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First published online September 5, 2008
Journal of Experimental Biology 211, 2901-2908 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020743
A surface lipid may control the permeability slump associated with entry into anhydrobiosis in the plant parasitic nematode Ditylenchus dipsaci
1 Department of Zoology, University of Otago, PO Box 56, Dunedin, New
Zealand
2 Department of Chemistry, University of Otago, PO Box 56, Dunedin, New
Zealand
3 Department of Human Nutrition, University of Otago, PO Box 56, Dunedin, New
Zealand
* Author for correspondence (e-mail: david.wharton{at}stonebow.otago.ac.nz)
Accepted 15 July 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: cuticle, desiccation, annulations, lipid, confocal microscopy, permeability, accessory layer, ATR–IR spectroscopy
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Wharton, 2002a;
Wharton, 2002b). A slow rate
of water loss is essential for anhydrobiotic survival
(Evans and Perry, 1976).
Anhydrobiotic nematodes have been divided into two broad groups:
slow-dehydration strategists, which rely on a slow rate of water loss from
their environment, and fast-dehydration strategists, which control their own
rate of water loss (Wharton,
2002b; Womersley,
1987). Ditylenchus dipsaci (Kühn) Filipjev is a
plant-parasitic nematode that can survive direct exposure to 0% relative
humidity (RH) and is thus a fast-rate strategist
(Perry, 1977a;
Wharton and Aalders,
1999).
There is a marked decrease in the rate of water loss from D.
dipsaci during the early stages of desiccation: `the permeability slump'
(Wharton, 1996). This must
involve a change in the permeability of the cuticle. Two mechanisms have been
suggested for the permeability slump. If the cuticular annulations are more
permeable areas of the cuticle, the permeability slump could be produced by a
decrease in annulation spacing and a deepening of the annulation grooves
(Rössner and Perry, 1975;
Wharton, 1996;
Wharton and Marshall, 2002).
Alternatively there could be a physical change in the properties of the
cuticle as it dries (Ellenby,
1968; Perry,
1977b).
Wharton and Marshall (Wharton and
Marshall, 2002), using cold-stage field-emission scanning electron
microscopy, were unable to demonstrate changes in annulation spacing that
correlated with the permeability slump. However, these measurements were
averages from different nematode samples frozen after differing periods of
desiccation and this technique could not follow changes in individual
specimens. Since both annulation spacing and the timing of the permeability
slump may vary between individuals, any changes could have been obscured.
Length changes during desiccation are not as clear if measurements from
averages over several nematodes are compared with changes in individual
nematodes (Wharton, 1996). We
have used confocal microscopy to follow changes in the annulation spacing of
individual nematodes during the early phases of desiccation. We have also used
lipid and carbohydrate probes, confocal microscopy and other microscopical and
analytical techniques to examine changes in the surface of the cuticle of
D. dipsaci during desiccation that may be related to changes in
cuticular permeability.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). When nematodes appeared in numbers on the sides of the
culture vessel they were harvested by macerating the carrot tissue in a
blender, separated using a modified Baermann funnel technique
(Hooper, 1986), washed into an
artificial tap water (ATW) (Greenaway,
1970) and stored at 4°C. When sufficient nematodes had been
accumulated they were transferred to a clay medium (Minsorb, OMYA NZ Ltd.,
Auckland, NZ), which was then dried at room temperature and stored at 4°C.
When required for experiments nematodes were rehydrated in tapwater, separated
from the Minsorb by allowing them to migrate through tissue paper, washed in
ATW and stored at 20°C for at least a week before use. These samples
comprise predominantly fourth-stage larvae (L4s)
(Wharton, 1996).
Nematodes were exposed to 50% RH, 20°C for 30 min in a controlled
humidity chamber, as described by Wharton and Aalders
(Wharton and Aalders, 1999).
This desiccation exposure is known to induce a lag phase, indicating entry
into anhydrobiosis (Wharton and Aalders,
1999), and to produce a water content of about 0.23 g
g–1 dry mass (Wharton,
1996). The samples were then immersed in ATW, the recovery counted
at intervals and the time taken for 50% to recover calculated by probit
analysis (Norusis, 1999).
Survival was measured as the percentage recovery after 24 h in ATW.
Changes in annulation spacing observed by confocal microscopy and reflected laser light
Changes in cuticular annulation spacing were observed using a Zeiss LSM510
Axiovert 200 confocal laser scanning microscope operated in reflection mode
using a 543 nm helium–neon laser. Nematodes were washed briefly in
distilled water and a single nematode transferred to a humidity-controlled
observation chamber, the relative humidity of which was controlled by an
81.77% (w/w) solution of glycerol in distilled water
(Grover and Nicol, 1940),
giving 50% RH. The temperature of the microscope room was controlled at
20°C. Most of the surface water was removed from the nematode and the
observation chamber sealed with Vaseline. The nematode was observed until the
remaining surface water evaporated and then images were taken at intervals
during desiccation. The annulation spacing was determined by measuring the
distance across ten annulations.
Staining with wheat germ agglutinin
Nematodes were stained with wheat germ (Tritium vulgaris)
agglutinin (WGA: Sigma-Aldrich, Uckland, NZ), conjugated to fluorescein
isothiocyanate (FITC), using 1 mg ml–1 in phosphate-buffered
saline (PBS, pH 7.4) for 15 min and then washed three times in PBS. They were
then observed using epifluorescence on a Zeiss Axiophot Photomicroscope with a
450–490 nm exciter filter and a 520 nm barrier filter or on the confocal
microscope with argon ion laser excitation at a wavelength of 488 nm.
Unstained controls were examined for autofluorescence under the Zeiss Axiophot
Photomicroscope. WGA/FITC-stained specimens were also used, after washing six
times in distilled water, to follow changes in annulation spacing during
desiccation in a humidity-controlled observation chamber on the confocal
microscope as described above.
Staining with Nile Red
Nematodes were stained with Nile Red
[9-diethylamino-5H-benzo(
)phenoxazine-5-one; Sigma]. Nile Red is a
benzophenoxazone dye that is intensely fluorescent in organic solvents, with
its fluorescence maxima depending upon the relative hydrophobicity of its
surrounding environment, but which is poorly soluble and is fully quenched in
water. It can thus be used as a selective and sensitive stain for lipids
(Fowler and Greenspan, 1985;
Greenspan et al., 1985). 10
µl of a stock solution of Nile Red (10 mg ml–1 in ethanol)
was added to 1 ml of PBS in an Eppendorf tube and vortexed to mix. This was
added to nematodes on a coverslip. The nematodes were stained for 15 min at
room temperature and then washed three times in PBS. The coverslip was
inverted onto a microscope slide for observation. Samples were observed using
epifluorescence on a Zeiss Axiophot Photomicroscope with a 546–558 nm
exciter filter and a 590 nm barrier filter or with a 450–490 nm exciter
filter and a 520 nm barrier filter. Samples were also observed using the
confocal microscope with laser excitation at a wavelength of 543 nm
(helium–neon laser). The staining of material left behind after the
removal of nematodes by gentle ATW washing was also examined.
Other observations of surface material
Nematodes were allowed to dry from a small droplet of ATW on a coverslip at
50% RH, 20°C for 3 days. The coverslip was then inverted onto a microscope
slide and the edges of the coverslip sealed with nail varnish. The contact
areas between the nematodes and the coverslip were observed using a Zeiss
Photomicroscope and differential interference contrast (DIC) optics. Similar
observations were made on nematodes dissected from fresh carrot tissue, washed
thoroughly in distilled water to ensure that they were free of any
contaminating material (migrated through tissue paper twice and then washed
three times in distilled water), allowed to dry at 0% RH (over silica gel),
50% RH or 98% RH (saturated solution of K2SO4)
(Winston and Bates, 1960), at
20°C for 24 h on a clean coverslip and then mounted and observed as
above.
Nematodes were also observed for the presence of surface material using confocal microscopy and reflected laser light at 543 nm (helium–neon laser) after desiccation in a controlled humidity chamber (50% RH, 20°C).
Attenuated total reflection infrared (ATR–IR) spectroscopy
Internal reflection spectroscopy is based on the existence of an evanescent
wave in a medium of lower index of refraction (n) in contact with an
optically denser medium in which a light is introduced
(Harrick, 1967). The
evanescent electric field decays exponentially in the rarer medium and a
penetration depth (dp) is defined as the decay of such an
electric field by 63%.
In this work, a three-reflection diamond-coated ZnSe prism (ASI SensIR
Technologies, Norwalk, CT, USA) of 3 mm diameter sampling area was used to
collect the infrared spectra. For a diamond prism [index of refraction
(n)=2.35] in contact with water (n=1.33)
dp is 1.35 µm at 1650 cm–1
(Vigano et al., 2005). Thus
the sampling depth is of the order of a few micrometers. This configuration
provides three contacts across the surface between the radiation and the
sample (Fig. 1). The prism
surface was cleaned prior to each experiment by polishing with 0.015 µm
Al2O3 powder (BDH, polishing grade) on a wet polishing
microcloth (Buehler, Lake Bluff, IL, USA) and then rinsed with deionised water
(Millipore, Milli-Q, Billerica, MA, USA). Infrared spectra were obtained using
a Digilab FTS 4000 infrared spectrometer equipped with a KBr beamsplitter and
a Peltier-cooled DTGS (Deuterated Tri-Glycine Sulfate) detector. Dried air was
used to purge the optical bench and contained a variable amount of
CO2 which is evident as a variable intensity band in the spectra at
about 2350 cm–1. All the infrared spectra show interferences
in the region 1800–2300 cm–1 due to the diamond prism
absorptions. Win-IR Pro version 3.4 software was used to analyze the spectra
recorded from 64 co-added scans at 4 cm–1 resolution. The
background spectrum was from the bare diamond prism. An air conditioning unit
in the room in which the spectrometer was located produced a controlled
temperature of 20°C and a measured relative humidity of 27–28%
(±1%).
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To isolate the nematode surface lipid material, nematodes were washed thoroughly with distilled water and transferred to a watchglass. The surface water was removed and the nematodes desiccated at 0%, 50% or 98% RH at 20°C for 24 h. Distilled water was then added, the nematodes allowed to rehydrate for 10 min and then carefully removed using a fine pipette. The remaining water was then gently removed and the watchglasses dried at 0%RH, 20°C for 1 h. Diethyl ether (100 µl) or chloroform:methanol (2:1 v/v; 100 µl) was added and the samples transferred to gas chromatography (GC) vials and sealed. Samples (5 µl) were transferred to the surface of the ATR prism, the solvent allowed to evaporate, and the spectrum collected.
Thin layer chromatography and gas chromatography
Thin layer chromatography (TLC) separation was carried out in order to
purify and to separate the nematode surface lipid material from any
phospholipids coming from the worms' cuticle outer layer. Nematode surface
lipid material was prepared as described above and taken up in 100 µl
methanol. The sample was applied to a silica-gel-coated aluminium plate.
The developing solvent system and staining reagent for neutral lipids were n-hexane:diethyl ether:acetic acid (85:15:1, v/v/v) and ANS 0.1% (8,1-anilinonaphthalene sulphonic acid). Phosphatidylcholine (PC) and glycerol triacetate (TAG) were used as standards. The lipid to be examined (50 µl) and each standard (50 µl) were applied to the TLC plate, left for 40 min and dried at room temperature. The triglyceride region was scraped off and poured into a test tube for the subsequent methylation reaction.
Three millilitres of a 6% (v/v) sulphuric acid in methanol solution were added to the isolated lipid fraction as a methylating reagent. Fatty acids were methylated by acid-catalysed transesterification at 80°C for 2 h. After cooling to room temperature, 2 ml of hexane followed by 1 ml of water were added to the sample with 2 min of vortexing separating each addition. The upper hexane layer containing the fatty acid methyl esters (FAMEs) was removed, dried under N2 flow, reconstituted in 70 µl of hexane and stored at –20°C until gas chromatographic analysis. Sulphuric acid was AnalaR grade (BDH Chemicals, Poole, England).
The hexane in which the FAMEs were dissolved was evaporated under a stream of nitrogen and the FAMEs reconstituted in 70 µl of hexane for analysis of fatty acid composition by GC. FAMEs were separated using a HP-225 capillary column, 30 mx0.25 mm i.d., 0.25 µm film (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector. The gas chromatographic system consisted of a 5890 GC equipped with an autosampler (HP7673) and Chem Station integration (all Hewlett Packard, Avondale, PA, USA). The column oven was held at a temperature of 180°C for 5 min, then programmed to increase at 1°C min–1 to 210°C and held for 5 min. The total runtime was 45 min.
Fatty acid peaks were identified by retention time matching with authentic standards. A composite standard was used, made from commercially available methyl esters (NuCheck Prep, Elysian, MN, USA and Sigma, St Louis, MO, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Changes in annulation spacing
Cuticular annulations could be observed using reflected laser light but
changes in annulation spacing were difficult to follow with this technique,
since nematodes often moved during the desiccation series. There was clear
labelling of the surface of the nematode after staining with WGA
(Fig. 2). The amphids and the
opening of the excretory duct were also stained. There was no autofluorescence
associated with the cuticle in unstained controls, although droplets in the
intestine did show autofluorescence. The WGA labelling observed by
epifluorescence or confocal microscopy appeared to be concentrated at, or
confined to, the annulations and enabled the major annulations (which extend
to the lateral alae) and the minor annulations (which do not) to be
distinguished (Fig. 2).
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Lipid material on the nematode surface
There was no staining with Nile Red in PBS of fully hydrated nematodes and
no autofluorescence observed in unstained specimens observed by
epifluorescence microscopy. Desiccated nematodes, however, became permeable to
the dye and showed extensive staining of internal structures. The staining of
surface material was noted, with the nematodes appearing to be covered with
droplets (Fig. 4). The staining
of this material was variable, appearing in 0–69% of nematodes in a
sample. No consistent relationship was found between the time of exposure to
desiccation and the appearance of this material. However, the greatest
proportion exhibiting these droplets was in samples desiccated at 50%RH,
20°C for 5 min on a coverslip, stained in Nile Red for 15 min and washed
twice in PBS; with care being taken to ensure that changes of liquid were
gentle. The droplets appeared to be associated with the cuticular annulations
and the grooves of the lateral alae (Fig.
4).
|
)
were observed. These also appeared to be made up of droplets but left clear
impressions of the cuticular annulations and lateral alae
(Fig. 5).
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),
along with the ester group peak at 1744 cm–1
(Nara et al., 2002), clearly
show the presence of lipids. This lipid signal first appeared after 7 min
desiccation. The with-worms spectrum (Fig.
7A) shows also the characteristic protein vibrational modes
peaking at 3289, 1633 and 1546 cm–1, which are assigned
respectively to amide A (N–H stretch), amide I (C=O stretch) and amide
II (N–H bend, C–N stretch)
(Barth, 2007). The band
centered at 1237 cm–1 arises from phosphodiester
(PO2–) asymmetric stretch observed in
phospholipids, and the corresponding PO2–
symmetric stretch is found at 1079 cm–1
(Wang et al., 2006). In
addition, the broad band in the 1200–1000 cm–1 region,
peaking at 1035 cm–1, is related to C–H, C–OH,
C=O and C–O–C stretching/bending modes in polysaccharides
(Kacurakova and Mathlouthi,
1996). Peaks in the regions 1900–2150 cm–1
and 2200–2400 cm–1 are variable and are due to
absorptions from the diamond prism and the CO2 gas,
respectively.
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Another infrared spectrum was recorded after removal of the nematodes from the diamond prism [without-worms spectrum (Fig. 7B)]. The characteristic peaks of lipids were still evident at 2955, 2923, 2853, 1460 and 1744 cm–1, whereas the signals resulting from proteins (1636 and 1546 cm–1) and polysaccharides (broad band peaking at 1022 cm–1) appeared only as weak peaks.
The diamond surface was subsequently washed with diethyl ether and the spectrum collected (ether-extraction spectrum; Fig. 7C). The bands arising from proteins and polysaccharides were still visible at 1638, 1546 and 1021 cm–1. Therefore, after the ether wash the lipid peaks disappeared but most of the protein and the polysaccharide peaks remained on the prism.
The infrared spectrum of the isolated lipid from the nematode surface exposed to 50% RH (Fig. 8A) had peaks at 2958, 2922, 2852, 1721, 1462 and 1377 cm–1, all associated with lipid characteristic absorptions and there were no peaks associated with protein, polysaccharide or phospholipid. The overlapped infrared spectra of the lipid material from the nematode surface produced at different relative humidities showed that more lipid is produced at 0% and 50% RH than at 98% RH (Fig. 8B).
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-linolenic). The main chain lengths of saturated
fatty acids detected, in order of abundance, were: C8 (caprylic), C16
(palmitic), C6 (caproic), C12 (lauric), C18 (stearic), C10 (capric) and C14
(myristic).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Wharton and Aalders, 1999).
The water content of D. dipsaci L4s after desiccation at 20°C for
24 h, determined gravimetrically (Wharton
and Aalders, 1999), was 0.45±0.06 g g–1
dry mass (mean ± s.e.m., N=4: 30.8% w/w water content) at 98%
RH, 0.02±0.01 g g–1 dry mass (2.3%) at 50% RH but was
not measurable at 0% or 33% RH. Some water is retained by this nematode at
98%, 76% and 50% RH but exposure to 0 or 33% RH results in the loss of all
measurable water (Wharton and Aalders,
1999).
D. dipsaci L4s undergo a permeability slump during the early
stages of desiccation (2–6 min), which slows their rate of water loss
and thus facilitates survival (Wharton,
1996; Wharton and Marshall,
2002). There was no consistent change in the annulation spacing of
individual nematodes, observed by confocal microscopy in unstained specimens,
by reflected laser light or in specimens labelled with WGA, during this period
that correlates with the permeability slump. There is a 4% change in length
during the first 2 min of desiccation
(Wharton, 1996). Any
corresponding change in annulation spacing may be too small to measure.
No change corresponding to the permeability slump was measured (the average
of several specimens) by cold-stage field-emission scanning electron
microscopy on unfixed, frozen samples observed at 0, 2 and 5 min of
desiccation (Wharton and Marshall,
2002). There were, however, changes in the appearance of the
annulations after longer periods of desiccation (10–30 min), with the
annulation grooves becoming more prominent and the minor annulations extending
closer to the margins of the lateral alae
(Wharton and Marshall, 2002).
Given the lack of change in annulation spacing, however, it seems unlikely
that the permeability slump involves the narrowing or deepening of the
annulation grooves (Rössner and
Perry, 1975; Wharton,
1996; Wharton and Marshall,
2002).
The labelling of the annulation grooves by WGA suggests that the material
in the grooves is different from that covering the general surface of the
cuticle or that it is concentrated at the grooves. A similar pattern of
labelling of D. dipsaci J4s has been observed using a monoclonal
antibody raised against surface antigens
(Palmer et al., 1992) and
using concanavalin A and limulin, which bind to surface carbohydrates
(Durschnerpelz and Atkinson,
1988). The presence of surface carbohydrates is also indicated by
WGA, which binds to N-acetylglucosamine and/or
N-acetylneurominic acid (sialic acid) residues
(Jansson et al., 1986;
Wright, 1984).
During desiccation, oily material that stains with Nile Red appears on the
surface of the cuticle. The fluorescence of Nile Red is greatly enhanced in
organic solvents and hydrophobic lipids but is fully quenched in water. It can
thus be used as a selective and sensitive stain for lipids
(Fowler and Greenspan, 1985),
suggesting that the droplets observed contain lipid. Nile Red also stains
hydrophobic proteins, although the fluorescent enhancement is weaker than that
produced by lipids (Sackett and Wolff,
1987). The lipid nature of this material in D. dipsaci is
also suggested by its oily appearance on the surface of desiccated specimens
and the formation of droplets by this material upon the addition of water. The
material is easily lost after immersion of the nematodes in water (and hence
during staining procedures) but is closely applied to the surface of
desiccated nematodes. Remnants of this material adhere to a coverslip from
which desiccated nematodes have detached after the addition of water. This
leaves impressions of the cuticular annulations and lateral alae. A similar
phenomenon was observed by Bird (Bird,
1988) in Anguina agrostis L2 after labelling with
WGA–FITC and termed `cuticle prints'. Cuticle prints were observed in
some WGA–FITC-labelled samples of D. dipsaci but these were not
as clear as in samples labelled with Nile Red.
The lipid nature of this material was confirmed by ATR–IR
spectroscopy and GC analysis. In contrast to staining with Nile Red, which
could not be reliably obtained, detection by ATR–IR spectroscopy was
reliable and reproducible, and was capable of detecting very small quantities
of material (using less than 20 nematodes). The lipid material was easily
purified by dissolving the material retained on a glass surface in an organic
solvent, following the desiccation of nematodes and their subsequent removal
by gentle washing with water. The lipid was identified by GC analysis as being
a triglyceride and its main chain lengths were found to be saturated fatty
acids, mainly caprylic, palmitic and caproic acid. In fact, the infrared
spectra (Fig. 8) show the
dominant presence of CH2 characteristic absorptions (stretching and
bending modes), indicating an elevated content of saturated lipids. In
contrast to the surface lipid material, a total lipid analysis of D.
dipsaci (by TLC–GC) is dominated by unsaturated fatty acids;
particularly oleic acid, which constitutes 66.6% of the total lipid content
(Krusberg, 1967).
The nematode epicuticle consists of lipid and protein and the surface coat of the nematode cuticle contains carbohydrates, as is shown in the with-worms spectrum (Fig. 7A). The surface lipid described here contains neither proteins nor carbohydrates. Only alkyl and ester moieties are observed by ATR–IR spectroscopy (Fig. 8) and the presence of esterified fatty acids was confirmed by GC analysis (Table 1). It thus does not appear to be part of the surface coat or the epicuticle but to form a material that overlies the cuticle but is not part of it. This is also suggested by the appearance of the oily material seen coating the surface of the desiccated nematodes when observed by DIC and confocal microscopy. In nematodes dried on the surface of the diamond prism (Fig. 7) the surface lipid signal first appeared after 7 min of desiccation. However, it was probably secreted earlier than this since the signal would have at first been obscured by the signal from water inside and surrounding the nematodes.
There are a few reports in the literature that suggest the presence of
extracuticular lipid material on the surface of some species of nematodes.
Water loss from infective larvae of Nippostrongylus brasiliensis may
be reduced by a thin layer of lipid, derived from the skin and hairs of the
host (Lee, 1972). The
accessory layer covering the cuticle of some of the larval stages of
Trichinella spiralis and T. pseudospiralis contains lipid
(Lee, 2002). A variety of
plant-parasitic cyst nematodes, of the genus Heterodera, produce
cuticular exudates that contain lipids and which appear to originate from
secretions of the epidermis (Endo and Wyss,
1992). Additional material on the surface of the cuticle [a
`lipophilic coating' (Womersley et al.,
1998)] is produced by desiccated larvae of the plant parasites
Anguina amsinckiae (Womersley et
al., 1998) and Ditylenchus myceliophagus
(Perry, 1999). The nature of
this material, and its origin, is uncertain but it could be similar to the
lipid coating described here in D. dipsaci. Although such evidence is
lacking, lipid coatings could be widespread in nematodes and play important
roles in desiccation survival and other aspects of nematode biology.
The oily material formed on the surface of D. dipsaci during
desiccation is perhaps best referred to as an accessory layer. This term is
used to refer to layers associated with the nematode cuticle but which are not
part of the cuticle itself (Lee,
2002). The production of this surface lipid material occurs
rapidly upon exposure to desiccation and may account for the permeability
slump observed during desiccation. Since this material is washed away by
immersion in water it does not impede the uptake of water during the
rehydration of desiccated nematodes. This may explain the paradox that
cuticular permeability decreases during the permeability slump
(Wharton, 1996) and yet
desiccated nematodes are more permeable, upon immersion in water, than are
fully hydrated nematodes (Wharton et al.,
1988).
LIST OF ABBREVIATIONS
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Acknowledgments |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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