BACKGROUND
barnacles are marine dominant fouling organisms accumulate on ship hulls, piers and under water structures; biofouling by these invertebrates results in an increase in power and fuel consumption  associated with marine transport.
removing biofouling barnacles is a major expense involving significant economic losses in shipping and other marine industries.
however, some barnacles are of great commercial importance; for example, the gooseneck barnacle achieves an average market price of 30– <dig> usd per kilogram, depending on the season  <cit> .
such species are highly exploited, involving harvesting of approximately 500 kg per season in spain and portugal, and hence there is an urgent need for strategies to protect the stocks.
the barnacle balanus amphitrite is a widely distributed dominant fouling organism and a major component of rocky shore benthic communities .
therefore, b. amphitrite is an excellent model organism for research on settlement biology, adaptation and biofouling.
nauplii are the earliest free-swimming stage in the development of most crustaceans, including barnacles  <cit> , and are extremely abundant in the marine ecosystems.
barnacle embryos are brooded by the parents, and the nauplii are released into the water column as swimming larvae  <cit> .
the nauplius i stage has an elongated and pear-shaped body, and the abdominal and caudal spines are partially differentiated.
the eye is located in a front medial position and occurs in all developmental stages.
the presence of posteriorly folded frontal horns and simple setae on the limbs are characteristics of the nauplius i stage.
the newly released larvae swim actively and molt to the next developmental stage  within 30 min  <cit> .
in the nauplius ii stage the carapace extends in all directions and the entire body develops a bell shape that has numerous small spines and a pair of prominent spines.
the nauplius ii molts to the nauplius iii stage in 38 h  <cit> .
the nauplius iii stage undergoes several moults before it transforms into a cyprid stage  <cit> .
molecular studies defining the role of gene or protein expression signatures in development and adaptive strategies of marine larvae in the natural marine environment are limited.
previous studies have used a broad range of proteomics and genomic tools to identify proteins and genes in larval stages of barnacles.
however, these studies have mainly focused on exploring settlement and attachment processes.
for example, chen et al.
identified  <dig> putative differentially-expressed genes in larvae and adults of b. amphitrite using  <dig> pyrosequencing  <cit> , and yan et al.
carried out in silico transcriptome data mining to study neuropeptides and their possible functional role in barnacle larval settlement  <cit> .
similarly, de gregoris et al.
generated deep sequencing est libraries for nauplii and the cyprid and adult stages of b. amphitrite and identified unique contigs for each developmental stage  <cit> .
more recently, lin et al.
performed illumina sequencing of a membranous-based barnacle and identified cement- and adhesion-related genes  <cit> .
thiyagarajan and qian  reported the first study of global proteome expression patterns in cyprids and metamorphosed larvae of b. amphitrite  <cit> , and zhang et al.
identified proteins from cyprid larvae using prefractionation methods to reduce sample complexity  <cit> .
chandramouli et al.
used multiplex proteomics to describe a distinct protein glycosylation pattern during barnacle larval metamorphosis  <cit> , and chen et al.
recently used label-free quantitative proteomics to identify  <dig> proteins from nauplii ii and vi, cyprids, and juvenile stages of b. amphitrite  <cit> .
in addition, several studies have demonstrated the feasibility of using proteomics tools to investigate the mode of action of antifoulant chemicals in preventing barnacle larval settlement .
these studies have provided a wealth of molecular information, but comprehensive genome and proteome datasets for nauplius larvae are still lacking.
thus, in the present study we revealed sequence information and investigated the dynamics of protein expression patterns of nauplius larvae.
patterns of differential protein expression between two larval populations from contrasting marine environments were screened to investigate the survival strategies of larvae.
extreme marine environments pose great challenges for the survival of marine larvae.
for example, the red sea  is one of the warmest, most saline bodies of water; it receives little rainfall and has reduced oxygen solubility and high evaporation rates  <cit> .
the ranges of salinity  and temperature  in the rs are considered extreme.
in contrast, in hong kong  marine water the salinity and temperature ranges are 28–31psu and 26–28 °c, respectively  <cit> .
the rs has a low concentration of dissolved oxygen  compared with that of hk marine water .
therefore, we hypothesized that changes in protein expression patterns would reflect the adaptation of barnacle larvae to the rs marine environment.
to test this hypothesis we adopted a population proteomics approach to compare protein expression in larvae from hk marine waters and the rs.
the specific objectives were:  to generate a comprehensive transcriptome dataset for the nauplius larvae of b. amphitrite;  to compare the proteomes of barnacle larvae populations from the rs and hk; and  to explore survival strategies of larvae in the rs environment.
RESULTS
transcriptome of rs barnacle nauplii
figure  <dig> shows the experimental design used for the transcriptome and proteome analyses.
sequencing data statistics, assembly and annotation information for newly released nauplii of rs b. amphitrite are shown in table  <dig>  the transcriptome data were deposited in the ncbi bioproject id: prjna <dig> .
approximately  <dig> million paired-end reads were obtained.
following trimming of low quality reads  and adapters, the number of reads was reduced to  <dig> million, with an average read length of 87 bp.
assembly of the reads using abyss v <dig> .
<dig> and trans-abyss v <dig> .
<dig> yielded  <dig>  contigs with an average contig length  of 471 bp.
using getorf we predicted  <dig> , <dig> open reading frames , of which  <dig>  appeared in at least one blast hit.
blastp against a non-redundant  database resulted in  <dig>  contigs having at least one sequence homologous with other species, while no homolog was found for  <dig>  contigs.
an interproscan v <dig>  search resulted in  <dig>  contigs having at least one annotated pfam domain, while no domain was found for  <dig>  contigs.
in total,  <dig>  contigs showed at least one gene ontology  annotation while  <dig>  contigs remained hypothetical, and  <dig>  contigs showed at least one reactome annotation, whereas no annotation was found for  <dig>  contigs.
a total of  <dig> , <dig> were assigned to ‘biological process’ , and  <dig>  and  <dig>  contigs were assigned to ‘molecular function’  and ‘cellular component’ , respectively .
figure  <dig> shows the top  <dig> categories of assembled contigs belonging to the bp, mf and cc categories.
the gos having the top  <dig> hits and contigs under the bp category were ‘embryo development’ , ‘transport’ , ‘reproduction’ , ‘transcription regulation’ , ‘larval development’ , ‘signal transduction’ , ‘metabolic process’ , ‘small molecule metabolic process’ , ‘transcription’  and ‘locomotion’ .fig.
<dig> schematic representation of the experimental design for the transcriptome and proteome analyses
fig.
<dig> gene ontology  of assembled contigs of newly-released nauplii larvae of b. amphitrite.
the top  <dig> categories for biological processes, molecular function and cellular components are shown
abundant transcripts, protein domains and reactome pathways
functional analysis of contigs revealed that transcripts related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant.
the top  <dig> abundant transcripts and the number of reads are shown in additional file 2: table s <dig>  elongation factor- <dig> alpha was ranked first , followed by nadh dehydrogenase subunit  <dig> .
notably, four transcripts encoding cytochrome oxidase subunit i were also well represented.
interestingly,  <dig> of the  <dig> most abundant transcripts encoded hypothetical or predicted proteins.
additional file 3: table s <dig> lists the top  <dig> pfam domains predicted in the transcriptome.
we were able to map  <dig> pathways present in the reactome database .
these pathways were highly overlapping in terms of the go bp  and differentially-expressed protein categories .table  <dig> up-regulated proteins involved in stress response and osmoregulation in red sea newly released nauplii larvae of b.amphitrite
a) refer to contig no.
in transcriptome database
b) averaged expression values derived from independent replicates of red sea /hong kong  samples
a large number of genes related to larval development were found to be expressed , among which those for brain , muscle , eye , digestive track  and cuticle  were dominant.
notably,  <dig> genes related to metamorphosis  were also identified.
a substantial number of genes assigned to signaling pathways involved in larvae development were also detected .
we found that a large number of genes involved in stress and osmoregulation were over-represented.
genes expressed in response to high salinity, high temperature, cellular stress and ultraviolet  light exposure were identified .
we also identified several genes involved in homeostasis .fig.
<dig> selected genes involved in  larval development and  signaling mechanisms.
the contig numbers inside the pie chart were obtained from the go biological functions listed in supplementary table s1
fig.
<dig> selected genes involved in  response to environment stressors and  osmoregulation.
the contig numbers inside the pie chart were obtained from the go biological functions listed in additional file 11: figure s1
the larvae proteome
a total of  <dig> proteins from hk and rs nauplii were identified based on  <dig> unique peptides and  <dig> unique spectra .
proteins were quantified using hk larvae as a reference .
among these proteins,  <dig> showed differential expression in rs larvae, based on an average of  <dig>  unique peptides,  <dig>  spectra and  <dig>  % sequence coverage.
of these proteins,  <dig> were up-regulated and  <dig> were down-regulated in rs larvae .
additional file 6: table s <dig>  additional file 7: table s <dig> and additional file 8: table s <dig> shows the number of peptides and the spectra, sequence coverage, peptide sequences and fold changes for each differential protein.table  <dig> down-regulated proteins involved in homeostasis and energy metabolism in red sea newly released nauplii larvae of b.amphitrite
a) refer to contig no.
in transcriptome database
b) averaged expression values derived from independent replicates of red sea  /hong kong  samples
differences in proteome expression between rs and hk larvae
principle component analysis  of the proteome datasets showed clear separation of the hk and rs samples , and hierarchical clustering analysis revealed two clusters of down-regulated  and up-regulated  proteins in the rs larvae that were distinct from those in the hk larvae .
a slight variation in expression pattern between the replicates  of hk larvae was observed.
it is important to note that the adult barnacles were collected from wild natural conditions.
habitat conditions could differ substantially because of factors including origin, age, gender and physiological state.
it is difficult to account for uncontrolled sources of variation among specimens collected from natural habitats at the same time in the same area.
however, such variability may not have a significant impact as the average individual protein expression was only slightly different among replicates.
furthermore, the average coefficient of variation  among replicate samples from hk was lower , and not enough to mask the overall difference in the expression patterns of hk samples.
table  <dig> and additional file 6: table s <dig> lists the up-regulated proteins and their fold change values.
nine stress-activated proteins were up-regulated in the rs larvae relative to the hk larvae .
the expression of osmoregulatory proteins was also significantly increased in the rs larvae .
additional file 7: table s <dig> lists some of the down-regulated proteins and their fold change values.
in rs larvae  <dig> proteins involved in cellular detoxification  and  <dig> involved in glycolysis and gluconeogenesis were down-regulated .
notably, all  <dig> actin-binding proteins, including gelsolins, vinculin, plastin- <dig> and villin- <dig> were also down-regulated.fig.
<dig> 2-d view of the principal component analysis of  <dig> identified proteins in two natural larval populations.
log2-transformed fold change ratios from six replicates were used for the analysis.
hkr: replicates of hong kong larvae; rsr: replicates of rs larvae
fig.
<dig> hierarchical clustering of differentially-expressed proteins in hong kong and red sea larvae of b. amphitrite.
a) hierarchical cluster  <dig>  b) hierarchical cluster  <dig>  red: up-regulation; green: down-regulation.
the horizontal axis indicates the replicates in the order r <dig> to r6
fig.
<dig> proteins involved in the stress response  and osmoregulation  were up-regulated in red sea b. amphitrite larvae, whereas detoxification  and glycolysis  proteins were down-regulated.
the full protein names are shown in tables  <dig> and 3
correlation between the proteome and transcriptome of rs larvae
the number of genes in rs larvae of b. amphitrite that were identified in each functional category, and their corresponding protein expression patterns, are shown in table  <dig> and additional file 9: table s <dig>  the profiles of the two datasets revealed overlap in the majority of abundant transcripts and differentially expressed proteins.
these categories, which included stress response, and development and signaling pathways, showed similar trends of expression at both the transcript and protein levels.
we identified  <dig> stress response proteins from the proteome analysis and  <dig>  contigs in the transcriptome; these belonged to various categories of stress.
similarly, the ‘developmental’ category showed  <dig> proteins and  <dig>  transcripts, while ‘signaling pathways’ showed  <dig> proteins and  <dig>  transcripts in the respective datasets.
further, the proteins of binding and catalytic activity were also equally represented in the proteome and the transcriptome.
overall, abundant proteins showed high degree of correlation with the corresponding transcripts.table  <dig> parallel comparison of protein regulation to their corresponding transcript abundance in red sea larvae of b.amphitrite
discussion
deep sequencing of the larval transcriptome
the transcriptome analysis yielded approximately  <dig> million reads and  <dig>  contigs  for the rs larvae, including  <dig>  unique predicted orfs, and constitutes the largest available sequence dataset for nauplii larvae.
chen et al.
obtained  <dig>  reads using  <dig> pyrosequencing and  <dig>  contigs from pooled larvae and adult samples of b. amphitrite from hk seawater  <cit> , while de gregoris et al.
generated  <dig>  reads from nauplius, cyprid and adult stages of b. amphitrite  <cit> .
more recently, lin et al.
obtained  <dig> , <dig> reads and  <dig>  contigs from the prosoma, and  <dig> , <dig> reads and  <dig>  contigs from the base of the barnacle tetraclita japonica formosana  <cit> .
the sequencing depth of the present study provides much greater read coverage than the previous studies.
using this in-house transcriptome database we successfully used mass spectrometry to identify  <dig> proteins; of these,  <dig> showed distinct expression patterns between the hk and rs larval populations.
we also identified a large number of transcripts related to development, locomotion and signal transduction.
molecular basis of larval development
the abundant expression of cuticular proteins in both the transcriptome and proteome datasets may reflect the importance of exoskeleton dynamics during development.
the barnacle body is covered with exoskeleton, which is composed of carbohydrates and proteins  <cit> .
during molting the larvae partially resorb the cuticle to provide space for newly forming tissues.
the larval proteome showed that several structural proteins were differentially expressed.
for example, myosins, titin, actin, tubulins, talin- <dig> and fasciclin- <dig> were up-regulated in rs larvae , whereas settlement pheromone, cement secretion protein  <dig>  retinol dehydrogenase  <dig> and nmda receptor protein  <dig> were down-regulated .
this is consistent with our previous reports showing differential expression of cytoskeletal proteins during development in polychaetes .
the myosins and titins contribute to muscle development and the contraction of striated muscle.
in barnacles, striated muscles in antennules and thoracopods help in locomotion and sensory detection for substratum attachment  <cit> .
barnacle cyprid-specific protein  <dig>  is abundantly expressed in cyprids, but decreases following larval attachment  <cit> .
the protein bcl- <dig> is less abundant in young cyprids, but gradually increases in older cyprids.
apoptosis and protein degradation occur spontaneously during larval development and metamorphosis  <cit> , and are critical in the removal and subsequent degradation of early larval structures, and in cellular and tissue remodeling.
in the present study we found several differentially expressed proteins related to apoptosis.
our previous proteomic analyses identified several apoptosis proteins in early stages of larval development in barnacles  <cit> .
signal transduction pathways
genes that participate in the mapk cascade, cgmp, nitric oxide  biosynthesis, calcium and wnt pathways were identified in the present study.
our previous studies showed that in barnacles these pathways are closely associated with larval settlement and metamorphosis.
for example, p38-mediated mapk signaling has been found to be crucial for larval attachment in barnacles  <cit> .
zhang et al.
found that no regulates attachment of barnacle larvae by modulating cgmp signaling  <cit> .
it appears that no signaling contributes to stress-activated responses , and thereby influences larval survival under unfavorable environmental conditions.
based on our findings, we hypothesize that mapk, no, cgmp and calcium signaling are important for larval development and stress tolerance in larvae of b. amphitrite.
furthermore, the proteome data showed up-regulation of six proteins related to g protein signaling or protein kinase.
among these, gtp-binding nuclear protein , gtp-binding protein sar1b  and guanine nucleotide-binding protein  belong to g protein signaling, whereas receptor for protein kinase c , innexin  <dig>  and epidermal growth factor receptor  belong to the protein kinase and calcium receptor-mediated signal transduction cascades.
chen et al.
reported up-regulation of the g protein-coupled receptor in barnacle cyprid larvae  <cit> , and g protein receptors bind to the metamorphic cue, while the protein kinase independent pathway participates in settlement signaling.
adaptive strategies under extreme marine conditions
adult barnacles live in various habitats and are subject to differing selection pressures that could have a major influence on the survival strategies of their offspring   <cit> .
temperature and salinity influence larval recruitment and the survival of marine invertebrates  <cit> .
in addition, the intertidal zone is highly variable with respect to temperature conditions and among different marine environments  <cit> .
for example, in summer the offshore rs and hk water temperatures average  <dig>  °c and 28 °c, respectively  <cit> .
nauplii may have developed remarkable adaptive mechanisms for survival in the pelagic stage and display a variety of survival strategies.
the higher expression levels among stress response genes in rs larvae, and the up-regulation of stress-activated proteins including heat shock proteins , chaperonins , major egg antigen , t-complex protein , annexin , cyclophilin  and collagen alpha  suggest the development of adaptive strategies to cope with extreme conditions.
this observation is consistent with previous studies showing significant up-regulation of hsps in barnacle larvae challenged with antifoulants  <cit> .
the cellular response of larvae to stress leads to abundant expression of genes encoding hsps.
some hsps act as molecular chaperons  by modulating the correct folding of other proteins under stress, and are involved in various developmental signaling pathways, and assembly and transport mechanisms  <cit> .
for instance, in ascidians and gastropods, hsp <dig> activity regulates the repression of larval metamorphosis by nitric oxide signaling  <cit> .
anxixa expression patterns have been shown to change significantly when cells undergo proliferation or differentiation  <cit> .
the differential expression of anxixa in rs nauplii and juveniles of b. amphitrite could contribute to anti-inflammatory responses.
cypa secreted by cells in response to inflammatory stimuli is known to play an important role in protein folding.
we identified a large number of genes involved in osmotic stress tolerance.
furthermore, the rs larval proteome displayed dramatic up-regulation of osmoregulatory proteins including h + k + αatpase, v-type proton atpase, phosphate transport protein , voltage-dependent anion channel , neuronal acetylcholine receptor , carbonic anhydrase  and selenoprotein  .
during barnacle larval development, biomineralization and osmoregulation facilitate larval tolerance to a wide range of osmotic stresses via active ion transport  <cit> .
ca is widely distributed in barnacle tissues, and is involved in the synthesis of calcium carbonate  <cit>  and the molting cycle in crustaceans.
in other marine species osmotic stress affects the expression patterns of the proteins atp6v0a <dig> and atpv0b, and na + −k + −atpase and v-h + −atpase are required for ion regulation  <cit> .
the sodium-phosphate symporter slc2c plays a role in osmoregulation by absorbing phosphate from interstitial fluid.
the ca2+ transport regulator vdac plays an important role in regulating the exchange of ions and solute molecules in mitochondria, and α7-nachr facilitates the opening of ion channels across the plasma membrane.
consequently, we believe that under extreme environmental conditions larvae may be protected by abundant expression of osmoregulatory proteins that maintain cellular homeostasis, and that the large number of genes involved in various homeostasis mechanisms  are probably involved in osmoregulation .
metabolic depression
under extreme environmental conditions marine invertebrates typically show greater metabolic depression by minimizing energy loss  <cit> .
it has been estimated that protein translation alone may consume up to 50 % of cellular energy; by suppressing metabolism, energy can be diverted to synthesis of stress tolerance proteins  <cit> .
metabolic depression may lead to functional cellular modification, post-translational modification of proteins , and maintenance of homeostasis by ion transport.
consistent with these observations, in rs larvae we found down-regulation of  <dig> proteins involved in various metabolic processes .
for instance, glutathione s-transferases  are antioxidant isoenzymes that provide cellular defense against oxidative stress and harmful chemicals  <cit> .
han et al.
reported increased expression of gsts in barnacle cyprid larvae following exposure to the antifoulant meleagrin  <cit> , suggesting a role in chemical detoxification.
gst also regulates apoptosis by inhibiting kinases, thereby affecting downstream signaling pathways.
it has been shown in barnacle cyprid larvae that proteins involved in glycolysis and gluconeogenesis are differentially expressed, suggesting roles in maintaining homeostasis  <cit> .
fatty acid binding proteins  and fructose  <dig> -bisphosphatase , which are involved in the synthesis of byproducts of glycolysis and gluconeogenesis, have been identified in barnacle larvae  <cit> .
membrane proteins play an important role in the transport of solutes and toxic materials across the cell membrane.
in particular, mitochondrial inner membrane proteins  transport metabolites across the membrane in a highly controlled manner  <cit> .
fabps act as carrier proteins for fatty acids and other lipophilic substances  <cit> .
the regulation of energetically expensive transport systems appears to be an important strategy used by rs larvae to survive in extreme conditions.
the actin binding proteins may regulate cytoskeletal dynamics in developing larvae.
it is hypothesized that larval tolerance of stress is regulated by complex stress response mechanism and strategies, and metabolic depression is probably more diverse and complicated than previously assumed.
we believe this study is of international significance in advancing marine biology, as most previous studies have focused on response to specific environmental factors in controlled environment; it also highlights the importance of studying the cumulative effects of environmental conditions in natural habitats.
CONCLUSIONS
the transcriptome and proteome data generated in this study provide a valuable resource that will enhance molecular studies of crustacean larvae, which are of ecological and economic importance.
the large differences found in the protein expression patterns of larval populations from the two contrasting environments were striking.
the genes, proteins and signaling pathways identified may play crucial roles in the development of nauplii in the rs environment.
for example, the abundant expression of cuticular and muscle proteins in nauplii is critical in the molting cycle, and in muscle development that influences swimming and sensory detection.
we also identified genes that participate in several biochemical pathways that are important for larval settlement, metamorphosis and the stress response in barnacles.
specifically, heat shock proteins, chaperonin, major egg antigen, annexin and no signaling participate in stress-activated responses, and thereby contribute to larval strategies for survival in the rs environment.
proteins involved in osmoregulation, including atpase, transport proteins, acetylcholine receptor and carbonic anhydrase facilitate larval tolerance to osmotic stress.
overall, the findings of this study contribute significantly to knowledge of larval development and adaptation in extreme conditions, and the settlement biology of crustaceans.

 barnacles are marine dominant fouling organisms accumulate on ship hulls, piers and under water structures; biofouling by these invertebrates results in an increase in power and fuel consumption associated with marine transport. removing biofouling barnacles is a major expense involving significant economic losses in shipping and other marine industries. however, some barnacles are of great commercial importance; for example, the gooseneck barnacle achieves an average market price of 30– usd per kilogram, depending on the season. such species are highly exploited, involving harvesting of approximately 500 kg per season in spain and portugal, and hence there is an urgent need for strategies to protect the stocks. the barnacle balanus amphitrite is a widely distributed dominant fouling organism and a major component of rocky shore benthic communities. therefore, b. amphitrite is an excellent model organism for research on settlement biology, adaptation and biofouling. nauplii are the earliest free-swimming stage in the development of most crustaceans, including barnacles, and are extremely abundant in the marine ecosystems. barnacle embryos are brooded by the parents, and the nauplii are released into the water column as swimming larvae. the nauplius i stage has an elongated and pear-shaped body, and the abdominal and caudal spines are partially differentiated. the eye is located in a front medial position and occurs in all developmental stages. the presence of posteriorly folded frontal horns and simple setae on the limbs are characteristics of the nauplius i stage. the newly released larvae swim actively and molt to the next developmental stage within 30 min. in the nauplius ii stage the carapace extends in all directions and the entire body develops a bell shape that has numerous small spines and a pair of prominent spines. the nauplius ii molts to the nauplius iii stage in 38 h. the nauplius iii stage undergoes several moults before it transforms into a cyprid stage. molecular studies defining the role of gene or protein expression signatures in development and adaptive strategies of marine larvae in the natural marine environment are limited. previous studies have used a broad range of proteomics and genomic tools to identify proteins and genes in larval stages of barnacles. however, these studies have mainly focused on exploring settlement and attachment processes. for example, chen et al. identified putative differentially-expressed genes in larvae and adults of b. amphitrite using pyrosequencing, and yan et al. carried out in silico transcriptome data mining to study neuropeptides and their possible functional role in barnacle larval settlement. similarly, de gregoris et al. generated deep sequencing est libraries for nauplii and the cyprid and adult stages of b. amphitrite and identified unique contigs for each developmental stage. more recently, lin et al. performed illumina sequencing of a membranous-based barnacle and identified cement- and adhesion-related genes. thiyagarajan and qian reported the first study of global proteome expression patterns in cyprids and metamorphosed larvae of b. amphitrite, and zhang et al. identified proteins from cyprid larvae using prefractionation methods to reduce sample complexity. chandramouli et al. used multiplex proteomics to describe a distinct protein glycosylation pattern during barnacle larval metamorphosis, and chen et al. recently used label-free quantitative proteomics to identify proteins from nauplii ii and vi, cyprids, and juvenile stages of b. amphitrite. in addition, several studies have demonstrated the feasibility of using proteomics tools to investigate the mode of action of antifoulant chemicals in preventing barnacle larval settlement. these studies have provided a wealth of molecular information, but comprehensive genome and proteome datasets for nauplius larvae are still lacking. thus, in the present study we revealed sequence information and investigated the dynamics of protein expression patterns of nauplius larvae. patterns of differential protein expression between two larval populations from contrasting marine environments were screened to investigate the survival strategies of larvae. extreme marine environments pose great challenges for the survival of marine larvae. for example, the red sea is one of the warmest, most saline bodies of water; it receives little rainfall and has reduced oxygen solubility and high evaporation rates. the ranges of salinity and temperature in the rs are considered extreme. in contrast, in hong kong marine water the salinity and temperature ranges are 28–31psu and 26–28 °c, respectively. the rs has a low concentration of dissolved oxygen compared with that of hk marine water. therefore, we hypothesized that changes in protein expression patterns would reflect the adaptation of barnacle larvae to the rs marine environment. to test this hypothesis we adopted a population proteomics approach to compare protein expression in larvae from hk marine waters and the rs. the specific objectives were: to generate a comprehensive transcriptome dataset for the nauplius larvae of b. amphitrite; to compare the proteomes of barnacle larvae populations from the rs and hk; and to explore survival strategies of larvae in the rs environment.  transcriptome of rs barnacle nauplii figure  shows the experimental design used for the transcriptome and proteome analyses. sequencing data statistics, assembly and annotation information for newly released nauplii of rs b. amphitrite are shown in table  the transcriptome data were deposited in the ncbi bioproject id: prjna. approximately million paired-end reads were obtained. following trimming of low quality reads and adapters, the number of reads was reduced to million, with an average read length of 87 bp. assembly of the reads using abyss v. and trans-abyss v. yielded contigs with an average contig length of 471 bp. using getorf we predicted, open reading frames, of which appeared in at least one blast hit. blastp against a non-redundant database resulted in contigs having at least one sequence homologous with other species, while no homolog was found for contigs. an interproscan v search resulted in contigs having at least one annotated pfam domain, while no domain was found for contigs. in total, contigs showed at least one gene ontology annotation while contigs remained hypothetical, and contigs showed at least one reactome annotation, whereas no annotation was found for contigs. a total of, were assigned to ‘biological process’, and and contigs were assigned to ‘molecular function’ and ‘cellular component’, respectively. figure  shows the top categories of assembled contigs belonging to the bp, mf and cc categories. the gos having the top hits and contigs under the bp category were ‘embryo development’, ‘transport’, ‘reproduction’, ‘transcription regulation’, ‘larval development’, ‘signal transduction’, ‘metabolic process’, ‘small molecule metabolic process’, ‘transcription’ and ‘locomotion’.fig. schematic representation of the experimental design for the transcriptome and proteome analyses fig. gene ontology of assembled contigs of newly-released nauplii larvae of b. amphitrite. the top categories for biological processes, molecular function and cellular components are shown abundant transcripts, protein domains and reactome pathways functional analysis of contigs revealed that transcripts related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. the top abundant transcripts and the number of reads are shown in additional file 2: table s elongation factor- alpha was ranked first, followed by nadh dehydrogenase subunit. notably, four transcripts encoding cytochrome oxidase subunit i were also well represented. interestingly, of the most abundant transcripts encoded hypothetical or predicted proteins. additional file 3: table s lists the top pfam domains predicted in the transcriptome. we were able to map pathways present in the reactome database. these pathways were highly overlapping in terms of the go bp and differentially-expressed protein categories.table up-regulated proteins involved in stress response and osmoregulation in red sea newly released nauplii larvae of b.amphitrite a) refer to contig no. in transcriptome database b) averaged expression values derived from independent replicates of red sea /hong kong samples a large number of genes related to larval development were found to be expressed, among which those for brain, muscle, eye, digestive track and cuticle were dominant. notably, genes related to metamorphosis were also identified. a substantial number of genes assigned to signaling pathways involved in larvae development were also detected. we found that a large number of genes involved in stress and osmoregulation were over-represented. genes expressed in response to high salinity, high temperature, cellular stress and ultraviolet light exposure were identified. we also identified several genes involved in homeostasis.fig. selected genes involved in larval development and signaling mechanisms. the contig numbers inside the pie chart were obtained from the go biological functions listed in supplementary table s1 fig. selected genes involved in response to environment stressors and osmoregulation. the contig numbers inside the pie chart were obtained from the go biological functions listed in additional file 11: figure s1 the larvae proteome a total of proteins from hk and rs nauplii were identified based on unique peptides and unique spectra. proteins were quantified using hk larvae as a reference. among these proteins, showed differential expression in rs larvae, based on an average of unique peptides, spectra and  % sequence coverage. of these proteins, were up-regulated and were down-regulated in rs larvae. additional file 6: table s additional file 7: table s and additional file 8: table s shows the number of peptides and the spectra, sequence coverage, peptide sequences and fold changes for each differential protein.table down-regulated proteins involved in homeostasis and energy metabolism in red sea newly released nauplii larvae of b.amphitrite a) refer to contig no. in transcriptome database b) averaged expression values derived from independent replicates of red sea /hong kong samples differences in proteome expression between rs and hk larvae principle component analysis of the proteome datasets showed clear separation of the hk and rs samples, and hierarchical clustering analysis revealed two clusters of down-regulated and up-regulated proteins in the rs larvae that were distinct from those in the hk larvae. a slight variation in expression pattern between the replicates of hk larvae was observed. it is important to note that the adult barnacles were collected from wild natural conditions. habitat conditions could differ substantially because of factors including origin, age, gender and physiological state. it is difficult to account for uncontrolled sources of variation among specimens collected from natural habitats at the same time in the same area. however, such variability may not have a significant impact as the average individual protein expression was only slightly different among replicates. furthermore, the average coefficient of variation among replicate samples from hk was lower, and not enough to mask the overall difference in the expression patterns of hk samples. table  and additional file 6: table s lists the up-regulated proteins and their fold change values. nine stress-activated proteins were up-regulated in the rs larvae relative to the hk larvae. the expression of osmoregulatory proteins was also significantly increased in the rs larvae. additional file 7: table s lists some of the down-regulated proteins and their fold change values. in rs larvae proteins involved in cellular detoxification and involved in glycolysis and gluconeogenesis were down-regulated. notably, all actin-binding proteins, including gelsolins, vinculin, plastin- and villin- were also down-regulated.fig. 2-d view of the principal component analysis of identified proteins in two natural larval populations. log2-transformed fold change ratios from six replicates were used for the analysis. hkr: replicates of hong kong larvae; rsr: replicates of rs larvae fig. hierarchical clustering of differentially-expressed proteins in hong kong and red sea larvae of b. amphitrite. a) hierarchical cluster b) hierarchical cluster red: up-regulation; green: down-regulation. the horizontal axis indicates the replicates in the order r to r6 fig. proteins involved in the stress response and osmoregulation were up-regulated in red sea b. amphitrite larvae, whereas detoxification and glycolysis proteins were down-regulated. the full protein names are shown in tables  and 3 correlation between the proteome and transcriptome of rs larvae the number of genes in rs larvae of b. amphitrite that were identified in each functional category, and their corresponding protein expression patterns, are shown in table  and additional file 9: table s the profiles of the two datasets revealed overlap in the majority of abundant transcripts and differentially expressed proteins. these categories, which included stress response, and development and signaling pathways, showed similar trends of expression at both the transcript and protein levels. we identified stress response proteins from the proteome analysis and contigs in the transcriptome; these belonged to various categories of stress. similarly, the ‘developmental’ category showed proteins and transcripts, while ‘signaling pathways’ showed proteins and transcripts in the respective datasets. further, the proteins of binding and catalytic activity were also equally represented in the proteome and the transcriptome. overall, abundant proteins showed high degree of correlation with the corresponding transcripts.table parallel comparison of protein regulation to their corresponding transcript abundance in red sea larvae of b.amphitrite discussion deep sequencing of the larval transcriptome the transcriptome analysis yielded approximately million reads and contigs for the rs larvae, including unique predicted orfs, and constitutes the largest available sequence dataset for nauplii larvae. chen et al. obtained reads using pyrosequencing and contigs from pooled larvae and adult samples of b. amphitrite from hk seawater, while de gregoris et al. generated reads from nauplius, cyprid and adult stages of b. amphitrite. more recently, lin et al. obtained, reads and contigs from the prosoma, and, reads and contigs from the base of the barnacle tetraclita japonica formosana. the sequencing depth of the present study provides much greater read coverage than the previous studies. using this in-house transcriptome database we successfully used mass spectrometry to identify proteins; of these, showed distinct expression patterns between the hk and rs larval populations. we also identified a large number of transcripts related to development, locomotion and signal transduction. molecular basis of larval development the abundant expression of cuticular proteins in both the transcriptome and proteome datasets may reflect the importance of exoskeleton dynamics during development. the barnacle body is covered with exoskeleton, which is composed of carbohydrates and proteins. during molting the larvae partially resorb the cuticle to provide space for newly forming tissues. the larval proteome showed that several structural proteins were differentially expressed. for example, myosins, titin, actin, tubulins, talin- and fasciclin- were up-regulated in rs larvae, whereas settlement pheromone, cement secretion protein retinol dehydrogenase and nmda receptor protein were down-regulated. this is consistent with our previous reports showing differential expression of cytoskeletal proteins during development in polychaetes. the myosins and titins contribute to muscle development and the contraction of striated muscle. in barnacles, striated muscles in antennules and thoracopods help in locomotion and sensory detection for substratum attachment. barnacle cyprid-specific protein is abundantly expressed in cyprids, but decreases following larval attachment. the protein bcl- is less abundant in young cyprids, but gradually increases in older cyprids. apoptosis and protein degradation occur spontaneously during larval development and metamorphosis, and are critical in the removal and subsequent degradation of early larval structures, and in cellular and tissue remodeling. in the present study we found several differentially expressed proteins related to apoptosis. our previous proteomic analyses identified several apoptosis proteins in early stages of larval development in barnacles. signal transduction pathways genes that participate in the mapk cascade, cgmp, nitric oxide biosynthesis, calcium and wnt pathways were identified in the present study. our previous studies showed that in barnacles these pathways are closely associated with larval settlement and metamorphosis. for example, p38-mediated mapk signaling has been found to be crucial for larval attachment in barnacles. zhang et al. found that no regulates attachment of barnacle larvae by modulating cgmp signaling. it appears that no signaling contributes to stress-activated responses, and thereby influences larval survival under unfavorable environmental conditions. based on our findings, we hypothesize that mapk, no, cgmp and calcium signaling are important for larval development and stress tolerance in larvae of b. amphitrite. furthermore, the proteome data showed up-regulation of six proteins related to g protein signaling or protein kinase. among these, gtp-binding nuclear protein, gtp-binding protein sar1b and guanine nucleotide-binding protein belong to g protein signaling, whereas receptor for protein kinase c, innexin and epidermal growth factor receptor belong to the protein kinase and calcium receptor-mediated signal transduction cascades. chen et al. reported up-regulation of the g protein-coupled receptor in barnacle cyprid larvae, and g protein receptors bind to the metamorphic cue, while the protein kinase independent pathway participates in settlement signaling. adaptive strategies under extreme marine conditions adult barnacles live in various habitats and are subject to differing selection pressures that could have a major influence on the survival strategies of their offspring. temperature and salinity influence larval recruitment and the survival of marine invertebrates. in addition, the intertidal zone is highly variable with respect to temperature conditions and among different marine environments. for example, in summer the offshore rs and hk water temperatures average  °c and 28 °c, respectively. nauplii may have developed remarkable adaptive mechanisms for survival in the pelagic stage and display a variety of survival strategies. the higher expression levels among stress response genes in rs larvae, and the up-regulation of stress-activated proteins including heat shock proteins, chaperonins, major egg antigen, t-complex protein, annexin, cyclophilin and collagen alpha suggest the development of adaptive strategies to cope with extreme conditions. this observation is consistent with previous studies showing significant up-regulation of hsps in barnacle larvae challenged with antifoulants. the cellular response of larvae to stress leads to abundant expression of genes encoding hsps. some hsps act as molecular chaperons by modulating the correct folding of other proteins under stress, and are involved in various developmental signaling pathways, and assembly and transport mechanisms. for instance, in ascidians and gastropods, hsp activity regulates the repression of larval metamorphosis by nitric oxide signaling. anxixa expression patterns have been shown to change significantly when cells undergo proliferation or differentiation. the differential expression of anxixa in rs nauplii and juveniles of b. amphitrite could contribute to anti-inflammatory responses. cypa secreted by cells in response to inflammatory stimuli is known to play an important role in protein folding. we identified a large number of genes involved in osmotic stress tolerance. furthermore, the rs larval proteome displayed dramatic up-regulation of osmoregulatory proteins including h + k + αatpase, v-type proton atpase, phosphate transport protein, voltage-dependent anion channel, neuronal acetylcholine receptor, carbonic anhydrase and selenoprotein. during barnacle larval development, biomineralization and osmoregulation facilitate larval tolerance to a wide range of osmotic stresses via active ion transport. ca is widely distributed in barnacle tissues, and is involved in the synthesis of calcium carbonate and the molting cycle in crustaceans. in other marine species osmotic stress affects the expression patterns of the proteins atp6v0a and atpv0b, and na + −k + −atpase and v-h + −atpase are required for ion regulation. the sodium-phosphate symporter slc2c plays a role in osmoregulation by absorbing phosphate from interstitial fluid. the ca2+ transport regulator vdac plays an important role in regulating the exchange of ions and solute molecules in mitochondria, and α7-nachr facilitates the opening of ion channels across the plasma membrane. consequently, we believe that under extreme environmental conditions larvae may be protected by abundant expression of osmoregulatory proteins that maintain cellular homeostasis, and that the large number of genes involved in various homeostasis mechanisms are probably involved in osmoregulation. metabolic depression under extreme environmental conditions marine invertebrates typically show greater metabolic depression by minimizing energy loss. it has been estimated that protein translation alone may consume up to 50 % of cellular energy; by suppressing metabolism, energy can be diverted to synthesis of stress tolerance proteins. metabolic depression may lead to functional cellular modification, post-translational modification of proteins, and maintenance of homeostasis by ion transport. consistent with these observations, in rs larvae we found down-regulation of proteins involved in various metabolic processes. for instance, glutathione s-transferases are antioxidant isoenzymes that provide cellular defense against oxidative stress and harmful chemicals. han et al. reported increased expression of gsts in barnacle cyprid larvae following exposure to the antifoulant meleagrin, suggesting a role in chemical detoxification. gst also regulates apoptosis by inhibiting kinases, thereby affecting downstream signaling pathways. it has been shown in barnacle cyprid larvae that proteins involved in glycolysis and gluconeogenesis are differentially expressed, suggesting roles in maintaining homeostasis. fatty acid binding proteins and fructose -bisphosphatase, which are involved in the synthesis of byproducts of glycolysis and gluconeogenesis, have been identified in barnacle larvae. membrane proteins play an important role in the transport of solutes and toxic materials across the cell membrane. in particular, mitochondrial inner membrane proteins transport metabolites across the membrane in a highly controlled manner. fabps act as carrier proteins for fatty acids and other lipophilic substances. the regulation of energetically expensive transport systems appears to be an important strategy used by rs larvae to survive in extreme conditions. the actin binding proteins may regulate cytoskeletal dynamics in developing larvae. it is hypothesized that larval tolerance of stress is regulated by complex stress response mechanism and strategies, and metabolic depression is probably more diverse and complicated than previously assumed. we believe this study is of international significance in advancing marine biology, as most previous studies have focused on response to specific environmental factors in controlled environment; it also highlights the importance of studying the cumulative effects of environmental conditions in natural habitats.  the transcriptome and proteome data generated in this study provide a valuable resource that will enhance molecular studies of crustacean larvae, which are of ecological and economic importance. the large differences found in the protein expression patterns of larval populations from the two contrasting environments were striking. the genes, proteins and signaling pathways identified may play crucial roles in the development of nauplii in the rs environment. for example, the abundant expression of cuticular and muscle proteins in nauplii is critical in the molting cycle, and in muscle development that influences swimming and sensory detection. we also identified genes that participate in several biochemical pathways that are important for larval settlement, metamorphosis and the stress response in barnacles. specifically, heat shock proteins, chaperonin, major egg antigen, annexin and no signaling participate in stress-activated responses, and thereby contribute to larval strategies for survival in the rs environment. proteins involved in osmoregulation, including atpase, transport proteins, acetylcholine receptor and carbonic anhydrase facilitate larval tolerance to osmotic stress. overall, the findings of this study contribute significantly to knowledge of larval development and adaptation in extreme conditions, and the settlement biology of crustaceans. 
we presented, for the first time, comprehensive transcriptome and proteome datasets for red sea nauplii. genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. the proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. the synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments. a nauplii larval transcript dataset, containing predicted open reading frames, was constructed and used as a reference for the proteome analysis. the barnacle balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. we also investigated itraq-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the red sea marine environment. we observed remarkable plasticity in the proteome of red sea larvae. however, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments.
 the barnacle balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. nauplii, the first larval stage of most crustaceans, are extremely abundant in the marine zooplankton. however, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments. we sequenced and constructed the genome dataset for nauplii to obtain comprehensive larval genetic information. we also investigated itraq-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the red sea marine environment.  a nauplii larval transcript dataset, containing predicted open reading frames, was constructed and used as a reference for the proteome analysis. genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. we observed remarkable plasticity in the proteome of red sea larvae. the proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. the synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments.  we presented, for the first time, comprehensive transcriptome and proteome datasets for red sea nauplii. the datasets provide a foundation for future investigations focused on the survival mechanisms of other crustaceans in extreme marine environments. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords b. amphitritered seatranscriptomicsproteomicsadaptationking abdullah university of science and technologysa-c0040/uk-c0016ravasi timothy issue-copyright-statement© the author 2015 