BACKGROUND
adhesion related to biological processes is often important to maintain proper function, and this is especially true of sessile marine organisms where substrate adhesion is critical for survival.
a prominent example is the barnacle, which first relies on temporary adhesion on a suitable substrate as a cyprid and then transitions to permanent attachment as the animal undergoes metamorphosis to a sessile juvenile and matures to an adult with either a calcareous or membranous base attached to the underlying surface  <cit> .
barnacles are arthropods in the crustacea subphylum and are partly characterized by their growth via molt cycles.
molting in adult acorn barnacles is evidenced by shedding of the exuvia as well as the formation of radial ecdysal lines underneath the animal.
much effort has been dedicated to understanding the intertwined processes of growth and adhesion as well as the composition and the molecular mechanisms that lead to a strongly adhered organism.
these efforts are complicated by the presence of a rigid outer calcareous shell, under which cement components are delivered and deposited by the barnacle under the leading edge of the barnacle/substrate interface and sheltered from the external marine environment.
as with growth, evidence suggests the process of adhesion occurs in a cyclic, multi-step manner synchronized with the molt cycle  <cit> .
the means by which cement precursor materials are delivered to the leading edge of the barnacle/substrate interface are not completely understood, yet a network of capillaries originating from cement glands and terminating at ducts at the substrate interface are thought to play a prominent role .
barnacle cement itself is composed predominantly of protein , though other materials, such as lipids, polysaccharides and biomineralization components, have also been found at the barnacle-substrate interface  <cit> .
several putative cement proteins have been identified and studied, mainly through solubilization and analysis of cement plaques that remained on substrates following removal of the barnacle shell and body .
these include cp100k, cp68k, cp52k, cp20k, cp19k, where the number corresponds with the protein molecular weight in daltons.
detailed structural information about each protein remains unknown, though amino acid composition and conserved and/or repeat subunits have shed light on potential function  <cit> .
additionally, advances in gene sequencing technology have aided in the development of cdna libraries as well as the discovery of genes that encode cement protein homologues in different barnacle species .
this knowledge continues to fill in the picture of how barnacles are able to create a robust adhesive interface in a relatively harsh marine environment.
with this backdrop, we set out to test the hypothesis that cement proteins are actively regulated at different stages of the molt cycle and in different tissues of adult barnacles.
molting of several adult amphibalanus amphitrite  barnacles was tracked based on shedding of the exuvia under controlled laboratory conditions.
protein and mrna were collected from the sub-mantle tissue  of barnacles in pre- and post-molt stages.
material was also collected from the main body of adult barnacles for comparison.
using rna-seq technology, the expression levels of cement protein genes in the sub-mantle tissue of pre- and post-molt samples were found to be the same, suggesting these proteins are not regulated at the transcript level when synchronized to the molt cycle.
comparison of cement protein gene expression in the sub-mantle tissue versus the main barnacle body revealed significantly elevated levels in sub-mantle tissue.
these results fit with the current understanding of barnacle anatomy, where cement pre-cursor material is thought to be produced and stored in cement glands located in the sub-mantle tissue.
we also report the discovery of a novel 114kd protein, named aacp114k, and found its transcript to be highly expressed in the sub-mantle tissue and the protein identified in barnacle cement secretions collected at the barnacle/substrate interface.
this protein shares significant homology to a previously identified 100kd cement protein in a. amphitrite as well as others identified in the barnacle species megabalanus rosa and teraclita japonica formosana  <cit> .
in the absence of a complete genome, the information collected from the comparison of pre- and post-molt samples allowed for a more general analysis of differential gene expression in these two stages.
a number of genes were identified as being up-regulated in the sub-mantle tissue predominantly in pre-molt barnacle samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.
collectively, these results add a novel and potentially prominent protein to the mix of cement-related components and further our understanding of the complex processes at play as barnacles grow via cyclic molting and extend their adhesive area to substrates.
RESULTS
sample collection and molt stage confirmation
to test our hypothesis that cement protein genes are differentially expressed during the molt cycle of barnacles and in separate regions of the body, stable laboratory conditions were established for barnacles.
adult a. amphitrite individually reattached on glass slides were maintained and monitored in an incubator providing control over several variables including temperature, humidity, salinity, and light/dark cycles to minimize stressful environmental variables.
barnacles exhibiting stable, regular molting were selected in either a pre-molt or post-molt stage and the cirri and sub-mantle tissue were collected for further analysis.
a barnacle schematic showing the cirri, main body, and sub-mantle tissue is shown in fig.
<dig> fig.
<dig> simplified schematic of adult acorn barnacle cross section.
the three sections used in this study are shown.
cirri were used to confirm the molt stage of each sample, while the main body and sub-mantle tissue were collected for protein and mrna analysis.
note the sub-mantle tissue contains the cement glands
each barnacle used in this study had the cirri removed for microscopic determination of the molt cycle based on the analysis of davis et al.
<cit> .
they reported significant temporal variability of the molt stages, yet the most common stages observed in the current study were the pre-molt, proecdysis  and the post- and interecdysis stages .
postecdysis stage a is a very short period of ~4 h immediately following a molt and was not observed in this study.
the most distinguishing feature of the cirri was the state of the setae, the finger-like projections used to capture food during cirral pumping.
as shown in fig.
2a-b, pre-molt samples  were readily identified by the formation of new setae within the existing ones and invaginations into the main tissue of the cirri.
post-molt samples were noted by the absence of these features and the setal matrix in an expanded state .
using these identifiable features, the molt stage of each barnacle sample was categorized as being either pre- or post-molt.fig.
<dig> rami from adult barnacle a. amphitrite highlighting the setae in pre- and post-molt stages.
a-b) images of several setae from barnacles collected in pre-molt  stage.
black arrows indicate the presence of new setae development prior to molting, and white arrows indicate invaginations of the newly developing setae.
c-d) images of setae from barnacles collected in post-molt stages as indicated by the absence of new setae or invaginations and an expanded setal matrix
rna-seq transcriptome analysis
to understand barnacle cement protein gene expression levels in pre- and post-molt growth stages, rna-seq technology was used to compare transcriptomic profiles of pre- and post-molt barnacles, specifically examining the sub-mantle tissue containing cement gland tissue.
we first provide a general summary of these results.
complementary dna  libraries were constructed from three independent samples of sub-mantle tissue collected in pre-molt or post-molt stages.
two additional cdna libraries were made from two pooled main body samples as pilot experiments for a total of eight separate samples.
paired-end sequencing technology  was performed on each of the eight cdna libraries.
a total of  <dig> , <dig> trimmed high-quality reads giving approximately  <dig>  gbp total sequences were generated with an average trimmed read length of 90 bp.
de novo assembly of the a. amphitrite transcriptome was performed from combined reads from all eight samples using the trinity program  <cit> .
the summary of transcriptome sequencing and assembly is shown in table  <dig>  a total of  <dig>  mbp transcriptome sequences were assembled into  <dig>  overlapping sequence data reads  with an average length of 697 bp.
a summary of the contig length distribution is presented in additional file  <dig>  the transcriptome was then annotated with the program trinotate  <cit> .
from these data,  <dig>  transcripts were predicted.
only  <dig>  transcripts  have annotated functions that were identified using the swissprot protein database and  <dig> are assigned with gene ontology  terms in  <dig> functional groups.
abundance profiles of functional groups  are very similar to those previously reported in a. amphitrite and t. j. formosana  <cit> .table  <dig> summary of a. amphitrite transcriptome
cement protein gene expression: pre-molt versus post-molt
there is strong evidence that cement proteins are required for permanent adhesion of barnacles to their substrate and are secreted by cement glands located in the sub-mantle tissue .
a number of studies have identified five genes encoding cement proteins  from several barnacles including m. rosa, a. amphitrite and t. j. formosana and these studies also demonstrated most were abundantly expressed in the main body of the barnacle  <cit> .
in this study, all of these cement protein transcripts were identified within the sub-mantle tissue .
additionally, a novel paralogue of cp100k, named aacp114k , was also identified.
this protein shares 58 % homology with the amino acid sequence of the previously identified cp100k in a. amphitrite .
see additional file  <dig> for sequence comparison and phylogeny analysis compared to other 100 k homologues in a. amphitrite, m. rosa and t. j. formosana.
to confirm the accuracy of transcriptome assembly of the aacp114k gene, the full length cdna  of this gene was amplified from the sub-mantle rna with rt-pcr, cloned into the pet22b vector and sequenced.
all but six nucleotides from the cloned fragment were identical to the assembled sequence , indicating that aacp114k gene is unique and was indeed present and expressed in the sub-mantle of a. amphitrite.
sequence discrepancy between the illumina sequencing and sanger sequencing indicate heterogeneity within the samples collected for this study.table  <dig> comparison of cement proteins in a. amphitrite
the transcriptomic data show that cement protein gene expression levels in the sub-mantle tissue did not have significant differences between pre- and post-molt stages, despite significant differences in the expression levels relative to one another .
these data suggest that expression of genes encoding cement proteins identified in the sub-mantle were independent of molting.
statistical analysis of a perceived difference in the pre- and post-molt transcript number of aacp20k- <dig> showed the difference between these two populations is not significant.
in terms of the general expression level of cement protein genes, aacp19k, aacp20k- <dig> and aacp114k were expressed significantly higher than the other three cement protein genes based on transcript counts.
it is noteworthy that the aacp20k- <dig> and aacp20- <dig> genes showed opposite expression patterns: aacp20k- <dig> exhibited negligible expression, whereas aacp20k- <dig> was highly expressed in the sub-mantle tissue.
unlike the report by lin et al.
that the cp52k gene was abundant in the basis of t. j. formosana  <cit> , the expression level of aacp52k in the sub-mantle tissue of a. amphitrite was relatively low .fig.
<dig> pre- and post-molt comparison of genes encoding cement proteins.
transcript numbers of cement protein genes in the pre- and post-molt conditions from rna-seq analysis.
gray and black bars represent pre- and post-molt gene expression, respectively.
values are expressed as mean ± sd from three experimental samples in each condition
cement protein gene expression: main body versus sub-mantle tissue
next, the expression level of cement protein genes was compared on the basis of tissue, examining main body versus the sub-mantle tissue of adult a. amphitrite samples.
mrna was isolated from the main body and the transcript levels of the cement protein genes measured with qrt-pcr using the cytochrome b gene  as an internal control.
in general, the gene expression levels of cement protein genes in the main body were very low as detected by rt-pcr.
this accentuates fig.
<dig>  which is a semi-log plot of the relative fold change in the gene expression level of cement proteins in the sub-mantle tissue compared to the main body.
of the cement protein genes examined, all except aacp20k- <dig> showed varying degrees of increased expression in sub-mantle tissue compared to the main body.
the gene for aacp20- <dig> was negligibly expressed in sub-mantle tissue and not expressed in the main body.
mass spectrometry was performed on sub-mantle tissue samples processed at the same time as the mrna collection with the intent to separate the proteinaceous content and target identification of cement proteins.
no cement proteins were identified using this technique, though an overwhelming amount of housekeeping and scaffold proteins identified could have masked their presence.fig.
<dig> cement protein gene expression comparison in main body and sub-mantle tissue.
relative fold change  in cement protein gene expression levels in sub-mantle tissue versus barnacle body.
data are based on qrt-pcr analysis and expressed as mean ± sd from two replicates
secretion analysis
in addition to examining cement protein gene expression levels, material secreted from barnacles at the substrate interface was analyzed using mass spectrometry.
there is not a standard method to collect barnacle secretions, so different approaches were employed to identify the presence of cement proteins and attempt to correlate their presence in the sub-mantle tissue and the barnacle/substrate interface.
see methods for detailed explanation of the coverslip, medallion, mortar, and bead collection techniques.
in all secretion samples, the only cement proteins detected with ms analysis were the higher molecular weight aacp100k and the newly identified aacp114k .
aacp114k was clearly present in the inverted coverslip, medallion, and microsphere samples.
two instances of aacp114k were identified in the mortar samples.
interestingly, the presence of aacp100k was confirmed in the medallion and microsphere samples, but not in either the coverslip or the mortar samples.table  <dig> number of unique peptides identified for cp100k
apeptide and protein identifications were accepted if they could be established at > 70 % and 90 % probability, respectively.
protein identifications also had to contain at least  <dig> identified peptides
extended analysis of differential gene expression: pre- and post-molt
one objective of this study was to examine the gene expression levels of cement proteins in the sub-mantle tissue and identify cement proteins from barnacle secretions.
however, the transcriptome data collected from the pre- and post-molt sub-mantle tissue samples allowed for a more general examination of differential gene expression.
the edger analysis package  <cit>  revealed that of all predicted transcripts discovered from the transcriptome,  <dig> were differentially expressed between pre- and post-molt stages  <  <dig> ).
a volcano plot representing highly expressed transcripts in the pre- and post-molt samples is shown in fig.
<dig>  among these,  <dig> were highly expressed in pre-molt samples and only  <dig> transcripts in post-molt samples.
these results indicate that barnacle molting is regulated by a limited number of genes with significantly more involved in preparing for and initiating molting.fig.
<dig> volcano plot of differential gene expression profile in pre-  and post-molt  conditions.
data points in red correspond to a false discovery rate  <  <dig>
to confirm the rna-seq result,  <dig> transcripts were randomly selected from the differentially expressed transcripts and subjected to real time rt-pcr analysis.
.
the expression changes between pre- and post-molt were indeed correlated to the rna-seq data.
annotated transcripts that were differentially expressed in pre- and post-molt samples are listed in table  <dig>  several of the highly expressed transcripts in the pre-molt samples were found to encode cuticular proteins, metalloproteases, c-type lectins, and antioxidant enzymes .
four of the transcripts encoding cuticle proteins and up-regulated in pre-molt samples were found to contain the rebers-riddiford  consensus motif   <cit> , either in its complete form or in the partial form missing the two c-terminal proline residues .table  <dig> differentially expressed transcripts in the pre- and post-molting conditions annotated from swisspro database
a
e value represents the transcript has the best hit in swiss-pro database; btranscript fold change  in log <dig> between pre-molt and post-molt samples; citems shaded in gray potentially linked to a pre-molt condition and most are discussed in further detail in main text; dgenes in bold indicate that their encoded proteins were identified in secretion analysis using mass spectrometry
of the  <dig> differentially expressed transcripts in the pre- and post-molt samples , proteins encoded by  <dig> of them  were identified in the secretion samples studied by proteomics, as highlighted in table  <dig>  additional file  <dig> provides detailed information about protein identifications from these samples.
interestingly, one of the encoding genes highly expressed in pre-molt samples, comp97185_c <dig>  shares 44 % sequence homology to the settlement inducing protein complex  of both a. amphitrite  <cit>  and m. rosa  <cit> .
peptides of this protein were recovered and identified in all four barnacle secretion collections.
discussion
in this study, we compare gene expression levels of pre- and post-molt barnacles while also examining the main body versus the sub-mantle tissue.
the first comparison was enabled by tracking the shed exuvia of several barnacles and collecting sub-mantle tissue at time intervals preceding or following molt.
the molt stage was confirmed by examining cirri anatomy via light microscopy and correlated with established stages of barnacle molting  <cit> .
the two comparisons presented in this study have resulted in a thorough analysis of principal genes differentially expressed in pre- and post-molt stages and also a more complete picture of the abundance and presence of putative barnacle cement proteins involved in adhesion to the underlying surface of an adult barnacle.
the assembled transcriptome size  in this study is more than four times greater than the number previously reported in a. amphitrite by chen et al.
<cit> .
in rna-seq analysis of differential gene expression in different conditions, it is very common that de novo assembling of eukaryotic transcriptome often reports fragmented transcripts of one gene or repeated contigs that only differ by a single-nucleotide polymorphism  or a base insertion or deletion .
such expanded transcriptome size is usually evidence of isoforms of the same gene and artifacts such as sequencing errors, repeats, and variations in coverage and genetics within diploid individuals and pooled samples  <cit> .
in our study, rna of the pre- and post-molt samples was from six individuals and rna of the whole bodies was collected from two pooled samples, each containing ten individual barnacles.
the transcriptome generated from those eight samples resulted in many paths in the graph construction during the assembly process, effectively fragmenting the assembly which ultimately produced  <dig>  assembled contigs.
the main contribution to the larger transcriptome size might be heterogeneities within input samples, which was indicated by following evidence:  when sequence reads from the pre- and post-molt conditions were assembled separately, the contig numbers were greatly reduced to  <dig>  and  <dig> , respectively, and the total transcriptome sizes were reduced to approximately  <dig> mbp .
further clustering these individual assemblies using a homology approach  resulted in an overall reduced assembly of approximately  <dig>  mbp in  <dig>  contig clusters .
clustering the individual assemblies results in ca.
60 k contig clusters in each condition, which might be closer to the expected gene number of the barnacle.
the fact that the contig sequences from the pre- and post-molting samples do not cluster to a greater degree at 95 % suggested that variation  played a greater role in the transcriptome assembly.
finding a six nucleotide difference between the assembled and the cloned aacp <dig> gene sequences  also indicated the heterogeneity phenomenon present within individuals.
further inspecting the reads mapping to the aacp114k gene, we observed many differences between the consensus sequences not shared across individual samples .
the illumina sequencing approach employed in the current study produced sequence coverage at ~114x, which is an order of magnitude higher than  <dig> x coverage using the  <dig> method reported by chen et al.
<cit> .
higher depth of sequencing would reveal more real alternative splicing transcripts and splicing variants, which contribute to greater numbers of assembled sequences and contigs.
on the other hand, higher coverage may also amplify issues with genomic contamination, partially spliced products, and other artifacts of the method.
in this study, we were only interested in genes changing within the conditions queried, negating many of these issues in the final differential analysis.
as such, reducing the putative transcript/gene  tally was not necessary.
here, only about 23 % of the ~ <dig>  predicted transcripts had annotated functions based on available database sequences, meaning the vast majority are unidentified.
this trend was consistent even among the differentially expressed proteins in the pre- and post-molt tissue samples, indicating the need for a much larger effort of creating a fully annotated barnacle genome.
the main purpose of targeting the sub-mantle tissue for temporal comparative analysis is that, anatomically, this region is where cement-related tissue of adult barnacles resides.
while it was straightforward to separate main body from sub-mantle tissue with little cross-contamination, collection of only cement gland tissue within the sub-mantle cavity proved more challenging.
ovarian tissue, identified by its distinct color and texture, was often intimately intertwined with the surrounding tissue, which included the cement glands  <cit> .
during sample collection, care was taken to remove the vast majority of ovarian tissue, though its complete removal was beyond the scope of this investigation given the limits of dissection.
therefore, potential contamination of a minimal amount of ovarian tissue in the sub-mantle samples is a possibility.
despite this, the aim was to collect as much of the non-ovarian sub-mantle tissue as possible in order to have an optimal sampling of material thought to contain cement precursor materials.
judging by the gene expression data comparison between sub-mantle tissues and the main body, the sub-mantle tissues clearly demonstrates elevated expression of cement proteins and resulted in the identification of a new cement-related protein.
in regard to temporal expression of cement protein genes, there have been previous claims of differential cement protein gene expression as a function of barnacle molting  <cit> , however, these are unsubstantiated and cite unpublished work.
in the current study, several barnacle samples with regular molt cycles were chosen in either a pre- or post-molt state accompanied by collection of cirri for molt cycle confirmation based on established criteria  <cit> .
for the detected cement protein genes , there was no significant difference in the pre- and post-molt gene expression levels despite that fact that several other genes were found to be differentially expressed .
it may be possible to increase the resolution of molt-dependent gene expression using qrt-pcr according to eight stages described by davis et al.
<cit> .
however, differential gene expression was intentionally limited to pre-molt and post-molt due to the considerable number of other parameters that, collectively, are difficult to control and monitor.
these include barnacle size, age, and season, time variability within each molt stage and transcript heterogeneity.
despite these variables, two phenotypes were observed in the present study and were consistent among themselves.
the presence of cement proteins aacp20- <dig> and − <dig> has been reported previously and were shown to localize and express in different types of secretory cells in the cyprid cement gland  <cit> .
in adult barnacles, he et al.
reported that protein aacp20k- <dig> was present in soft tissue, whereas protein aacp20k- <dig> was dominant in the basal shell of adult barnacles, which agrees with our gene expression results .
interestingly, aacp20k- <dig> is the only cement protein with a low pi of ~ <dig>  and its gene expression was detected at very low levels in the sub-mantle tissue and not at all in the body.
it is possible that transcripts of aacp20k- <dig> are not stable and were degraded, making it difficult to assess if these results are coincident.
nevertheless, the questions of how and why this transcript is regulated are of interest.
we report high levels of gene expression of a novel paralogue cp100k protein termed aacp114k.
irrespective of the pre- or post-molt stage, this newly identified protein had the highest gene expression compared to all other putative cement proteins located in the sub-mantle tissue based on transcript numbers .
these results and the relative change in expression level in the sub-mantle tissue versus body  suggest that this protein is localized in sub-mantle tissue.
proteomic analysis confirms this protein is found in material secreted by the barnacle to a greater extent than the aacp100k paralogue, as observed in coverslip, medallion, microsphere and mortar samples .
based upon our gene expression and mass spectrometry results, we suggest the aacp114k paralogue identified in the current study is a prominent protein component of barnacle cement in a. amphitrite.
interestingly, phylogenetic analysis of the various cp <dig> homologues show a closer relation between the cp <dig> homologue in a. amphitrite first identified by chen et al.
<cit>  and in t. j. formosana  <cit>  than between the two paralogues found in a. amphitrite .
a previous report on m. rosa claimed cp52k, cp68k, and cp100k as being the major protein components of barnacle cement  <cit> .
however, we were not able to identify a cp68k-related gene from the transcriptome in a. amphitrite mainly because the cp68k sequence is unavailable in public domains  despite references to it having been identified and its sequence  <cit> .
using n-terminal sequences of eight cyanogen bromide  peptides determined from the insolubilized cement fraction of m. rosa  <cit>  as queries, we found that only five of those peptides were homologous to either aacp52k or aacp100k in our transcriptome database.
therefore, more studies are required to verify whether cp68k exists as single protein in a. amphitrite and, if so, whether the expression level is differentiated throughout molting.
in comparing the main body to the sub-mantle tissue using rt-pcr, cement protein genes were predominantly expressed in sub-mantle tissue, which is the expected location of barnacle cement precursor materials.
proteomic analysis of barnacle secretions collected using different techniques indicate that aacp114k and aacp100k are secreted by the animal at the barnacle/substrate interface.
of course, the inability to detect other putative cement proteins from the secreted materials does not necessarily mean they are not present.
two factors could contribute to this finding.
first, the amount of secreted material is expected to be relatively low since the collection methods used in this study are targeted at newly secreted material over a limited time period with a maximum of 2 weeks and minimum of  <dig> day.
.
in addition, ms analysis may lie outside the limits of detection or may indicate the proteins have been cross-linked prior to enzymatic digestion, escaping detection.
combined, these factors are likely to contribute to the limited detection of cement proteins in the various barnacle secretion collections.
nevertheless, the newly described variations to the collection methods of cement material over relatively short time frames using four different approaches  allowed for proteomic comparison with sub-mantle tissue and offer a fresh means of analyzing recently secreted material and newly formed barnacle cement.
we expand upon the insights gained from cement protein  detection and analysis to the more general findings of differential gene expression in the pre- and post-molt samples of the sub-mantle tissue.
most highly expressed genes were found in pre-molt samples and, of those identified, generally encoded proteins in four categories: cuticular proteins, metalloproteases, c-type lectins, and antioxidants.
these categories can be associated with molting, biomineralization, morphogenesis, and cell adhesion.
here, we provide a more in-depth discussion of some of the highly expressed protein genes identified in the sub-mantle tissue of pre-molt barnacle samples.
the increased expression of cuticular protein genes was not unexpected since molting requires significant production of cuticular material and tissue scaffolding to replace the soon-to-be-shed exuvia.
several cuticle-related protein genes in the pre-molt samples were identified and four were found to have the chitin-binding rebers-riddiford consensus motif  <cit> .
multiple transcripts of rr-containing cuticle proteins were reported to be temporally and spatially expressed in the decapod crustacean callinectes sapidus and involved in biomineralization  <cit> .
more than  <dig> homologs of this type of cuticle protein gene were identified from the transcriptome data in this study, indicating a variety of functions at different growth stages or conditions.
rr-containing cuticle protein genes highly expressed in the pre-molt sub-mantle tissue of barnacles strongly suggest an involvement with chitin and possibly a role in biominerialization, both of which have a role in barnacle growth  <cit> .
two genes encoding c-type lectins, bra- <dig> and nattectin, were also found to be highly expressed in the pre-molt condition.
c-type lectins usually bind carbohydrate structures in a ca2+-dependent manner and are well-recognized immune response initiator proteins  <cit> .
they have been found to participate in mineralization  <cit>  and inhibit the growth of calcium carbonate crystals  <cit> .
high expression of these two genes in the pre-molt condition suggests that they have significant roles in biomineralization during growth/molting and coordinating post-molt responses.
coincident differential expression of genes encoding peroxiredoxin- <dig> and thioredoxin domain-containing protein  <dig> in pre-molt samples suggests that the barnacle experiences oxidative stress during or following molting, since peroxiredoxin uses thioredoxin to recharge after reducing hydrogen peroxide  so as to restore its catalytic activity  <cit> .
antioxidant proteins like peroxiredoxin are often coupled with others  to limit damage of oxidative stress to host tissue  <cit> .
increased expression of genes encoding proteases  in the pre-molt stage points toward proteolytic activation of proteins, including contributors to barnacle cement, having an essential role for effective cement cross-linking and co-adhesion within the interface.
zinc metalloproteinases have a significant role in inflammation as enzymes that rework extracellular matrices and remodeling of structural material  <cit> .
in addition, it has previously been suggested that barnacle cement cross-linking involves proteolytic activation of structural proteins in order to maximize the potential for bonding interactions with other proteins and with the surface  <cit> .
a glycoprotein, settlement inducing protein complex  has been reported as the species-specific cue to settlement of barnacles  <cit>  and expressed in the cuticle of adults  <cit> .
it has been reported as a complex of three proteins  but retains some biological activity as separate subunits  <cit> .
in the transcriptomic data, we found sipc that is 100 % identical to the previously reported one  <cit>  and its gene expressed at similar levels in both pre- and post-molt conditions.
interestingly, we also found a homologous sipc gene, comp97185_c <dig> , highly expressed in the pre-molt condition and its encoded peptides were identified in all secretion samples analyzed with ms. this finding suggests that the barnacle may secrete a new and different type or combination of sipc molecule with alternative  ligands as a molting signal, and its function is worth further investigation.
CONCLUSIONS
this work advances the understanding of the complex process of barnacle molting/growth by defining the temporal and tissue distribution of various proteins, including those reported to be major components of barnacle cement.
our analysis suggests there is not differential expression of cement protein genes in pre- and post-molt barnacles and most appear to be highly expressed in the sub-mantle tissue compared to the main body.
in addition, we identify a novel cement protein homologue, aacp114k, with high gene expression levels in the sub-mantle tissue and confirmed its presence in secretions at the barnacle/substrate interface.
in the absence of a fully annotated genome, our transcriptomic analysis reveals highly expressed genes identified in pre-molt barnacle samples and paints a picture of extensive cuticular development, biominerialization, and proteolytic activity that may be associated with the precursor elements of barnacle cement.
our results also highlight the fact that the majority of differentially expressed genes in the pre- and post-molt stage are unidentified and potentially unique to barnacles.
the description of several new cement collection techniques opens opportunity to build a profile of the macromolecules present and active at the barnacle/substrate interface with the goal of elucidating the mechanism of the cementing process.
future directions include developing approaches to collect and profile pre-secreted material as well as establishing protocols to identify the location of various cement components prior to secretion.
we note that in situ hybridization studies have been carried out in a. amphitrite cyprids  <cit>  and offer great potential for identifying the tissue  specific expression of particular genes relevant to the current study.
given the increased complexity of the adult barnacle anatomy and the fact that all tissue is surrounded by an opaque calcified shell, there are challenges with in situ hybridization that are beyond the scope of the current work yet are a promising future direction.
ultimately, the research goals are to identify the various precursor components of barnacle cement, understand the mechanism of delivery, and how the components interact at the substrate interface.
a complete understanding of this process can lead to targeted strategies to mitigate barnacle adhesion as well as mimic its functionality in underwater environments.

 adhesion related to biological processes is often important to maintain proper function, and this is especially true of sessile marine organisms where substrate adhesion is critical for survival. a prominent example is the barnacle, which first relies on temporary adhesion on a suitable substrate as a cyprid and then transitions to permanent attachment as the animal undergoes metamorphosis to a sessile juvenile and matures to an adult with either a calcareous or membranous base attached to the underlying surface. barnacles are arthropods in the crustacea subphylum and are partly characterized by their growth via molt cycles. molting in adult acorn barnacles is evidenced by shedding of the exuvia as well as the formation of radial ecdysal lines underneath the animal. much effort has been dedicated to understanding the intertwined processes of growth and adhesion as well as the composition and the molecular mechanisms that lead to a strongly adhered organism. these efforts are complicated by the presence of a rigid outer calcareous shell, under which cement components are delivered and deposited by the barnacle under the leading edge of the barnacle/substrate interface and sheltered from the external marine environment. as with growth, evidence suggests the process of adhesion occurs in a cyclic, multi-step manner synchronized with the molt cycle. the means by which cement precursor materials are delivered to the leading edge of the barnacle/substrate interface are not completely understood, yet a network of capillaries originating from cement glands and terminating at ducts at the substrate interface are thought to play a prominent role. barnacle cement itself is composed predominantly of protein, though other materials, such as lipids, polysaccharides and biomineralization components, have also been found at the barnacle-substrate interface. several putative cement proteins have been identified and studied, mainly through solubilization and analysis of cement plaques that remained on substrates following removal of the barnacle shell and body. these include cp100k, cp68k, cp52k, cp20k, cp19k, where the number corresponds with the protein molecular weight in daltons. detailed structural information about each protein remains unknown, though amino acid composition and conserved and/or repeat subunits have shed light on potential function. additionally, advances in gene sequencing technology have aided in the development of cdna libraries as well as the discovery of genes that encode cement protein homologues in different barnacle species. this knowledge continues to fill in the picture of how barnacles are able to create a robust adhesive interface in a relatively harsh marine environment. with this backdrop, we set out to test the hypothesis that cement proteins are actively regulated at different stages of the molt cycle and in different tissues of adult barnacles. molting of several adult amphibalanus amphitrite barnacles was tracked based on shedding of the exuvia under controlled laboratory conditions. protein and mrna were collected from the sub-mantle tissue of barnacles in pre- and post-molt stages. material was also collected from the main body of adult barnacles for comparison. using rna-seq technology, the expression levels of cement protein genes in the sub-mantle tissue of pre- and post-molt samples were found to be the same, suggesting these proteins are not regulated at the transcript level when synchronized to the molt cycle. comparison of cement protein gene expression in the sub-mantle tissue versus the main barnacle body revealed significantly elevated levels in sub-mantle tissue. these results fit with the current understanding of barnacle anatomy, where cement pre-cursor material is thought to be produced and stored in cement glands located in the sub-mantle tissue. we also report the discovery of a novel 114kd protein, named aacp114k, and found its transcript to be highly expressed in the sub-mantle tissue and the protein identified in barnacle cement secretions collected at the barnacle/substrate interface. this protein shares significant homology to a previously identified 100kd cement protein in a. amphitrite as well as others identified in the barnacle species megabalanus rosa and teraclita japonica formosana. in the absence of a complete genome, the information collected from the comparison of pre- and post-molt samples allowed for a more general analysis of differential gene expression in these two stages. a number of genes were identified as being up-regulated in the sub-mantle tissue predominantly in pre-molt barnacle samples with a range of functions including cuticular development, biominerialization, and proteolytic activity. collectively, these results add a novel and potentially prominent protein to the mix of cement-related components and further our understanding of the complex processes at play as barnacles grow via cyclic molting and extend their adhesive area to substrates.  sample collection and molt stage confirmation to test our hypothesis that cement protein genes are differentially expressed during the molt cycle of barnacles and in separate regions of the body, stable laboratory conditions were established for barnacles. adult a. amphitrite individually reattached on glass slides were maintained and monitored in an incubator providing control over several variables including temperature, humidity, salinity, and light/dark cycles to minimize stressful environmental variables. barnacles exhibiting stable, regular molting were selected in either a pre-molt or post-molt stage and the cirri and sub-mantle tissue were collected for further analysis. a barnacle schematic showing the cirri, main body, and sub-mantle tissue is shown in fig.  fig. simplified schematic of adult acorn barnacle cross section. the three sections used in this study are shown. cirri were used to confirm the molt stage of each sample, while the main body and sub-mantle tissue were collected for protein and mrna analysis. note the sub-mantle tissue contains the cement glands each barnacle used in this study had the cirri removed for microscopic determination of the molt cycle based on the analysis of davis et al.. they reported significant temporal variability of the molt stages, yet the most common stages observed in the current study were the pre-molt, proecdysis and the post- and interecdysis stages. postecdysis stage a is a very short period of ~4 h immediately following a molt and was not observed in this study. the most distinguishing feature of the cirri was the state of the setae, the finger-like projections used to capture food during cirral pumping. as shown in fig. 2a-b, pre-molt samples were readily identified by the formation of new setae within the existing ones and invaginations into the main tissue of the cirri. post-molt samples were noted by the absence of these features and the setal matrix in an expanded state. using these identifiable features, the molt stage of each barnacle sample was categorized as being either pre- or post-molt.fig. rami from adult barnacle a. amphitrite highlighting the setae in pre- and post-molt stages. a-b) images of several setae from barnacles collected in pre-molt stage. black arrows indicate the presence of new setae development prior to molting, and white arrows indicate invaginations of the newly developing setae. c-d) images of setae from barnacles collected in post-molt stages as indicated by the absence of new setae or invaginations and an expanded setal matrix rna-seq transcriptome analysis to understand barnacle cement protein gene expression levels in pre- and post-molt growth stages, rna-seq technology was used to compare transcriptomic profiles of pre- and post-molt barnacles, specifically examining the sub-mantle tissue containing cement gland tissue. we first provide a general summary of these results. complementary dna libraries were constructed from three independent samples of sub-mantle tissue collected in pre-molt or post-molt stages. two additional cdna libraries were made from two pooled main body samples as pilot experiments for a total of eight separate samples. paired-end sequencing technology was performed on each of the eight cdna libraries. a total of, trimmed high-quality reads giving approximately gbp total sequences were generated with an average trimmed read length of 90 bp. de novo assembly of the a. amphitrite transcriptome was performed from combined reads from all eight samples using the trinity program. the summary of transcriptome sequencing and assembly is shown in table  a total of mbp transcriptome sequences were assembled into overlapping sequence data reads with an average length of 697 bp. a summary of the contig length distribution is presented in additional file the transcriptome was then annotated with the program trinotate. from these data, transcripts were predicted. only transcripts have annotated functions that were identified using the swissprot protein database and are assigned with gene ontology terms in functional groups. abundance profiles of functional groups are very similar to those previously reported in a. amphitrite and t. j. formosana.table summary of a. amphitrite transcriptome cement protein gene expression: pre-molt versus post-molt there is strong evidence that cement proteins are required for permanent adhesion of barnacles to their substrate and are secreted by cement glands located in the sub-mantle tissue. a number of studies have identified five genes encoding cement proteins from several barnacles including m. rosa, a. amphitrite and t. j. formosana and these studies also demonstrated most were abundantly expressed in the main body of the barnacle. in this study, all of these cement protein transcripts were identified within the sub-mantle tissue. additionally, a novel paralogue of cp100k, named aacp114k, was also identified. this protein shares 58 % homology with the amino acid sequence of the previously identified cp100k in a. amphitrite. see additional file for sequence comparison and phylogeny analysis compared to other 100 k homologues in a. amphitrite, m. rosa and t. j. formosana. to confirm the accuracy of transcriptome assembly of the aacp114k gene, the full length cdna of this gene was amplified from the sub-mantle rna with rt-pcr, cloned into the pet22b vector and sequenced. all but six nucleotides from the cloned fragment were identical to the assembled sequence, indicating that aacp114k gene is unique and was indeed present and expressed in the sub-mantle of a. amphitrite. sequence discrepancy between the illumina sequencing and sanger sequencing indicate heterogeneity within the samples collected for this study.table comparison of cement proteins in a. amphitrite the transcriptomic data show that cement protein gene expression levels in the sub-mantle tissue did not have significant differences between pre- and post-molt stages, despite significant differences in the expression levels relative to one another. these data suggest that expression of genes encoding cement proteins identified in the sub-mantle were independent of molting. statistical analysis of a perceived difference in the pre- and post-molt transcript number of aacp20k- showed the difference between these two populations is not significant. in terms of the general expression level of cement protein genes, aacp19k, aacp20k- and aacp114k were expressed significantly higher than the other three cement protein genes based on transcript counts. it is noteworthy that the aacp20k- and aacp20- genes showed opposite expression patterns: aacp20k- exhibited negligible expression, whereas aacp20k- was highly expressed in the sub-mantle tissue. unlike the report by lin et al. that the cp52k gene was abundant in the basis of t. j. formosana, the expression level of aacp52k in the sub-mantle tissue of a. amphitrite was relatively low.fig. pre- and post-molt comparison of genes encoding cement proteins. transcript numbers of cement protein genes in the pre- and post-molt conditions from rna-seq analysis. gray and black bars represent pre- and post-molt gene expression, respectively. values are expressed as mean ± sd from three experimental samples in each condition cement protein gene expression: main body versus sub-mantle tissue next, the expression level of cement protein genes was compared on the basis of tissue, examining main body versus the sub-mantle tissue of adult a. amphitrite samples. mrna was isolated from the main body and the transcript levels of the cement protein genes measured with qrt-pcr using the cytochrome b gene as an internal control. in general, the gene expression levels of cement protein genes in the main body were very low as detected by rt-pcr. this accentuates fig.  which is a semi-log plot of the relative fold change in the gene expression level of cement proteins in the sub-mantle tissue compared to the main body. of the cement protein genes examined, all except aacp20k- showed varying degrees of increased expression in sub-mantle tissue compared to the main body. the gene for aacp20- was negligibly expressed in sub-mantle tissue and not expressed in the main body. mass spectrometry was performed on sub-mantle tissue samples processed at the same time as the mrna collection with the intent to separate the proteinaceous content and target identification of cement proteins. no cement proteins were identified using this technique, though an overwhelming amount of housekeeping and scaffold proteins identified could have masked their presence.fig. cement protein gene expression comparison in main body and sub-mantle tissue. relative fold change in cement protein gene expression levels in sub-mantle tissue versus barnacle body. data are based on qrt-pcr analysis and expressed as mean ± sd from two replicates secretion analysis in addition to examining cement protein gene expression levels, material secreted from barnacles at the substrate interface was analyzed using mass spectrometry. there is not a standard method to collect barnacle secretions, so different approaches were employed to identify the presence of cement proteins and attempt to correlate their presence in the sub-mantle tissue and the barnacle/substrate interface. see methods for detailed explanation of the coverslip, medallion, mortar, and bead collection techniques. in all secretion samples, the only cement proteins detected with ms analysis were the higher molecular weight aacp100k and the newly identified aacp114k. aacp114k was clearly present in the inverted coverslip, medallion, and microsphere samples. two instances of aacp114k were identified in the mortar samples. interestingly, the presence of aacp100k was confirmed in the medallion and microsphere samples, but not in either the coverslip or the mortar samples.table number of unique peptides identified for cp100k apeptide and protein identifications were accepted if they could be established at > 70 % and 90 % probability, respectively. protein identifications also had to contain at least identified peptides extended analysis of differential gene expression: pre- and post-molt one objective of this study was to examine the gene expression levels of cement proteins in the sub-mantle tissue and identify cement proteins from barnacle secretions. however, the transcriptome data collected from the pre- and post-molt sub-mantle tissue samples allowed for a more general examination of differential gene expression. the edger analysis package revealed that of all predicted transcripts discovered from the transcriptome, were differentially expressed between pre- and post-molt stages  <  ). a volcano plot representing highly expressed transcripts in the pre- and post-molt samples is shown in fig.  among these, were highly expressed in pre-molt samples and only transcripts in post-molt samples. these results indicate that barnacle molting is regulated by a limited number of genes with significantly more involved in preparing for and initiating molting.fig. volcano plot of differential gene expression profile in pre- and post-molt conditions. data points in red correspond to a false discovery rate  <  to confirm the rna-seq result, transcripts were randomly selected from the differentially expressed transcripts and subjected to real time rt-pcr analysis.. the expression changes between pre- and post-molt were indeed correlated to the rna-seq data. annotated transcripts that were differentially expressed in pre- and post-molt samples are listed in table  several of the highly expressed transcripts in the pre-molt samples were found to encode cuticular proteins, metalloproteases, c-type lectins, and antioxidant enzymes. four of the transcripts encoding cuticle proteins and up-regulated in pre-molt samples were found to contain the rebers-riddiford consensus motif, either in its complete form or in the partial form missing the two c-terminal proline residues.table differentially expressed transcripts in the pre- and post-molting conditions annotated from swisspro database a e value represents the transcript has the best hit in swiss-pro database; btranscript fold change in log between pre-molt and post-molt samples; citems shaded in gray potentially linked to a pre-molt condition and most are discussed in further detail in main text; dgenes in bold indicate that their encoded proteins were identified in secretion analysis using mass spectrometry of the differentially expressed transcripts in the pre- and post-molt samples, proteins encoded by of them were identified in the secretion samples studied by proteomics, as highlighted in table  additional file provides detailed information about protein identifications from these samples. interestingly, one of the encoding genes highly expressed in pre-molt samples, comp97185_c shares 44 % sequence homology to the settlement inducing protein complex of both a. amphitrite and m. rosa. peptides of this protein were recovered and identified in all four barnacle secretion collections. discussion in this study, we compare gene expression levels of pre- and post-molt barnacles while also examining the main body versus the sub-mantle tissue. the first comparison was enabled by tracking the shed exuvia of several barnacles and collecting sub-mantle tissue at time intervals preceding or following molt. the molt stage was confirmed by examining cirri anatomy via light microscopy and correlated with established stages of barnacle molting. the two comparisons presented in this study have resulted in a thorough analysis of principal genes differentially expressed in pre- and post-molt stages and also a more complete picture of the abundance and presence of putative barnacle cement proteins involved in adhesion to the underlying surface of an adult barnacle. the assembled transcriptome size in this study is more than four times greater than the number previously reported in a. amphitrite by chen et al.. in rna-seq analysis of differential gene expression in different conditions, it is very common that de novo assembling of eukaryotic transcriptome often reports fragmented transcripts of one gene or repeated contigs that only differ by a single-nucleotide polymorphism or a base insertion or deletion. such expanded transcriptome size is usually evidence of isoforms of the same gene and artifacts such as sequencing errors, repeats, and variations in coverage and genetics within diploid individuals and pooled samples. in our study, rna of the pre- and post-molt samples was from six individuals and rna of the whole bodies was collected from two pooled samples, each containing ten individual barnacles. the transcriptome generated from those eight samples resulted in many paths in the graph construction during the assembly process, effectively fragmenting the assembly which ultimately produced assembled contigs. the main contribution to the larger transcriptome size might be heterogeneities within input samples, which was indicated by following evidence: when sequence reads from the pre- and post-molt conditions were assembled separately, the contig numbers were greatly reduced to and, respectively, and the total transcriptome sizes were reduced to approximately mbp. further clustering these individual assemblies using a homology approach resulted in an overall reduced assembly of approximately mbp in contig clusters. clustering the individual assemblies results in ca. 60 k contig clusters in each condition, which might be closer to the expected gene number of the barnacle. the fact that the contig sequences from the pre- and post-molting samples do not cluster to a greater degree at 95 % suggested that variation played a greater role in the transcriptome assembly. finding a six nucleotide difference between the assembled and the cloned aacp gene sequences also indicated the heterogeneity phenomenon present within individuals. further inspecting the reads mapping to the aacp114k gene, we observed many differences between the consensus sequences not shared across individual samples. the illumina sequencing approach employed in the current study produced sequence coverage at ~114x, which is an order of magnitude higher than x coverage using the method reported by chen et al.. higher depth of sequencing would reveal more real alternative splicing transcripts and splicing variants, which contribute to greater numbers of assembled sequences and contigs. on the other hand, higher coverage may also amplify issues with genomic contamination, partially spliced products, and other artifacts of the method. in this study, we were only interested in genes changing within the conditions queried, negating many of these issues in the final differential analysis. as such, reducing the putative transcript/gene tally was not necessary. here, only about 23 % of the ~ predicted transcripts had annotated functions based on available database sequences, meaning the vast majority are unidentified. this trend was consistent even among the differentially expressed proteins in the pre- and post-molt tissue samples, indicating the need for a much larger effort of creating a fully annotated barnacle genome. the main purpose of targeting the sub-mantle tissue for temporal comparative analysis is that, anatomically, this region is where cement-related tissue of adult barnacles resides. while it was straightforward to separate main body from sub-mantle tissue with little cross-contamination, collection of only cement gland tissue within the sub-mantle cavity proved more challenging. ovarian tissue, identified by its distinct color and texture, was often intimately intertwined with the surrounding tissue, which included the cement glands. during sample collection, care was taken to remove the vast majority of ovarian tissue, though its complete removal was beyond the scope of this investigation given the limits of dissection. therefore, potential contamination of a minimal amount of ovarian tissue in the sub-mantle samples is a possibility. despite this, the aim was to collect as much of the non-ovarian sub-mantle tissue as possible in order to have an optimal sampling of material thought to contain cement precursor materials. judging by the gene expression data comparison between sub-mantle tissues and the main body, the sub-mantle tissues clearly demonstrates elevated expression of cement proteins and resulted in the identification of a new cement-related protein. in regard to temporal expression of cement protein genes, there have been previous claims of differential cement protein gene expression as a function of barnacle molting, however, these are unsubstantiated and cite unpublished work. in the current study, several barnacle samples with regular molt cycles were chosen in either a pre- or post-molt state accompanied by collection of cirri for molt cycle confirmation based on established criteria. for the detected cement protein genes, there was no significant difference in the pre- and post-molt gene expression levels despite that fact that several other genes were found to be differentially expressed. it may be possible to increase the resolution of molt-dependent gene expression using qrt-pcr according to eight stages described by davis et al.. however, differential gene expression was intentionally limited to pre-molt and post-molt due to the considerable number of other parameters that, collectively, are difficult to control and monitor. these include barnacle size, age, and season, time variability within each molt stage and transcript heterogeneity. despite these variables, two phenotypes were observed in the present study and were consistent among themselves. the presence of cement proteins aacp20- and − has been reported previously and were shown to localize and express in different types of secretory cells in the cyprid cement gland. in adult barnacles, he et al. reported that protein aacp20k- was present in soft tissue, whereas protein aacp20k- was dominant in the basal shell of adult barnacles, which agrees with our gene expression results. interestingly, aacp20k- is the only cement protein with a low pi of ~ and its gene expression was detected at very low levels in the sub-mantle tissue and not at all in the body. it is possible that transcripts of aacp20k- are not stable and were degraded, making it difficult to assess if these results are coincident. nevertheless, the questions of how and why this transcript is regulated are of interest. we report high levels of gene expression of a novel paralogue cp100k protein termed aacp114k. irrespective of the pre- or post-molt stage, this newly identified protein had the highest gene expression compared to all other putative cement proteins located in the sub-mantle tissue based on transcript numbers. these results and the relative change in expression level in the sub-mantle tissue versus body suggest that this protein is localized in sub-mantle tissue. proteomic analysis confirms this protein is found in material secreted by the barnacle to a greater extent than the aacp100k paralogue, as observed in coverslip, medallion, microsphere and mortar samples. based upon our gene expression and mass spectrometry results, we suggest the aacp114k paralogue identified in the current study is a prominent protein component of barnacle cement in a. amphitrite. interestingly, phylogenetic analysis of the various cp homologues show a closer relation between the cp homologue in a. amphitrite first identified by chen et al. and in t. j. formosana than between the two paralogues found in a. amphitrite. a previous report on m. rosa claimed cp52k, cp68k, and cp100k as being the major protein components of barnacle cement. however, we were not able to identify a cp68k-related gene from the transcriptome in a. amphitrite mainly because the cp68k sequence is unavailable in public domains despite references to it having been identified and its sequence. using n-terminal sequences of eight cyanogen bromide peptides determined from the insolubilized cement fraction of m. rosa as queries, we found that only five of those peptides were homologous to either aacp52k or aacp100k in our transcriptome database. therefore, more studies are required to verify whether cp68k exists as single protein in a. amphitrite and, if so, whether the expression level is differentiated throughout molting. in comparing the main body to the sub-mantle tissue using rt-pcr, cement protein genes were predominantly expressed in sub-mantle tissue, which is the expected location of barnacle cement precursor materials. proteomic analysis of barnacle secretions collected using different techniques indicate that aacp114k and aacp100k are secreted by the animal at the barnacle/substrate interface. of course, the inability to detect other putative cement proteins from the secreted materials does not necessarily mean they are not present. two factors could contribute to this finding. first, the amount of secreted material is expected to be relatively low since the collection methods used in this study are targeted at newly secreted material over a limited time period with a maximum of 2 weeks and minimum of day.. in addition, ms analysis may lie outside the limits of detection or may indicate the proteins have been cross-linked prior to enzymatic digestion, escaping detection. combined, these factors are likely to contribute to the limited detection of cement proteins in the various barnacle secretion collections. nevertheless, the newly described variations to the collection methods of cement material over relatively short time frames using four different approaches allowed for proteomic comparison with sub-mantle tissue and offer a fresh means of analyzing recently secreted material and newly formed barnacle cement. we expand upon the insights gained from cement protein detection and analysis to the more general findings of differential gene expression in the pre- and post-molt samples of the sub-mantle tissue. most highly expressed genes were found in pre-molt samples and, of those identified, generally encoded proteins in four categories: cuticular proteins, metalloproteases, c-type lectins, and antioxidants. these categories can be associated with molting, biomineralization, morphogenesis, and cell adhesion. here, we provide a more in-depth discussion of some of the highly expressed protein genes identified in the sub-mantle tissue of pre-molt barnacle samples. the increased expression of cuticular protein genes was not unexpected since molting requires significant production of cuticular material and tissue scaffolding to replace the soon-to-be-shed exuvia. several cuticle-related protein genes in the pre-molt samples were identified and four were found to have the chitin-binding rebers-riddiford consensus motif. multiple transcripts of rr-containing cuticle proteins were reported to be temporally and spatially expressed in the decapod crustacean callinectes sapidus and involved in biomineralization. more than homologs of this type of cuticle protein gene were identified from the transcriptome data in this study, indicating a variety of functions at different growth stages or conditions. rr-containing cuticle protein genes highly expressed in the pre-molt sub-mantle tissue of barnacles strongly suggest an involvement with chitin and possibly a role in biominerialization, both of which have a role in barnacle growth. two genes encoding c-type lectins, bra- and nattectin, were also found to be highly expressed in the pre-molt condition. c-type lectins usually bind carbohydrate structures in a ca2+-dependent manner and are well-recognized immune response initiator proteins. they have been found to participate in mineralization and inhibit the growth of calcium carbonate crystals. high expression of these two genes in the pre-molt condition suggests that they have significant roles in biomineralization during growth/molting and coordinating post-molt responses. coincident differential expression of genes encoding peroxiredoxin- and thioredoxin domain-containing protein in pre-molt samples suggests that the barnacle experiences oxidative stress during or following molting, since peroxiredoxin uses thioredoxin to recharge after reducing hydrogen peroxide so as to restore its catalytic activity. antioxidant proteins like peroxiredoxin are often coupled with others to limit damage of oxidative stress to host tissue. increased expression of genes encoding proteases in the pre-molt stage points toward proteolytic activation of proteins, including contributors to barnacle cement, having an essential role for effective cement cross-linking and co-adhesion within the interface. zinc metalloproteinases have a significant role in inflammation as enzymes that rework extracellular matrices and remodeling of structural material. in addition, it has previously been suggested that barnacle cement cross-linking involves proteolytic activation of structural proteins in order to maximize the potential for bonding interactions with other proteins and with the surface. a glycoprotein, settlement inducing protein complex has been reported as the species-specific cue to settlement of barnacles and expressed in the cuticle of adults. it has been reported as a complex of three proteins but retains some biological activity as separate subunits. in the transcriptomic data, we found sipc that is 100 % identical to the previously reported one and its gene expressed at similar levels in both pre- and post-molt conditions. interestingly, we also found a homologous sipc gene, comp97185_c, highly expressed in the pre-molt condition and its encoded peptides were identified in all secretion samples analyzed with ms. this finding suggests that the barnacle may secrete a new and different type or combination of sipc molecule with alternative ligands as a molting signal, and its function is worth further investigation.  this work advances the understanding of the complex process of barnacle molting/growth by defining the temporal and tissue distribution of various proteins, including those reported to be major components of barnacle cement. our analysis suggests there is not differential expression of cement protein genes in pre- and post-molt barnacles and most appear to be highly expressed in the sub-mantle tissue compared to the main body. in addition, we identify a novel cement protein homologue, aacp114k, with high gene expression levels in the sub-mantle tissue and confirmed its presence in secretions at the barnacle/substrate interface. in the absence of a fully annotated genome, our transcriptomic analysis reveals highly expressed genes identified in pre-molt barnacle samples and paints a picture of extensive cuticular development, biominerialization, and proteolytic activity that may be associated with the precursor elements of barnacle cement. our results also highlight the fact that the majority of differentially expressed genes in the pre- and post-molt stage are unidentified and potentially unique to barnacles. the description of several new cement collection techniques opens opportunity to build a profile of the macromolecules present and active at the barnacle/substrate interface with the goal of elucidating the mechanism of the cementing process. future directions include developing approaches to collect and profile pre-secreted material as well as establishing protocols to identify the location of various cement components prior to secretion. we note that in situ hybridization studies have been carried out in a. amphitrite cyprids and offer great potential for identifying the tissue specific expression of particular genes relevant to the current study. given the increased complexity of the adult barnacle anatomy and the fact that all tissue is surrounded by an opaque calcified shell, there are challenges with in situ hybridization that are beyond the scope of the current work yet are a promising future direction. ultimately, the research goals are to identify the various precursor components of barnacle cement, understand the mechanism of delivery, and how the components interact at the substrate interface. a complete understanding of this process can lead to targeted strategies to mitigate barnacle adhesion as well as mimic its functionality in underwater environments. 
the main body and sub-mantle tissue of the barnacle amphibalanus amphitrite were collected in pre- and post-molt stages. while no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue compared to the main barnacle body. we report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. our results reveal a novel and potentially prominent protein to the mix of cement-related components in a. amphitrite. rna-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry was used to analyze the protein content of barnacle secretions. despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. we report the discovery of a novel 114kd cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity. the expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. a complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate. cement proteinmolt cycletranscriptomeproteomebarnacleamphibalanus amphitriteissue-copyright-statement© the author 2015 little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion.
 a complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion. methods the main body and sub-mantle tissue of the barnacle amphibalanus amphitrite were collected in pre- and post-molt stages. rna-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry was used to analyze the protein content of barnacle secretions.  we report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. while no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue compared to the main barnacle body. we report the discovery of a novel 114kd cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.  the expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. our results reveal a novel and potentially prominent protein to the mix of cement-related components in a. amphitrite. despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cement proteinmolt cycletranscriptomeproteomebarnacleamphibalanus amphitriteissue-copyright-statement© the author 2015 