BACKGROUND
the development of high throughput techniques supplies a rich source of information for the protein-protein interaction network  research.
the interpretation of such information is a key to understand the complex world of biological processes inside the cell  <cit> .
knowledge of pins helps researchers to solve many problems such as signaling pathways identification  <cit> , recognition of functional modules  <cit>  and prediction of protein functions  <cit> .
given the significant importance of pins, proteome-wide interaction networks based on protein interactions has been constructed for many organisms  <cit> .
the early study of pin mostly focused on saccharomyces cerevisiae.
schwikowski et al.
performed a global analysis of published proteins interactions in s. cerevisiae and predicted the functions of  <dig> previously uncharacterized proteins  <cit> .
some interesting sub-networks were extracted from the pins of s. cerevisiae and analyzed, for example, the spindle pole body related sub-network in ito t’s work  <cit>  and dna damage response data set in ho y’s work  <cit> .
construction and analysis of pins for other microorganisms has been subsequently performed, such as the pins of drosophila melanogaster  <cit> , helicobacter pylori  <cit>  and bacillus subtilis  <cit> .
in the decades-long development of pin, interest has shifted from microbial systems  <cit>  to mammalian  <cit>  and more kinds of organisms  <cit> .
however, to date, there is no large-scale pin available for the study of aquatic crustacean.
although much effort has been made on the phenotype or physiological study of aquatic animals and crustaceans  <cit> , an important ongoing problem is that the original inducement of all the phenotype and physiological features is the expression of genes and interaction of proteins.
however, the expression and interaction of genes and proteins are still indistinct in most aquatic animals.
as the protein interactions based on the gene expression has a significant role in the in-depth exploration of the biological process mechanism in cells, a pin is necessary and important for the systematic study of aquatic crustaceans.
the chinese mitten crab   is one of the most important aquaculture species in china with high commercial value as a food source  <cit> .
many studies have been performed focusing on single or several genes  <cit> , proteins  <cit>  or a specific pathway  <cit>  to accelerate the growth or improve the immune and signal transduction system of e. sinensis.
however, the genome sequence of any e. sinensis species is still unavailable.
therefore, a whole map of the protein interactions in e. sinensis is still fragmentary and different signaling pathways implicated in growth and immune response also remain incomplete.
recently, illumina rna-seq, the next-generation deep-sequencing technique, provides new approaches to obtain a whole transcriptome sequencing  <cit> , which makes it possible to get huge amounts of knowledge on e. sinensis proteins and subsequently obtain a systematic overview of the protein-protein interaction system.
in this work we sequenced the transcriptional rna sequences in the eyestalk, y-organ and hepatopancreas of e. sinensis and presented a substantial resource of affinity-tagged proteins.
a pin of e. sinensis was generated based on the transcriptome sequencing.
the network covers hundreds of previously-uncharacterized proteins, thus providing functional associations and biological context for the proteins that previously lacked annotation.
the signaling sub-network was extracted from the global pin and the evolution paths of known signaling pathways were examined, which represents a new global view of the signaling systems in e. sinensis.
functional assignment of the unclassified proteins and unigenes supplies significant guidance for the in vivo investigation of proteins/genes related to specific function.
to our knowledge, the pin of e. sinensis is the first large-scale aquatic crustacean protein interaction network, thereby providing a systems biology view of an aquatic crustacean proteome.
RESULTS
transcriptome sequencing of e. sinensis
to obtain the e. sinensis transcriptome data, rna from eyestalks, y-organs and hepatopancreas mixed samples of e. sinensis were sequenced with the illumina hiseqtm <dig>  in total  <dig> , <dig>  nt clean nucleotides were found with q <dig> and gc percentages of  <dig> % and  <dig> %, respectively.
<dig> , <dig> clean reads were then obtained.
from these clean reads,  <dig>  contigs  were assembled and then  <dig>  unigenes  were constructed from contigs with soap de novo, including  <dig>  distinct singletons and  <dig>  distinct clusters.
the sequenced unigenes were subsequently aligned against the nr database using blastn and blastx searching with e-value < 1*e- <dig>  finally  <dig>  unigenes  were matched.
with nr annotation, go annotations of unigenes were obtained with the blast2go program.
among the total  <dig>  unigenes of e. sinensis,  <dig>  unigenes  were annotated to the go database with confident matches, including  <dig>  assigned to the biological process category,  <dig> assigned to the cellular component category and  <dig>  assigned to the molecular function category.
after the go annotation of each unigene, wego software was used to obtain the go functional classification for all unigenes in biological process category and to understand the distribution of gene functions from the macro level.
in the biological process category, unigenes were divided into  <dig> different biological processes.
cellular process  and metabolic process  were most highly represented among them, other processed such as biological regulation , developmental process , localization , multicellular organismal process , regulation of biological process  and response to stimulus  were also included in biological process.
the transcriptome sequencing and go annotation results can be found in additional file  <dig>
the protein information of model organisms
the protein sequence data of six model organisms  were downloaded from the uniprot database.
the number of protein sequences in each organism is shown in table  <dig>
the protein interaction information of the above six model organisms was obtained from the pina database.
usually, protein names were used in the protein interactions for presenting proteins, whereas for some interactions, the proteins in different model organisms were presented in different ways, which needed to be normalized for the convenience of the following analysis.
two special situations between protein ids and protein names should be pretreated.
firstly, when there is no related protein name for a protein, the protein id was used to mark the protein instead of a protein name.
secondly, sometimes the same protein is represented in different ways in different interactions.
some are marked with the protein name and the others are marked with the protein id.
in this situation, the protein name was used instead of the protein id.
for example, vha36- <dig> is a protein in d. melanogaster.
in the interaction pair: uniprotkb:q7plp5-uniprotkb:a1z8v <dig>  it is represented with the protein id a1z8v <dig> and in another interaction pair: uniprotkb:rpl15- uniprotkb:vha36- <dig>  it is represented with its protein name vha36- <dig>  we replaced a1z8v <dig> with vha36- <dig> in the first interaction to represent the protein.
in this way,  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> protein names were pretreated in c. elegans, d. melanogaster, h. sapiens, m. musculus, r. norvegicus and s. cerevisiae, respectively.
the number of proteins and protein-protein interactions of the six model organisms from pina are shown in table  <dig>
construction of model-organism-based protein-protein interaction sub-network
in order to construct the pin network of e. sinensis, the model-organism-based protein-protein interaction sub-networks were first constructed.
the sub-networks were constructed based on the sequence alignment of model organisms and e. sinensis.
according to the alignment result, the homologous sequence existing in each protein interaction of a model organism was marked.
if two proteins in an interaction can both be matched with the unigenes in e. sinensis, then these two proteins and their interaction were considered as part of the model-organism-based sub-network.
in this way,  <dig> different protein-protein interaction sub-networks were constructed based on the protein information of  <dig> model organisms respectively.
the numbers of proteins, related unigenes and interactions in each model-organism-based protein-protein interaction sub-network are shown in table  <dig>  the unigenes and proteins in the sub-networks are not in one-to-one relationships.
for example, protein yrt in d. melanogaste based sub-network is related to two unigenes  in e. sinensis.
e. sinensis
unigene
a
b
c
athe number in bracket shows the proportion of related unigenes accounting for all the unigenes in the model organism.
bthe number in brackets shows the proportion of proteins in the sub-network accounting for all the proteins in the model organism.
cthe number in brackets shows the proportion of interaction pairs in the sub-network accounting for all the interaction pairs in the model organism.
construction of pin for e. sinensis
the pin of e. sinensis was constructed according to the method described in the methods section.
the organism with a closer relationship to e. sinensis was integrated preferentially.
d. melanogaster belongs to the arthropoda-insecta, which has the closest genetic relationship with e. sinensis  compared with the other  <dig> organisms.
c. elegans belongs to protocoelomata-nematomorpha, which is more primitive than arthropoda-crustacea, but has a closer genetic relationship with e. sinensis than the other  <dig> organisms.
therefore, d. melanogaster and c. elegans based sub-networks were used as the target and query networks respectively in the first turn integration.
subsequently the h. sapiens, r. norvegicus, m. musculus and s. cerevisiae based sub-network were aligned and integrated in order.
one thing to mention is that r. norvegicus and m. musculus actually have similar genetic relationship with h. sapiens.
however, the protein and interaction information of h. sapiens from uniprot and pina database is much more than that of r. norvegicus and m. musculus.
considering the attribution of the organisms to the pin reconstruction, the h. sapiens based sub-network was integrated prior to r. norvegicus and m. musculus.
various numbers of protein interactions were added to the integrated network in each turn of integration and thus the pin of e. sinensis was expanded based on the model organisms.
finally the pin of e. sinensis was obtained after five-turn integrations of the above  <dig> sub-networks.
the final e. sinensis pin contained  <dig>  proteins and  <dig>  interaction pairs .
the scale of each intermediate integrated network after each turn of integration is shown in table  <dig>
the pin of e. sinensis is composed of a largest weakly connected component  and several small components.
as the lwcc contains most of the nodes and edges in the global pin, the topological features of the lwcc were analyzed and compared with that of the model-organism-based sub-networks.
the basic features of lwccs are shown in table  <dig>  actually, the features of lwccs from different sub-networks are quite different from each other.
for example, the number of nodes and edges in d. melanogaster based sub-network is much more than that in c. elegans based sub-network, and the diameter is smaller, which means the connectivity of the d. melanogaster based sub-network is stronger than that of the c. elegans based sub-network.
the smaller path length further proved this conclusion.
the s. cerevisiae based sub-network obviously has the strongest connectivity.
generally the topological parameter values of e. sinensis are between that of s. cerevisiae and the other five organisms, indicating that the pin of e. sinensis assimilates the information in the six model organisms as well as eliminating the redundant information.
e. sinensis
pin
score of protein-protein interaction pair
as the proteins  and interactions  in the pin came from different model organisms with various genetic relationships with e. sinensis, in order to represent the confidence of the interaction pairs, the score of each protein-protein interaction pair was evaluated according to the method described in the methods section.
the highest score is  <dig>  reached by  <dig> interaction pairs.
the highest score of edges and nodes are  <dig> and  <dig>  respectively.
figure  <dig> shows the score distribution of the interaction pairs in the pin of e. sinensis.
<dig> % interaction pairs distribute in scores of 11– <dig>  the interaction pairs with scores of 6– <dig> and 16– <dig> make up  <dig> % and  <dig> % of all the interaction pairs respectively.
the score of each interaction pair is shown in additional file  <dig>
e. sinensis
protein-protein interaction pairs.
the number of interaction pairs with scores above or equal to  <dig> is only  <dig> .
these interactions exist in most of the model organisms, indicating their conservatism.
for example, proteins pros <dig> , mov <dig>  pros <dig>  tbp- <dig> and rpn <dig> in these interactions are part of 26s protease.
mov <dig> is a regulation subunit and rpn <dig> is a metal protease component of 26s protease.
26s protease is a huge protease complex widely found in many organisms  <cit> .
pros <dig>  mov <dig>  cg <dig>  tcp-1zeta, lwr and skpa are the hub proteins with their degree larger than  <dig>  which exist in at least four model organisms and have important biological function in the metabolism by influencing many of their neighbor proteins.
identification of signaling sub-network in e. sinensis
signaling pathways are significant mediates of cell growth, development, immune and other life activities, which play a crucial role in almost all growth stages.
to better understand the signaling systems of e. sinensis, the proteins with go annotation “signaling”  and their related interactions were extracted from the global pin to generate the signaling sub-network.
there are in total  <dig> proteins with go: <dig> in e. sinensis, in which  <dig> are isolated and  <dig> interacted with other protein, the number of protein-protein interaction relationships is  <dig> .
seven known signal transduction pathways in the kegg database were found in e. sinensis: hippo, jak-stat mtor, wnt, mapk, the notch signal transduction pathway and the protein assembly process in endoplasmic reticulum.
some proteins are active in two or more signal pathways.
proteins in the above  <dig> pathways also interacted with other proteins in the pin and finally the sub-network contains  <dig> proteins and  <dig>  interaction pairs, including the  <dig> proteins in known signaling pathways and their neighboring proteins.
the  <dig> proteins with  <dig> interaction pairs in known signaling pathways are shown in figure 3a, in which  <dig> proteins and  <dig> interaction pair are isolated from the fully connected sub-network.
the hippo signal transduction pathway is responsible for the growth inhibition of cells, which is a highly conservative pathway.
it was first found in d. melanogaster and has been found in many mammals such as r. norvegicus and h. sapiens.
the hippo signal transduction pathway has significant function in organ size control, stem cell self-renewal, cancer inhibition and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction  <cit> .
proteins wts, hpo and sav in this pathway are found to be responsible for cancer inhibition.
the interaction of hpo and sav is able to phosphorylate and activate the complex composed of wts and mats  <cit> .
two top cell skeleton signal proteins mer and ex can be reciprocally activated with kibra to further activate the hippo pathway  <cit> .
in addition, wts can directly phosphorylate, and thus inhibit the activating transcription factor yki.
and yki is closely related with cell multiplication and apoptosis  <cit> .
the hippo related proteins wts, hpo, sav, mats, mer, yki and kibra were found in the signaling sub-network of e. sinensis , indicating that the hippo signal transduction pathway also exists in e. sinensis.
the growth control and cell self-renewal of e. sinensis is probably dominated by the hippo pathway.
the jak-stat signal transduction pathway is composed of the ptk related receptor, ptk jak and transcription factor.
it is simulated by cytokine and participates in many important biological processes such as cell multiplication, differentiation, apoptosis and immunoregulation  <cit> .
the pias protein in this pathway can inhibit activation of the stat protein by blocking the binding activity of the transcription factor and dna.
in addition, it is reported that pias can interact with more than  <dig> proteins, many of which are immune-system-related  <cit> .
the stat and pias proteins were found in the signal network of e. sinensis,coming from the h. sapiens based sub-network.
the other proteins in the jak-stat pathway came from the d. melanogaster based sub-network, such as the suppressor of cytokine signaling , which inhibits the phosphorylation of stat by combining and blocking jak or competing for the phosphotyrosine site on the cytokine receptor with stat .
the multiplication, differentiation and apoptosis of e. sinensis are possibly controlled by the jak-stat pathway.
the different source of proteins indicated that the integration process provided more information for the pin of e. sinensis.
in addition, the mtor, wnt, mapk, notch and protein processing in endoplasmic reticulum were also found in the signaling sub-network of e. sinensis.
the mtor pathway is a central regulator for both cell proliferation and cell growth  <cit> .
the wnt pathway is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal in a number of adult tissues.
many studies report that mutation of the wnt pathway is closely related to several hereditary diseases and cancers  <cit> .
the notch pathway is first found in d. melanogaster and participates in the regulation of cell multiplication, differentiation, and apoptosis, and acts as an important regulator of immune cells development  <cit> .
the seven signaling transduction pathways found in e. sinensis represent the regulation of basic cell life activity about growth, development, reproduction and disease-resistance.
the signaling sub-network of e. sinensis provides substantial information of the signal transduction pathways and unknown proteins which need to be further studied.
evolution path of e. sinensis signaling network
the signaling network has been used to understand evolution in multicellular animals  <cit> .
as the e. sinensis signaling sub-network was obtained from the integration of six model organisms and these organisms are located in different evolutionary stages, in order to promote understanding of the evolution of the signaling sub-network in e. sinensis, we examined the evolution path by comparing the e. sinensis signaling network with the six model organisms, and investigated the original organisms and preferred evolution paths of the e. sinensis signaling network.
the six species were classified into three groups: primitive, bilaterian and vertebrate groups as described in lei li’s work  <cit> .
the primitive group included s. cerevisiae.
the bilaterian lineage was composed of d. melanogaster and c. elegans.
all three vertebrate species were placed in the vertebrate group.
based on this group partition, we identified each protein interaction in the e. sinensis signaling sub-network.
for each protein interaction, the origin of two proteins and an interaction were defined separately using the principle in lei li’s work  <cit> .
if a protein/interaction exists in a primitive organism, it is assigned to a primitive  origin.
if a protein/interaction exists in a bilaterian organism but not in a primitive organism, it is assigned to a bilaterian  origin.
if a protein/interaction exists only in vertebrate organism, it is assigned to a vertebrate  origin.
finally, the origin of a complete protein interaction  was assigned to the evolutionary stage in which the last component in the protein interaction appeared.
the origin groups of seven known signaling pathways were examined .
we found that different signaling pathways had various evolutionary paths.
protein processing in er is relatively complete in the primitive stage with more than half of the interactions in this stage.
most interactions in the hippo pathway exist in the bilateria and vertebrate stages, which is consistent with its first discovery in d. melanogaster  <cit> .
several interactions in the hippo pathway originated from the primitive stage but no complete path was formed until it was found in d. melanogaster.
the same situation also appears in the jak-stat, mtor and wnt pathway, indicating that these pathways may grow in the primitive stage and mature in the bilateria stages.
mapk and notch have the latest evolution origin, with all interactions found in the biliteria and vertebrate stages.
function assignment of unclassified proteins and unigenes
according to the go biological process annotation of proteins in pin, the functions of  <dig>  proteins related to  <dig>  unigenes were annotated, whereas the functions of the other  <dig> proteins related to  <dig>  unigenes were still unknown, which makes up approximately 23% of all the proteins and 19% of all the unigenes.
in order to investigate the functions of these unclassified proteins and unigenes, the method described in the methods section was used to assign go annotations.
finally,  <dig> unclassified proteins related to  <dig> unclassified unigenes were annotated , making up 76% and 73% of all the unknown proteins and unigenes .
e. sinensis.
as the go terms are organized in a treelike structure, we further analyzed the number distribution of newly-annotated proteins and unigenes in different go depths .
as go depth increases, the annotation becomes more detailed.
as shown in figure  <dig>  the fifth layer of the go treelike structure contains the most assigned proteins and unigenes.
as proteins or unigenes may have multiple functions, the go annotation and proteins/unigenes are not in one-to-one relationship.
<dig> proteins were annotated as immune response-related proteins.
they functioned in innate immune response , humoral immune response , mucosal immune response , regulation of innate immune response  and unclear immune response .
furthermore,  <dig> signaling-related proteins were found.
they acted as receptors of signaling factor or the regulators of signaling pathways.
for example,  <dig> proteins  were annotated as hippo signaling cascade and  <dig> other proteins  functioned as regulators of hippo signaling cascade.
although the functions of the proteins/unigenes still need to be further validated, the assignment of functions provides important reference for identification of the targets in the in vivo experiment.
CONCLUSIONS
with the improvement in high-throughput sequencing technology, rna sequencing and annotation are possible for further analysis and detection in the pursuit of certain biological goals.
in present work we constructed a pin of e. sinensis on the basis of transcriptomics sequencing and the proteome of six model organisms.
the pin defines a primary protein interaction landscape for e. sinensis cells that allows study of sub-networks with specific function.
seven known pathways were identified in the signaling sub-network extracted from the global pin.
with the analysis of evolution paths for these pathways, we found their differences in evolution origin.
more proteins identified as neighbors of the proteins in seven identified pathways were prepared for further confirmation.
furthermore, the function assignment of unclassified proteins offers a new reference in protein function exploration.
it is the first large-scale pin of aquatic crustaceans, thereby providing necessary experience for the exploration of pin for other aquatic crustacean species, as well as supplying a systems biological view of an aquatic crustacean interactome.

 the development of high throughput techniques supplies a rich source of information for the protein-protein interaction network research. the interpretation of such information is a key to understand the complex world of biological processes inside the cell. knowledge of pins helps researchers to solve many problems such as signaling pathways identification, recognition of functional modules and prediction of protein functions. given the significant importance of pins, proteome-wide interaction networks based on protein interactions has been constructed for many organisms. the early study of pin mostly focused on saccharomyces cerevisiae. schwikowski et al. performed a global analysis of published proteins interactions in s. cerevisiae and predicted the functions of previously uncharacterized proteins. some interesting sub-networks were extracted from the pins of s. cerevisiae and analyzed, for example, the spindle pole body related sub-network in ito t’s work and dna damage response data set in ho y’s work. construction and analysis of pins for other microorganisms has been subsequently performed, such as the pins of drosophila melanogaster, helicobacter pylori and bacillus subtilis. in the decades-long development of pin, interest has shifted from microbial systems to mammalian and more kinds of organisms. however, to date, there is no large-scale pin available for the study of aquatic crustacean. although much effort has been made on the phenotype or physiological study of aquatic animals and crustaceans, an important ongoing problem is that the original inducement of all the phenotype and physiological features is the expression of genes and interaction of proteins. however, the expression and interaction of genes and proteins are still indistinct in most aquatic animals. as the protein interactions based on the gene expression has a significant role in the in-depth exploration of the biological process mechanism in cells, a pin is necessary and important for the systematic study of aquatic crustaceans. the chinese mitten crab is one of the most important aquaculture species in china with high commercial value as a food source. many studies have been performed focusing on single or several genes, proteins or a specific pathway to accelerate the growth or improve the immune and signal transduction system of e. sinensis. however, the genome sequence of any e. sinensis species is still unavailable. therefore, a whole map of the protein interactions in e. sinensis is still fragmentary and different signaling pathways implicated in growth and immune response also remain incomplete. recently, illumina rna-seq, the next-generation deep-sequencing technique, provides new approaches to obtain a whole transcriptome sequencing, which makes it possible to get huge amounts of knowledge on e. sinensis proteins and subsequently obtain a systematic overview of the protein-protein interaction system. in this work we sequenced the transcriptional rna sequences in the eyestalk, y-organ and hepatopancreas of e. sinensis and presented a substantial resource of affinity-tagged proteins. a pin of e. sinensis was generated based on the transcriptome sequencing. the network covers hundreds of previously-uncharacterized proteins, thus providing functional associations and biological context for the proteins that previously lacked annotation. the signaling sub-network was extracted from the global pin and the evolution paths of known signaling pathways were examined, which represents a new global view of the signaling systems in e. sinensis. functional assignment of the unclassified proteins and unigenes supplies significant guidance for the in vivo investigation of proteins/genes related to specific function. to our knowledge, the pin of e. sinensis is the first large-scale aquatic crustacean protein interaction network, thereby providing a systems biology view of an aquatic crustacean proteome.  transcriptome sequencing of e. sinensis to obtain the e. sinensis transcriptome data, rna from eyestalks, y-organs and hepatopancreas mixed samples of e. sinensis were sequenced with the illumina hiseqtm in total,  nt clean nucleotides were found with q and gc percentages of % and %, respectively., clean reads were then obtained. from these clean reads, contigs were assembled and then unigenes were constructed from contigs with soap de novo, including distinct singletons and distinct clusters. the sequenced unigenes were subsequently aligned against the nr database using blastn and blastx searching with e-value < 1*e- finally unigenes were matched. with nr annotation, go annotations of unigenes were obtained with the blast2go program. among the total unigenes of e. sinensis, unigenes were annotated to the go database with confident matches, including assigned to the biological process category, assigned to the cellular component category and assigned to the molecular function category. after the go annotation of each unigene, wego software was used to obtain the go functional classification for all unigenes in biological process category and to understand the distribution of gene functions from the macro level. in the biological process category, unigenes were divided into different biological processes. cellular process and metabolic process were most highly represented among them, other processed such as biological regulation, developmental process, localization, multicellular organismal process, regulation of biological process and response to stimulus were also included in biological process. the transcriptome sequencing and go annotation results can be found in additional file the protein information of model organisms the protein sequence data of six model organisms were downloaded from the uniprot database. the number of protein sequences in each organism is shown in table  the protein interaction information of the above six model organisms was obtained from the pina database. usually, protein names were used in the protein interactions for presenting proteins, whereas for some interactions, the proteins in different model organisms were presented in different ways, which needed to be normalized for the convenience of the following analysis. two special situations between protein ids and protein names should be pretreated. firstly, when there is no related protein name for a protein, the protein id was used to mark the protein instead of a protein name. secondly, sometimes the same protein is represented in different ways in different interactions. some are marked with the protein name and the others are marked with the protein id. in this situation, the protein name was used instead of the protein id. for example, vha36- is a protein in d. melanogaster. in the interaction pair: uniprotkb:q7plp5-uniprotkb:a1z8v it is represented with the protein id a1z8v and in another interaction pair: uniprotkb:rpl15- uniprotkb:vha36- it is represented with its protein name vha36- we replaced a1z8v with vha36- in the first interaction to represent the protein. in this way, and protein names were pretreated in c. elegans, d. melanogaster, h. sapiens, m. musculus, r. norvegicus and s. cerevisiae, respectively. the number of proteins and protein-protein interactions of the six model organisms from pina are shown in table  construction of model-organism-based protein-protein interaction sub-network in order to construct the pin network of e. sinensis, the model-organism-based protein-protein interaction sub-networks were first constructed. the sub-networks were constructed based on the sequence alignment of model organisms and e. sinensis. according to the alignment result, the homologous sequence existing in each protein interaction of a model organism was marked. if two proteins in an interaction can both be matched with the unigenes in e. sinensis, then these two proteins and their interaction were considered as part of the model-organism-based sub-network. in this way, different protein-protein interaction sub-networks were constructed based on the protein information of model organisms respectively. the numbers of proteins, related unigenes and interactions in each model-organism-based protein-protein interaction sub-network are shown in table  the unigenes and proteins in the sub-networks are not in one-to-one relationships. for example, protein yrt in d. melanogaste based sub-network is related to two unigenes in e. sinensis. e. sinensis unigene a b c athe number in bracket shows the proportion of related unigenes accounting for all the unigenes in the model organism. bthe number in brackets shows the proportion of proteins in the sub-network accounting for all the proteins in the model organism. cthe number in brackets shows the proportion of interaction pairs in the sub-network accounting for all the interaction pairs in the model organism. construction of pin for e. sinensis the pin of e. sinensis was constructed according to the method described in the methods section. the organism with a closer relationship to e. sinensis was integrated preferentially. d. melanogaster belongs to the arthropoda-insecta, which has the closest genetic relationship with e. sinensis compared with the other organisms. c. elegans belongs to protocoelomata-nematomorpha, which is more primitive than arthropoda-crustacea, but has a closer genetic relationship with e. sinensis than the other organisms. therefore, d. melanogaster and c. elegans based sub-networks were used as the target and query networks respectively in the first turn integration. subsequently the h. sapiens, r. norvegicus, m. musculus and s. cerevisiae based sub-network were aligned and integrated in order. one thing to mention is that r. norvegicus and m. musculus actually have similar genetic relationship with h. sapiens. however, the protein and interaction information of h. sapiens from uniprot and pina database is much more than that of r. norvegicus and m. musculus. considering the attribution of the organisms to the pin reconstruction, the h. sapiens based sub-network was integrated prior to r. norvegicus and m. musculus. various numbers of protein interactions were added to the integrated network in each turn of integration and thus the pin of e. sinensis was expanded based on the model organisms. finally the pin of e. sinensis was obtained after five-turn integrations of the above sub-networks. the final e. sinensis pin contained proteins and interaction pairs. the scale of each intermediate integrated network after each turn of integration is shown in table  the pin of e. sinensis is composed of a largest weakly connected component and several small components. as the lwcc contains most of the nodes and edges in the global pin, the topological features of the lwcc were analyzed and compared with that of the model-organism-based sub-networks. the basic features of lwccs are shown in table  actually, the features of lwccs from different sub-networks are quite different from each other. for example, the number of nodes and edges in d. melanogaster based sub-network is much more than that in c. elegans based sub-network, and the diameter is smaller, which means the connectivity of the d. melanogaster based sub-network is stronger than that of the c. elegans based sub-network. the smaller path length further proved this conclusion. the s. cerevisiae based sub-network obviously has the strongest connectivity. generally the topological parameter values of e. sinensis are between that of s. cerevisiae and the other five organisms, indicating that the pin of e. sinensis assimilates the information in the six model organisms as well as eliminating the redundant information. e. sinensis pin score of protein-protein interaction pair as the proteins and interactions in the pin came from different model organisms with various genetic relationships with e. sinensis, in order to represent the confidence of the interaction pairs, the score of each protein-protein interaction pair was evaluated according to the method described in the methods section. the highest score is reached by interaction pairs. the highest score of edges and nodes are and respectively. figure  shows the score distribution of the interaction pairs in the pin of e. sinensis. % interaction pairs distribute in scores of 11– the interaction pairs with scores of 6– and 16– make up % and % of all the interaction pairs respectively. the score of each interaction pair is shown in additional file e. sinensis protein-protein interaction pairs. the number of interaction pairs with scores above or equal to is only. these interactions exist in most of the model organisms, indicating their conservatism. for example, proteins pros, mov pros tbp- and rpn in these interactions are part of 26s protease. mov is a regulation subunit and rpn is a metal protease component of 26s protease. 26s protease is a huge protease complex widely found in many organisms. pros mov cg tcp-1zeta, lwr and skpa are the hub proteins with their degree larger than which exist in at least four model organisms and have important biological function in the metabolism by influencing many of their neighbor proteins. identification of signaling sub-network in e. sinensis signaling pathways are significant mediates of cell growth, development, immune and other life activities, which play a crucial role in almost all growth stages. to better understand the signaling systems of e. sinensis, the proteins with go annotation “signaling” and their related interactions were extracted from the global pin to generate the signaling sub-network. there are in total proteins with go: in e. sinensis, in which are isolated and interacted with other protein, the number of protein-protein interaction relationships is. seven known signal transduction pathways in the kegg database were found in e. sinensis: hippo, jak-stat mtor, wnt, mapk, the notch signal transduction pathway and the protein assembly process in endoplasmic reticulum. some proteins are active in two or more signal pathways. proteins in the above pathways also interacted with other proteins in the pin and finally the sub-network contains proteins and interaction pairs, including the proteins in known signaling pathways and their neighboring proteins. the proteins with interaction pairs in known signaling pathways are shown in figure 3a, in which proteins and interaction pair are isolated from the fully connected sub-network. the hippo signal transduction pathway is responsible for the growth inhibition of cells, which is a highly conservative pathway. it was first found in d. melanogaster and has been found in many mammals such as r. norvegicus and h. sapiens. the hippo signal transduction pathway has significant function in organ size control, stem cell self-renewal, cancer inhibition and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. proteins wts, hpo and sav in this pathway are found to be responsible for cancer inhibition. the interaction of hpo and sav is able to phosphorylate and activate the complex composed of wts and mats. two top cell skeleton signal proteins mer and ex can be reciprocally activated with kibra to further activate the hippo pathway. in addition, wts can directly phosphorylate, and thus inhibit the activating transcription factor yki. and yki is closely related with cell multiplication and apoptosis. the hippo related proteins wts, hpo, sav, mats, mer, yki and kibra were found in the signaling sub-network of e. sinensis, indicating that the hippo signal transduction pathway also exists in e. sinensis. the growth control and cell self-renewal of e. sinensis is probably dominated by the hippo pathway. the jak-stat signal transduction pathway is composed of the ptk related receptor, ptk jak and transcription factor. it is simulated by cytokine and participates in many important biological processes such as cell multiplication, differentiation, apoptosis and immunoregulation. the pias protein in this pathway can inhibit activation of the stat protein by blocking the binding activity of the transcription factor and dna. in addition, it is reported that pias can interact with more than proteins, many of which are immune-system-related. the stat and pias proteins were found in the signal network of e. sinensis,coming from the h. sapiens based sub-network. the other proteins in the jak-stat pathway came from the d. melanogaster based sub-network, such as the suppressor of cytokine signaling, which inhibits the phosphorylation of stat by combining and blocking jak or competing for the phosphotyrosine site on the cytokine receptor with stat. the multiplication, differentiation and apoptosis of e. sinensis are possibly controlled by the jak-stat pathway. the different source of proteins indicated that the integration process provided more information for the pin of e. sinensis. in addition, the mtor, wnt, mapk, notch and protein processing in endoplasmic reticulum were also found in the signaling sub-network of e. sinensis. the mtor pathway is a central regulator for both cell proliferation and cell growth. the wnt pathway is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal in a number of adult tissues. many studies report that mutation of the wnt pathway is closely related to several hereditary diseases and cancers. the notch pathway is first found in d. melanogaster and participates in the regulation of cell multiplication, differentiation, and apoptosis, and acts as an important regulator of immune cells development. the seven signaling transduction pathways found in e. sinensis represent the regulation of basic cell life activity about growth, development, reproduction and disease-resistance. the signaling sub-network of e. sinensis provides substantial information of the signal transduction pathways and unknown proteins which need to be further studied. evolution path of e. sinensis signaling network the signaling network has been used to understand evolution in multicellular animals. as the e. sinensis signaling sub-network was obtained from the integration of six model organisms and these organisms are located in different evolutionary stages, in order to promote understanding of the evolution of the signaling sub-network in e. sinensis, we examined the evolution path by comparing the e. sinensis signaling network with the six model organisms, and investigated the original organisms and preferred evolution paths of the e. sinensis signaling network. the six species were classified into three groups: primitive, bilaterian and vertebrate groups as described in lei li’s work. the primitive group included s. cerevisiae. the bilaterian lineage was composed of d. melanogaster and c. elegans. all three vertebrate species were placed in the vertebrate group. based on this group partition, we identified each protein interaction in the e. sinensis signaling sub-network. for each protein interaction, the origin of two proteins and an interaction were defined separately using the principle in lei li’s work. if a protein/interaction exists in a primitive organism, it is assigned to a primitive origin. if a protein/interaction exists in a bilaterian organism but not in a primitive organism, it is assigned to a bilaterian origin. if a protein/interaction exists only in vertebrate organism, it is assigned to a vertebrate origin. finally, the origin of a complete protein interaction was assigned to the evolutionary stage in which the last component in the protein interaction appeared. the origin groups of seven known signaling pathways were examined. we found that different signaling pathways had various evolutionary paths. protein processing in er is relatively complete in the primitive stage with more than half of the interactions in this stage. most interactions in the hippo pathway exist in the bilateria and vertebrate stages, which is consistent with its first discovery in d. melanogaster. several interactions in the hippo pathway originated from the primitive stage but no complete path was formed until it was found in d. melanogaster. the same situation also appears in the jak-stat, mtor and wnt pathway, indicating that these pathways may grow in the primitive stage and mature in the bilateria stages. mapk and notch have the latest evolution origin, with all interactions found in the biliteria and vertebrate stages. function assignment of unclassified proteins and unigenes according to the go biological process annotation of proteins in pin, the functions of proteins related to unigenes were annotated, whereas the functions of the other proteins related to unigenes were still unknown, which makes up approximately 23% of all the proteins and 19% of all the unigenes. in order to investigate the functions of these unclassified proteins and unigenes, the method described in the methods section was used to assign go annotations. finally, unclassified proteins related to unclassified unigenes were annotated, making up 76% and 73% of all the unknown proteins and unigenes. e. sinensis. as the go terms are organized in a treelike structure, we further analyzed the number distribution of newly-annotated proteins and unigenes in different go depths. as go depth increases, the annotation becomes more detailed. as shown in figure  the fifth layer of the go treelike structure contains the most assigned proteins and unigenes. as proteins or unigenes may have multiple functions, the go annotation and proteins/unigenes are not in one-to-one relationship. proteins were annotated as immune response-related proteins. they functioned in innate immune response, humoral immune response, mucosal immune response, regulation of innate immune response and unclear immune response. furthermore, signaling-related proteins were found. they acted as receptors of signaling factor or the regulators of signaling pathways. for example, proteins were annotated as hippo signaling cascade and other proteins functioned as regulators of hippo signaling cascade. although the functions of the proteins/unigenes still need to be further validated, the assignment of functions provides important reference for identification of the targets in the in vivo experiment.  with the improvement in high-throughput sequencing technology, rna sequencing and annotation are possible for further analysis and detection in the pursuit of certain biological goals. in present work we constructed a pin of e. sinensis on the basis of transcriptomics sequencing and the proteome of six model organisms. the pin defines a primary protein interaction landscape for e. sinensis cells that allows study of sub-networks with specific function. seven known pathways were identified in the signaling sub-network extracted from the global pin. with the analysis of evolution paths for these pathways, we found their differences in evolution origin. more proteins identified as neighbors of the proteins in seven identified pathways were prepared for further confirmation. furthermore, the function assignment of unclassified proteins offers a new reference in protein function exploration. it is the first large-scale pin of aquatic crustaceans, thereby providing necessary experience for the exploration of pin for other aquatic crustacean species, as well as supplying a systems biological view of an aquatic crustacean interactome. 
the pin generated in this work is the first large-scale pin of aquatic crustacean, thereby providing a paradigmatic blueprint of the aquatic crustacean interactome. furthermore, the function assignment of unclassified proteins based on the pin offers a new reference in protein function exploration. however, in recent years, the development of next-generation deep-sequencing techniques makes it possible to get high throughput data of e. sinensis tanscriptome and subsequently obtain a systematic overview of the protein-protein interaction system. by investigating the evolution paths of the seven pathways, we found that these pathways got mature in different evolutionary stages. more importantly, the construction of the e. sinensis pin provides necessary experience for the exploration of pins in other aquatic crustacean species. eriocheir sinensis is a highly-commercial aquaculture species with an unclear proteome background which hinders the construction and development of pin for e. sinensis. the signaling sub-network, representing the signal transduction pathways in e. sinensis, was extracted from the global network, which depicted a global view of the signaling systems in e. sinensis. seven basic signal transduction pathways were identified in e. sinensis. signaling sub-network extracted from the global pin depicts the interaction of different signaling proteins and the evolutionary paths of the identified signal transduction pathways. in this work we sequenced the transcriptional rna of eyestalk, y-organ and hepatopancreas in e. sinensis and generated a pin of e. sinensis which included proteins and interactions. moreover, the functions of unclassified proteins and unigenes in the pin of e. sinensis were predicted. each protein-protein interaction in the network was scored according to the homology and genetic relationship.
 the protein-protein interaction network is an effective information tool for understanding the complex biological processes inside the cell and solving many biological problems such as signaling pathway identification and prediction of protein functions. eriocheir sinensis is a highly-commercial aquaculture species with an unclear proteome background which hinders the construction and development of pin for e. sinensis. however, in recent years, the development of next-generation deep-sequencing techniques makes it possible to get high throughput data of e. sinensis tanscriptome and subsequently obtain a systematic overview of the protein-protein interaction system.  in this work we sequenced the transcriptional rna of eyestalk, y-organ and hepatopancreas in e. sinensis and generated a pin of e. sinensis which included proteins and interactions. each protein-protein interaction in the network was scored according to the homology and genetic relationship. the signaling sub-network, representing the signal transduction pathways in e. sinensis, was extracted from the global network, which depicted a global view of the signaling systems in e. sinensis. seven basic signal transduction pathways were identified in e. sinensis. by investigating the evolution paths of the seven pathways, we found that these pathways got mature in different evolutionary stages. moreover, the functions of unclassified proteins and unigenes in the pin of e. sinensis were predicted. specifically, the functions of unclassified proteins related to unclassified unigenes were assigned, which respectively covered 76% and 73% of all the unclassified proteins and unigenes in the network.  the pin generated in this work is the first large-scale pin of aquatic crustacean, thereby providing a paradigmatic blueprint of the aquatic crustacean interactome. signaling sub-network extracted from the global pin depicts the interaction of different signaling proteins and the evolutionary paths of the identified signal transduction pathways. furthermore, the function assignment of unclassified proteins based on the pin offers a new reference in protein function exploration. more importantly, the construction of the e. sinensis pin provides necessary experience for the exploration of pins in other aquatic crustacean species. eriocheir sinensischinese mitten crabnetwork integrationsignal transductionevolution path 