BACKGROUND
spiders  use silk throughout their lives for functions such as prey capture, sperm webs, egg cases, safety draglines, and dispersal  <cit> .
orb-web weavers and their relatives  spin six or more task-specific silk fibers.
each of these task-specific fibers is synthesized in its own set of specialized abdominal glands  <cit> .
for example, dragline silk is synthesized in the major ampullate  glands and egg-case silk is synthesized in the tubuliform glands.
each type of silk has a unique suite of impressive mechanical properties  <cit> .
the dragline silk of orb-weavers approaches the tensile strength of steel and is more extensible than rubber or tendon collagen  <cit> .
this combination of strength and extensibility makes dragline silk incredibly tough.
tubuliform silk, by contrast, is a stiff fiber that can store a large amount of energy  <cit> .
there is interest in capitalizing on the remarkable variation and combination of material properties of spider silks for medical and industrial applications such as tendon replacements, sutures, and as an environmentally friendly alternative to synthetic fibers  <cit> .
spider silks are composed primarily of large structural proteins termed spidroins  that are encoded by members of a single gene family.
silk types differ from each other in their constituent spidroins  <cit> .
for instance, ma glands express masp <dig> and masp <dig>  <cit>  and the outer egg case silk synthesized in tubuliform glands includes tusp <dig>  <cit> .
other spidroin paralogs have been identified from each of the gland types except for the aggregate gland  <cit> .
spidroins are large proteins  made up almost entirely of repeated blocks of amino acids , flanked by non-repetitive amino  and carboxy  terminal domains.
these terminal domains are conserved in length  and sequence among spidroin types and across species  <cit> .
the majority of spider silk genetic research has focused on silk synthesized in the ma glands because of the high tensile strength of dragline fibers  <cit> .
these efforts include characterizing masp <dig> and masp <dig> sequences from multiple species  and creating recombinant proteins expressed in transgenic organisms .
however, the mechanical properties of fibers spun from recombinant spider silk proteins do not yet accurately mimic natural spider silk fibers , suggesting more extensive work is needed to understand the genetics and spinning of spider silks.
synthesis, assembly, and material properties of dragline silk likely rely on the relative expression of spidroin paralogs as well as the influence of non-spidroin genes.
for instance, the relative proportion of masp <dig> and masp <dig> in dragline silk varies among species and even among individuals of the same species, which could potentially explain inter-and intra-specific level differences in mechanical properties  <cit> .
the extensibility of dragline silk correlates positively with its proline content  <cit> , an amino acid that is present in higher proportion in masp <dig> than in masp <dig>  <cit> .
the western black widow, latrodectus hesperus , synthesizes draglines with a low proline content and thus masp <dig> is predicted to be approximately three times more abundant than masp <dig> in the fiber  <cit> .
the extensibility of black widow dragline silk is correspondingly lower than other species that have draglines primarily composed of masp <dig>  <cit> .
additionally, masp <dig> is encoded by multiple loci in l. hesperus and the golden orb-weaver nephila clavipes <cit> .
masp <dig> loci are similar to each other, but not identical, within a species.
variation in the expression patterns of each locus could contribute to variation in mechanical properties of dragline silks within or among species, but such variation in expression has yet to be documented.
non-spidroin genes can also play important roles in the synthesis and assembly of spider silks, although only a few cases have been described .
for instance, the egg case proteins, ecp- <dig> and ecp- <dig>  are expressed primarily in the tubuliform glands and are predicted to form cross-links with tusp <dig>  <cit> .
exploring the genetics of spider silk synthesis has been limited to a gene-by-gene approach because of the minimal genomic information available for spiders.
genomic methods have only recently become feasible for non-model organisms, such as spiders, with the advent of next generation sequencing technologies  <cit> .
next generation sequencing and de novo assembly were used to characterize  <dig>  “uni-genes” expressed in the silk glands of two spider species, a tarantula and an orb-weaver  <cit> .
this approach identified several major functional classes of genes expressed in silk glands, and discovered new spidroin paralogs, but did not measure relative expression rates.
these results represent a major advance in characterizing spider genes, but due to the lack of comparisons with other tissues, it is unclear which genes or functional classes are important for silk synthesis  <cit> .
here, we use massively parallel signature sequencing  to profile expression patterns in ma glands and cephalothoraxes  of the western black widow.
we targeted this species for several reasons.
black widows synthesize dragline silks that are among the strongest measured  <cit> .
as descendants of orb-web weaving ancestors  <cit> , black widows possess all the gland types of true orb-web spiders despite building three-dimensional cobwebs  <cit> .
six of the seven known spidroin paralogs have been described for the western black widow.
furthermore, because the western black widow has a relatively small genome for a spider , we have arrayed a genomic library and completely sequenced the dragline silk genes masp <dig> and masp <dig> <cit>  and the prey-wrapping gene acsp <dig> <cit> .
our aims were to 1) determine relative levels of transcript abundance within ma glands compared to cephalothorax control tissue and 2) identify novel candidate genes of importance to dragline silk production.
our findings demonstrate that dragline silk synthesis involves a much greater transcriptional complexity than previously known, laying the foundation for further studies of silk gland functional genomics and recombinant silk production.
RESULTS
generation of “tags” and construction of reference database
we generated more than  <dig> million tags, which are 3′-anchored  <dig> base cdna fragments, from mrna transcripts found in the ma glands and cephalothoraxes of adult female western black widows using mpss .
mpss allows for highly quantitative comparison between tissues, even in organisms with poorly characterized genomes, by anchoring sequencing to the 3′ end of transcripts.
different transcripts are discriminated by their 3′-most tags and expression levels estimated with higher accuracy than could be achieved by random rna-seq  <cit> .
sequencing more than  <dig> million tags from each of four libraries  resulted in  <dig>  unique tag sequences.
of these,  <dig>  unique tags were found at levels greater than one count per million total tags  in two or more libraries .
a
acpm = counts per million.
to determine the gene identity of tags, we compiled a non-redundant set of  <dig> “reference” protein-coding sequences for the western black widow from  <dig> newly sequenced cdnas ,  <dig> published cdna sequences, and  <dig> published gene sequences .
the only sequences available that included untranscribed regions were for the complete gene sequences of masp <dig> and masp <dig>  for initial expression profiling, we thus limited representatives of masp <dig> and masp <dig> to the longest cdna for each.
the mpss method we used targeted the 3′-most occurrence of the dpnii recognition sequence, 5′-gatc-3′.
we determined tag identity by first predicting all possible tag sequences from the coding or sense strand and the complementary or antisense strand  in the reference genes.
predicted tags were then matched to the sequenced tags from our four libraries.
of the  <dig> sequences in our reference database ,  <dig> contained a predicted tag and  <dig> were represented by at least one observed tag.
identification of differentially expressed transcripts in western black widows
an exact test implemented in edger  <cit>  identified approximately 23% of tags were significantly more abundant in one tissue type:  <dig>  unique tags were significantly more abundant in the ma glands and  <dig>  tags were more abundant in the cephalothoraxes .
each of these differentially expressed tags was at least  <dig> times more abundant in one tissue than the other .
we assigned  <dig> unique tags to  <dig> genes in our reference database .
in terms of abundance and fold change, all tag sequences that matched a reference gene  as well as tags that matched the 3′-most position  appear to be an unbiased subset of all observed tags.
abundance levels of these tags were also highly correlated between individual samples .
approximately 46% of the reference genes represented by a sequenced tag were represented by both sense and antisense tags, 45% were represented by only a sense tag, and 9% were represented by only an antisense tag .
most reference genes were represented by one sense tag and/or one anti-sense tag .
we observed instances of multiple tags aligning to a single gene, but the 3′-most sense tag was almost always considerably more abundant than other sense tags, as expected .
genes represented by multiple sense tags were usually represented by multiple antisense tags, but antisense tags did not show a consistent relationship between position on the gene and tag abundance .
alternative tags could result from incomplete digestion with dpnii, or from degradation of mrnas.
however, in many cases multiple tags represent genuine alternative transcripts .
an example of these alternative transcripts is described for masp <dig> below.
because the origin of multiple tags per gene is not always clear, we estimated transcript abundance two ways.
first, under the assumption that all tags that matched a gene represent transcription of some kind, we summed the average cpm of all tags from the sense strand, or separately, from the antisense strand .
second, to account for the possibility that multiple tags represent alternative transcripts, we estimated transcript abundance based solely on the 3′-most position of the sense strand, or its matched position on the antisense strand .
these two estimates are tightly correlated .
because we selected for polyadenylated mrna, we did not expect to capture antisense transcripts.
total abundance of a transcript estimated from summing all tags that matched that gene sequence is correlated between sense and antisense strands, with total abundance of sense tags almost always higher than total abundance of antisense tags .
for instance, expression levels based on sense tags varied from  <dig> to  <dig>  cpm in ma glands while antisense counts varied from  <dig> to  <dig>  cpm.
despite lower abundance of antisense tags, differences in gene expression levels between tissues estimated by sense and antisense tags are tightly correlated .
thus, even if our antisense tags represent an experimental error associated with the reverse transcription phase of cdna synthesis  <cit> , they provide consistent information regarding differential expression.
it is also possible that antisense transcription is widespread in spiders.
in metazoan genomes investigated thus far, 5-22% of genes are represented by both sense and antisense transcripts  <cit> .
in mice and humans, more than 40% of the transcript pool could result from antisense transcription  <cit> .
furthermore, expression patterns of sense and antisense transcripts generated from the same genomic locus tend to be correlated  <cit> , similar to the pattern seen here .
variable expression of spidroin paralogs in ma glands
our database contained  <dig> unique sequences encoding masp <dig>  including one complete protein-coding sequence for locus  <dig> and several thousand base pairs of non-coding flanking sequence  <cit> .
it additionally included representatives of two other loci  and partial length cdnas.
from these sequences, including non-coding regions, we predicted  <dig> sense tags  and  <dig> antisense tags .
full-length masp <dig> has  <dig> predicted sense tags  and  <dig> predicted antisense tags  that matched an observed tag.
importantly, all of the tags predicted in the coding region of the full-length gene were observed as well as two downstream of the coding region .
as expected, there were no observed tags that corresponded to intergenic genomic dna.
in addition to the eight unique sense tags that matched the full-length masp <dig> gene, there were five other unique sense tags that aligned to alternative masp <dig> sequences .
alignment of all masp <dig> sequences indicated that the three loci and the cdnas share an identical observed sense tag in the c-terminal encoding region .
this tag is the 3′-most predicted one in the cdnas and was the most abundant observed masp <dig> tag and the second most abundant observed tag in ma glands overall .
the seven sense tags found throughout the repetitive encoding region of the full-length masp <dig> were much less abundant , consistent with selecting for the 3′-most occurrence of the dpnii recognition sequence.
although the 3′-most tag from the coding sequence cannot discriminate expression levels of each masp <dig> locus, there are other indices that are diagnostic for particular loci .
prior to this study, only locus  <dig> was supported by expression data  <cit> .
here, we observed unique tags for all three loci.
locus  <dig> has an observed unique tag  <dig> bases downstream of the stop codon .
loci  <dig> and  <dig> share an observed tag  <dig> bases downstream of the stop codon that is not found in locus  <dig> .
yet another observed unique tag occurs in locus  <dig>   <dig> bases downstream of the stop codon .
a different tag was predicted in the same location in locus  <dig>  but was not observed.
however, locus  <dig> has a unique observed tag in the n-terminal coding region .
thus, we have evidence for the co-expression all three loci at the same time in the same tissue.
the observed tags found downstream of the masp <dig> stop codon also suggest that both loci  <dig> and  <dig>  and possibly  <dig>  are alternatively polyadenylated.
consistent with this hypothesis is the occurrence of a polyadenylation signal  downstream of an observed tag in each of these loci .
both 3′ utr length, and specific sequence elements contained therein, have been shown to be important for a variety of regulatory events including control of mrna stability and translational efficiency  <cit> .
our results thus raise the possibility that differential polyadenylation of masp <dig> transcripts may be involved in locus-specific stability or translational control.
although the 3′-most coding region tag of masp <dig> is identical among all three loci , the sequences of downstream tags are unique and can therefore be used to compare locus-specific expression levels.
analysis of these tags reveals that the isoform of locus  <dig> carrying an elongated 3′ utr is ~ <dig> times more abundant than the alternatively polyadenylated forms of loci  <dig> and  <dig> combined .
in the absence of strong, locus-specific alternative 3′ end regulation, this finding suggests that locus  <dig> is dominant in the ma gland tissue of adult females.
our data are therefore consistent with the previously suggested hypothesis that masp <dig> locus variants could be incorporated in differing proportions into draglines  <cit> .
because masp <dig> encoded by each locus differs in proportion and ordering of ensemble repeats, varying the proportion of each could affect silk properties.
importantly, prior to this study there was no direct evidence for simultaneous expression from all three loci.
our database also contained seven sequences encoding masp <dig>  including a complete gene with several thousand base pairs of non-coding flanking sequence  <cit> .
from these masp <dig> sequences,  <dig> tag locations  were predicted from the sense strand representing  <dig> unique tag sequences .
in the full-length gene,  <dig> tag locations were observed .
four of these observations were well outside the coding region and appeared at low frequencies in all tissues , leaving seven unique observed sense tags within the masp <dig> coding region .
alignment of the complete masp <dig> gene with other masp <dig> sequences including partial cdnas showed that all sequences shared a 3′-most predicted sense tag.
this tag was the most abundant in masp <dig> sequences .
all other observed masp <dig> tags were much less abundant .
there was only one additional unique sense tag observed from other masp <dig> sequences.
this tag is in the n-terminal encoding region of a partial masp <dig> cdna .
a tag that only differed by one base pair from this tag was predicted from the corresponding coding region in the full-length gene, but was not observed .
the ratio of masp1:masp <dig> transcript abundance based on the 3′-most coding tags varied between the two individuals, but the ratio based on the average cpm in ma glands is 3: <dig> masp1:masp <dig> consistent with the amino acid composition of ma fibers collected from western black widows  <cit> .
thus, despite the potential for complex locus-specific translational control, transcript abundance may accurately reflect overall protein abundance of masp <dig> and masp <dig>
variation in proportion of masp <dig> and masp <dig> could explain variation in fiber properties among individual black widow spiders  <cit> .
in three other species, varying the amount of protein in the spider’s diet significantly alters mechanical properties of ma fibers  <cit> .
specifically, extensibility of ma fibers, a property highly correlated with the proline content of the fiber  <cit> , decreased with a low protein or protein-less diet  <cit> .
since proline is in higher proportion in masp <dig> than in masp <dig>  <cit> , an increased extensibility in ma fibers is likely due to an increase in the proportion of masp <dig> in the fiber.
individual spiders could have widely varying diets that overtime could change the proportion of spidroins in the silk fibers and thus create fibers with varying mechanical properties.
tusp <dig>  which encodes the primary component of outer egg-cases, was unexpectedly expressed in ma glands.
our non-redundant database contained two sequences predicted to encode tusp <dig>  but both shared an identical observed tag in the c-terminal encoding region .
tusp <dig> was surprisingly abundant, ranking 10th most represented gene of the ones that are more abundant in ma glands than cephalothoraxes, directly following masp <dig> .
however, one individual transcribed much more tusp <dig>  than the other .
the difference in expression levels between the two individuals could be explained by individual differences in readiness to make egg-cases.
the number of transcripts encoding tusp <dig> and other egg-case proteins increase in tubuliform silk glands as the individual nears egg-case production  <cit> .
the individual with lower levels of tusp <dig> in ma glands did not have well-developed eggs.
however, the individual with high levels of tusp <dig> had large, mature eggs, suggesting that tusp <dig> transcription increases in ma glands at the same time as increasing in tubuliform glands.
it is possible tusp <dig> becomes incorporated in ma fibers at the time of egg-case production, which could contribute to variation in mechanical properties.
alternatively, translational control of tusp <dig> prevents protein synthesis in ma glands despite the presence of tusp <dig> transcripts.
we also cannot exclude the possibility that our detection of tusp <dig> in ma glands is due to leakage from mrnas transcribed in the tubuliform glands.
novel candidate genes involved in silk synthesis
in addition to spidroin encoding genes, we identified  <dig>  other genes represented by tags that were significantly more abundant in ma glands than cephalothoraxes .
when translated,  <dig>  of these genes aligned to functionally annotated proteins or conserved domains .
of the remaining ten genes, one did not match any entries in the ncbi nr database and nine aligned only to hypothetical proteins previously predicted for western black widows.
all but one of these ten encode proteins with signal peptides predicted by signalp  <cit>  , suggesting they are secreted.
secreted proteins could be components of ma fibers or involved with fiber assembly.
an unanticipated finding was that the 3′-most sense transcript tag of one of the hypothetical protein-encoding genes is three times more abundant in ma glands than that of masp <dig> .
this gene, “contig5”, has a short  open reading frame  and signalp predicts an even smaller mature peptide .
the first  <dig> bases, which includes the first  <dig> aa, of “contig5” is nearly identical to that of another gene overrepresented in ma glands, “lnc168”, as well as one  that was not differentially expressed between ma and cephalothorax.
each of these sequences differs in the last few amino acids and most of the putative 3′ utrs, including the tag locations.
“lnc168” is much less abundant in ma glands than “contig5” .
these sequences may represent transcripts from paralogous genes, alternatively spliced genes, or a combination of both scenarios.
alignment revealed two regions of almost exact identity among all three sequences, and another region of exact identity between two, interspersed with unalignable regions.
these patterns are consistent with both hypotheses of paralogous genes and alternatively spliced genes.
alternative splicing is an important mechanism for generating functionally distinct proteins with tissue specific expression  <cit>  as well as unique 3′ utrs.
isoforms caused by 3′ utr alternative splicing can modify the production of the protein through altering locations for microrna binding and activating decay pathways such as the nonsense-mediated decay pathway  <cit> .
among the most highly expressed genes in ma glands that could be annotated were ones predicted to encode fasciclin, translation elongation factor 1-alpha, and lectin.
each of these was more abundant than masp <dig> .
abundance ranking of genes expressed in ma glands was almost identical if estimates were based solely on the 3′-most tags .
tags corresponding to elongation factor 1-alpha were only slightly more abundant in ma glands than cephalothoraxes .
in fact, the 3′-most sense tag of this gene was not significantly different between the two tissues.
thus, the higher abundance in ma glands probably reflects the very high rates of translation to produce dragline spidroins, masp <dig> and masp <dig>  in contrast, tags representing fasciclin and lectin-encoding genes were consistently highly abundant in ma glands but either absent or extremely low in cephalothoraxes .
in various insect species, fasciclins are cell adhesion glycoproteins that play a role in neuronal development .
lectins are sugar-binding proteins involved in the innate immune response in drosophila <cit> .
however, it is not immediately clear how these particular functions might be involved with silk synthesis.
it is possible that these genes are expressed in specialized cells within the ma glands that are not secreting silk proteins.
the ma glands are divided into multiple regions including a tail and ampullate region where spidroins are expressed, the lower ampullate region where spidroins are stored, and the duct where spidroins begin assembly for ultimate extrusion as fibers  <cit> .
there may well be cells within any of these regions that have currently unknown functions not found in the cephalothorax.
thirteen of the  <dig> genes overrepresented in ma glands that were annotated by sequence similarity are predicted to encode proteins that can be grouped into three functional classes .
these include protease inhibitors , proteins with transferase activity , and transcription factors .
the protease inhibitors appear to be derived from two large gene families.
the putative serine protease inhibitor genes encode  <dig> aa domains that align to each other with 47% pairwise identity.
they also align to four additional translated genes in our reference database that were not overrepresented in ma glands.
similarly, four of the genes overrepresented in ma glands encode thyroglobulin type- <dig> conserved domains, which function as cysteine protease inhibitors.
these four genes align with each other with 85-89% pairwise nucleotide identity over their entirety.
at the amino acid level, they align to an additional four sequences in our reference database.
the numerous protease inhibitors expressed in ma glands likely reflect the importance of maintaining protein integrity in the storage compartment of the ma glands.
widow spiders regularly spin dragline silk and need a ready supply of functional spidroins to incorporate into fibers.
these upregulated gene family members may be specialized for maintaining spidroins.
one of the genes predicted to encode a protein with transferase activity is homologous to a phosphoserine aminotransferase, which is part of the cascade for serine biosynthesis as shown in escherichia coli <cit> .
another two transcripts encode gamma-glutamyltransferases also known as gamma-glutamyltranspeptidases , which in mammals, are primarily utilized in the glutathione pathway  <cit> .
this pathway is implicated in protection against oxidative stress and redox regulation of gene expression  <cit> .
because spider ma silk processing involves a series of ph changes in the gland  <cit> , oxidation regulation is likely to be crucial and could be mediated by γ-gt-like proteins.
CONCLUSIONS
through cdna tag profiling, we describe previously unrecognized gene expression complexity in ma glands of the western black widow.
as expected, masp <dig> and masp <dig> were among the most highly expressed genes in ma glands.
surprisingly, the most abundant transcript in ma glands was one with unknown function.
also unexpected, was the discovery of high levels of tusp <dig> expression and an additional six genes that were more abundantly represented in ma glands than masp <dig> .
in addition, we demonstrated the simultaneous, but unequal expression of three masp <dig> loci.
alternative polyadenlyation of masp <dig> and alternative splicing of other genes upregulated in ma glands may also increase the complexity of individual variation of ma proteins.
we propose that modulating the composition of masp <dig> variants, masp <dig>  and possibly tusp <dig> within silk glands will alter the material properties of dragline silk.
hence, if the ratios of these spidroins change as a consequence of ontogeny, physiology, or the environment, this can contribute to variation in the properties of silks spun by the same spider  or different  spiders.
we identified  <dig> unique gene sequences represented by tags that were significantly more abundant in ma glands than cephalothoraxes.
besides spidroin genes, we found  <dig> new candidate genes for dragline silk synthesis.
approximately 1/ <dig> of these genes have no known homolog outside of latrodectus.
it is possible that these genes resulted from black widow specific evolutionary events.
however, due to the paucity of genomic resources for spiders we cannot exclude the possibility that these genes have homologs in other spiders.
identifying homologs expressed in silk glands of other spider species would strengthen the argument that these genes are involved in silk synthesis.
of the genes predicted to encode proteins with functional homologs some may simply reflect the high rates of translation and protein storage in silk glands such as translation factors, amino transferases, and protease inhibitors.
others are likely transcription factors important for regulating gland-specific spidroin expression.
gamma-glutamyl-transferases may be important regulators of ph changes in the ma gland that are necessary for fiber assembly.
an overriding theme for many of the genes overrepresented in ma glands is that they are members of larger gene families.
neofunctionalization of gene copies expressed in silk glands may be even more important for silk synthesis than the previously recognized diversity of the spidroin gene family.
as such, silk glands represent a model system for understanding the evolution of tissue specific functions.
furthermore, our increased understanding of gene regulation in spider silk glands will ultimately lead to improved recombinant silk production.

 spiders use silk throughout their lives for functions such as prey capture, sperm webs, egg cases, safety draglines, and dispersal. orb-web weavers and their relatives spin six or more task-specific silk fibers. each of these task-specific fibers is synthesized in its own set of specialized abdominal glands. for example, dragline silk is synthesized in the major ampullate glands and egg-case silk is synthesized in the tubuliform glands. each type of silk has a unique suite of impressive mechanical properties. the dragline silk of orb-weavers approaches the tensile strength of steel and is more extensible than rubber or tendon collagen. this combination of strength and extensibility makes dragline silk incredibly tough. tubuliform silk, by contrast, is a stiff fiber that can store a large amount of energy. there is interest in capitalizing on the remarkable variation and combination of material properties of spider silks for medical and industrial applications such as tendon replacements, sutures, and as an environmentally friendly alternative to synthetic fibers. spider silks are composed primarily of large structural proteins termed spidroins that are encoded by members of a single gene family. silk types differ from each other in their constituent spidroins. for instance, ma glands express masp and masp and the outer egg case silk synthesized in tubuliform glands includes tusp. other spidroin paralogs have been identified from each of the gland types except for the aggregate gland. spidroins are large proteins made up almost entirely of repeated blocks of amino acids, flanked by non-repetitive amino and carboxy terminal domains. these terminal domains are conserved in length and sequence among spidroin types and across species. the majority of spider silk genetic research has focused on silk synthesized in the ma glands because of the high tensile strength of dragline fibers. these efforts include characterizing masp and masp sequences from multiple species and creating recombinant proteins expressed in transgenic organisms. however, the mechanical properties of fibers spun from recombinant spider silk proteins do not yet accurately mimic natural spider silk fibers, suggesting more extensive work is needed to understand the genetics and spinning of spider silks. synthesis, assembly, and material properties of dragline silk likely rely on the relative expression of spidroin paralogs as well as the influence of non-spidroin genes. for instance, the relative proportion of masp and masp in dragline silk varies among species and even among individuals of the same species, which could potentially explain inter-and intra-specific level differences in mechanical properties. the extensibility of dragline silk correlates positively with its proline content, an amino acid that is present in higher proportion in masp than in masp. the western black widow, latrodectus hesperus, synthesizes draglines with a low proline content and thus masp is predicted to be approximately three times more abundant than masp in the fiber. the extensibility of black widow dragline silk is correspondingly lower than other species that have draglines primarily composed of masp. additionally, masp is encoded by multiple loci in l. hesperus and the golden orb-weaver nephila clavipes. masp loci are similar to each other, but not identical, within a species. variation in the expression patterns of each locus could contribute to variation in mechanical properties of dragline silks within or among species, but such variation in expression has yet to be documented. non-spidroin genes can also play important roles in the synthesis and assembly of spider silks, although only a few cases have been described. for instance, the egg case proteins, ecp- and ecp- are expressed primarily in the tubuliform glands and are predicted to form cross-links with tusp. exploring the genetics of spider silk synthesis has been limited to a gene-by-gene approach because of the minimal genomic information available for spiders. genomic methods have only recently become feasible for non-model organisms, such as spiders, with the advent of next generation sequencing technologies. next generation sequencing and de novo assembly were used to characterize “uni-genes” expressed in the silk glands of two spider species, a tarantula and an orb-weaver. this approach identified several major functional classes of genes expressed in silk glands, and discovered new spidroin paralogs, but did not measure relative expression rates. these results represent a major advance in characterizing spider genes, but due to the lack of comparisons with other tissues, it is unclear which genes or functional classes are important for silk synthesis. here, we use massively parallel signature sequencing to profile expression patterns in ma glands and cephalothoraxes of the western black widow. we targeted this species for several reasons. black widows synthesize dragline silks that are among the strongest measured. as descendants of orb-web weaving ancestors, black widows possess all the gland types of true orb-web spiders despite building three-dimensional cobwebs. six of the seven known spidroin paralogs have been described for the western black widow. furthermore, because the western black widow has a relatively small genome for a spider, we have arrayed a genomic library and completely sequenced the dragline silk genes masp and masp and the prey-wrapping gene acsp. our aims were to 1) determine relative levels of transcript abundance within ma glands compared to cephalothorax control tissue and 2) identify novel candidate genes of importance to dragline silk production. our findings demonstrate that dragline silk synthesis involves a much greater transcriptional complexity than previously known, laying the foundation for further studies of silk gland functional genomics and recombinant silk production.  generation of “tags” and construction of reference database we generated more than million tags, which are 3′-anchored base cdna fragments, from mrna transcripts found in the ma glands and cephalothoraxes of adult female western black widows using mpss. mpss allows for highly quantitative comparison between tissues, even in organisms with poorly characterized genomes, by anchoring sequencing to the 3′ end of transcripts. different transcripts are discriminated by their 3′-most tags and expression levels estimated with higher accuracy than could be achieved by random rna-seq. sequencing more than million tags from each of four libraries resulted in unique tag sequences. of these, unique tags were found at levels greater than one count per million total tags in two or more libraries. a acpm = counts per million. to determine the gene identity of tags, we compiled a non-redundant set of “reference” protein-coding sequences for the western black widow from newly sequenced cdnas, published cdna sequences, and published gene sequences. the only sequences available that included untranscribed regions were for the complete gene sequences of masp and masp for initial expression profiling, we thus limited representatives of masp and masp to the longest cdna for each. the mpss method we used targeted the 3′-most occurrence of the dpnii recognition sequence, 5′-gatc-3′. we determined tag identity by first predicting all possible tag sequences from the coding or sense strand and the complementary or antisense strand in the reference genes. predicted tags were then matched to the sequenced tags from our four libraries. of the sequences in our reference database, contained a predicted tag and were represented by at least one observed tag. identification of differentially expressed transcripts in western black widows an exact test implemented in edger identified approximately 23% of tags were significantly more abundant in one tissue type: unique tags were significantly more abundant in the ma glands and tags were more abundant in the cephalothoraxes. each of these differentially expressed tags was at least times more abundant in one tissue than the other. we assigned unique tags to genes in our reference database. in terms of abundance and fold change, all tag sequences that matched a reference gene as well as tags that matched the 3′-most position appear to be an unbiased subset of all observed tags. abundance levels of these tags were also highly correlated between individual samples. approximately 46% of the reference genes represented by a sequenced tag were represented by both sense and antisense tags, 45% were represented by only a sense tag, and 9% were represented by only an antisense tag. most reference genes were represented by one sense tag and/or one anti-sense tag. we observed instances of multiple tags aligning to a single gene, but the 3′-most sense tag was almost always considerably more abundant than other sense tags, as expected. genes represented by multiple sense tags were usually represented by multiple antisense tags, but antisense tags did not show a consistent relationship between position on the gene and tag abundance. alternative tags could result from incomplete digestion with dpnii, or from degradation of mrnas. however, in many cases multiple tags represent genuine alternative transcripts. an example of these alternative transcripts is described for masp below. because the origin of multiple tags per gene is not always clear, we estimated transcript abundance two ways. first, under the assumption that all tags that matched a gene represent transcription of some kind, we summed the average cpm of all tags from the sense strand, or separately, from the antisense strand. second, to account for the possibility that multiple tags represent alternative transcripts, we estimated transcript abundance based solely on the 3′-most position of the sense strand, or its matched position on the antisense strand. these two estimates are tightly correlated. because we selected for polyadenylated mrna, we did not expect to capture antisense transcripts. total abundance of a transcript estimated from summing all tags that matched that gene sequence is correlated between sense and antisense strands, with total abundance of sense tags almost always higher than total abundance of antisense tags. for instance, expression levels based on sense tags varied from to cpm in ma glands while antisense counts varied from to cpm. despite lower abundance of antisense tags, differences in gene expression levels between tissues estimated by sense and antisense tags are tightly correlated. thus, even if our antisense tags represent an experimental error associated with the reverse transcription phase of cdna synthesis, they provide consistent information regarding differential expression. it is also possible that antisense transcription is widespread in spiders. in metazoan genomes investigated thus far, 5-22% of genes are represented by both sense and antisense transcripts. in mice and humans, more than 40% of the transcript pool could result from antisense transcription. furthermore, expression patterns of sense and antisense transcripts generated from the same genomic locus tend to be correlated, similar to the pattern seen here. variable expression of spidroin paralogs in ma glands our database contained unique sequences encoding masp including one complete protein-coding sequence for locus and several thousand base pairs of non-coding flanking sequence. it additionally included representatives of two other loci and partial length cdnas. from these sequences, including non-coding regions, we predicted sense tags and antisense tags. full-length masp has predicted sense tags and predicted antisense tags that matched an observed tag. importantly, all of the tags predicted in the coding region of the full-length gene were observed as well as two downstream of the coding region. as expected, there were no observed tags that corresponded to intergenic genomic dna. in addition to the eight unique sense tags that matched the full-length masp gene, there were five other unique sense tags that aligned to alternative masp sequences. alignment of all masp sequences indicated that the three loci and the cdnas share an identical observed sense tag in the c-terminal encoding region. this tag is the 3′-most predicted one in the cdnas and was the most abundant observed masp tag and the second most abundant observed tag in ma glands overall. the seven sense tags found throughout the repetitive encoding region of the full-length masp were much less abundant, consistent with selecting for the 3′-most occurrence of the dpnii recognition sequence. although the 3′-most tag from the coding sequence cannot discriminate expression levels of each masp locus, there are other indices that are diagnostic for particular loci. prior to this study, only locus was supported by expression data. here, we observed unique tags for all three loci. locus has an observed unique tag bases downstream of the stop codon. loci and share an observed tag bases downstream of the stop codon that is not found in locus. yet another observed unique tag occurs in locus bases downstream of the stop codon. a different tag was predicted in the same location in locus but was not observed. however, locus has a unique observed tag in the n-terminal coding region. thus, we have evidence for the co-expression all three loci at the same time in the same tissue. the observed tags found downstream of the masp stop codon also suggest that both loci and and possibly are alternatively polyadenylated. consistent with this hypothesis is the occurrence of a polyadenylation signal downstream of an observed tag in each of these loci. both 3′ utr length, and specific sequence elements contained therein, have been shown to be important for a variety of regulatory events including control of mrna stability and translational efficiency. our results thus raise the possibility that differential polyadenylation of masp transcripts may be involved in locus-specific stability or translational control. although the 3′-most coding region tag of masp is identical among all three loci, the sequences of downstream tags are unique and can therefore be used to compare locus-specific expression levels. analysis of these tags reveals that the isoform of locus carrying an elongated 3′ utr is ~ times more abundant than the alternatively polyadenylated forms of loci and combined. in the absence of strong, locus-specific alternative 3′ end regulation, this finding suggests that locus is dominant in the ma gland tissue of adult females. our data are therefore consistent with the previously suggested hypothesis that masp locus variants could be incorporated in differing proportions into draglines. because masp encoded by each locus differs in proportion and ordering of ensemble repeats, varying the proportion of each could affect silk properties. importantly, prior to this study there was no direct evidence for simultaneous expression from all three loci. our database also contained seven sequences encoding masp including a complete gene with several thousand base pairs of non-coding flanking sequence. from these masp sequences, tag locations were predicted from the sense strand representing unique tag sequences. in the full-length gene, tag locations were observed. four of these observations were well outside the coding region and appeared at low frequencies in all tissues, leaving seven unique observed sense tags within the masp coding region. alignment of the complete masp gene with other masp sequences including partial cdnas showed that all sequences shared a 3′-most predicted sense tag. this tag was the most abundant in masp sequences. all other observed masp tags were much less abundant. there was only one additional unique sense tag observed from other masp sequences. this tag is in the n-terminal encoding region of a partial masp cdna. a tag that only differed by one base pair from this tag was predicted from the corresponding coding region in the full-length gene, but was not observed. the ratio of masp1:masp transcript abundance based on the 3′-most coding tags varied between the two individuals, but the ratio based on the average cpm in ma glands is 3: masp1:masp consistent with the amino acid composition of ma fibers collected from western black widows. thus, despite the potential for complex locus-specific translational control, transcript abundance may accurately reflect overall protein abundance of masp and masp variation in proportion of masp and masp could explain variation in fiber properties among individual black widow spiders. in three other species, varying the amount of protein in the spider’s diet significantly alters mechanical properties of ma fibers. specifically, extensibility of ma fibers, a property highly correlated with the proline content of the fiber, decreased with a low protein or protein-less diet. since proline is in higher proportion in masp than in masp, an increased extensibility in ma fibers is likely due to an increase in the proportion of masp in the fiber. individual spiders could have widely varying diets that overtime could change the proportion of spidroins in the silk fibers and thus create fibers with varying mechanical properties. tusp which encodes the primary component of outer egg-cases, was unexpectedly expressed in ma glands. our non-redundant database contained two sequences predicted to encode tusp but both shared an identical observed tag in the c-terminal encoding region. tusp was surprisingly abundant, ranking 10th most represented gene of the ones that are more abundant in ma glands than cephalothoraxes, directly following masp. however, one individual transcribed much more tusp than the other. the difference in expression levels between the two individuals could be explained by individual differences in readiness to make egg-cases. the number of transcripts encoding tusp and other egg-case proteins increase in tubuliform silk glands as the individual nears egg-case production. the individual with lower levels of tusp in ma glands did not have well-developed eggs. however, the individual with high levels of tusp had large, mature eggs, suggesting that tusp transcription increases in ma glands at the same time as increasing in tubuliform glands. it is possible tusp becomes incorporated in ma fibers at the time of egg-case production, which could contribute to variation in mechanical properties. alternatively, translational control of tusp prevents protein synthesis in ma glands despite the presence of tusp transcripts. we also cannot exclude the possibility that our detection of tusp in ma glands is due to leakage from mrnas transcribed in the tubuliform glands. novel candidate genes involved in silk synthesis in addition to spidroin encoding genes, we identified other genes represented by tags that were significantly more abundant in ma glands than cephalothoraxes. when translated, of these genes aligned to functionally annotated proteins or conserved domains. of the remaining ten genes, one did not match any entries in the ncbi nr database and nine aligned only to hypothetical proteins previously predicted for western black widows. all but one of these ten encode proteins with signal peptides predicted by signalp, suggesting they are secreted. secreted proteins could be components of ma fibers or involved with fiber assembly. an unanticipated finding was that the 3′-most sense transcript tag of one of the hypothetical protein-encoding genes is three times more abundant in ma glands than that of masp. this gene, “contig5”, has a short open reading frame and signalp predicts an even smaller mature peptide. the first bases, which includes the first aa, of “contig5” is nearly identical to that of another gene overrepresented in ma glands, “lnc168”, as well as one that was not differentially expressed between ma and cephalothorax. each of these sequences differs in the last few amino acids and most of the putative 3′ utrs, including the tag locations. “lnc168” is much less abundant in ma glands than “contig5”. these sequences may represent transcripts from paralogous genes, alternatively spliced genes, or a combination of both scenarios. alignment revealed two regions of almost exact identity among all three sequences, and another region of exact identity between two, interspersed with unalignable regions. these patterns are consistent with both hypotheses of paralogous genes and alternatively spliced genes. alternative splicing is an important mechanism for generating functionally distinct proteins with tissue specific expression as well as unique 3′ utrs. isoforms caused by 3′ utr alternative splicing can modify the production of the protein through altering locations for microrna binding and activating decay pathways such as the nonsense-mediated decay pathway. among the most highly expressed genes in ma glands that could be annotated were ones predicted to encode fasciclin, translation elongation factor 1-alpha, and lectin. each of these was more abundant than masp. abundance ranking of genes expressed in ma glands was almost identical if estimates were based solely on the 3′-most tags. tags corresponding to elongation factor 1-alpha were only slightly more abundant in ma glands than cephalothoraxes. in fact, the 3′-most sense tag of this gene was not significantly different between the two tissues. thus, the higher abundance in ma glands probably reflects the very high rates of translation to produce dragline spidroins, masp and masp in contrast, tags representing fasciclin and lectin-encoding genes were consistently highly abundant in ma glands but either absent or extremely low in cephalothoraxes. in various insect species, fasciclins are cell adhesion glycoproteins that play a role in neuronal development. lectins are sugar-binding proteins involved in the innate immune response in drosophila. however, it is not immediately clear how these particular functions might be involved with silk synthesis. it is possible that these genes are expressed in specialized cells within the ma glands that are not secreting silk proteins. the ma glands are divided into multiple regions including a tail and ampullate region where spidroins are expressed, the lower ampullate region where spidroins are stored, and the duct where spidroins begin assembly for ultimate extrusion as fibers. there may well be cells within any of these regions that have currently unknown functions not found in the cephalothorax. thirteen of the genes overrepresented in ma glands that were annotated by sequence similarity are predicted to encode proteins that can be grouped into three functional classes. these include protease inhibitors, proteins with transferase activity, and transcription factors. the protease inhibitors appear to be derived from two large gene families. the putative serine protease inhibitor genes encode aa domains that align to each other with 47% pairwise identity. they also align to four additional translated genes in our reference database that were not overrepresented in ma glands. similarly, four of the genes overrepresented in ma glands encode thyroglobulin type- conserved domains, which function as cysteine protease inhibitors. these four genes align with each other with 85-89% pairwise nucleotide identity over their entirety. at the amino acid level, they align to an additional four sequences in our reference database. the numerous protease inhibitors expressed in ma glands likely reflect the importance of maintaining protein integrity in the storage compartment of the ma glands. widow spiders regularly spin dragline silk and need a ready supply of functional spidroins to incorporate into fibers. these upregulated gene family members may be specialized for maintaining spidroins. one of the genes predicted to encode a protein with transferase activity is homologous to a phosphoserine aminotransferase, which is part of the cascade for serine biosynthesis as shown in escherichia coli. another two transcripts encode gamma-glutamyltransferases also known as gamma-glutamyltranspeptidases, which in mammals, are primarily utilized in the glutathione pathway. this pathway is implicated in protection against oxidative stress and redox regulation of gene expression. because spider ma silk processing involves a series of ph changes in the gland, oxidation regulation is likely to be crucial and could be mediated by γ-gt-like proteins.  through cdna tag profiling, we describe previously unrecognized gene expression complexity in ma glands of the western black widow. as expected, masp and masp were among the most highly expressed genes in ma glands. surprisingly, the most abundant transcript in ma glands was one with unknown function. also unexpected, was the discovery of high levels of tusp expression and an additional six genes that were more abundantly represented in ma glands than masp. in addition, we demonstrated the simultaneous, but unequal expression of three masp loci. alternative polyadenlyation of masp and alternative splicing of other genes upregulated in ma glands may also increase the complexity of individual variation of ma proteins. we propose that modulating the composition of masp variants, masp and possibly tusp within silk glands will alter the material properties of dragline silk. hence, if the ratios of these spidroins change as a consequence of ontogeny, physiology, or the environment, this can contribute to variation in the properties of silks spun by the same spider or different spiders. we identified unique gene sequences represented by tags that were significantly more abundant in ma glands than cephalothoraxes. besides spidroin genes, we found new candidate genes for dragline silk synthesis. approximately 1/ of these genes have no known homolog outside of latrodectus. it is possible that these genes resulted from black widow specific evolutionary events. however, due to the paucity of genomic resources for spiders we cannot exclude the possibility that these genes have homologs in other spiders. identifying homologs expressed in silk glands of other spider species would strengthen the argument that these genes are involved in silk synthesis. of the genes predicted to encode proteins with functional homologs some may simply reflect the high rates of translation and protein storage in silk glands such as translation factors, amino transferases, and protease inhibitors. others are likely transcription factors important for regulating gland-specific spidroin expression. gamma-glutamyl-transferases may be important regulators of ph changes in the ma gland that are necessary for fiber assembly. an overriding theme for many of the genes overrepresented in ma glands is that they are members of larger gene families. neofunctionalization of gene copies expressed in silk glands may be even more important for silk synthesis than the previously recognized diversity of the spidroin gene family. as such, silk glands represent a model system for understanding the evolution of tissue specific functions. furthermore, our increased understanding of gene regulation in spider silk glands will ultimately lead to improved recombinant silk production. 
we also identified transcription of tusp in ma glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. our results demonstrate previously unrecognized transcript complexity in spider silk glands. we sequenced millions of 3′-anchored “tags” of cdnas derived either from ma glands or control tissue mrnas, then associated the tags with genes by compiling a reference database from our newly constructed normalized l. hesperus cdna library and published l. hesperus sequences. in addition to the spidroin paralogs, we identified genes that are more abundantly represented in ma glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis. modulating expression rates of masp variants as well as masp and tusp could lead to differences in mechanical properties of dragline fibers. here, we used deep sequencing to profile gene expression patterns in the western black widow, latrodectus hesperus. orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized. we were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding masp the ratio of masp1:masp transcripts varied between individuals, but on average was similar to the estimated ratio of masp1:masp in dragline fibers. many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly.
 orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. draglines are primarily composed of masp and masp two spidroins expressed in the major ampullate silk glands. prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized.  here, we used deep sequencing to profile gene expression patterns in the western black widow, latrodectus hesperus. we sequenced millions of 3′-anchored “tags” of cdnas derived either from ma glands or control tissue mrnas, then associated the tags with genes by compiling a reference database from our newly constructed normalized l. hesperus cdna library and published l. hesperus sequences. we were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding masp the ratio of masp1:masp transcripts varied between individuals, but on average was similar to the estimated ratio of masp1:masp in dragline fibers. we also identified transcription of tusp in ma glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. in addition to the spidroin paralogs, we identified genes that are more abundantly represented in ma glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis.  modulating expression rates of masp variants as well as masp and tusp could lead to differences in mechanical properties of dragline fibers. many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly. our results demonstrate previously unrecognized transcript complexity in spider silk glands. spidroinmajor ampullate glandsalternative polyadenylationmasp1spidertag profiling 