Molecular snapshot of an intracellular freezing event in an Antarctic nematode

30 Pages·2017·3.34 MB·English

Accepted Manuscript
Molecular snapshot of an intracellular freezing event in an Antarctic nematode
Michael A.S. Thorne, Anna Seybold, Craig Marshall, David Wharton
PII: S0011-2240(16)30407-2
DOI: 10.1016/j.cryobiol.2017.01.003
Reference: YCRYO 3805
To appear in: Cryobiology
Received Date: 8 November 2016
Revised Date: 19 December 2016
Accepted Date: 8 January 2017
Please cite this article as: M.A.S. Thorne, A. Seybold, C. Marshall, D. Wharton, Molecular snapshot
of an intracellular freezing event in an Antarctic nematode, Cryobiology (2017), doi: 10.1016/
j.cryobiol.2017.01.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT

1 MMMMoooolllleeeeccccuuuullllaaaarrrr ssssnnnnaaaappppsssshhhhooootttt ooooffff aaaannnn iiiinnnnttttrrrraaaacccceeeelllllllluuuullllaaaarrrr ffffrrrreeeeeeeezzzziiiinnnngggg eeeevvvveeeennnntttt iiiinnnn aaaannnn AAAAnnnnttttaaaarrrrccccttttiiiicccc nnnneeeemmmmaaaattttooooddddeeee
2
3 Michael A.S. Thorne1*, Anna Seybold2, Craig Marshall2, David Wharton3
4 T
5 1British Antarctic Survey, Cambridge, UK
P
6 2Department of Biochemistry, and Genetics Otago, University of Otago, Dunedin, New Zealand
I
7 3Department of Zoology, University of Otago, Dunedin, New Zealand
R
8 *Corresponding author, [email protected]
9 C
10 TTTThhhheeee AAAAnnnnttttaaaarrrrccccttttiiiicccc nnnneeeemmmmaaaattttooooddddeeee,,,, PPPPaaaannnnaaaaggggrrrroooollllaaaaiiiimmmmuuuussss sssspppp.... DDDDAAAAWWWW1111 ((((ffffoooorrrrmmmmeeeerrrrllllyyyy ccccaaaalllllllleeeedddd PPPPaaaannnnaaaaggggrrrroooollllaaaaiiiimmmmuuuussss
S
11 ddddaaaavvvviiiiddddiiii)))),,,, iiiissss tttthhhheeee bbbbeeeesssstttt ddddooooccccuuuummmmeeeennnntttteeeedddd eeeexxxxaaaammmmpppplllleeee ooooffff aaaannnn oooorrrrggggaaaannnniiiissssmmmm aaaabbbblllleeee ttttoooo ssssuuuurrrrvvvviiiivvvveeee
U
12 iiiinnnnttttrrrraaaacccceeeelllllllluuuullllaaaarrrr iiiicccceeee ffffoooorrrrmmmmaaaattttiiiioooonnnn iiiinnnn aaaallllllll ooooffff iiiittttssss ccccoooommmmppppaaaarrrrttttmmmmeeeennnnttttssss.... NNNNooootttt oooonnnnllllyyyy iiiissss iiiitttt aaaabbbblllleeee ttttoooo ssssuuuurrrrvvvviiiivvvveeee
13 ssssuuuucccchhhh eeeexxxxttttrrrreeeemmmmeeee pppphhhhyyyyssssiiiioooollllooooggggiiiiccccaaaallll ddddiiiissssrrrruuuuppppttttiiiioooonnnn,,,, bbbbuuuutttt iiiitttt iiiissss aaaabbbbllllNeeee ttttoooo pppprrrroooodddduuuucccceeee pppprrrrooooggggeeeennnnyyyy oooonnnncccceeee
14 tttthhhhaaaawwwweeeedddd ffffrrrroooommmm ssssuuuucccchhhh aaaa ssssttttaaaatttteeee.... IIIInnnn aaaaddddddddiiiittttiiiioooonnnn,,,, uuuunnnnddddeeeerrrr sssslllloooowwwweeeerrrr rrrraaaatttteeeessss,,,, oooorrrr lllleeeessssssss eeeexxxxttttrrrreeeemmmmeeee
A
15 ddddeeeeggggrrrreeeeeeeessss,,,, ooooffff ccccoooooooolllliiiinnnngggg,,,, iiiittttssss bbbbooooddddyyyy rrrreeeemmmmaaaaiiiinnnnssss uuuunnnnffffrrrroooozzzzeeeennnn aaaannnndddd tttthhhheeee vvvvaaaappppoooouuuurrrr pppprrrreeeessssssssuuuurrrreeee ddddiiiiffffffffeeeerrrreeeennnncccceeee
M
16 bbbbeeeettttwwwweeeeeeeennnn tttthhhheeee ssssuuuuppppeeeerrrrccccoooooooolllleeeedddd bbbbooooddddyyyy fffflllluuuuiiiiddddssss aaaannnndddd tttthhhheeee ssssuuuurrrrrrrroooouuuunnnnddddiiiinnnngggg iiiicccceeee lllleeeeaaaaddddssss ttttoooo aaaa pppprrrroooocccceeeessssssss
17 tttteeeerrrrmmmmeeeedddd ccccrrrryyyyoooopppprrrrooootttteeeeccccttttiiiivvvveeee ddddeeeehhhhyyyyddddrrrraaaattttiiiioooonnnn.... IIIInnnn ccccoooonnnnttttrrrraaaasssstttt ttttoooo aaaa ffffaaaaiiiirrrrllllyyyy llllaaaarrrrggggeeee bbbbooooddddyyyy ooooffff wwwwoooorrrrkkkk iiiinnnn

18 bbbbuuuuiiiillllddddiiiinnnngggg uuuupppp oooouuuurrrr mmmmoooolllleeeeccccuuuullllaaaarrrr uuuunnnnddddeeeerrrrssssttttaaaannnnddddiiiinnnnDgggg ooooffff ccccrrrryyyyoooopppprrrrooootttteeeeccccttttiiiivvvveeee ddddeeeehhhhyyyyddddrrrraaaattttiiiioooonnnn,,,, nnnnoooo
19 ccccoooommmmppppaaaarrrraaaabbbblllleeee wwwwoooorrrrkkkk hhhhaaaassss bbbbeeeeeeeennnn uuuunnnnddddeeeerrrrttttaaaakkkkeeeennnn oooonnnn iiiinnnnttttrrrraaaacccceeeelllllllluuuullllaaaarrrr ffffrrrreeeeeeeezzzziiiinnnngggg.... TTTThhhhiiiissss ppppaaaappppeeeerrrr
E
20 ddddeeeessssccccrrrriiiibbbbeeeessss aaaannnn eeeexxxxppppeeeerrrriiiimmmmeeeennnntttt ssssuuuubbbbjjjjeeeeccccttttiiiinnnngggg ccccuuuullllttttuuuurrrreeeessss ooooffff PPPPaaaannnnaaaaggggrrrroooollllaaaaiiiimmmmuuuussss sssspppp.... DDDDAAAAWWWW1111 ttttoooo aaaa
21 rrrraaaannnnggggeeee ooooffff tttteeeemmmmppppeeeerrrraaaattttuuuurrrreeeessss iiiinnnncccclllluuuuddddiiiinnnnggggT aaaa rrrraaaappppiiiidddd ddddeeeesssscccceeeennnntttt ttttoooo ----11110000ººººCCCC,,,, iiiinnnn aaaa mmmmeeeeddddiiiiuuuummmm jjjjuuuusssstttt pppprrrriiiioooorrrr
22 ttttoooo,,,, aaaannnndddd aaaafffftttteeeerrrr,,,, ffffrrrreeeeeeeezzzziiiinnnngggg.... TTTThhhhrrrroooouuuugggghhhh ddddeeeeeeeepppp sssseeeeqqqquuuueeeennnncccciiiinnnngggg ooooffff RRRRNNNNAAAA lllliiiibbbbrrrraaaarrrriiiieeeessss wwwweeee hhhhaaaavvvveeee ggggaaaaiiiinnnneeeedddd
P
23 aaaa ssssnnnnaaaappppsssshhhhooootttt ooooffff wwwwhhhhiiiicccchhhh ggggeeeennnneeeessss aaaarrrreeee hhhhiiiigggghhhhllllyyyy aaaabbbbuuuunnnnddddaaaannnntttt wwwwhhhheeeennnn PPPP.... sssspppp.... DDDDAAAAWWWW1111 iiiissss uuuunnnnddddeeeerrrrggggooooiiiinnnngggg
E
24 aaaannnn iiiinnnnttttrrrraaaacccceeeelllllllluuuullllaaaarrrr ffffrrrreeeeeeeezzzziiiinnnngggg eeeevvvveeeennnntttt.... TTTThhhheeee oooonnnnsssseeeetttt ooooffff ffffrrrreeeeeeeezzzziiiinnnngggg ccccoooorrrrrrrreeeellllaaaatttteeeedddd wwwwiiiitttthhhh aaaa hhhhiiiigggghhhh
25 pppprrrroooodddduuuuccccttttiiiioooonnnn ooooffff ggggeeeennnneeeessss iiiinnnnCvvvvoooollllvvvveeeedddd iiiinnnn ccccuuuuttttiiiicccclllleeee ffffoooorrrrmmmmaaaattttiiiioooonnnn aaaannnndddd ssssuuuubbbbsssseeeeqqqquuuueeeennnnttttllllyyyy,,,, aaaafffftttteeeerrrr 22224444 hhhhoooouuuurrrrssss
26 iiiinnnn aaaa ffffrrrroooozzzzeeeennnn ssssttttaaaatttteeee,,,, pppprrrrooootttteeeeaaaasssseeee pppprrrroooodddduuuuccccttttiiiioooonnnn.... IIIInnnn aaaaddddddddiiiittttiiiioooonnnn ttttoooo tttthhhheeee mmmmaaaappppppppiiiinnnngggg ooooffff RRRRNNNNAAAA
C
27 sssseeeeqqqquuuueeeennnncccciiiinnnngggg,,,, wwwweeee hhhhaaaavvvveeee ffffooooccccuuuusssseeeedddd oooonnnn aaaa sssseeeelllleeeecccctttt sssseeeetttt ooooffff ggggeeeennnneeeessss aaaarrrriiiissssiiiinnnngggg bbbbooootttthhhh ffffrrrroooommmm tttthhhheeee
A
28 eeeexxxxpppprrrreeeessssssssiiiioooonnnn pppprrrrooooffffiiiilllleeeessss,,,, aaaassss wwwweeeellllllll aaaassss iiiimmmmpppplllliiiiccccaaaatttteeeedddd ffffrrrroooommmm ooootttthhhheeeerrrr ccccoooolllldddd ttttoooolllleeeerrrraaaannnncccceeee ssssttttuuuuddddiiiieeeessss,,,, ttttoooo
29 uuuunnnnddddeeeerrrrttttaaaakkkkeeee qqqqPPPPCCCCRRRR.... AAAAmmmmoooonnnngggg tttthhhheeee mmmmoooosssstttt aaaabbbbuuuunnnnddddaaaannnnttttllllyyyy rrrreeeepppprrrreeeesssseeeennnntttteeeedddd ttttrrrraaaannnnssssccccrrrriiiippppttttssss iiiinnnn tttthhhheeee RRRRNNNNAAAA
30 mmmmaaaappppppppiiiinnnngggg iiiissss tttthhhheeee zzzziiiinnnncccc----mmmmeeeettttaaaallllllllooooeeeennnnzzzzyyyymmmmeeee,,,, nnnneeeepppprrrriiiillllyyyyssssiiiinnnn,,,, wwwwhhhhiiiicccchhhh aaaallllssssoooo sssshhhhoooowwwwssss aaaa ppppaaaarrrrttttiiiiccccuuuullllaaaarrrrllllyyyy
31 ssssttttrrrroooonnnngggg uuuupppprrrreeeegggguuuullllaaaatttteeeedddd ssssiiiiggggnnnnaaaallll tttthhhhrrrroooouuuugggghhhh qqqqPPPPCCCCRRRR oooonnnncccceeee tttthhhheeee nnnneeeemmmmaaaattttooooddddeeeessss hhhhaaaavvvveeee ffffrrrroooozzzzeeeennnn....
32
33 keywords: Panagrolaimus davidi, Antarctic, nematode, intracellular freezing,
34 neprilysin, leucine-rich repeat.
35

1
ACCEPTED MANUSCRIPT

36 IIIInnnnttttrrrroooodddduuuuccccttttiiiioooonnnn
37
38 Apart from the work of Salt in describing intracellular freezing in the large fat body
39 cells and labial glands of the goldenrod gall fly, Eurosta solidaginis, in theT late 50's
40 [44,45,46,50], the study of the phenomenon of intracellular freezing in vivo has
P
41 remained little understood, and little explored. However, some recent physiological
I
42 studies on the Antarctic nematode, P. sp. DAW1 [67,68,38,69,70,72,73,39,40],
R
43 provides the necessary background to now bring molecular techniques to bear on
44 an extraordinary adaptation. C
45
S
46 A free-living bacteriovorous nematode, Panagrolaimus sp. DAW1 [61] (previously
U
47 called Panagrolaimus davidi, see Raymond & Wharton [40]), was first isolated from
48 the McMurdo Sound region of the Antarctic in 198N9 and cultured at the University
49 of Otago [66]. The culture was found to survive intracellular freezing [68], with the
A
50 ability to produce progeny afterwards. To date, P. sp. DAW1 remains the best
51 documented case of such survival, even amoMng other Antarctic nematodes
52 [51,74,30]. P. sp. DAW1 also has the ability to undergo cryoprotective

53 dehydration when subjected to high sub-zero temperatures or slower rates of
D
54 cooling [71,73]. However, while an increasingly detailed molecular picture is
55 emerging of cryoprotective dehydraEtion [8,9,59,57,18,58], no comparable
56 molecular work has been undertTaken on the survival of intracellular freezing at the
57 whole organism level. The study of P. sp. DAW1 provides an increasingly detailed
P
58 physiological understanding of intracellular ice formation on which to build up a
E
59 molecular picture of the process.
60 C
61 While there are many organisms able to withstand ice formation in extracellular
C
62 spaces [1,53,64,17,20,16,65,26,10,54], the ability to withstand such disruption
63 within the cellsA, and survive with the ability to produce progeny afterwards, is
64 much rarer. In P. sp. DAW1, although the cuticle is a sufficient barrier to ice, at
65 low sub-zero temperatures inoculative freezing can spread through the excretory
66 pore and other orifices, with the ice seeding the body fluid and freezing all,
67 including intracellular, compartments [68,73]. One of the ideas on the more lethal
68 consequences of this freezing is the physical damage to the cell membranes once
69 the smaller ice crystals that have initially formed, themselves begin to cluster
70 together, or recrystallise [23,38,73]. Traditionally the term antifreeze protein has

2
ACCEPTED MANUSCRIPT

71 been used to designate any protein involved in the inhibition of freezing. Recently,
72 some authors have argued for a more general term for proteins that play a role in
73 ice formation and in combating ice recrystallisation [72,12]. These ice-active
74 proteins (IAP) can then be classed according to one of three types: Ice nTucleating
75 proteins (INP), which help to initiate ice formation; antifreeze proteins (AFP),
P
76 which prevent ice nucleation by creating a thermal hysteresis (TH) between the
I
77 melting and freezing point of a sample in the presence of an ice crystal; and
R
78 recrystallisation-inhibition proteins (RIP), which inhibits recrystallisation, the
C
79 aforementioned process in which larger ice crystals form at the expense of smaller
80 more numerous crystals. Although most RIPs also have at least low levels of
S
81 thermal hysteresis activity, in some cases extracts of organisms with RIP activity
U
82 have to be concentrated to a high degree before TH activity can be detected. RIPs
83 tend to be found in freeze tolerant species while AFNPs (with more substantive TH)
84 are mostly present in freeze avoiding species. However, in Panagrolaimus sp.
A
85 DAW1, given the extreme freezing disruption that it can experience, and the fact
M
86 that it is freeze tolerant, it is expected that its IAPs will not include AFPs [38],
87 but rather RIPs. A further clue on the possible importance of RIPs in the survival

88 of intracellular freezing, comes from a Drecent study by Raymond and Wharton [40],
89 where the smaller the sizes of the ice crystals is correlated positively with the
E
90 survival rate. But to date, no IAP of any kind has been isolated in P. sp. DAW1
T
91 [60], even though they have been shown to play a role in freezing tolerance [72].
92
P
93 Among other important classes of genes are those involved in the sugar
E
94 biosynthetic pathways (such as those producing trehalose), Late Embryogenesis
95 Abundant (LEA) proteCins, chaperone proteins (such as the heat shock proteins),
96 and enzymes involved in antioxidant metabolism. These all play a role in the
C
97 process of cryoprotective dehydration, and have been detected in P. sp. DAW1
A
98 [60]. While it is not clear what role, if any, these play in the nematodes' survival of
99 intracellular freezing, it would be highly surprising if they were not involved.
100
101 The extent of survival of P. sp. DAW1 when subjected to intracellular freezing is
102 dependent, however, on at least two properties. Wharton and To [69] have shown
103 decreased survival ability when under either hyper- or hypo-osmotic stress. While
104 Raymond and Wharton [39,40] have shown that nutritional status is an important
105 factor, possibly related to the fact that glycogen levels decrease when under

3
ACCEPTED MANUSCRIPT

106 nourished. These two studies imply that unless in a relatively stress-free state,
107 Panagrolaimus sp. DAW1 is unable to endure the extreme physiological disruption
108 of intracellular freezing, suggesting that the physiological conditions that allow for
109 a sudden rate of cooling and intracellular freezing are at the very limit ofT its
110 physiological endurance.
P
111
I
112 With all the physiological work that has been undertaken a number of key insights
R
113 have been gained, yet there remains little understanding at the molecular level. To
114 address this, we have generated sets of transcripts expressedC in P. sp. DAW1
115 when the nematodes have been cooled rapidly to a relatively low sub-zero
S
116 temperature (-10°C), both before and after (immediately and post 24 hours) the
U
117 medium has frozen. These treatments were compared with controls that include
118 stages in which P. sp. DAW1 is expected to undergNo both cold acclimation and
119 cryoprotective dehydration. This approach provides a snapshot of highly abundant
A
120 transcripts from genes novel to the intracellular freezing process and distinct from
M
121 those generally involved in responses to stress. We have further validated the
122 results through qPCR analysis on genes selected both from within the transcript

123 analysis, as well as those implicated in Dother cold tolerance studies.
124
E
125 MMMMaaaatttteeeerrrriiiiaaaallllssss aaaannnndddd mmmmeeeetttthhhhooooddddssss
126 T
127 CCCCuuuullllttttuuuurrrriiiinnnngggg aaaannnndddd eeeexxxxppppeeeerrrriiiimmmmeeeennnnttttaaaallll ddddeeeessssiiiiggggnnnn
P
128
E
129 The culturing and experimental treatments of the nematodes consisted of control
130 (culturing temperatureC and cold acclimation), cryoprotective dehydration and
131 intracellular freezing stages. The control and cryoprotective stages consisted of
C
132 the following:
A
133
134 Treatment 1, culture temperature: P. sp. DAW1 were cultured in S medium at
135 20°C for 3 weeks and fed every 3-4 days with Escherichia coli [39].
136
137 Treatment 2, cold acclimation: After treatment 1, the nematodes were exposed to
138 cold acclimation by keeping them at +5°C for 3 days. This duration at +5°C was
139 used because longer periods of acclimation can be detrimental to the ability to
140 survive intracellular freezing, due to the effects of starvation. This acclimation

4
ACCEPTED MANUSCRIPT

141 regime is known to produce about 80% survival in nematodes frozen at -10°C, at
142 which temperature all individuals freeze intracellularly [39, 40].
143
144 T
145 Nematodes from treatments 1 and 2 were extracted for the subsequent
P
146 experiments using a modified Baermann technique [19] and transferred to 1.5 ml
I
147 microcentrifuge tubes in a balanced salt solution (BSS) [36] before snap freezing in
R
148 liquid nitrogen and storage at -70°C.
C
149
150 Treatment 3, cryoprotective dehydration: After cold acclimation, nematodes were
S
151 extracted and transferred to 1 ml of BSS in 1.5 ml microcentrifuge tubes and
U
152 placed in an aluminium holder immersed in the bath of a refrigerated circulator.
153 The sample was cooled from +1°C to -1°C at 0.N5°C min-1, and the sample
154 frozen by adding a small ice crystal and maintained at -1°C for 24 h. Under these
A
155 conditions, the medium freezes but not the nematodes.
M
156
157 Treatment 4, cryoprotective dehydration thawing: After treatment 3 the

158 nematodes were warmed to +1°C at 0D.5°C min-1 and allowed to recover for 24 h
159 at 20°C.
E
160
T
161 The intracellular freezing stages (the stages in which ice is formed inside the cells
162 of P. sp DAW1) consisted of the following:
P
163
E
164 Treatment 5U, -10°C in unfrozen media: The samples were cooled to -10°C at
165 0.5°C min-1. When tChis temperature was reached, it was noted whether the
166 medium had frozen or not, and those in unfrozen medium removed for extraction.
C
167
A
168 Treatment 5F, -10°C in frozen media: The samples were cooled to -10°C at
169 0.5°C min-1 as with Treatment 5U. When this temperature was reached samples
170 that had frozen spontaneously during cooling were removed and labelled.
171
172 Treatment 6, -10°C for 24h: Samples subjected to treatment 5F were kept frozen
173 at -10°C for 24 h.
174
175 Treatment 7, thawing and recovery from intracellular freezing: Samples subjected

5
ACCEPTED MANUSCRIPT

176 to treatment 6 were warmed to +1°C at 0.5°C min-1 and then kept at 5°C for
177 24 h. Subsamples (four 2µl samples diluted 4× with BSS and incubated at 5°C)
178 were taken to determine survival and the proportion moving, after a physical
179 stimulus (expelling from a pipette), counted at intervals up to 24 hours, aTfter which
180 no additional increase in survival rate would be expected. The time taken for 50%
P
181 of the nematodes to recover, and 95% confidence limits (CL), was determined using
I
182 probit analysis in SPSS after log transformation of time [31].
10 R
183
C
184 After treatment, nematodes were snap frozen in liquid nitrogen before storage at -
185 70°C, ready for RNA extraction.
S
186
U
187 IIIIlllllllluuuummmmiiiinnnnaaaa lllliiiibbbbrrrraaaarrrryyyy ccccoooonnnnssssttttrrrruuuuccccttttiiiioooonnnn aaaannnndddd sssseeeeqqqquuuueeeennnncccciiiinnnngggg
188 N
189 Thorne et al. [60] describe the process of extraction, library construction, and
A
190 sequencing on an Illumina HiSeq 2000. After quality control, this resulted in 70,897,520
M
191 paired-end reads of 100bp from Treatment 1 (deposited in the NCBI SRA repository
192 (www.ncbi.nlm.nih.gov/sra) under SRR5091936 ), 73,894,280 from treatment 2

193 (SRR5092011), 73,249,548 from treatmDent 3 (SRR5092012), 69,830,160 from treatment 4
194 (SRR5092013), 72,354,956 from treatment 5U (SRR5092108), 69,940,196 from treatment
E
195 5F (SRR5092338), 69,304,572 from treatement 6 (SRR5094587) and 73,423,192 from
T
196 treatment 7 (SRR5094588).
197
P
198 SSSSeeeeppppaaaarrrraaaatttteeee lllliiiibbbbrrrraaaarrrryyyy aaaasssssssseeeemmmmbbbbllllyyyy aaaannnndddd ffffuuuunnnnccccttttiiiioooonnnnaaaallll aaaannnnaaaallllyyyyssssiiiissss
E
199
200 Illumina reads from allC the different treatments were assembled separately and
201 annotated to identify any broad patterns of difference in the transcripts. The
C
202 program Soapdenovo [28] was used to assemble the transcripts using default
A
203 (genome style) parameters. Contigs greater than 300bp in length were selected
204 from each treatment and annotated against the non-redundant database housed at
205 Genbank (www.ncbi.nlm.nih.gov/genbank). Each transcript set was then
206 functionally annotated using the SEED subsystem [34].
207
208 MMMMaaaappppppppiiiinnnngggg tttthhhheeee RRRRNNNNAAAA lllliiiibbbbrrrraaaarrrriiiieeeessss aaaannnndddd ddddeeeetttteeeerrrrmmmmiiiinnnniiiinnnngggg tttthhhheeee hhhhiiiigggghhhhllllyyyy aaaabbbbuuuunnnnddddaaaannnntttt ttttrrrraaaannnnssssccccrrrriiiippppttttssss
209
210 Using a transcriptome backbone (previously described in [60]), the paired-end

6
ACCEPTED MANUSCRIPT

211 Illumina sequencing reads from the treatments were mapped back using Maq [27]
212 and normalised by a transcripts per million approach. All the treatments taken
213 together have allowed for the comparison of intracellular freezing against not only
214 control cultures of actively reproducing (20°C) and cold acclimated (5°TC)
215 nematodes, but also those in which P sp. DAW1 would be expected to undergo
P
216 cryoprotective dehydration (-1°C). We compared each of the intracellular stages
I
217 (-10°C unfrozen; -10°C frozen; -10°C frozen 24 h; and after freezing to -
R
218 10°C, thawing and recovery), against all the preceding stages so as to enrich for
C
219 those transcripts that are more likely to be expressed during intracellular freezing.
220 Each intracellular treatment comparison required at least a 2-fold change in
S
221 expression vis-a-vis all the previous stages, as well as a ratio test [37] with a
U
222 multiple correction cutoff of 0.01 [4]. The sets of transcripts from the comparisons
223 were additionally annotated with the C. elegans datNabase (wormbase.org), and the
224 results used in STRING [56], which provided not only the gene network analysis,
A
225 but also the enrichment for various functional representations such as GO, PFAM,
M
226 KEGG and INTERPRO. Comparison of the neprilysin transcripts was aided by
227 Clustal [25], T-Coffee [32] and Boxshade (sourceforge.net/projects/boxshade/).

228 D
229 qqqqPPPPCCCCRRRR
E
230
T
231 Primer design
232
P
233 Both the genomic DNA and cDNA sequences from P. sp. DAW1 [60] were used to
E
234 develop the qPCR primers. To identify and target introns, as well as to avoid
235 amplification of any coCntaminating genomic DNA, both cDNA and genomic DNA
236 sequences were aligned through in-house homology search tools and Spidey
C
237 (www.ncbi.nlm.nih.gov/spidey). Primers were designed using Primer3web
A
238 (primer3.ut.ee), and potential primers analyzed for possible dimer formation by
239 Beacon Designer Free Edition (www.premierbiosoft.com) and for specificity by
240 NCBI Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast).
241
242 Quantitative polymerase chain reaction (qPCR)
243
244 Quantitative PCR was performed using the BioRad CFX96 System (Hercules, CA,
245 USA) and theBioRad SSoFast EVA Green Supermix with Low Rox. A 20 μL

7
ACCEPTED MANUSCRIPT

246 reaction contained a 5 μL sample (total of 50 ng cDNA), 10 μL SYBR green mix,
247 1.2 μL primer mix and 3.2 μL mQ water. Primers were developed as described
248 above and details are shown in Supplementary file 1. Specificity and efficiency
249 assays were performed for all genes and details of these are shown in T
250 Supplementary file 1. Of six reference genes tested, the combination of Pd-gpd-2
P
251 and Pd-tba-1 (see Table 2 in Supplementary file 1) were defined as the most stable
I
252 and used for all qPCR experiments [7].
R
253
C
254 Data analysis
255 S
256 The qPCR data were analyzed using the BioRad CFX MUanager. Means, standard
257 deviations (s.d.), P-values and relative expressions of the normalized expression
N
258 values were calculated in MS Excel. P-values were assessed using a t-test
259 (parametric, two samples, equal variance). The AΔΔCt (Livak) method was used to
260 determine the relative difference in expression level of the target gene in different
M
261 samples. In the first step, the Ct of the target gene was normalized to that of the
262 reference gene, for both the test sample and the control sample (normalized
D
263 relative expression value). Then, the ΔCt of the test sample was normalized to
264 that of the control sample (relativeE expression).
265
T
266 RRRReeeessssuuuullllttttssss aaaannnndddd DDDDiiiissssccccuuuussssssssiiiioooonnnn
267 P
268 SSSSuuuurrrrvvvviiiivvvvaaaallll aaaafffftttteeeerrrr ffffrrrreeeeeeeezzzziiiinnnngggg E
269
C
270 Survival after freezing at -10°C, followed by thawing and recovery at 5°C was
271 83.9 ± 3.5% (meaCn ± 1 s.e., N = 4), and the nematodes took several hours to
272 recover from the freezing stressor (Figure 1), with a 50% recovery time of 7.89 h
A
273 (95% CL = 7.22, 8.69). These values are in agreement with previous survival
274 analyses post freezing [39].
275
276 FFFFuuuunnnnccccttttiiiioooonnnnaaaallll aaaannnnaaaallllyyyyssssiiiissss ooooffff sssseeeeppppaaaarrrraaaatttteeee ttttrrrreeeeaaaattttmmmmeeeennnntttt ttttrrrraaaannnnssssccccrrrriiiippppttttoooommmmeeee aaaasssssssseeeemmmmbbbblllliiiieeeessss
277
278 Illumina data from each of the treatments were assembled separately using identical
279 criteria. The resulting assemblies contained similar numbers of transcripts and
280 annotation rates: Treatment 1 produced 14,742 transcripts, of which 54.7% were

8
ACCEPTED MANUSCRIPT

281 annotated; treatment 2, 16,349 transcripts with 52.5% annotated; treatment 3,
282 14,804 transcripts with 53.2% annotated; treatment 4, 14,718 transcripts with 53%
283 annotation; treatment 5U, 13,481 transcripts of which 55% were annotated;
284 treatment 5F 16,670 transcripts with 52% annotated; treatment 6, 14,493T with 54%
285 annotated; and treatment 7, 14,179 transcripts with 54% annotated. The
P
286 annotations by library can be seen in the Supplementary files 2-9. SEED
I
287 subsystem analysis was carried out on all the separate treatment assemblies to
R
288 assess overall functional differences and is depicted in Supplementary Table 1. The
C
289 breakdown by function is almost identical betweeen the libraries, and indicates a
290 broad distribution of functions. Protein Metabolism has the highest percentage in
S
291 all the libraries, the same as reflected in an assembly of the entire transcriptome
U
292 [60]. A subtle difference is that in treatments 2, 3 and 7, there are small
293 representations of the functional category DormancNy and Sporulation, which the
294 others do not have. Apart from this slight functional difference, the overall
A
295 comparison indicates that the machinery to undergo all the respective physiological
M
296 changes seems present in all stages of the nematodes that we have looked at. From
297 this, we conclude that it is either the abundance of particular transcripts that plays

298 a role, or that the ability to withstand Dany of the environmental disruptions tested
299 here is constitutive. This possibility was reinforced by searches in each of the
E
300 annotation files for selected relevant genes that that are discussed below.
T
301
302 MMMMoooolllleeeeccccuuuullllaaaarrrr rrrreeeessssppppoooonnnnsssseeee ttttoooo iiiinnnnttttrrrraaaacccceeeelllllllluuuullllaaaarrrr ffffrrrreeeeeeeezzzziiiinnnngggg
P
303
E
304 OOOOnnnnsssseeeetttt ooooffff ffffrrrreeeeeeeezzzziiiinnnngggg
305 C
306 As the medium temperature was brought down to -10°C, the samples either froze
C
307 spontaneously or they remained suspended in an unfrozen medium. By sampling
A
308 the nematodes in the medium that did or did not freeze spontaneously, we were
309 able to look for any large differences in the highly represented genes induced at
310 this particular transitional point. Supplementary Table 2 lists those transcripts that
311 were shown to be highly abundant that were commonly shared both before and
312 after the medium froze, and those unique to each sampling point. The overriding
313 response in both stages is collagen production with 42 (44%) and 36 (55%)
314 transcripts represented in the 5U and 5F treatments respectively. By contrast,
315 after 24 hours at -10°C, only one collagen transcript is represented. Expression

9