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BMC Genomics

BioMed Central

Open Access

Research article

Desiccation survival in an Antarctic nematode: molecular analysis
using expressed sequenced tags
Bishwo N Adhikari*1,3, Diana H Wall2 and Byron J Adams1,3
Address: 1Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA, 2Department of Biology and Natural
Resource Ecology Laboratory, Colorado State University, Fort Collins, CO, USA and 3Department of Biology and Evolutionary Ecology
Laboratories, Brigham Young University, Provo, UT, USA
Email: Bishwo N Adhikari* - adhikaribn@hotmail.com; Diana H Wall - diana@nrel.colostate.edu; Byron J Adams - bjadams@byu.edu
* Corresponding author

Published: 9 February 2009
BMC Genomics 2009, 10:69

doi:10.1186/1471-2164-10-69

Received: 15 September 2008
Accepted: 9 February 2009

This article is available from: http://www.biomedcentral.com/1471-2164/10/69
© 2009 Adhikari et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Nematodes are the dominant soil animals in Antarctic Dry Valleys and are capable
of surviving desiccation and freezing in an anhydrobiotic state. Genes induced by desiccation stress
have been successfully enumerated in nematodes; however we have little knowledge of gene
regulation by Antarctic nematodes which can survive multiple environmental stresses. To address
this problem we investigated the genetic responses of a nematode species, Plectus murrayi, that is
capable of tolerating Antarctic environmental extremes, in particular desiccation and freezing. In
this study, we provide the first insight into the desiccation induced transcriptome of an Antarctic
nematode through cDNA library construction and suppressive subtractive hybridization.
Results: We obtained 2,486 expressed sequence tags (ESTs) from 2,586 clones derived from the
cDNA library of desiccated P. murrayi. The 2,486 ESTs formed 1,387 putative unique transcripts of
which 523 (38%) had matches in the model-nematode Caenorhabditis elegans, 107 (7%) in
nematodes other than C. elegans, 153 (11%) in non-nematode organisms and 605 (44%) had no
significant match to any sequences in the current databases. The 1,387 unique transcripts were
functionally classified by using Gene Ontology (GO) hierarchy and the Kyoto Encyclopedia of
Genes and Genomes (KEGG) database. The results indicate that the transcriptome contains a
group of transcripts from diverse functional areas. The subtractive library of desiccated nematodes
showed 80 transcripts differentially expressed during desiccation stress, of which 28% were
metabolism related, 19% were involved in environmental information processing, 28% involved in
genetic information processing and 21% were novel transcripts. Expression profiling of 14 selected
genes by quantitative Real-time PCR showed 9 genes significantly up-regulated, 3 down-regulated
and 2 continuously expressed in response to desiccation.
Conclusion: The establishment of a desiccation EST collection for Plectus murrayi, a useful model
in assessing the structural, physiological, biochemical and genetic aspects of multiple stress
tolerance, is an important step in understanding the genome level response of this nematode to
desiccation stress. The type of transcript analysis performed in this study sets the foundation for
more detailed functional and genome level analyses of the genes involved in desiccation tolerance
in nematodes.

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Background
The Dry Valleys of Antarctica are one of the most extreme
terrestrial environments on Earth [1]. Soils in this cold
desert ecosystem are subjected to freezing temperatures,
desiccation and salt accumulation that affect biological
water availability [2,3]. Soil communities in Antarctic Dry
Valleys are simple; primary production is largely limited
to algae, and fauna are almost exclusively microbial grazers (mostly protozoa, rotifers, tardigrades and nematodes
[4]). Nematodes are the dominant soil animals, present in
65% of the 415 soils sampled by Wall Freckman & Virginia [3] across four McMurdo Dry Valleys (MCM). Nematodes have been isolated from soil in an inactive coiled
state called anhydrobiosis [5]. Anhydrobiosis is a survival
strategy employed by nematodes, rotifers, and tardigrades
in response to desiccation [6]. Nematodes in anhydrobiosis lose 95–99% of their body water content and can cease
metabolic activity at any stage in their life cycle [7]. While
in an anhydrobiotic state, nematodes are capable of surviving desiccation [8] as well as extreme cold [9]. Though
Antarctic ecosystems are simple and have low species
diversity compared to temperate ecosystems, nematodes
are the most widely distributed and biologically diverse
invertebrates in the Dry Valleys [5], with four species; Scottnema lindsayae, Eudorylaimus antarcticus, Plectus antarcticus, and Geomonhystera antarcticola [10]. It has been
suggested that specimens identified as P. antarcticus de
Man 1904 in MCM are P. murrayi [11,12] and we accept
this nomenclature for the present paper.
Plectus murrayi, a bacteria feeding nematode [13], inhabits
both semi-aquatic and terrestrial biotopes in the Dry Valleys, but is also reported from other parts of the Antarctica
[11]. Similar to S. lindsaye, another nematode endemic to
the Southern continent [14], P. murrayi has a multiple
year life cycle [15]. The distribution of these nematodes in
Antarctica is dependent on organic carbon and soil moisture [16] with high abundance in stream sediments [5]. P.
murrayi from the MCM are freeze tolerant, and can tolerate repeated freeze-thaw cycles in the laboratory (data not
shown). Although adapted to the extreme desiccation and
freezing encountered in its habitat [12], the biology and
environmental tolerance of this nematode has not been
well studied.
Despite recent work on behavioral, biochemical and
molecular stress response mechanisms [17-19] the molecular mechanisms governing anhydrobiosis in nematodes
are not fully understood. Anhydrobiosis in nematodes is
reported to involve the biosynthesis of low molecular
weight carbohydrates, proteins and glycerol [20,21].
Recent research suggests anhydrobiotes synthesize many
other compounds (primarily proteins) that are essential
for survival [22-24]. Studies on these desiccation responsive compounds have resulted in the identification of
many genes that play important roles in stress acclimation

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and survival. These responses include up-regulation of
transcriptional regulators, molecular chaperones, antioxidants, hydrophilic proteins, and proteins involved in cell
cycle regulation [19,25-27]. The anhydrobiotic nematode
Aphelenchus avenae synthesizes large amounts of trehalose
in response to desiccation [28]. However, it has become
clear that such sugars are not sufficient for anhydrobiosis
[29] and, indeed, that some anhydrobiotic organisms
seem not to use them [30]. As an effort to identify other
adaptations required for anhydrobiosis Goyal et al. [31]
characterised genes in the nematode A. avenae that
requires a period of preconditioning to enter anhydrobiosis. During this preconditioning period, several genes,
including trehalose synthase [31], hydrophilins (highly
hydrophilic proteins), anhydrin, and a polypeptide, AavLEA-1, related to plant Group 3 late embryogenesis abundant (LEA) proteins were induced [23,19]. Although similar gene classes were found to be associated with
desiccation stress in many nematodes, none of the ESTs or
proteins detected in these studies were encoded by the
same gene [26] and their expression level was quite variable [32]. To understand such molecular mechanisms activated during anhydrobiosis, a condition induced by slow
dehydration, we identified gene expression patterns by
gradually desiccating nematodes at relative humidity
(RH) values reflective of the Antarctic environment.
To understand the mechanisms of desiccation survival we
have initiated a genomic level analysis of gene expression
during anhydrobiosis of P. murrayi. The first step in this
process was to establish an EST collection that is representative of the desiccation induced transcripts and to
identify the transcripts differentially expressed during desiccation stress. Here we present bioinformatics and molecular analysis of 2,486 ESTs from the gradually desiccated
and anhydrobiotically induced nematode P. murrayi and
80 transcripts differentially expressed during the anhydrobiotic process. The bioinformatics approaches include
EST cluster analyses, transcript abundancy estimations,
and functional classifications based on Inter-Pro
domains, Gene Ontology hierarchy, and KEGG biochemical classifications. The genetic information derived from
P. murrayi informs the characterization of genes responding to desiccation stress, and is expected to further our
understanding of the potential genetic determinants of
desiccation tolerance in nematodes and perhaps other
metazoans.

Results
Sequencing and assembly of ESTs
A directionally cloned cDNA library of desiccated nematodes was constructed and a total of 2,586 of clones were
subjected to single pass sequencing from their 5' ends.
Trimming of vector sequences, poly A/T tails, low quality,
adaptor, and contaminating sequences provided a data set
of 2,486 high quality (hq) ESTs with a minimum length
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BMC Genomics 2009, 10:69

of 100 base pairs (bp) (Table 1). Among 2,486 hq ESTs,
1,423 were assembled into a total of 324 contigs, and the
remaining 1,063 ESTs were classified as singletons, suggesting a combined total of 1,387 putative unique transcripts (Table 1). The number of ESTs in the 324 contigs
varied from 2 to 37, with the highest number of contigs
being two ESTs, followed by more than 3 ESTs and the
least number with more than 21 ESTs (Fig. 1). These hq
ESTs ranged from 90–1125 bp with average lengths of 545
± 156 bp. The average length of the contigs was higher
than for singletons. The average GC content was higher in
P. murrayi (44%) than in C. elegans (36%). All sequences
have been deposited in the dbEST division of DDBJ/
EMBL/GenBank under accession numbers [GenBank:
FG618921] – [GenBank: FG621295], [GenBank:
FG647736] – [GenBank: FG647869].
Comparison against public nematode ESTs
We used the 1,387 unique sequences to search a nonredundant protein data base using BLASTX [33,34] (Table
1) and the Wormpep 190 database consisting of extensively curated C. elegans proteins from WormBase [35]. A
total of 782 unique sequences (56%) matched known
proteins, including 523 unique sequences (38%) with significant match to C. elegans proteins at a cut-off expectation (E)-value of 10-5 or below. The remainder of the
unique sequences (44%) had no meaningful matches (E
> 10-5). We compared our unique sequences with the ESTs
from other nematodes as well as non-nematodes using
BLAST searches. Only 107 unique sequences (7%)
matched other nematode ESTs and 153 unique sequences
(11%) matched organisms other than nematodes at E <
10-5 (Table 1). Of 1,387 unique sequences, 36 had homologues in C. elegans which could be silenced by RNAi. The
RNAi phenotypes (as described by WormBase) included
mig-15 (migration), lin-8 (lineage), unc-16, 89 (uncoordinated), dpy-6 (dumpy), rde-1 (RNAi defective), drh-2
(dicer related), nhr-67 (nuclear hormone receptor) and
ard-1 (alcohol/ribitol dehydrogenase).

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Identification of differentially expressed genes
To identify transcripts differentially expressed (DE) during desiccation stress, subtractive hybridization was conducted between cDNA from gradually desiccated and
fresh active nematodes (control). Two rounds of hybridization were done and DE clones were sequenced which
resulted in 80 quality sequences above 100 bp (Table 2).
The nucleotide sequences were analyzed and their putative functions identified by BLASTX search. The DE transcripts included 22 ESTs (28%) similar to metabolism
related genes, 15 (19%) similar to environmental information processing genes, 23 (28%) similar to genetic
information processing genes, 3 (4%) similar to hypothetical proteins of other organisms and 17 (21%) novel transcripts that had no identifiable similarity to known
sequences in GenBank [36]. Among the metabolism
related genes, 13 ESTs (68%) were involved in carbohydrate metabolism, 2 transcripts (10%) of each in lipid
metabolism, amino acid metabolism and protein folding,
sorting and degradation. The environmental information
processing category was dominated (53%) by stress
related proteins. In the genetic information processing
category, ribosomal proteins were the most abundant
(42%) group followed by translation elongation factor
(19%) (Table 2). All sequences have been deposited in the
dbEST division of DDBJ/EMBL/GenBank under accession
numbers [GenBank: FK670236] – [GenBank: FK670315]
and [GenBank: GH196899; GH229101].

The most unexpected discovery among DE ESTs was Type
II antifreeze protein (AFP) [GenBank: FK670242 which
showed high similarity to that of Clupea harengus (Table
2), the Atlantic herring. This finding represents another
case of an ice structuring protein from an Antarctic nematode, suggesting the possibility that Antarctic nematodes
may use similar antifreeze proteins for stress adaptation
heretofore observed only in one Antarctic nematode [37],
some fishes [38], insects [39], plants [40] and fungi and
bacteria [41].

Table 1: Plectus murrayi EST summary

Total number of high quality sequences‡
Average length of sequences (bp)†
Number of contigs§
Number of singletons
Number of putative unique transcripts¶
Unique transcripts with similarity to C. elegans database
Unique transcripts with similarity to other nematode database
Unique transcripts with similarity to other organisms
Total unique transcripts with significant similarity
Unique transcripts with no significant similarity

2,486
545 ± 156
324
1,063
1,387
523 (38%)*
106 (7%)
153 (11%)
782 (56%)
605 (44%)

‡A sequence is considered high quality if it's trimmed PHRED 20 length is >100 bases after vector only, low-quality and contaminating sequences are
removed.
†Calculated from the total ESTs.
§A contig (contiguous sequence) contains two or more ESTs.
¶Number of putative unique transcripts equals the number of contigs plus the number of singletons.
*Calculated as percentage of total unique transcripts with similarity at E < 10-5.

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Figure 1
Distribution of Plectus murrayi ESTs by cluster size
Distribution of Plectus murrayi ESTs by cluster size.

Abundant transcripts expressed during desiccation
A total of 23 contigs containing 384 ESTs were highly
redundant. This accounted for more than 15% of the total
high quality ESTs. The minimum and maximum number
of ESTs that made up these highly redundant contigs was
7 and 37 respectively (Table 3). More than one third (9)
of the highly redundant contigs, totalling 127 ESTs, had
significant similarity to various genes involved in metabolism. One third (8) of the highly redundant contigs,
totalling 139 ESTs, had significant similarity to various
environmental information processing related genes,
indicating high transcript abundance of stress related
genes, as expected. Two of the contigs, totalling 48 ESTs,
had significant similarity to ribosomal proteins while
three of the contigs, totalling 37 ESTs, had significant similarity to genetic information processing related genes.
One of the highly redundant contigs totalling 33 ESTs
matched similar sequences derived from the mitochondrial cytochrome oxidase subunit (Table 3). The most
redundant group of contigs were composed of 37 ESTs
and had significant similarity to ribosomal protein from
C. elegans, indicating higher activities of ribosomal protein genes during desiccation stress.
Functional classification based on gene ontology
assignments
To categorize transcripts by putative function, we utilized
the GO classification scheme (April 2008 release of GO
database, Gene Ontology Consortium). GO provides a
dynamic controlled vocabulary and hierarchy that unifies
descriptions of biological, cellular and molecular functions across genomes [42]. In this report, we relied on
well-annotated GO information of C. elegans and other
nematodes. GO representation of P. murrayi clusters is
shown for each organizing principle of GO: molecular
functions (Additional file 1a; Fig. 1a), cellular compo-

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nents (Additional file 1b; Fig. 1b), and biological processes (Additional file 1c; Fig. 1c). Additional file 1 and Fig.
2 provide a breakdown of representation by major GO
categories. The highest GO term for molecular functions
was protein binding, under 'ligand binding and carrier'
categories, which had 87 unique sequences accounting for
18% of the total unique sequences matched in this category and 6% of the total unique sequences. The highest
final GO term in cellular components was mitochondria
(under the 'cytoplasm' category) with a total of 28 unique
sequences, 17 of which are encoded on the mitochondrial
genome, accounting for 12% of the total in this category.
Similarly, the highest final GO term for biological processes was protein metabolism, under 'metabolism' categories, which had 44 unique sequences accounting for 12%
of the total in this category and more than 3% of the total
unique sequences. We found 13 unique sequences showing significant similarity to C. elegans signal transduction
factors; 8 of them belonged to the receptor binding group
and 5 sequences belonged to receptor and receptor signalling proteins (Additional file 1).
Functional classification based on KEGG analysis
As an alternative method of categorizing unique
sequences by biochemical functions, sequences were
assigned to metabolic pathways via KEGG [43] using
enzyme commission (EC) numbers as the basis for assignment. Only 281 unique sequences (36% of total) were
assigned EC numbers and had 158 unique mappings to
KEGG biochemical pathways (Table 4). The KEGG metabolic pathways that are well represented by P. murrayi
unique sequences are carbohydrate metabolism (18
enzymes), amino acid metabolism (9 enzymes), lipid
metabolism (8 enzymes), xenobiotic and bio-degradation
metabolism (5 enzymes), and biosynthesis of secondary
metabolites (3 enzymes). Of these, 12% of the unique
sequences belonged to the environmental information
processing (EIP) category, indicating higher activities of
stress and chaperone related genes during desiccation. The
KEGG pathways well-represented under EIP are membrane transport (15 enzymes), ligand-receptor interaction
(15 enzymes), signal transduction (8 enzymes) and signalling molecules and interaction (9 enzymes). About
11% of the unique sequences belonged to the genetic
information processing (GIP) category with most of them
having roles in folding, sorting and degradation. The
KEGG pathways well-represented under GIP are folding,
sorting and degradation (25 enzymes), transcription (9
enzymes), translation (8 enzymes) and replication and
repair (6 enzymes). Most of the sequences (49%)
remained unassigned to any known functional pathway
and 15% of the sequences were similar to C. elegans hypothetical proteins (Table 4). The lowest number of
sequences mapped to the cellular processes category
(3%), suggestive of developmental arrest during anhydro-

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Table 2: Listing of ESTs differentially expressed during desiccation of Plectus murrayi and their homologs in GenBank.

Clone ID

GenBank accession number Homolog accession

Metabolism (22)
SH_Pa.AA.01
FK670236
SH_Pa.AB.02
SH_Pa.AF.06

FK670237
FK670241

SH_Pa.AH.08

FK670243

SH_Pa.AI.09
SH_Pa.EA.014

FK670244
FK670249

SH_Pa.FA.015
SH_Pa.IA.018
SH_Pa.BD.021
SH_PA_Ssh.056

FK670250
FK670253
FK670254
FK670287

SH_Pa.BH.025
SH_Pa.BI.026
SH_Pa.CC.027
PA_Sh_Ab.062

FK670258
FK670259
FK670260
FK670293

SH_Pa.CD.019

FK670261

SH_Pa.CH.031

FK670264

SH_Pa.CI.032
SH_Pa.DG.038

FK670265
FK670269

SH_Pa.DH.039

FK670270

SH_Pa.DH.030
SH_Pa.AA.036
PA_Sh_bb.067

FK670263
FK670268
FK670298

Annotation (Organism) number

E-value

ref|NP_496237.1|

GPD family member [Caenorhabditis
elegans]
ref|NP_498081.2|
ALDH family member [C. elegans]
gb|AAF81283.1|
Glutathione S-transferase [Haemonchus
contortus]
ref|NP_496161.1|
Lipid Transfer protein family member [C.
elegans]
gb|AAC47996.1|
Aspartyl protease protein 6 [C. elegans]
WBGene00002263
Plant LEA related family member [C.
elegans]
gb|EDP32297.1|
TPS6 protein 1 [Brugia malayi]
gb|EDP36623.1|
FBA1, putative [B. malayi]
gb|AAC97508.1|
Thymidylate synthase [C. elegans]
WBGene00006975
Zinc finger protein family member [C.
elegans]
ref|NP_494721.1|
Probable glycerol kinase [C. elegans]
ref|NP_001006395.1| MDH1, NAD (soluble) [Gallus gallus]
ref|ZP_00056387.1|
IDH [Magnetospirillum magnetotacticum]
ref|NP_498111.2|
ATP synthase subunit family member [C.
elegans]
gb|EDP37408.1|
NADH-ubiquinone oxidoreductase [B.
malayi]
ref|NP_496736.1|
Glycogen synthase family member [C.
elegans]
emb|CAA53718.1|
ADP/ATP translocase [C. elegans]
ref|NP_503306.1|
Bi-functional glyoxylate cycle protein [C.
elegans]
gb|AAC19750.1|
Putative glutamate dehydrogenase [H.
contortus]
gb|AAC97508.1|
Thymidylate synthase [C. elegans]
WBGene00009165
Glutathione peroxidase [C. elegans]
ref|NP_498111.2|
ATP synthase sub unit family member [C.
elegans]

Environmental information processing (15)
SH_Pa.AC.03
FK670238
gb|AAM55195.1|
SH_Pa.AD.04

FK670239

ref|NP_508913.1|

SH_Pa.AE.05

FK670240

WBGene00004930

SH_Pa.AG.07

FK670242

gb|ABA41369.1|

SH_Pa.AA.010

FK670245

gb|AAN78300.1|

SH_Pa.DA.013

FK670248

ref|NP_495536.1|

SH_Pa.GA.016
SH_Pa.HA.017

FK670251
FK670252

ref|NP_496549.1|
gb|EDP28446.1|

SH_Pa.BF.023

FK670256

ref|NP_509019.1|

SH_Pa.BG.024

FK670257

gb|AAD00182.1|

SH_Pa.CF.029
SH_Pa.DD.034

FK670262
FK670266

gb|EDP35652.1|
ref|NP_499889.2|

PA_Sh_Ab.063
PA_Sh_Ib.070

FK670294
FK670300

gb|AAO44907.1|
gb|EDP30373.1|

PA_Sh_bB.072

FK670302

gb|EDP31428.1|

Cathepsin L cysteine protease [H.
contortus]
JNK kinase family member jkk-1 [C.
elegans]
Superoxide dismutase family member
[C.elegans]
Type II antifreeze protein [Clupea
harengus]
Heat shock protein 70 A [Heterodera
glycines]
Small heat shock protein family member
[C. elegans]
RAB family member [C. elegans]
Ras-related protein Rab-11B, putative [B.
malayi]
Heat shock protein family member [C.
elegans]
Inhibitor of apoptosis homolog [C.
elegans]
Heat shock protein 90 protein [B. malayi]
DumPY: shorter than wild-type
[C.elegans]
Collagen protein 170 [C. elegans]
Leucine rich repeat family protein [B.
malayi]
Laminin receptor 1 [Xenopus laevis]

Percentage similarity

1e-40

86%

2e-80
3e-51

74%
49%

6e-42

72%

2e-51
1e-33

53%
28%

1e-55
1e-73
1e-28
2e-28

51%
84%
62%
53%

4e-62
1e-71
4e-86
8e-110

71%
68%
73%
93%

2e-57

60%

1e-35

50%

6e-93
4e-95

90%
90%

9e-111

88%

1e-26
3e-63
8e-110

49%
79%
93%

2e-81

77%

2e-06

41%

4e-80

81%

2e-35

69%

2e-59

89%

2e-115

90%

7e-103
1e-53

88%
77%

8e-78

91%

5e-53

52%

8e-59
7e-47

53%
63%

4e-30
3e-27

62%
40%

1e-73

71%

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Table 2: Listing of ESTs differentially expressed during desiccation of Plectus murrayi and their homologs in GenBank. (Continued)

Genetic information processing (23)
SH_Pa.BA.011
FK670246
SH_Pa.CA.012 FK670247

gb|AAG50205.1|
emb|CAJ57642.1|

SH_Pa.DE.035
PA_Sh_Cb.064
PA_Sh_bH.078
SH_Pa.DI.040

FK670267
FK670295
FK670308
FK670271

gb|EDP39185.1|
gb|AAT28331.1|
ref|NP_956267.1|
ref|NP_491416.1|

SH_Pa.EE.041

FK670272

ref|NP_502794.1|

SH_Pa.EF.042

FK670273

ref|NP_502794.1|

SH_Pa.EG.043

FK670274

ref|NP_501167.1|

PA_Sh_EH.044

FK670275

ref|NP_741371.2|

PA_Sh_EI.045

FK670276

ref|NP_498660.1|

PA_Sh_Ab.046

FK670277

ref|NP_740944.1|

PA_Sh_Bb.047

FK670278

ref|NP_496375.1|

PA_Sh_Cb.048

FK670279

gb|EDP38710.1|

PA_Sh_Db.049

FK670280

gb|EDP38220.1|

PA_Sh_Lb.057

FK670288

gb|EDP29175.1|

PA_Sh_Eb.050
PA_Sh_Fb.051
PA_Sh_Gb.052
PA_Sh_Hb.053

FK670281
FK670282
FK670283
FK670284

ref|NP_492457.1|
gb|EDP34276.1|
ref|NP_492457.1|
ref|NP_524808.2|

PA_Sh_Ib.054
SH_Pa.BE.022
PA_Sh_Mb.058

FK670285
FK670255
FK670289

7e-41
2e-74

62%
97%

3e-33
5e-89
5e-40
7e-104

94%
83%
44%
85%

8e-73

94%

3e-67

81%

9e-85

84%

5e-57

77%

1e-67

88%

2e-40

82%

9e-51

97%

5e-54

83%

9e-21

94%

1e-25

86%

2e-66
1e-117
2e-66
5e-45

89%
88%
89%
68%

ref|NP_498520.1|
WBGene00003623
gb|EDP33960.1|

AP inhibitor [Parelaphostrongylus tenuis]
Putative E2 enzyme [Oesophagostomum
dentatum]
Histone H2B 2, putative [B. malayi]
Peroxiredoxin [H. contortus]
Ubiquitin specific protease 14 [D. rerio]
Ribosomal protein, LSU family member
[C. elegans]
Ribosomal protein, SSU family member
[C. elegans]
Ribosomal protein, SSU family member
[C. elegans]
Ribosomal protein, SSU family member
[C. elegans]
Ribosomal protein, LSU family member
[C. elegans]
Ribosomal protein, LSU family member
[C. elegans]
Ribosomal protein, SSU family member
[C. elegans]
Ribosomal protein, LSU family member
[C. elegans]
60S ribosomal protein L27a, putative [B.
malayi]
60S ribosomal protein L39, putative [B.
malayi]
40S ribosomal protein S6, putative [B.
malayi]
EF family member [C. elegans]
EF1-alpha, putative [B. malayi]
EF family member [C. elegans]
EF1 beta, isoform A [Drosophila
melanogaster]
EF family member [C. elegans]
NHR family member [C. elegans]
Transcription factor, putative [B. malayi]

4e-100
2e-06
9e-16

89%
38%
87%

Hypothetical proteins (3)
PA_Sh_Jb.055
FK670286
PA_Sh_Ob.060 FK670291
PA_Sh_bC.073 FK670303

ref|XP_001666153.1|
ref|XP_001676045.1|
ref|XP_001631386.1|

Hypothetical protein [C. briggsae]
Hypothetical protein [C. briggsae]
Predicted protein [X. laevis]

4e-41
3e-32
2e-34

78%
55%
42%

Novel proteins (17)
PA_Sh_Nb.059 FK670290
PA_Sh_Pb.061
FK670292
PA_Sh_Db.065 FK670296
PA_Sh_Eb.066
FK670297
PA_Sh_Gb.068 FK670299
PA_Sh_bA.071 FK670301
PA_Sh_bD.074 FK670304
PA_Sh_bE.075
FK670305
PA_Sh_bF.076
FK670306
PA_Sh_bG.077 FK670307
PA_Sh_bI.079
FK670309
PA_Sh_bA.080 FK670310
PA_Sh_bC.028 FK670311
PA_Sh_bD.020 FK670312
PA_Sh_bF.033
FK670313
PA_Sh_bG.037 FK670314
PA_Sh_bG.069 FK670315

n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a

Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel

n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.

Pathway assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) classification.

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Table 3: The most abundantly represented transcripts in the cDNA library

Contig No

Tentative annotation§

Contig_57
Contig_132
Contig_9
Contig_312
Contig_87
Contig_231
Contig_32
Contig_198
Contig_54
Contig_56
Contig_67
Contig_301
Contig_287
Contig_93
Contig_74
Contig_63
Contig_152
Contig_242
Contig_131
Contig_29
Contig_149
Contig_313
Contig_112

Ribosomal protein
Cytochrome c oxidase subunit 2
Small heat shock protein family
Rab-family member
Aquaporin
DNA-binding protein
Y-box family member
Zinc finger protein
Cu/Zn superoxide dismutase
Translation initiation factor
Chaperonin containing subunit
Glutamate synthase
NADH-dehydrogenase subunit 1
Cathespin b -like cysteine proteinase
Elongation factor 1 alpha
Glutathione S-transferase
Heat shock 70 kda protein
60S ribosomal protein
Elongation factor family member
Aldehyde dehydrogenase
Heat shock 90 kda protein
ATP synthase subunit
Nuclear hormone protein family

Number of ESTs
37
33
27
25
24
23
22
18
17
17
16
13
13
13
13
12
11
11
11
7
7
7
7

Percentage (%)
1.48
1.32
1.08
1.00
0.96
0.92
0.88
0.72
0.68
0.68
0.64
0.52
0.52
0.52
0.52
0.48
0.44
0.44
0.44
0.28
0.28
0.28
0.28

§Annotation based on most significant BLAST alignment for each cluster.
Percentage based on total number of high quality sequences.

biosis. The cell growth and death (5 enzymes) and cell
communication (4 enzymes) pathways were also the wellrepresented categories under cellular processes (Table 4).
Refined gene-specific expression using quantitative realtime PCR
In order to validate our differential gene expression results
and obtain more refined gene expression data, we
designed gene-specific primers for 14 transcripts selected
from Table 2 and analyzed their expression using quantitative real-time PCR (qRT-PCR) (Fig. 3). These genes were
chosen to represent a variety of functional classifications.
Among these 14 transcripts, 9 were significantly induced
(fold-change > 2.0×, P value < 0.05) and 3 genes were
reduced (fold-change > 2.0×, P value < 0.05) in response
to desiccation. Significant desiccation-induced gene
expression change ranged from -6.50-fold for antifreeze
protein to 26.77-fold for trehalose-6-phosphate synthase.
Heat shock protein 70 and 90 were also weakly induced
(fold-change 1.7 and 1.94×), but lacked significant statistical support P > 0.05). Among the DE transcripts, putative
homologs to trehalose 6-phosphate synthase protein
showed highest induction (fold-change 26.77×, P value <
0.05) followed by the putative homolog to glycerol kinase
(fold-change 25.04×, P value < 0.05). Interestingly, there
was significant reduction and induction on the level of
expression of two novel transcripts, [GenBank: FK670306

and FK670310] (fold-change -10.3× and 16.71× respectively, P < 0.05), suggestive of their possible roles in desiccation tolerance (Fig. 3). The mRNA copy number was
calculated using an absolute quantification method [44].
There was no significant difference (P value < 0.05) in
copy number of transcripts encoding Hsp70 and Hsp90
on desiccated and control nematodes (Additional file 2).

Discussion
The expressed genome of P. murrayi showed that anhydrobiotic survival in nematodes involves a suite of genes from
diverse functional areas, such as hormone signaling transduction, transcription regulation, ROS scavenging, reestablishment of homeostasis, molecular chaperoning and
transcriptional regulation of ribosomal proteins and other
genes. The 2,486 sequences comprised 1,387 putative
unique sequences from P. murrayi and represent the first
EST analysis from an Antarctic nematode. The number of
ESTs analyzed in the study were relatively few; nonetheless, 44% of the unique transcripts (605) expressed by
desiccated nematodes represent novel genes, which falls
within the range reported from several other nematode
EST analyses [45]. Comparison of ESTs presented in this
study with ESTs from GenBank showed that 56% of the
unique transcripts (1,387) isolated from P. murrayi have
been previously isolated from other organisms, including
the model organism C. elegans (Table 1). Subtractive

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BMC Genomics 2009, 10:69

(a)

Enzyme
regulator
4%
Other
enzymes
2%
Phosphatase
3%

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Translation
regulation
2%

Transcription
al regulation
2%
Protein
binding
18%

Transferase
5%

Nucleotide
binding
11%

Hydrolase
9%
Response to
stimulus
7%
Structual
molecule
4%
Transporter
6%

(b)
Plasma
membrane
4%
Mitochondria
l membrane
5%

Molecular
tranducer
6%

Nucleic acid
bindng
10%
Calcium
binding
Cofactor 5%
binding
3%

Extracellular
8%
Mitochondria
12%
Ribosome
8%

Integral
membrane
6%

Nucleus
12%
Ribonucleoprotein
5%
Cell fraction
1%

Other
intracellular
24%
Protein
complex
15%

(c)
Behavior
4%
Signal
transduction
4%
Response to
stimulus
7%

Cell death
2%

Protein
metabolism
12%
Biosynthesis
5%
Catabolism
4%
Nucleic acid
metabolism
4%

Development
17%

Other
metabolism
15%

Others
10%
Response to
stress
4%

Localization
6%
Transport
6%

Figure 2
for Plectus representation
Percentagemurrayi clusters of gene ontology (GO) mappings
Percentage representation of gene ontology (GO)
mappings for Plectus murrayi clusters. (A) Molecular
functions; (B) Cellular components; and (C) Biological process. More detailed information is provided in Additional files
2a, 2b and 2c. Note that individual GO categories can have
multiple mappings.

hybridization of cDNAs from desiccated and undesiccated
nematodes used in library construction was done to
enrich for the rare transcripts that are differentially
expressed (DE). A total of 80 DE transcripts were identified, some of which are involved in genetic information
processing followed by metabolism, and presumably are
involved in stress survival of nematodes (Table 2). The
expression level of 14 transcripts DE during desiccation
survival using qRT-PCR indicates that these genes were DE
between desiccated and undesiccated nematodes. The
results from subtractive hybridization coupled with qRTPCR showed that we appropriately enriched for the DE
transcripts (Fig. 3). Our results showed that expressed
sequence tag (EST) analysis coupled with subtractive
hybridization is a powerful method to identify the genes
involved in nematode desiccation stress. EST analysis is a
commonly-used approach to identify genes involved in
specific biological functions, especially in organisms
where genomic data are not available [46].
Functional analysis of expressed genes
Gene ontology has been widely used to characterize gene
function annotation and classification [42]. GO describes
gene function using controlled vocabulary and hierarchy,
including molecular function, biological processes, and
cellular components (Fig. 2; Additional file 1). A large
number of the unique sequences from our study mapped
to molecular functions and ligand binding, enzymes,
molecular transducer and transporter subcategories. Each
one of these subcategories represents catalytic activities
that could be argued are important for a cell to survive
major metabolic perturbation during desiccation. The second most represented category was biological process,
which includes unique sequences associated with cell
growth or maintenance, development and cellular communications. Within the cell growth or maintenance category most unique sequences were associated with
metabolism, localization, transport and response to
stress. This distribution is not surprising for ESTs (unique
sequences) derived from an organism undergoing metabolic changes such as desiccation. The subcategories
under the least represented category, cellular process, also
seem to reflect the nature of the cellular disturbances that
result from desiccation. Under this category there is significant representation under intracellular and membrane
subcategories. Within these subcategories representation
is most significant in mitochondria, cytosol, ribosome,
nucleus and ribonucleoprotein complex. The importance
of protein synthesis and cellular homeostastasis during
desiccation may explain the preponderance of unique
sequences associated with mitochondrial and ribosomal
structural components.

As an alternative method of categorizing clusters by biochemical function, clusters were assigned to metabolic

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Table 4: KEGG biochemical mappings for Plectus murrayi clusters.

KEGG categories represented
Metabolism
Carbohydrate metabolism
Amino acid metabolism
Lipid metabolism
Xenobiotics biodegradation and metabolism
Biosynthesis of secondary metabolites
Energy metabolism
Nucleotide metabolism
Metabolism of other amino acids
Glyoxylate and dicarboxylate metabolism
Genetic information processing
Folding, sorting and degradation
Transcription
Translation
Replication and repair
Environmental information processing
Membrane transport
Ligand-receptor interaction
Signal transduction
Signalling molecules and interaction
Cellular processes
Cell growth and death
Cell communication
Cell motility
Development
Unassigned‡
Hypothetical
§Percentage
‡Unassigned

Unique sequences (Number of enzymes)
84 (52)

Percentage§
11%

29(18)
14(9)
13(8)
8(5)
6(3)
5(2)
3(2)
3(2)
3(3)

4%
2%
2%
1%
<1%
<1%
<1%
<1%
<1%

83 (48)

11%
42(25)
17(9)
16(8)
8(6)

6%
2%
2%
1%

95 (47)

12%
30(15)
28(15)
14(8)
13(9)

4%
4%
2%
2%

19 (11)

3%
8(5)
6(4)
3(1)
2(1)

1%
<1%
<1%
<1%

385
116

49%
15%

based on total unique transcripts (782) with significant similarity to sequences in database.
sequences are those that have significant similarity to known sequences whose functions are unclear.

pathways using the KEGG database [44] (Table 4). Only
36% of the unique sequences mapped to the currently
known KEGG pathways with 158 unique mappings. The
paucity of EC assignments limits this aspect of analysis
but the mapping of unique sequences to the KEGG metabolic and other pathways still presents some useful perspectives on the metabolic, protein folding and
degradation and cellular repair emphasis of desiccating
cells. Most of the unique transcripts belonged to genetic
information processing (GIP) with protein folding, sorting and degradation. Transcripts encoding a cathespin Llike protease [GenBank: FK670238] enzyme found in this
category have long been recognized for their role in intracellular and extracellular protein degradation in a range of
cellular processes. In C. elegans cathespin L-protease (Cecpl) has been demonstrated to play a critical role during
embryogenesis, larval development and moulting [47].
Considering the putative homology of the transcript
encoding P. murrayi cathespin L-protease (Pm-cpl) with
C. elegans, it might have a similar role in nematode molting and thus its possible role during desiccation survival
needs further investigation.
Metabolism was the second most represented category
and pathways well-represented by the P. murrayi clusters

were carbohydrate metabolism, amino acid metabolism,
lipid metabolism, biosynthesis of secondary metabolites,
glyoxylate, and decarboxylation metabolism (Table 4).
The lipid metabolism pathway in anhydrobiotes is one of
the most active pathways as lipids are the main reservoir
of energy and the most likely source of carbon for the synthesis of trehalose [21]. The GIP category included the
pathways involved in protein folding, sorting and degradation, transcription, translation, replication and repair.
Desiccation stress coupled with oxidative stress results in
lipid and protein damage, leading to impaired cellular
survival and functioning. Nematodes are reported to
respond to these damages and repair them with increased
expression of stress-protective genes and antioxidant
enzymes [48]. The cellular processes category has the least
number of transcripts. This finding is suggestive of developmental arrest during anhydrobiosis. Indeed, anhydrobiosis may be an exaptation that allows the individual to
survive unfavourable conditions by staying in an arrested
state of development and reproduction (Table 4).
Abundantly expressed transcripts during anhydrobiosis
A high level of representation in a cDNA library generally
correlates with high transcript abundance in the original
biological sample [49], although artefacts of library con-

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35
Relative expression level

30

*

*

25
20

*

*

*
*

15
10

*

*

*

5
0
-5
-10

*

-15

*
*

Figure 3
Quantitative Real-time PCR analysis of gene expression in Plectus murrayi in response to desiccation
Quantitative Real-time PCR analysis of gene expression in Plectus murrayi in response to desiccation. Values
were determined using qRT-PCR and represents relative expression of genes from desiccated to undesiccated nematodes
(control). The relative expression of the target gene (Pm-alh: aldehyde dehydrogenase; Pm-tps: trehalose-6-phosphate synthase;
Pm-gpx: glutathione peroxidise; Pm-afp: antifreeze protein; Pm-hsp-70: heat shock protein 70; Pm-lea: late embryogenesis abundant protein; Pm-gk: glycerol kinase; Pm-ms: malate synthase; Pm-gsy: glycogen synthase; Pm-hsp-90: heat shock protein 90; Pmrpl-4: ribosomal protein-4; Pm-desc-1: novel protein I; Pm-desc-2: novel protein II; Pm-gst-1: glutathione s-transferase 1,) normalized to Pm-18s:18S rRNA and relative to the expression of control, was calculated for each sample using the 2-ΔΔCT method
[87]. Gene expression was determined in each sample using three independent technical replicates. A transcript with relative
abundance of one is equivalent to the abundance of 18S rRNA. Bars represent standard errors calculated from three replicates
of each experiment. *Significant difference (P < 0.05) from control.
struction can result in selection for or against representation of some transcripts. The genes with the most
abundant transcripts were mostly involved in metabolism, molecular chaperones, reactive oxygen species scavenging and genetic information processing (Table 3).
Ribosomal protein (RP) (GenBank: FG618944) was the
most abundant transcript expressed during desiccation,
suggesting the possible involvement of transcriptional
regulation of ribosomal proteins during desiccation stress.
Differential expression of plant ribosomal protein genes
has been observed during development and following
various stress or hormone treatments, including desiccation stress [50]. The 'ribosomal filter hypothesis' of Mauro
and Edelman [51] proposes that particular combinations
of ribosomal proteins or rRNA could favour the translation of specific mRNAs, thereby providing a mechanism
for translational control. Interestingly, many ribosomal
proteins were DE and one of them was up-regulated (Pmrpl-4), suggesting that RPs may provide a means for selec-

tively translating specific mRNAs required for the desiccation response as suggested for the insect parasitic
nematode Steinernema carpocapsae [28]. Similarly, it may
be part of an adaptive response to maintain ribosomal
function in dehydrated cytoplasm.
A number of metabolism related transcripts encoding
cytochrome c oxidase subunit 2 (GenBank: FG618989),
glutamate synthase (GenBank: FG620419), NADH-dehydrogenase subunit 1 (GenBank: FG619004) and aldehyde
dehydrogenase (GenBank: FG619103) were also abundantly expressed during desiccation stress. Several lines of
evidence have suggested that nematodes activate metabolic pathways in response to desiccation shortly after
exposure to dehydrating conditions [26].
Transcripts of a number of stress related genes were also
abundantly expressed, including heat shock proteins, a
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sive zinc finger proteins and oxidative stress responsive
genes. Desiccation stress in nematodes reportedly produces a large number of molecular chaperones to facilitate
the synthesis, folding, assembly and intracellular transport of proteins, reduce protein denaturation and aggregation, and aid in protein renaturation [19,52]. Transcripts
of several oxidative stress related genes like glutathione Stransferase (GenBank: FG618951) and copper/zinc superoxide dismutase (GenBank: FG618942) were also abundantly expressed. Reactive oxygen species and other toxins
produced by oxidative stress during desiccation of nematodes can damage membrane systems, proteins and
nucleic acids. Therefore, the transcript abundance of several proteins that contribute to cellular survival after oxidative damage is not surprising.
Genes of general and secondary metabolism
Nematodes activate their metabolic pathways in response
to desiccation shortly after exposure to dehydration [26].
Several transcripts encoding metabolism related genes are
differentially expressed by desiccation stress in P. murrayi.
These genes include aldehyde dehydrogenase [GenBank:
FK670237], trehalose-6-phosphate synthase [GenBank:
FK670250], thymidylate synthase [GenBank: FK670263],
glycerol kinase [GenBank: FK670258], glycogen synthase
[GenBank: FK670264], ATP synthase [GenBank:
FK670293], ADP/ATP translocase [GenBank: FK670265],
and malate dehydrogenase [GenBank: FK670259]. Interestingly, a transcript encoding a bifunctional glyoxylate
cycle protein (malate synthase), a distinct and anaplerotic
variant of the tricarboxylic acid cycle, was also found to be
highly expressed during desiccation (Table 2).

Nematodes are unique among animals in utilizing the glyoxylate cycle to generate carbohydrates from the beta-oxidation of fatty acids [53]. The glyoxylate pathway, generally
found in plants and micro-organisms, is similar to the citrate cycle, but relies on two critical enzymes, malate synthase and isocitrate lyase, to bypass two decarboxylation
steps. Interestingly, the anhydrobiotic nematode A. avenae
has been reported to use the glyoxylate cycle during induction of anhydrobiosis [54]. One sequence unique to P. murrayi that mapped to a glyoxylate cycle protein [GenBank:
FK670269] includes putative homologs of malate synthase
(Pm-ms). The abundant expression of malate synthase transcripts in our EST collection and its up-regulation upon desiccation stress may provide experimental support for an
active role of glyoxylate cycle proteins during induction of
anhydrobiosis by P. murrayi.
Transcriptional regulation and signalling affected by
desiccation
Transcriptional regulation and intracellular signalling cascades for nematode stress response in general and secondary metabolism in particular, are poorly understood. A
number of desiccation responsive P. murrayi ESTs encode

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putative signalling molecules or transcription factors. One
of the P. murrayi ESTs was most similar to the unc-16 gene
of C. elegans, which encodes a c-Jun N-terminal kinase
(JNK)-interacting protein [55], and one of the members of
JNK kinase family (jkk-1) [GenBank: FK670239] was DE
during desiccation. JNK (also known as stress-activated
MAP kinases or SAPK) is a member of the mitogen-activated protein kinases (MAPKs) that regulate cellular
responses to a variety of extracellular signals, including
desiccation stress [56-58]. Transcripts homologous to
genes encoding Zinc finger protein (GenBank:
FG619080) were abundantly present in the expressed
genome of P. murrayi. Zinc finger proteins are cellular proteins which play a major role in transcriptional regulation
by binding with high affinity to specific regions of DNA.
In conjunction with leucine zipper domains these proteins may form hetero- or homodimers and activate transcription, either constitutively, or in a regulatory manner,
through post-translational modifications in response to
external stimuli (although some may also be cell specific
or developmentally regulated) [59]. The abundance of
genes encoding zinc finger protein in response to desiccation suggests that these proteins may regulate further
events in the stress-response cascade of P. murrayi. Three
transcripts from P. murrayi were most similar to the C. elegans gene encoding a predicted neurotransmitter gated
ion-channel (GenBank: FG620960) protein. Neuronal
signal transduction in response to desiccation stress
would be required to initiate the coiling response [60,70]
of desiccating P. murrayi.
Stress response genes expressed during anhydrobiosis
Nematodes respond to desiccation stress by synthesizing
a conserved set of proteins [23-25]. Our results demonstrate that for P. murrayi desiccation stress can significantly
elevate stress related genes encoding trehalose 6-phosphate synthase, late embryogenesis abundant proteins,
heat shock proteins, ubiquitin, c-type lectins, chaperone
related proteins, and other stress responsive genes (Table
3). Three transcripts encoding trehalose 6-phosphate synthase, which synthesize the storage carbohydrate trehalose, were expressed during desiccation stress. A
characteristic feature of anhydrobiotic organisms is their
synthesis of high concentrations of non-reducing sugars
during the induction of anhydrobiosis [29,61]. Trehalose
protects membranes and proteins from desiccation damage by replacing structural water [61], and contributes to
the formation of an intracellular organic glass [62] which
is thought to stabilize the cell's contents. The up-regulation of trehalose during desiccation stress of P. murrayi
could be part of an adaptive response to desiccation
(Fig. 3).

Protein aggregation during desiccation is likely to be a
major potential hazard for anhydrobiotes; late embryogenesis abundant (LEA) proteins acting as molecular
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chaperones or molecular shields play an important role in
prevention of this aggregation [63]. Transcripts similar to
plant LEA related family members [GenBank: FK670249]
of C. elegans were up-regulated during desiccation stress of
P. murrayi. An LEA group 3 gene Aav-lea-1 was strongly
induced in A. avenae during the induction of anhydrobiosis [24]. The C. elegans genome encodes three LEA genes
[20] and silencing of the lea-1 gene by RNA interference
(RNAi) caused a marked reduction in desiccation resistance in dauer larvae [64]. We thus assume that LEA proteins contribute to protection and recovery from
desiccation stress in anhydrobiotic nematodes.
Molecular chaperones, such as the Hsp70 family and the
Hsp60 chaperonin complexes, are commonly perceived
as heat shock proteins (Hsps), being up-regulated by
stress. The P. murrayi transcripts of 70 [GenBank:
FK670245] and 90 kda heat shock protein [GenBank:
FK670262] and small heat shock proteins were abundantly expressed following desiccation stress. Heat shock
proteins have been implicated in response to desiccation
in many nematodes [18], but they appear to be constitutively expressed in P. murrayi (Additional file 2). Though
Hsps may contribute to enhanced stress resistance overall,
our results showed no evidence that expression levels of
these Hsps were altered by desiccation. It has been shown
that other Antarctic organisms constitutively express
Hsp70 and Hsp90, showing no or modest up-regulation
of this gene in response to thermal stress [65-67]. Characteristically, Hsps are expressed not at the animal's normal
habitat condition, but only as part of the organism's stress
response. In fact, the expression of these genes is thought
to be incompatible with ongoing protein synthesis and
the progression of development [68,69]. It may be that P.
murrayi has evolved a mechanism to maintain Hsp function without disrupting normal metabolism and growth
that requires synthesis of other proteins. Consistent with
the observed constitutive expression, it is possible that
desiccation stress did not activate these genes, and the
mild desiccation failed to boost Hsp expression. An alternative explanation for the continuous up-regulation of
Hsps is that because Antarctic nematodes are frequently,
although unpredictably, exposed to a variety of environmental stressors such as desiccation, high pH, extreme
osmotic shock, freezing, and anoxia as well as temperature [70], their survival depends on maintaining continuous expression of molecular chaperones. Furthermore,
because of the unpredictability and the potential rapidity
of exposure to diverse environmental stresses, the continuous production of these molecular chaperones may be
energetically justified.
Unexpectedly, one of the ESTs encoded a protein similar
to the type II antifreeze protein (AFP) [GenBank:
FK670242] of Atlantic herring (Clupea harengus). This

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finding is somewhat surprising since antifreeze proteins
from phylogenetically divergent organisms (insects, fish,
plants, etc.,) generally have very little similarity to each
other [71]. The transcript encoding antifreeze protein
(AFP) (Pm-afp) was down-regulated during desiccation
stress of P. murrayi. It is found that Pm-afp is up-regulated
only during freezing and once nematodes are exposed to
desiccation other stress response mechanisms are activated by down-regulating AFP (B.N. Adhikari, unpublished work). Many overwintering organisms, including
insects, fish, bacteria, fungi, and plants, accumulate AFPs
that bind to the faces of ice crystals during freezing and
inhibit their growth [71]. While the high degree of AFP
similarity between P. murrayi and C. harengus raises the
idea that it originated in either of the two organisms via
horizontal gene transfer, we suggest that such speculation
is premature, particularly given their geographic, ecological, and phylogenetic disparity, but also because such a
small fraction of the phylogenetic diversity of nematode
genomes has been explored. Further sequencing and characterization are currently underway to better understand
the origin and evolution of this gene in P. murrayi.
Oxidative stress genes expressed during anhydrobiosis
Desiccation stress induces the generation of reactive oxygen species (ROS) in nematodes, and therefore it is important for nematodes to have effective ROS-scavenging
mechanisms. A number of ESTs encoding proteins which
detoxify reactive oxygen species like superoxide dismutase
(SOD) [GenBank: FK670240], Ras-related protein [GenBank: FK670252], and glutathione S-transferase (GST)
(GenBank: FK670241) were expressed in P. murrayi in
response to desiccation (Table 2). The SOD enzymes are a
family of metalloenzymes responsible for quenching the
potentially deleterious effects of superoxide radicals.
There was abundant expression of ESTs similar to C. elegans sod-1 that encode a copper/zinc superoxide dismutase (Table 3). Three transcripts encoding glutathione
s-transferase 1 were expressed in the ESTs of P. murrayi
and one of them was up-regulated following desiccation
stress. GSTs are a diverse super-family of multifunctional
proteins that play prominent roles in detoxification
metabolism in nematodes [72]. More than a dozen different GSTs have been isolated from C. elegans [73] and these
detoxifying enzymes are reported to be involved in several
functions, including xenobiotic detoxification and oxidative stress tolerance [72,74]. Differential expression and
up-regulation of detoxifying enzymes like Pm-sod-1 and
Pm-gst-1 suggests that P. murrayi has efficient ROS scavenging mechanisms under desiccation stress.
Membrane and transport-related protein expressed during
desiccation
In this study, we identified several ESTs encoding proteins
involved in transport facilitation. A total of 15 genes

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encoding ion and water transporters included ABC transporter proteins (GenBank: GH229101), water channel
proteins (GenBank: GH196899), ATPase (GenBank:
FG618924) and lipid transfer proteins (LTP) [GenBank:
FK670243]. Though the mechanism of coupling ion and
water flow through membrane channels is not well studied, there is evidence that up-regulation of such genes is
correlated with sensitivity to different types of stress in
nematodes [75]. Sixty ABC transporters have been identified and functionally characterized in C. elegans. Members of this protein family are responsible for resistance to
heavy metals [76]. In addition to short-term response and
regulatory mechanisms, a functional system for reestablishing homeostasis is vital to desiccation tolerance. We
found transcripts encoding proteins involved in ion
homeostasis, such as vacuolar H+-ATPase and ATP synthase [GenBank: FK670298], showing that P. murrayi has
efficient homeostatic pathways. Desiccation tolerance
involves changes in the levels and composition of fatty
acids of the major glycerolipids in nematodes [21]. Our
data showed that gene encoding non-specific LTP was differentially regulated following desiccation, suggesting an
active lipid metabolism and desiccation resistance.
Refined gene-specific expression using quantitative realtime PCR
In general, we observed larger changes in gene expression
using qRT-PCR, likely reflecting the greater dynamic range
of detection and sensitivity of this method for gene
expression profiling. Desiccation stress caused significant
up-regulation of transcripts encoding stress related genes
like Pm-lea (late embryogenesis abundant protein) and
Pm-tps (trehalose-6-phosphate synthase) and down-regulation of Pm-afp (antifreeze protein). Similarly, there was
significant up-regulation of the ROS scavenging enzyme
Pm-gst-1(glutathione s-transferase 1) and metabolism
related genes like Pm-gpx (glutathione peroxidise) and
Pm-ms (malate synthase), and down-regulation of Pm-afp
and Pm-gsy (glycogen synthase). Similar gene expression
patterns, except for antifreeze protein, were reported for
the free-living mycophagous nematode A. avenae [20] and
insect parasitic nematode Steinernema feltiae [26] during
desiccation acclimation and survival (Fig. 3). Although
the observed changes in gene expression are common to
many nematodes (except for the antifreeze protein and
heat shock proteins), we observed significant variability in
the magnitude of transcript abundance in P. murrayi. It is
likely that the expression of these genes in response to desiccation stress is influenced by the thermal history of the
organism, including seasonal variation experienced in the
field, and by phylogenetic constraints.

Conclusion
Nematodes have an important yet poorly understood
place in the study of stress response survival, and desicca-

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tion tolerance in particular. Nematodes are the most
abundant metazoans on earth [77] and prominent drivers
of Antarctic ecosystem functioning [78]. Similar to the
Antarctic nematode Panagrolaimus davidi [79], P. murrayi
are desiccation as well as freeze tolerant, which establishes
them as a useful model in assessing the structural, physiological, biochemical and genetic aspects of multiple stress
tolerance, and the mechanisms by which organisms
respond to and survive in extreme environments. To date
very few studies have focused on the molecular aspects of
desiccation tolerance in Antarctic nematodes and no
genomic data is available in existing databases. The progression of Antarctic nematode genomics will be critical to
the development of more useful and manipulable models
for understanding desiccation tolerance and the nature of
extremophiles. It is to these ends that we have initiated
this study into the transcriptome of P. murrayi as they
respond to a major stress event, in this case desiccation.
In the present study of 2,486 ESTs, a significant portion of
transcripts had no known homologue in any nematode or
other organisms for which sequence data are currently
available in public databases. These molecules are particularly interesting as they may represent genes that are species/lineage specific or unique to Antarctic nematodes.
There is considerable scope in exploring such molecules
in the future and using a combination of genomic and
proteomic approaches could provide valuable information on the survival strategy of extremophiles. Although
the number of transcripts analyzed under this study is not
large, we have identified and validated several genes differentially expressed during desiccation stress. It is crucial
to emphasize that changes in mRNA accumulation may
not necessarily correlate with protein/enzyme activity
level [80]. However, the expression profiles provide starting points for more in-depth studies on candidate genes
using additional genetic approaches. The annotation of
transcripts with significant fold change and detection of
consistently DE transcripts by SSH and qRT-PCR strongly
suggest that these putative genes have an important role in
P. murrayi desiccation stress response. In combination
with the bioinformatics analysis presented here, further
functional genomics analyses are required to generate a
more complete picture of the cellular response of a freeliving metazoan to extreme abiotic stress and determine
the biological roles of these genes within the larger context of its Antarctic ecosystem.

Methods
Nematode culture
Live nematodes (P. murrayi) from Antarctica were reared
in culture in order to produce sufficient quantities for
experiments. Nematodes were cultured in sand agar plates
containing Bold Modified Basal Freshwater Nutrient
Media (BMBFN) (Sigma Aldrich Inc. USA) and stored at

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15°C until used. Briefly, 20 ml/L BMBFN solution was
mixed with 15 g/L Bacto-Agar in deionised water and
autoclaved. The autoclaved Agar media was poured into
Petri dishes and Standard Ottawa Sand (EMD Chemicals
Inc., Gibbstown, NJ) was spread over each plate before the
media solidified. About 5–10 nematodes were released
into each plate, and a thin layer of water was maintained
on the surface of the media for the duration of culture. The
plates were incubated at 26°C for one to two weeks followed by 16°C for 3–5 weeks. In the laboratory culture at
16°C, nematodes completed their life cycle in about 6–8
weeks. Nematodes were also cultured in Nematode
growth medium (NGM) plates with Escherichia coli OP50.
About 15–20 nematodes were placed in one NGM plate
and incubated at 16°C for about 5–8 weeks. Nematodes
were sub-cultured every 3–4 weeks by chunking the agar
with a sterile scalpel and moving a chunk of agar from an
old plate to a fresh plate containing a lawn of E. coli OP50.
To harvest the nematodes, the Agar media was cut into
small pieces and poured into #40 standard testing sieve
(Advantech Manufacturing, New Berlin, WI). Agar was
removed by sieving and nematodes were washed twice
with deionized water before collection by centrifugation.
Desiccation treatment
Nematodes (about 3,000–5,000) were desiccated in glass
desiccation chambers. The relative humidity (RH) was
controlled by using saturated salt solutions as described
by Winston & Bates [81] with some modifications. The
requisite RH was maintained at 23°C ± 1°C in the desiccation chamber for 3 days prior to the addition of nematodes for equilibration. Relative humidity was maintained
as 100% RH with distilled water vapour; 97% with K2SO4,
87% RH with HCl and 75% with NaCl. The treatments
were an initial desiccation at 97% RH for 3 days followed
by exposure to 87% for 2 days. All experiments were
repeated under identical conditions using mixed stage
populations of P. murrayi.
RNA isolation
The desiccated and control nematodes were transferred to
10 volumes of Trizol Reagent (Molecular Research Center
Inc., Cincinnati, OH) and exposed to freeze thaw cycles
using liquid nitrogen and a 37°C water bath. The suspension was ground using mortar and pestle and vortexed.
RNA was phase separated using chloroform, precipitated
by isopropanol and pelleted. Total RNA was quantified
and quality checked by spectrophotometer (NanoDrop,
ND-1000 Spectrophotometer) and running agarose gel
with RNA Century™ Plus Marker (Ambion Inc., Austin,
TX).
cDNA Library Construction and Sequencing
Total RNA was extracted from the gradually desiccated
nematodes and used as starting material for complementary deoxyribonucleic acid (cDNA) library construction. A

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full-length cDNA library was prepared using Creator™
SMART™ cDNA library construction kit (Clontech, Mountain View, CA) according to the manufacturer's protocol.
Briefly, 2 μg of total RNA was reverse transcribed to cDNA
using SMART™ IV Oligonucleotide and PowerScript
Reverse Transcriptase and amplified using long distance
(LD) PCR. The cycling parameters were 95°C, 20 s; two 20
cycles of 95°C, 5 s; and 68°C, 6 min. cDNA was analyzed
using agarose gel electrophoresis, proteinase K digested
and restriction digested by SfiI. cDNA was size fractionated using CHROMA SPIN-400 columns and analyzed via
agarose gel electrophoresis. The size fractionated cDNA
was directionally cloned into pDNR-LIB vector and incubated at 16°C for 15 h. Transformation was done at 37°C
overnight, and colonies were picked and grown for 18 h.
Template DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA), amplified, cleaned
using ExoSAP-IT® (USB Corporation, Cleveland, Ohio)
and sequenced using an ABI PRISM 377 automated DNA
sequencer (Applied Biosystems, CA) at the DNA Sequencing Center (DNASC) at Brigham Young University (BYU).
Sequencing reactions were performed with 2 μl of ABI
PRISM BigDye Terminators v3.0 (Applied Biosystems)
and 3.5 pmol of primer in 12 μl reaction volumes, followed by a sequencing reaction clean up to remove residual dye and enzyme. Sequencing was with M13 forward
(GTAAAACGACGGCCAG)
and
reverse
(CAGGAAACAGCTATGAC) primers for 25 cycles of 96°C, 10 s;
50°C, 5 s; and 60°C, 4 min. The sequenced products were
sorted and analyzed with Sequencher™ 4.8 (Gene Codes
Corporation, Ann Arbor, MI).
Subtractive Hybridization
Subtractive hybridization was performed with the PCRSelect™ cDNA subtraction kit (Clontech, Palo Alto, CA).
Random hexamer primers (Clontech) were used to convert mRNA that had been extracted from desiccated and
control nematodes into cDNA. The cDNA under investigation was designated tester, and the reference cDNA was
designated driver. In the forward run cDNA from desiccated samples was designated as tester, in a reverse run
control nematode cDNA was used as tester to investigate
both up- and down-regulation of mRNA molecules. Second strands of driver and tester DNA were synthesized
using T4 DNA polymerase and restriction digested by RsaI
to create blunt-ended double stranded cDNA for subtraction. The tester cDNAs were subdivided into two portions
and each group ligated to different adaptor sequence and
subjected to two levels of subtractive hybridizations as
described in the manufacturer's protocol. The DE cDNAs
were selectively amplified by two PCR reactions using 50×
Advantage cDNA Polymerase mix. The first PCR amplified
double stranded cDNA with different adaptor sequences
and the second PCR further reduced background and
enriched for DE sequences. The PCR-amplified cDNA
fragments generated by SSH were then ligated into the
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TOPO® TA Cloning® vector (Invitrogen Corporation, USA)
and sequenced using ABI 3730xl DNA Analyzer (Applied
Biosystems, CA) at the BYU DNASC.
Sequence analysis and EST clustering
Base calling was performed using PHRED software (versions 0.000925.c) [82,83] with the quality cut-off set at
PHRED 20. Raw sequences were then imported into the
Vector NTI Advance™ 10 software (Invitrogen Corporation) and subjected to trimming of vector sequences and
5' adapter sequences (for subtractive hybridization) using
default settings. Sequences with less than 100 quality
bases (PHRED 20 or better) after trimming were discarded. Sequences having polyA tails of 100 bases or more
were eliminated from analysis. EST sequences representing contamination from bacterial, yeast or fungal sources
were identified using BLASTN algorithm [34,35] and
removed from further analyses. ESTs were aligned and
assembled into contigs using CAP3 software [84] when
the criterion of a minimum identity of 95% over 50 bp
was met. When an EST could not be assembled with others in a contig, it remained as a "singleton". The contigs
and the singletons should thus correspond to sequences
of unique genes. The consensus sequences of the contigs
and the sequences of the singlets were compared to the
sequences in GenBank's non-redundant (nr) and Uniprot
database using the TBLASTX and the BLASTX [38,39] algorithms and C. elegans Wormpep 190 database [36] using
the BLASTX algorithm [35]. The cut-off for sequence similarity was E-value < 10-5 for all analyses.
Functional analysis and pathway assignments
Gene ontology (GO) term annotation and function-based
analysis [85] of unique sequences were performed using
Blast2go (V 1.6.2) [86], a sequence-based tool to assign GO
terms, extracting them for each BLAST hit obtained by mapping to extant annotation associations. GO terms for each
of the three main categories (biological process, molecular
function, and cellular component) was obtained from
sequence similarity using the application default parameters. From these annotations, pie charts were made using
2nd level GO terms based on biological process, molecular
function, and cellular component. Pathway assignments
were carried out according to Kyoto encyclopedia of genes
and genomes (KEGG) mapping [43]. Enzyme commission
(EC) numbers were assigned to unique sequences that had
BLASTX scores with a cut-off value of E = 10-5 or less upon
searching protein databases. The sequences were mapped
to KEGG biochemical pathways according to the EC distribution in the pathway database.
Primer Design
All the primers used in quantitative real-time PCR (qRTPCR) were designed with IDT SciTools (Integrated DNA
Technologies, Coralville, IA) by aligning EST sequences

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with similar sequences from NCBI. All the primers used in
this study were synthesized by Operon (Operon Biotechnologies Inc., Huntsville, AL). The sequences of the primers and product sizes are listed in Additional file 3.
Quantitative Real-time PCR
Total RNA extracted from dehydrated and control P. murrayi nematodes was reverse transcribed using ImProm-II™
reverse transcriptase (Promega corporation, Madison, WI)
and subjected to qRT-PCR analysis using Light cycler 480
SYBER Green I mastermix and gene specific primers in a
Light cycler 480 RT-PCR system (Roche Applied Science,
Mannheim, Germany) equipped with light cycler 480
software. High-resolution gel electrophoresis was used to
verify that the qRT-PCR amplification product from each
examined gene was a single-band product. Thermal
cycling was performed in accordance with the manufacturer's instructions for a total of 40 cycles at an annealing
temperature of 58°C for each primer pair. Quantitative
RT-PCR analysis was performed with Lightcycler 480 software, the threshold cycle was automatically calculated by
the second-derivative maximum method, and the copy
number of the specific mRNA in the experimental samples
was calculated by extrapolation from the gene-specific
standard curve.
Data analyses
The copy number of specific cDNA molecules present in
the samples was determined by absolute quantification
method of qPCR analysis [45]. A range of six dilutions
(107–102 copies) of the cDNA was made and a gene-specific external standard curve was generated by using cDNA
standards that were run simultaneously with the experimental samples. Change in target gene expression was calculated using equation 2-ΔΔCT [87]. The fold change in the
target gene, normalized to 18S rRNA (Pm-18s) and relative to the expression of control, was calculated for each
sample. A gene with a relative abundance of one is equal
to the abundance of 18S rRNA in the same sample. An Ftest at a significance level of P < 0.05 was used to compare
the ratio of the mean gene expression of desiccated samples with that of the control. To minimize mRNA quantification errors, genomic DNA contamination biases and
to correct for inter-sample variations, we used 18s ribosomal RNAs (rRNAs) of P. murrayi as an internal control.

Authors' contributions
BNA carried out most of the work described here including conception and design of experiments, acquisition of
data, analysis and interpretation of data and drafting the
manuscript. DHW and BJA contributed to sample and culture collection, conception and design of experiments,
supervision of the work and critical review of the manuscript. All authors read and approved the final manuscript.

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http://www.biomedcentral.com/1471-2164/10/69

Additional material
Additional File 1

3.

Distribution of (a) molecular functions, (b) cellular components, and
(c) biological process categories based on gene ontology for Plectus
murrayi unique sequences. Distribution of (a) molecular functions, (b)
cellular components, and (c) biological process categories based on gene
ontology for Plectus murrayi unique sequences. More information provided in Figure 2a, b and 2c. Note that individual GO categories can have
multiple mappings.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-69-S1.doc]

7.

Additional File 2

8.

Analysis of mRNA copy number (×107) of Pm-hsp-90: heat shock protein 90 and Pm-hsp-70: heat shock protein 70 gene in Plectus murrayi under desiccated and normal condition. Analysis of mRNA copy
number (×107) of Pm-hsp-90: heat shock protein 90 and Pm-hsp-70:
heat shock protein 70 gene in Plectus murrayi under desiccated and normal condition. The experiment was performed using an absolute quantitation method of quantitative real-time PCR analysis with each value
represents the mean ± SE of three replicates. Nematode samples were
exposed to 97 and 85% RH for 3 and 2 days respectively prior to RNA
extraction. Controls received no treatment. *Significant difference (P <
0.05) from control.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-69-S2.doc]

4.
5.
6.

9.
10.

11.
12.
13.
14.
15.

Additional File 3
List of the gene specific primer sequences used for quantitative realtime PCR analysis. List of the gene specific primer sequences used for
quantitative real-time PCR analysis. Primers were designed by aligning
the EST sequences with their putative homologue from GenBank using
IDT SciTools (Integrated DNA Technologies, Coralville, IA, USA) and
synthesized by Operon (Operon Biotechnologies Inc., Huntsville, AL,
USA).
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-69-S3.doc]

Acknowledgements
This work was supported by the National Science Foundation McMurdo
Long Term Ecological Research Project (OPP #98-10219) to DHW, the
United States Department of Agriculture CSREES NRI (2005-00903) to
BJA, and a Brigham Young University Mentored Environment Grant to BJA.
We thank C-Y Lin, J. Griffitts, and G. F. Burton for their critical advice during the planning and execution of these experiments. We are grateful to J.
A. Udall for bioinformatics assistance, and three anonymous reviewers
whose constructive criticism significantly improved the paper. Funding for
the Open Access publication charges for this article was provided by the
Brigham Young University College of Life Sciences and Department of Biology.

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