Musca Autumnalis) Russell A

206 Jurenka et al. Archives of Insect Biochemistry and Physiology 37:206–214 (1998) Hydrocarbon Profiles of Diapausing and Reproductive Adult Face Flies (Musca 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, Musca autumnalis, 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 diapausing 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/fly, 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 differences 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 alkanes 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 insects. The most important insects. Recently it has been demonstrated that roles are in chemical communication and in pro- the puparium from a diapausing pupa 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 determined 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 housefly (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.

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