The anhydrobiotic potential and molecular phylogenetics of species and strains of Panagrolaimus (Nematoda, Panagrolaimidae)
Institute of Bioengineering and Agroecology, Department of Biology, National University of Ireland Maynooth, Maynooth, Co Kildare, Ireland
Author for correspondence (e-mail:
ann.burnell{at}nuim.ie)
Accepted 31 March 2005
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Summary |
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Key words: Panagrolaimus, nematode, anhydrobiosis, desiccation, trehalose, phylogeny, rDNA ITS, rDNA D3
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Introduction |
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Anhydrobiotic organisms have the ability to tolerate conditions ranging
from those in which there is no continuous aqueous phase within the cell
cytoplasm to those (at 15% of normal hydration levels) in which the
hydration shell of molecules is gradually lost
(Barrett, 1991;
Clegg, 1979
). Womersley
(1987
) recognised two broad
categories of anhydrobiotic nematodes: slow-dehydration and fast-dehydration
strategists. Fast-dehydration strategists are able to withstand rapid
dehydration, whereas slow-dehydration strategists are unable to survive
exposure to extreme desiccation unless they have first experienced a period of
preconditioning to moderate reductions in relative humidity. This
categorisation has general applicability to all anhydrobiotic organisms, with
most anhydrobiotic animals belonging to the slow-dehydration group.
Nematodes are aquatic animals and a moisture film is necessary for normal
nematode activity. However, nematodes occupy a great diversity of terrestrial
habitats, ranging from the relative protection of the lower soil profile to
highly exposed surfaces such as plant foliage. These habitats are often the
subject of either partial or severe drought and thus many nematodes have
become adapted to withstand desiccation. A large number of plant and animal
parasitic nematodes have anhydrobiotic eggs or infective juvenile stages
(Antoniou, 1989;
Perry, 1999
). Some nematodes
can survive immediate and prolonged exposure to rapid dehydration
(Nicholas, 1984
). These
include Panagrolaimus rigidus
(Ricci and Pagani, 1997
),
which inhabits extremely exposed environments on the surface of moss cushions.
Such fast dehydration strategist nematodes are probably pre-adapted at a
cellular level to survive desiccation. Nematodes that experience slow rates of
water loss have the time needed to induce the biochemical changes necessary to
survive in an anhydrobiotic state. The majority of anhydrobiotic nematodes are
believed to fall into this latter group of slow dehydration strategists
(Womersley, 1987
). Many
nematode species that are unable to enter into cryptobiotic anhydrobiosis can
nevertheless survive more modest water losses by entering a quiescent state in
which metabolic activity is reduced but not suspended
(Womersley, 1990
).
One of the best characterised metabolic changes that occurs during the
induction of anhydrobiosis in slow-dehydration strategists is the accumulation
of high concentrations of disaccharides; sucrose is the predominant sugar
accumulated in plants, while animals and yeast accumulate trehalose. Trehalose
accumulation has been associated with the successful induction of
anhydrobiosis in invertebrates such as brine shrimps
(Clegg, 1965), tardigrades
(Westh and Ramlov, 1991
)
larvae of the chironomid Polypedilum vanderplanki
(Watanabe et al., 2003
) and
nematodes such as Aphelenchus avenae
(Madin and Crowe, 1975
),
Anguina tritici and Ditylenchus dipsaci
(Womersley and Smith, 1981
).
Trehalose accumulation is believed to protect membranes and proteins from
desiccation damage by replacing structural water
(Carpenter et al., 1987
;
Crowe et al., 1984
). This
sugar may also contribute to the formation of an intracellular glass
(Crowe et al., 1998
), which is
believed to stabilise the cell contents and prevent damage associated with
water loss. While the accumulation of trehalose in A. avenae is
believed to be necessary for anhydrobiotic survival, it is not sufficient. A
further period of preconditioning following maximum trehalose accumulation is
needed before maximum survival is seen, suggesting that other changes must
also occur in A. avenae before it can successfully enter the
anhydrobiotic state (Higa and Womersley,
1993
; Browne et al.,
2004
). An extensive series of studies has revealed that
desiccation tolerance in plants requires the co-ordinated expression of a
large array of genes at the onset of desiccation. This leads to the
accumulation of various osmolytes and to the synthesis of a variety of
proteins, particularly hydrophilic proteins, as well as proteins involved in
cellular protection and repair and in the detoxification of reactive oxygen
species (Bartels and Sunkar,
2005
; Collett et al.,
2004
; Ingram and Bartels,
1996
). Similarly to anhydrobiotic plants, there is also a
dehydration-specific induction of hydrophilic protein genes in the
anhydrobiotic nematode A. avenae (Browne et al.,
2002
,
2004
). Data from
microorganisms (Potts, 1994
,
1996
) and brine shrimps
(Liang et al., 1997
) also
demonstrate that successful entry into anhydrobiosis requires a coordinated
set of biochemical and cellular adaptations in these organisms.
We have initiated a research programme aimed at gaining a deeper
understanding of the molecular biology of anhydrobiosis in nematodes. Our
initial studies on the molecular biology of anhydrobiosis in nematodes were
carried out with A. avenae, one of the best characterised
anhydrobiotic nematodes. A. avenae, a soil dwelling fungivore, was
originally selected by J. H. Crowe as an ideal model in which to study the
biochemical changes associated with anhydrobiosis because it can be cultured
in large quantities on autoclaved wheat seed inoculated with the fungus
Rhizoctonia solani (Evans,
1970). Many anhydrobiotic soil nematodes are bacterial feeders and
such nematodes can be cultured in the laboratory using methods developed for
the model nematode Caenorhabditis elegans
(Brenner, 1974
). Members of
the genus Panagrolaimus are bacterial feeding nematodes that occupy a
diversity of niches ranging from Antarctic, temperate and semi-arid soils to
terrestrial mosses, and includes both fast dehydration and slow dehydration
anhydrobiote strategists (Aroian et al.,
1993
; Ricci and Pagani,
1997
; Wharton and Barclay,
1993
). We have assembled a collection of Panagrolaimus
species and strains and have investigated their anhydrobiotic phenotypes. Our
data show that these strains fall into three broad categories: fast and slow
dehydration strategists and desiccation sensitive strains; the majority being
slow desiccation strategists. Using this panel of nematodes we investigated
the effect of preincubation at high relative humidity (RH), on the
accumulation of trehalose and on the nematodes' anhydrobiotic potential. Our
data indicate that there is a strong correlation between trehalose induction
and anhydrobiotic survival in Panagrolaimus. Indeed the high
trehalose levels observed in fully hydrated P. superbus (10% dry
mass) suggest that constitutive expression of high levels of trehalose
pre-adapt this fast dehydration strategist to combat desiccation. Phylogenetic
analyses were carried out to investigate whether the observed anhydrobiotic
phenotypes were the result of convergent evolution or represent a single
phylogenetic lineage. Our analyses show that the strongly anhydrobiotic
strains of Panagrolaimus form a single phylogenetic lineage which is
separate from the phylogenetically divergent weakly anhydrobiotic strains.
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Materials and methods |
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Axenization of nematodes from plate cultures
Gravid worms were rinsed from the agar surface with S Basal buffer. The
worms were centrifuged (2000 g for 5 min) at room temperature
and washed three times with S Basal buffer in a final volume of 30 ml. Worms
were allowed to settle and then treated for 4 min at room temperature with 8
ml of 1:3 NaOCl solution and 2 ml freshly prepared 10 mol l1
NaOH, during which time the samples were vigorously shaken to assist the
disintegration of the worms and release of eggs. Released eggs and embryos
were washed three times with S Basal buffer, collected by centrifugation (2000
g), and added directly to liquid culture
(Lewis and Fleming, 1995). A
sample of the embryos was transferred to a Petri dish containing S Basal
buffer and incubated at room temperature overnight. The following day, the
embryos were examined under the microscope to ensure that they developed into
L1 larvae and that the axenization procedure had been successful.
Control of relative humidity
Relative humidity (RH) was controlled by the use of saturated salt
solutions as described by Solomon
(1951) and Winston and Bates
(1960
). Saturated salt
solutions (200 ml) were maintained at 20°C in the dark in desiccation
chambers for 3 d prior to the addition of nematodes in order to allow them to
equilibrate. A hygrometer was used to confirm that the correct relative
humidity had been established. The required relative humidities were
maintained as follows: 100% RH, distilled water vapour; 98% RH, potassium
dichromate (K2Cr2O7); 95% RH, sodium hydrogen
phosphate (Na2HPO4.7H2O); 90% RH, magnesium
sulphate (MgSO4.7H2O) and
10% RH, freshly activated
silica gel. All experiments were performed at 20°C using mixed stage
populations.
Desiccation tolerance
A 1 ml suspension of nematodes in S Basal buffer (concentration 2000
nematodes ml1) was vacuum filtered onto a 2.5 cm
Supor®-450 filter (0.45 µm, Gelman Science East Hills, New York, USA)
using a Sartorius funnel (25 mm glass vacuum filter holder with a 30 ml funnel
(cat. no. 5m16315, Sartorius AG, Goettingen, Germany) and a vacuum flask
attached to a pump. The filters were then transferred to 3 cm Petri dishes
without lids and placed in a 2.5 l desiccation chamber containing
approximately 200 ml of the appropriate saturated salt solution required to
maintain the desired relative humidity. The nematode dishes were then
transferred to sealed plastic boxes containing freshly activated silica gel
for 48 h. Following this drying period the nematodes were prehydrated at 100%
RH over distilled water for 24 h before immersion in S Basal buffer.
Percentage survival was assessed by microscopic observation of motility and
response to probing 24 h after rehydration. Five replicates were prepared for
each experiment. In a preliminary experiment, Panagrolaimus sp. WS94
nematodes were exposed to various relative humidities (100%, 98%, 95% and 90%
RH) for 48 h before exposure for 48 h to freshly activated silica gel (<10%
RH). Nematodes pre-treated at 98% RH had the highest survival
(KruskalWallis test, P<0.001; data not shown). Thus 98% RH
was used as standard in all subsequent preconditioning experiments.
Measurement of length
Nematodes were vacuum filtered onto Supor®-450 membrane filters as
described above. The control group was maintained at 98% RH for 96 h,
rehydrated for 24 h in S Basal buffer and then allowed to migrate through two
layers of Kleenex® tissue paper in a wide meshed sieve in order to exclude
dead nematodes. The experimental group was treated as follows: 98% RH for 48
h, <10% RH (silica gel) for 24 h, 100% RH for 24 h, followed by rehydration
for 24 h in S Basal buffer, after which the animals were allowed to migrate
though tissue to exclude dead nematodes. The recovered nematodes were heat
killed and straightened by plunging a 0.5 mlsuspension of nematodes in an
Eppendorf tube into a water bath at 60°C. Animals were then fixed in TAF
(triethanolamine, formalin, distilled water, 2:7:91), and measured using a
micrometer eyepiece, previously calibrated using a micrometer slide
(Perry, 1976).
Carbohydrate extraction
For biochemical analyses, a 1 ml suspension containing 10 000 nematodes per
ml was filtered onto a 2.5 cm filter and placed in a 3 cm Petri dish. The
Petri dishes containing the nematodes were exposed to 98% RH for the
appropriate time period (096 h) and then transferred to silica gel for
24 h. The filter papers containing the nematode samples were placed into
individual 1.5 ml Eppendorf tubes and stored at 80°C until
carbohydrates were extracted. Three replicates were prepared for each
experiment and fresh untreated nematodes were used as controls.
The samples (nematodes and filter) were suspended in 1 ml of 30% ethanol for 30 min. Then 0.75 ml of 95% ethanol was added along with 0.75 g of glass beads (<106 µm; cat. no. G 8893; Sigma-Aldrich, St Louis, MO, USA). The samples were homogenised (Mini-beadbeater, Biospec Products, Bartlesville, OK, USA) on ice for six 30 s pulses at high speed. 83% ethanol (1 ml) was then added and the sample was centrifuged at 10 000 g for 30 min at 4°C. The supernatant was decanted into 2x 1.5 ml Eppendorf tubes, which were placed in a Speedivac and dried for approximately 12 h at room temperature. The samples were then resuspended by the addition of 50 µl of deionised water to each tube. The samples were then recombined and filtered using 0.22 µm centrifuge filter tubes (Spin-X, Costar, Baar, Germany). These samples were then used for low molecular mass carbohydrate analysis. Dry mass was determined by weighing identical control samples of nematodes (minus the mass of the dry filter paper), which had been incubated over silica gel in a small airtight plastic box at 70°C for 3 days.
Analysis of carbohydrate content
Carbohydrates were analysed using high performance liquid chromatography
(HPLC). The HPLC instrument (Spectra Physics; model SP 8800, Mountain View,
CA, USA) was attached to a Shodex Rl SE-61 refractive index detector. Samples
were run through a hypersil hyperREZ carbohydrate H+ column (300 mm
x 7.8 mm; Shandon, Cheshire, UK) which separated the carbohydrates
(range 1, attenuation 64, running time 15 min) using 2.5 mmol
l1 H2SO4 as the mobile phase (flow
rate 1 ml min1). Carbohydrates were identified and
quantified by running serial dilutions of known carbohydrate standards through
the column.
Scanning electron microscopy
Scanning electron microscopy (SEM) was used to investigate the morphology
of desiccated Panagrolaimus sp. The nematodes were washed repeatedly
using S Basal buffer and filtered on a Supor®-450 (Pall Corporation,
Portsmouth, Hampshire, UK) membrane filter (100 nematodes per filter) using
vacuum filtration. The nematodes were then preconditioned at 98% RH for 48 h,
followed by exposure for 48 h to freshly activated silica gel. Specimens
treated in this way did not require fixation and were mounted on aluminium
stubs using double-sided Sellotape®, coated with gold using a Polaron
SC500 sputter coater (Polaron plc, Watford, UK) for 3 min at 20 mV and viewed
at 20 kV using a Hitachi S4300 field emission scanning electron microscope
(Hitachi Ltd, Tokyo, Japan).
Phylogenetic analyses
Gemomic DNA was prepared using standard phenol chloroform extraction
described by Maniatis et al.
(1982). PCR amplification of
the D3 expansion region of the 28S rDNA subunit was achieved using the D3A and
D3B primers (Nunn et al.,
1996
) and each strain of Panagrolaimus yielded a single
PCR product of approximately 320 bp. PCR cycling conditions were: 94°C, 3
min; 94°C, 1 min; 52°C, 1 min; 72°C, 1 min; 35 cycles followed by
72°C, 10 min. Each PCR amplification was performed in 25 µl volumes
containing 0.25 U of Hi-Fidelity Platinum Taq polymerase (Invitrogen,
Carlsbad, CA, USA; 2.5 µl 10x High Fidelity PCR Buffer (Invitrogen);
1.5 µl 50 mmol l1 MgSO4; 1 µl 10 mmol
l1 dNTP solution; 10 pmol of each primer; 1001500 ng
of genomic DNA (which was optimised for each DNA sample) and sterile distilled
H2O to 25 µl. All reactions were analysed by agarose gel
electrophoresis.
A fragment of approximately 1.1 kb comprising the rDNA ITS1, 5.8S rDNA and
rDNA ITS2 was amplified from Panagrolaimus PS1159 using the rDNA1 and
rDNA2 primers (Vrain et al.,
1992). This DNA fragment was cloned and sequenced as described
below. The resulting DNA sequence was aligned with that of Caenorhabditis
elegans rDNA (accession number X03680.1) to identify conserved regions
within the 5.8S rDNA. This information was used to design the PCR primers
Pana5.8R (5'-GGTAAGTAACGCAGCAAGC-3') and Pana5.8F
(5'-GCTTGCTGCGTTACTTACC-3'). Pana5.8R was used with the rDNA1
primer to amplify the rDNA ITS1 region (
690 bp) and Pana5.8F rDNA was used
with the ITS2 to amplify the rDNA ITS2 region (
590 bp) from all
Panagrolaimus strains. The PCR reagents and their concentrations were
as described above. The PCR cycling conditions were: 94°C, 3 min;
94°C, 1 min; 50°C, 2 min; 72°C, 2 min; 35 cycles followed by
72°C, 10 min.
PCR products were excised from the gel and gel purified using the Montage DNA Gel extraction Kit (Millipore, Billerica, MD, USA). The purified PCR products were cloned into the pCR2.1 TOPO vector using the TOPO-TA Cloning kit (Invitrogen). The insert sizes of the recombinant clones were confirmed by colony PCR using M13 forward and reverse primers. Selected clones were sequenced (AGOWA, Berlin, Germany). These sequences have been deposited in GenBank with the following accession numbers: rDNA D3 sequences, AY878376 to AY878386; rDNA ITS1 sequences, AY878387 to AY878397 and rDNA ITS2 sequences, AY878398 to AY878408.
Alignments of the rDNA D3, ITS1 and ITS2 fragments from the Panagrolaimus strains were made using ClustalW 1.8 at the University of Aberdeen (http://www.abdn.ac.uk/~mbi094/SEQANAL.htm#DNA). The rDNA D3, ITS1 and ITS2 sequences from C. elegans were included in the alignment and used as an outgroup when constructing phylogenetic trees. For pairwise alignments the gap opening penalty was set to 10.0 and the gap extension penalty was set to 6. For multiple alignments the gap opening penalty was set to 12.0 and the gap extension penalty was set to 5. The alignments were then edited manually and the three sequences were concatenated into one sequence of approximately 1.4 kb for phylogenetic analysis.
To ensure that there was phylogenetic signal within the alignment a
permutation tail probability (PTP) test
(Archie, 1989) as implemented
in PAUP 4.0b10 (Swofford,
2003
) was performed. Phylogenetic analyses were performed using
the maximum parsimony (MP), maximum likelihood (ML), Bayesian and distance
based criteria. Distance based trees were constructed using the
neighbor-joining (NJ) program (Saitou and
Nei, 1987
), with distances corrected by Kimura's two parameter
model (Kimura, 1980
). MP, ML
and distance based analyses were performed in PAUP 4.0b10. MP trees were
created using the random stepwise addition option of the heuristic search for
1000 replicates with tree bisection-reconnection branch swapping. Characters
were treated as unordered and unweighted. For the ML analysis the optimal
model of sequence substitution was found by comparing substitution model
likelihood scores using Modeltest 3.04
(Posada and Crandall, 1998
).
The optimal model of substitution was found to be HKY
(Hasegawa et al., 1985
). The
statistical robustness of the MP, NJ and ML tree topologies were assessed
using the bootstrap resampling technique
(Felsenstein, 1985
), with 1000
replicates for the MP and NJ trees and 100 replicates for ML tree.
A phylogeny was also reconstructed using the Bayesian framework as
implemented in MR BAYES 3.0B4 (Huelsenbeck
and Ronquist, 2001). Model parameters were estimated during the
run and four Markov chains were used. The Markov chain Monte Carlo was set to
ten million generations and the chain was sampled every 100th generation. The
trees generated after the initial burn in period of two hundred thousand
generations were summarised using the majority rule consensus method
implemented in PAUP 4.0b10. The first 500 trees were discarded and the
remaining trees were combined into a single file which was imported into
PAUP* 4.0b10 to compute the 50% majority rule consensus tree. The
statistical robustness of branch support was determined by calculating
Bayesian posterior probabilities (BPPs) using the sumt command of MR
BAYES 3.0B4. Two tests: the Shimodaira Hasegawa test
(Shimodaira and Hasegawa,
1999
) and the approximately unbiased test
(Shimodaira, 2002
) were used
to compare the robustness of phylogenetic trees inferred by the four
phylogenetic reconstruction methods.
Statistical analyses
All statistical analyses were carried out using MinitabTM version 13.1
(Minitab Inc., State College, PA, USA). Simple linear regression was carried
out alongside fitted line plots and correlation was considered significant at
P<0.05 in all cases. Analysis of variance (ANOVA) was carried out
with confidence intervals at the 95% level to test for differences between
means. Means were considered to be significantly different at
P<0.05. The 2-tailed MannWhitney U-test was used
as a non-parametric method to test for difference between population means.
The null hypothesis that population means are all equal was tested at the
P=0.05 level of significance. The KruskalWallis method was
used as a non-parametric test of the equality of medians. The null hypothesis
that medians are all equal was tested at the P=0.05 level of
significance.
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Results |
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Following 48 h preincubation the survival capacity of three of the four poor desiccators (P. detritophagus, P. rigidus AF40 and Panagrolaimus sp. BW287) had improved significantly and this trend was maintained for these strains when preconditioned at 98% RH for 72 h and 96 h. Nevertheless, the maximal survival for these strains was less than 50%, ranging from 23% P. rigidus AF40 to 48% Panagrolaimus sp. BW287. The survival capacity of the poor desiccator Panagrolaimus sp. PS1732 did not improve significantly upon preincubation at 98% RH at any of the time points tested over a 96 h period, its maximal survival value never exceeding 4%.
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Scanning electron microscopy of desiccated Panagrolaimus
When exposed directly to silica gel, individuals of P. superbus, a
fast dehydration strategist nematode, displayed the typical coiled morphology
of anhydrobiotic nematodes (Fig.
5A). This nematode does not appear shrunken or distorted and seems
structurally intact after exposure to activated silica gel for 48 h without
preconditioning. The slow dehydration strategist nematode
Panagrolaimus sp. PS1159 (Fig.
5B) was exposed to 72 h preconditioning at 98% RH prior to
exposure to silica gel for 48 h. This individual also appears to be
structurally intact. There are, however, small areas where the cuticle has
fused to the filter surface. Panagrolaimus sp. BW287 (whose maximal
survival following 72 h preconditioning at 98% RH is 49%) shows moderate
shrinkage and, in addition, large areas of its cuticle have fused to the
filter surface (Fig. 5C).
Panagrolaimus sp. PS1732 (Fig.
5D) was also exposed to 72 h preconditioning at 98% RH prior to
exposure to silica gel for 48 h. This nematode shows extreme structural
collapse and severe cuticle and membrane damage and leakage of body fluids,
despite being coiled. This poor desiccator has a very limited ability to
synthesise trehalose upon preincubation at 98% RH
(Fig. 3) and does not show an
increase in desiccation survival following preincubation at 98% RH
(Fig. 1).
|
Phylogenetic analyses
Two competing topologies were consistently inferred by the four
phylogenetic reconstruction methods applied to the data
(Fig. 6). Both topologies were
in agreement regarding the branching order of C. elegans, P.
detritophagus, P. rigidus AF40 and Panagrolaimus spp. BW287 and
PS1732. The MP and NJ analysis grouped clade 1 (Panagrolaimus spp.
WS94 and PS443) and clade 2 (P. davidi, Panagrolaimus spp. PS1159 and
PS1579) as sister taxa to the exclusion of P. superbus and P.
rigidus AF36 (clade 3) with relatively high bootstrap support
(Fig. 6, Tree A). However, the
ML and Bayesian methods inferred clades 1 and 3 as sister taxa
(Fig. 6, Tree B). Branch
supports for this tree derived from Bayesian posterior probabilities (BPP)
were found to be highly significant with all internal nodes having support
values equal to 1. BPPs have been shown to be excessively liberal
(Suzuki et al., 2002). Because
of this, only the bootstrap supports derived from the ML analysis were
considered. ML bootstrap support (56%) is weak for the grouping of clades 1
and 3 as sister taxa in Tree B.
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Discussion |
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The ten strains of Panagrolaimus investigated in this study
display great variability in their anhydrobiotic ability. The 93% survival
observed for P. superbus following direct exposure to activated
silica gel for 48 h is remarkable and provides and excellent example of a fast
dehydration strategist. This nematode was isolated from the mosses making up a
gull's nest in a small lava cavity in a nunatak on Surtsey island, Iceland
(Bostrom, 1988;
Sohlenius, 1988
).
Surprisingly, we have found that the P. detritophagus strain, which
was also isolated at the same site from the same nest material has a very
limited ability to survive rapid desiccation and is a slow desiccation
strategist. Womersley (1987
)
distinguished between slow-dehydration and fast-dehydration strategist
nematodes on the basis that slow-dehydration strategists could not survive
direct exposure to harsh desiccation. Examples of such slow dehydration
strategists are A. avenae (Crowe
and Madin, 1975
), Rotylenchus reniformis
(Womersley and Ching, 1989
)
and P. davidi (Wharton and
Barclay, 1993
). All the Panagrolaimus strains
investigated in this study show some survival upon direct exposure to silica
gel, so they could all be described as fast dehydration strategists
sensu Womersley. However, with the exception of the fast dehydration
strategist P. superbus and the poor desiccator Panagrolaimus
sp. PS1732, all of the other strains show substantial improvements in their
desiccation tolerance to low RH following preincubation at 98% RH. Thus it
appears that within the genus Panagrolaimus there is a continuum of
strains from those that are unable to survive exposure to low RH without prior
preconditioning at high RH such as P. davidi
(Wharton and Barclay, 1993
),
through strains that have limited ability to survive rapid desiccation but
whose anhydrobiotic ability improves upon preconditioning (e.g. P.
detritophagus, P. sp. PS1159), to strains such as P. superbus
that can survive immediate exposure to severe desiccation.
Trehalose accumulation has been associated with the successful induction of
anhydrobiosis in invertebrates such as brine shrimps, tardigrades, larvae of
the chironomid Polypedilum vanderplanki and the nematodes A.
avenae (Madin and Crowe,
1975), Anguina tritici and Ditylenchus dipsaci
(Womersley and Smith, 1981
).
However, data on trehalose accumulation in response to desiccation in other
anhydrobiotic nematodes are lacking (reviewed by Behm, 1977). While the
accumulation of trehalose in A. avenae is believed to be necessary
for anhydrobiotic survival, it is not sufficient. A further period of
preconditioning following maximum trehalose accumulation is needed before
maximum survival is seen, suggesting that other changes must also occur in
A. avenae before it can successfully enter the anhydrobiotic state
(Higa and Womersley, 1993
;
Browne et al., 2004
). Data,
especially from plants (reviewed by Ingram
and Bartels, 1996
; Hoekstra et
al., 2001
; Ramanjulu and
Bartels, 2002
) and microorganisms (Potts,
1994
,
1996
), but also from brine
shrimps (Liang et al., 1997
)
and nematodes (Browne et al.,
2004
) demonstrate that successful entry into anhydrobiosis
requires a coordinated set of biochemical and cellular adaptations. Such
studies show that the accumulation of trehalose or other disaccharides
represents one possible anhydrobiotic mechanism, but alternative biochemical
adaptations may also be utilised by organisms to achieve desiccation tolerance
(reviewed by Crowe et al.,
2001
; Oliver et al.,
2001
).
In fully hydrated A. avenae trehalose levels are 1.52.0%
dry mass, but these levels rise to a maximum of 1112% dry mass
following 72 h preconditioning at high RH
(Madin and Crowe, 1975;
Browne et al., 2004
). A.
avenae is unable to survive immediate exposure to activated silica gel,
however, all of the Panagrolaimus strains tested in this study (even
the poor desiccator Panagrolaimus. sp. PS1732) showed some ability to
survive direct exposure to silica gel. The mean concentration of trehalose in
the 10 Panagrolaimus strains when fully hydrated was 4.5% dry mass
more than twice the levels recorded in undesiccated A.
avenae. We found that the correlation between preconditioning at high RH
and trehalose accumulation was strongest in the slow dehydration strategist
strains of Panagrolaimus, but trehalose accumulation also occurred in
the fast dehydration strategist P. superbus following preincubation
although no further improvement on the initial survival (93%) of this strain
was detected. The two poorest desiccator strains show only moderate levels of
trehalose accumulation (which increased by
1.5%). The high trehalose
levels observed in fully hydrated P. superbus (10% dry mass) suggest
that constitutive expression of high levels of trehalose pre-adapt this fast
dehydration strategist to combat desiccation.
Overall, these data indicate that trehalose accumulation is required for successful induction of anhydrobiosis in Panagrolaimus. However it is also apparent that Panagrolaimus strains that accumulate similar levels of trehalose during preincubation (e.g. P. detritophagus, Panagrolaimus sp. BW287 and Panagrolaimus sp. PS1732) show different levels of anhydrobiotic survival. This suggests that the anhydrobiotic capacity resulting from a given level of trehalose accumulation in Panagrolaimus may be strain specific and that other factors in addition to trehalose accumulation are also involved in anhydrobiotic survival in this genus. The behavioural responses of the strains of Panagrolaimus in response to desiccation appear to be more conserved than the production of trehalose. All the strains observed, regardless of survival rates, undertook both coiling and clumping, which have the effect of reducing surface area and slowing the rate of water loss during desiccation. It can be seen, however, that the ability to coil is not sufficient to enable nematodes to survive extreme desiccation especially in the absence of sufficient levels of trehalose, as in the case of Panagrolaimus sp. PS1732.
Our phylogenetic analyses indicate that the strongly anhdyrobiotic strains
of Panagrolaimus form a single phylogenetic lineage, which is
separate from the four poor desiccation strains. It is also clear that these
poor desiccators are phylogenetically divergent from each other. Clade 3
contains P. superbus a fast desiccation strategist, and P.
rigidus AF36 whose desiccation tolerance improves upon preconditioning,
but whose capacity for fast desiccation tolerance results in over 30% survival
without preconditioning. Whether this fast desiccation strategist clade
evolved from within the slow desiccation clades, as indicated by Tree B, or
whether it represents a distinct sister taxon to the slow desiccation
strategists in clades 1 and 2 (Tree A) cannot be elucidated from our data. The
phylogenetic trees presented here are derived from alignments of the rDNA ITS
and D3 sequences. These fast evolving regions have been found to provide
phylogenetically informative data for closely related genera and species in a
range of invertebrates, including nematodes
(Adams et al., 1998;
Al-Banna et al., 1997
;
Carta et al., 2001
; Livaitis
et al., 1994; Nguyen et al.,
2001
). However, it will be necessary to find other more
polymorphic molecular markers to fully resolve the phylogenetic relationships
between Panagrolaimus clades 1-3. Few species within the genus
Panagrolaimus possess diagnostic morphological characters
(Andrássy, 1984
;
Bostrom, 1995
;
Williams, 1987
) and species
diagnosis of Panagrolaimus is difficult, being based predominantly on
morphometric data. Thus many Panagrolaimus strains remain unassigned
to species level. The positioning of the two strains of P. rigidus in
our phylogram suggests that the taxonomic designation of these strains needs
to be reevaluated.
Oliver et al. (2000) have
analysed the evidence for differing mechanisms of vegetative desiccation
tolerance (i.e. anhydrobiosis) in land plants. They hypothesise that
vegetative desiccation tolerance was primitively present in land plants, but
was lost in the evolution of the tracheophytes and subsequently re-evolved at
least eight times in the angiosperms. They postulate that this re-evolution of
desiccation tolerance in angiosperm `resurrection plants' was achieved by the
expression in vegetative tissues of genes involved in seed development (and
seed desiccation tolerance). Thus each time the desiccation tolerant phenotype
re-evolved in an angiosperm lineage, the response patterns and biochemical
adaptations differed. Some free-living nematodes can undergo anhydrobiosis at
all stages of their life cycle, and in addition, a large number of plant and
animal parasitic nematodes have anhydrobiotic egg, cyst or infective juvenile
stages. In a phylogenetic analysis based on rDNA small subunit sequences
Blaxter et al. (1998, 2000
)
recognise six clades within the phylum Nematoda, referred to as clades I to V,
the sixth clade, the Chromadorida, is considered by these authors as basal to
clades III, IV and V. Clade IV contains the best described anhydrobiotic
nematodes, including Panagrolaimus and Aphelencus and the
plant parasitic order Tylenchida (which frequently has anhydrobiotic encysted
infective stages, e.g. Globodera, Meliodogyne). Clade III contains
four animal parasitic orders, one of which, the Ascaridida, contains members
with anhydrobiotic encysted infective stages. There has been no systematic
investigation of anhydrobiotic phenotypes across the phylum Nematoda, but all
the remaining clades in the phylogeny of Blaxter et al.
(2000
) contain some
terrestrial and parasitic forms (either plant or animal parasites), thus it is
possible that nematodes with anhydrobiotic capabilities may also occur in
these taxa. The nematode families contained in clade IV are phylogenetically
divergent (Blaxter et al.,
2000
) and in addition to containing many anhydrobiotic taxa, this
clade also has many desiccation sensitive nematode species. A more detailed
phylogenetic investigation will be required to determine whether the
anhydrobiotic members of clade IV are derived from an anhydrobiotic ancestor,
or whether they independently acquired or re-evolved an anhydrobiotic
capability. In the plant parasitic order Tylinchida of clade IV, taxa with the
best developed anhydrobiotic capabilities are not closest relatives,
suggesting independent evolutionary modifications
(Baldwin et al., 2004
).
However, it is noteworthy that all the anhydrobiotic taxa that have been
investigated to date in clade IV accumulate trehalose in response to
desiccation.
In comparison with the large research effort expended in investigating anhydrobiosis and desiccation tolerance in land plants, research in nematodes has been limited. Our data indicate that Panagrolaimus has the potential to be an excellent model system for the investigation of molecular aspects of nematode anhydrobiosis. This genus contains closely related nematodes that possess a diverse range of anhydrobiotic abilities. This should facilitate researchers in correlating gene expression data with anhydrobiotic phenotypes and thereby help in the elucidation of the key molecular strategies employed by anhydrobiotic nematodes.
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