 0.01 0.4 ± 0.04 0.5 ± 0.02 1.2 ± 0.2 0.6 ± 0.02 1.3 ± 0.1 11. 3-me-25 19.3 ± 2.0 3.2 ± 0.2 3.5 ± 0.2 11.5 ± 0.7 4.7 ± 0.1 10.4 ± 0.3 12. 5,x-dime-25 0.8 ± 0.03 nd 0.3 ± 0.1 2.9 ± 0.1 0.4 ± 0.03 3.0 ± 0.3 13. n-hexacosane 1.1 ± 0.1 0.6 ± 0.1 0.6 ± 0.04 0.6 ± 0.1 0.7 ± 0.02 0.5 ± 0.1 14. 3,x-dime-25 0.9 ± 0.04 nd 0.2 ± 0.1 3.2 ± 0.3 0.3 ± 0.03 3.3 ± 0.05 15. Heptacosenes 3.9 ± 0.2 19.8 ± 1.3 21.1 ± 0.8 1.9 ± 0.3 17.8 ± 0.5 1.9 ± 0.2 16. n-heptacosane 12.8 ± 0.8 12.6 ± 1.3 10.8 ± 0.5 4.7 ± 0.5 12.0 ± 0.8 4.2 ± 0.3 17. 9-,11-,13-me-27 4.3 ± 0.3 1.8 ± 0.6 2.2 ± 0.1 9.1 ± 0.6 3.1 ± 0.2 10.3 ± 1.4 18. 5-me-27 2.1 ± 0.1 0.6 ± 0.1 0.5 ± 0.03 1.1 ± 0.1 0.6 ± 0.1 0.8 ± 0.03 19. 11,15-dime-27 0.9 ± 0.1 0.9 ± 0.2 1.1 ± 0.1 1.4 ± 0.1 0.7 ± 0.1 1.5 ± 0.2 20. 3-me-27 5.3 ± 0.4 1.7 ± 0.2 2.0 ± 0.1 3.5 ± 0.2 2.0 ± 0.1 3.1 ± 0.4 21. 5,x-dime-27 2.1 ± 0.2 nd 0.1 ± 0.03 4.0 ± 0.5 0.1 ± 0.02 4.2 ± 0.8 22. n-octacosane 0.6 ± 0.02 0.3 ± 0.1 0.4 ± 0.04 0.1 ± 0.1 0.4 ± 0.02 0.2 ± 0.1 23. 3,x-dime-27 1.3 ± 0.1 nd nd 1.8 ± 0.4 0.2 ± 0.02 2.2 ± 0.4 24. Nonacosenes 2.0 ± 0.3 13.6 ± 0.7 11.6 ± 0.9 0.6 ± 0.1 8.0 ± 0.8 0.3 ± 0.05 25. n-nonacosane 2.4 ± 0.2 2.9 ± 0.4 3.2 ± 0.3 0.6 ± 0.05 4.3 ± 0.4 0.6 ± 0.1 26. 9-,11-,13-me-29 2.3 ± 0.2 1.2 ± 0.3 1.9 ± 0.3 1.0 ± 0.1 1.9 ± 0.3 0.8 ± 0.2 27. 5-me-29 0.4 ± 0.04 0.2 ± 0.02 0.3 ± 0.03 0.1 ± 0.04 0.2 ± 0.02 nd 28. 11,15-dime-29 0.8 ± 0.1 0.5 ± 0.1 0.9 ± 0.1 0.2 ± 0.1 0.5 ± 0.1 0.1 ± 0.1 29. 3-me-29 3.1 ± 0.2 1.0 ± 0.1 1.8 ± 0.3 1.0 ± 0.1 1.2 ± 0.1 0.7 ± 0.1 30. 5,x-dime-29 0.8 ± 0.1 nd 0.1 ± 0.1 0.6 ± 0.1 nd 0.4 ± 0.1 % alkenes 7.0 ± 0.5 45.0 ± 2.8 45.3 ± 1.0 3.7 ± 0.4 41.2 ± 1.2 3.4 ± 0.7 % n-alkanes 40.1 ± 2.7 40.1 ± 1.8 33.4 ± 1.0 24.5 ± 2.6 32.9 ± 1.3 23.0 ± 3.0 % monomethyl 45.0 ± 2.4 13.2 ± 1.2 18.0 ± 1.2 56.6 ± 2.6 22.9 ± 1.2 57.6 ± 1.9 % dimethyl 7.9 ± 0.4 1.7 ± 0.4 3.2 ± 0.5 15.2 ± 0.6 2.9 ± 0.2 16.0 ± 1.7 µg/fly 13.8 ± 0.5 9.7 ± 1.1 9.6 ± 1.1 7.3 ± 0.6 9.5 ± 1.0 8.2 ± 0.5 aNumbers refer to Fig. 1. The methyl-branched hydrocarbon abbreviations have the first number(s) indicating the position of the methyl group(s), and the last number indicates the number of carbons in the chain. me = methyl; dime = dimethyl; x = 9, 11, 13, 15. bNumber in parentheses = n. nd = not detected. sected to determine the physiological state. Fe- of heptacosane. Diapausing females caught in male flies caught in early April and late Sep- April had 3-methylpentacosane as the major tember were determined to be in a state of monomethyl alkane, while those in September diapause, as indicated by hypertrophied fat body were found to have 9-, 11-, and 13-methylpenta- and previtellogenic ovaries. These flies were cosanes as the major monomethyl alkanes with characterized by a high percentage of saturated 3-methylpentacosane as the second most abun- n-alkanes and monomethylalkanes. Although dant. The flies caught in April also contained these two types of hydrocarbons make up the almost twice as much total hydrocarbon as those greatest amounts, differences were found in in- caught in September. Both groups of flies also dividual hydrocarbon profiles. Females in dia- had fairly high levels of dimethyl alkanes, with pause in both April and September had the 3,x- and 5,x-dimethyl pentacosanes and pentacosane as the major n-alkane; however, heptacosanes predominating. those found in April also had a high percentage The flies caught in May and June were Face Fly Hydrocarbons 211 found to be reproductive. The reproductive state mating behavior and were positively phototrophic. is characterized by unhypertrophied fat body and In September both reproductive and diapause the presence of vitellogenic ovaries. In contrast males could be found. Dissection of males indi- to diapause flies, reproductive flies contained cated that those categorized as in diapause had higher levels of alkenes and lower levels of hypertrophied fat bodies and before capture monomethyl and dimethyl alkanes. The total per- were not displaying mating behavior. Hydrocar- cent composition of straight chain alkanes showed bon profiles in males followed that of females the least variation amongst the different groups. in the comparison between diapause and repro- Reproductive flies were also characterized by an ductive physiological states. Diapause males had increase in n-nonacosane, and the amount of greater amounts of mono- and dimethyl- methylbranched nonacosanes did not decrease. branched alkanes and lower levels of alkenes This is especially the case in a comparison of than the reproductive males. reproductive flies with diapause flies sampled in We also tested a strain of lab-reared flies September. Flies caught in April, although deter- (Table 3). Lab-reared flies maintained under re- mined to be in diapause based on the amount of productive or diapausing conditions had similar hypertrophied fat body, were breaking diapause hydrocarbon profiles as those of wild flies. Repro- and becoming reproductive (Krafsur et al., 1985). ductive flies had high levels of alkenes and low They had similar profiles of the 29 hydrocarbon levels of mono- and dimethylalkanes. These find- series as reproductives, except for the nonacosenes. ings indicate that hydrocarbon profiles can be Reproductive flies had lesser amounts of al- altered under these two laboratory conditions and most all methyl-branched hydrocarbons and that they mimic what was found in wild-caught higher levels of alkenes with 25, 27, and 29 car- flies. Therefore, we should be able to study bons. The unsaturated hydrocarbons were found changes in hydrocarbon profiles under controlled in equal amounts in both May and June repro- conditions. ductive flies. These flies are different in that those caught in May were flies that overwintered in Physiological Significance adult diapause, emerged from hibernacula in the These findings indicate that dramatic spring, and became reproductive. Flies caught in changes in hydrocarbon profiles occur in flies that June were from the first generation and in the wild are destined for adult reproductive diapause. The would not enter diapause. The flies caught in April data presented in this paper indicate that a were immediately postdiapause, and their hydro- switch occurs in flies that are destined to dia- carbon profile is in the process of transition from pause. A change in biosynthesis occurs with a the diapause profile to a reproductive profile. This reduction in the biosynthesis of alkenes and an is characterized by slightly lower levels of mono- increase in the production of mono- and dim- methyl-branched hydrocarbons, especially 9-, 11-, ethyl-branched alkanes. We do not yet know and 13-methylpentacosanes, and slightly in- when this switch occurs in the life of the adult creased levels of alkenes and the 29 carbon series fly. In late summer and early autumn, eclosing based on percent composition. face flies enter reproductive diapause (Krafsur et Levels of total dimethyl-branched hydrocar- al., 1986). During this time, hydrocarbon biosyn- bons changed appreciably between diapause and thesis must also be programmed to produce the reproductive flies. Reproductive flies had lesser diapause profile. amounts of 3,x- and 5,x-dimethyl–branched 25, 27, Is there a physiological significance of the and 29 hydrocarbons, whereas the levels of the different hydrocarbon profiles for reproductive internally branched 11,15-dimethyl hydrocarbons and diapausing face flies? Reproductive face flies did not change very much. Flies caught in April were thought to utilize the alkenes as a sex had, on a percent composition basis, lesser pheromone (Uebel et al., 1975). Several alkenes, amounts of the dimethylalkanes; however, on a including (Z)-14-nonacosene, (Z)-13-nonacosene, weight basis it was very similar to what was found and (Z)-13-heptacosene, were shown to increase mating strikes by males. However, these com- in diapause flies caught in September. pounds were found in both male and female face Wild-caught males had similar hydrocarbon flies, suggesting that these are not true sex profiles to females (Table 2). Dissections showed pheromones. We did not find large differences that all first generation males (June) were repro- in the hydrocarbon profiles between the sexes ductive because they did not have hypertrophied in the present study. This suggests that the hy- fat body. Before capture they also demonstrated drocarbons are not being utilized as pheromones. 212 Jurenka et al.

TABLE 3. Percent Compositionof the Hydrocarbons From Male and Female Adults Reared in the Lab and Placed Under Diapause or Reproductive Conditions Female Male Hydrocarbonsa Diapause (5)b Reproductive (3) Diapause (5) Reproductive (3) 1. Tricosenes nd nd nd nd 2. n-tricosane 0.7 ± 0.04 3.9 ± 0.7 0.6 ± 0.1 3.5 ± 1.1 3. 9-,11-,13-me-23 0.1 ± 0.01 0.3 ± 0.04 0.1 ± 0.03 0.4 ± 0.1 4. 3-me-23 0.7 ± 0.1 0.2 ± 0.02 0.5 ± 0.1 0.3 ± 0.1 5. n-tetracosane 0.4 ± 0.03 2.6 ± 0.3 0.4 ± 0.1 2.2 ± 0.6 6. Pentacosenes 1.5 ± 0.2 4.8 ± 1.0 1.2 ± 0.3 4.7 ± 1.5 7. n-pentacosane 28.2 ± 1.7 21.6 ± 2.6 25.2 ± 1.8 21.0 ± 6.1 8. 9-,11-,13-me-25 6.6 ± 0.7 3.3 ± 0.5 6.5 ± 0.7 5.2 ± 1.9 9. 5-me-25 4.0 ± 0.3 0.2 ± 0.03 4.4 ± 0.4 0.5 ± 0.2 10. 11,15-dime-25 0.2 ± 0.1 0.3 ± 0.04 0.3 ± 0.1 0.3 ± 0.1 11. 3-me-25 14.3 ± 0.4 3.0 ± 0.2 16.1 ± 0.7 4.2 ± 1.3 12. 5,x-dime-25 1.0 ± 0.1 nd 0.9 ± 0.1 nd 13. n-hexacosane 0.8 ± 0.03 2.1 ± 0.2 0.9 ± 0.04 1.9 ± 0.6 14. 3,x-dime-25 1.4 ± 0.1 0.1 ± 0.02 1.3 ± 0.1 0.2 ± 0.1 15. Heptacosenes 3.7 ± 0.2 16.6 ± 1.3 6.0 ± 1.3 15.8 ± 5.0 16. n-heptacosane 11.1 ± 0.7 14.3 ± 1.4 10.6 ± 0.6 14.1 ± 4.3 17. 9-,11-,13-me-27 6.8 ± 0.4 2.2 ± 0.6 6.3 ± 1.5 3.2 ± 1.1 18. 5-me-27 2.5 ± 0.1 0.7 ± 0.1 2.7 ± 0.2 0.9 ± 0.3 19. 11,15-dime-27 0.5 ± 0.03 0.5 ± 0.05 0.4 ± 0.1 0.5 ± 0.2 20. 3-me-27 4.0 ± 0.2 1.3 ± 0.2 4.0 ± 0.1 1.7 ± 0.5 21. 5,x-dime-27 2.7 ± 0.2 nd 2.6 ± 0.2 nd 22. n-octacosane 0.3 ± 0.03 0.9 ± 0.04 0.6 ± 0.2 0.8 ± 0.2 23. 3,x-dime-27 1.1 ± 0.1 0.1 ± 0.02 0.6 ± 0.2 0.1 ± 0.04 24. Nonacosenes 1.1 ± 0.2 16.8 ± 1.5 1.4 ± 0.4 14.4 ± 4.7 25. n-nonacosane 1.1 ± 0.1 2.4 ± 0.5 1.1 ± 0.1 2.2 ± 0.7 26. 9-,11-,13-me-29 2.0 ± 0.2 0.9 ± 0.2 2.3 ± 0.2 1.0 ± 0.3 27. 5-me-29 0.3 ± 0.03 0.1 ± 0.03 0.5 ± 0.1 0.1 ± 0.04 28. 11,15-dime-29 0.3 ± 0.02 0.3 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 29. 3-me-29 1.7 ± 0.1 0.4 ± 0.1 1.7 ± 0.1 0.5 ± 0.2 30. 5,x-dime-29 0.7 ± 0.1 nd 0.7 ± 0.1 nd % alkenes 6.4 ± 0.3 38.2 ± 1.3 8.6 ± 1.1 34.9 ± 11.1 % n-alkanes 42.7 ± 2.3 47.9 ± 2.5 39.4 ± 1.7 45.7 ± 13.4 % monomethyl 43.1 ± 1.6 12.6 ± 1.9 45.1 ± 1.9 18.0 ± 5.8 % dimethyl 7.9 ± 0.5 1.3 ± 0.1 7.0 ± 0.5 1.4 ± 0.4 µg/fly 7.7 ± 1.0 8.3 ± 0.2 6.2 ± 0.7 7.8 ± 2.3 aNumbers refer to Fig. 1. Abbreviations are the same as Table 1. bNumber in parentheses = n. nd = not detected.

However, the alkenes could be utilized as cues vide a waterproofing mechanism primarily be- for reproductive status. It will be interesting to cause of their relatively high melting tempera- determine hydrocarbon profiles after mating. tures (Hadley, 1981). The introduction of double The mating history of the wild caught flies was bonds and methyl groups lowers the melting unknown in this study; however, the lab-reared temperature compared to straight chain alkanes flies were all virgins. (Gibbs and Pomonis, 1995). In the face fly, re- The other major physiological role of hy- productive flies had about 45% of the total hy- drocarbons is to protect against desiccation. It drocarbon composition as alkenes, and this has been demonstrated that several insects in- would presumably produce an overall lower crease hydrocarbon amounts during diapause melting temperature compared to hydrocarbons (Bell et al., 1975; Hegdekar 1979; Yoder et al., found in diapausing flies. However, a study con- 1992), although the hydrocarbon profile did not ducted using the housefly, Musca domestica, change in at least S. crassipalpis (Yoder et al., demonstrated that 4-day-old male flies and 1- 1995). The increase in hydrocarbon content pre- day-old flies had the same total hydrocarbon sumably would help to protect the insect from melting temperature despite the fact that the desiccation during diapause. Hydrocarbons pro- older flies contained 54% alkenes compared with Face Fly Hydrocarbons 213 6% for 1-day-old flies (Gibbs et al., 1995). It was Blomquist GJ, Tillman-Wall JA, Guo L, Quilici DR, Gu P, concluded that hydrocarbon melting tempera- Schal C (1993): Hydrocarbon and hydrocarbon derived sex pheromones in insects: Biochemistry and endocrine tures were due to the overall composition and regulation. In Stanley-Samuelson DW, Nelson DR (eds): cannot be predicted from percent composition of Insect Lipids: Chemistry, Biochemistry and Biology. Lin- individual components (Gibbs et al., 1995). coln: University of Nebraska Press, pp 317–351. Therefore, the large percent composition of alk- Dillwith JW, Blomquist GJ, Nelson DR (1981): Biosynthesis enes found in reproductive face flies may not of the hydrocarbon components of the sex pheromone lower the overall melting temperature com- of the housefly, Musca domestica L. Insect Biochem pared to diapause flies. 11:247–253. If they are to survive subzero temperatures, Francis GW, Veland K (1981): Alkylthiolation for the determi- diapausing face flies probably must do so at –8°C nation of double bond location in linear alkenes. J to +8°C ambient temperatures (Rosales et al., Chromatogr 219:379–384. 1994). Very little is known about the biophysical properties of hydrocarbons at these temperatures, Gibbs A, Pomonis JG (1995): Physical properties of insect cuticular hydrocarbons: The effects of chain length, and the protection they provide against desicca- methyl-branching and unsaturation. Comp Biochem tion is therefore not understood. Presumably, most Physiol [B] 112:243–249. hydrocarbons with a chain length of 23 carbons or more and either saturated or with one double Gibbs A, Kuenzli M, Blomquist GJ (1995): Sex- and age-re- lated changes in the biophysical properties of cuticular bond have melting temperatures at or above 0°C lipids of the housefly, Musca domestica. Arch Insect (Gibbs and Pomonis, 1995). Therefore, face flies Biochem Physiol 29:87–97. that have hydrocarbons with chain lengths of 25 and 27 carbons would have hydrocarbons that Hadley NF (1981): Cuticular lipids of terrestrial plants and : A comparison of their structure, composi- provide protection during winter. We also found tion and waterproofing function. Biol Rev 56:23–47. an increase in hydrocarbon content between flies caught in April and those in September (Table Hadley NF (1994): Water Relations of Terrestrial Arthropods. 2). This may indicate that overwintering flies con- San Diego: Academic Press. tinue to produce hydrocarbon or that flies emerg- Hegdekar BM (1979): Epicuticular wax secretion in diapause ing from hibernacula in the spring begin to and non-diapause pupae of the Bertha armyworm. Ann produce increased amounts of hydrocarbon. Since Entomol Soc Am 72:13–15. it remains unclear exactly where flies in the wild Howard RW (1993): Cuticular hydrocarbons and chemical com- overwinter (Krafsur and Moon, 1997), determin- munication. In Stanley-Samuelson DW, Nelson DR ing the hydrocarbon amount during the winter (eds): Insect Lipids: Chemistry, Biochemistry and Biol- is not easy. Utilizing lab-reared flies should an- ogy. Lincoln: University of Nebraska Press, pp 179–226. swer some of these questions. Howard RW, Blomquist GJ (1982): Chemical ecology and bio- chemistry of cuticular hydrocarbons. Annu Rev Ento- mol 27:149–172. ACKNOWLEDGMENTS Musca This is journal paper J-17429 of the Iowa Krafsur ES, Moon RD (1997): Bionomics of the face fly, autumnalis. Annu Rev Entomol 42:503–523. Agriculture and Home Economics Experiment Station, Ames, Iowa, project 3304 and 3457, and Krafsur ES, Moon RD, Church CJ (1985): Age structure and was supported by the Hatch Act and State of reproductive history of some overwintering face fly Iowa funds. (Diptera: ) populations in . Ann Entomol Soc Am 78:480–487.

LITERATURE CITED Krafsur ES, Evans LD, Black WC (1986): Autumn phenology of face fly (Diptera: Muscidae) populations in Iowa, USA. Bell RA, Nelson DR, Borg TK, Cardwell DL (1975): Wax se- J Med Entomol 23:389–395. cretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J Insect Physiol Read NR, Moon RD (1986): Diapause induction and commit- 21:1725–1729. ment in face fly, Musca autumnalis (Diptera: Muscidae). Environ Entomol 15:669–677. Blomquist GJ, Nelson DR, de Renobales M (1987): Chemis- try, biochemistry, and physiology of insect cuticular lip- Roelofs WL, Cardé RT (1971): Hydrocarbon sex pheromones ids. Arch Insect Biochem Physiol 6:227–265. in tiger moths (Arctiidae). Science 217:684–686. 214 Jurenka et al.

Rosales AL, Krafsur ES, Kim Y (1994): Cryobiology of the face Yoder JA, Denlinger DL, Dennis MW, Kolattukudy PE (1992): fly and house fly (Diptera: Muscidae). J Med Entomol Enhancement of diapausing flesh fly puparia with ad- 31:671–680. ditional hydrocarbons and evidence for alkane biosyn- thesis by a decarbonylation mechanism. Insect Biochem Stoffolano JG, Matthysse JG (1967): Influence of photoperiod Mol Biol 22:237–243. and temperature on diapause in the face fly, Musca autumnalis (Diptera: Muscidae). Ann Entomol Soc Am 60:1242–1246. Yoder JA, Blomquist GJ, Denlinger DL (1995): Hydrocarbon profiles from puparia of diapausing and nondiapausing Uebel EC, Sonnet PE, Miller RW, Beroza M (1975): Sex flesh flies (Sarcophaga crassipalpis) reflect quantitative pheromone of the face fly, Musca autumnalis De Geer rather than qualitative differences. Arch Insect Biochem (Diptera: Muscidae). J Chem Ecol 1:195–202. Physiol 28:377–385.