Cuticular lipids for species recognition of mole crickets OrthopteraGryllotalpidaeII. Scapteriscus abbreviatus S. acletus S. vicinus S. sp. and Neocurtilla hexadactylaкод для вставкиСкачать
Archives of Insect Biochemistry and Physiology 3:127-134 (1986) Cuticular Lipids for Species Recognition of Mole Crickets (Orthoptera: Gryllotalpidae): II. Scapteriscus abbreviatus, S. acletus, S. vicinus, S. sp., and Neocurtilla hexadactyla James L. Castner and James L. Nation Department of Entomology and Nematology, University of Florida, Gainesville Qualitative analyses were made from whole-body cuticular extracts of Scapteriscus abbreviatus, Scapteriscus acletus, Scapteriscus vicinus, and Neocurtilla hexadactyla by isothermal and temperature-programmed gas chromatography. Adults of both sexes and nymphs of each species were collected in Florida. The chromatographic profiles of peaks were distinct and easily recognizable for each species, regardless of sex or developmental stage. Distinct sexual differences were found in S. acletus and S. abbreviatus. Specimens of S. abbreviatus from Puerto Rico and S. vicinus from Bolivia produced gas chromatography (GC) traces very similar to those of conspecifics collected in Florida. Evidence is presented to illustrate the potential importance of volatile cuticular lipid analysis as a tool for mole cricket identification. Cuticular extracts of an undescribed short-winged species of Scapteriscus from Bolivia were also examined and produced GC traces unlike those of any other species analyzed to date. Key words: Scapferiscus, cuticular lipids, hydrocarbons, gas chromatography, mole cricket INTRODUCTION Seven species of mole crickets (Orthoptera: Gryllotalpidae) have been introduced into the United States, Puerto Rico, and the Virgin Islands, Acknowledgments: T h e authors thank Dr. Chris Pruett for logistical support in Bolivia. Dr. Harry Fowler aided in the field collection of mole crickets. Dr. Tom Walker and Dr. Bob VanderMeer reviewed the manuscript and made helpful suggestions. We thank Kathy Dennis for technical assistance, Sue Wineriter for drawing the figures, and JaniceCastner and Glinda Burnett for typing the manuscript. This work was supported in part by USDA Cooperative Agreement 58-7B30-2-420. This is No. 6154 of the Florida Agricultural Experiment Station Journal Series. Received April 2,1985; accepted July 22,1985. Address reprint requests to James L. Castner, 3103 McCarty Hall, Department of Entomology and Nematology, University of Florida, Cainesville, FL 32611. 0 1986 Alan R. Liss, Inc. 128 Castner and Nation presumably from northern and central South America [l]. Four species now exist in Florida, of which only the northern mole cricket, Neocurtilla hexadactyla (Perty), is native. The three introduced species in Florida belong to the genus Scapteriscus and are the tawny mole cricket, S. vicinus Scudder; the southern mole cricket, S. acletus Rehn and Hebard; and the short-winged mole cricket, S. abbreviatus Scudder. Walker and Nickle  and Walker  traced the entry and spread of introduced mole crickets in the southeastern United States, demonstrating that the ranges of long-winged species have been greatly extended through dispersal flights. Feeding habits among species differ in the amount of plant and animal matter consumed , but all probably cause mechanical damage to subterranean plant structures by tunneling. Lack of specialized natural enemies has allowed mole crickets to become important economic pests of turf and agriculture in Florida. The estimate of total losses and costs of control for 1980 due to mole crickets in Florida was $35 million . The primary morphological features used to distinguish mole cricket species are wing length, pronotal patterns, and the number and spacing of the dactyls on the forelegs . Species differentiation has also been accomplished by analysis and comparison of calling songs produced by adult males . Morphological analyses do not work well with juvenile mole crickets, however, because in many species diagnostic morphological characteristics do not develop until later instars. Calling-song analysis necessitates sophisticated equipment and is only useful in comparing live adult male specimens, because females and juveniles do not call. The analysis of cuticular lipids by GC* has been used by Carlson and Service [7J,Carlson and Walsh [S], and Howard and Blomquist  to distinguish a variety of closely related insect species. Results by Castner and Nation [lo] suggested that this method would be useful in differentiating morphologically similar species of mole crickets. The present study was undertaken 1)to determine if a unique GC profile could be obtained from all mole cricket species present in Florida, 2) to evaluate the consistency of the GC pattern obtained from males, females, and nymphs of the same species, and 3) to examine conspecific specimens collected from widely separated geographic populations. MATERIALS AND METHODS Biological Material The four mole cricket species occurring in Florida were examined from specimens collected in 1983-84 by sound traps [ll], linear pitfall traps , and digging. Table 1gives the number, sex or developmental stage, location, and method of capture for each species analyzed. S. acletus occurs in two distinct forms in Florida, as characterized by the pronotum . One form has an ovate, mottled pronotum (mottled); the other has a dark pronotum with *Abbreviations: gas chromatography = GC; Kovats index = KI. Cuticular Lipids of Mole Crickets 129 TABLE 1. Collection Data of Mole Crickets Utilized in Cuticular Lipid Analyses No. examined and sexistage Location Neocurt illa hexadactyla Scapteriscus abbreviat us 90' 7 Q 9 nymphsb Gainesville, FL LPT 40' 8 Q 2 nymphs 30' 6 nymphs 1Q 2 nymphs Scapferiscus acfetus (4-dot) Scapteriscus aclefus (mottled) Scapteriscus vicinus 60'69 5 nymphs 10'20 Boynton Beach, FL Ft. Lauderdale, FL Arecibo, PR Boynton Beach, FL Gainesville, FL Defuniak Springs, FL LPT D LPT ST LPT ST Jacksonville, FL Gainesville, FL Santa Cruz, Bolivia ST LPT D Scapteriscus sp. 30' 4 Q Santa Cruz, Bolivia D Svecies 100' 1 O Q 10 nymphs Collection methoda 100'4 Q 'LPT = linear pitfall trap; ST = sound trap; D = digging. bThe sex of nymphs cannot be determined from external morphology. four light dots and the sides subparallel (4-dot). Both forms of S. acletus were analyzed in this study. Specimens were killed by freezing and maintained frozen until extraction. Analyses The following method of extraction, used in previous analyses [lo] with qualitatively and quantitatively reproducible results, was repeated here. Whole bodies of frozen mole crickets were immersed in 5-10 ml of pentane and gently agitated for 1min. The pentane was evaporated to approximately 0.5 ml at room temperature with a gentle stream of nitrogen. From 1to 4 pl of the concentrated extract was injected into a Varian 3700 gas chromatograph equipped with a flame ionization detector. Data were collected and processed directly from the chromatograph by a Hewlett-Packard 3390A integrator. Volatile components were separated on a 2 mm x 1.83 m coiled glass column packed with 3% OV-1 coated on 1001120 mesh Gas Chrom Q. The carrier gas was nitrogen at a flow of 28 mllmin. Representative samples from each species, including the samples used to derive Figures 1 and 2 (except Fig. lb), were compared with respect to GC pattern before and after passing an aliquot of the sample through 0.5-g BioSil HA, minus 325 mesh (BioRad Corporation) in a Pasteur pipette, and collecting the first 1 ml eluted with pentane. A synthetic standard consisting of a mixture of hexadecanol, methyl tetradecanoate, methyl hexadecanoate, methyl octadecanoate, eicosane, docosane, tetracosane, and hexacosane was used to test the ability of the BioSil HA column to separate hydrocarbons from other lipid classes. Kovats indices were calculated according to Kovats 1131, using isothermal chromatography at 190°C with hexacosane (C26 alkane) and octacosane (C28 alkane) hydrocarbon standards for the known species of mole crickets. The injector port was held at 200°C and the detector at 210°C. KI values of the longer-chain hydrocarbons found in the newly discovered 130 Castner and Nation short-winged species from Bolivia were calculated similarly but with the column at 250°C and the injector port and detector maintained at 260°C. Temperature-programmed analyses were carried out from 150°C to 270°C at 2"lmin and holding at 270°C for 10 min. The injector port was maintained at 270°C and the detector at 280°C for temperature-programmed chromatography. RESULTS Representative GC traces from temperature programming are shown in Figures 1 and 2 with calculated KI values for mole cricket hydrocarbons. Comparison of GC records before and after the BioSil HA column treatment showed that all peaks indicated by a KI value are hydrocarbons, having passed through the BioSil column in the first milliliter of eluate. Hydrocarbons in the test mixture also passed through the BioSil HA column in the first milliliter of eluate, but the oxygenated compounds in the test mixture remained on the BioSil column. A qualitative sexual difference in hydrocarbons present on the cuticle was consistently found between the adult sexes of S. ubbreviutus and S. acletus. Sexual differences were not noted in N. hexuductylu or in the Scupteviscus sp. from Bolivia. Nymphs at various stages of development were available for all species, except Scupteviscus sp. from Bolivia. Chromatographic profiles of the immature mole crickets did not differ substantially from those of adults. The sex of nymphs was not determined. Cuticular components of both sexes of S. abbreviatus are shown in Figure la,b. The major compounds are indicated by two pairs of peaks with calculated KI values (Fig. la) of 2298, 2337, 2498, and 2540. The peak with KI = 2540 ( 0 )or KI = 2546 ( Q ) actually consists of at least two components (see arrow on shoulder of peak, Fig. lb) that do not completely separate under the chromatographic conditions utilized. All samples also showed minor peaks with calculated KI values (Fig. la) at 2211, 2263, 2398, 2436, 2583,2608,2642,2661,2698,2745, and 2779. No peaks with a KI value greater than 3000 were recorded from males or nymphs, but all females examined (n = 10) showed a prominent peak with a KI value of approximately 3008, with the exception of the two specimens collected from Puerto Rico. Minor peaks with values of 2914 and 3110 were also recorded from female S. abbueviutus, but not observed in males. The compounds present on the cuticle of N. hexudactyla (Fig. lc) in greatest amounts had KI values of 2098, 2262, 2294, and 2694. Minor constituents were found with KI values of 2195, 2401, 2493, 2594, and 2888. Only minor differences were recorded on the chromatograms of N. hexuductylu, regardless of sex or developmental stage. The volatile cuticular components of both sexes of S. ucletus are similar, although consistent sexual differences were found (Fig. 2a,b). Major peaks in 4-dot and mottled females have KI values of 2664, 2703, 2843, and 2862. Males of both 4-dot and mottled S. acletus do not show the peak at 2862, unless it is represented by the small shoulder (arrow in Fig. 2b) on the 2842 S. abbrevlatus 13' I 0 I 10 20 30 40 60 50 70 Retention Time (Min.1 S. abbrevlatus $! I 0 10 20 30 40 50 60 70 Retention Time (Min.1 m C N - o) mo) 0 N N N N N N. hexadactyla $! W C fn 0 a fn W U L 0 +- 0 W W L3 1 I 0 10 20 I 30 40 50 60 70 Retention Time (Min.1 Fig. 1. Temperature programmed gas chromatographic traces of the volatile cuticular lipids of individual mole crickets (a) S. abbreviatus,o (b) S. abbreviatusq (arrow indicates the probable presence of an additional component that did not separate completely), (c) N. hexadactyla Q . Numbers above peaks refer to calculated Kovats index. 132 Castner and Nation a S.acletus $? 14-dotl m c 0 fn a m a: L 0 0 m m c n I 0 10 20 30 40 50 60 70 Retention Time (Min.) b S. ecletus 8 [rl-dotl m fn C 0 a fn m a: L 0 0 m 4- m 0 0 10 20 30 40 50 60 70 50 60 70 Retention Time (Min.1 C L 0 I 0 10 20 30 40 Retention Time (Min.1 Fig. 2. Temperature programmed gas chromatographic traces of the volatile cuticular lipids of individual mole crickets (a) S. acletus (4dot), Q (b) S. acletus ( 4 d o t ) u (arrow indicates a component that has not separated completely and that may represent the 2862 peak observed in females), (c) Scapteriscus sp. Q Numbers above peaks refer to calculated Kovats index. Cuticular Lipids of Mole Crickets 133 peak. Peaks were also seen at 2462, 2496, 2570, and 2598 on the chromatograms of males, but not of females. The 4-dot and mottled morphological populations did exhibit some differences. In all 4-dot females (n = 6), the peak at 2843 was larger than the peak at 2862 (Fig. 2a). However, in the two mottled females examined, this situation was reversed. Small peaks on the leading edge of the large 2842 peak (Fig. 2b) were more prominent on the mottled male chromatogram than on the chromatograms of 4-dot males. GC data for S. vicinus collected in Florida were initially reported by Castner and Nation [lo]. The GC traces produced from mole crickets collected in Bolivia and identified as S. vicinus showed the same array of peaks, with some variation in relative abundances. Primary peaks from the Bolivian S. vicinus had KI values of 2201, 2245, 2300, 2324, and 2444. Although the 2444 peak is present in Florida individuals, it does not represent a major cuticularcomponent asit didinall(n = 14)Bolivianindividualssampled. Florida S. vicinus consistently showed a much larger peak at 2324 than at 2300 [lo], whereas the relative magnitudes of these two peaks were reversed in Bolivian specimens. Minor peaks were seen in samples from both locations at 2104, 2400, 2499, 2601, 2699, 2798, and 2893. The last species analyzed was an undescribed species of Scapteriscus collected from Santa Cruz, Bolivia. Specimens were 25-35 mm long with extremely short wings. The pronotum was dark with a pattern of two oval light areas on each side, which sometimes combined to form two parallel light areas extending the length of the pronotum. The GC traces were different from all previous species analyzed in the large number of high-molecularweight compounds representing components of 36 carbons or more (Fig. 2c). Major peaks had KI values of 2500, 2695, 3677, and 3890. Minor peaks occurred at 2901, 3599, 3694, 3783, and 3847. DISCUSSION The lipids of insect cuticle are located primarily on the surface of the integument and serve physiological, behavioral, and ecological roles. Physiologically they aid in permeability relationships and help control water loss through the cuticle [14-161. Hadley  found seasonal and acclimation temperature-induced changes as a probable adaptation against water loss in the cuticular lipids of a desert-dwelling tenebrionid beetle. Numerous examples in which cuticular lipids serve as recognition pheromones, kairomones, and in other behavioral contexts have been reviewed by Howard and Blomquist . Because cuticular lipids are often closely integrated with the specific behavior, physiology, and ecology of an insect, it seems logical that they may serve as reliable indicators of species, or even of races and strains [17l. There are 55 known species of mole crickets divided among five genera [1,18]. Although we have examined only seven species, it is clear that gas chromatographic analysis of cuticular lipids is a useful tool for species identification of mole crickets. In the seven species examined, a simple visual comparison of the qualitative profile is sufficient for characterization. Closely 134 Castner and Nation related species, however, may necessitate a more detailed quantitative comparison of marker peaks andlor peak ratios [7l. The 4-dot and mottled morphological forms of S. ucletus occurring in Florida showed only minor qualitative and quantitative differences in their cuticular lipid patterns. This suggests that they represent only one species. Additional analyses of the mottled form should be performed, however, because of the small sample size available for the present study. LITERATURE CITED 1. Nickle DA, Castner JL: Introduced species of mole crickets in the United States, Puerto Rico, and the Virgin Islands (Orthoptera: Gryllotalpidae). 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