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Effect of temperature on the composition of fatty acids in total lipids and phospholipids of entomopathogenic nematodes
Pergamon PII:
J. therm. Bid. Vol. 22, No. 415, pp. 245-251, 1997 ~0 1997 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain s03&-4565(97)ooo19-3 0306-4565/97 $17.00 + 0.00
EFFECT OF TEMPERATURE ON THE COMPOSITION OF FATTY ACIDS IN TOTAL LIPIDS AND PHOSPHOLIPIDS OF ENTOMOPATHOGENIC NEMATODES GANPAT
B. JAGDALE’
and ROGER
GORDON’
‘Agriculture and Agri-food Canada, Pest Management Research Centre, P.O.B. 186, Delhi, Ontario, Canada, N4B 2W9 and 2Department of Biology, University of Prince Edward Island, Charlottetown. Prince Edward Island, Canada, CIA 4P3 (Received 7 December
1996; accepted in revised form 3 Ma>’ 1997)
Abstract-l. Gas liquid chromatography was used to determine the composition of fatty acids in total lipids and phospholipids of the entomopathogenic nematodes. Steinernema feltiae Umel strain, S. carpocapsae All strain, S. riobravis TX strain and S. filfiae NF strain that had been recycled or stored at 5, 10, 15, 20 and 25°C. 2. In all nematode strains, the unsaturation indices of total lipids increased as recycling or storage temperatures decreased. The unsaturation indices increased in the phospholipids of all strains except the TX strain of S. riobravis. 3. Increased unsaturation indices at low temperatures was due to an increase in polyunsaturated fatty acids with concomitant decline in the proportion of saturated fatty acids, especially palmitic (16:O)and/or stearic (18:O) acids 4. Sfeinernema riobrauis displayed a lower degree of physiological adaptation to cold temperatures than the other strains. The saturated fatty acids and unsaturation index of this nematode’s phospholipid did not change in response to recycling or storage temperatures. 0 1997 Published by Elsevier Science Ltd. All rights reserved. Key Word Index: Entomopathogenic nematodes; fatty acids; Galleria mellonella; gas liquid chromatography; phospholipids; Steinernema feltiae; Steinernema carpocapsae; Steinernema riobravis; steinernematids; temperature; thin-layer chromatography
INTRODUCTION
Entomopathogenic and
nematodes
Heterorhabditidae)
(Hominick
are
( f. Steinernematidae globally
distributed
et al., 1996) and are being commercially
for use in insect pest management (Smart, 1995). The soil dwelling infective juveniles of these nematodes infect susceptible insect species via the host’s natural openings, then kill the host by reteasing a mutualistic bacterium; nematode development and reproduction then occur within the host cadaver (Poinar, 1990). The commercialization of entomopathogenic nematodes involves continual recycling, so it is important to determine the degree to which they are affected by the recycling regimes at the whole organism and physiological levels. There is evidence to suggest that in certain species, the temperature range over which infection occurs may be modified by the temperature at which recycling is carried out (Grewal ef al., 1996). Both the infectivity (Jagdale and Gordon, 1997a) and capacities to tolerate produced
temperature extremes (Jagdale and Gordon, 1997b) were adaptively modified in four strains of steinernematid nematodes by recycling them at various temperatures. The changes in the physiology of steinemematids that are responsible for modifying whole organism characteristics (e.g. temperature tolerance, infectivity) at various recycling temperatures require determination. Many poikilothermic animals adapt to changing environmental temperatures by modifying the degree of unsaturation of their lipids. At low ambient temperatures, the proportion of unsaturated: saturated fatty acids increases in phospholipids to maintain cell membrane fluidity and normal cellular functions (Hazel, 1995; Hazel and Williams, 1990). Information on the degree of lipid unsaturation relative to temperature adaptation mechanisms of nematodes is sparse. It has been suggested that the synthesis of unsaturated fatty acids in the cysts of the plant parasitic nematode, Globodera rostochiensis (Gibson et al., 1993, and in the storage organs of 245
G. B. Jagdale and R. Gordon
246
mermithid (insect parasitic) nematodes (Gordon et al.. 1979) may constitute a low temperature adaptation mechanism. Experimental evidence in support
of such a hypothesis
free-living showed
nematode,
an increased
(20:5) fatty
was obtained
Cuenorhabditis
proportion
25 C (Tanaka,
moiety
to nematodes
1996). In the Mexican
entomopathogenic
nematode,
which when
recycled at strain
Steinernema
of these nematodes
S. riohraz~is from 20 C) and stored
in tissue culture
bottles
at temperatures
(600 ml) for
three
E.uraclion
oj‘ lipids
Freshly emerged
(048
h old) infective juveniles
acids and membrane
dilute
formalin
Kaya.
1988) to separate
including
entomopathogenic
c’t al., 1994). nematodes,
ones, adaptively
the degree of unsaturation
modify
of their lipids in response
rinsed
ef al., Using
1996). then a Gilson”
beakers
matodes
into
ascertain
whether
changes
boreal
capacities
strains
that
to determine composition phospholipids had
periods
to
such as
to effect such changes represent
climatic zones. The purpose
that
it is important adaptations
a broad
vary
range
of
of the present study was
whether there was a change in the of fatty acids in total lipids and of four such strains of Steinevnenzu
been
recycled
or stored
of time at various
for
temperature
formed confined samples stored
in fatty acids occur at lower temperatures
and whether among
habitats.
physiological
prolonged
of each
by Plant Products
Ltd., Brampton,
S. riobraris
strain
by Dr.
was provided
Ontario,
Canada;
H. E. Cabanillas.
USDA, ARS. Crop Insects Research Unit, Weslaco. TX.. USA. Steirzernema ,fe/fiae Umes strain was
strain
that
of the
so that they
had been
recycled
regimes
or
(each
sample 50 and 100 mg wet weight for total lipid and phospholipid into
analysis.
separate
(I .5 ml), then samples were Labconco”
respectively)
polypropylene
freeze dry system,
conco Corp..
were transferred
microcentrifuge
tubes
frozen ( - 2O’C) overnight. then freeze-dried for 24 h
Kansas
Lypho-lock
The in a
6 (Lab-
City. MO, USA) and stored at of fatty acids. Each
as a separate
of rotal Lipid ,fh/rv
gas liquid
chromatography.
replicate
(12= 3).
acids
was then transferred
Biologic
Biocontrol
Willow Hill, PA.. USA. Sfeinernema
felriae
using
were extracted
Freeze dried samples were homogenized in 200 ~1 methanol containing a few crystals of hydroquinone (antioxidant agent) in a Potter-Elvehjem tissue grinder with a motor-driven
from
was determined Lipids
from infective juveniles using the method described by Bligh and Dyer (I 959). with some modifications.
been
Products,
the bottom
temperature
provided by Dr. R. West, Canadian Forest Service, St. John’s. NF. Canada from a stock colony that had initially
from
Lipid fatty acid composition
carpocappsae All strain
obtained
water
AND METHODS
Sources of’ nematodes
TX
distilled
no. 4 filter papers,
at the specified
.4w/~:vix
Steinernemu
with
and
containing
allowed to settle (ca. pipetman, infective
~ 20 C until used for analysis
regimes.
(Woodring
blobs on the papers. Three different
tube was considered MATERIALS
twice
were transferred to Whatman
traps
of
from the
250 ml beakers
water.
juveniles
ne-
in the White
(Gordon IO min).
commercial
of entomopathogenic
were transferred
distilled
to recycling temperatures. no studies have been done involving recycling below 15’ C. To expand the exploitation
at
5. IO and 15°C).
each of the four strains
that certain
(S. jkltiue
at 5 C, S. c’arpocapsae at 5 and IO-C, S. riobrah
the recycling temperature from 25 to 18 C caused an increase in the unsaturation of fatty fluidity (Fodor
weeks
of the
carpocap-
sue, decreasing
While these data suggest
were collected
where recycling of strains was not possible
of polyunsaturated
acids in its phospholipid
recycled at I5 C, compared
for the
elegans,
Infective juveniles
from their lowest recycling temperature regimes (S. /h/the from IO C. S. carpocapsae from I5 C.
microcentrifuge
pestle. Each homogenate
into a separate
polypropylene
tube (I .5 ml), then 250 pl distilled
NF strain is a new strain (Jagdale e/ al.. 1996) that we isolated in Summer 1993 from soil on a farm site
water. 250 pl chloroform and 550 11 methanol added; samples were vortexed and incubated at 4’C for I h.
close to St. John’s, NF. Canada. using Galleria traps (Woodring and Kaya. 1988).
The homogenate was centrifuged at I3 I50 RPM for 2 min at room temperature (25°C). supernatant
bait
transferred Recycling/storage
All nematode (May, Galleria
tempernture
strains
1994May. mellonella
regimes
were recycled
for two years
1996) by propagation through larvae (Woodring and Kaya,
1988): NF and Umei strains of S. ftiltiue at IO. 15. 20 and 25 C: S. carpocapsae All strain at 15, 20 and 75 C and S. riohark TX strain at 20 and 25 C.
to further
microcentrifuge
tubes,
then
300 ~1 distilled water and 300 ~1 of chloroform added. Samples were vortexed and incubated at room temperature Following
(25°C) for 30 min. centrifugation (13,150
RPM;
2 min;
25 C), the lower lipid-containing layer was transferred into separate 6.0 ml capacity transmethylation vials. Solvents in each vial were evauorated under a
Effect of temperature on phospholipids of nitrogen, then 2.0 ml transmethylating reagent (94.0 ml methanol, 6.0 ml sulfuric acid) and a few crystals of hydroquinone added. Vials were incubated for 5 h at 7O”C, 1.0 ml distilled water and 1.5 ml hexane added, then shaken and allowed to stand for 10 min to form two separate layers. The upper lipid layers (hexane extract) containing the fatty acid methyl esters (FAMES) were transferred into small screw cap vials (1 .O ml capacity), then blown dry with nitrogen. Extracted FAMES were dissolved in 20 ul carbon disulphide and 0.5 ul of the solution injected into the GLC apparatus, a Hewlett Packard 5890 series II gas liquid chromatograph equipped with a flame ionization detector. The column used was a 30 m Supelcowax lo/O.53 mm (Supelco, Supelco Park, Bellefonte, PA, USA). The inert carrier gas was helium with a flow rate of 3.27 ml min- ‘. The oven, injector and detector temperatures were set at 180, 225 and 225”C, respectively. Commercial standards of FAMES, obtained from Supelco and Sigma Chemical Co. (St. Louis, MO, USA), were run under identical conditions and the chromatograms evaluated with reference to the retention times of the standards. stream
Analysis
of phospholipid
fatty
acid composition
The procedure used for the extraction, transmethylation and subsequent analysis of phospholipids was
of entomopathogenic
nematodes
247
the same as that used for total lipids, except that thin-layer chromatography (TLC) was first used to separate phospholipids from the 100 mg wet weight infective juvenile samples. Following freeze-drying and chloroform: methanol extraction, the lipid extracts were transferred into screw cap vials (ca. 1 ml), blown dry under nitrogen, then re-dissolved in 50 pl chloroform: methanol (2:l u/v). Each sample (25 11) was applied on silica gel plates, the sample spots allowed to dry at room temperature (25°C) for l-2 min, then plates were transferred into the developing tank containing the solvent mixture (mobile phase) hexanediethyl ether-acetic acid (85:15:2 v/v/u). The TLC plates were developed for 30 min then dried at room temperature (25°C) for l-2 min. Phospholipid spots were visualized by placing the TLC plates for a few seconds in a separate developing tank containing a few crystals of iodine. The phospholipid spots outlined on the TLC plates were scraped off into separate transmethylation vials. Statistical
analysis
All fatty acids were expressed as mol% of the total lipid and phospholipid fractions. Unsaturation indices, which are a measure of the degree of unsaturation in terms of the number of double bonds/mole, were computed (Sumner and Morgan,
Table 1. Effect of recycling and storage temperatures on the proportions of saturated and unsaturated fatty acids in the total lipids of four strains of Steinernema Recycling/Storage Temperature (“C)
Fatty acids 25
20
15
10
S. feltiae NF strain SFA” MUFA” PUFA” UP
48.5 34.5 18.6 0.8 f 0.1’
41.3 31.9 21.5 0.9 f 0.1’
46.8 30.5 24.5 1.1 * 0.1’
34.8 22.2 44.4 1.5 + O.ld
30.5 21.3 48.5 1.7 & O.Od
S. feltiae Umea strain SFA MUFA PUFA UI
51.0 30.3 , 21.0 0.8 + 0.1’
27.9 51.8 23.0 I.1 + O.Od
35.1 49.6 19.4 I .o + o.04
32.9 23.3 44.0 1.6 + 0.0
29.3 24.7 41.4 1.7 f 0.1’
S. carpocapsae All strain SFA MUFA PUFA UI
48.7 24.8 26.4 1.0 * 0.1’
36.5 23.1 39.9 I.4 * O.ld
34.9 20.2 44.9 1.6 + O.Od
34.1 18.4 47.3 1.6 + O.Od
26.5 21.5 44.1 I .7 & O.Od
S. riobrauis TX strain SFA MUFA PUFA UI
47.2 31.7 22.8 I.0 f 0.1’
41.2 32.1 28.3 1.1 f 0.0
39.2 33.0 29.5 1.2 f O@
38.1 29.1 32.9 I .3 f O.Od’
32.3 26.5 41.9 I.5 f 0.1’
5
“SFA = Saturated fatty acids, MUFA = Monounsaturated fatty acids, PUFA = Polyunsaturated fatty acids. Values are mean mol% of total lipids (n = 3). bUI = Unsaturation index. Means with the same lower case superscript letter (across the columns) are not significantly different (P > 0.05) by Student Newman-Keul; test.
Ganpat B. Jagdale and Roger Gordon
248 1969). Mole and
percentage
unsaturation
one-way (Jandel
indices
ANOVA, Carp”.
significance
data
(arcsine were
analysed
using
Student-Newman-Keuls
Sigma
Stat,
was defined
unaffected
transformed) test
1992). The
level
of
in the total lipids of the other three strains.
In the phospholipids,
rated fatty acids remained temperature;
as P < 0.05.
however,
the NF strain was the
only strain in which the proportion this fraction
of monounsatu-
unaltered
by such a low
decreased
in the phospho-
lipids of the other three strains. Analysis of the fatty acid profiles (data not shown)
RESULTS The
recycling
or
storage
revealed that the increased temperature
influenced the fatty acid composition the entomopathogenic nematodes. general decline in the content and
a concomitant
indices
in
both
the
phospholipids
(Table
S. riobraris
TX strain,
saturated
of saturated
increase total
in the lipids
(Table
1) and
except
for of
according
the unsaturato recycling
or
(l&O)
acids
acids,
47.5-50.8
fatty
and 32.7-37.4
that
had
moieties, been
low
fatty
temperature
phospholipid (i.e. other
Decreases
in
acids,
caused
by storing
at
were
attributable
oleic
acid
of S. jeltiae)
total lipids and phospholipids
acids in the total lipids of S. fdtiae
Changes
storage
in proportions
of all four strains as the temperatures
decreased.
of monounsaturated
acids were less consistently
fatty
related to temperature.
At
the 5’C storage temperature, the monounsaturated fatty acids decreased as a proportion of the total lipids
in the
NF
strain
Table 2. Effect of recycling
Fatty
of
S. ,fkdtiue, but
was
storage
at 5”C, the proportions
(20: 1)
NF strain. After
of oleic acid in the
phospholipids of S. feltiae Umea strain (9.5 mol%), S. carpocapsae All strain (12.8 mol%) and S. riohraois TX strain mately
half those
nematodes.
Recycling/Storage
(17.2 mol%)
pertaining
The combined
Temperature
were approxi-
in the 25”C-recycled
mol%
of palmitoleic
and unsaturated
fatty acids
(/ C)
25
20
15
10
5
S. /k/rise NF strain SFA” MUFA” PUFA” UP
39.5 14.1 44.8 1.6 i 0.1’
36.5 12.2 51.5 1.8 f 0.1’”
32.0 14.6 53.6 I .8 + 0.0’”
31.0
25.7 15.0 59.5 2.1 f 0.1”
S. f&t& SFA MUFA PUFA UI
40.6 17.6 42.0 I.5 + 0.1’
38.0
19.4 42.9 I .6 + 0.0’”
32. I 13.0 52.9 I .7 * O.Od
12.8 56.3 I .9 -+ 0.0”
29.8 10.4 59.5 2.0 f 0.0’
40.7 24.8 34.8 I.3 * 0.1‘
36. 19.7 43.2 1.5 + 0.1.
33.5 22.0 45.2 1.5 + 0.1’
37.1 16.1 46.5 I.5 + 0.1’
27.7 13.4 58.5 1.9 + O.Od
34.8 31.5 34.4 I .4 f 0.0
35.x 2x.1 35.8 I.5 f 0.1’
33.2 19.5 47.5 1.6 & 0.0’
30.9 27.3 41.3 1.7 + 0.0’
34.4 17.6 48.1 I .6 _+ 0. I’
Umel
S. ccrrpocapstrr SFA MUFA PUFA UI S. riohruris SFA MUFA PUFA UI
a the
and to
(16: 1) and eicosenoic
in palmitoleic
and storage temperatures on the proportions of saturated in the phospholipids of four strains of Steinernema
acids
to
(18: 1) in
of three of the four isolates
than the NF strain
decreases
and
in nematodes
25°C.
in
fractions
comprised
at
(5’C),
decrease
together,
of the total lipid and
respectively,
recycled
monounsaturated significant
that mol%
storage temperatures. There was an increase in the proportions of polyunsaturated fatty acids in the recycling
of fatty acids
in total lipids or phospholipids of palmitic (16:0) and/or stearic
phospholipid
the proportion
fatty acids and consequently,
tion index, did not change
fatty acids unsaturation
2) of all strains, in which
regimes
of lipids in all There was a
unsaturation
at low temperatures was at the expense
13.2 55.7 I .9 * O.O’d
strain 30.2
All strain
TX strain
“SFA = Saturated fatty acids, MUFA = Monounsaturated fatty acids. PUFA = Polyunsaturated fatty acids. Values are mean mol% of phospholipids (17= 3). WI = Unsaturation index. Means with the same lower case superscript letter (across the columns) are not significantly different (P > 0.05) by Student Newman-Keuls test.
and
Effect of temperature on phospholipids of entomopathogenic nematodes eicosenoic acids was 10.4 in the total lipids of S. feltiae NF strain, but was reduced to 0.9 in infective juveniles stored at 5°C. The increase in polyunsaturated fatty acids at reduced temperatures was attributable to significantly greater percentages of linoleic acid (182) in total lipids and phospholipids of all strains. Depending on the strain, linoleic acid increased as a proportion of the total lipids from 15.3-18.3 mol% at 25°C to 27.4-29.2 mol% at 5°C. The comparable increase in this fatty acid in the phospholipids was from 19.9-24.6 mol% to 29.0-37.2 mol% over the same drop in temperature. In all except S. riobrauis, this was augmented by significantly increased proportions of eicosapenic acid (20:5w3) at 5°C. The mole percentages of this polyunsaturated fatty acid in the total lipids of the two strains of S. feltiae were 2.4-2.6 in nematodes recycled at 25°C and 15.2-15.5 after storage at 5°C; comparable values for S. carpocupsae All strain were 6.6 (25°C) and 13.0 mol% (SC). Eicosapenic acid increased as a mole percentage of the phospholipids from 13.6-14.4 in the two strains of S. feltiae recycled at 25°C to 20.4 after storage at 5°C; comparable values for S. carpocapsue All strain were 10.4 (25°C) and 16.0 mol% (SC). In S. riobravis stored at 5°C arachidonic acid (20:4), rather than eicosapenic acid, was elevated in the phospholipid moiety. This polyunsaturated fatty acid comprised 6.3 mol% of the phospholipids of nematodes recycled at 25°C compared to 8.5 mol% of this lipid moiety in nematodes stored at 5°C. In S. feltiae UmeP strain, arachidonic acid (20:4) actually decreased as a proportion of the phospholipids at temperatures _< 10°C. This polyunsaturated fatty acid comprised 3.64.7 mol% of phospholipids in S. riobruuis that had been recycled at 2 15°C and 2.9 mol% at those recycled or stored at I 10°C. In the two strains of S. feltiae and S. riobravis TX strain, the temperature below which significant increases in unsaturation indices of total lipids occurred was 15”C, while the comparable temperature was 25°C for S. curpocupsae All strain (Table 1). With respect to phospholipids, the unsaturation indices were significantly increased for S. feltiue NF strain and S. carpocupsue All strain at < 10°C and for the UmeP strain of S. feltiae at < 20°C (Table 2).
DISCUSSION
This study has shown that the composition of saturated and unsaturated fatty acids in the lipids of entomopathogenic nematodes was influenced by recycling and storage temperatures from 25 to 5°C. Recycling or storing at colder temperatures increased
249
the proportions of polyunsaturated fatty acids in the total lipids and phospholipids of all four strains of Steinernema. Except for the phospholipids of S. riobruvis TX strain, this was accompanied by a reduction in the proportions of saturated fatty acids, resulting in increases in unsaturation indices. The increased production of polyunsaturated fatty acids at low temperatures was due to linoleic acid (18:2) and eicosapenic acid (20:5w3) or, in S. riobravis phospholipids (5”C), arachidonic acid (20:4). In a related study, embodying only two warmer rearing temperatures (18 and 25’Q Fodor et al. (1994) also reported that nematodes recycled for an unspecified time at 18°C contained a higher proportion of eicosapenic acid (20:5w3) in their phospholipids. Adaptation to environmental temperatures by shifting the proportions of saturated: unsaturated fatty acids is a widespread phenomenon in poikilotherms. High levels of unsaturated fatty acids in phospholipids increase the membrane fluidity to maintain normal cellular functions at low environmental temperatures (Hazel, 1995). The cyst stages of the plant parasitic nematode, G. rostochiensis, were found to contain high levels of polyunsaturated fatty acids in their lipids, principally triacylglycerols and phospholipids, as an overwintering adaptation (Gibson et al., 1995). Among entomopathogenic nematodes, the proportion of unsaturated fatty acids in the phospholipids of the Mexican strain of S. carpocapsue was greater when nematodes were reared at 18°C than when reared at 25°C and this was accompanied by a less ordered arrangement of phospholipids within the cell membranes at the lower temperatures (Fodor et al., 1994). Thus, in the present study, the changes in unsaturation of phospholipids that occurred in all strains other than S. riobruvis TX strain, consequent to recycling or storing at various temperatures, are probably adaptive in helping to preserve membrane integrity from 25 to 5°C. In all four strains, there was a temperature-induced shift in the unsaturation indices of total lipids that parallelled those of the phospholipids. Infective juveniles of entomopathogenic nematodes contain high levels of lipids, presumably neutral lipids, and utilize them as an energy substrate (Selvan et al., 1993). Gordon et al. (1979) reported that the unsaturation index of stored triacylglycerols was greater in a boreally adapted mennithid nematode, Neomesomermis flumenulis, than in the tropical culicivorux, and Romanomermis mermithid, suggested that such differences in unsaturation were necessary to maintain fluidity within the nematode’s storage organ and permit accessibility of enzymes to
Ganpat
250
B. Jagdale
lipid energy reserves at the temperatures prevailing within the respective habitats of the nematodes. It is possible
that the four strains
present
study
adjusted
of Steinernema in the
the physical
storage nutriment to permit energy occur over a broad range of temperatures. adaptive
A shift toward
in permitting
state
metabolism to environmental
unsaturation
enzyme accessibility
from habitats
two strains
of S. ,feltiae are boreal, while the All strain
and
S. ,feltiae represents
for this nematode appeared
which
to be no obvious
significant
indices occurred.
elevations
the strains displayed
increased
in their total lipids at
by three of the strains
unsaturation
findings appear at et al. (1993). who
species, Steinemema scupter-
that a tropical
isci, contained
a higher proportion
acids
its
within
total
entomopathogenic temperature.
lipids
nematodes
Their data
such a conclusion,
of saturated than
several
recycled
of
a temperate
strain of S. ,/kltiae contained
the highest
proportion of polyunsaturated fatty acids. According to our study, the two boreally
of S. fhlriae, as well as the warm temperate
strains
S. carpocupsae All strain
increased
the unsaturation
indices of both total lipids and phospholipids as recycling and storage temperatures declined. However,
S. riobravis TX
the subtropical
adjusted (mostly
the unsaturation neutral
index
strain
of its total
lipids) in such a manner.
only lipids
Although
this nematode displayed, in common with the other strains, decreases in proportions of monounsaturated fatty acids and compensatory increases in polyunsaturated fatty acids, it was the only strain in which the saturated
fatty acids, and consequently
indices, did not significantly recycling
or storage
unsaturation
increase due to reduced
temperatures.
Such an inability
of this nematode to fully adapt physiologically to colder temperatures is in keeping with the perception that it is a warm adapted species, capable of infecting hosts at 2 lO”C, reproducing at 2 20°C (Grewal et al., 1994) and surviving poorly when stored at 5 ‘C. the preferred storage temperature for the other isolates (Jagdale and Gordon: unpublished observations). Paradoxically, this nematode is capable of tolerating freezing (Brown and Gaugler, 1994) an attribute of questionable relevance in its subtropical southern
Texas habitat.
The combination
15°C. recycling
of freezing
this
temperature.
was not possible.
Also.
in
rearing
At other tempera-
that some artificial selection may resulting in genetically altered
occurred,
synthetic
capacities
for fatty
acids adaptive
to the
temperatures.
We have shown that the upper and lower thermal tolerances
of these
entomopathogenic
nematodes
were influenced by the temperature at which they were recycled. Recycling at warmer temperatures increased
adapted
at
S. carpocapsae at IO‘C and S. riobravis at 10 and
have
as the proportion
were
at 5°C.
in all strains, since no recycling
out
other
does not seem to support
however,
carried
tures, it is possible
fatty acids in S. scapterisci was only higher than some of the other species and
saturated marginally
was
fatty
at the same
of phospholipids
Many of the shifts in lipid unsaturation reported in this study were environmentally induced. This applies to 5 ‘C determinations
in their total lipids and phospholipids
when recycled at 25°C. These variance with those of Selvan reported
of
that all
I 10°C and that comparable
displayed
degrees
however,
levels of unsaturation
extensively subcultured, has a warm temperate origin (Poinar. 1979). Despite such differences in origin, similar
or
in unsaturation
It was apparent,
in the unsaturation
possessed
strain
trend with respect to the temperatures
increases
nematodes
the et al..
family (Hominick
of S. carpocapsae,
these
to
distributed
prototype
1996). There
S. riobravis is
adjustment
and is in keeping with the suggestion
habitat-related
The
physiological
boreal ancestory
would be
climates.
partial
that the globally
to storage
in this study
with diverse
subtropical,
tolerance
cold temperatures in a species with a restricted subtropical distribution suggests a cold temperate to
below
lipid at cold temperatures. The four nematode strains examined originated
of their
and Roger Gordon
the
upper
lethal
temperatures
creased survival time in Conversely, when nematodes
and
de-
the frozen condition. were recycled at colder
temperatures, their upper lethal temperatures were decreased, while their freezing survival times were lengthened
(Jagdale
of physiological
and Gordon,
mechanisms
temperature-induced
shifts
1997b). The array
responsible in
includes
modifications
in
(Jagdale
and
1997~) and
Gordon,
the
thermal specific
for such tolerances activities
capacities
to
synthesize isozymes of metabolic enzymes (Jagdale and Gordon, 1997d). The present study has extended the warm temperature (1994).
based
demonstrating
range studies of Fodor
on a single that adaptive
subtropical
e/ a/.
species,
shifts in the degree
by of
unsaturation of lipids also occur in response to recycling temperatures, but that such capacities are not equally expressed among all isolates. Implementation of pest management
programs,
particularly
in
cold climates, should recognize the biological and physiological effects of continuous recycling on the selected
nematode
strain.
Acknowledgements-We acknowledge
financial support for the study from the Natural Sciences and Engineering Research Council of Canada. The authors thank Dr. P. Davis, Department of Biochemistry, Memorial University
Effect of temperature
on phospholipids
of Newfoundland. for the use of his gas liquid chromatography apparatus and valuable guidance in lipid analysis. Authors also thank Ms. Joanne Evans for technical assistance with respect to gas liquid chromatography. This study was conducted at Memorial University of Newfoundland, Canada.
REFERENCES
Bligh, E. G. and Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. and Ph#ol. 37, 91 l-917. Brown, I. and Gaugler, R. (1994) Cold tolerance of steinernematid and heterorhabditid nematodes. J. Nematol. 24, 539. Fodor, A., Dey, I., Farkas, T. and Chitwood, D. J. (1994) Effects of temperature and dietary lipids on phospholipid fatty acids and membrane fluidity in Steinernema carpocapsae. J. Nematol. 26, 278-285. Gibson, D. M., Moreau, R. A., McNeil, G. P. and Brodie, B. B. (1995) Lipid composition of cyst stages of Globodera roslochiensis. J. Nematol. 27, 3043 I 1. Gordon, R.. Finney, J. R., Condon, W. J. and Rusted, T. N. (1979) Lipids in the storage organs of three mermithid nematodes and in the hemolymph of their hosts. Camp. Biochem. Physiol. 64B, 369-374. Gordon, R., Chippett, J. and Tilley, J. (1996) Effects of two carbamates on infective juveniles of Steinernema carpocapsae All strain and Steinernema feltiae Umea strain. J. Nematol. Zg, 31&317. Grewal, P. S.. Selvan, S. and Gaugler, R. (1994) Thermal adaptation of entomopathogenic nematodes: niche breadth for infection, establishment, and reproduction. 1. Therm. Biol. 19, 245-253. Grewal, P. S., Gaugler, R. and Shupe, C. (1996) Rapid changes in thermal sensitivity of entomopathogenic nematodes in response to selection at temperature extremes. J. Inverrebr. Pathol. 68, 65-73. Hazel, J. R. (1995) Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu. Ret]. Phvsiol. 57, 1942. Hazel, J. R. and Williams, E. E. (1990) The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog. Lipid Res. 29, 167-227.
of entomopathogenic
nematodes
251
Hominick. W. M., Reid, A. P., Bohan, D. A. and Briscoe, B. R. (1996) Entomopathogenic nematodes: biodiversity, geographical distribution and convention on biological diversity. Biocont. Sci. Technol. 6, 317-331. Jagdale, G. B. and Gordon, R. (1997a). Effect of recycling temperature on the infectivity of entomopathogenic nematodes. Can. 1. Zool. (in press). Jagdale, G. B. and Gordon, R. (1997b) Effect of propagation temperatures on temperature tolerances of entomopathogenic nematodes. Fundam. Appl. Nemarol. (in press). Jagdale, G. B. and Gordon, R. (1997~). Effect of temperature on the activities of glucose-&phosphate dehydrogenase and hexokinase in entomopathogenic nematodes. Camp. Biochem. Physiol. (in press). Jagdale, G. B. and Gordon, R. (1997d). Variable expression of isozymes in entomopathogenic nematodes following laboratory recycling. Fundam. Appl. Nematol. (in press). Jagdale, G. B.. Gordon, R. and Vrain. T. C. (1996) Use of cellulose acetate electrophoresis in the taxonomy of steinernematids (Rhabditida, Nematoda). J. Nematol. 28, 301-309. Poinar, G. 0. Jr. (1979). Nematodes for bioiogical control of insects. CRC Press, Boca Raton, FL. Poinar, G. 0. Jr. (1990). Taxonomy and biology of Steinernematidae and Heterorhabditidae. In Entomopathogenic nematodes in biological control, ed. R. Gaugler and H. K. Kaya, CRC Press, Boca Raton, FL. Selvan, S., Gaugler, R. and Lewis, E. E. (1993) Biochemical reserves of entomopathogenic nematodes. energy J. Parasitol. 79, 167-172. Smart, G. C. Jr. (1995) Entomopathogenic nematodes for the biological control of insects. Suppl. J. Nemafol. 27, 529-534. Sumner, J. L. and Morgan, E. D. (1969) The fatty acid composition of sporangiospores and vegetative mycelium of temperature-adapted fungi in the order Mucorales. J. Gen. Microbial. 59, 215-219. Tanaka, T. (1996) Effects of growth temperature on the fatty-acid composition of the free-living nematode Caenorhabditis elegans. Lipids 31, 1173-l 178. Woodring, J. L. and H. K. Kaya. 1988. Steinernematid and Heierorhabditid nematodes: a handbook of techniques. Southern Cooperative Series Bulletin 331. Arkansas Agricultural Experiment Station. Fayetteville. AR.
J. therm. Bid. Vol. 22, No. 415, pp. 245-251, 1997 ~0 1997 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain s03&-4565(97)ooo19-3 0306-4565/97 $17.00 + 0.00
EFFECT OF TEMPERATURE ON THE COMPOSITION OF FATTY ACIDS IN TOTAL LIPIDS AND PHOSPHOLIPIDS OF ENTOMOPATHOGENIC NEMATODES GANPAT
B. JAGDALE’
and ROGER
GORDON’
‘Agriculture and Agri-food Canada, Pest Management Research Centre, P.O.B. 186, Delhi, Ontario, Canada, N4B 2W9 and 2Department of Biology, University of Prince Edward Island, Charlottetown. Prince Edward Island, Canada, CIA 4P3 (Received 7 December
1996; accepted in revised form 3 Ma>’ 1997)
Abstract-l. Gas liquid chromatography was used to determine the composition of fatty acids in total lipids and phospholipids of the entomopathogenic nematodes. Steinernema feltiae Umel strain, S. carpocapsae All strain, S. riobravis TX strain and S. filfiae NF strain that had been recycled or stored at 5, 10, 15, 20 and 25°C. 2. In all nematode strains, the unsaturation indices of total lipids increased as recycling or storage temperatures decreased. The unsaturation indices increased in the phospholipids of all strains except the TX strain of S. riobravis. 3. Increased unsaturation indices at low temperatures was due to an increase in polyunsaturated fatty acids with concomitant decline in the proportion of saturated fatty acids, especially palmitic (16:O)and/or stearic (18:O) acids 4. Sfeinernema riobrauis displayed a lower degree of physiological adaptation to cold temperatures than the other strains. The saturated fatty acids and unsaturation index of this nematode’s phospholipid did not change in response to recycling or storage temperatures. 0 1997 Published by Elsevier Science Ltd. All rights reserved. Key Word Index: Entomopathogenic nematodes; fatty acids; Galleria mellonella; gas liquid chromatography; phospholipids; Steinernema feltiae; Steinernema carpocapsae; Steinernema riobravis; steinernematids; temperature; thin-layer chromatography
INTRODUCTION
Entomopathogenic and
nematodes
Heterorhabditidae)
(Hominick
are
( f. Steinernematidae globally
distributed
et al., 1996) and are being commercially
for use in insect pest management (Smart, 1995). The soil dwelling infective juveniles of these nematodes infect susceptible insect species via the host’s natural openings, then kill the host by reteasing a mutualistic bacterium; nematode development and reproduction then occur within the host cadaver (Poinar, 1990). The commercialization of entomopathogenic nematodes involves continual recycling, so it is important to determine the degree to which they are affected by the recycling regimes at the whole organism and physiological levels. There is evidence to suggest that in certain species, the temperature range over which infection occurs may be modified by the temperature at which recycling is carried out (Grewal ef al., 1996). Both the infectivity (Jagdale and Gordon, 1997a) and capacities to tolerate produced
temperature extremes (Jagdale and Gordon, 1997b) were adaptively modified in four strains of steinernematid nematodes by recycling them at various temperatures. The changes in the physiology of steinemematids that are responsible for modifying whole organism characteristics (e.g. temperature tolerance, infectivity) at various recycling temperatures require determination. Many poikilothermic animals adapt to changing environmental temperatures by modifying the degree of unsaturation of their lipids. At low ambient temperatures, the proportion of unsaturated: saturated fatty acids increases in phospholipids to maintain cell membrane fluidity and normal cellular functions (Hazel, 1995; Hazel and Williams, 1990). Information on the degree of lipid unsaturation relative to temperature adaptation mechanisms of nematodes is sparse. It has been suggested that the synthesis of unsaturated fatty acids in the cysts of the plant parasitic nematode, Globodera rostochiensis (Gibson et al., 1993, and in the storage organs of 245
G. B. Jagdale and R. Gordon
246
mermithid (insect parasitic) nematodes (Gordon et al.. 1979) may constitute a low temperature adaptation mechanism. Experimental evidence in support
of such a hypothesis
free-living showed
nematode,
an increased
(20:5) fatty
was obtained
Cuenorhabditis
proportion
25 C (Tanaka,
moiety
to nematodes
1996). In the Mexican
entomopathogenic
nematode,
which when
recycled at strain
Steinernema
of these nematodes
S. riohraz~is from 20 C) and stored
in tissue culture
bottles
at temperatures
(600 ml) for
three
E.uraclion
oj‘ lipids
Freshly emerged
(048
h old) infective juveniles
acids and membrane
dilute
formalin
Kaya.
1988) to separate
including
entomopathogenic
c’t al., 1994). nematodes,
ones, adaptively
the degree of unsaturation
modify
of their lipids in response
rinsed
ef al., Using
1996). then a Gilson”
beakers
matodes
into
ascertain
whether
changes
boreal
capacities
strains
that
to determine composition phospholipids had
periods
to
such as
to effect such changes represent
climatic zones. The purpose
that
it is important adaptations
a broad
vary
range
of
of the present study was
whether there was a change in the of fatty acids in total lipids and of four such strains of Steinevnenzu
been
recycled
or stored
of time at various
for
temperature
formed confined samples stored
in fatty acids occur at lower temperatures
and whether among
habitats.
physiological
prolonged
of each
by Plant Products
Ltd., Brampton,
S. riobraris
strain
by Dr.
was provided
Ontario,
Canada;
H. E. Cabanillas.
USDA, ARS. Crop Insects Research Unit, Weslaco. TX.. USA. Steirzernema ,fe/fiae Umes strain was
strain
that
of the
so that they
had been
recycled
regimes
or
(each
sample 50 and 100 mg wet weight for total lipid and phospholipid into
analysis.
separate
(I .5 ml), then samples were Labconco”
respectively)
polypropylene
freeze dry system,
conco Corp..
were transferred
microcentrifuge
tubes
frozen ( - 2O’C) overnight. then freeze-dried for 24 h
Kansas
Lypho-lock
The in a
6 (Lab-
City. MO, USA) and stored at of fatty acids. Each
as a separate
of rotal Lipid ,fh/rv
gas liquid
chromatography.
replicate
(12= 3).
acids
was then transferred
Biologic
Biocontrol
Willow Hill, PA.. USA. Sfeinernema
felriae
using
were extracted
Freeze dried samples were homogenized in 200 ~1 methanol containing a few crystals of hydroquinone (antioxidant agent) in a Potter-Elvehjem tissue grinder with a motor-driven
from
was determined Lipids
from infective juveniles using the method described by Bligh and Dyer (I 959). with some modifications.
been
Products,
the bottom
temperature
provided by Dr. R. West, Canadian Forest Service, St. John’s. NF. Canada from a stock colony that had initially
from
Lipid fatty acid composition
carpocappsae All strain
obtained
water
AND METHODS
Sources of’ nematodes
TX
distilled
no. 4 filter papers,
at the specified
.4w/~:vix
Steinernemu
with
and
containing
allowed to settle (ca. pipetman, infective
~ 20 C until used for analysis
regimes.
(Woodring
blobs on the papers. Three different
tube was considered MATERIALS
twice
were transferred to Whatman
traps
of
from the
250 ml beakers
water.
juveniles
ne-
in the White
(Gordon IO min).
commercial
of entomopathogenic
were transferred
distilled
to recycling temperatures. no studies have been done involving recycling below 15’ C. To expand the exploitation
at
5. IO and 15°C).
each of the four strains
that certain
(S. jkltiue
at 5 C, S. c’arpocapsae at 5 and IO-C, S. riobrah
the recycling temperature from 25 to 18 C caused an increase in the unsaturation of fatty fluidity (Fodor
weeks
of the
carpocap-
sue, decreasing
While these data suggest
were collected
where recycling of strains was not possible
of polyunsaturated
acids in its phospholipid
recycled at I5 C, compared
for the
elegans,
Infective juveniles
from their lowest recycling temperature regimes (S. /h/the from IO C. S. carpocapsae from I5 C.
microcentrifuge
pestle. Each homogenate
into a separate
polypropylene
tube (I .5 ml), then 250 pl distilled
NF strain is a new strain (Jagdale e/ al.. 1996) that we isolated in Summer 1993 from soil on a farm site
water. 250 pl chloroform and 550 11 methanol added; samples were vortexed and incubated at 4’C for I h.
close to St. John’s, NF. Canada. using Galleria traps (Woodring and Kaya. 1988).
The homogenate was centrifuged at I3 I50 RPM for 2 min at room temperature (25°C). supernatant
bait
transferred Recycling/storage
All nematode (May, Galleria
tempernture
strains
1994May. mellonella
regimes
were recycled
for two years
1996) by propagation through larvae (Woodring and Kaya,
1988): NF and Umei strains of S. ftiltiue at IO. 15. 20 and 25 C: S. carpocapsae All strain at 15, 20 and 75 C and S. riohark TX strain at 20 and 25 C.
to further
microcentrifuge
tubes,
then
300 ~1 distilled water and 300 ~1 of chloroform added. Samples were vortexed and incubated at room temperature Following
(25°C) for 30 min. centrifugation (13,150
RPM;
2 min;
25 C), the lower lipid-containing layer was transferred into separate 6.0 ml capacity transmethylation vials. Solvents in each vial were evauorated under a
Effect of temperature on phospholipids of nitrogen, then 2.0 ml transmethylating reagent (94.0 ml methanol, 6.0 ml sulfuric acid) and a few crystals of hydroquinone added. Vials were incubated for 5 h at 7O”C, 1.0 ml distilled water and 1.5 ml hexane added, then shaken and allowed to stand for 10 min to form two separate layers. The upper lipid layers (hexane extract) containing the fatty acid methyl esters (FAMES) were transferred into small screw cap vials (1 .O ml capacity), then blown dry with nitrogen. Extracted FAMES were dissolved in 20 ul carbon disulphide and 0.5 ul of the solution injected into the GLC apparatus, a Hewlett Packard 5890 series II gas liquid chromatograph equipped with a flame ionization detector. The column used was a 30 m Supelcowax lo/O.53 mm (Supelco, Supelco Park, Bellefonte, PA, USA). The inert carrier gas was helium with a flow rate of 3.27 ml min- ‘. The oven, injector and detector temperatures were set at 180, 225 and 225”C, respectively. Commercial standards of FAMES, obtained from Supelco and Sigma Chemical Co. (St. Louis, MO, USA), were run under identical conditions and the chromatograms evaluated with reference to the retention times of the standards. stream
Analysis
of phospholipid
fatty
acid composition
The procedure used for the extraction, transmethylation and subsequent analysis of phospholipids was
of entomopathogenic
nematodes
247
the same as that used for total lipids, except that thin-layer chromatography (TLC) was first used to separate phospholipids from the 100 mg wet weight infective juvenile samples. Following freeze-drying and chloroform: methanol extraction, the lipid extracts were transferred into screw cap vials (ca. 1 ml), blown dry under nitrogen, then re-dissolved in 50 pl chloroform: methanol (2:l u/v). Each sample (25 11) was applied on silica gel plates, the sample spots allowed to dry at room temperature (25°C) for l-2 min, then plates were transferred into the developing tank containing the solvent mixture (mobile phase) hexanediethyl ether-acetic acid (85:15:2 v/v/u). The TLC plates were developed for 30 min then dried at room temperature (25°C) for l-2 min. Phospholipid spots were visualized by placing the TLC plates for a few seconds in a separate developing tank containing a few crystals of iodine. The phospholipid spots outlined on the TLC plates were scraped off into separate transmethylation vials. Statistical
analysis
All fatty acids were expressed as mol% of the total lipid and phospholipid fractions. Unsaturation indices, which are a measure of the degree of unsaturation in terms of the number of double bonds/mole, were computed (Sumner and Morgan,
Table 1. Effect of recycling and storage temperatures on the proportions of saturated and unsaturated fatty acids in the total lipids of four strains of Steinernema Recycling/Storage Temperature (“C)
Fatty acids 25
20
15
10
S. feltiae NF strain SFA” MUFA” PUFA” UP
48.5 34.5 18.6 0.8 f 0.1’
41.3 31.9 21.5 0.9 f 0.1’
46.8 30.5 24.5 1.1 * 0.1’
34.8 22.2 44.4 1.5 + O.ld
30.5 21.3 48.5 1.7 & O.Od
S. feltiae Umea strain SFA MUFA PUFA UI
51.0 30.3 , 21.0 0.8 + 0.1’
27.9 51.8 23.0 I.1 + O.Od
35.1 49.6 19.4 I .o + o.04
32.9 23.3 44.0 1.6 + 0.0
29.3 24.7 41.4 1.7 f 0.1’
S. carpocapsae All strain SFA MUFA PUFA UI
48.7 24.8 26.4 1.0 * 0.1’
36.5 23.1 39.9 I.4 * O.ld
34.9 20.2 44.9 1.6 + O.Od
34.1 18.4 47.3 1.6 + O.Od
26.5 21.5 44.1 I .7 & O.Od
S. riobrauis TX strain SFA MUFA PUFA UI
47.2 31.7 22.8 I.0 f 0.1’
41.2 32.1 28.3 1.1 f 0.0
39.2 33.0 29.5 1.2 f O@
38.1 29.1 32.9 I .3 f O.Od’
32.3 26.5 41.9 I.5 f 0.1’
5
“SFA = Saturated fatty acids, MUFA = Monounsaturated fatty acids, PUFA = Polyunsaturated fatty acids. Values are mean mol% of total lipids (n = 3). bUI = Unsaturation index. Means with the same lower case superscript letter (across the columns) are not significantly different (P > 0.05) by Student Newman-Keul; test.
Ganpat B. Jagdale and Roger Gordon
248 1969). Mole and
percentage
unsaturation
one-way (Jandel
indices
ANOVA, Carp”.
significance
data
(arcsine were
analysed
using
Student-Newman-Keuls
Sigma
Stat,
was defined
unaffected
transformed) test
1992). The
level
of
in the total lipids of the other three strains.
In the phospholipids,
rated fatty acids remained temperature;
as P < 0.05.
however,
the NF strain was the
only strain in which the proportion this fraction
of monounsatu-
unaltered
by such a low
decreased
in the phospho-
lipids of the other three strains. Analysis of the fatty acid profiles (data not shown)
RESULTS The
recycling
or
storage
revealed that the increased temperature
influenced the fatty acid composition the entomopathogenic nematodes. general decline in the content and
a concomitant
indices
in
both
the
phospholipids
(Table
S. riobraris
TX strain,
saturated
of saturated
increase total
in the lipids
(Table
1) and
except
for of
according
the unsaturato recycling
or
(l&O)
acids
acids,
47.5-50.8
fatty
and 32.7-37.4
that
had
moieties, been
low
fatty
temperature
phospholipid (i.e. other
Decreases
in
acids,
caused
by storing
at
were
attributable
oleic
acid
of S. jeltiae)
total lipids and phospholipids
acids in the total lipids of S. fdtiae
Changes
storage
in proportions
of all four strains as the temperatures
decreased.
of monounsaturated
acids were less consistently
fatty
related to temperature.
At
the 5’C storage temperature, the monounsaturated fatty acids decreased as a proportion of the total lipids
in the
NF
strain
Table 2. Effect of recycling
Fatty
of
S. ,fkdtiue, but
was
storage
at 5”C, the proportions
(20: 1)
NF strain. After
of oleic acid in the
phospholipids of S. feltiae Umea strain (9.5 mol%), S. carpocapsae All strain (12.8 mol%) and S. riohraois TX strain mately
half those
nematodes.
Recycling/Storage
(17.2 mol%)
pertaining
The combined
Temperature
were approxi-
in the 25”C-recycled
mol%
of palmitoleic
and unsaturated
fatty acids
(/ C)
25
20
15
10
5
S. /k/rise NF strain SFA” MUFA” PUFA” UP
39.5 14.1 44.8 1.6 i 0.1’
36.5 12.2 51.5 1.8 f 0.1’”
32.0 14.6 53.6 I .8 + 0.0’”
31.0
25.7 15.0 59.5 2.1 f 0.1”
S. f&t& SFA MUFA PUFA UI
40.6 17.6 42.0 I.5 + 0.1’
38.0
19.4 42.9 I .6 + 0.0’”
32. I 13.0 52.9 I .7 * O.Od
12.8 56.3 I .9 -+ 0.0”
29.8 10.4 59.5 2.0 f 0.0’
40.7 24.8 34.8 I.3 * 0.1‘
36. 19.7 43.2 1.5 + 0.1.
33.5 22.0 45.2 1.5 + 0.1’
37.1 16.1 46.5 I.5 + 0.1’
27.7 13.4 58.5 1.9 + O.Od
34.8 31.5 34.4 I .4 f 0.0
35.x 2x.1 35.8 I.5 f 0.1’
33.2 19.5 47.5 1.6 & 0.0’
30.9 27.3 41.3 1.7 + 0.0’
34.4 17.6 48.1 I .6 _+ 0. I’
Umel
S. ccrrpocapstrr SFA MUFA PUFA UI S. riohruris SFA MUFA PUFA UI
a the
and to
(16: 1) and eicosenoic
in palmitoleic
and storage temperatures on the proportions of saturated in the phospholipids of four strains of Steinernema
acids
to
(18: 1) in
of three of the four isolates
than the NF strain
decreases
and
in nematodes
25°C.
in
fractions
comprised
at
(5’C),
decrease
together,
of the total lipid and
respectively,
recycled
monounsaturated significant
that mol%
storage temperatures. There was an increase in the proportions of polyunsaturated fatty acids in the recycling
of fatty acids
in total lipids or phospholipids of palmitic (16:0) and/or stearic
phospholipid
the proportion
fatty acids and consequently,
tion index, did not change
fatty acids unsaturation
2) of all strains, in which
regimes
of lipids in all There was a
unsaturation
at low temperatures was at the expense
13.2 55.7 I .9 * O.O’d
strain 30.2
All strain
TX strain
“SFA = Saturated fatty acids, MUFA = Monounsaturated fatty acids. PUFA = Polyunsaturated fatty acids. Values are mean mol% of phospholipids (17= 3). WI = Unsaturation index. Means with the same lower case superscript letter (across the columns) are not significantly different (P > 0.05) by Student Newman-Keuls test.
and
Effect of temperature on phospholipids of entomopathogenic nematodes eicosenoic acids was 10.4 in the total lipids of S. feltiae NF strain, but was reduced to 0.9 in infective juveniles stored at 5°C. The increase in polyunsaturated fatty acids at reduced temperatures was attributable to significantly greater percentages of linoleic acid (182) in total lipids and phospholipids of all strains. Depending on the strain, linoleic acid increased as a proportion of the total lipids from 15.3-18.3 mol% at 25°C to 27.4-29.2 mol% at 5°C. The comparable increase in this fatty acid in the phospholipids was from 19.9-24.6 mol% to 29.0-37.2 mol% over the same drop in temperature. In all except S. riobrauis, this was augmented by significantly increased proportions of eicosapenic acid (20:5w3) at 5°C. The mole percentages of this polyunsaturated fatty acid in the total lipids of the two strains of S. feltiae were 2.4-2.6 in nematodes recycled at 25°C and 15.2-15.5 after storage at 5°C; comparable values for S. carpocupsae All strain were 6.6 (25°C) and 13.0 mol% (SC). Eicosapenic acid increased as a mole percentage of the phospholipids from 13.6-14.4 in the two strains of S. feltiae recycled at 25°C to 20.4 after storage at 5°C; comparable values for S. carpocapsue All strain were 10.4 (25°C) and 16.0 mol% (SC). In S. riobravis stored at 5°C arachidonic acid (20:4), rather than eicosapenic acid, was elevated in the phospholipid moiety. This polyunsaturated fatty acid comprised 6.3 mol% of the phospholipids of nematodes recycled at 25°C compared to 8.5 mol% of this lipid moiety in nematodes stored at 5°C. In S. feltiae UmeP strain, arachidonic acid (20:4) actually decreased as a proportion of the phospholipids at temperatures _< 10°C. This polyunsaturated fatty acid comprised 3.64.7 mol% of phospholipids in S. riobruuis that had been recycled at 2 15°C and 2.9 mol% at those recycled or stored at I 10°C. In the two strains of S. feltiae and S. riobravis TX strain, the temperature below which significant increases in unsaturation indices of total lipids occurred was 15”C, while the comparable temperature was 25°C for S. curpocupsae All strain (Table 1). With respect to phospholipids, the unsaturation indices were significantly increased for S. feltiue NF strain and S. carpocupsue All strain at < 10°C and for the UmeP strain of S. feltiae at < 20°C (Table 2).
DISCUSSION
This study has shown that the composition of saturated and unsaturated fatty acids in the lipids of entomopathogenic nematodes was influenced by recycling and storage temperatures from 25 to 5°C. Recycling or storing at colder temperatures increased
249
the proportions of polyunsaturated fatty acids in the total lipids and phospholipids of all four strains of Steinernema. Except for the phospholipids of S. riobruvis TX strain, this was accompanied by a reduction in the proportions of saturated fatty acids, resulting in increases in unsaturation indices. The increased production of polyunsaturated fatty acids at low temperatures was due to linoleic acid (18:2) and eicosapenic acid (20:5w3) or, in S. riobravis phospholipids (5”C), arachidonic acid (20:4). In a related study, embodying only two warmer rearing temperatures (18 and 25’Q Fodor et al. (1994) also reported that nematodes recycled for an unspecified time at 18°C contained a higher proportion of eicosapenic acid (20:5w3) in their phospholipids. Adaptation to environmental temperatures by shifting the proportions of saturated: unsaturated fatty acids is a widespread phenomenon in poikilotherms. High levels of unsaturated fatty acids in phospholipids increase the membrane fluidity to maintain normal cellular functions at low environmental temperatures (Hazel, 1995). The cyst stages of the plant parasitic nematode, G. rostochiensis, were found to contain high levels of polyunsaturated fatty acids in their lipids, principally triacylglycerols and phospholipids, as an overwintering adaptation (Gibson et al., 1995). Among entomopathogenic nematodes, the proportion of unsaturated fatty acids in the phospholipids of the Mexican strain of S. carpocapsue was greater when nematodes were reared at 18°C than when reared at 25°C and this was accompanied by a less ordered arrangement of phospholipids within the cell membranes at the lower temperatures (Fodor et al., 1994). Thus, in the present study, the changes in unsaturation of phospholipids that occurred in all strains other than S. riobruvis TX strain, consequent to recycling or storing at various temperatures, are probably adaptive in helping to preserve membrane integrity from 25 to 5°C. In all four strains, there was a temperature-induced shift in the unsaturation indices of total lipids that parallelled those of the phospholipids. Infective juveniles of entomopathogenic nematodes contain high levels of lipids, presumably neutral lipids, and utilize them as an energy substrate (Selvan et al., 1993). Gordon et al. (1979) reported that the unsaturation index of stored triacylglycerols was greater in a boreally adapted mennithid nematode, Neomesomermis flumenulis, than in the tropical culicivorux, and Romanomermis mermithid, suggested that such differences in unsaturation were necessary to maintain fluidity within the nematode’s storage organ and permit accessibility of enzymes to
Ganpat
250
B. Jagdale
lipid energy reserves at the temperatures prevailing within the respective habitats of the nematodes. It is possible
that the four strains
present
study
adjusted
of Steinernema in the
the physical
storage nutriment to permit energy occur over a broad range of temperatures. adaptive
A shift toward
in permitting
state
metabolism to environmental
unsaturation
enzyme accessibility
from habitats
two strains
of S. ,feltiae are boreal, while the All strain
and
S. ,feltiae represents
for this nematode appeared
which
to be no obvious
significant
indices occurred.
elevations
the strains displayed
increased
in their total lipids at
by three of the strains
unsaturation
findings appear at et al. (1993). who
species, Steinemema scupter-
that a tropical
isci, contained
a higher proportion
acids
its
within
total
entomopathogenic temperature.
lipids
nematodes
Their data
such a conclusion,
of saturated than
several
recycled
of
a temperate
strain of S. ,/kltiae contained
the highest
proportion of polyunsaturated fatty acids. According to our study, the two boreally
of S. fhlriae, as well as the warm temperate
strains
S. carpocupsae All strain
increased
the unsaturation
indices of both total lipids and phospholipids as recycling and storage temperatures declined. However,
S. riobravis TX
the subtropical
adjusted (mostly
the unsaturation neutral
index
strain
of its total
lipids) in such a manner.
only lipids
Although
this nematode displayed, in common with the other strains, decreases in proportions of monounsaturated fatty acids and compensatory increases in polyunsaturated fatty acids, it was the only strain in which the saturated
fatty acids, and consequently
indices, did not significantly recycling
or storage
unsaturation
increase due to reduced
temperatures.
Such an inability
of this nematode to fully adapt physiologically to colder temperatures is in keeping with the perception that it is a warm adapted species, capable of infecting hosts at 2 lO”C, reproducing at 2 20°C (Grewal et al., 1994) and surviving poorly when stored at 5 ‘C. the preferred storage temperature for the other isolates (Jagdale and Gordon: unpublished observations). Paradoxically, this nematode is capable of tolerating freezing (Brown and Gaugler, 1994) an attribute of questionable relevance in its subtropical southern
Texas habitat.
The combination
15°C. recycling
of freezing
this
temperature.
was not possible.
Also.
in
rearing
At other tempera-
that some artificial selection may resulting in genetically altered
occurred,
synthetic
capacities
for fatty
acids adaptive
to the
temperatures.
We have shown that the upper and lower thermal tolerances
of these
entomopathogenic
nematodes
were influenced by the temperature at which they were recycled. Recycling at warmer temperatures increased
adapted
at
S. carpocapsae at IO‘C and S. riobravis at 10 and
have
as the proportion
were
at 5°C.
in all strains, since no recycling
out
other
does not seem to support
however,
carried
tures, it is possible
fatty acids in S. scapterisci was only higher than some of the other species and
saturated marginally
was
fatty
at the same
of phospholipids
Many of the shifts in lipid unsaturation reported in this study were environmentally induced. This applies to 5 ‘C determinations
in their total lipids and phospholipids
when recycled at 25°C. These variance with those of Selvan reported
of
that all
I 10°C and that comparable
displayed
degrees
however,
levels of unsaturation
extensively subcultured, has a warm temperate origin (Poinar. 1979). Despite such differences in origin, similar
or
in unsaturation
It was apparent,
in the unsaturation
possessed
strain
trend with respect to the temperatures
increases
nematodes
the et al..
family (Hominick
of S. carpocapsae,
these
to
distributed
prototype
1996). There
S. riobravis is
adjustment
and is in keeping with the suggestion
habitat-related
The
physiological
boreal ancestory
would be
climates.
partial
that the globally
to storage
in this study
with diverse
subtropical,
tolerance
cold temperatures in a species with a restricted subtropical distribution suggests a cold temperate to
below
lipid at cold temperatures. The four nematode strains examined originated
of their
and Roger Gordon
the
upper
lethal
temperatures
creased survival time in Conversely, when nematodes
and
de-
the frozen condition. were recycled at colder
temperatures, their upper lethal temperatures were decreased, while their freezing survival times were lengthened
(Jagdale
of physiological
and Gordon,
mechanisms
temperature-induced
shifts
1997b). The array
responsible in
includes
modifications
in
(Jagdale
and
1997~) and
Gordon,
the
thermal specific
for such tolerances activities
capacities
to
synthesize isozymes of metabolic enzymes (Jagdale and Gordon, 1997d). The present study has extended the warm temperature (1994).
based
demonstrating
range studies of Fodor
on a single that adaptive
subtropical
e/ a/.
species,
shifts in the degree
by of
unsaturation of lipids also occur in response to recycling temperatures, but that such capacities are not equally expressed among all isolates. Implementation of pest management
programs,
particularly
in
cold climates, should recognize the biological and physiological effects of continuous recycling on the selected
nematode
strain.
Acknowledgements-We acknowledge
financial support for the study from the Natural Sciences and Engineering Research Council of Canada. The authors thank Dr. P. Davis, Department of Biochemistry, Memorial University
Effect of temperature
on phospholipids
of Newfoundland. for the use of his gas liquid chromatography apparatus and valuable guidance in lipid analysis. Authors also thank Ms. Joanne Evans for technical assistance with respect to gas liquid chromatography. This study was conducted at Memorial University of Newfoundland, Canada.
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