Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus)

Abstract

BackgroundOrb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. Draglines are primarily composed of MaSp1 and MaSp2, two spidroins (spider fibrous proteins) expressed in the major ampullate (MA) 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.ResultsHere, 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 (cephalothorax) 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 MaSp1. The ratio of MaSp1:MaSp2 transcripts varied between individuals, but on average was similar to the estimated ratio of MaSp1:MaSp2 in dragline fibers. We also identified transcription of TuSp1 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 30 genes that are more abundantly represented in MA glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis.ConclusionsModulating expression rates of MaSp1 variants as well as MaSp2 and TuSp1 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.

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

“…Every silk gland that we profiled was found to express broad combinations of spidroin transcripts representing multiple classes. Together with analogous non-gland-specific expression patterns seen for individual spidroins in other spider species 27,53 , these data argue for complex, gland-specific models of spidroin expression and silk production. Future studies that measure the levels of spidroin proteins in silk glands would be useful to validate the correspondence of transcript abundance to translational output and will provide an additional dimension to understanding of silk production.…”

“…Previous experimental findings have suggested that spidroin expression is not exclusively gland specific [50][51][52][53][54] . Our RNA sequencing studies also suggested broader patterns of spidroin transcript expression across silk glands ( Supplementary Fig.…”

“…To identify non-spidroin candidate genes potentially involved in silk production, we cataloged transcripts that were (i) highly expressed and/or (ii) uniquely expressed in N. clavipes silk glands, resulting in a list of 649 candidates from our RNA sequencing data ( Supplementary Fig. 12 and Supplementary Table 15); 183 of these genes exhibited homology to documented silk gland-specific transcripts [50][51][52][53][54] . The candidates included catalytic enzymes such as kinases, proteases, dehydrogenases, acetyltransferases, and synthases, many of which are active in eukaryotic secretory systems 57 .…”

“…The candidates included catalytic enzymes such as kinases, proteases, dehydrogenases, acetyltransferases, and synthases, many of which are active in eukaryotic secretory systems 57 . We expect this catalog to include genes encoding proteins involved in the conversion of liquid silk dope to solid silk thread 52,53 , such as enzymes that maintain the pH gradient along the gland body to spigots on the spinnerets 50,54 . Candidates that might generate ions for the pH gradient include three carbonic anhydrase orthologs (Ca10, Ca13, and Ca14), four thyroid peroxidase (Tpo) paralogs, and five chorion peroxidase (Pxt) paralogs (Supplementary Table 15).…”

“…The normalized expression data set of 14,025 'gold' genes was filtered to identify putative SST loci that could be categorized as (i) 'HighInSilk' , with >1,000 absolute normalized mapped RNA reads in ≥1 silk gland and <200 reads in non-silk tissues; (ii) 'ExclusiveToSilk' , with >100 absolute normalized mapped RNA reads in ≥1 silk gland and zero reads in non-silk tissues; (iii) 'GlandEnriched' , with >400 absolute normalized mapped reads in only a single silk gland and <350 in all non-silk tissues; and (iv) 'Literature' , corresponding to the BLASTP 84 homologs (e value: ≤ 1 × 10 −6 ) of 282 unique SSTs from studies of spider silk gland transcriptomes and proteomes [50][51][52][53][54] plus peroxidase or anhydrase gene family members hypothesized to be involved in silk production 50,54 ( Supplementary Fig. 12 and Supplementary Table 15).…”