The New Zealand glowworm is the larva of a carnivorous fungus gnat that produces bioluminescence to attract prey. The bioluminescent system of the glowworm is evolutionarily distinct from other well-characterised systems, especially that of the fireflies, and the molecules involved have not yet been identified. We have used high throughput sequencing technology to produce a transcriptome for the glowworm and identify transcripts encoding proteins that are likely to be involved in glowworm bioluminescence.
Results
Here we report the sequencing and annotation of the first transcriptome of the glowworm, and a differential analysis of expression from the glowworm light organ compared with non-light organ tissue. The analysis identified six transcripts encoding proteins that are potentially involved in glowworm bioluminescence. Three of these proteins are members of the ANL superfamily of adenylating enzymes, with similar amino acid sequences to that of the luciferase enzyme found in fireflies (31 to 37 % identical), and are candidate luciferases for the glowworm bioluminescent system. The remaining three transcripts encode putative aminoacylase, phosphatidylethanolamine-binding and glutathione S-transferase proteins.
Conclusions
This research provides a basis for further biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm.
Background
In caves and forested river gorges across New Zealand, an abundance of star-like lights can be seen when it is dark. The creature responsible for this light, known locally as a glowworm, is the carnivorous larva of a fungus gnat, Arachnocampa luminosa. The larva lies in a mucous hammock, hanging long sticky silk threads below it like fishing lines, and luminesces from a small light organ located at the end of its tail. Small flying insects are attracted by the light, become entangled in the sticky lines, and are then consumed by the glowworm [1, 2].
Despite their common name, glowworms are actually a type of fly (order Diptera, family Keroplatidae) [3]. Confusingly, some firefly larvae and adults are also referred to as glowworms, but the well-studied bioluminescent beetles (including fireflies, click beetles and railroad worms) are members of a different order – Coeloptera (superfamily Elateroidea) [4]. Diptera and Coleoptera diverged about 330 million years ago, with no known bioluminescent species intervening [5–7], which indicates that bioluminescence evolved independently in these insects.
The ability to bioluminesce has evolved many times. One recent estimate suggests that bioluminescence has evolved at least 40 times across extant organisms, possibly more than 50 times, when counting the number of distinct light-producing chemical mechanisms across monophyletic lineages [6]. Despite their differences and separate evolutionary origins, all bioluminescent systems that have been studied produce light by oxidation of a light-emitting substrate (generically referred to as a luciferin) catalyzed by an enzyme (a luciferase). Luciferase enzymes have extremely varied structures, mechanisms and substrate specificities [8].
Researchers have studied the biochemistry used by Arachnocampa to produce light [9–11], but many details remain elusive, including the identities of the luciferase and luciferin. Although both the glowworm and firefly systems use ATP to bioluminesce, the chemistries of bioluminescence in the two creatures are distinct. When mixed, the substrate for the firefly system (luciferin) and the glowworm luciferase do not produce light [9], which implies that they use different luciferase enzymes and substrates. The physiology of the glowworm light organ is also unique. It is made up of the swollen distal tips of the four Malpighian tubules [12, 13]. Malpighian tubules are part of the insect excretory system, analogous to the vertebrate kidneys, and are not part of any other insect bioluminescence system [14].
The ability of firefly, bacteria (such as Vibrio harveyi) and sea pansy (Renilla) luciferases and luciferins to produce easily-measured light has led to the use of these systems as tools in biomedical and biological research, for example as genetic reporters, drug screening assays, bioluminescence imaging, and assays for the presence of ATP or calcium [15, 16]. Understanding the molecular basis of light generation in glowworms will not only expand our understanding of how bioluminescence works, but may also lead to novel bioluminescent applications. For example, the glowworm system uses a different luciferin substrate and produces a different bioluminescent spectra maximum than currently used bioluminescent research tools, therefore the glowworm system could be used in conjunction with existing bioluminescent applications, for example, to detect several compounds in one sample or monitor expression of multiple genes simultaneously.
One approach to revealing the molecular physiology of glowworm bioluminescence is to sequence the transcriptome of the organism. Transcriptome sequencing is a relatively cheap and easy way of providing genome-wide sequence data for non-model organisms for which no genome sequence data is available [17, 18].
Sequencing on a genome-wide scale is still a new approach for investigating bioluminescence. Although reported high-throughput sequencing of bioluminescent creatures so far include four genomes (the ctenophore Mnemiopsis leidyi [19], various strains of V. fischeri [20–22], the luminous mushroom Mycena chloropho [23], and the European brittle star Amphiura filiformis [24]), and several transcriptomes (the European brittle star [24], cypridinid ostracods or seed shrimp [25], the Oplophorid shrimp [26], the luminous mushroom [23], and the dinoflagellate Lingulodinium polyedrum [27, 28]), none of these studies have reported any detailed analyses specifically looking at the differences in transcripts produced between luminous and non-luminous tissues. It should be noted that a limited transcriptional profiling study has been carried out on A. luminosa [29]. The authors of this study sequenced 537 cDNAs that were constructed from light organ expressed sequence tags using the Sanger method, and did not include a comparison between tissues. The same research group also carried out a small transcriptional survey on a cDNA library from Macrolampis sp2 firefly lanterns [30].
Here we present the first in-depth sequencing of polyadenylated RNAs from the New Zealand glowworm, and a detailed analysis at the transcriptomic level of luminescent versus nonluminescent tissue, through which we have identified six proteins that are likely to be involved in bioluminescence. This research provides a basis for biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm.
Results and discussion
Experimental plan
We carried out two separate transcriptome sequencing experiments, on biological replicates, using the most appropriate bioinformatic processing and analyses for each approach. We first performed 454 GS-FLX sequencing because there was no genomics information available for the species, and then used Illumina sequencing to validate those results, and to provide greater sequencing depth to support a differential gene expression analysis. RNA was extracted from the light organ and from the rest of the body (non-light organ). The two experiments differed in the use of biological replicates, sequencing platform, and analysis software (Additional file 1: Figure S1).
Sequencing, read cleaning and de novo assembly
454
mRNA was isolated from two samples: one prepared from non-light organ tissue (~200 mg of tissue from eight glowworms) and one prepared from light organ tissue (~420 mg of tissue from 172 glowworms). After cDNA synthesis, 454 GS-FLX sequencing for each library was carried out on one-half of a pico-titer plate. A total of about 1.12 million high quality reads were obtained, amounting to almost 400 Mbp (Table 1; Fig. 1). Reads, including singletons, were merged from both libraries for de novo transcript assembly, which was carried out using CLC Genomics Workbench 5.1, and yielded a reference transcriptome of 18,794 transcripts, with an N50 of 897 (Table 2).
Table 1
Summary statistics for reads from 454 GS-FLX sequencing
Total HQ reads
Total HQ sequence (bp)
Average read length
% Mixed
% Dots
Light organ tissue
559 773
192 760 671
344
16.13
11.25
Non-light organ tissue
564 835
194 425 594
344
12.07
11.37
Combined
1 124 608
387 186 265
344
14.09
11.31
HQ = high quality; % Mixed = percentage of reads filtered out by the mixed filter, where a mixed read is the result of simultaneously sequencing a mixture of different DNA molecules; % Dots = percentage of reads filtered by the dots filter, where a dot is an instance of three successive nucleotide flows that record no incorporation
Fig. 1
Distribution of read lengths from 454 sequencing of light organ (blue) and non-light organ (red) cDNA libraries
Table 2
De novo assembly statistics
Input
Assembly software
Number of transcripts
Number of bases assembled
N50 (bp)
Number of singleton reads
Median contig length (bp)
Mean contig length (bp)
Maximum contig length (bp)
454 reads (two libraries merged)
CLC Genomics Workbench
18 794
14 257 486
897
94 753
586
759
11 217
Illumina reads (six libraries merged)
Trinity
196 766
187 289 921
1 828
NA
447
952
30 278
N50 size = the length such that 50 % of the assembled genome lies in N50 size or greater; NA = not applicable to results from this assembly program
Illumina
We sequenced six cDNA libraries using the Illumina HiSeq-2000 sequencer, each prepared from either the light organ or non-light organ mRNA of three individual glowworms. The increased number of biological replicates in the second experiment provided it with increased statistical power for the subsequent RNA-seq analysis [31]. The Illumina platform also provided us with information on strand origin, i.e. from which of the two DNA strands a given RNA transcript was derived. This information can increase the percentage of alignable reads, thereby improving transcript reconstruction compared with non-strand specific data of known strand origin [32].
The Illumina machine generated 37.1 to 44.7 million pairs of 200 base length paired-end reads for each library (Table 3). In order to improve the quality of the data, we removed adapter sequences, trimmed low quality bases (Q < 20) from both ends of reads and discarded reads less than 50 bases in length. The resulting 29.5 to 35.7 million high quality reads per library (79 to 80 % of total raw reads) were merged together for de novo transcript assembly using the Trinity package, producing a de novo assembly containing 187,289,921 bases and a total of 196,766 transcripts (Table 2). A graph of the contig length distribution highlights the differences in contig size and number between the two assemblies (Fig. 2).
Table 3
Summary statistics for reads from Illumina sequencing
Sample
Raw reads
Trimmed, quality filtered reads
Trimmed, quality filtered reads (% of raw reads)
Glowworm 1 light organ
44 776 272
35 671 872
80 %
Glowworm 1 non-light organ
39 450 268
31 259 202
79 %
Glowworm 2 light organ
41 122 414
32 769 962
80 %
Glowworm 2 non-light organ
37 092 452
29 526 444
80 %
Glowworm 3 light organ
40 295 844
32 042 384
80 %
Glowworm 3 non-light organ
39 697 568
31 624 498
80 %
Combined
242 434 818
192 894 362
80 %
Fig. 2
Distribution of contig lengths for 454/CLC Genomics Workbench (green) and Illumina/Trinity (orange) assemblies
Functional annotation and gene ontology of the glowworm transcriptome
In order to provide descriptions of the functions and properties of as many glowworm gene products as possible, BLASTX searches were performed for each transcript above 1 kb in size from the Illumina/Trinity transcriptome assembly against the Drosophila RefSeq non-redundant database at the National Centre for Biotechnology Information (NCBI). 38,259 of the 55,997 transcripts matched to a known protein within the database with a score of E < 10−6). 32 % of the transcripts (17,738) did not have a BLAST result. Some of these sequences may not have a homolog in Drosophila; others may be from non-coding RNA sequences that are polyadenylated. Some sequences may be unmatched due to assembly errors. We used Blast2GO to assign Gene Ontology (GO) terms to transcripts with BLASTX matches. 34,332 transcripts were assigned GO terms (Fig. 3).
Fig. 3
Gene ontology annotations. Pictured are the top ten GO terms for each of the three GO categories
We also carried out an analysis to find out which metabolic processes predominate in the light organ. First of all we mapped all of the Illumina light organ reads back to the full Illumina transcriptome, then used an FDR adjusted enrichment test for the light organ over the whole transcriptome with 10 % as the cutoff (so that transcripts with less than 10 % of their length mapped by fragments were counted as not expressed). We used Blast2GO to assign GO terms to this subset of light organ transcripts. The results showed that there are a large range of metabolic processes occurring in the light organ, although none that could be confidently linked to its bioluminescent function (see Additional file 2: Table S1).
Read mapping, measurement of gene expression and differential expression analyses
For each experiment we assembled a database of transcripts de novo, since there is no reference genome available for Arachnocampa.
454
90.78 and 87.41 % of reads from the 454 light organ and non-light organ libraries, respectively, uniquely mapped to transcripts in the 454/CLC Genomics Workbench assembly. We normalized expression values for each sample by scaling so that the median values were made equal. A comparison of normalized expression values provided us with a list of 34 transcripts found at ≥ 10-fold higher levels in the light organ sample than in the rest of the glowworm body sample (Table 4). In addition, 4616 different transcripts were expressed in the light organ sample, but not in the rest of the glowworm body sample. The top 30 of these, ranked according to normalised expression levels, are listed in Table 5.
Table 4
Differential expression analysis for 454 data: transcripts expressed ≥ 10-fold more highly in glowworm light organ tissue than in non-light organ tissue
Light organ
Rest of body
Contig number
Gene length (bp)
Total gene reads
Normalized expression values
RPKM
Total gene reads
Normalized expression values
RPKM
Difference (normalized values)
Fold change (normalized values)
Putative identity from BLASTX search hits
16656
711
955
2387.2
2876.3
2
7.4
5.9
2379.8
322.6
phosphatidylethanolamine-binding protein
12303
1832
1702
1651.2
1989.5
9
13
10.3
1638.2
127.0
luciferin 4-monooxygenase
10195
478
2588
9622.5
11594.2
14
77.4
61.6
9545.1
124.3
luciferin 4-monooxygenase
13441
1452
13014
15929.3
19193.3
87
158.4
126.0
15770.9
100.6
luciferin 4-monooxygenase
12283
1279
446
619.8
746.7
3
6.2
4.9
613.6
100.0
glutathione s-transferase
4637
1047
273
463.4
558.4
2
5.1
4.0
458.3
90.9
sulfotransferase
12325
1830
2569
2495
3006.2
21
30.3
24.1
2464.7
82.3
luciferin 4-monooxygenase
2377
1303
339
462.4
557.1
4
8.1
6.5
454.3
57.1
aminoacylase-1
2684
2024
65
57.1
68.8
1
1.3
1.0
55.8
43.9
otopetrin
17552
746
513
1222.2
1472.6
8
28.4
22.5
1193.8
43.0
cleavage stimulation factor 64 kDa subunit
11885
2198
49
39.6
47.7
1
1.2
1.0
38.4
33.0
ATP-binding cassette transporter
3008
1206
49
72.2
87.0
1
2.2
1.7
70
32.8
GST-containing flywch zinc-finger protein
16267
1647
42
45.3
54.6
1
1.6
1.3
43.7
28.3
protein lsm14 homolog b-like
12345
961
293
541.9
652.9
7
19.3
15.3
522.6
28.1
carboxylesterase
2668
928
72
137.9
166.1
2
5.7
4.5
132.2
24.2
tpa_inf: hdc07468
12310
1030
452
779.9
939.7
13
33.4
26.5
746.5
23.4
carboxylesterase
4414
2035
138
120.5
145.2
4
5.2
4.1
115.3
23.2
sodium-dependent phosphate transporter
4376
704
32
80.8
97.3
1
3.8
3.0
77
21.3
carbonic anhydrase
11948
1363
29
37.8
45.6
1
1.9
1.5
35.9
19.9
facr2_drome ame: full = fatty acyl- reductase cg8303
16727
1498
30
35.6
42.9
1
1.8
1.4
33.8
19.8
short-chain dehydrogenase
16747
1127
28
44.2
53.2
1
2.3
1.9
41.9
19.2
cofilin actin-depolymerizing factor homolog
3316
1969
21
19
22.8
1
1.3
1.1
17.7
14.6
isoform a
9802
1035
22
37.8
45.5
1
2.6
2.0
35.2
14.5
kynurenine aminotransferase
9665
1782
19
18.9
22.8
1
1.5
1.2
17.4
12.6
transposable element tc3 transposase
11695
1953
56
51
61.4
3
4.1
3.2
46.9
12.4
protein yellow
12223
1142
36
56
67.5
2
4.6
3.7
51.4
12.2
ganglioside-induced differentiation-associated protein 1
10010
267
18
119.8
144.4
1
9.9
7.9
109.9
12.1
hypothetical protein Sulku_2095
3263
1410
17
21.4
25.8
1
1.9
1.5
19.5
11.3
multiple inositol polyphosphate phosphatase
4985
851
16
33.4
40.3
1
3.1
2.5
30.3
10.8
ubiquitin fusion degradaton protein
13509
589
32
96.6
116.3
2
9
7.1
87.6
10.7
kda midgut protein
16372
1493
16
19
22.9
1
1.8
1.4
17.2
10.6
cytochrome p450
4731
700
31
78.7
94.8
2
7.6
6.0
71.1
10.4
isoform b
7053
817
15
32.6
39.3
1
3.2
2.6
29.4
10.2
No significant similarity found
16679
960
45
83.3
100.4
3
8.3
6.6
75
10.0
hypothetical conserved protein
Transcripts are ranked according to normalized expression values; read counts were scaled so that the median values were made equal. RPKM = reads per kilobase of exon model per million mapped reads
Table 5
Differential expression analysis for 454 data: top 30 transcripts expressed in glowworm light organ tissue but not in non-light organ tissue
Contig number
Gene length (bp)
Total gene reads
Normalized expression values
RPKM
Putative identity from BLASTX search hits
13443
427
347
1444.3
1740.2
No significant similarity found
10414
304
64
374.2
450.8
luciferin 4-monooxygenase
2392
1330
156
208.5
251.2
heat shock protein 70
13448
565
65
204.5
246.4
sugar transporter sweet1-like isoform 2
13375
816
82
178.6
215.2
No significant similarity found
14967
142
10
125.2
150.8
No significant similarity found
94
215
15
124
149.4
No significant similarity found
860
68
4
104.5
126.0
No significant similarity found
4416
1031
54
93.1
112.2
No significant similarity found
10178
78
4
91.1
109.8
No significant similarity found
10298
119
6
89.6
108.0
hypothetical protein Sulku_2095
14957
504
25
88.2
106.2
No significant similarity found
161
142
7
87.6
105.6
No significant similarity found
17729
168
8
84.6
102.0
No significant similarity found
706
85
4
83.6
100.8
No significant similarity found
10061
152
7
81.8
98.6
troponin i
14955
181
8
78.6
94.6
No significant similarity found
2879
1600
70
77.8
93.7
protein maelstrom homolog
80
70
3
76.2
91.8
No significant similarity found
14930
118
5
75.3
90.7
No significant similarity found
15799
49
2
72.5
87.4
No significant similarity found
10252
74
3
72.1
86.8
No significant similarity found
7300
228
9
70.2
84.5
No significant similarity found
10131
104
4
68.4
82.4
No significant similarity found
1453
55
2
64.6
77.9
No significant similarity found
15071
83
3
64.2
77.4
No significant similarity found
14973
194
7
64.1
77.3
No significant similarity found
9
201
7
61.9
74.6
No significant similarity found
15207
173
6
61.6
74.3
No significant similarity found
788
117
4
60.8
73.2
No significant similarity found
Transcripts areranked according to normalized expression values; read counts were scaled so that the median values were made equal. RPKM = reads per kilobase of exon model per million mapped reads
Illumina
We mapped reads for each of the six samples in this experiment onto the Illumina/Trinity reference transcriptome assembly. 91 % of the reads from all six samples were matched to transcripts from the assembly set. Since there were three separate samples for each tissue type, we performed inter-sample normalization, so that cross sample comparison could be carried out without being biased by sequencing depth. We used a TMM method (trimmed-mean of M values) to accommodate the difference in sequencing depth between replicates by finding a scaling factor for each library that minimizes the log-fold changes between the samples. The scaling factor is used to normalise the expression values for each sample.
Differential expression analysis was carried out on transcript expression values from all six Illumina-sequenced samples after adjusting for library size. Only six transcripts were considered to be expressed to a significantly higher level in the light organ than in the non-light organ tissue (false discovery rate of < 0.1); these are listed in Table 6.
Table 6
Transcripts from the Illumina sequenced samples that are significantly more highly expressed in the light organ relative to the rest of the body in the glowworm
Transcript number
Rank
Log2 fold change
Log2 of read count per million
P value
False discovery rate
Putative identity from BLASTX search hits
64201-seq1
1
9.90
11.88
1.68E-06
0.054
acyl-CoA synthetase/luciferin 4-monooxygenase
62762
2
10.15
15.44
3.05E-06
0.054
acyl-CoA synthetase/luciferin 4-monooxygenase
60014
3
9.91
9.09
3.14E-06
0.054
aminoacylase
51138
4
9.71
10.98
4.75E-06
0.054
phosphatidylethanolamine-binding protein
64201-seq2
5
10.05
11.12
5.13E-06
0.054
acyl-CoA synthetase/luciferin 4-monooxygenase
56768
6
9.81
10.63
5.56E-06
0.054
glutathione S-transferase
A positive value for log2 fold change indicates over-expression in light organ relative to non-light organ tissue
Differential expression analysis validation
When comparing the sequences of differentially expressed transcripts in the two experiments, it became apparent that the six transcripts from the Illumina experiment were all found to be in the top eight ranked transcripts from the 454 experiment (Table 7), with one transcript (annotated as 62762 in the Illumina experiment) listed twice in the top eight. The close matching of the results from these two separate experiments, despite differences in samples, sequencing platforms and analytical algorithms, effectively validates these results.
Table 7
Common differentially expressed transcripts from 454 and Illumina sequencing analyses
Rank (Illumina)
Transcript number (Illumina)
Rank (454)
Contig number (454)
Protein size (kDa)
Putative identity from BLASTX search hits
Speculative function
1
64201-seq1
7
12325
58.6
acyl-CoA synthetase/luciferin 4-monooxygenase
bioluminescence catalysis
2
62762
3,4
10195, 13441
59.0
acyl-CoA synthetase/luciferin 4-monooxygenase
bioluminescence catalysis
3
60014
8
2377
45.0
aminoacylase
processing of bioluminescent substrate
4
51138
1
16656
20.1
phosphatidylethanolamine-binding protein
ATP binding
5
64201-seq2
2
12303
58.2
acyl-CoA synthetase/luciferin 4-monooxygenase
bioluminescence catalysis
6
56768
5
12283
25.0
glutathione S-transferase
directly or indirectly involving glutathione in bioluminescence
The number of differentially expressed transcripts is small in both analyses. This may be because there are Malpighian tubules in both tissue types: the light organ tissue sample contains the modified Malpighian tubule tips that produce light, and the non-light organ sample contains the remaining non-luminescent parts of the Malpighian tubules. Presuming the modified Malpighian tubule tips retain some of the same functionality as the remainder of the tubules, both samples would share many of the same transcripts.
Functional annotation of genes highly expressed in the light organ
The proteins encoded by these six transcripts are likely to play important roles in the bioluminescence of the glowworm, assuming that the transcript levels are equivalent to protein levels. In order to find out what these roles might be, we searched for annotated sequence homologs in the publically available non redundant Genbank protein sequence database using the BLASTX algorithm. The resulting annotations will need to be confirmed using biochemical investigation of both the native and recombinant forms of the encoded proteins.
64201-seq1, 64201-seq2, 62762
The proteins encoded by these three transcripts all display the signature motifs of the ANL superfamily of adenylating enzymes [33] (Fig. 4). The three main subfamilies in the ANL superfamily include the Acyl-CoA synthetases, the NRPS adenylation domains, and the beetle (firefly) Luciferase enzymes. Despite catalyzing a wide range of different overall reactions, ANL enzymes all use a two-step reaction where the first step is always the activation of a carboxylate substrate with ATP to form an adenylate intermediate. These three glowworm proteins are very similar to firefly luciferase (31 to 37 % identical with luciferase from Photinus pyralis). The glowworm proteins are very similar and two appear to be isoforms. 64201_seq1 and 64201_seq2 are 79 % identical (the Trinity software labelled them with the same number and ‘seq1’ or ‘seq2’ to reflect this), and 62762 is 43 and 45 % identical to 64201_seq2 and 64201_seq1, respectively. The differences between these three proteins do not appear to be due to alternative splicing, since the differences in sequence are scattered throughout the proteins (Fig. 4).
Fig. 4
Alignment of amino acid sequences encoded by transcripts 64201-seq1, 64201-seq2, and 62762. The alignment was carried out using Clustal Omega (http://www.ebi.ac.uk) and visualised using Jalview (http://www.jalview.org). Residues are colored according to conservation of sequence identity (dark blue: 100 % conserved). Black boxes represent positions of ATP-binding motifs conserved throughout the ANL superfamily [33], and red boxes represent luciferin-binding residues from the beetle luciferase [62, 63]. The residue marked with a ‘#’ plays a key role in the firefly luciferase adenylation half reaction, and the residue marked with a ‘*’ plays a key role in the oxidation (light-producing) half reaction [64]
60014
The protein encoded by this transcript has 44 % amino acid sequence identity with human aminoacylase-1. Amino acids can be stored with an acyl group attached to their N-terminus, which makes them more stable. Aminoacylase-1 removes the acyl group, making the amino acid available for protein synthesis and other metabolic roles [34, 35], and acts specifically on mercapturic acids (S-conjugates of N-acetyl-L-cysteine) and neutral aliphatic N-acyl-α-amino acids. If the glowworm luciferin is revealed to be a derivative of an amino acid, as it appears to be for the unrelated Siberian luminous earthworm Fridericia heliota [36], it is possible that the glowworm might store the luciferin substrate in a stable acylated form and 60014 could deacetylate the substrate, making it available for the bioluminescent reaction. There is, however, no other evidence for this involvement, and the identity of the luciferin is unclear at this stage.
51138
This transcript encodes a member of the phosphatidylethanolamine-binding protein superfamily. Proteins in this family generally play roles in modulating cellular signaling [37]. At a molecular level they have been found to bind nucleotides, opioids and phosphatidylethanolamine. They can also bind kinases, leading to inhibition or activation of signalling pathways. From this information we can infer that 51138 may be involved in the modulation of a bioluminescence signaling pathway.
56768
This protein is a member of the glutathione S-transferase (GST) family of proteins, which play an important role in insecticide resistance and protection against oxidative stress. Members of this family catalyze the conjugation of reduced glutathione to a variety of exogenous and endogenous hydrophobic electrophiles for the purpose of detoxification [38]. 56768 has closest homology with the Delta class of insect GSTs, and has 45 % identity with a mosquito Delta GST that has DDT dehydrochlorinase activity [39]. Therefore glutathione may play a role in glowworm bioluminescence, either directly or indirectly, although it is unclear at this stage what this role might be.
Evolution of bioluminescence in glowworms
It is interesting that the firefly and glowworm luciferase enzymes belong to the same family of enzymes (assuming that one or more of the three ANL proteins from the light organ are confirmed biochemically to be the glowworm luciferases). In one way it is not unexpected as both are from the same class (Insecta), but because of the evolutionary distance between the glowworm and the firefly, and because the two bioluminescent systems use different substrates [9], it seemed likely that the proteins would differ significantly. However, these differential transcriptomic analyses, and the observation that both the glowworm and the firefly use ATP as a cofactor [10], suggest that the two luciferases may indeed have evolved from the same ancestral non-bioluminescent enzyme. Other explanations for both glowworms and fireflies having a similar luciferase enzyme are unlikely, such as horizontal gene transfer, or the possibility that the insect ancestral to both Coleoptera and Diptera was bioluminescent and passed the bioluminescent gene to both fireflies and glowworms but not to the vast majority of other non-bioluminescent Coleoptera and Diptera that exist today. The independent evolution of the two bioluminescent enzymes from a non-bioluminescent ancestral acyl-CoA synthetase enzyme is most likely, because it has been well established that the beetle luciferases evolved from non-bioluminescent acyl-CoA synthetases [40–42]. In addition, acyl-CoA synthetases from two nonluminous insects, the mealworm Zophobas morio [43] and the fruit fly Drosophila melanogaster [44], have been shown to bioluminesce faintly in the presence of either the firefly luciferin substrate or a synthetic analog of this luciferin. A firefly luciferase-like gene has also been identified from an animal unrelated to insects: the siliceous sponge Suberites domuncula [45]; however, this claim needs to be confirmed [46], especially since the native luciferase protein itself has not yet been isolated from the sponge tissue.
We carried out a phylogenetic analysis of the three glowworm luciferase-like proteins along with known firefly luciferases and other luciferase-like proteins from various insects. Notably, the three glowworm proteins are grouped together and are placed closer to proteins from non-bioluminescent dipteran insects (D. melanogaster and A. aegypti) than to firefly luciferases (see Fig. 5 and Table 8).
Fig. 5
Unrooted phylogenetic tree of luciferases and related proteins. Details for each protein are provided in Table 8
Table 8
Details of luciferases and related proteins included in the phylogenetic analysis (Fig. 5)
It is uncertain why there are three isoforms of the firefly-luciferase-like protein expressed in the glowworm light organ. The firefly Luciola cruciata expresses three isoforms, where only one has bioluminescent activity [42], so it is possible that the glowworm may also follow a similar pattern of gene duplication. Gene duplication followed by enzyme diversification and the development of novel functions has been a feature previously observed in vertebrate acyl-CoA synthetase genes [47]. There appear to be many duplication events in the evolution of the acyl-CoA synthetase enzyme family in insects. We used tBLASTn to search the total glowworm transcriptome from the Illumina experiment and found that the three glowworm proteins have numerous paralogs: between 65 and 68 transcripts have 20 to 50 % identity with the three protein sequences. In addition, the limited transcriptional profiling studies by Silva et al. mentioned previously found about 1 % of the 537 A. luminosa light organ cDNAs and 2 % of the 572 firefly lantern cDNAs sequenced were members of the acyl-CoA synthetase enzyme family (also known as AMP/CoA-ligases) [29, 30].
Conclusions
This report describes the first high-throughput transcriptome sequencing of the New Zealand glowworm, and the use of comparative RNAseq to identify genes expressed in luminescent tissue that are involved in bioluminescence. Two separate differential expression analyses have identified six genes that are significantly more highly expressed in the light organ than in non-luminescent tissue. These genes encode putative aminoacylase, GST and phosphatidylethanolamine-binding proteins, and, most notably, three proteins that are homologs of firefly luciferase, at least one of which we expect to be the glowworm luciferase.
Interestingly, in the Silva et al. study of glowworm light organ cDNAs [29], one of the members of the acyl-CoA synthetase enzyme family sequenced showed 44–45 % identity with railroad worm luciferases, and 2.1 % of the transcripts sequenced were putative GST proteins. This, combined with our research underlines the potential importance of these sequences in glowworm bioluminescence.
Further biochemical studies are required to confirm that one or more of the candidate luciferases are able to produce light. These studies should include two approaches: firstly, produce these proteins in a recombinant form and assay them for bioluminescent activity using the native luciferin substrate extracted from the glowworm; secondly, isolate the native luciferase protein(s) from the light organ tissue, using the same assay to track bioluminescent activity, and identify the isolated protein(s) using mass spectrometry and the transcriptome database generated in the current study. If the candidate luciferase(s) is/are confirmed, then this will show that this enzyme has independently evolved the ability to produce light at least twice in extant organisms, in New Zealand glowworms and in fireflies, but with different substrates. Once the identity of the glowworm enzyme has been confirmed, and the chemistry of the glowworm substrate has been revealed, potential applications of the novel glowworm bioluminescence system can be explored.
Methods
Sample collection and RNA extraction
A. luminosa is an endemic species and is of particular interest to the indigenous Maori people of New Zealand. Therefore local Maori were consulted about this research through Te Komiti Rakahau ki Kāi Tahu (the Ngāi Tahu Research Consultation Committee). As glowworms are invertebrates, the approval of an ethics committee was not required. A. luminosa is not protected, and no experiment was performed on living animals.
A. luminosa specimens were collected from locations around New Zealand, including Dunedin, Te Anau and Palmerston North. Live glowworms were snap frozen by being placed on foil above dry ice, and then stored until required at −80 °C. Using a razor, light organs were removed from the glowworm bodies while still frozen. Light organ samples contained only white light organ tissue, and non-light organ samples contained the rest of the glowworm body and head (darker tissue) with white light organ tissue removed entirely. Tissues were homogenised with TRIzol® Reagent (Invitrogen) and a glass dounce homogeniser; total RNA was extracted using UltraPure™ (Phenol: Chloroform:Isoamyl Alcohol; Invitrogen), and then further purified using the RNeasy Kit (Qiagen). RNA quantification and integrity assessment were performed for each sample on an RNA chip (Bioanalyzer 2100, Agilent Technologies).
cDNA library construction, sequencing and quality control
454
Two hundred μg of non-light organ RNA (prepared from ~200 mg of non-light organ tissue from eight glowworms) and 65 μg of light organ RNA (prepared from ~420 mg of light organ tissue from 172 glowworms) were sent to the 454 Life Sciences Sequencing Center (Roche, Branford, Connecticut, USA) for mRNA enrichment, cDNA library construction (including mRNA fragmentation using a zinc chloride solution, cDNA synthesis using random hexamer primers and adaptor ligation) and subsequent 454 sequencing on a Roche GS FLX Titanium Genome Sequencer. Each sample was sequenced on one half of a picotiter plate. Low quality reads were discarded (quality limit of 0.05). We also removed: ambigious nucleotides (maximum of 5 nucleotides allowed), terminal nucleotides (1 each from both the 5′ and 3′ ends), adapter sequences and sequences less than 20 nucleotides in length.
Illumina
Light organ and non-light organ total RNA was prepared separately from three individual glowworms (0.6 to 1.2 μg of RNA for each light organ sample, and 8.8 to 27.0 μg of RNA for each non-light organ sample). The six samples, each with an RNA Integrity Number (RIN) of over 6, were sent to the Otago Genomics and Bioinformatics Facility, where mRNA was isolated using oligo-dT magnetic beads, and cDNA libraries were constructed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Sequencing was carried out on an Illumina HiSeq-2000 sequencer, generating 100 bp paired-end reads. Each sample was run on one eighth of a sequencing lane. Adaptor sequences were trimmed from reads using fastq-mcf [48], and bases with low quality phred scores trimmed (cut-off phred score of Q20). Adapter and quality trimmed reads less than 50 nucleotides in length were discarded using the SolexaQA package [49]. Reads were quality assessed using FASTQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc).
De novo assembly
454
We used the CLC Genomics Workbench v5.1 (http://www.clcbio.com) to de novo-assemble 454 reads from the combined light organ and non-light organ samples into a single, non-redundant contig dataset. Singletons were merged into the contig set. Assembly parameters were set at the program default settings.
Illumina
Reads combined from all light organ and non-light organ samples were assembled using the Trinity software package [50]. Assembly parameters were adjusted for Illumina stranded paired end sequencing (i.e. –left and –right for both R1 and R2 using --SS_lib_type RF for the stranded library type).
Read mapping and measurement of gene expression
454
We used the CLC Genomics Workbench to map reads from each of the 454 light organ and non-light organ samples back onto the CLC Genomics Workbench/454 assembly dataset. Expression of genes was represented by the abundance of reads uniquely mapped to each contig in the assembly. Abundance was expressed as RPKM (reads per kilobase of exon model per million mapped reads) or normalized expression values (the contig read counts were scaled so that the median values were made equal).
Illumina
For each of the six Illumina samples, reads were mapped onto the Trinity/Illumina assembly using Bowtie 2 [51], and transcript abundance for each sample was estimated using the RSEM package [52]. Per sample relative abundance was estimated using FPKM (fragments per kilobase of transcript per million fragments mapped), EC (expected counts) and TPM (transcripts per million). For cross-sample comparisons, we normalized raw read counts for these samples using the TMM method (trimmed-mean of M values; a weighted trimmed mean of the log expression ratios [53]) as implemented in the edgeR package [54] (http://www.bioconductor.org).
Differential expression analyses
To identify candidate bioluminescence-related genes from our datasets, we compared normalized transcript abundance values between corresponding light organ and non-light organ samples.
454
In order to detect the differentially expressed genes between the two 454-sequenced samples, a two-group differential analysis was performed in CLC Genomics Workbench on normalized expression values.
Illumina
Genes with significantly different levels of expression between light organ and non-light organ samples (a single factor design) were identified using the quantile-adjusted Conditional Maximum Likelihood method (qCML) in the edgeR package [55]. We considered transcripts to be differentially expressed to a significant level at a false discovery rate at < 0.1.
Functional annotation
Transcripts that were expressed at significantly higher levels in light organ compared with non-light organ tissue were annotated by searching the GenBank non-redundant database at the NCBI (http://www.ncbi.nlm.nih.gov) using the BLASTX software [56] with default parameters. Automated annotation of the transcriptome database was carried out using BLASTX searches to the NCBI non-redundant database within the Blast2GO program v2.8.0 (http://www.blast2go.com) [57]. An E-value cut-off of 1E−6 was used to identify similar annotated proteins from which function could be inferred.
Phylogenetic analysis
Multiple alignments were produced using MUSCLE [58, 59]. A phylogenetic analysis was carried out in MrBayes [60], using the WAG model of protein evolution [61] which was found to be the most appropriate after tests with mixed models. Monte-Carlo Markov chains were run for 1 000 000 generations with the initial 25 % of trees discarded as burn-in. The consensus trees produced were visualized with CLC Genomics Workbench.
Availability of supporting data
The raw sequence data from the Illumina experiment was submitted in FASTQ format to the NCBI Sequence Read Archive (SRA) database (accessions: SRR2241413, SRR2283829, SRR2283830, SRR2283831, SRR2283975 and SRR2284246) and are also accessible through the BioProject accession PRJNA290397 (http://www.ncbi.nlm.nih.gov/bioproject/290397). The Illumina/Trinity transcriptome assembly has been deposited at the NCBI Transcriptome Shotgun Assembly (TSA) database and is available at the DNA DataBank of Japan (DDBJ), the European Molecular Biology Laboratory (EMBL), and GenBank at NCBI under the accession GDQV00000000.
Abbreviation
RPKM:
Reads per kilobase per million
Declarations
Acknowledgements
Funding for this study was provided by the Foundation for Research, Science and Technology (now the New Zealand Ministry of Business, Innovation, and Employment) and the Division of Health Sciences, University of Otago. New Zealand government subsidised sequencing services were provided by New Zealand Genomics Limited (www.nzgenomics.co.nz).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
(1)
Department of Biochemistry, University of Otago
(2)
Otago Genomics & Bioinformatics Facility, University of Otago
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