Fatty Acid Composition of Neutral Lipid Energy Reserves in Infective Juveniles of Entomopathogenic Nematodes

Comp. Biochem. Physiol. Vol. 118B, No. 2, pp. 341–348, 1997 Copyright  1997 Elsevier Science Inc. All rights reserved.

ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00057-6

Fatty Acid Composition of Neutral Lipid Energy Reserves in Infective Juveniles of Entomopathogenic Nematodes Mavji N. Patel and Denis J. Wright Department of Biology, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire SL5 7PY, U.K. ABSTRACT. The fatty acid composition of neutral lipids from infective juveniles (IJs) of Steinernema carpocapsae strain All, S. riobravis strain Biosys 355, S. feltiae strain UK76, and S. glaseri strain NC stored in distilled water at 25°C was determined. Newly emerged IJs of all four species had similar neutral lipid fatty acid profiles and of the 18 fatty acids identified, C18:1n29 (43–49 mol %), C16:0 (18–23%), C18:2n26 (8–14%) and C18: 0 (4–8%) were the most abundant. Unsaturated fatty acids predominated, with about 50% being monoene and 14–22% polyene; the unsaturation index ranged from 91.6 in S. glaseri to 111.6 in S. carpocapsae. The fatty acid composition of the total lipid and the free fatty acid fraction mirrored that of the neutral lipids. During storage, the relative levels (%) of C16:0, C18:0, and C18:1n29 in the neutral lipids declined significantly, suggesting they were preferentially utilised. comp biochem physiol 118B;2:341–348, 1997.  1997 Elsevier Science Inc. KEY WORDS. Infective juvenile, Steinernema, entomopathogenic nematodes, neutral lipids, fatty acids, GLC

INTRODUCTION The dauer, third stage infective juvenile (IJ) of insect parasitic (entomopathogenic) nematodes in the genera Steinernema and Heterorhabditis (Steinernematidae and Heterorhabditidae) is free-living and responsible for locating and infecting a new insect host (16). The aerobic habitat of IJs means that they can utilise fatty acids to yield energy and, in common with the free-living stages of a number of animal parasitic nematode species (2,11), stored neutral lipids (mainly triacylglycerols) provide the main source of fatty acids for this purpose (15). Neutral lipids comprise between 24 and 31% dry weight of the IJs of steinernematid nematodes (15). With such large amounts, their fatty acid composition could have profound effects on nematode survival by affecting the fluidity of the lipids within the nematode and the amount of potential energy derived from them. The fatty acid composition of neutral lipids from IJs of entomopathogenic nematodes has only been reported for Steinernema carpocapsae (5), although total lipids have been examined for several other species. Selvan et al. (18,19) reported that the total lipids fatty acid of newly emerged IJs Address reprint requests to: Mavji Patel, Department of Biology, Imperial College of Science, Technology & Medicine, Silwood Park, Ascot, Berkshire SL5 7PY, United Kingdom; Tel. 144 1344 294303; Fax 144 1344 294339; Messages: 144 1344 294243; E-mail: [email protected]. Received 8 January 1997; revised 12 March 1997; accepted 14 April 1997.

of S. carpocapsae, S. feltiae, S. glaseri, and S. scapterisci mostly contained saturated fatty acids (50–66%), while the total lipids of the heterorhabditid nematodes, Heterorhabditis bacteriophora and H. megidis contained proportionately more unsaturated fatty acids (57% and 62%, respectively). In contrast, Wijbenga and Rodgers (26) and Stolinski (21) found unsaturated fatty acids accounted for over 70% of the total lipid composition in S. feltiae, and Fodor et al. (5) found a similar situation in the neutral lipids of S. carpocapsae. The conflicting results for the steinernematid species are of particular interest, since within nematodes and helminths in general, unsaturated fatty acids are reported to predominate (2,12). In the present study, we determined the fatty acid compositions of neutral lipids from newly emerged and stored IJs of four steinernematid species: S. carpocapsae, S. riobravis, S. feltiae and S. glaseri. In addition, the compositions of the total lipids and free fatty acids from newly emerged IJs were examined for comparison. MATERIALS AND METHODS Nematodes and Insects Infective juveniles of S. carpocapsae strain All, S. riobravis strain Biosys 355, S. feltiae strain UK76, and S. glaseri strain NC were cultured at 20°C in late instar larvae of the wax moth, Galleria mellonella (27) and only IJs emerging within 4 days after first emergence were used in the study. Infective

M. N. Patel and D. J. Wright

342

juveniles were stored in distilled water in plastic culture boxes (15 3 11 3 6 cm; 60 boxes per species) and incubated in the dark at 25°C (200 ml per box at 2000 IJs ml 21 ). The 60 boxes were numbered and divided randomly into three groups of 20 for each nematode species. The storage water was changed weekly and dead nematodes were removed by allowing nematodes to pass through filter paper (dead nematodes were retained on the filter paper). The culture boxes were manually shaken twice daily for 10 min. Nematode Fatty Acid Analysis Infective juveniles were sampled at various intervals from all 60 boxes (one pooled sample of .100,000 IJs per 20 boxes; giving three independent replicates). The first sample of nematodes (day 0) was taken on the first day the experiment was set up; these nematodes were no more than 4 days old following emergence from the insect. Dead nematodes were removed by filtration (see above) and the number of IJs in each sample was estimated by dilution. Differences in the survivorship of the four species (15) meant that S. carpocapsae and S. riobravis were studied for 100 days, and S. feltiae and S. glaseri for 260 days. The extraction of lipids was based on the technique described by Christie (3). Nematodes were homogenized by sonication and then freeze-

dried. Lipids were extracted with 19 volumes of chloroform: methanol (2:1 v/v) at 4°C for 48 hr. Contaminants were removed by adding 0.04% (w/v) CaCl 2 , corresponding to 22% by volume of the combined solvent. The mixture was centrifuged at 681 g for 10 min to give two clear phases. The upper phase was discarded and the lower phase (chloroform) was washed with an equal volume of equilibrated solvents containing chloroform :methanol:0.04% (w/v) CaCl 2 (3:48 :47 v/v). The mixture was centrifuged at 681 g for 10 min and the upper phase discarded. Absolute ethanol (200 µl) was added to the lower phase and the solvents removed by rotary film evaporation. The dried lipids were then dissolved in a small volume of chloroform. A small sample (10%) of the extracted lipid was set aside for the analysis of total lipids while the remainder was fractionated to give neutral lipids and free fatty acids using solid-phase column chromatography (NH 2-aminopropyl, 500 mg, 3 ml; Bond Elut, International Sorbent Technology Ltd., Mid Glamorgan, UK) (9). The neutral lipids were eluted with 4 ml chloroform:propan-2-ol (2:1 v/v) and the free-fatty acids were eluted with 4 ml 2% (v/v) acetic acid in diethyl ether. The lipid fractions were then dried by rotary film evaporation. The lipids were saponified and converted to fatty acid methyl esters (FAMEs) using a mixture of 1 ml toluene and

TABLE 1. Changes in neutral lipid fatty composition (mol % 6 SE) of infective juveniles of Steinernema carpocapsae stored

in distilled water at 25°C Storage time (days)* Fatty acid C14 :0 C14 :1 C16 :0 C16 :1n27 C16 :3n23 C16 :4 C18 :0 C18 :1n29 C18 :2n26 C18 :3n23 C20 :0 C20 :1 C20 :2 C20 :3n26 C20 :4n26 C20 :5n23 C22 :6n23 C24 :0 Saturated Monoene Polyene U.I. Neutral lipid (ng IJ21)†

0 6 0.0 6 0.1 6 0.6 6 0.1 6 0.1 6 0.1 6 0.3 6 1.7 6 0.7 6 0.0 nd 1.6 6 0.3 nd 1.5 6 0.2 1.6 6 0.1 1.7 6 0.2 nd nd 24.9 53.1 21.6 111.6 34.8 6 0.7

0.4 2.0 18.8 3.2 1.2 1.4 5.7 46.3 12.7 1.5

30

a a a a a

t 1.7 6 0.1 10.6 6 0.3 2.3 6 0.1 1.0 6 0.0 1.4 6 0.1 6.7 6 0.2 49.9 6 1.5 12.9 6 0.8 1.6 6 0.1 3.1 6 0.3 0.6 6 0.1 0.6 6 0.0 2.4 6 0.1 2.1 6 0.3 2.2 6 0.1 0.5 6 0.1 t 20.4 54.5 24.7 124.5 34.0 6 3.1

60

b b b a a

a

0.4 6 0.0 1.5 6 0.2 8.1 6 0.2 5.7 6 0.1 1.4 6 0.1 2.6 6 0.2 4.3 6 0.1 43.8 6 2.2 16.6 6 1.1 nd 3.9 6 0.2 2.1 6 0.2 1.2 6 0.2 0.3 6 0.0 2.4 6 0.3 2.5 6 0.2 2.9 6 0.2 t 16.7 53.1 29.9 143.7 11.4 6 2.1

100

c c c b b

b

6 0.1 6 0.1 6 0.1 6 0.2 6 0.2 6 0.3 6 0.8 6 0.9 6 1.3 nd 2.9 6 0.1 2.9 6 0.2 0.7 6 0.1 0.3 6 0.0 1.6 6 0.3 2.1 6 0.0 4.7 6 0.2 nd 9.5 56.5 33.8 160.7 3.7 6 0.3

0.7 1.6 3.6 8.4 1.4 3.3 2.3 43.6 19.7

d d d b c

c

*n 5 3; t 5 trace (,0.1%); nd 5 not detectable; U.I. 5 unsaturation index, defined as S(% of fatty acid 3 number of double bonds); totals do not necessarily add up to 100%; Mol percentage refers to the amount of each fatty acid as a percentage of the total fatty acid, expressed on a molar basis. Means within the same row followed by the same letter are not significantly different (P . 0.05). †Data from Patel et al. (15).

Steinernema Neutral Lipid Fatty Acids

343

TABLE 2. Changes in neutral lipid fatty composition (mol % 6 SE) of infective juveniles of Steinernema riobravis stored in

distilled water at 25°C Storage time (days)* Fatty acid C14 :0 C14 :1 C16 :0 C16 :1n27 C16 :3n23 C16 :4 C18 :0 C18 :1n29 C18 :2n26 C18 :3n23 C20 :0 C20 :1 C20 :2 C20 :3n26 C20 :4n26 C20 :5n23 C22 :6n23 C24 :0 Saturated Monoene Polyene U.I. Neutral lipid (ng IJ21)†

0 6 0.0 6 0.2 6 0.8 6 0.1 6 0.1 6 0.1 6 0.3 6 0.9 6 0.6 6 0.0 nd 3.0 6 0.0 nd 0.9 6 0.1 0.9 6 0.2 1.0 6 0.1 t nd 28.8 55.7 14.3 96.9 46.9 6 2.5 0.2 1.7 22.8 2.2 1.2 1.2 5.8 48.8 9.1 1.0

30

a a a a a a

t 6 0.3 6 0.3 6 0.3 6 0.2 6 0.3 6 0.6 6 1.3 6 1.1 6 0.2 nd 2.8 6 0.1 0.2 6 0.0 0.9 6 0.1 1.0 6 0.1 1.0 6 0.1 1.0 6 0.3 nd 25.9 53.2 20.6 113.2 30.1 6 1.2 1.4 19.7 2.4 2.5 2.6 6.2 46.6 10.2 1.2

60

b a a a ab a

a

t 6 0.1 6 0.3 6 0.1 6 0.2 6 0.2 6 0.3 6 1.1 6 0.6 6 0.2 nd 3.4 6 0.3 1.5 6 0.2 1.5 6 0.2 1.3 6 0.1 1.2 6 0.2 1.4 6 0.2 nd 17.2 52.4 30.2 143.5 16.7 6 3.3

4.0 12.6 3.5 1.5 6.1 4.6 41.5 12.0 3.7

100

c b b b bc b

b

t 5.6 6 0.3 9.4 6 0.1 4.5 6 0.3 1.8 6 0.1 7.3 6 0.2 4.8 6 0.2 37.7 6 0.2 13.2 6 0.6 3.9 6 0.8 nd 5.0 6 0.2 1.1 6 0.1 1.0 6 0.1 0.9 6 0.0 0.5 6 0.0 3.1 6 0.2 nd 14.2 52.8 32.8 155.4 3.7 6 0.9

d c b c c c

c

*n 5 3; t 5 trace (,0.1%); nd 5 not detectable; U.I. 5 unsaturation index, defined as S(% of fatty acid 3 number of double bonds); totals do not necessarily add up to 100%; Mol percentage refers to the amount of each fatty acid as a percentage of the total fatty acid, expressed on a molar basis. Means within the same row followed by the same letter are not significantly different (P . 0.05). †Data from Patel et al. (15).

1% (v/v) sulphuric acid in 2 ml of methanol (3). The dry FAMEs were dissolved in a known volume of hexane and analysed by gas-liquid chromatography (GLC) using a Varian 6000 equipped with a capillary column (Carbowax EconoCap 30 m 3 0.32 mm internal diameter, 0.25 µm film thickness, Alltech Associates, Lancashire, UK) and a flame ionization detector (270°C). Injections (at 250°C) were made in split mode (50:1) with N 2 as the carrier gas. The temperature program was isothermal at 100°C for 2 min, 10°C min 21 to 160°C and 2°C min 21 to 235°C and held for 1 min. GLC peak areas were quantified using a Varian 401 Vista integrator. FAMEs were identified by reference to authentic standards and by using the ‘‘equivalent chain-length’’ method (3). The addition of an internal standard to the lipid samples allowed quantification of FAMEs and hence the quantitative determination lipids. Fatty acid compositions are expressed as mol percentages in text and tables. Statistical Analysis The generalised linear modelling package GLIM v3.77 ( 1985, Royal Statistical Society, London; Numerical Al-

gorithms Group, Oxford) was used for statistical analysis (4). The fatty acid data were subject to arcsine square root or log transformation before ANOVA. Significance was tested at the 5% level. RESULTS Fatty Acid Compositions of Neutral Lipids from Newly Emerged Infective Juveniles Infective juveniles of the four Steinernema species were found to have similar fatty acid compositions for their neutral lipids (Tables 1–4). In newly emerged IJs (day 0), oleic acid (C18 :1n29; 43–49 mol %), palmitic acid (C16:0; 18– 23%), linoleic acid (C18 :2n26; 8–14%) and stearic acid (C18:0; 4–8%) predominated. The remaining fatty acids detected were present at low levels, typically between 0.5 to 4%. Unsaturated fatty acids predominated in all four species, with almost 50% of fatty acids being monoene and 15– 20% polyene. The unsaturation index of S. carpocapsae, S. riobravis, S. feltiae, and S. glaseri was 111.6, 96.9, 108.7, and 91.6, respectively. The major polyene fatty acids belonged to the C 20 n23 and n26 families and accounted for 3–6% of the total composition in the four species.

M. N. Patel and D. J. Wright

344

TABLE 3. Changes in neutral lipid fatty composition (mol % 6 SE) of infective juveniles of Steinernema feltiae stored in

distilled water at 25°C Storage time (days)* Fatty acid C14 :0 C14 :1 C16 :0 C16 :1n27 C16 :3n23 C16 :4 C18 :0 C18 :1n29 C18 :2n26 C18 :3n23 C20 :0 C20 :1 C20 :2 C20 :3n26 C20 :4n26 C20 :5n23 C22 :6n23 C24 :0 Saturated Monoene Polyene U.I. Neutral lipid (ng IJ21)†

0 6 0.0 6 0.2 6 1.3 6 0.1 6 0.0 6 0.1 6 0.2 6 3.2 6 1.1 6 0.1 6 0.2 6 0.1 6 0.0 6 0.0 6 0.1 6 0.2 6 0.0 nd 27.0 51.1 21.8 108.7 52.2 6 1.0

0.2 4.1 20.1 2.1 1.2 1.4 4.8 43.1 13.3 0.9 1.9 1.8 0.9 1.1 1.2 1.6 0.2

80

a a a a a

a

t 6 0.4 6 0.4 6 0.4 6 0.2 6 0.3 6 0.4 6 2.0 6 0.8 6 0.5 6 0.1 6 0.4 6 0.4 6 0.3 6 0.3 6 0.2 6 0.2 nd 24.8 49.6 25.3 120.5 48.1 6 3.0 3.7 18.4 3.4 1.3 2.4 5.4 39.7 13.5 1.9 1.0 2.8 1.4 1.0 1.3 1.3 1.2

210

a b b a a

b

t 4.3 6 0.4 14.1 6 0.1 3.5 6 0.2 1.5 6 0.3 4.8 6 0.1 4.6 6 0.2 40.4 6 1.0 14.8 6 1.3 1.6 6 0.2 0.2 6 0.0 1.9 6 0.2 0.8 6 0.0 1.2 6 0.2 1.1 6 0.3 1.6 6 0.4 3.6 6 0.2 nd 18.7 50.1 30.9 147.2 28.1 6 2.6

260

b b ac a a

c

6 0.0 6 0.4 6 0.3 6 0.2 6 0.4 6 0.2 6 0.3 6 0.9 6 1.5 6 0.2 nd 2.0 6 0.3 1.6 6 2.2 1.1 6 0.0 1.3 6 0.8 3.0 6 0.8 3.1 6 0.2 nd 18.5 44.6 36.9 158.2 13.9 6 1.3 0.1 4.9 13.3 3.9 1.8 5.8 5.1 33.8 17.9 1.3

c b bc b b

d

*n 5 3; t 5 trace (,0.1%); nd 5 not detectable; U.I. 5 unsaturation index, defined as S(% of fatty acid 3 number of double bonds); totals do not necessarily add up to 100%; Mol percentage refers to the amount of each fatty acid as a percentage of the total fatty acid, expressed on a molar basis. Means within the same row followed by the same letter are not significantly different (P . 0.05). †Data from Patel et al. (15).

Fatty Acid Compositions of the Total Lipid and Free Fatty Acid Fractions from Newly Emerged Infective Juveniles The fatty acid composition of total lipids from newly emerged IJs (Table 5) mirrored in general their neutral lipid compositions. The one obvious difference was the significantly greater (P , 0.05) proportion (%) of lignoceric acid (C24 :0) in the total lipids compared with the neutral lipids, particularly in the case of S. feltiae and S. glaseri. Analysis of the free fatty acid composition of newly emerged IJs (Table 6) also showed strong similarities to the composition of neutral lipids in each species. Notable differences were the significantly greater (P , 0.05) proportions of stearic acid and significantly lower (P , 0.05) proportions of linoleic acid in the free fatty acid fraction, and the absence of lignoceric acid and C22:6n23. In the case of S. feltiae, the proportion of C16:0 was also significantly greater (P , 0.05) in the free fatty acid fraction compared with the neutral lipids. The unsaturation index of the free fatty acid fraction was different for each species compared with those for their respective neutral lipid fractions, more so in the case of S. feltiae. Steinernema feltiae and S. glaseri had greater lev-

els (on a % basis) of saturated fatty acids compared with S. carpocapsae and S. riobravis. The relatively lower unsaturation index of the free fatty acid fraction of S. feltiae (83.2) was due largely to the presence of a lower proportion of oleic acid compared with the other three species. Changes in Fatty Acid Compositions of Neutral Lipids from Infective Juveniles During Storage In all four nematode species the percentage proportion of the four major fatty acids in the neutral lipids significantly changed during storage (Tables 1–4). The proportion of palmitic, stearic and oleic acids declined during the storage period, while linoleic acid, C22:6n23 and C20:1 increased. Of the two most utilised fatty acids, palmitic (C16 : 0) and oleic (C18:1n29), the latter was used faster in S. feltiae and S. glaseri while in S. carpocapsae palmitic acid was used at a greater rate; there was no distinction in the rates of utilisation of these fatty acids in S. riobravis. In all four species, the unsaturation index increased with storage time; by almost 50% in some cases. During the storage period, the fatty acid composition of the free fatty acid fraction did

Steinernema Neutral Lipid Fatty Acids

345

TABLE 4. Changes in neutral lipid fatty composition (mol % 6 SE) of infective juveniles of Steinernema glaseri stored in

distilled water at 25°C Storage time (days)* Fatty acid C14 :0 C14 :1 C16 :0 C16 :1n27 C16 :3n23 C16 :4 C18 :0 C18 :1n29 C18 :2n26 C18 :3n23 C20 :0 C20 :1 C20 :2 C20 :3n26 C20 :4n26 C20 :5n23 C22 :6n23 C24 :0 Saturated Monoene Polyene U.I. Neutral lipid (ng IJ21)†

0

80

6 0.0 6 0.1 6 1.1 6 0.2 6 0.1 6 0.1 6 1.1 6 2.3 6 0.9 6 0.1 6 0.2 6 0.1 6 0.0 6 0.0 6 0.1 6 0.2 6 0.0 nd 33.1 52.0 14.8 91.6 115 6 4.3

0.5 6 0.1 2.0 6 0.4 19.9 6 1.3 1.5 6 0.6 1.3 6 0.3 2.5 6 0.7 6.3 6 0.8 42.6 6 3.4 10.4 6 2.6 1.8 6 0.4 2.9 6 0.3 3.3 6 0.3 1.1 6 0.2 0.9 6 0.0 1.1 6 0.1 1.0 6 0.1 0.9 6 0.1 nd 29.6 49.4 21.0 109.2 111 6 3.4

0.4 1.2 21.7 2.1 1.5 0.6 7.2 46.0 8.1 1.2 3.8 2.7 0.5 0.9 1.0 0.8 0.2

a a a a a

a

210

a a b a a

b

t 1.2 6 0.1 15.1 6 1.0 3.0 6 0.2 1.1 6 0.0 7.3 6 0.2 4.4 6 0.6 43.1 6 1.6 10.4 6 1.1 1.2 6 0.1 nd 6.9 6 0.2 0.8 6 0.1 1.7 6 0.2 1.4 6 0.0 1.1 6 0.1 0.9 6 0.0 nd 19.5 54.2 25.9 134.3 73.8 6 5.7

260

b b b a a

b

t 6 0.2 6 0.3 6 0.2 6 0.2 6 0.4 6 0.2 6 0.6 6 0.7 6 0.3 nd 7.4 6 0.3 0.9 6 0.2 1.3 6 0.3 1.6 6 0.3 1.3 6 0.4 1.0 6 0.2 nd 19.0 48.8 32.0 144.9 52.8 6 0.7

1.5 14.9 3.2 1.8 8.1 4.1 36.7 14.3 1.7

b b b b b

b

*n 5 3; t 5 trace (,0.1%); nd 5 not detectable; U.I. 5 unsaturation index, defined as S(% of fatty acid 3 number of double bonds); totals do not necessarily add up to 100%; Mol percentage refers to the amount of each fatty acid as a percentage of the total fatty acid, expressed on a molar basis. Means within the same row followed by the same letter are not significantly different (P . 0.05). †Data from Patel et al. (15).

not change significantly (P . 0.05) in each species (data not shown). DISCUSSION The major fatty acids present in the neutral lipids of all four Steinernema species were oleic acid (C18:1n29) . palmitic acid (C16:0) . linoleic acid (C18 :2n26) . stearic acid (C18 :0), accounting between them for 81–87% of total composition in newly emerged IJs, and the proportion of unsaturated fatty acids to saturated fatty acids was more than three-fold greater. This was also the case in the total lipid and free fatty acid fractions. Wijbenga and Rodgers (26) and Fodor et al. (5) also found that unsaturated fatty acids dominated in S. feltiae and S. carpocapsae, respectively; with oleic and linoleic acids accounting for more than 50% of the total composition. Thus, although the nematodes used in both of the latter studies were cultured in vitro, their results together with ours are in agreement with the general consensus that unsaturated fatty acids predominate in nematodes (1,8,10,21). These results contrast with the findings of Selvan et al. (18,19) who found the reverse to be true in S. carpocapsae, S. feltiae, S. glaseri, and S. scapterisci (all cultured in G. mellonella), where stearic acid (C18:0) was re-

ported to account for up to 50% of the total composition in some species. Although the latter authors analysed total rather than neutral lipids, the present study shows how similar the fatty acid profiles are for total lipid and neutral lipids. This is to be expected, since neutral lipids in Steinernema species comprise between 74% and 77% of the total lipids (15). There are two main factors that can influence the fatty acid composition of lipids: temperature and diet (2). In the present context, temperature was unlikely to have been a contributory factor since the culturing temperature in our study and that of Selvan et al. (18,19) was similar. With respect to diet, the wax moth larvae used in the present study were purchased from a commercial supplier and had been reared on an artificial diet. Similarly, Selvan et al. (18,19) also used commercially reared G. mellonella (P. S. Grewal, personal communication). Analysis of the fatty acid composition of the total lipids from wax moth larvae used in the present study (data not shown) found the insect to have a composition similar to that found by other workers and was typical of lepidopteran larvae (24). Although the nutritional quality of the insect is certain to change following degradation caused by the action of the symbiotic bacteria released into the insect. Even so, the bacteria upon

M. N. Patel and D. J. Wright

346

TABLE 5. Total lipid fatty composition (mol % 6 SE)* of newly emerged infective juveniles of four Steinernema species

Fatty acid C14 :0 C14 :1 C16 :0 C16 :1n27 C16 :3n23 C16 :4 C18 :0 C18 :1n29 C18 :2n26 C18 :3n23 C20 :0 C20 :1 C20 :2 C20 :3n26 C20 :4n26 C20 :5n23 C22 :6n23 C24 :0 Saturated Monoene Polyene U.I. Total lipid (ng IJ21)†

S. carpocapsae t 1.7 6 0.2 17.6 6 0.8 3.3 6 0.2 1.1 6 0.1 2.9 6 0.9 7.4 6 0.3 45.0 6 1.9 11.2 6 1.0 0.7 6 0.1 0.6 6 0.1 1.8 6 0.2 0.5 6 0.1 1.7 6 0.1 2.1 6 0.0 1.9 6 0.2 0.3 6 0.0 t 25.7 51.8 22.4 117.0 44.6 6 2.5

a a a a a a a a a

S. riobravis t 6 0.2 6 1.7 6 0.3 6 0.0 6 0.0 6 0.1 6 0.7 6 0.1 6 0.2 t 2.9 6 0.4 0.2 6 0.0 1.1 6 0.1 0.9 6 0.1 1.1 6 0.1 t 0.8 6 0.1 32.0 53.2 14.6 91.6 59.7 6 2.8

1.8 25.8 2.1 1.2 0.2 5.3 46.4 8.8 1.1

b b b a b b b b b a

S. feltiae

S. glaseri

t 3.1 6 0.3 23.4 6 2.4 2.3 6 0.1 1.0 6 0.1 1.3 6 0.1 7.2 6 0.2 36.3 6 1.2 10.7 6 1.0 0.2 6 0.0 2.6 6 0.3 2.1 6 0.3 0.3 6 0.0 1.3 6 0.1 1.5 6 0.3 1.8 6 0.3 0.7 6 0.2 4.0 6 1.1 37.2 43.8 18.8 97.7 70.4 6 2.2

t 6 0.1 6 1.5 6 0.1 6 0.0 6 0.2 6 0.5 6 2.2 6 0.5 nd t 2.8 6 0.2 0.2 6 0.0 1.1 6 0.1 1.5 6 0.1 1.2 6 0.1 1.2 6 0.1 2.9 6 0.2 30.7 51.0 18.1 104.1 149 6 3.8

b b a b a a b c c b

2.4 21.1 4.3 1.2 1.5 6.6 41.5 10.2

c c c c c b b c b c

*n 5 3; t 5 trace (,0.1%); nd 5 not detectable; U.I. 5 unsaturation index, defined as S(% of fatty acid 3 number of double bonds); totals do not necessarily add up to 100%; Mol percentage refers to the amount of each fatty acid as a percentage of the total fatty acid, expressed on a molar basis. Means within the same row followed by the same letter are not significantly different (P . 0.05). †Data from Patel et al. (15).

TABLE 6. Free fatty acid composition (mol % 6 SE)* of newly emerged infective juveniles of four Steinernema species

Fatty acid C14 :0 C14 :1 C16 :0 C16 :1n27 C16 :3n23 C16 :4 C18 :0 C18 :1n29 C18 :2n26 C18 :3n23 C20 :0 C20 :1 C20 :2 C20 :3n26 C20 :4n26 C20 :5n23 Saturated Monoene Polyene U.I. Free fatty acids (ng IJ21)

S. carpocapsae 6 0.0 6 0.1 6 0.9 6 0.1 6 0.6 6 0.9 6 0.4 6 1.5 6 0.6 6 0.3 t 2.2 6 0.3 0.9 6 0.5 1.9 6 0.2 2.7 6 0.3 2.7 6 0.4 28.2 48.6 22.8 116.5 1.6 6 0.4

0.2 1.8 18.7 3.6 1.6 1.9 9.3 41.0 9.6 1.5

a a a a ab a a a a

S. riobravis t 6 0.3 6 2.8 6 0.1 6 0.1 nd 6.5 6 0.5 44.4 6 1.0 9.8 6 0.1 0.3 6 0.1 t 3.6 6 0.6 0.6 6 0.0 1.5 6 0.1 1.1 6 0.1 1.5 6 0.1 32.1 51.6 15.9 93.0 2.8 6 0.2 1.7 25.5 1.9 1.1

a b b a a

S. feltiae 4.7 31.1 2.5 1.1

b

8.0 34.3 8.1 0.9 2.2 2.6

b b b

1.2 1.3 1.6

6.5

t 6 0.7 6 1.8 6 0.5 6 0.2 nd 6 0.8 6 3.5 6 1.0 6 0.1 6 0.3 6 0.3 t 6 0.2 6 0.1 6 0.4 41.3 44.1 14.2 83.2 6 1.6

S. glaseri

b c c b bc a b b b

0.4 6 0.0 1.6 6 0.1 25.2 6 1.1 2.0 6 0.5 1.6 6 0.1 0.7 6 0.1 4.8 6 0.2 45.5 6 2.2 7.5 6 0.5 0.7 6 0.1 2.7 6 0.2 2.8 6 0.0 t 1.4 6 0.2 2.0 6 0.1 1.1 6 0.0 33.0 51.9 15.0 94.4 4.6 6 0.9

a b d a c a b c c

*n 5 3; t 5 trace (,0.1%); nd 5 not detectable; U.I. 5 unsaturation index, defined as S(% of fatty acid 3 number of double bonds); totals do not necessarily add up to 100%; Mol percentage refers to the amount of each fatty acid as a percentage of the total fatty acid, expressed on a molar basis. Means within the same row followed by the same letter are not significantly different (P . 0.05).

Steinernema Neutral Lipid Fatty Acids

which the nematodes feed (Xenorhabdus in the case of steinernematids) have a predominately unsaturated fatty acid composition (14,22). Diet has been shown to have some influence on the fatty acid composition of entomopathogenic nematodes [(5), P. S. Grewal, personal communication], and on a wider perspective, acanthocephalans [e.g., (23,25)], rotifers [e.g., (17)] and insects [e.g., (6)], but in all cases, unsaturated fatty acids remained dominant. In general, organisms rely on obtaining essential fatty acids, particularly those of longer chain lengths, from their diet but have the ability to regulate the relative proportions of each fatty acid. The main point raised in the present study is that the steinernematids examined had a typical, predominately unsaturated fatty acid composition and that the results of Selvan et al, (18,19) deviate strongly from the norm. Since the fatty acid profiles obtained for the heterorhabditid species studied by Selvan et al. (18,19) show patterns typical of nematodes (i.e., greater proportion of unsaturated fatty acids), and are similar to profiles obtained more recently for heterorhabditid nematodes (H. Menti, unpublished data), an obvious explanation for their results on steinernematids is still lacking. Selvan et al. (19) concluded that the relatively ‘‘poorer’’ survival of heterorhabditid nematodes compared with steinernematids was a consequence of the former having a greater proportion of unsaturated fatty acids, which provide less (net) energy compared with saturated fatty acids during aging. This statement is too much of a generalisation and the fact that steinernematids can have a high proportion of unsaturated fatty acids suggests that the difference in survivorship between some heterorhabditid and steinernematid IJs cannot be attributed to their fatty acid composition. The possession of a predominately unsaturated store of fatty acids seems to be widespread within invertebrates although platyhelminth and aschelminth parasites of mammals, like their hosts, tend to have a greater proportion of saturated fatty acids (2). This reflects the environmental conditions in which the parasite finds itself. The relatively higher body temperature of a mammalian host means that the parasite would not need unsaturated fatty acids to keep its lipids in a fluid state. In contrast, soil-dwelling organisms would generally require a greater proportion of unsaturated fatty acids in order to keep their lipids fluid. However, it is important to point out that S. riobravis is a subtropical species from southern Texas, where soil temperatures can be high. Fatty acid analysis of neutral lipids in the Steinernema species examined in the present study detected 18 fatty acids, from myristic acid (C14:0) to lignoceric acid (C24:0). The major fatty acids, oleic and palmitic along with stearic acid declined as the nematodes aged, suggesting their preferential utilisation. A few of the minor fatty acids identified were long chain polyenes (C 20 and C 22 ). Studies on the synthesis of fatty acids in nematodes have suggested that certain nem-

347

atodes, particularly free-living, may be able to synthesise polyene fatty acids de novo (2). Fodor et al. (5) found that diet influenced the fatty acid composition of the phospholipids in S. carpocapsae to some extent, but suggested de novo synthesis also occurred, particularly of eicosapentaenoic acid (C20:5n23) because it was present even when its precursor, linolenic acid (C18 :3n23), was undetectable in the diet. The function of minor polyene fatty acids has not been established to any great extent in nematodes or any other invertebrate. The polyene C 20 and C 22 fatty acids are considered to be important components of phospholipids (7) and in entomopathogenic nematodes these fatty acids have been found in greater proportions in the phospholipids compared with the neutral lipids and the free fatty acid fraction [(5); M. N. Patel, unpublished data]. The presence of long chain polyenes in the free fatty acid fraction suggests that they may have functions other than as structural components. For example, arachidonic acid (C20: 4n26), dihomo-γ -linolenic acid (C20 :3n26) and eicosapentaenoic acid (C20: 5n23) are precursors of eicosanoids which have profound physiological effects in mammalian systems (20). Reports of these compounds are on the increase in invertebrates, including insects (20) and trematodes (13). We thank B. Briscoe (CAB International Institute of Parasitology, St Albans, UK) for providing the initial nematode cultures. M. N. Patel was supported by a Biotechnology and Biological Sciences Research Council (UK) CASE studentship, Biosys, Inc. (Columbia, MD, USA) and CAB International Institute of Parasitology.

References 1. Badhwar, R.; Raheja, R.K.; Ahuja, S.; Ahuja, S.P. Lipid composition of the seed gall nematode, Anguina tritici. Nematologica 41:584–591;1995. 2. Barrett, J. Biochemistry of Parasitic Helminths. London: MacMillan Publishers Ltd; 1981. 3. Christie, W.W. Gas Chromatography and Lipids. Ayr, Scotland: The Oily Press; 1989. 4. Crawley, M.J. GLIM for Ecologists. Methods in Ecology. Oxford: Blackwell Scientific Publications; 1993. 5. Fodor, F.; Dey, I.; Farkas, T.; Chitwood, D.J. Effects of temperature and dietary lipids on phospholipid fatty acids and membrane fluidity in Steinernema carpocapsae. J. Nematol. 26:278– 285;1994. 6. Ghioni, C.; Bell, J.G.; Sargent, J.R. Polyunsaturated fatty acids in neutral lipids and phospholipids of some freshwater insects. Comp. Biochem. Physiol. 114B:161–170;1996. 7. Hansen, H.S. Linoleic Acid—Essential Fatty Acid and Eicosanoid Precursor. Herlev, Denmark: Bondegaard; 1989. 8. Holz, R.A.; Wright, D.J.; Perry, R.N. The lipid content and fatty acid composition of different lipid classes of hatched second stage juveniles of Globodera rostochiensis and G. pallida. Fundam. Appl. Nematol. 20:291–298;1997. 9. Kaluzny, M.A.; Duncan, L.A.; Merritt, M.V.; Epps, D.E. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J. Lipid Res. 26:135–140;1985. 10. Krusberg, L.R. Chemical composition of nematodes. In: Zuckerman, B.M.; Mai, W.F.; Rohde, R.A. (eds). Plant Parasitic

M. N. Patel and D. J. Wright

348

11. 12. 13. 14.

15.

16.

17.

18.

Nematodes, Vol. 2. New York: Academic Press; 1971:213– 234. Lee, D.L.; Atkinson, H.J. The Physiology of Nematodes. London: Macmillan Press; 1976. Monteoliva, M.; Molinero, A.C.; Valero, A.; MonteolivaSanchez, M. Acidos grasos fijos en helmintos para´sitos. Rev. Ibe´r. Parasitol. 46:81–87;1986. Nevhutalu, P.A.; Salafsky, B.; Haas, W.; Conway, T. Schistosoma mansoni and Trichobilharzia ocellata: Comparison of secreted cercarial eicosanoids. J. Parasitol. 79:130–133;1993. Nishimura, Y.; Hagiwara, A.; Suzuki, T.; Yamanaki, S. Xenorhabdus japonicus sp-nov associated with the nematode Steinernema kushidai. World J. Microbiol. Biotechnol. 10:207–210; 1994. Patel, M.N.; Stolinski, M.; Wright, D.J. Neutral lipids and the assessment of infectivity in entomopathogenic nematodes: Observations on four Steinernema species. Parasitology 114: 489–496;1997. Poinar, G.O., Jr. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Gaugler, R.; Kaya, H.K. (eds). Entomopathogenic Nematodes in Biological Control. Boca Raton, FL: CRC Press; 1990:23–61. Rainuzzo, J.R.; Reitan, K.I.; Jørgensen, L.; Olsen, Y. Lipid composition in turbot larvae fed live feed cultured by emulsions of different lipid classes. Comp Biochem. Physiol. 107A: 699–710;1994. Selvan, S.; Gaugler, R.; Lewis, E.E. Biochemical energy reserves of entomopathogenic nematodes. J. Parasitol. 79:167– 172;1993.

19. Selvan, S.; Gaugler, R.; Grewal, P.S. Water content and fatty acid composition of infective juveniles entomopathogenic nematodes during storage. J. Parasitol. 79:510–516;1993. 20. Stanley-Samuelson, D.W.; Pedibhotla, V.K. What can we learn from prostaglandins and related eicosanoids in insects? Insect Biochem. Molec. Biol. 26:223–234;1996. 21. Stolinski, M. Lipid utilization in free-living stages of soil nematodes. Doctoral thesis, University of London; 1994. 22. Suzuki, T.; Yamanaka, S.; Nishimura, Y. Chemotaxonomic study of Xenorhabdus species—cellular fatty acids, ubiquinone and DNA-DNA hybridization. J. Gen. Appl. Microbiol. 36: 393–401;1990. 23. Taraschewski, H.; Aitzetmuller, K.; Werner, G.; Kuhs, K. Lipids of fish parasites and their hosts—fatty acid fingerprints of 4 species of acanthocephalans and of their hosts intestinal tissues. Parasitol. Res. 81:522–526;1995. 24. Thompson, S.N. A review and comparative characterization of the fatty acid compositions of seven insect orders. Comp. Biochem. Physiol. 45B:467–482;1973. 25. Weber, N.; Vosmann, K.; Aitzetmuller, K.; Filipponi, C.; Taraschewski, H. Sterol and fatty acid composition of neutral lipids of Paratenuisentis ambiguus and its host eel. Lipids 29:421– 427;1994. 26. Wijbenga, J.; Rodgers, P.B. Lipid content of insect parasitic nematodes. IOBC/WPRS Bull. 17:155–158;1994. 27. Woodring, J.L.; Kaya, H.K. Steinernematid and Heterorhabditid nematodes: A handbook of biology and techniques. Southern Cooperative Series Bulletin 331, Arkansas Agricultural Experimental Station; 1988.