PreprintPDF Available

Towards Spider Glue: Long-read scaffolding for extreme length and repetitious silk family genes AgSp1 and AgSp2 with insights into functional adaptation

December 2018

DOI:10.1101/492025

Authors:

Sarah Stellwagen

University of North Carolina at Charlotte

Rebecca L Renberg

Army Research Laboratory

The aggregate gland glycoprotein glue coating the prey-capture threads of orb weaving and cobweb weaving spider webs is comprised of silk protein spidroins (spider fibroins) encoded by two members of the silk gene family. It functions to retain prey that make contact with the web, but differs from solid silk fibers as it is a viscoelastic, amorphic, wet adhesive that is responsive to environmental conditions. Most spidroins are extremely large, highly repetitive genes that are impossible to sequence using only short-read technology. We sequenced for the first time the complete genomic Aggregate Spidroin 1 (AgSp1) and Aggregate Spidroin 2 (AgSp2) glue genes of Argiope trifasciata by using error-prone long reads to scaffold for high accuracy short reads. The massive coding sequences are 42,270 bp (AgSp1) and 20,526 bp (AgSp2) in length, the largest silk genes currently described. The majority of the predicted amino acid sequence of AgSp1 consists of two similar but distinct motifs that are repeated ~40 times each, while AgSp2 contains ~48 repetitions of an AgSp1-similar motif, interspersed by regions high in glutamine. Comparisons of AgSp repetitive motifs from orb web and cobweb spiders show regions of strict conservation followed by striking diversification. Glues from these two spider families have evolved contrasting material properties in adhesion, extensibility, and elasticity, which we link to mechanisms established for related silk genes in the same family. Full-length aggregate spidroin sequences from diverse species with differing material characteristics will provide insights for designing tunable bio-inspired adhesives for a variety of unique purposes.

Aggregate spider glue from three spider types. (A) Orb weavers coat the web's sticky capture spiral with glue, (B) the bolas spider creates a large droplet of glue specialized for capturing moths, and (C) cobweb weavers cover the lower portion of their triplines with glue to create 'gumfoot' threads. (D) A stretching glue droplet after contacting a probe that was subsequently withdrawn at a constant rate. Inset images courtesy of Brent Opell.

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Repeat motif and phylogenetic comparisons for AgSp1 across species. (A) Repetitive AgSp1 predicted amino acid motifs for seven species, three orb web weavers and four cobweb weavers. Each repeat consists of 4 subgroups (SG) with a conserved number of amino acids (aa) followed by a variable length tail. Conserved amino acid residues are within boxes. Grey highlight within species repeats indicates hydrophobic residues. (B) Aligned A.trifasciata major ampullate spidroin 2 (MaSp2) repeat (accession: AF350267.1) and AgSp1 repeat tail predicted proteins; grey highlight indicates shared amino acid residues. (C) Maximum likelihood tree with bootstrap support of repeat region encoding nucleotides (alignment Supplementary Fig. S5). Tree was rooted using the repeat region of major ampullate silk (MaSp1) from N. clavipes (slashed lines indicate this branch was arbitrarily shortened).

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Figure S4: Putative splice sites for A. trifasciata AgSp1 (A) and AgSp2 (B) exons. Introns are capped with standard GT and AG dinucleotides (bold).

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Figure S6: L. geometricus (A) and L. hesperus (B) AgSp2 predicted amino acid sequences.

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Figure S7: L. geometricus (A) and L. hesperus (B) AgSp2 aligned to A. trifasciata AgSp2 and A. argentata FLAG. (C) Alignment percent identity matrix.

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TOWARDS SPIDER GLUE: LONG-READ SCAFFOLDING FOR

EXTREME LENGTH AND REPETITIOUS SILK FAMILY GENES

AGSP1AND AGSP2WITH INSIGHTS INTO FUNCTIONAL

ADAPTATION

A PR EPR INT

Sarah D. Stellwagen

Department of Biological Sciences

University of Maryland Baltimore County

Baltimore, MD 21250

sstellw@umbc.edu

Rebecca L. Renberg

General Technical Services

Adelphi, MD 20783

rebecca.l.renberg.ctr@mail.mil

December 10, 2018

ABS TR ACT

The aggregate gland glycoprotein glue coating the prey-capture threads of orb weaving and cobweb

weaving spider webs is comprised of silk protein spidroins (spider ﬁbroins) encoded by two members

of the silk gene family. It functions to retain prey that make contact with the web, but differs from

solid silk ﬁbers as it is a viscoelastic, amorphic, wet adhesive that is responsive to environmental

conditions. Most spidroins are extremely large, highly repetitive genes that are impossible to sequence

using only short-read technology. We sequenced for the ﬁrst time the complete genomic Aggregate

Spidroin 1 (AgSp1) and Aggregate Spidroin 2 (AgSp2) glue genes of Argiope trifasciata by using

error-prone long reads to scaffold for high accuracy short reads. The massive coding sequences are

42,270 bp (AgSp1) and 20,526 bp (AgSp2) in length, the largest silk genes currently described. The

majority of the predicted amino acid sequence of AgSp1 consists of two similar but distinct motifs

that are repeated ~40 times each, while AgSp2 contains ~48 repetitions of an AgSp1-similar motif,

interspersed by regions high in glutamine. Comparisons of AgSp repetitive motifs from orb web

and cobweb spiders show regions of strict conservation followed by striking diversiﬁcation. Glues

from these two spider families have evolved contrasting material properties in adhesion, extensibility,

and elasticity, which we link to mechanisms established for related silk genes in the same family.

Full-length aggregate spidroin sequences from diverse species with differing material characteristics

will provide insights for designing tunable bio-inspired adhesives for a variety of unique purposes.

Keywords spidroin ·aggregate glue ·long reads ·full-length gene ·spider silk ·Argiope trifasciata

1 Introduction

Spiders use a suite of remarkable silk and silk-derived materials for various applications during their life cycles, from

wrapping prey and egg cases, to creating webs, lifelines, and prey capture glues. Aggregate gland glue is produced

by spiders within the superfamily Araneoidea and functions to retain prey that get caught in a spider’s silken trap.

This material is an adhesive glycoprotein (Tillinghast, 1981) that coats the capture spiral silk of orb web weavers, the

silk trip lines of cobweb weavers, and the globule of glue at the end of the bolas of bolas spiders (Fig. 1). The glue

is produced within a spider’s abdomen by two pairs of aggregate glands that terminate with valved spigots on the

spinnerets (Sekiguchi, 1952).

In orb weavers, each pair of semi-circle-shaped glue spigots surrounds a ﬂagelliform silk spigot, which produces the

supporting thread as the glue is concurrently extruded. The amorphous glue is deposited on the ﬂagelliform threads as

a continuous cylinder, however as the two coated threads merge to form a single strand, hygroscopic low molecular

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mass compounds (LMMC) and salts absorb atmospheric moisture. Surface tension then causes the cylinder of glue to

separate into equally spaced droplets (Fig. 1A)(Plateau, 1873; Boys, 1889; Edmonds and Vollrath, 1992). A single

droplet contains a core of adhesive glycoprotein (Opell and Hendricks, 2010; Sahni et al., 2010) surrounded by an outer

aqueous layer containing the LMMC and salts that retain water and lubricate the core of glue (Fischer and Brander,

1960; Anderson and Tillinghast, 1980; Tillinghast and Christenson, 1984; Townley et al., 1991; Opell et al., 2011, 2013;

Sahni et al., 2014).

Orb weaving spider glue acts as a viscoelastic solid, exhibiting viscous properties at more rapid extension rates, and

elasticity at slow extension rates (Sahni et al., 2010). This allows the glue to adhere to fast-moving insects at interception,

and retain insects while a spider travels to and subdues them. The stickiness of the glue droplets, however, is constrained

by the force required to break the ﬂagelliform support ﬁbers, as glue that does not preemptively release results in

damaged webs as an insect pulls free by breaking the support ﬁbers (Agnarsson and Blackledge, 2009). Capture spiral

threads also exhibit a suspension bridge mechanism, allowing forces to be distributed across multiple droplets and

reducing thread peeling that would occur if only droplets on the ends of a contacted thread contributed to adhesion

(Opell and Hendricks, 2007).

Orb weaver glue responds instantly to humidity, which changes droplet volume and inﬂuences the performance of the

glue (Opell et al., 2011; Sahni et al., 2011a). Orb weavers produce glue optimized for their habitat’s humidity, for

example the glue of species from drier habitats becomes over-lubricated when exposed to higher humidity, while glue

from species occupying wetter habitats becomes too viscous in drier conditions. Moreover, temperature and ultraviolet

exposure also inﬂuence performance optima (Stellwagen et al., 2014, 2015).

In contrast to orb weavers, cobweb weavers connect major ampullate silk threads from their webs to the ground,

depositing aggregate glue on the lower portions to produce trip lines aimed at ambulatory prey (Fig. 1B). The trip lines,

or ‘gumfoot’ threads, easily release from the ground, pulling insects into the air when accidentally intercepted, and the

prey hang suspended until the spider can subdue them (Argintean et al., 2006). Unlike orb weaver glue, cobweb glue is

considered a viscoelastic liquid, is resistant to changes in humidity, and is less extensible (Sahni et al., 2011a). Gumfoot

droplets lack the glycoprotein core visible in those of orb weavers, and tend to ﬂow together and coalesce rather than

simply swelling when exposed to increased humidity (see ﬁgure 1 in Sahni et al. (2011a)). Cobweb aggregate gland

pairs are morphologically distinct, and have likely diverged in function as well (Kovoor, 1977; Townley and Tillinghast,

2013; Clarke et al., 2017).

Bolas spiders capture male moths using a single large glue droplet that hangs at the end of a silk strand (Fig. 1C). By

mimicking female moth pheromones, the bolas spider lures the males to within striking range, and then swings their

sticky snare until contact is made (Eberhard, 1977). The remarkable glue of these spiders is able to adhere in spite

of a moth’s scales, which easily detach allowing them to escape other forms of predation (Sahni et al., 2011b). The

aggregate glue of related moth specialist Cyrtarachne akirai Tanikawa, 2013 was recently characterized and was shown

to have unique properties that allow the glue to spread and penetrate a moth’s scales (Diaz et al., 2018).

The proteins that form spider silks and glues are from the same family termed spidroins (‘spider ﬁbroins’) (Hinman and

Lewis, 1992), and are often encoded by very long (>5kb), repetitive, single exon genes. Functional specialization of

spidroin paralogs is associated with the evolutionary success of spiders (Schultz, 1987), particularly the central repetitive

motifs that are responsible for the diversity of silk material properties (Gosline et al., 1999). The ﬁrst full-length

spidroin cDNAs were isolated from the orb weaving spider Argiope bruennichi (Scopoli, 1772) in 2006 (Zhao et al.,

2006). The ~9 kb A. bruennichi cylindrical gland spidroins (CySp1 and CySp2; synonymous with tubiliform spidroins,

TuSp), encode for silk that is used to wrap egg sacs. The gene that encodes for the most well known spidroin, major

ampullate silk (more colloquially known as dragline silk), was fully sequenced from the black widow Latrodectus

hesperus Chamberlin & Ivie (1935) genomic DNA the following year (Ayoub et al., 2007). To date, 19 large (>5kb),

full-length, and classiﬁed spidroins (i.e., assigned to a speciﬁc silk gland source) have been described (Table 1). Several

other smaller or unclassiﬁed silk gland genes have also been characterized (Babb et al., 2017).

Initial evidence for aggregate glue gene sequences was derived from what were thought to be two full-length transcripts

encoding the glue proteins of orb weaver Nephila clavipes (Linnaeus, 1767), and were named ASG1 (Aggregate

Spider Glue; 1,221 bp) and ASG2 (2,145 bp) (Choresh et al., 2009). Several years later, it was determined that ASG1

expression is not unique to aggregate glands, and that the ASG2 sequence, while mostly correct, contained a short

insertion (Collin et al., 2016). Collin et al. (2016) also discovered that the predicted ASG2 C-terminus aligns with

other spidroins, and it was suggested ASG2 be renamed to AgSp1 (Aggregate Spidroin 1) to follow gene family

convention, which we have adopted here. This study extended the known sequence to 2,821 bp, however the complete

gene including the 5’ end encoding the predicted N-terminus still remained unidentiﬁed. During sequencing of the ﬁrst

orb weaving spider genome, which focused on describing spidroins from N. clavipes, the 5’ sequence for AgSp1 was

discovered (Babb et al. (2017); called AgSp-c, following the study’s own spidroin naming scheme). While Babb et al.

(2017) were unable to bridge a gap between the 5’ (7,660 bp) and 3’ (3,446 bp) ends of the gene, the known length of

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

Figure 1. Aggregate spider glue from three spider types. (A) Orb weavers coat the

web’s sticky capture spiral with glue, (B) the bolas spider creates a large droplet of

glue specialized for capturing moths, and (C) cobweb weavers cover the lower

portion of their triplines with glue to create ‘gumfoot’ threads. (D) A stretching glue

droplet after contacting a probe that was subsequently withdrawn at a constant rate.

Inset images courtesy of Brent Opell.

Figure 1: Aggregate spider glue from three spider types. (A) Orb weavers coat the web’s sticky capture spiral with glue,

(B) the bolas spider creates a large droplet of glue specialized for capturing moths, and (C) cobweb weavers cover the

lower portion of their triplines with glue to create ‘gumfoot’ threads. (D) A stretching glue droplet after contacting a

probe that was subsequently withdrawn at a constant rate. Inset images courtesy of Brent Opell.

AgSp1 was extended to at least 11,106 bp. This study further reported three other aggregate gland spidroins (AgSp-a,

AgSp-b, and AgSp-d).

We combined Illumina and Oxford Nanopore technologies to sequence AgSp1 from orb weaving spider Argiope

trifasciata (Forskål, 1775), a large cosmopolitan species that is abundant in our location, and which has had many

aspects of its biology extensively researched. During gene expression analyses to conﬁrm high expression of AgSp1,

we discovered and subsequently assembled another highly expressed spidroin, which we call AgSp2. Furthermore, we

analyzed aggregate glue transcript data from three orb weavers and four cobweb weavers to discover and compare the

repetitive motifs of AgSp1. While hygroscopicity is the result of the LMMC and salts, which provide tunable hydration

and lubrication of the glycoprotein in orb weavers, less is known about what is responsible for differences in other

functional properties of aggregate glues across species. It is not clear whether it is changes in the protein sequence, the

glycosylations that are added to the protein during post-translational modiﬁcation, or more likely a combination of

these and other variables.

We hypothesize that described differences in material properties will be reﬂected in the predicted protein backbone of

the glue of orb weaving and cobweb weaving spiders, as inferred from mechanisms established for related spidroins.

Spider glue droplets exhibit 1) adhesion, typically to insect prey, 2) extensibility, to remain in contact with struggling

prey, and 3) elasticity, to retain prey after extension. Glycosylations have been attributed to glue adhesion (Singla et al.,

2018), however potential mechanisms for extensibility and elasticity have only been described for other silks. Orb web

weaver glue droplets are more extensible (i.e. stretch farther, Fig. 1D) than cobweb weaver droplets (Sahni et al., 2011a),

and we expect to ﬁnd sequence similarity related to increased extensibility in paralogous genes from the spidroin family

to be more prominent in orb weaver glue. Elasticity, or the ability of a material to resume its original shape, is an

important property for spider glues. As prey struggle and glue droplets extend, glue elasticity causes droplets to return

to their original shape, retaining prey the web. We expect AgSp1 to contain sequence that may contribute to the elastic

properties of the predicted protein.

Understanding the underlying mechanisms that provide aggregate spider glue with its diverse properties will provide

insight into designing novel bio-inspired adhesives. Synthetic spider glues would not require replicating the spinning

process that transforms liquid protein dope into solid threads within a spider’s gland duct, traits that have made synthetic

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

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Table 1: Full length, classiﬁed spidroins with >5 kb complete coding sequences (including start and stop codons),

ordered from smallest to largest. Asterisk indicates a correction reported in Han and Nakagaki (2013) but not corrected

on GenBank. CySp is synonymous with TuSp.

Species Spidroin bp (cds) Introns Reference Accession #

Araneus ventricosus MiSp 5298 Yes Chen et al. (2012) JX513956.1

Araneus ventricosus TuSp1 5766 No Wen et al. (2017) MF192838.1

Latrodectus hesperus MiSp_v1 6564 No Vienneau-Hathaway et al. (2017) KX584003.1

Nephila clavipes MaSp-g 7398 Yes Babb et al. (2017) MWRG01015344.1

Nephila clavipes TuSp 8631 No Babb et al. (2017) MWRG01000785.1

Argiope bruennichi CySp1 8952 No Zhao et al. (2006) AB242144.1

Latrodectus hesperus MaSp1 9390 No Ayoub et al. (2007) EF595246.1

Argiope argentata TuSp1 9462 No Chaw et al. (2018) MF962652.2

Nephila clavipes AcSp 9636 No Babb et al. (2017) MWRG01010975.1

Argiope bruennichi CySp2* 9657 No Zhao et al. (2006) AB242145.1

Nephila clavipes MaSp-h 10014 No Babb et al. (2017) MWRG01038219.1

Argiope bruennichi MaSp2 10083 No Zhang et al. (2013) JX112872.1

Araneus ventricosus AcSp 10338 No Wen et al. (2018) MG021196.1

Nephila clavipes PiSp 10554 No Babb et al. (2017) MWRG01012117.1

Latrodectus hesperus MaSp2 11340 No Ayoub et al. (2007) EF595245.1

Argiope argentata AcSp1 13440 No Chaw et al. (2014) KJ206620.1

Argiope argentata PySp1 17280 No Chaw et al. (2017) KY398016.1

Nephila clavipes AgSp-d 17820 No Babb et al. (2017) MWRG01016148.1

Latrodectus hesperus AcSp1 18999 No Ayoub et al. (2013) JX978171.1

silks challenging to scale. The aggregate glue is extruded without the same processing (Townley and Tillinghast, 2013),

and synthetic versions will provide water-soluble and biodegradable solutions to problems ranging from pest control to

temporary sealants.

2 Results

We sequenced, assembled, and corrected two highly expressed spidroin genes from the aggregate glue glands of orb

weaving spider species Argiope trifasciata. To achieve full-length sequences, we used high-accuracy RNAseq short

reads from Illumina to correct error-prone gDNA long reads from Oxford Nanopore. Aggregate Spidroin 1 (AgSp1) is

encoded by 42,270 bp from two similarly sized exons spanning ~48,960 bp of genomic DNA, which includes a single

~6,690 bp central intron (Fig. 2; accession: MK138561). AgSp2 is encoded by 20,526 bp from a short 5’ and large 3’

exon, and contains an ~31,455 bp intron, in total spanning ~51,981 bp of gDNA (Fig. 3, accession: MK138559). We

collected RNAseq short reads for long read correction, resulting in highly accurate coding sequences for these two

genes, however apart from a few reads from pre-mRNA, RNAseq reads do not cover genomic intron sequence and is

therefore uncorrected. Long read scaffolds and pre-mRNA reads did allow identiﬁcation of putative splice sites for both

aggregate spidroins (Supplementary Fig. S4).

AgSp1 and AgSp2 are members of the spidroin protein family (Collin et al., 2016) and are encoded by the same

general N-terminus/Repeat(n)/C-terminus pattern found in this family, however the size and internal organization are

distinct. We have broken the predicted protein of each spidroin into several regions. AgSp1 regions include: 1) an

N-terminal domain (NTD), 2) a short region of N-terminal repeats (NTR), 3) an N-terminal transition (NTT, region with

degenerate, repeat-similar structure), 4) 43 iterations of repeat motif 1 (RM1), 5) 38 iterations of repeat motif 2 (RM2),

6) a C-terminal transition (CTT, region with degenerate, repeat-similar structure), and 7) a C-terminal domain (CTD)

(Fig. 2and Supplementary Fig. S1). AgSp2 consists of an NTD and areas rich in glutamine (QRR - glutamine-rich

region) that intersperse variable sized blocks of an iterated single repeat motif (RM) (Fig. 3and Supplementary Fig.

S2).

Current RNA sequencing technology does not provide reliable full-length sequencing of extreme-sized transcripts,

however we were able to sequence a ~16.5 kb continuous RNA read from each aggregate spidroin using Oxford

Nanopore’s MinION and direct RNA sequencing kit (Figs. 2C and 3C). These enormous reads terminated with their 5’

ends still in the repetitive regions a few iterations before the genes’ respective introns. While most classiﬁed spidroins

>5 kb in length are single exon (Table 1), AgSp1 and AgSp2 have large, single introns (Figs. 2and 3).

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Intron

NTR

NTD

24,815

Repeat Motif 2

NTT CTT

Repeat Motif 1

Figure 2. A. trifasciata Aggregate Spidroin 1 (AgSp1) schematic, aligned Oxford Nanopore gDNA reads, longest alignable mRNA read,

and mapped read coverage. (A) AgSp1 consists of 42,270 bp of coding sequence and ~6,690bp of intronic sequence, totalling ~48,960 bp

of genomic sequence (intronic sequence could not be corrected with short reads derived from mRNA). Abbreviations correspond to

regions of the predicted protein: NTD = N-terminal domain; NTR = N-terminal repeats; NTT = N-terminal transition; MRT = mid-repeat

transition; CTT = C-terminal transition, CTD = C-terminal domain. (B) Individual alignment of four Oxford Nanopore reads to the

consensus AgSp1 together cover the entirety of the gene. (C) Alignment of a 16.4 kb mRNA transcript to the consensus AgSp1. (D) Log

read coverage of Illumina RNASeq data generated from aggregate gland tissue mapped to AgSp1.

CTD

5,000

5,000 10,000 15,000 20,000

20,000 25,000 30,000

25,000

30,000

35,000

35,000 40,000 45,000

MRT

35,000 40,000 45,000

mRNA

Figure 2: A. trifasciata Aggregate Spidroin 1 (AgSp1) schematic, aligned Oxford Nanopore gDNA reads, longest

alignable mRNA read, and mapped read coverage. (A) AgSp1 consists of 42,270 bp of coding sequence and ~6,690 bp

of intronic sequence, totalling ~48,960 bp of genomic sequence (intronic sequence could not be corrected with short

reads derived from mRNA). Abbreviations correspond to regions of the predicted protein: NTD = N-terminal domain;

NTR = N-terminal repeats; NTT = N-terminal transition; MRT = mid-repeat transition; CTT = C-terminal transition,

CTD = C-terminal domain. (B) Individual alignment of four Oxford Nanopore reads to the consensus AgSp1 together

cover the entirety of the gene. (C) Alignment of a 16.4 kb mRNA transcript to the consensus AgSp1. (D) Log read

coverage of Illumina RNAseq data generated from aggregate gland tissue mapped to AgSp1.

AgSp1:

The 5’ end of AgSp1 encodes for the N-terminal domain (NTD; Spidroin_N domain superfamily BLASTx

e-value 3.36e-14). Following the NTD, AgSp1 contains a short ~1,758 bp region that consists of translated

TGSYITGESGSYD

repetitions before connecting to the N-terminal transition encoding region. The N- and C- ter-

minal transition regions that ﬂank the iterative central repeat motif blocks (discussed below), consist of sequence that

is unique enough to be assembled using only short reads, however the amino acid translation clearly resembles and

aligns with the repeat motifs. These degenerate repeats become increasingly less conserved as the sequence approaches

either terminus (Supplementary Fig. S1). The C-terminal transition region is shorter than the N-terminal transition, and

maintains the same organization as the internal repeats, with the majority of variation occurring towards the end of each

repeat iteration. The C-terminus has been previously described (Choresh et al., 2009; Collin et al., 2016).

The central repetitive region of AgSp1 contains at least two distinct repeat motifs. There are 43 iterations of repeat motif

1 (RM1, 387 bp) and 38 iterations of repeat motif 2 (RM2, 339 bp); RM1 and RM2 predicted amino acid sequences are

mostly conserved except for a few short regions (Fig. 4). These results demonstrate that previously reported AgSp1

repeat motifs were representative of a portion the C-terminal transition region, rather than the dominant repeat motifs

described here (Supplementary Fig. S1, underlined sequence aligns to reported repeat of Argiope argentata (Fabricius,

1775) from Collin et al. (2016).

In addition to A. trifasciata, we de novo assembled Illumina RNAseq data from the aggregate glands of Argiope

aurantia Lucas (1833), and assembled publicly available aggregate gland RNAseq data sets from GenBank to discover

the repeat motifs of aggregate spidroin genes from several other species: an additional orb weaver, N. clavipes, and

three cobweb weavers, Steatoda grossa (C. L. Koch, 1838), Latrodectus hesperus, and Latrodectus geometricus Koch

(1841) (SRR accession numbers in Supplementary Table S1). AgSp1 from a fourth cobweb species, Parasteatoda

tepidariorum (C. L. Koch, 1841), was discovered after a BLASTp search using AgSp1 RM2 as a query (~14.8 kb;

accession: XM_021148663.1). This sequence was assembled using short read data and is likely incomplete, though

many repeat motifs are present and therefore included in our comparison.

Across species, repeat motifs can be classiﬁed into four subgroups with many conserved residues and completely

conserved lengths, followed by a variable ‘tail’ (Fig. 5). Subgroups from all species begin with a

GPXG

group preceding

a high proportion of hydrophobic amino acids (Fig. 5A, highlight). The ﬁrst three subgroups contain regions high in

serine/threonine residues, which are likely glycosylated, and are ﬂanked by proline and glycine. The AgSp1 repeat

tail region of orb weaving species is distinct from the cobweb species, which is both longer and contains stretches of

poly-threonine on either end. The tail regions also contain

GGQ

PGG

GPG

, and

QGP

motifs found frequently in other

spidroins, and the tail of A. trifasciata aligns well to repeats of major ampullate silk spidroin 2 (MaSp2; Fig. 5B,

highlight). All species have several variations of their repeats, however the subgroups remain conserved with most

variation again lying in the tail region (Supplementary Figure S3). Without full length sequences from the other species,

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

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Table 2: Percentage of glycosylated and glycosylation-associated amino acids (aa) in the repeat motifs of three orb web

and four cobweb species. Bold indicates signiﬁcant differences (P< 0.5).

orb weavers cobweb weavers average

aa Ncl Atr Aau Pte Sgr Lhe Lge orb cob

G24.1 21.2 21.2 20 25 20.8 20.8 22.2 21.7

P23.3 23 23 18.9 19.8 19.8 19.8 23.1 19.6

T14.7 15 15 10.5 10.4 9.4 11.5 14.9 10.4

S3.4 1.8 1.8 6.3 7.3 8.3 7.3 2.3 7.3

S+T 18.1 16.8 16.8 16.8 17.7 17.7 18.8 17.2 17.8

it is difﬁcult to assess if these repeat motifs are part of transitional regions or if dominating motifs are present as in A.

trifasciata. However, the motifs chosen for comparison were the most similar to repeat motifs of the Argiope species.

Percentages of the most prominent or glycosylation-associated amino acids that make up the AgSp1 glue repeats vary

between orb web weavers and cobweb weavers (Fig. 5A, Table 2). The percent of glycine is similar between the repeats

motifs of the two glue types (~22%, P = 0.7847), however orb web repeats have a higher percent of proline than cobweb

repeats (23.1% vs 19.6%, respectively, P < 0.0001). The total amount of potentially O-glycosylated residues (serine +

threonine) is the same between the two glue type repeats (17.8%), however orb web repeats contain more threonine

than cobweb repeats (14.9% vs 10.4%, respectively, P = 0.0003) and cobweb repeats contain more serine than orb web

repeats (7.3% vs 2.3%, respectively, P = 0.0006).

We conducted maximum likelihood analyses of AgSp1 repetitive motifs for the three orb web and four cobweb weaving

species rooted with MaSp1 repeats from N. clavipes (accession: M92913.1), a spidroin found in non-orb weaving

spider groups as well (Fig. 5C). Bootstrap support for phylogenetic analysis reﬂects the most recent described spider

relationships (Garrison et al., 2016; Bond et al., 2014; Fernández et al., 2014), as well as analyses based on the AgSp1

carboxyl-terminus (Collin et al., 2016).

AgSp2:

AgSp2 is half the size of AgSp1 and is organizationally distinct. The 5’ end is also a member of the Spidroin_N

domain superfamily (BLASTx e-value 3.49e-13), however it lacks the subsequent N-terminal repeats found in AgSp1.

Instead, the N-terminus leads directly into a short glutamine-rich region, and similar regions intersperse iterated AgSp2

repeat motif blocks along the length of the gene. AgSp2 has one main repeat motif (RM), which forms two main blocks

of 14 and 27 iterations, as well as several smaller blocks (Fig. 3; Supplementary Fig. S2). As in AgSp1, there are repeat

motifs that appear to be degenerate, and are usually located at ends of repeat blocks. The repeat motif of AgSp2 is

similar to those of AgSp1 (Fig. 4, with four conserved subgroups and a unique tail. AgSp2 corresponds to AgSp-b of N.

clavipes reported in Babb et al. (2017), however we did not ﬁnd evidence for aggregate spidroins AgSp-a or AgSp-d

from their report.

We identiﬁed the putative AgSp2 from cobweb weavers L. geometricus (accession: MK138562) and L. hesperus

(accession: MK138563; Supplementary Fig. S6) after de novo assembly of online data sets (Supplementary Table S1).

The assembly software was able to completely reconstruct AgSp2 in both species, with no evidence of truncation as

seen in AgSp1. AgSp2 is extremely reduced to less than 2 kb in these species, and has lost the repetitious nature of

most spidroins. BLASTp results identify them as ﬂagelliform (FLAG) spidroin N-termini, however after N-terminal

alignment with A.trifasciata AgSp2 and A. argentata FLAG, the encoding sequences have ~10% more bases in common

with A. trifasciata AgSp2 (Supplementary Fig. S7).

Gene Expression Analyses:

We performed gene expression analyses using transcript data sequenced from A. trifasciata

aggregate gland tissue to compare AgSp1 and AgSp2 expression, and from major ampullate silk gland and fat body

tissues as controls. AgSp1 and AgSp2 are among the most highly expressed genes within the aggregate glands, with

signiﬁcantly (q < 0.05) less expression detected in the major ampullate or fat body tissues (Fig. 6A). Within the

aggregate glands, expression of AgSp1 and AgSp2 is not signiﬁcantly different (Fig. 6B).

3 Discussion

The aggregate glue spidroins AgSp1 and AgSp2 of A. trifasciata have coding sequences over 42 kb and 20 kb in length

(respectively), both larger than the previous record-holding 19 kb aciniform prey-wrapping silk spidroin AcSp1 (Ayoub

et al. (2013); Table 1). Both predicted proteins are structured as others in the family, containing repetitive central

regions capped by conserved termini. The repetitive motifs are notably similar between the two spidroins, and predicted

protein alignments of the repeat motifs from these two spidroins supports paralogous origins (Fig. 4). Interestingly,

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Intron

NTD

55,987

Repeat Motif

Figure 3. A. trifasciata Aggregate Spidroin 2 (AgSp2) schematic, aligned Oxford Nanopore gDNA reads, longest alignable mRNA read,

and mapped read coverage. (A) AgSp2 consists of 20,526 bp of coding sequence and ~31,455 bp of intronic sequence, totalling ~51,981bp

of genomic sequence (intronic sequence could not be corrected with short reads derived from mRNA). Abbreviations correspond to

regions of the predicted protein: NTD = N-terminal domain; QRR = glutamine-rich region (all regions in green); CTD = C-terminal

domain. (B) Individual alignment of four Oxford Nanopore reads to the consensus AgSp2 together cover the entirety of the gene. (C)

Alignment of a 16.5 kb RNA transcript to the consensus AgSp2. (D) Log read coverage of Illumina RNASeq data generated from

aggregate gland tissue mapped to AgSp2.

CTD

5,000

10,000 15,000 20,000

50,00030,000

25,000 30,000

35,000 40,000

50,000

45,000

40,000 45,000 50,000

35,000 40,000

QRR

mRNA

Figure 3: A. trifasciata Aggregate Spidroin 2 (AgSp2) schematic, aligned Oxford Nanopore gDNA reads, longest

alignable mRNA read, and mapped read coverage. (A) AgSp2 consists of 20,526 bp of coding sequence and ~31,455

bp of intronic sequence, totalling ~51,981bp of genomic sequence (intronic sequence could not be corrected with short

reads derived from mRNA). Abbreviations correspond to regions of the predicted protein: NTD = N-terminal domain;

QRR = glutamine-rich region (all regions in green); CTD = C-terminal domain. (B) Individual alignment of four Oxford

Nanopore reads to the consensus AgSp2 together cover the entirety of the gene. (C) Alignment of a 16.5 kb RNA

transcript to the consensus AgSp2. (D) Log read coverage of Illumina RNASeq data generated from aggregate gland

tissue mapped to AgSp2.

Figure 4: Predicted amino acid sequence of repeat motif 1 (RM1) and repeat motif 2 (RM2) from AgSp1 aligned with

the repeat motif (RM) from AgSp2 of A. trifasciata. Conserved residues are highlighted.

AgSp2 is highly reduced in cobweb weavers (Supplementary Fig. S6). It is unknown how AgSp1 and AgSp2 interact to

form the sticky capture glue, or what properties each imparts, particularly considering the differences between AgSp2

in orb web and cobweb weavers. However we are able to make inferences about the molecular function of aggregate

spidroins based on extensive research of related spidroins in the silk family.

Orb web glue is much more extensible than cobweb glue (Sahni et al., 2011a), and, interestingly, the tail region of orb

web AgSp1 repeats contains a higher proportion of short motifs similar to those that contribute to the extensibility

of major ampullate (MaSp) and ﬂagelliform (FLAG) silks (Gosline et al., 1999; Hayashi et al., 1999; Adrianos et al.,

2013; Malay et al., 2017). Similar to MaSp1 and FLAG, orb weaver AgSp1 repeat tails contain

GGQ

groups, and share

PGG

GPG

, and

QGP

groups in common with MaSp2 (Fig. 5). Furthermore,

GPG

motifs of minor ampullate spidroin

proteins are signiﬁcantly correlated with extensibility (Vienneau-Hathaway et al., 2017). Not surprisingly, the stretches

of poly-alanine that impart silk strength to MaSp silk (Gosline et al., 1999) are lacking in the much less strong aggregate

glue. The

GPG

and

QGP

regions are also found in the cobweb weaving species’ repeat motif tail regions, though the

tails in these species are shorter and contain much fewer of these groups than their orb weaving counterparts (Fig. 5A,

Supplementary Figure S3). If the protein backbone is responsible for imparting extensibility (how far the glue stretches)

reduction in the tail region may be a response to differing functional needs for cobweb prey capture. Cobweb weavers

intercept ambulatory prey that accidentally trip their gumfoot lines at low speeds. In contrast, orb weavers capture

highly mobile ﬂying or saltatory prey, and the glue may need to be more extensible in order to remain in contact with

higher velocity prey items.

Notably, AgSp2 of A. trifasciata has 60% more total glutamine than AgSp1 despite being only half the size. A quarter

of glutamine in AgSp2 is part of

motifs located in high density between blocks of iterated repeats (Supplementary

Fig. S2). Similar expansion regions are also found in pyriform spidroins (Chaw et al., 2017), and

motifs have been

shown to allow self-aggregation of these silk proteins into ﬁbers (Geurts et al., 2010). AgSp2 is dramatically reduced

in Latrodectus cobweb weavers, consisting of less than 2000 bp (Supplementary Fig. S6). Differences between orb

weaving and cobweb weaving pyriform spidroins have also been noted, including a loss of the

motifs (Chaw et al.,

2017).

Aggregate spider glue has been compared to the mucin family of proteins because of their glycosylated structure,

viscoelastic properties (Shogren et al., 1989; Tillinghast et al., 1993; Choresh et al., 2009; Sahni et al., 2010), and highly

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Figure 5. Repeat motif and phylogenetic comparisons for AgSp1 across species. (A) Repetitive AgSp1 predicted amino acid motifs for seven species,

three orb web weavers and four cobweb weavers. Each repeat consists of 4 subgroups (SG) with a conserved number of amino acids (aa) followed by a

variable length tail. Conserved amino acid residues are within boxes. Grey highlight within species repeats indicates hydrophobic residues. (B) Aligned

A.trifasciata major ampullate spidroin 2 (MaSp2) repeat (accession#: AF350267.1) and AgSp1 repeat tail predicted proteins; grey highlight indicates

shared amino acid residues. (C) Maximum likelihood tree with bootstrap support of repeat region encoding nucleotides (alignment Supplementary Fig. 4).

Tree was rooted using the repeat region of major ampullate silk (MaSp1) from N. clavipes (slashed lines indicate this branch was arbitrarily shortened).

cobweb

GP G P P GPG TPG VT GPDG P G TTPGS GP G P PAG GTTPG T GPDG P

Ncl GPDGKPLQIVPAGPGTTPGTVT GPDGKPSKFVVPDGAFTTPGSIP GPDGKPLPVEPAGSGTTPGLQT GPDGKPSKLVVP TTTKGPGGPMGPGGPMGPGGPMGPGGQTTTTTPAPVK

Atr GPDGKPLQIEPAGPGTTPGTVT GPDGKPKKFVLPKGAFTTPGSIP GPDGKPIHVEPAGPGTTPGAQT GPDGKINKLVVP TTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

Aau GPDGKPLQIEPAGPGTTPGTVT GPDGKPNKFVLPKGAFTTPGSIP GPDGKPIHVEPAGPGTTPGAQT GPDGKINKLVVP TTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

Pte GPGGRPIQLIPQGPGSTPGAVT GPDGVIIYIILPRGSGTTPGSVE GPDGQPIKIMPAGPGTTPGAET GPDGNIRIIYIP SHPRTRPDGSTPSQVT

Sgr GPGGRPISLIPGGPGNTPGAVT GPDGNIYYIILPSGSGTTPGSVE GPGGQPIRIVPAGPGTTPGAQT GPDGKINVIYVP RSPSPQGPSSSTPSQVT

Lhe GPGGRPISLIPSGPGNTPGAVT GPDGKIYYIVLPSGSGTTPGSIE GPGGKPIKIVPAGPGTTPGAET GPDGKINKIYVP KGPEPQGPSRSTPSQVT

Lge GPGGRPISLIPSGPGNTPGAVT GPDGKIYYIVLPSGSGTTPGSIE GPGGKPIKIVPAGPGTTPGAET GPDGKINKIYIP KSPEPQGPTRSTPSQVT

SG1, 22 aa SG2, 23 aa SG3, 22 aa SG4, 12 aa Tail, 17-37 aa

orb web

cobweb

BAtr MaSp2 AAAAAAAAGPGYGPGAGQQGPGSQGPGSGGQQGPGGQGPYGPSD

Atr AgSp1 TTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

N.clavipes MaSp1

S.grossa

L.geometricus

L.hesperus

A.trifasciata

A.aurantia

N.clavipes

0.2 substitutions/site

P.tepidariorum

Figure 5: Repeat motif and phylogenetic comparisons for AgSp1 across species. (A) Repetitive AgSp1 predicted amino

acid motifs for seven species, three orb web weavers and four cobweb weavers. Each repeat consists of 4 subgroups

(SG) with a conserved number of amino acids (aa) followed by a variable length tail. Conserved amino acid residues

are within boxes. Grey highlight within species repeats indicates hydrophobic residues. (B) Aligned A.trifasciata

major ampullate spidroin 2 (MaSp2) repeat (accession: AF350267.1) and AgSp1 repeat tail predicted proteins; grey

highlight indicates shared amino acid residues. (C) Maximum likelihood tree with bootstrap support of repeat region

encoding nucleotides (alignment Supplementary Fig. S5). Tree was rooted using the repeat region of major ampullate

silk (MaSp1) from N. clavipes (slashed lines indicate this branch was arbitrarily shortened).

repetitive central domains (Perez-Vilar and Hill, 1999). Our study supports similarities of aggregate spider glue to

this family of secretory glycoproteins, however distinct differences can also be noted. Particularly, mucins contain

cysteine-rich regions, which are joined via disulﬁde bonds between protein monomers resulting in the mucous net

barrier or gel-like characteristics common to mucins (Desseyn, 2009; Bansil and Turner, 2006). Mucins with more

cysteine residues produce stronger barriers, protecting underlying epithelial layers (Gouyer et al., 2015). It has been

hypothesized that the conserved 2-3 cysteine residues in each AgSp1 terminus may form disulﬁde bridges between

silk monomers (Collin et al., 2016), however gel-forming mucins contain cys-rich domains between the glycosylated

regions as well, which are lacking in AgSp1 and AgSp2. Groups of nonpolar and hydrophobic amino acids, however,

could contribute to multimerizing the individual glycoprotein monomers. Furthermore, these residues may enhance the

elasticity of the glue as they pull together and exclude water (Gosline, 1978; Li et al., 2001; Bansil and Turner, 2006),

which is hypothesized to contribute to elasticity in hydration-dependent ﬂagelliform capture spiral silk (Hayashi and

Lewis, 1998; Vollrath and Edmonds, 1989; Bonthrone et al., 1992). Dense hydrophobic groupings (Fig. 5A, highlight)

in subgroups two and four of A. trifasciata AgSp1 RM2 correspond to distinct hydrophobic pockets in Kyte-Doolittle

hydropathicity plots (Fig. 7). These hydrophobic residues are conserved across AgSp1 and AgSp2 repeat motifs, and

both orb web and cobweb weavers. In contrast to extensibility, the glue needs to maintain elasticity, or the ability to

resume its original shape, regardless of web type in order to retain prey that has contacted the threads.

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AgSp1 AgSp2

0.5

1.5

2.5

3.5

4.5

Normalized Read Counts (LOG)

AgSp1

agg

AgSp2

maj fat agg maj fat

q < 0.0000 *

q = 0.0116 *

q = 0.2436

q = 0.0024 *

q = 0.1563

Figure 6. Gene expression analyses for AgSp1 and AgSp2 of A. trifasciata. (A) Log number of

normalized reads that align to AgSp1 and AgSp2 (each manually edited to 999 bp of the 3’ ends) from

aggregate gland tissue (agg), major ampullate gland tissue (maj) and fat body tissue (fat). Line ends

correspond to statistical comparisons between tissues. (B) Transcripts per million (TPM) for AgSp1 and

AgSp2 averaged across six aggregate gland tissue samples. Asterisks indicate significant q- and p-values

(<0.05).

AgSp1 AgSp2

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

TPM

p = 0.0699

A B

q < 0.0000 *

Figure 6: Gene expression analyses for AgSp1 and AgSp2 of A. trifasciata. (A) Log number of normalized reads that

align to AgSp1 and AgSp2 (each manually edited to 999 bp of the 3’ ends) from aggregate gland tissue (agg), major

ampullate gland tissue (maj) and fat body tissue (fat). Line ends correspond to statistical comparisons between tissues.

(B) Transcripts per million (TPM) for AgSp1 and AgSp2 averaged across six aggregate gland tissue samples. Asterisks

indicate signiﬁcant q- and p-values (<0.05).

Figure 7. Hydrophobicity plot for repeat motif 2 of A. trifasciata AgSp1. Positive values indicate hydrophobicity.

Arrows indicate peaks produced from triplets of hydrophobic amino acids in repeat subunits 2 and 4. Window size: 3.

SG1, 22 aa SG2, 23 aa SG3, 22 aa SG4, 12 aa Tail, 17-37 aa

Figure 7: Hydrophobicity plot for repeat motif 2 of A. trifasciata AgSp1. Positive values indicate hydrophobicity.

Arrows indicate peaks produced from triplets of hydrophobic amino acids (aa) in repeat subgroups (SG) 2 and 4 (see

ﬁgure 5). Window size: 3.

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Glycosylations are responsible for the adhesive qualities of spider glue (Singla et al., 2018), and at least 80% of

threonine residues in Argiope glues are likely O-glycosylated with mainly N-acetylgalactosamine (GalNAc)(Dreesbach

et al., 1983; Tillinghast et al., 1993). The threonine residues are also ﬂanked by glycine and proline, which is consistent

with studies that show there is an increase in these residues around O-linked glycosylated threonines (Christlet and

Veluraja, 2001; Yoshida et al., 1997). Glycine is likely more prominent due to its small, non-interfering side-chains,

whereas proline is hypothesized to play a structural role, kinking the protein in the form of

-turns. Furthermore,

mucin-type glycosylated residues tend to occur in adjacent multiples, and this is consistent in the orb weaving AgSp

repeats, where most threonines are directly adjacent to or within one residue from each other (Fig. 4)(Christlet and

Veluraja, 2001). O-glycosylations prevent proteins from forming globules, and instead allow the protein to maintain an

expanded conformation, as well as hydrating and solubilizing the protein (Shogren et al., 1989; Perez-Vilar and Hill,

1999). Electron micrographs from earlier studies of spider glue proteins showed extended, ﬁlament-like molecular

structure and allowed estimations of size from 450 to 1400 kDa (ﬁgure 4 in Tillinghast et al. (1993)); our estimations

from the predicted AgSp1 (1,383 kDa) and AgSp2 (676 kDa) correspond to the upper and lower range values from the

previous estimate.

There are differences in the percentages of amino acids that make up the AgSp1 repeats of orb webs and cobwebs (Fig.

5A). Interestingly, repeats from both glues contain a similar number of O-glycosylation sites, however orb weaver glue

has a higher percentage of threonine residues than cobweb glue, while cobweb glue contains more serine residues.

Changes in humidity differentially affects orb and cobweb glue adhesion (Sahni et al., 2011a), and, in addition to low

molecular weight compounds and salts, this contrast in function could be due to differences between the serine and

threonine glycosidic linkages (Corzana et al., 2007). These linkages structure the surrounding water in different ways,

forming water pockets and bridges between the sugar and protein backbone. Additionally, threonine is more readily

glycosylated than serine (O’Connell and Tabak, 1993), which could also contribute to the material differences observed

between the two types of glues.

Spider silk has been notoriously difﬁcult to synthetically produce due to the difﬁculty in replicating the transition

from liquid protein dope to the solid ﬁber, which occurs within the duct of a spider’s silk gland. Aggregate spider

glue is extruded as an amorphous liquid that does not undergo the same processing, however the post-translational

glycosylation may provide its own challenges for future synthesis. Encouragingly, there has been success using cloned

and expressed glycosyltransferases to in vitro O-link GalNAc residues to synthetic peptides (Yoshida et al., 1997; Hagen

et al., 1997). We have identiﬁed an N-acetylgalactosaminyltransferase (pp-GalNAc-T, BLAST e-value = 3.71e-174;

accession: MK138560) from A. trifasciata, which is part of the family of transferases that facilitates the addition of

mucin-type, O-linked glycans by catalyzing the transfer of GalNAc from the sugar donor, UDP-GalNAc, to the hydroxyl

groups of serine or threonine residues of the core protein, forming GalNAc--1-O-Ser/Thr. This speciﬁc GalNAc-T is

differentially expressed in the aggregate glands compared to major ampullate gland and fat body tissues (q-value =

3.9e-8 and 7.8e-17, respectively). Furthermore, glue function does not seem to be limited by droplet size, as bolas

spiders in the genus Mastophora produce droplets that are visibly large and specialized for capturing fast-moving

moths whose wings are covered in fragile scales. In vitro glycosylation as well as the potential for bulk production are

encouraging attributes for synthetic versions of aggregate spider glue.

4 Conclusion

We used Illumina short reads aligned to long reads from Oxford Nanopore’s MinION to resolve aggregate spidroin

encoding sequences AgSp1 and AgSp2 from orb weaving spider Argiope trifasciata. AgSp1 (42,270 bp) and AgSp2

(20,526 bp) are the largest spidroins currently described, each containing a large intron, and are of the most highly

expressed genes within the aggregate glands. The predicted proteins consist of dominant central repetitive motifs and

comparisons of AgSp1 repeat motifs across three orb web and four cobweb species suggests differences in the peptide

backbone are related to observed functional differences of these two aggregate glue types. Aggregate spider glue must

function in three ways: 1) it must adhere to surfaces, usually insect prey, 2) it must be extensible, to remain in contact

with struggling prey, and 3) it must be elastic, to retain prey after extension. The tail regions of orb weaver repeats

resemble regions in other spidroins that impart extensibility, while the shortened tail of cobweb weaver repeats may

reﬂect observed reductions in this property. Conserved hydrophobic residues across the repeats of glue spidroins from

both web types may contribute the elastic nature of the droplets.

As sequencing technology continues to improve, the genetics of complex biomaterials like silk spidroins will continue

to be described. The similarities and differences between the orb web and cobweb glue repeat backbones are striking

and provide useful insight into the relationship between form and function. There is ample opportunity to learn from

differences in glue function not only between orb web and cobweb weavers, but also across the orb weavers themselves.

Understanding contrasting performance optima at different humidities will provide another level of tunability to

synthetic spider glues. Examining glue diversity from more distantly related species will allow an understanding of how

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differences in spider glue glycopeptides inﬂuence performance, contributing to the development of novel bio-inspired

materials.

5 Methods

Specimen collection and dissection:

Adult female A. aurantia and A. trifasciata were collected from Schooley Mill

Park in Highland, Maryland during September 2016 and August 2017. Specimens were kept in cages and allowed to

build webs overnight. After building a fresh capture spiral in the morning, specimens were euthanized and dissected

in Invitrogen RNALater (cat.no. AM7021) before 1200h. Aggregate and major ampullate glands, as well as fat body

tissue samples were collected and stored in RNALater at -20°C.

Illumina RNA sequencing and assembly:

Twenty-four RNA samples were selected for Illumina sequencing: ag-

gregate gland samples from 6 individuals of each species, and major ampullate glands and fat body tissue from 3

individuals of each species. Samples were processed according to the Qiagen RNeasy Mini Kit protocol (cat.no. 74104)

including the optional on column DNAse treatment. Puriﬁed RNA was prepared for sequencing using the Illumina

TruSeq Stranded mRNA library prep kit (cat.no. RS-122-2101) and run in-house on a NextSeq500 using a High Output

kit with 2 x 150 PE cycles on 31 October 2016.

Sequence data sets obtained from this study and publicly available online data sets (Supplementary Table S1;

http://www.ncbi.nlm.nih.gov/sra) were trimmed and ﬁltered for quality using Trimmomatic v.0.36 (Bolger et al.,

2014) and de novo assembled using Trinity (Grabherr et al., 2011) set with default parameters. Resultant contigs were

searched to select any sequences that contained similar regions and motifs to aggregate spidroins previously published.

Oxford Nanopore RNA Sequencing:

Messenger RNA was extracted and pooled using the NEB Magnetic mRNA

Isolation Kit (cat.no. S1550S) from 3 adult female A. trifasciata aggregate glands. The mRNA was then prepared

for Oxford Nanopore sequencing using the Direct RNA Sequencing Kit (cat.no. SQK-RNA001). We replaced the

recommended RT and buffer with those from the Thermo Scientiﬁc Maxima H Minus First Strand cDNA Synthesis

kit (cat.no. K1681). The cDNA produced during library preparation is not sequenced, but provides stability for direct

sequencing of the RNA molecules. Samples were run on a SpotON Flow Cell (R9.5; cat.no. FLO-MIN107), and

resultant fast5 ﬁles were basecalled using Oxford Nanopore’s program Albacore v2.1.3.

Oxford Nanopore gDNA Sequencing:

Two juvenile A. trifasciata were collected from Schooley Mill park on July 23,

2018 and kept overnight in containers. High molecular weight DNA was gently extracted and pooled from whole, fresh

specimens the next day using the MasterPure Complete DNA and RNA Puriﬁcation Kit following the DNA Puriﬁcation

section protocol. A total of 10

g of the gDNA extraction (pooled from both individuals) was loaded onto a Sage

Science BluePippin cassette (cat.no. BLF7150) and run with a 20 kb high pass threshold overnight. The resultant

elution was used directly in Oxford Nanopore’s 1D Genomic DNA by Ligation protocol (SQK-LSK109). A total of four

runs were completed using SpotON Flow Cells (R9.4; cat.no. FLO-MIN106) and resultant fast5 ﬁles were basecalled

using Oxford Nanopore’s program Albacore v2.2.7.

Analyses:

Sequence alignment and analysis was conducted using Geneious v11.0.5 (Kearse et al., 2012) with the

Geneious alignment and ClustalW tools (Thompson et al., 1994). Maximum likelihood phylogenetic analysis was

performed using the RAxML v8.2.11 Geneious plugin (Stamatakis, 2014) with the GTRGAMMA model with 10,000

bootstrap replicates. RSEM (Li and Dewey, 2011), EdgeR (Robinson et al., 2010), and R (R Core Team, 2014) were

used for differential gene expression analyses. Statistical signiﬁcance of amino acid percentages and TPM comparisons

was calculated using Student’s t-tests. Hydrophobicity was predicted using the Kyte and Doolittle scale from online

Expasy tools and a window size of 3.

List of Abbreviations:

AgSp1: Aggregate Spidroin 1

AgSp2: Aggregate Spidroin 2

RM: Repeat Motif

NTD: N-terminal domain

NTR: N-terminal repeats

NTT: N-terminal transition

CTT: C-terminal transition

CTD: C-terminal domain

QRR: Glutamine rich region

Funding:

This work was supported by the U.S. Army Research Laboratory Postdoctoral Fellowship Program adminis-

tered by the Oak Ridge Associated Universities through a contract with the U.S. Army Research Laboratory.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Authors’ contributions:

SDS contributed to experimental design, data collection, and manuscript preparation. RLR

contributed to experimental design, led data collection, and reviewed and edited the manuscript.

Acknowledgments:

We thank Nathan Schwalm and Mercedes Burns for helpful comments during manuscript prepara-

tion.

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Figure S1: Predicted AgSp1 amino acid sequence from A. trifasciata. Line breaks were inserted to emphasize different

regions. Abbreviations: NTD = N-terminal domain; NTR = N-terminal repeats; NTT = N-terminal transition; RM1 =

repeat motif 1; MRT = mid-repeat transition; RM2 = repeat motif 2; CTT = C-terminal transition; CTD = C-terminal

domain. Bolded glycine in the MRT indicates putative splice site. Underlined sequence in the C-terminal transition

corresponds to the A. argentata repeat reported in (Collin et al., 2016). Figure is split over two pages.

NTD MGWISHVIVLLCLVGTQFTKAYPRQNYQPGIGVVQDDPLLDSDTGENFANVFAQSLENCGAFSHEEAKEYKEIIQVLTRAFGSFPDLQKAPPGVLKATAAGFASAVAERTAADIEQGELYAKTRCVIQALRK

SFIVTTGVSNPYFISEVERLIEVFYLTRGVPRPTFEQPFEQAGQIQSQGQPPAQPQGQPPAQPQGQDLGGGIQGTQHAGGNLGEAAPSEPIGKAIRGKKREPSNDLGGLISGAFNGIFGGGGGGGSQQSGDD

NTR YYTYETESYYTGSYETYETSSYETGSYETYETESYDTGPYGTFETESYDTGPYGTYETDSYDPGSYVTGSYDTYETGSSATGEYETYEDGSYVTGEYDTYEDGSYETGEYETYETESYETGEYQTGEYSSYE

TGSYETGESGSYETGEYQGEYSSYETGSYETGEYQTGEYSSYETGSYETGESGSYETGESGSYETGESGSYETGESGSYVTGSYGTGSYVTGSYGTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGS

YDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYD

TGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYDTGSYITGESGSYVTDESGSYVTDESGSYVTDESGSYVTDESGSYLTGESGSYVT

GESSSFETASYDTGNYLTDEYATDEF

NTT GTYETASSEYDTGEYTTREYVSGEYGTGEIPVIEGGATPGAITNPDGELNRVILPDGSGAVPGTIS

GPDGQPIKIIPARPGTTPGAIMGPDGKISKIIVPQVPSPRSDEPKFVGGKGVQPVRIVTAKPGTTPGAVTG

PNGRLNKLVLPDGTGTTPGIVTGPYGEFLQVVLPVGAGFTPGTVT

APGGKPILVEPAQYGTTPGVVTDPDGKIKKLIVPHAPTPGPQRPSTIRGPEGRPLIIIPGGPTPGAVTGPDGNIKRIIYPEGAGTTPGTVK

GPDGNPIQIVPESPGTTPGAITDQDGKISKIVLPTPGPRGIITGPKGQPIKIAPGAHVKAPKAVISSSGNLKKILLPKGSGNTPGSVR

GPDGNPIQIIPAGPGTTPGAVTDTNGKLSKIIIPTPGPVGRTAIQGPNDQPIL

LEPEGSTPGAVTGPDGKISKIVLPVQSSEATSTIS

GPDGEPIHVVPAGKGTTPGVVTGPDGKIKKIVVPTPGPEGTIR

GPNGRPIQIIPEQPGTTPGTVTDPDGNIVRVVVPEGSNTTPGTVR

GPDGQPIKIIPAGPGTTPGIVTGPDGQINQIIVPIGPPRPGSGTYTLVGPSGEVIDLIPSGLTPGYIT

GPKGKPIHVVPADPGTTPGIQTDHHGVISKIAVPQGTTPNLRTVKGPDGQKVLLIPNGPTPGTVTGPGGNVLRFIVPEGAGITPSMVK

GPEGQQIPVVPEGPGTTPGIVTAPDGRVIEIIVPTPPPLGAVKDPDSRPIEFFVGGPSNRPQAITAPDGNLAYVLPYGSATTPGTAE

GPDGKPVQLEPAGSGTTPGVVTGPDGQPSKIVFPTDGPRGDVPQGPGKKPIQILRGGPGTTPATITDPDGNIIRLILPEEAATTPGSVE

GPDGQPIHIEPAGPGTTPGVVTDSDGHIRKIIVPVPGPQGIVKGPGGHPIQIVPGGRGSTPQALTDDDGNLDRIIMPGGGAGTTPGSIT

GPDGKPIKLIPESPGTTPGVVTGPDGDIQTIVVPKGPTTTTKGPGSLLIGLGSTPGKTSIE

GPDGKPIKIIPVGEGTTPGVVTDKHGKPKEIYVPKGSFTTPGKIRGPHGKPIHIIPAGPGTTPGAKTGPHGNINKVIIPTTPDAPGSPEGTPGSPNGPGRQKPGIG

GPQGQPIQIVPAGPGTTPGTVTGPDGQLVRFIVPNGAFMTPGSIPGPDGRPIHVEPAGPGTTPGAETGPDGNINKIILPTTPFRPSPQPPTTTTKIE

GPQGKPILILPAGPGTTPGTVTGPDGKPTKFIVPLGAFTTPGSIQGPDGKPIHVEPAGPGTTPGTITDSNGNVKEIILPTTPKGQQFYFPPTPSPTPTPVT

GTEGKPIRIIPAGPGTTPGTVTGSDGKPTRFIVPTGAFTTPGSIPGPDGRPIHVEPAGPGTTPGAQTGPDGKINKIFLPTTPKGAGGPGINFGKPATTPTPVP

GPKGRPIQIVPAGPGTTPGTITGPDGKPTKFIIPLGAFTTPGSIPGPDGRPIHVQPAGPGTTPGAETGPDGKINKIIVPTTPKTPQGSDGQPMYPFGPGSPYGPGEQTTTTPIP

RM1 GPDGKPLPIEPAGPGTTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVQPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPVGPGGMPLSPYSPQGPGGQPMYPFGPGSPYGPGEQTTTTPIP

GPDGKPLPIEPAGPGTTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVEPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPVGPGGMPLSPYSPQGPGGQPMYPFGPGSPYGPGEQTTTTPIP

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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MRT GPDGKPLPIEPAGPGTTQGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVQPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPVGPGGMPLSPYSPQGPGGQPMYPSGPQGPGGQTTTTPIP

GPDGKPLQIEPAGPGTTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVEPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

GPDGKPLQIEPAGPGTTPGTMTGPDGKPNKFVIPKGAFTTPGSIPGPDGKPIHVQPAGPGSTPGTVTGPDGSINQVFVPTTPKSPETSGGKKGTGENPANTNNAIGPLQPGSATTPTSIS

GPDGQPERIVPAGPGSTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVEPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

RM2 GPDGKPLQIEPAGPGTTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVEPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

GPDGKPLQIEPAGPGTTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVEPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPLGPGGQPMYPSGPQGPGGQTTTTPIP

GPDGKPLQIEPAGPGTTPGTVTGPDGKPKKFVLPKGAFTTPGSIPGPDGKPIHVEPAGPGTTPGAQTGPDGKINKLVVPTTTTPKGPLGPGGQPMYPSGPQGTGGQTTTTPIP

CTT GPDGNPIQIEPAGPGTTPGTMTGPDGKPNKFVIPKGAFTTPGSIPGPDGKPIHVQPAGPGSTPGTVTGPDGSINQVFVPTTPKSSEASGGKKGTGENPANSNNALGPLQPASTTTPTSIS

GPDGQPVQVVPAGPGSTPGTVTGPDGKPKKFVVPQGAFTTPGSVPGPDGNPIPVEPAGPGTTPGTQTGPDGKINKVVVPTTTPNPKGPESQPGNPLSPNGQTTPGSEGPGFNFQTPRPIK

GPKGKPIQIIPAGPGTTPGTVTGRNGRPKRFVVPKGAFTTPGSIPGPNGKPIHVKPAGPGTTPGVITGSDGSIDSIILPSTPKESQPGFPTYQFQTPKPIK

GPKGKRIQVIPAGPGTTPGVVTGPDGKPVKFVVPFGAFTTPGSIPGPNGKPIHVEPAGPGTTPGAETDSDGDIDSIVLPSTPKGPSPGLQFQTPRPIK

GPLGKPLQIIPAFPGTTPGVVTGPDGVPVEYIVPFGAFTTPGSIPGPNGKPIHVKPAGPGTTPGAKTDSDGNIDSIVLPSTPKGPSPGLQFQTPRSVT

KPDGQPIQVIPAGPGTTPGVVTGPDGRPVKFIVPQGAFTTPGSFMGPNGKPIHVGPAGPGTTPGAKTDSDGNIDSIVLPRTPRGAGPGFQFSTPGPIP

QPDGQPIQIVPAGVGTTPGVVTGPDGQPVKFIVPNGAFSTPGYIPGPHGKPIRVGPAGPGTTPGAKTDSDGDVSEILLPSTPSPPAPKPANPTTPTDVQ

GPQGGPIMIIPAGPGTTPGTITGPDGNPTKFIVPLGAFTTPGSIPGPDGKPIHVQPAGAGTTPGAETGPDGKINKIILPTTPKRPSYTVPTTTTPI

PGPGQPIQILPAGPGTTPGTVTGPDGQPVKFIVPQGAFVTPGTILGPDGKPIPVEPQGPGTTPGAELGPDGKINKIILPTTPPNPPSS

CTD GPLNPKGEPIPPFGPGNSPNSPQPPGNYPGSAFQFPGFPDAPGANGPIGYYDFSQLPSGESPEMEGPIGFLPNFSPEIGGPFPGFPGGPGSSGPGGFLNVNSLPDFINQGYGFPGSPQDPLGYLN

FSMLPPGYSPDFSGQFVYPGYPGSPGSGGQFPGGFLSFSELPQGVTDKINGTFSLPQLMQALQPLFPGQNINIGAIPKDQMQNIPGFKGTYDNLRLANFGSDNQPTGGVFYLPELVRLTSYLPVG

SFGNGPGTINQNGGFGDPFNFPGLNGAPGYICDYPDNGHATTGGSQNLGGEIKGNDNGPSGDVADAAPGNNLGGAAPGNNLGSAAPGNNLGSAAPGNNLGSAAPENDLGGAAPENNPAPQSDTPS

SPTDKGCEEDVQGAFSKARQALMNVASSTGEETVSQLLHDIISGINISGNYVDYNDFFNQLSSLLSQVRTDPTDSPSKQLIKILMEGLIAALEALNSAKVTGFRSDVSIDSDLPVYTSFLNEILY

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

Figure S2: Predicted AgSp2 amino acid sequence from A. trifasciata. Line breaks were inserted to emphasize different

regions. Abbreviations: NTD = N-terminal Domain; QRR = glutamine-rich region; RM = repeat motif; CTD =

C-terminal Domain. Highlighted serine in the ﬁrst QRR indicates putative splice site.

NTD MGYLSHVFIAVCLVALSSKSTSGQVNPAKDDPMEFNETEQKFASVFIDNIINSGAFGNLSEDEISEVTQILLKAIQILKKFHDGDASQKKTLMVAFASSMAELI

LDRTDTNLDLEQKTEIVTNALRQAYLKTTGKPNKALIKEVKFLVALFLTLEKPGFLDPLWNPYGRRPAFGPPPPFGVPLYPTPPPLPFPFDQIRRGNFKLYYLN

GTEVPQSEIQNLFPAGQR

QRR QPQFPSNIRFPNLNPFVPGPGGNSYQPRYSGSRQGYTYRSQYPGGRSEYQQYPYGQNSYGPGRQQYPYGPGSQPQGPDGQPQYSYGLDGQPQYSYGPDGQPQY

SYGPDGQPQYSYGPDGQPQYSYGPGGQPQYPYGPDGQPQGPGGQPQGPGGQPQGTGGQPQGPGGQPEGQPHYSYGPDGQPQYSYGPGGQPQYPYGPDGQPQ

GPGGQPQGPGGQPQGPGGQPQGPGRQPQSSGGQPLGPGGQPQYPSSTPLGPGSQPTSAGPAPIPGAGLPGSKPTPAGAKPTKGPQVMNPTPSTTQKIP

RM QSGGQPIEVQPSKPGTTPGVVTGPYNKPSKVILPPGGKSTPGNIPGPGGKPIEVQPAKPGETPGAITGPDHQVSKIILPSTTAGKGGAPQNPSKKMEPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVKPAKPGTTPGAITGPDSQVSKIILPSSPGNAPQNKGPGQQTT

QRR PAGGPQQPGQPMGPGSGPQQPEQQTTPASAPQQPGQPMGPEGVPQQPGQQTTTAVPPQQPGQPMGPGGGPQRPGQQTTPAGGSQKPGASPSNPNKPSAQEGNPKTGTNP

FGAPDAQPSSGSKATGQNQPGSSQNIGPLPSSTPSQQQSPTGTTPQKPGSIPQQSGQPTKQGGVPQQPGQQTTPEGAPQQPEQQTTIAGAPQQPGQPMGPGGQTTQMIP

RM QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVKPAKPGTTPGAITGPDSQVSKIILPSSPGNAPQNKGPGQQTT

QRR PAGGPQQPGQPMGPGSGPQQPEQQTTPASAPQPGQPMGPEGVPQQPGQQTTTAGPPQQPGQPMGPGGGPQRPGQQTTQAGGSQKPGASPSNLNKPSAQEGNPKTGTNPF

GAPDAQPSSGSKATGQNQPGSSQNIGPLPSSTPSQQQSPTGTTPQKPGSIPQPSGQPTKQGGVPQQPGQQTTPEGAPQQPEQQTTIAGAPQQPGQPMGPGGQTTQMIP

RM QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQQPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQQPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGKAPQKPLGPGQPTQMIP

QPGTQPIQVKPEQPGTTPGVVTGPDKKPSQVIVPQGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKQSQVIVPQGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKQSQVIVPQGGGSTPGTLPGPGGKPVQVKPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKQSQVIVPQGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGYTPGTLPGQGGKPVQVAPAKPGTTQGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQQPLGPGQTTQMIP

QPGGQPIQVKPAQPGTTPGVVTGPDQKPSQVYLPPGGESTPGTLPGPGGKPIQVVPAKPGTTPGAVTGPDNQVHKLILPEPTTGSMYGPEGPNAPESTTA

QRR FIPRPSGQPIKVVPSQPGTTPGAITGPDRKISTVVLPPGGASTPGNVFGPNGQPINVQPAEPGTTPGAVTGPDHNVHRLVLPTHRPRPMKPSQTPAPGN

APNNNPTVPSSSTNTPSPLNKPSAQQGNPKTGTDPFGAPEGQPSSGRKATGQNPTGSSQNIGSLPSSTPSPNAPISGPQNPTGTNPQQPGQPMGPGGVAQ

QPGQQTTPAGAPQQPGQQTTPASAPQQPGQPMGKGGAPQQPGQQTTPAGAPQQPGQQTTPAGAPQQQGQPMGPGVPQQQPGQQTTPVSAPQQPGRQTTPAGAP

KQPGKQTTPAGGQQQPGQQMGPGGVPQQPGQRTTPAGAPQQPGKKMGPGVPQQPGTQTTPAGAPQQPSQPMGPGGIPQQPGQQTTPAGAPQQPGQQTTPAGAP

QQQGQPMGPGVPQQPGQQTTPAGAPQQPEKQPTPAGGQQQPGQQMGPTVPQQPGHQTTPAGGAPQQPGQQTTPASSPQQPGQPTRPGGVPQQPGQPTTPAGAP

QQPGEQTTPAGAPQQPGQPMGPEGVPQQPGQQTPPGGAPQQPGQQTTPAGAPQQPGQQTTPAGAPQQPGQPIGPGAPQQPGQQTTPAGAPQQPGQQTTPAGGSQ

KPGASPSNLNKPSAQEGNPKTGTNPFGAPDAQPSSGSKATGQNQPGSSQNIGPLPSSTPSQQQSPTGTTPQKPGSIPQQSGQPTKQGGVPQQPGQQTTPAGAPQ

QPGQQTTTAGAPQQPEQQTTPAGAPQQPGQPMGPGGQTTQMIP

RM QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPQGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPQGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQQPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPQGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDRQVSKIILPTGPGNAPQKPLGPGQTTQMIP

QPGSQPIQVKPAQPGTTPGVVTGPDQKPSQVIVPPGGGSTPGTLPGPGGKPVQVEPAKPGTTPGAITGPDREVSKIILPSSPGNAPQPMEPGVPQQPGQL

CTD TTPGASQEPGQPTGPGGAPQQPGQPMGPGGSPQQPGQPLGPGNAPMKPGQTPKPVAPVSPPQVIPQPGGLPIEILPAMPGTTPGAITGPDNKISQIVLPPG

GGSTPGSVPGPGGRPIYIEPGKPGNTPGAVTGPDHQVTKIVLPDQPATANAPLPGPGSSLVGPPGGTTAGYFSPQTVTPAPNAAAYNQQNPYNIGLGFPGL

PNNQNLFQYLPNFETIFRQIVNRGNTRIPSLIQGVMQSKGQSPDTFDLREFIRNLSAVFGVMRYENSNMTYIEMYMELMMETLIGALEIIRVSDPSCLKQP

TTLGGIPKYVNAFYDILI

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

Figure S3: Additional AgSp1 repeat motifs from three orb weaving and three cobweb weaving species.

Ncl Motif 2 GPGGKPINIVPAGPGTTPGTVT GPDGKPSKFVVPDGAFTTPGSIP GPDGKPLPVEPAGPGTTPGLQT GPDGKPSKLVVP TTTKGPGGPMGPGGPMGPGGPMGPGGPMGPGGPMGPGGQTTTTTPAPVK

Ncl Motif 3 GPDGKPLQIVPAGPGTTPGTVT GPDGKPSKFVVPKGAFTTPGSIP GPDGKPLPVEPAGPGTTPGLET GPDGKPSKLVVP TTPKRPRGPGGSPLGPGTQFQTGTTPTPVP

Atr Motif 1 GPDGKPLPIEPAGPGTTPGTVT GPDGKPKKFVLPKGAFTTPGSIP GPDGKPIHVQPAGPGTTPGAQT GPDGKINKLVVP TTTTPKGPVGPGGMPLSPYSPQGPGGQPMYPFGPGSPYGPGEQTTTTPIP

Aau Motif 1 GPDGKPLQIEPAGPGTTPGTVT GPDGKPKKFVVPKGAFTTPGSIP GPDGKPIHVEPAGPGTTPGAET GPDGKINKIVVP TTTTPKGPVGPGGIPLSPYYPQGPGGQPMYPFGPGSPYGPGEQTTTTPIP

Pte Motif 2 GPGGRPIQLIPSRPGQTPGTVT GPDGYPIYIILPSGSGSTPGSFE GPDGQPIKVVPVGPGTTPGAET RPDGSIKIIFIP QSGSTPSGSTPSQVT

Pte Motif 3 GPGGNPIQLIPQGPGTTPGAVT GPDGTIIYIILPKGSGTTPGSVE GPGGKPIKILPAKPGTTPGAET GPDGTIEIIYIP QYPTTRRDGSTPSQIT

Sgr Motif 2 GPGGKPIQLVPSGPGSTPGVVT GPDGRPIKFILPSGSGSTPGSIE GPNGKPIKIMPAGPGTTPGAKT GPDGSVSIIYVP SGPSGQTTPGSQRPGGNTPSQIT

Lhe Motif 2 GPSGKPIQLVPSGPGSTPGVVT GPDGSPIKFILPKGSGSTPGSFE GPNGQPIKVMPVGPGSTPGAKT GPDGSVSIIYVP SQPSGQTTPGSGGPGGSTPSQIT

Lge Motif 2 GPSGKPIQLVPSGPGSTPGVVT GPDGSPIRFILPKGSGSTPGSFE GPNGQPIKVVPVGPGSTPGAKT GPDGSVSIIYVP QQPSGQTTPGSEGPGGSTPSQIT

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

CCAGCTGGACCAGGTATGTGACGTTAA...GTCTCTTAAAAAGGTACTACTCAAGGA

P A G P G M * R * ... V S * K G T T Q G

splice site

intron cdscds

CCAGCTGGACCAGGTACTACTCAAGGA

P A G P G T T Q G

Supplementary Figure 3. Putative splice sites for AgSp1 (A) and AgSp2 (B) exons of A.

trifasciata. Introns are capped with standard GT and AG dinucleotides (bold).

spliced cds

predicted protein

CCTCAATATCCAAGGTAAGTTATTAGT...TATTTCTTTGTTAGTAGCACACCGCTC

P Q Y P R * V I S ... Y F F V S S T P L

splice site

intron cds

cds

CCTCAATATCCAAGTAGCACACCGCTC

P Q Y P S S T P L

spliced cds

predicted Protein

Figure S4: Putative splice sites for A. trifasciata AgSp1 (A) and AgSp2 (B) exons. Introns are capped with standard GT

and AG dinucleotides (bold).

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

1 10 20 30 40 50 60 70 80 90 100 110 120

| | | | | | | | | | | | |

Aau RM ------------------------------------------------------------------------------------------------------------------------

Atr RM ------------------------------------------------------------------------------------------------------------------------

Ncl RM ------------------------------------------------------------------------------------------------------------------------

Lge RM ------------------------------------------------------------------------------------------------------------------------

Lhe RM ------------------------------------------------------------------------------------------------------------------------

Sge RM ------------------------------------------------------------------------------------------------------------------------

Pte RM ------------------------------------------------------------------------------------------------------------------------

Ncl MaSp GTTATGGACCAGGTCAACAAGGACCTGGAGGATATGGACCAGGACAACAAGGACCATCTGGACCCGGCAGTGCCGCTGCAGCAGCAGCCGCGGCAGCAGGACCTGGACAACAAGGCTCAG

Aau RM -----------------------GGACCTG-ACGGTA--AACCACTTCAGATTGAGCCAGCTGGACCAGGT--ACCACTCCAGGAACAGTGACAGGACCTGACGGAAAACCG--AATAAA

Atr RM -----------------------GGACCGG-ACGGTA--AACCACTACAGATTGAGCCAGCTGGACCAGGT--ACCACTCCAGGAACCGTGACTGGACCTGACGGAAAACCA--AAGAAA

Ncl RM -----------------------GGACCAG-ATGGTA--AACCATTACAAATTGTTCCCGCTGGTCCAGGT--ACAACCCCCGGAACAGTGACCGGCCCAGACGGCAAACCA--AGTAAA

Lge RM -----------------------GGACCTG-GTGGTA--GACCTATATCACTCATACCGAGCGGCCCCGGA--AACACACCCGGTGCCGTAACTGGACCAGATGGAAAAATT-TACTATA

Lhe RM -----------------------GGTCCTG-GTGGTA--GACCTATATCGCTCATACCAAGCGGCCCCGGA--AACACACCAGGTGCCGTAACTGGGCCAGATGGAAAAATT-TACTATA

Sge RM -----------------------GGACCAG-GAGGTC--GACCTATATCTCTGATTCCAGGAGGTCCAGGA--AACACTCCCGGAGCAGTAACAGGTCCAGACGGAAATATA-TACTATA

Pte RM -----------------------GGTCCAG-GTGGAA--GGCCAATTCAACTTATTCCTCAAGGACCTGGC--AGTACTCCAGGCGCAGTTACAGGTCCAGATGGCGTTATAATA-TATA

Ncl MaSp GAGGTTATGGACCAGGTCAACAAGGACCAGTAGGATATGGACCAGGACAACAAGGTCCAGGAGGATATGGACCAGGACAACAAGGCCCAT--CTGGACCAGGCAGTGCCGCTGCGGCAGC

Aau RM TTCGTCCTTC-CCAAAGGAGCATTC--ACTACACC----AGGAT------CAA-TC--CCCGGACCCGATGGCAA---ACCAATCCATGTTGAGCCTGCTGGTCCCGGAA---CCACA-C

Atr RM TTCGTTCTCC-CCAAAGGAGCTTTC--ACTACACC----TGGAT------CAA-TC--CCCGGACCCGATGGCAA---ACCAATCCATGTTGAGCCTGCTGGTCCCGGAA---CCACT-C

Ncl RM TTCGTCGTTC-CCGACGGAGCATTC--ACGACACC----AGGCT------CAA-TC--CCCGGTCCAGACGGAAA---ACCGCTCCCAGTTGAACCAGCTGGTTCAGGCA---CCACT-C

Lge RM T-CGTATTAC-CTTCAGGATCAGGA--ACAACGCC----AGGTT------CAA-TT--GAAGGCCCCGGAGGCAA---ACCAATAAAAATAGTACCGGCAGGCCCAGGAA---CTACT-C

Lhe RM T-CGTATTAC-CATCAGGATCAGGA--ACAACGCC----AGGTT------CAA-TT--GAAGGACCAGGAGGCAA---ACCAATAAAAATAGTACCCGCAGGCCCAGGAA---CTACT-C

Sge RM T-CATATTAC-CCTCTGGATCTGGA--ACAACACC----AGGAT------CTG-TT--GAAGGACCAGGAGGACA---ACCAATAAGAATTGTGCCAGCAGGACCAGGAA---CAACA-C

Pte RM T-TATTCTGC-CGAGAGGTTCTGGG--ACAACTCC----AGGCT------CCG-TT--GAAGGGCCAGATGGACA---ACCTATCAAAATAATGCCAGCTGGACCAGGTA---CTACA-C

Ncl MaSp AGCAGCCGCCGCATCAGGACCTGGACAACAAGGCCCAGGAGGTTATGGACCAGGTCAACAAGGACCTGGAGGATATGGACCAGGACAACAAGGACCATCTGGACCAGGCAGTGCCGCTGC

Aau RM C-----TGGCGCTCAGACTGGACCTG-ACGGTAAA-ATAAACAAACTTGTTGTCC--CAA--CCACTAC---AACACCC-AAA-GGCCCTCTAGGACCCGGAGGTCAACCTATGTATCC-

Atr RM C-----TGGCGCTCAGACTGGACCTG-ACGGCAAA-ATAAACAAACTTGTTGTCC--CAA--CCACTAC---AACACCC-AAA-GGCCCGCTAGGACCCGGAGGTCAACCTATGTATCC-

Ncl RM C-----GGGCCTCCAAACCGGACCCG-ATGGCAAA-CCAAGTAAACTCGTCGTTC--CAA--CGACAACCA-AAGGACC-AGGTGGTCCGATGGGACCAGGAGGTC---CGATGGGACC-

Lge RM C-----AGGAGCAGAAACTGGCCCCG-ACGGAAAA-ATAAATAAAATTTATATTC--CAA--AGAGTCC---CGAACCT-CAA-GGTCCAACGCG--AAGTACTCCTTCTCAAGTTACA-

Lhe RM C-----AGGAGCAGAAACGGGTCCCG-ACGGAAAA-ATAAATAAGATATACGTTC--CAA--AGGGTCC---CGAACCT-CAA-GGTCCAAGTCG--AAGTACTCCTTCTCAAGTTACC-

Sge RM C-----GGGTGCGCAAACGGGACCAG-ATGGGAAG-ATAAATGTAATATATGTTC--CAA--GAAGTCC---AAGTCCT-CAA-GGTCCAAGTAG--CAGTACTCCATCTCAAGTTACA-

Pte RM C-----AGGTGCTGAGACGGGACCTG-ATGGCAAC-ATCCGAATTATATACATAC--CAT--CACATCC---AAGAACT---A-GA-CCGGATGG--TAGTACGCCTTCTCAGGTAACT-

Ncl MaSp CGCAGCAGCCGCCGCATCAGGACCTGGACAACAAGGAGCAGGAGGATATGGACCCGGCAGTGCAGCTGCAGCAGCAGCCGCCGCAGCAGGACCTGGACAACAAGGCTTAGGAGGTTATGG

Aau RM TTCTGGACCCCAAGGACCTGGAGGACA--GACAACAACGAC----TCCAATTCCC----------------

Atr RM TTCCGGACCCCAAGGACCTGGAGGACA--GACAACAACGAC----TCCAATTCCA----------------

Ncl RM AGGAGGTCCGATGGGACCAGGAGGACA--AACCACGACGACCAC-TCCAGCGCCCGTTAAA----------

Lge RM -----------------------------------------------------------------------

Lhe RM -----------------------------------------------------------------------

Sge RM -----------------------------------------------------------------------

Pte RM -----------------------------------------------------------------------

Ncl MaSp ACCAGGTCAACAAGGACCTGGAGGATATGGACCAGGACAACAAGGTCCAGGAGGATATGGACCAGGACAAC

Figure S5: AgSp1 repeat motif nucleotide alignment from three orb weaving and four cobweb weaving spider species,

and the repeat motif of major ampullate spidroin 2 (MaSp2) of orb weaver N. clavipes.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

Figure S6: L. geometricus (A) and L. hesperus (B) AgSp2 predicted amino acid sequences.

Figure S7: L. geometricus (A) and L. hesperus (B) AgSp2 aligned to A. trifasciata AgSp2 and A. argentata FLAG. (C)

Alignment percent identity matrix.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

APR EPR INT - DEC EMBER 10, 2018

Table S1: Sequencing runs used to analyze AgSp sequences. A. aurantia and A. trifasciata data were generated for this

study; other species data were accessed via NCBI. All samples were sequenced using the Illumina platform except

AtriAg7-13, which were sequenced using the Oxford Nanopore MinION. Ag=aggregate, Ma=Major ampullate, Fb=Fat

body, A=Anterior, P=Posterior.

Argiope trifasciata (Atr) Argiope aurantia (Aau) Latrodectus hesperus (Lhe) Latrodectus geometricus (Lge) Nephila clavipes (Ncl) Steatoda grossa (Sgr)

SRR7310653 (AtriAg1) SRR7310651 (AaurAg1) SRR5285106 (AgA1) SRR5285086 (AgA1) SRR5139355 (silk gland pair 03) SRR5285128 (AgA1)

SRR7310658 (AtriMa1) SRR7310650 (AaurMa1) SRR5285107 (AgA2) SRR5285087 (AgA2) SRR5139343 (silk gland pair 04) SRR5285125 (AgP1)

SRR7310659 (AtriFb1) SRR7310667 (AaurFb1) SRR5285102 (AgP1) SRR5285082 (AgP1) SRR5139327 (silk gland pair 05)

SRR7310656 (AtriAg2) SRR7310666 (AaurAg2) SRR5285103 (AgP2) SRR5285083 (AgP2)

SRR7310657 (AtriMa2) SRR7310665 (AaurMa2)

SRR7310652 (AtriFb2) SRR7310664 (AaurFb2)

SRR7310647 (AtriAg3) SRR7310663 (AaurAg3)

SRR7310646 (AtriMa3) SRR7310662 (AaurMa3)

SRR7310649 (AtriFb3) SRR7310661 (AaurFb3)

SRR7310648 (AtriAg4) SRR7310660 (AaurAg4)

SRR7310655 (AtriAg5) SRR7310669 (AaurAg5)

SRR7310654 (AtriAg6) SRR7310668 (AaurAg6)

SRR7346453 (AtriAg7-9)

SRR8167878(Atri_gDNA_10)

SRR8167879(Atri_gDNA_11)

SRR8167876(Atri_gDNA_12)

SRR8167877(Atri_gDNA_13)

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/492025doi: bioRxiv preprint first posted online Dec. 10, 2018;

Long-read transcriptomic analysis of orb-weaving spider Araneus ventricosus indicates transcriptional diversity of spidroins

Article

Dec 2020
INT J BIOL MACROMOL

Spider silk, which is composed of diverse silk proteins (spidroin), is a kind of natural high-mass biomaterial with great potential. However, due to the complexity of both the structure and the composition of the spidroins in natural spider silk, application of this valuable biomass is still limited to date. There are diverse kinds of spider silk in the orb-weaving spider with different mechanical and structural characteristics. In order to systematically illustrate the landscape of all the different spidrons, here we chose Araneus ventricosus, an orb-weaving spider with superior silk mechanical features and genome information, to generate a long-read whole body transcriptome. We deciphered the repeat arrangements of each kind of spidroin, based on which we found that there are substantially transcriptional diversity of each spidroin gene. Some repeat motifs are not documented before. Specifically, we discovered novel full-lengh MaSp transcript as well as a relatively small full-length AcSp isoforms, which are potential promising materials for bioengineering of recombinant spidroin. Our study provided a batch of new spidron resources with detail sequential information. The finding of transcriptional diversity may provide cues in understanding of within-species variation of the mechanical properties of the natural spider silk and further molecular designing of recombinant spidroin.

Supersaturation with water explains the unusual adhesion of aggregate glue in the webs of the moth-specialist spider, Cyrtarachne akirai

Article

Full-text available

Nov 2018

Orb webs produced by araneoid spiders depend upon aggregate glue-coated capture threads to retain their prey. Moths are challenging prey for most spiders because their scales detach and contaminate the glue droplets, significantly decreasing adhesion. Cyrtarachne are moth-specialist orb-weaving spiders whose capture threads adhere well to moths. We compare the adhesive properties and chemistry of Cyrtarachne aggregate glue to other orb-weaving spiders to test hypotheses about their structure, chemistry and performance that could explain the strength of Cyrtarachne glue. We show that the unusually large glue droplets on Cyrtarachne capture threads make them approximately 8 times more adhesive on glass substrate than capture threads from typical orb-weaving species, but Cyrtarachne adhesion is similar to that of other species after normalization by glue volume. Glue viscosity reversibly changes over 1000-fold in response to atmospheric humidity, and the adhesive strength of many species of orb spiders is maximized at a viscosity of approximately 10⁵–10⁶ cst where the contributions of spreading and bulk cohesion are optimized. By contrast, viscosity of Cyrtarachne aggregate glue droplets is approximately 1000 times lower at maximum adhesive humidity, likely facilitating rapid spreading across moth scales. Water uptake by glue droplets is controlled, in part, by hygroscopic low molecular weight compounds. NMR showed evidence that Cyrtarachne glue contains a variety of unknown low molecular weight compounds. These compounds may help explain how Cyrtarachne produces such exceptionally large and low viscosity glue droplets, and also why these glue droplets rapidly lose water volume after brief ageing or exposure to even slightly dry (e.g. < 80% RH) conditions, permanently reducing their adhesion. We hypothesize that the combination of large glue droplet size and low viscosity helps Cyrtarachne glue to penetrate the gaps between moth scales.

Egg Case Silk Gene Sequences from Argiope Spiders: Evidence for Multiple Loci and a Loss of Function Between Paralogs

Article

Full-text available

Nov 2017

Spiders swath their eggs with silk to protect developing embryos and hatchlings. Egg case silks, like other fibrous spider silks, are primarily composed of proteins called spidroins (spidroin = spider-fibroin). Silks, and thus spidroins, are important throughout the lives of spiders, yet the evolution of spidroin genes has been relatively understudied. Spidroin genes are notoriously difficult to sequence because they are typically very long (10 or more kilobases of coding sequence) and highly repetitive. Here, we investigate the evolution of spider silk genes through long-read sequencing of BAC clones. We demonstrate that the silver garden spider Argiope argentata has multiple egg case spidroin loci with a loss of function at one locus. We also use degenerate PCR primers to search the genomic DNA of congeneric species and find evidence for multiple egg case spidroin loci in other Argiope spiders. Comparative analyses show that these multiple loci are more similar at the nucleotide level within a species than between species. This pattern is consistent with concerted evolution homogenizing gene copies within a genome. More complicated explanations include convergent evolution or recent independent gene duplications within each species.

Analysis of repetitive amino acid motifs reveals the essential features of spider dragline silk proteins

Article

Full-text available

Aug 2017
PLOS ONE

The extraordinary mechanical properties of spider dragline silk are dependent on the highly repetitive sequences of the component proteins, major ampullate spidroin 1 and 2 (MaSp2 and MaSp2). MaSp sequences are dominated by repetitive modules composed of short amino acid motifs; however, the patterns of motif conservation through evolution and their relevance to silk characteristics are not well understood. We performed a systematic analysis of MaSp sequences encompassing infraorder Araneomorphae based on the conservation of explicitly defined motifs, with the aim of elucidating the essential elements of MaSp1 and MaSp2. The results show that the GGY motif is nearly ubiquitous in the two types of MaSp, while MaSp2 is invariably associated with GP and di-glutamine (QQ) motifs. Further analysis revealed an extended MaSp2 consensus sequence in family Araneidae, with implications for the classification of the archetypal spidroins ADF3 and ADF4. Additionally, the analysis of RNA-seq data showed the expression of a set of distinct MaSp-like variants in genus Tetragnatha. Finally, an apparent association was uncovered between web architecture and the abundance of GP, QQ, and GGY motifs in MaSp2, which suggests a co-expansion of these motifs in response to the evolution of spiders' prey capture strategy.

Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider silk glands

Article

Full-text available

Dec 2017

Spider silk synthesis is an emerging model for the evolution of tissue-specific gene expression and the role of gene duplication in functional novelty, but its potential has not been fully realized. Accordingly, we quantified transcript (mRNA) abundance in seven silk gland types and three non-silk gland tissues for three cobweb-weaving spider species. Evolutionary analyses based on expression levels of thousands of homologous transcripts and phylogenetic reconstruction of 605 gene families demonstrated conservation of expression for each gland type among species. Despite serial homology of all silk glands, the expression profiles of the glue-forming aggregate glands were divergent from fiber-forming glands. Also surprising was our finding that shifts in gene expression among silk gland types were not necessarily coupled with gene duplication, even though silk-specific genes belong to multi-paralog gene families. Our results challenge widely accepted models of tissue specialization and significantly advance efforts to replicate silk-based high-performance biomaterials.

The Nephila clavipes genome highlights the diversity of spider silk genes and their complex expression

Article

Full-text available

May 2017

Spider silks are the toughest known biological materials, yet are lightweight and virtually invisible to the human immune system, and they thus have revolutionary potential for medicine and industry. Spider silks are largely composed of spidroins, a unique family of structural proteins. To investigate spidroin genes systematically, we constructed the first genome of an orb-weaving spider: the golden orb-weaver (Nephila clavipes), which builds large webs using an extensive repertoire of silks with diverse physical properties. We cataloged 28 Nephila spidroins, representing all known orb-weaver spidroin types, and identified 394 repeated coding motif variants and higher-order repetitive cassette structures unique to specific spidroins. Characterization of spidroin expression in distinct silk gland types indicates that glands can express multiple spidroin types. We find evidence of an alternatively spliced spidroin, a spidroin expressed only in venom glands, evolutionary mechanisms for spidroin diversification, and non-spidroin genes with expression patterns that suggest roles in silk production.

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders

Article

Full-text available

Dec 2017
BMC EVOL BIOL

Background Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. Results We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. Conclusions MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0927-x) contains supplementary material, which is available to authorized users.

Complete gene sequence of spider attachment silk protein (PySp1) reveals novel linker regions and extreme repeat homogenization

Article

Full-text available

Jan 2017

Spiders use a myriad of silk types for daily survival, and each silk type has a unique suite of task-specific mechanical properties. Of all spider silk types, pyriform silk is distinct because it is a combination of a dry protein fiber and wet glue. Pyriform silk fibers are coated with wet cement and extruded into “attachment discs” that adhere silks to each other and to substrates. The mechanical properties of spider silk types are linked to the primary and higher-level structures of spider silk proteins (spidroins). Spidroins are often enormous molecules (>250 kDa) and have a lengthy repetitive region that is flanked by relatively short (∼100 amino acids), non-repetitive amino- and carboxyl-terminal regions. The amino acid sequence motifs in the repetitive region vary greatly between spidroin types, and motif length and number underlie the remarkable mechanical properties of spider silk fibers. Existing knowledge of pyriform spidroins is fragmented, making it difficult to define links between the structure and function of pyriform spidroins. Here, we present the full-length sequence of the gene encoding pyriform spidroin 1 (PySp1) from the silver garden spider Argiope argentata. The predicted protein is similar to previously reported PySp1 sequences but the A. argentata PySp1 has a uniquely long and repetitive “linker”, which bridges the amino-terminal and repetitive regions. Predictions of the hydrophobicity and secondary structure of A. argentata PySp1 identify regions important to protein self-assembly. Analysis of the full complement of A. argentata PySp1 repeats reveals extreme intragenic homogenization, and comparison of A. argentata PySp1 repeats with other PySp1 sequences identifies variability in two sub-repetitive expansion regions. Overall, the full-length A. argentata PySp1 sequence provides new evidence for understanding how pyriform spidroins contribute to the properties of pyriform silk fibers.

Hygroscopic compounds in spider aggregate glue remove interfacial water to maintain adhesion in humid conditions

Article

May 2018

Adhesion in humid environments is fundamentally challenging because of the presence of interfacial bound water. Spiders often hunt in wet habitats and overcome this challenge using sticky aggregate glue droplets whose adhesion is resistant to interfacial failure under humid conditions. The mechanism by which spider aggregate glue avoids interfacial failure in humid environments is still unknown. Here, we investigate the mechanism of aggregate glue adhesion by using interface-sensitive spectroscopy in conjunction with infrared spectroscopy. We demonstrate that glycoproteins act as primary binding agents at the interface. As humidity increases, we observe reversible changes in the interfacial secondary structure of glycoproteins. Surprisingly, we do not observe liquid-like water at the interface, even though liquid-like water increases inside the bulk with increasing humidity. We hypothesize that the hygroscopic compounds in aggregate glue sequester interfacial water. Using hygroscopic compounds to sequester interfacial water provides a novel design principle for developing water-resistant synthetic adhesives.

Molecular cloning and analysis of the full-length aciniform spidroin gene from Araneus ventricosus

Article

Dec 2017
INT J BIOL MACROMOL

Orb-web spiders produce more than seven differen protein-based silks/glues by specialized abdominal glands for different uses. Prey-wrapping silk is secreted by aciniform glands for wrapping prey and forming the inner layer of egg case, and is almost twice as tough as other silks because of high strength and extensibility. So far, only two complete gene sequences have been obtained for aciniform spidroins (AcSp1). Here we describe the AcSp1 full-length gene sequence from the spider species Araneus ventricosus, using a long-distance PCR (LD-PCR) approach. The full-length AcSp1 gene is a single enormous exon of 10,338 bp in size, and the predicted protein sequence is 3,445 amino acids long and consists of a conserved nonrepetitive N-terminal domain, a central predominantly repetitive region composed of fifteen repeat units, and a conserved nonrepetitive C-terminal domain.

Characterization of full-length tubuliform spidroin gene from Araneus ventricosus

Article

Jul 2017

Orb-web spiders produce at least seven different protein-based silk that exhibit different mechanical properties. Egg case silk is spun by tubuliform glands and used to form the outer shell of egg case. To date, only two complete cDNA sequences have been reported for tubuliform spidroin (TuSp, also known as CySp for cylindrical spidroin). Here, we report the complete genomic DNA sequence for a tubuliform spidroin (TuSp1) gene from orb-web weaving spider species Araneus ventricosus, which was obtained by using a long distance PCR approach. The primary component of Araneus ventricosus TuSp1 gene contains a single large exon (5736 bp) and encodes 1921 amino acid residues dominated by 9 tandem repeats flanked by conserved non-repetitive N- and C-terminal domains. These repeats (each 176–155 amino acids long) found in Araneus ventricosus TuSp1 are highly conserved (>96% identical at amino acid level).

Towards Spider Glue: Long-read scaffolding for extreme length and repetitious silk family genes AgSp1 and AgSp2 with insights into functional adaptation

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