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. 2018 Oct 17;13(10):e0204243. doi: 10.1371/journal.pone.0204243

Major ampullate silk gland transcriptomes and fibre proteomes of the golden orb-weavers, Nephila plumipes and Nephila pilipes (Araneae: Nephilidae)

Alessandra D Whaite 1, Tianfang Wang 1, Joanne Macdonald 1,2,*, Scott F Cummins 1,*
Editor: Paulo Lee Ho3
PMCID: PMC6192577  PMID: 30332416

Abstract

Natural spider silk is one of the world’s toughest proteinaceous materials, yet a truly biomimetic spider silk is elusive even after several decades of intense focus. In this study, Next-Generation Sequencing was utilised to produce transcriptomes of the major ampullate gland of two Australian golden orb-weavers, Nephila plumipes and Nephila pilipes, in order to identify highly expressed predicted proteins that may co-factor in the construction of the final polymer. Furthermore, proteomics was performed by liquid chromatography tandem-mass spectroscopy to analyse the natural solid silk fibre of each species to confirm highly expressed predicted proteins within the silk gland are present in the final silk product. We assembled the silk gland transcriptomes of N. plumipes and N. pilipes into 69,812 and 70,123 contigs, respectively. Gene expression analysis revealed that silk gene sequences were among the most highly expressed and we were able to procure silk sequences from both species in excess of 1,300 amino acids. However, some of the genes with the highest expression values were not able to be identified from our proteomic analysis. Proteome analysis of “reeled” silk fibres of N. plumipes and N. pilipes revealed 29 and 18 proteins, respectively, most of which were identified as silk fibre proteins. This study is the first silk gland specific transcriptome and proteome analysis for these species and will assist in the future development of a biomimetic spider silk.

Introduction

Spider silk is an outstanding proteinaceous fibre that outperforms other natural and synthetic fibres in tensile strength analyses. In addition to fibre strength, spider silks can also be tough, lightweight, highly extensible/flexible, biodegradable and stable across a broad temperature range [1]. Studies have also demonstrated the biocompatibility of spider silk: spider silk can be implanted into living tissue without eliciting an immune response [2, 3]. The potential of all these qualities present in one fibre type is unique and has led to intensive research interest in the molecular structure of the various silks spiders produce.

Orb-weaving spiders have up to seven different glands associated with producing silks and glues used for web architecture [major ampullate gland (MA), minor ampullate (Mi), flagelliform (Flag), aggregate (Ag), and pyriform (Py) glands], protection of eggs [tubuliform glands (Tu)], and prey wrapping [aciniform glands (Ac)] [4]. The molecular structure underpinning each of these silks and glues is the key to understanding the differing inherent mechanical properties of each type [5]. The major constituents in each silk gland are spider silk fibroins, called spidroins (Sp). While spidroins were named according to the gland from which they were primarily identified (for example the major ampullate spidroin (MaSp) from the MA gland), spidroins are not exclusively produced in their namesake gland, and proteomes have revealed silk to contain hundreds of proteins other than those derived from the spidroin gene family [6–11].

In the silk glands, silk proteins are secreted and then stored in the sac-like section of the gland in a water-soluble “molten fibril” or “aqua-melt” state, before processing occurs between the gland, duct and spinneret to produce the insoluble fibre we recognise within webs [12, 13]. Spidroin sequences are very large (approx. >10 kb) and code for a sequence that includes many repetitions of several repeat motifs, and is flanked by non-repetitive, highly conserved N- and C- termini [5,14–16]. Typical repeat motifs in MA protein residues include stretches of poly-alanine (poly-A) and repeat glycine (G) motifs, such as GGX (where X could be A, glutamine, or tyrosine) [14]. An alternate MA spidroin contains stretches of poly-A and contains GPGGX motifs, where proline (P) is believed to contribute to the greater extensibility of this spidroin [10, 17].

Beyond spidroins, much remains to be understood about the tertiary and quaternary interactions of the various proteins and glycoproteins from storage in the soluble phase within the gland, to assembly via processing steps within the duct, and finally, the solid phase fibre as it leaves the spinnerets [18–20]. Synthetic silks have not obtained the level of mechanical performance of their natural counterparts [21–23]. The factors inhibiting successful biomimicry include the inability to mimic the natural production process in current expression systems, including the inability to express full-length recombinant spidroins, and, possibly, the lack of concurrent expression of a set of spidroins and other molecular components found within a silk thread. The non-repetitive N- and C- termini of ampullate silks have been found to play a role in fibre storage and assembly [24]. Further, the N- and C-termini are also thought to aid assembly of the secondary structures of the repeat regions within the processing duct. Indeed, thus far, the best example of a biomimetic spidroin has included these important highly conserved regions flanking a short repeat region [25].

The total primary MaSp sequence has been identified within 3 species; Latrodectus hesperus, also known as the Western Black Widow spider, the golden orb-weaving banana spider, Nephila clavipes, and Argiope bruennichi, the Wasp Spider [14, 26, 27]. Of more than 44,000 spider species, entire spider genomes have only been described for 6 species: L. hesperus (GenBank: JJRX00000000.1); the brown recluse, Loxosceles reclusa (JJRW000000000.1); the common house spider, Parasteatoda tepidariorum (AOMJ00000000.2); the African social velvet spider, Stegodyphus mimosarum (AZAQ00000000.1); the Brazilian white-knee tarantula, Acanthoscurria geniculata (GCA_000661875.1); and, most recently, N. clavipes (MWRG00000000.1)[27–29]. In combination with their genome assembly for N. clavipes, Babb et al. characterised spidroin expression within the distinct silk glands [27]. Significant transcriptome coverage has also been achieved by Prosdocimi and colleagues in their infraorder comparative study of expressed RNAs of the spinning glands [30]; by Clark et al., based on multi-tissue de novo transcriptome assemblies of closely related cob-weavers [31–33]; and Correa-Garhwal et al. examined silk expression in male spiders of the family Theridiidae [34].

In Queensland, Australia, Nephila plumipes (Latreille, 1804) and Nephila pilipes (Fabricius, 1793) are commonly encountered golden orb-weaving species [35]. Studies on N. plumipes have reported on the mechanical properties of their silk, the relationship between protein secondary structure and primary amino acid sequence, populations in the urban environment, and copulation behaviour, however, no transcriptome data is available on the MA gland of this species, and only a handful of silk sequences are available for N. pilipes in online databases [36–42]. In this study, we report on a silk-gland specific transcriptome analysis for these golden orb-weaving species, N. plumipes and N. pilipes. Furthermore, proteomic analysis of silk fibres from these species was undertaken to compare predominant proteins within the silk fibre to predominant proteins expressed within the MA gland transcriptome. This study found that the silk gland transcriptome of N. plumipes and N. pilipes could be assembled into contiguous sequences, and proteome analysis of “reeled” silk fibres could confirm, and be used to mine for, spidroins within the transcriptomes. Novel proteins, which may be important constituents in the structure of spider silk, were also discovered in the silk proteome.

Methods

Animals and preparation of RNA

Golden orb spiders of the genus Nephila plumipes and Nephila pilipes were collected from the Sunshine Coast (26°41'43.1"S 153°05'56.7"E) and the Cooloola Coast (25°54'02.8"S 153°05'25.6"E) regions of Queensland, Australia, between the months of March and June 2013, and February and April 2016. Specimens were dissected immediately after sacrifice and each major ampullate gland were removed and either stored in RNAlater (Invitrogen) or immediately frozen in liquid nitrogen.

RNA was isolated from a pair of MA glands from an individual spider from each species using two different methods; the PicoPure RNA Isolation kit (Arcturus) and TRIzol Reagent (Invitrogen), according to manufacturer’s instructions, with the following changes:

For the PicoPure RNA extraction, an additional RNA purification step was performed using LiCl precipitation. Total RNA (30 μL) was divided into two aliquots for each species, and volume restored to 30 μL with 15 μL of RNase-free water so that samples were in duplicate. All RNA samples were incubated in 2.5 M LiCl (9.4 μL of 8 M LiCl) and 2.5 volumes (98.5 μL) of 100% EtOH for 1.5 h at -20°C. After incubation, the RNA was pelleted at 20,000 x g for 20 min at 4°C and the supernatant discarded. The pellet was washed with 70% EtOH and spun at 20,000 x g for 10 min at 4°C. The pellet was dried for 10 min at 37°C. The RNA concentration of a second aliquot from each species was estimated spectrophotometrically (NanoDrop 2000) after rehydration with 30 μL RNase-free water to ensure OD260/OD280 range was between 1.8 and 2.0. Dried samples of the first aliquot were stored at -80°C until shipment to BGI (China) for de novo RNA sequencing and bioinformatics.

For the TRIzol RNA extraction, all centrifugation steps were performed at 12,000 rpm at 4°C. RNA concentration and purity was estimated spectrophotometrically (NanoDrop 2000) to ensure OD260/OD280 range was between 1.8 and 2.0. Resuspended RNA was stored at -80°C until shipment to AGRF (Australia) for de novo RNA sequencing.

Next-generation sequencing, assembly and annotation

Total RNA from paired MA glands from individuals of both spider species were provided to the BGI for de novo RNA sequencing and bioinformatics using Illumina HiSeq 2000, and to the AGRF for library construction and paired-end sequencing using an Illumina HiSeq 2500 platform. Raw sequences were assembled into contigs using the Genomic CLC Workbench 9 software (default settings). Protein-coding regions were determined using the open reading frame (ORF) predictor [http://bioinformatics.ysu.edu/tools/OrfPredictor.html]. Blast2GO was utilised for functional annotation of protein-coding regions against the NCBI nr database [43]. Relative expression of genes in the transcriptome was determined based on reads per kilobase of transcript per million mapped reads (RPKM) values, utilizing the de novo RNA-seq CLC Genomic Workbench 9 software: transcripts per kilobase million mapped reads (TPM) are also reported.

Spidroin sequences of the genus Nephila were obtained from NCBI, compiled and used in a BLASTp search to identify homologous proteins derived from the N. plumipes and N. pilipes gland transcriptomes. Matches were manually assessed to determine conservation. Further, a “spidroin-like” database was created by examining the six translated nucleotide reading frames for the following spidroin-like amino acid motifs: AAAAA, GGYGG, GYGPG, GQQGP, and GAGAGG. Finally, CD-search [https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi?] was utilised to identify specific hits to spidroin protein domains and superfamilies.

Spider silk preparation for proteomics

Spider silk threads were obtained by hand-reeling silk straight from live N. plumipes and N. pilipes spiders. Proteins were extracted from the silk by homogenisation in 100 μL protein extraction buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT), to which 100 μL of 30 mM Tris (pH 7.5) was added. Homogenate was vortexed and pulse-centrifuged several times to mix the dissolution buffer and Tris solution. The homogenate was vortexed for 15 min, pulse centrifuged, and then incubated in a sonicating water bath at ambient temperature for 20 min. Undissolved substances were pelleted by centrifugation at 12,000 x g for 8 min, and the supernatant containing the dissolved proteins were collected and stored at -80°C.

Sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel trypsin digestion

The spider silk proteins were separated by SDS-PAGE using a 4–20% polyacrylamide gradient gel (Amersham ECL Gel, GE Healthcare Life Sciences) according to manufacturer’s instructions. Samples were prepared 1:1 with 2x SDS sample buffer and incubated at 95°C for 5 min, then cooled to room temperature prior to pulse centrifugation. Samples were run for 1 h at 160 V (45 mA). Separated proteins were visualised by Coomassie Brilliant Blue (Bio-Rad) according to the staining process recommended by GE Healthcare. Upon completion of electrophoresis the proteins were precipitated with fixing solution [400 mL of EtOH, 100 mL of acetic acid, 500 mL of distilled water (DI)] for 30 min, followed by immersion in staining solution (1 tablet of PhastGel Blue-R 350, 400 mL of destaining solution; 250 mL of EtOH, 80 mL of acetic acid, 670 mL DI) for 10 min. The gel was subsequently preserved by immersion in preserving solution (25 mL of (87% v/v) glycerol with DI, 225 mL destaining solution) for 30 min. Protein sizes were estimated using a Pierce Blue Molecular Weight marker (Thermo Scientific).

Excised protein bands were washed in 50 mM NH4HCO3 at room temperature for 5 min, and then de-stained by incubating gel pieces for 30 min in 50 mM NH4HCO3 in 30% acetonitrile in a sonication water bath to remove Coomassie Brilliant Blue. Gel pieces were subsequently collected by in-gel trypsin digestion using the method described elsewhere [44]. The samples were reconstituted in 0.1% formic acid and stored at -20°C until mass spectroscopy.

NanoLC tandem TripleTof MS/MS analyses and protein identification

The spider silk extracts were analysed by LC-MS/MS on a Shimadzu Prominence Nano HPLC (Japan) coupled to a Triple ToF 5600 mass spectrometer (ABSCIEX, Canada) equipped with a nano electrospray ion source. Each extract (7 μL) was injected onto a 50 mm x 300 μm C18 trap column (Agilent Technologies, Australia) at 30 μL/min. The samples were de-salted on the trap column for 5 minutes using 0.1% formic acid (aq) at 30 μL/min. The trap column was then placed in-line with the analytical nano HPLC column, a 150 mm x 75 μm 300SBC18, 3.5 μm (Agilent Technologies, Australia) for mass spectrometry analysis. Linear gradients of 1–40% solvent B over 35 min at 300 nL/minute flow rate, followed by a steeper gradient from 40% to 80% solvent B in 5 min were used for peptide elution. Solvent B was held at 80% for 5 min for washing the column and returned to 1% solvent B for equilibration prior to the next sample injection. Solvent A consisted of 0.1% formic acid (aq) and solvent B contained 90/10 acetonitrile/ 0.1% formic acid (aq). The ionspray voltage was set to 2400V, declustering potential 100V, curtain gas flow 25, nebuliser gas 1 (GS1) 12 and interface heater at 150°C. The mass spectrometer acquired 500 ms full scan TOF-MS data followed by 20 by 50 ms full scan product ion data in an Information Dependant Acquisition mode. Full scan TOF-MS data was acquired over the mass range 350–1800 and for product ion ms/ms 100–1800. Ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion, MS/MS spectra of the resultant 20 most intense ions. The data was acquired and processed using Analyst TF 1.5.1 software (AB SCIEX, Concord, Canada).

The LC-MS/MS data were imported to the PEAKS studio (Bioinformatics Solutions Inc., Waterloo, ON, Canada, version 7.0) with the assistance of MS Data Converter (Beta 1.3, http://sciex.com/software-downloads-x2110). The database search included our own Nephila sp. transcriptome-derived protein databases, our “spidroin-like” database made by motif-searching the six translated nucleotide reading frames, and non-redundant protein databases (GenBank and UniProt). De novo sequencing of peptides, database search and characterising specific PTMs were used to analyse the raw data; false discovery rate (FDR) was set to ≤ 1%, and [-10*log(p)] was calculated accordingly where p is the probability that an observed match is a random event. The PEAKS used the following parameters: (i) precursor ion mass tolerance, 0.1 Da; (ii) fragment ion mass tolerance, 0.1 Da (the error tolerance); (iii) tryptic enzyme specificity with two missed cleavages allowed; (iv) monoisotopic precursor mass and fragment ion mass; (v) a fixed modification of cysteine carbamidomethylation; and (vi) variable modifications including lysine acetylation, deamidation on asparagine and glutamine, oxidation of methionine and conversion of glutamic acid and glutamine to pyroglutamate.

Results and discussion

Nephila plumipes and N. pilipes (Fig 1A) were collected and the MA glands were removed (Fig 1B) for RNA isolation and sequencing. MA gland reference transcriptomes were constructed for each species by combining next-generation sequence (NGS) data from sequencing runs produced in 2013, with data produced in 2016 (GenBank Accession: SRR6747912, SRR6747911). The combined MA gland transcriptomes for N. plumipes and N. pilipes produced 42,351,802 and 46,060,170 total paired reads, respectively. Paired reads were assembled into 69,812 contiguous sequences (contigs) for N. plumipes with an average length of 685 nucleotides, and into 70,123 contigs for N. pilipes with an average nucleotide length of 672. The data returned from ORF prediction were 67,862 and 67,942 sequences, and Blast2Go annotation allowed for the annotation of approximately 25% and 29% of all transcripts plus identification of 48 and 35 spidroin contigs, for N. plumipes and N. pilipes, respectively. The total spidroin count for each species was increased to 73 and 60 spidroins for N. plumipes and N. pilipes (Table 1 and 2), upon mining for spidroin sequences identified within each corresponding transcriptome-derived silk proteome (S1 and S2 Tables), by analysing the six translated nucleotide reading frames for spidroin-like repeat motifs, and by BLAST searching unique silk sequences found in the related N. clavipes genome reported by Babb et al. [27].

Fig 1. The golden orb-weaving spiders and a representative MA gland.

Fig 1

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(A) Nephila plumipes (photo by Alessandra Whaite) and Nephila pilipes (photo by Amos T Fairchild). (B) The major ampullate gland of Nephila pilipes, indicating the secretory section and the sac that stores silk proteins.

Table 1. Total spidroins in each Nephila plumipes data set (2013 and 2016).

ID Predicted Spidroin Length Domain/ Superfamily RPKM 2013 RPKM 2016 TPM 2013 TPM 2016
U_9113 AcSp1 97 0.00 0.31 0.00 0.22
U_25766 AcSp1 156 0.00 1.11 0.00 0.82
U_397 AgSp1 319 59.00 149.92 48.18 110.17
U_1689 AgSp1 338 0.00 39.15 0.00 28.77
U_1891 AgSp1 183 2.01 853.75 1.64 627.39
U_25614 AgSp2 48 26.25 3.78 21.43 2.78
U_7370 AgSp-a 523 Spidroin_N superfamily 0.00 8.89 0.00 6.54
U_1394 AgSp-c 1391 0.16 11.22 0.13 8.25
U_85 AgSp-c 761 30.92 413.08 25.25 303.56
U_6753 AgSp-c 127 0.00 143.05 0.00 105.12
U_6110 AgSp-c 182 Spidroin_N superfamily 0.00 7.40 0.00 5.44
U_2628 ECP-1† 198 0.00 4088.26 0.00 3004.33
U_4754 Flag1 226 43.48 2.19 35.51 1.61
U_20913 Flag2 107 0.00 0.48 0.00 0.35
U_18953 MaSp 89 8697.28 85599.66 7102.42 62904.26
U_10053 MaSp 88 41232.70 10475.45 33671.66 7698.05
U_11966 MaSp 99 2879.08 16061.76 2351.13 11803.24
U_17526 MaSp 71 1395.00 11596.29 1139.19 8521.72
U_1088 MaSp 103 4472.41 4888.34 3652.28 3592.28
U_47 MaSp1 57 Spidroin_MaSp 16143.09 6830.23 13182.85 5019.30
U_28059 MaSp1 112 Spidroin_MaSp 106.66 10.81 87.10 7.94
U_56 MaSp1 217 Spidroin_N 6718.57 27671.27 5486.55 20334.67
U_82* MaSp1 189 Spidroin_N superfamily 6.10 6223.69 4.98 4573.57
U_172 MaSp1 60 Spidroin_MaSp 22158.38 581.88 18095.09 427.61
U_2048 MaSp1 60 Spidroin_N superfamily 0.00 32.84 0.00 24.13
U_4815 MaSp1 41 16.48 81.39 13.45 59.81
U_15285 MaSp1 248 Spidroin_MaSp 169.59 934.33 138.49 686.61
U_49434 MaSp1 130 Spidroin_N superfamily 0.00 0.25 0.00 0.18
U_999* MaSp1 126 5864.84 50336.66 4789.37 36990.68
U_6045* MaSp1 96 5861.11 7092.78 4786.33 5212.24
U_9403* MaSp1 92 52042.69 55197.89 42499.36 40563.04
U_10063* MaSp1 88 5068.79 30452.03 4139.30 22378.15
U_27155* MaSp1 73 1217.71 1221.18 994.41 897.41
U_8956* MaSp1 48 202.72 4187.95 165.55 3077.58
U_25731* MaSp1 119 39.68 328.95 32.40 241.74
U_33 MaSp1 85 Spidroin_MaSp 39225.86 7270.57 32032.82 5342.90
U_18230 MaSp2 154 Spidroin_N 0.89 6.39 0.73 4.70
U_22058* MaSp-c 68 Spidroin_N superfamily 15.46 1191.73 12.62 875.76
U_14382* MaSp-d 125 190.96 9074.23 155.94 6668.34
U_6066* MaSp-d 114 536.54 46701.72 438.15 34319.50
U_46611 MaSp-f 140 0.00 0.59 0.00 0.43
U_10737 MaSp-f 239 Spidroin_MaSp 13.74 50.18 11.22 36.88
U_1060* MaSp-g 228 317.41 560.02 259.21 411.54
U_17514* MaSp-g 68 3181.41 1064.47 2598.02 782.25
U_2249* MaSp-g 135 2005.01 1405.81 1637.35 1033.08
U_679* MaSp-g 220 406.52 428.27 331.97 314.72
U_24021 MaSp-h 82 Spidroin_N superfamily 31.96 525.52 26.10 386.19
U_17901 MiSp1 153 Spidroin_MaSp 0.00 2.99 0.00 2.20
U_11571 MiSp1 75 Spidroin_MaSp 224.25 100.05 183.13 73.52
U_12772 MiSp1 253 RP1-2 18.62 22.26 15.21 16.36
U_1636* MiSp1 336 RP1-2 5.21 58.80 4.26 43.21
U_27015 Sp-5803 111 4.31 0.50 3.52 0.37
U_25915 Sp-74867 118 Spidroin_MaSp 3.79 0.66 3.09 0.48
U_17052 Sp-74867 93 Spidroin_MaSp 9185.22 7487.22 7500.88 5502.10
U_37 Sp-8175 920 1.40 47.08 1.15 34.59
U_6378 Sp-907 277 Spidroin_N superfamily 0.33 32.09 0.27 23.58
U_4542* Sp-907 69 90.31 588.96 73.75 432.80
U_368* Sp-907 259 29.90 787.06 24.42 578.38
U_17587* Sp-907 108 1107.42 2025.01 904.35 1488.11
U_1699* Sp-907 110 55.68 259.41 45.47 190.63
U_2859* Sp-907 108 293.37 1928.52 239.57 1417.20
U_15734* Sp-907 124 65.56 2816.43 53.53 2069.70
U_6377* Sp-907 102 0.63 25.35 0.52 18.63
U_3* Sp-907 117 422.86 3432.85 345.32 2522.68
U_319 Sp-907 164 1330.27 12939.36 1086.33 9508.69
U_21571 TuSp 179 Spidroin_MaSp 0.00 8676.94 0.00 6376.38
U_586 TuSp1 241 Spidroin_N 0.00 1486.26 0.00 1092.20
U_897 TuSp1 152 RP1-2 0.00 74973.89 0.00 55095.74
U_3907 TuSp1 183 0.00 43.55 0.00 32.01
U_7486 TuSp1 116 RP1-2 0.00 3.58 0.00 2.63
U_23917 TuSp1 149 Spidroin_N superfamily 0.00 1.32 0.00 0.97
U_60428 TuSp1 119 Spidroin_N superfamily 0.00 0.84 0.00 0.62
U_86 TuSp1 89 0.00 40804.39 0.00 29985.74
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RPKM, Reads Per Kilobase of transcript per Million mapped reads

TPM, Transcripts Per Kilobase Million

† ECP-1, egg case protein-1

* Also identified in the corresponding proteome

Table 2. Total spidroins in each Nephila pilipes data set (2013 and 2016).

ID Predicted Spidroin Length Domain/ Superfamily RPKM 2013 RPKM 2016 TPM 2013 TPM 2016
I_65000 AcSp 106 0.00 0.16 0.00 0.13
I_55 AcSp1 240 Spidroin_N superfamily 0.00 9455.94 0.00 7450.90
I_29651 AgSp1 143 0.82 1.24 0.55 0.98
I_69410 AgSp1 131 1.04 0.91 0.69 0.72
I_17302 AgSp1 114 5.10 5.89 3.41 4.64
I_4597 AgSp-a 281 0.46 4.64 0.31 3.65
I_17659 AgSp-a 359 Spidroin_N superfamily 0.00 2.94 0.00 2.32
I_1837 AgSp-c 1609 Spidroin_MaSp 18.38 41.31 12.31 32.55
I_31481 AgSp-c 214 0.00 0.69 0.00 0.54
I_13893 AgSp-c 148 2.71 5.22 1.82 4.11
I_1450 AgSp-c 104 10.90 37.31 7.30 29.40
I_67816 AgSp-c 112 Spidroin_N superfamily 1.24 0.47 0.83 0.37
I_18284 Flag 277 23.42 0.72 15.68 0.57
I_14698 Flag1 203 Spidroin_N superfamily 0.00 4.80 0.00 3.78
I_4468 Flag2 245 6.51 93.38 4.36 73.58
I_12595 MaSp 116 17160.99 2766.09 11489.93 2179.57
I_33885 MaSp 110 44532.47 979.27 29816.18 771.62
I_7068 MaSp 79 Spidroin_MaSp 242027.26 87.87 162046.41 69.24
I_228 MaSp1 221 Spidroin_MaSp 689.40 4201.47 461.58 3310.59
I_304* MaSp1 277 7.87 786.60 5.27 619.81
I_820 MaSp1 173 Spidroin_MaSp 25576.95 2260.04 17124.73 1780.82
I_27560* MaSp1 105 44.86 3339.47 30.03 2631.37
I_141 MaSp1 306 Spidroin_N superfamily 10.72 3181.16 7.18 2506.63
I_478* MaSp1 498 Spidroin_N 133.27 362.93 89.23 285.98
I_11111 MaSp1 169 Spidroin_N superfamily 25.36 14.10 16.98 11.11
I_1327* MaSp1 89 2.63 446.18 1.76 351.57
I_262* MaSp1 173 2058.66 1616.88 1378.35 1274.04
I_2904* MaSp1 92 9607.91 2971.56 6432.86 2341.47
I_17778* MaSp1 79 17.66 1186.59 11.82 934.99
I_2074* MaSp2 120 138074.73 62.50 92446.26 49.25
I_272* MaSp2 96 Spidroin_MaSp 238666.86 1906.18 159796.50 1502.00
I_16679 MaSp2 93 60136.69 377.02 40263.79 297.07
I_1340* MaSp-f 328 Spidroin_MaSp 56.92 344.75 38.11 271.65
I_9111* MaSp-h 143 53644.90 35.16 35917.29 27.70
I_63355 MaSp-h 104 Spidroin_MaSp 8.03 0.34 5.38 0.26
I_359* MaSp-h 392 32815.12 2140.32 21970.96 1686.49
I_550 MaSp-h 166 18520.57 5149.23 12400.23 4057.39
I_4156 MiSp 133 10236.26 252.33 6853.56 198.83
I_12574 MiSp 84 0.00 323.96 0.00 255.27
I_10698 MiSp 129 60.56 15064.25 40.55 11870.03
I_20587 MiSp 153 7.17 9866.14 4.80 7774.13
I_4316 MiSp 83 6.03 8829.59 4.04 6957.37
I_26363 MiSp 113 66.04 8262.29 44.22 6510.35
I_3787 MiSp1 66 67.69 13233.55 45.32 10427.51
I_21222 MiSp1 119 Spidroin_MaSp 110.32 7302.47 73.86 5754.06
I_9 MiSp1 136 Spidroin_MaSp 207.34 13762.70 138.82 10844.46
I_7810 MiSp1 95 87.22 9654.94 58.40 7607.71
I_7754 MiSp-a 164 52.36 11827.42 35.05 9319.54
I_8908* MiSp-d 125 1939.86 211.64 1298.81 166.76
I_61631 Sp-1339 134 0.00 0.50 0.00 0.40
I_52113 Sp-5803 111 19.22 0.84 12.87 0.67
I_20950 Sp-907 147 Spidroin_N superfamily 30.53 19.48 20.44 15.35
I_12762 TuSp1 183 RP1-2 0.00 3.78 0.00 2.98
I_22088 TuSp1 78 Spidroin_MaSp 0.00 14056.52 0.00 11075.98
I_23940 TuSp1 187 0.00 8958.45 0.00 7058.90
I_48 TuSp1 97 0.00 978.98 0.00 771.40
I_1415 TuSp1 112 RP1-2 0.00 6772.23 0.00 5336.25
I_4212 TuSp1 118 0.00 221762.64 0.00 174740.14
I_9940 TuSp1 109 RP1-2 superfamily 0.00 20210.87 0.00 15925.36
I_8400 TuSp1 88 0.00 77285.64 0.00 60898.01
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RPKM, Reads Per Kilobase of transcript per Million mapped reads

TPM, Transcripts Per Kilobase Million

* Also identified in the corresponding proteome

A recent paper reporting on the genome and tissue transcriptomes of the golden-orb spider, N. clavipes, identified 28 spidroins [27]. Our transcriptomes included partial sequences with homology to several of the unique spidroins found in this closely related species, including AgSp-a, AgSp-c, Sp-5803, Sp8175, Sp74867 and, MaSp-c, -d -g and -h (Table 1 and 2). In N. plumipes, our study uncovered numerous matches to the N. clavipes spidroin Sp-907, potentially non-overlapping contigs aligning to different regions on the same gene. In N. pilipes, our study found sequences with homology to MiSp-a and -d, and Sp-1339. Our longest assembled spidroin contigs in both species matched to the N. clavipes the glue-like aggregate spidroin, AgSp-c. Aggregate spidroins, which form evenly-spaced droplets along flagelliform prey capture threads, have also been characterised in three other species from the family Araneidae, and three species from the family Theridiidae [45, 46], These spidroins vary greatly in length among species and it appears we have recovered a full-length aggregate spidroin (1609 aa) from N. pilipes. However, aggregate spidroins were not evident in the proteome of either species.

The N. pilipes and N. plumipes silk proteins were obtained after several predominant bands were excised from the Coomassie stained SDS-PAGE gel (Fig 2), followed by trypsin digestion and LC-MS/MS analysis. This analysis identified 29 and 18 proteins for N. plumipes and N. pilipes (Table 3 and 4), respectively (≥20.00 -10lgP, ≥ 2 peptide matches). For the silk proteins of N. plumipes mapped to the transcriptome, 24 were spidroins, mostly MaSp1-like spidroins, MiSp-like and novel N. clavipes spidroin, Sp-907, confirming the abundance of this spidroin in the transcriptome. Besides spidroins, a cuticle protein and a coiled-coil domain-containing protein were identified in the proteome along with two proteins which could not be identified. Cuticle proteins have been described previously in the MA gland [7, 47]. The coiled-coil domain protein found within the transcriptome and proteome of N. plumipes is potentially interesting as coiled-coil structures are also found to form in the silk of the Japanese yellow hornet, Vespa simillima [48]. N. pilipes silk proteins accounted for 13 of 18 proteins mapped back to the corresponding transcriptome. Again, most of these spidroins were MaSp-like, one MiSp and the remaining 5 proteins were not able to be annotated at this stage.

Fig 2. SDS-PAGE and Coomassie stain indicating prominent protein bands that were excised for LC-MS/MS analysis.

Fig 2

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(A-F) Nephila plumipes, (G-K) Nephila pilipes. MWM, molecular weight marker.

Table 3. Nephila plumipes proteins within the silk matching to the transcriptome.

ID -10lgP Cover-age (%) No. of Peptides Unique Peptides PTM Average Mass Example Peptides BLAST Similarity
U_999* 484.05 63 27 15 Amidation; Deamidation (NQ); Pyro-glu from Q 12348 R.GAGAAAAAAGGAGQGGYGGLGSQGAGR.G, G.QGAAAAAAGGAGQGGYGGLGGQGAGR.G Major ampullate spidroin 1 [Nephila clavipes]
U_1060 403.82 66 17 16 Amidation; Deamidation (NQ); Pyro-glu from Q 19573 R.GPGGYGPGQQGPAQQGPGQQGPGGAGAAAAAGR.G, R.GPGSYGPGQQGPGQQGPR.Q Major ampullate spidroin protein MaSp-g [Nephila clavipes]
U_434 282.78 23 15 15 Acetylation (K); Deamidation (NQ); Oxidation (M) 80294 R.GGGGGFNVPSGGGGLNIPSGGGR.G, R.DISSSATSASSASAGDAGGIGQGR.N N/A
U_368 237.58 39 8 7 21576 R.GGDSGAAAAAAAADGGR.G, R.GGDTGAAAAAAAADGGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_3 172.59 16 7 6 38371 R.GGDSGAAAAAAAADSGR.G, R.GGYGGLGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_679 141.03 24 4 3 Deamidation (NQ) 18685 R.YGPSGPGSAAAAAAAAGAGSR.G, G.GYGPGQQGPGQQ(+.98)GPGQQG Major ampullate spidroin protein MaSp-g [Nephila clavipes]
U_1081 102.56 18 4 4 Acetylation (K); Deamidation (NQ); Pyro-glu from Q 16054 K.GYDNDFVR.F, R.Q(-17.03)FDHPYK.R Hypothetical Protein NCL1 41264 [Nephila clavipes]
U_4542 132.07 29 3 3 5670 L.GGDSGAAAAAAAAADGGR.G, R.GLGGDSGAAAAAAAAADGGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_82* 90.27 18 3 3 Acetylation (K); Deamidation (NQ); Pyro-glu from Q 18384 K.AFYQTTGTEDSR.F, G.Q(-17.03)VTPWSNAK.L Major ampullate spidroin 1 variant 1, partial [Nephila clavipes]
U_6066* 159.66 46 2 2 Deamidation (NQ) 10109 R.FGSGGPGGDSAAAAAASGGNGGR.F, R.FGSGGPGGDSAAGAAASGGNGGNGGN(+.98)GGR.F Major ampullate spidroin protein MaSp-d [Nephila clavipes]
U_6045* 121.31 41 2 2 Deamidation (NQ) 10812 R.AAAAAAGGAGQGGYGSLGSQGAGR.G, G.AGGAAAAAGGAGQ(+.98)GG Spidroin 1 [Nephila clavipes]
U_22058 116.5 39 2 1 Oxidation (M) 7566 R.TGAFTADQLDDMSTIGDTLK.T, R.TGAFTADQLDDM(+15.99)STIGDTLK.T Major ampullate spidroin protein MaSp-c [Nephila clavipes]
U_14382* 133.02 35 2 2 7519 R.FGSEGPGGDSAAAAAASGGDGGR.F, R.FGTGGPESDSAAASGGNGGNR.Q Major ampullate spidroin protein MaSp-d [Nephila clavipes]
U_17587 73.53 23 2 1 8766 G.SSGAAAAAAAADGGIGR.G, S.GGYGGIGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_1699 55.99 16 2 1 9285 R.GGYGGLGR.G, A.AAAAAAEGGRGGYGGLGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_1669 27.36 3 2 2 85262 L.DVINSNESR.L, K.QKLSELEVQK.Q Coiled-coil domain-containing protein [Nephila clavipes]
U_9403 236.31 100 43 36 Acetylation (Protein N-term); Deamidation (NQ); Ethylation; Methyl ester; Acetylation (N-term); 7 more 7117 Q.GAGAAAAAAGGAGQGGYGGLGSQGAGR.G, G.AAAAAAGGAGQGGYGGLGSQGAGR.G Dragline silk fibroin [Nephila clavipes]
U_2859 151.32 99 30 29 Acetylation (Protein N-term); Deamidation (NQ); Ethylation; Methyl ester; Octanoyl; 6 more 8958 GLGGDSAAAAAAAADGGR.G, G.GDSAAAAAAAADGGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_8956* 99.15 56 19 19 Carbamidomethylation; Deamidation (NQ); Ethylation; Methyl ester; Octanoyl; 9 more 10187 R.GAGAAAAAAAGGAGQGGYRS(-18.01)EE(+14.02).H, R.GAGAAAAAAAGGAGQGGYRS(-18.01)E(+14.02)E.H Dragline silk fibroin [Nephila clavipes]
U_2249 57.34 77 14 7 Deamidation (NQ); Ethylation; Methyl ester; Hydroxylation; Dihydroxy; Dehydration 10986 G.GPGGYGPGQQGPGQQGPGQ(+.98)QG.P, P.GGAAAAAAAAGGPGGYGPGQQGP.G Major ampullate spidroin protein MaSp-g [Nephila clavipes]
U_10063* 98.84 92 12 7 Acetylation (Protein N-term); Deamidation (NQ); Ethylation; Octanoyl 6547 Q.GGYGGQGAGAAAAAGGAGQ.G, Q.GGYGGQGAGAAAAAGGAGQGGQ.G Major ampullate spidroin-like protein, partial [Nephilengys cruentata]
U_27155 116.09 89 11 8 Acetylation (Protein N-term); Deamidation (NQ) 6231 G.AGAGAAAAAAGGAGQGGYGGLGGQ(+.98)GAGQG.G, G.AGAGAAAAAAGGAGQGGYGGLGGQGAGQ(+.98)G.G Dragline silk spidroin 1 [Nephila pilipes]
U_17514 68.91 88 11 3 Acetylation (Protein N-term); Deamidation (NQ); Dehydration 5594 Q.Q(+42.01)(+.98)GPSGPGGAAAAAAAA.G, A.GPGGYGPGQQGPGQQGPGQ(+.98)QG.P Major ampullate spidroin protein MaSp-g [Nephila clavipes]
U_15734 128.72 36 4 3 10272 R.GGDSGAAAAAAAADGGR.G, G.DSGAAAAAAAADGGR.G Spidroin protein Sp-907 [Nephila clavipes]
U_29163 22.58 12 3 3 39198 G.GAGGGRGGGAGGNYPPQPYN.F, Q.VSIVVAALV.G Cuticle protein 10.9 [Nephila clavipes]
U_1636 22.56 24 3 3 Deamidation (NQ) 29130 A.AAGGAAGYGRGAGAGAGAAAG.A, S.GAGGGAVAGAGAAAGAV.S Chain A, 3D structure of RP domain of MiSp
U_6377 46.76 30 2 1 9218 R.GGYGGLGR.G, R.IGYGPGGVSGAAAVAAAADSGKG.S Spidroin protein Sp-907 [Nephila clavipes]
U_97 23.18 1 2 2 Deamidation (NQ) 361672 V.DASVPGGRHK.S, C.RDISLQ(+.98)NVQK.M N/A—short sequence
U_25731 20.94 19 2 1 9468 H.GGLGGQGAAAAAAGGAGQGGLGG.L, G.QGAAAAAAGGAGQGGLGG.L Dragline silk fibroin [Nephila clavipes]
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Proteins from the corresponding transcriptome with 2 or more peptide matches were BLAST annotated (E-value cut-off 10−3). Example matching peptides are shown (full list, see S3 Table). PTM, posttranslational modifications.

Table 4. Nephila pilipes proteins within the silk matching to the transcriptome.

ID -10lgP Cover-age (%) No. of Peptides Unique Peptides PTM Average Mass Example Peptides BLAST Similarity
I_478 288.93 25 8 8 Deamidation (NQ); Oxidation (M); Pyro-glu from Q 42362 R.QGGQGAGAATAAASGAGQGGYGR.Q, R.QGGQGAGAAAAGAGGAGR.G Major ampullate spidroin 1 variant 3 [Nephila clavipes]
I_304 261.74 21 2 2 22373 R.NAAVAAAAAGGLGGYGLGGQGSGQR.S, R.PSGAGGQGAQAPGGYGTGSGSTIVITAGGQR.G Spidroin 1 [Nephila clavipes]
I_33* 233.73 35 8 8 Acetylation (K); Deamidation (NQ); Oxidation (M) 24343 K.DAGGVM(+15.99)QGALGDFKDDLR.E, K.DAGGVMQGALGDFKDDLR.E N/A
I_67* 179.9 46 5 5 Deamidation (NQ); Oxidation (M) 11923 R.AISESMANTGGGGLGGSR.A, R.AISESM(+15.99)ANTGGGGLGGSR.A N/A
I_2074* 135.34 31 4 3 Amidation 9600 A.SYGPGPQASAAASR.L, Y.GPGPQASAAASR.L Major ampullate spidroin 2 [Nephila senegalensis]
I_272* 122.08 33 4 3 Amidation 9371 Y.AAASQSAQVVSR.S, N.YAAASQSAQVVSR.S Major ampullate spidroin 2 variant 1 [Nephila clavipes]
I_359* 188.07 51 28 28 Acetylation (Protein N-term); Deamidation (NQ); Ethylation; Dehydration; Dihydroxy 34638 A.GGLGGYGPGQQGPGQGGR.G, A.AAGGLGGYGP(+15.99)GQQGPGQQGPGQR.G Major ampullate spidroin protein MaSp-h [Nephila clavipes]
I_1340 178.26 24 18 18 Carbamidomethylation; Deamidation (NQ); Ethylation; Dehydration; Dihydroxy; 4 more 49394 R.LSAPEAGTR.V, I.LSGP(+31.99)GR(+15.99)QASAAASR.L Major ampullate spidroin protein MaSp-f isoform 1 [Nephila clavipes]
I_1327 147.26 40 12 12 Acetylation (Protein N-term); Deamidation (NQ); Ethylation; Dehydration; Hydroxylation; O-Diethylphosphorylation 14116 R.GQGGQGPSGQLAQAPSGYGQGSGAAAASGGLGGYGGQGGQR.S, G(+42.01)S(-18.01)GT(-18.01)AIAITAGGQR.G Major ampullate spidroin 1 [Argiope trifasciata]
I_262 89.5 39 7 6 Acetylation (Protein N-term); Deamidation (NQ); Ethylation; Hydroxylation; O-Diethylphosphorylation 12276 G.Q(+28.03)GSGAAAAGAGQGGY(+15.99)GR.Q, G.AGAAAAAAGGAGQGGYGGLG.G Dragline silk spidroin 1 [Nephila pilipes]
I_27560 129.55 71 5 2 Deamidation (NQ) 7995 R.SLGANSGEADAAGDR.G, G.AGAAAAAAGGAGQGGYGGLG.G Dragline silk spidroin 1 [Nephila pilipes]
I_8908 111.49 30 3 3 9045 R.GYGPGSGAGAAAAGGAGEGGR.G, A.AAAAGGAGGEGGR.G Minor ampullate spidroin protein MiSp-d [Nephila clavipes]
I_1660 34.34 1 3 3 Acetylation (Protein N-term) 438757 K.IALHLEQ.L, I.VATPDIAGV.H N/A
I_9111* 33.72 44 3 3 Acetylation (Protein N-term); Deamidation (NQ) 11976 P(+42.01)GGYGPGQQGPGGYGPGQQ(+.98)GPGGAGAAAAAAAAGG.S, P(+42.01)GGYGPGQQGPGGYGPGQ(+.98)QGPGGAGAAAAAAAAGG.S Major ampullate spidroin protein MaSp-h [Nephila clavipes]
I_3749 26.1 1 3 3 Deamidation (NQ) 269920 R.GGKRGN(+.98)THTKK.I, E.TLLSMN(+.98)PTR.G N/A
I_23726 45.13 4 2 2 53637 L.LAADDFR.L, K.QNVKVRVASSSK.N N/A
I_2904* 30.94 40 2 1 7155 G.GQGAGAAGAAAAAGGAGQGGYGGLG.G, G.QGGYGGLGGQGT.E Dragline silk spidroin 1 [Nephila pilipes]
I_17778 28.19 31 2 1 Deamidation (NQ) 8397 G.YGGLGGQ(+.98)GTGAGGAAAA.G, S.ASLGGYGGLG.G Major ampullate spidroin 1A precursor [Nephila clavipes]
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Proteins from the corresponding transcriptome with 2 or more peptide matches were BLAST annotated (E-value cut-off 10−3). Example supporting peptides are shown (full list, see S4 Table). PTM, posttranslational modifications.

Beyond the matches made to the transcriptome, from the silk of N. plumipes and N. pilipes a further 2,420 and 2,658 de novo only peptides, respectively, were identified with high confidence (average local confidence above 70) that did not match any sequences within the transcriptome. This de novo dataset of unmatched potential proteins was BLASTed against NCBI protein databases, however the focus in this study is on those proteins relevant to their corresponding transcriptomes. Unlike a genome, a transcriptome can only provide us with genes transcribed at the time of RNA isolation and this may partly explain the discrepancy between matched and de novo peptides. A further explanation is that the hand-reeled silk also contains silk from different silk glands. Each silk gland ends at its own spigot on the surface of a spinneret. It is possible that as the silk thread passes past other spigots during collection, it also collects fibres from other silk glands. However, the spigots closest to the major ampullate spigot on the anterior lateral spinnerets produce pyriform spidroins and there was no evidence of these spidroins in the proteome [4]. Further, our gel-based extraction method might have missed proteins with relatively low molecular weight or low abundance, such as the cysteine-rich proteins identified by Pham et al. which were found to co-localise with spidroins in the MA silk of Latrodectus hesperus [8].

Quantitative analyses were undertaken and based on reads per kilobase of transcript per million mapped reads (RPKM) values by mapping the 2013 and 2016 data back to a combined de novo reference transcriptome. The 50 most highly expressed sequences of N. pilipes and N. plumipes were manually selected for further annotation (Tables 5 and 6). These abundant sequences were matched to sequences found in the NCBI or Uniprot public protein databases (accessed Oct-Dec 2017). Spidroins were, as expected, among the most highly expressed sequences of both datasets, numbering 23 and 26 spidroins for N. plumipes and N. pilipes, respectively. In both species, major and minor ampullate, and tubuliform spidroins were highly expressed in the MA gland. Interestingly, the N. plumipes sequence with the highest RPKM value could not be characterised based on BLAST protein prediction. Uncharacterised highly expressed sequences will be selected for functional annotation in future works.

Table 5. Nephila plumipes silk gland most abundantly expressed genes.

ID RPKM TPM Match Description E-value
U_3789 346459.71 282927.68 Uncharacterised protein [Latrodectus hesperus] 9.40E-05
U_18953 85599.66 62904.26 Major ampullate spidroin-like protein [Nephilengys cruentata] 1.50E-31
U_897 74973.89 55095.74 Cylindrical silk protein 1 [Nephila clavata] 4.40E-54
U_9403 * 55197.89 40563.04 Dragline silk fibroin [Nephila clavipes] 4.80E-29
U_999 †* 50336.66 36990.68 Major ampullate spidroin 1 [Nephila clavipes] 1.00E-33
U_20 49180.92 36141.37 N/A
U_6066 * 46701.72 34319.50 Major ampullate spidroin protein MaSp-d [Nephila clavipes] 1.00E-37
U_10053 41232.70 33671.66 Major ampullate spidroin-like protein [Nephilengys cruentata] 4.70E-16
U_86 40804.39 29985.74 Tubuliform spidroin protein TuSp [Nephila clavipes] 4.70E-25
U_33 39225.86 32032.82 Major ampullate spidroin 1 [Nephila clavipes] 4.90E-49
U_440 32127.85 23609.65 Hypothetical protein NCL1_22245 [Nephila clavipes] 7.40E-38
U_10063 * 30452.03 22378.15 Major ampullate spidroin-like protein, partial [Nephilengys cruentata] 5.40E-32
U_56 † 27671.27 20334.67 Major ampullate spidroin 1 variant 2 [Nephila clavipes] 9.70E-102
U_137 † 24578.13 18061.63 Hypothetical protein NCL1_28494 [Nephila clavipes] 1.30E-69
U_172 22158.38 18095.09 Major ampullate spidroin 1A precursor, partial [Nephila clavipes] 9.30E-33
U_19886 22132.89 18074.27 Hypothetical protein NCL1_19751 [Nephila clavipes] 3.40E-08
U_63 22118.45 18062.48 N/A
U_30462 † 19965.96 14672.30 N/A
U_4890 18864.55 13862.91 N/A
U_47 16143.09 13182.85 Dragline silk spidroin 1 [Cyrtophora moluccensis] 4.50E-18
U_11966 16061.76 11803.24 Major ampullate gland dragline silk protein-2, partial [Araneus ventricosus] 4.20E-17
U_22856 † 14927.17 12189.90 Uncharacterized protein LOC107452916 isoform X1 [Parasteatoda tepidariorum] 1.10E-12
U_339 13236.17 9726.81 N/A
U_319 12939.36 9508.69 Spidroin protein Sp-907 [Nephila clavipes] 1.20E-56
U_17526 11596.29 8521.72 Major ampullate spidroin-like protein [Nephilengys cruentata] 4.10E-11
U_594 9427.11 7698.42 Tubulin alpha chain [Stegodyphus mimosarum] 0.00E+00
U_17052 9185.22 7500.88 Spidroin protein Sp-74867 [Nephila clavipes] 7.40E-43
U_14382 * 9074.23 6668.34 Major ampullate spidroin protein MaSp-d [Nephila clavipes] 2.40E-45
U_32 † 8925.67 7288.93 Hypothetical protein NCL1_19751 [Nephila clavipes] 1.30E-43
U_21571 8676.94 6376.38 Tubuliform spidroin-like protein [Nephilengys cruentata] 7.30E-42
U_16121 8344.15 6131.83 N/A
U_6045 * 7092.78 5212.24 Spidroin 1 [Nephila clavipes] 3.00E-38
U_24501 6484.79 5295.65 Hypothetical protein NCL1_37350 [Nephila clavipes] 4.90E-13
U_460 6471.84 5285.06 Ferritin [Stegodyphus mimosarum] 2.60E-114
U_570 6287.70 4620.62 N/A
U_82 †* 6223.69 4573.57 Major ampullate spidroin 1 variant 1, partial [Nephila clavipes] 9.20E-85
U_152 6087.77 4971.43 Elongation factor 1-alpha [Stegodyphus mimosarum] 0.00E+00
U_17648 5967.76 4385.50 N/A
U_134 † 5059.94 4132.07 Cathepsin B [Araneus ventricosus] 0.00E+00
U_1088 4888.34 3592.28 Dragline silk protein spidroin 2 [Nephila clavata] 3.30E-45
U_346 † 4649.24 3416.57 N/A
U_94 4571.75 3733.40 Nucleoside diphosphate kinase [Latrodectus hesperus] 3.50E-93
U_197 † 4458.19 3276.17 Putative fasciclin [Latrodectus hesperus] 9.40E-42
U_18 4188.49 3077.98 N/A
U_8956 † * 4187.95 3077.58 Dragline silk fibroin [Nephila clavipes] 9.20E-07
U_30 4175.22 3068.23 Cytochrome c oxidase subunit I [Cyclosa argenteoalba] 0.00E+00
U_12055 4175.03 3068.08 N/A
U_2628 4088.26 3004.33 Egg case protein variant 1 [Argiope argentata] 1.50E-38
U_318 † 4079.46 2997.85 N/A
U_924 † 4009.24 2946.25 N/A
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E-value cut-off 10−3

† Genes found to exhibit a signal sequence

* Highly expressed genes also found within the silk proteome

Table 6. Nephila pilipes silk gland most abundantly expressed genes.

ID RPKM TPM Match Description E-value
I_7068 242027.26 162046.41 Major ampullate spidroin 2 variant 1 [Nephila clavipes] 7.70E-39
I_272 * 238666.86 159796.50 Major ampullate spidroin 2 variant 1 [Nephila clavipes] 7.70E-39
I_4212 † 221762.64 174740.14 Tubuliform spidroin 1 [Araneus ventricosus] 5.20E-11
I_2074 * 138074.73 92446.26 Major ampullate spidroin 2 [Nephila senegalensis] 5.00E-13
I_8400 77285.64 60898.01 Tubuliform spidroin 1 [Agelenopsis aperta] 5.90E-18
I_16679 60136.69 40263.79 Major ampullate spidroin 2 [Nephila clavipes] 3.40E-35
I_9111 * 53644.90 35917.29 Major ampullate spidroin protein MaSp-h [Nephila clavipes] 1.30E-59
I_33885 † 44532.47 29816.18 Dragline silk fibroin [Araneus ventricosus] 1.40E-20
I_33 * 44168.19 34802.78 NA
I_52 34336.68 22989.71 Spider venom protein NPTX_B154 [Nephila pilipes] 3.20E-10
I_5 † 33908.87 26718.84 NA
I_359 * 32815.12 21970.96 Major ampullate spidroin protein MaSp-h [Nephila clavipes] 1.60E-136
I_67 * 26171.69 20622.25 NA
I_820 25576.95 17124.73 Dragline silk spidroin 1 [Nephila pilipes] 1.80E-94
I_1332 † 23250.68 15567.21 Spider venom protein NPTX_C786 [Nephila pilipes] 1.50E-12
I_3479 † 21411.41 14335.75 Hypothetical protein NCL1_21799 [Nephila clavipes] 3.00E-04
I_9940 20210.87 15925.36 Cylindrical silk protein 1 [Nephila clavata] 4.90E-12
I_2337 † 20187.99 13516.63 NA
I_77 19281.52 12909.71 ART2 [Enterospora canceri] 4.40E-57
I_550 18520.57 12400.23 Major ampullate spidroin protein MaSp-h [Nephila clavipes] 1.20E-58
I_12595 17160.99 11489.93 Dragline silk fibroin, partial [Araneus ventricosus] 1.80E-42
I_79 15849.11 12488.47 Hypothetical protein NCL1_39416 [Nephila clavipes] 2.10E-14
I_178 * 15167.42 11951.32 NA
I_16079 15154.62 10146.59 NA
I_10698 15064.25 11870.03 Minor ampullate spidroin-like protein [Nephilengys cruentata] 4.10E-63
I_5861 † 14879.35 9962.29 Hypothetical protein NCL1_19751 [Nephila clavipes] 3.40E-39
I_22088 14056.52 11075.98 Tubuliform spidroin 1 variant 1 [Araneus diadematus] 6.70E-07
I_9 13762.70 10844.46 Minor ampullate fibroin 1 [Nephila antipodiana] 1.90E-45
I_12551 † 13639.82 10747.64 Hypothetical protein NCL1_19751 [Nephila clavipes] 3.60E-31
I_3787 13233.55 10427.51 Minor ampullate silk protein MiSp1 [Nephila clavipes] 8.00E-27
I_576 13206.17 10405.94 Bm3878 [Brugia malayi] 4.50E-40
I_37 † 12356.47 9736.41 Venom allergen 5 [Stegodyphus mimosarum] 1.10E-41
I_7754 11827.42 9319.54 Minor ampullate spidroin protein MiSp-a [Nephila clavipes] 6.00E-16
I_60663 11758.95 9265.58 NA
I_2057 † 10260.75 6869.96 Hypothetical protein NCL1_37703 [Nephila clavipes] 8.10E-15
I_4156 * 10236.26 6853.56 Minor ampullate spidroin [Argiope argentata] 7.10E-27
I_2304 10148.69 6794.93 Hypothetical protein THAOC_21441 [Thalassiosira oceanica] 2.50E-22
I_20587 9866.14 7774.13 Minor ampullate spidroin-like protein [Nephilengys cruentata] 2.00E-56
I_8429 9848.94 7760.57 NA
I_7810 * 9654.94 7607.71 UniProt BLAST Minor ampullate silk protein MiSp1 [Nephila clavipes] 4.50E-39
I_2904 * 9607.91 6432.86 Dragline silk spidroin 1 [Nephila pilipes] 6.00E-33
I_31392 9573.32 7543.40 CRISP/Allergen/PR-1 [Parasteatoda tepidariorum] 6.40E-07
I_55 † 9455.94 7450.90 Cylindrical silk protein 1 [Nephila clavata] 6.40E-60
I_55059 9033.46 6048.25 NA
I_23940 8958.45 7058.90 Cylindrical silk protein 1 [Nephila clavata] 1.80E-66
I_251 8938.21 5984.47 Putative tumor differentially expressed protein [Latrodectus hesperus] 2.10E-21
I_4316 8829.59 6957.37 Minor ampullate spidroin-like protein [Nephilengys cruentata] 1.70E-30
I_710 8760.58 5865.54 NA
I_7 † 8494.88 6693.63 Hypothetical protein X975_26006 [Stegodyphus mimosarum] 1.90E-05
I_26363 8262.29 6510.35 Minor ampullate spidroin-like protein [Nephilengys cruentata] 1.40E-57
Open in a new tab

E-value cut-off 10–3

† Genes found to exhibit a signal sequence

* Highly expressed genes also found within the silk proteome

This study found the MA gland alone produces six of the seven classes of silk products: MA, minor ampullate, flagelliform, tubuliform (also at times referred to as cylindriform silk), aciniform and aggregate silk products. Several other studies have also found multiple spidroin types expressed in a single gland [6, 7, 49, 50]. The only silk product not found to be produced by the MA gland of both N. pilipes and N. plumipes was pyriform adhesive silk, which is used to attach threads to objects and to each other [51]. The processing duct of the pyriform gland is shorter than most other ducts suggesting other silks require more extensive processing, which may explain why this silk is absent from the MA gland transcriptome. However, pyriform products are the least intensively studied of the spider silk repertoire, and the lack of pyriform annotation in our MA databases may be a reflection of poor representation in the public databases at the present time [51, 52].

Interestingly, in both N. plumipes and N. pilipes, tubuliform spidroins were found to be more highly expressed in the 2016 MA gland transcriptomes yet not expressed in the 2013 transcriptomes (see Table 1 and 2). Tubuliform silk is produced during reproduction for the formation of egg sacs [4, 53]. While no spiders were gravid at the time of dissection, it is possible they were collected and dissected just after the production of an egg sac, or just prior to vitellogenesis, and the 2016 transcriptomes reflect this in their relatively high expression of tubuliform silk transcripts. Expression of tubuliform spidroins in the MA gland has been previously noted in transcriptomic studies [50]. Vasanthavada et al. suggest that spiders can downregulate the production of various silks to maintain MA spidroin synthesis as an energetic trade-off, and Larracas et al. suggest that female spiders may shift synthesis of MA gland spidroins to tubuliform spidroins during the reproductive stage [6, 54, 55]. Our study did not find tubuliform spidroins in the silk proteome, however, the silk was collected and digested at the same time as the 2013 transcriptomes. It would be interesting to see if tubuliform spidroins could be found within the dragline silk of spiders prior to, during, or just post egg sac production.

This study is the first silk gland-specific transcriptome and proteome analysis in these Australian golden orb-weaving species. Major ampullate transcriptome analysis procured sequences for all silk types thus far known for golden orb spiders with the exception of pyriform adhesive silk. We found differential expression of tubuliform silk in the MA gland, suggesting a greater role for this gland producing tubuliform silks during spider reproduction. The silk proteome analysis resulted in 29 and 18 proteins for N. plumipes and N. pilipes that match to their corresponding MA gland transcriptomes.

Supporting information

S1 Table. Nephila plumipes proteins with homology to Nephila clavipes spidroins.

(XLSX)

Click here for additional data file. (10.2KB, xlsx)
S2 Table. Nephila pilipes proteins with homology to Nephila clavipes spidroins.

(XLSX)

Click here for additional data file. (9.9KB, xlsx)
S3 Table. List of peptides that were mapped back to the Nephila plumipes transcriptome.

(XLSX)

Click here for additional data file. (43.4KB, xlsx)
S4 Table. List of peptides that were mapped back to the Nephila pilipes transcriptome.

(XLSX)

Click here for additional data file. (28KB, xlsx)

Acknowledgments

We gratefully thank Dr Alun Jones (Institute for Molecular Bioscience, the University of Queensland) for advice and assistance with the LC-MS/MS. We also thank Dr Owen Seeman and Dr Robert Raven (Queensland Museum, Brisbane) for training in species identification and gland dissection. Thanks to Dr Min Zhao for assistance with bioinformatics.

Data Availability

All gene files are available from the NCBI database using the accession numbers SRR6747912, SRR6747911.

Funding Statement

The authors received no specific funding for this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Nephila plumipes proteins with homology to Nephila clavipes spidroins.

(XLSX)

Click here for additional data file. (10.2KB, xlsx)
S2 Table. Nephila pilipes proteins with homology to Nephila clavipes spidroins.

(XLSX)

Click here for additional data file. (9.9KB, xlsx)
S3 Table. List of peptides that were mapped back to the Nephila plumipes transcriptome.

(XLSX)

Click here for additional data file. (43.4KB, xlsx)
S4 Table. List of peptides that were mapped back to the Nephila pilipes transcriptome.

(XLSX)

Click here for additional data file. (28KB, xlsx)

Data Availability Statement

All gene files are available from the NCBI database using the accession numbers SRR6747912, SRR6747911.

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