crustaceans like other invertebrates only have innate immunity , including many immune molecules to eliminate exogenous pathogens . to date
, many studies have verified that hemocyanin is an important nonspecific innate immune defense molecule and can provide an effective immune defense in arthropods [ 28 ] .
its primary function is to transport and store oxygen and also to participate in osmoregulation , molt cycle , exoskeleton formation , and melanin synthesis [ 1012 ] .
so far , studies on the immunologic function in crustacean are mainly focused on the hemocyanin itself or its degraded peptides , which have hemolytic activity , agglutination property , and antiviral function [ 2 , 4 , 5 , 7 , 13 ] .
hemocyanin genes have been cloned and characterized in certain crustacean species such as litopenaeus vannamei , eriocheir sinensis , cherax quadricarinatus , homarus americanus , and caridina multidentata and atyopsis moluccensis .
copper is a central component of hemocyanin and a cofactor of many other enzymes , like superoxide dismutase , cytochrome oxidase , and lysyl oxidase [ 19 , 20 ]
. the dietary copper level can affect crustacean immune responses in penaeus monodon   and e. sinensis .
it was found that the expression of hemocyanin mrna in e. sinensis was affected by dietary copper level .
investigation of the functional relationship between dietary copper and hemocyanin mrna expressions can provide better understanding on crustacean innate immunity and offer insight into disease control through dietary management in shrimp farming .
we hypothesize that copper as a major component of hemocyanin can impact prawn innate immunity through dietary copper manipulation . oriental river prawn ( macrobrachium nipponense ) is an important aquaculture species in china and other southeast asian countries .
various diseases have been found in the m. nipponense farming population due to intensive culture and environmental pollution .
for example , the bacterial disease induced by aeromonas hydrophila is one of the major diseases which can cause 30% death of prawn , sometimes as high as 70% . as a result
, the investigation on the m. nipponense innate immune mechanism against a. hydrophila has become a key issue of health management in crustacean farming .
the hemocyanin subunit is a functional group for crustacean immunity , and a hemocyanin subunit has been cloned and characterized in the freshwater prawn m. nipponense . in this study , we discovered another hemocyanin subunit in m. nipponense ( mnhc-1 ) . to understand the role of mnhc-1 in m. nipponense immunization , ( 1 ) the full - length cdnas of mnhc-1
was cloned , ( 2 ) the distribution of mnhc-1 in different tissues was examined , ( 3 ) the mrna expression of mnhc-1 in immune defense after prawn challenge with a. hydrophila was examined , and ( 4 ) the relative expression level of mnhc-1 in the hepatopancreas of m. nipponense fed different levels of copper was quantified .
all adult oriental river prawns ( m. nipponense ) were obtained from a local farm in shanghai .
the hepatopancreas , muscle , gill , ovary , intestine , heart , and stomach were collected from healthy prawn , flash - frozen in liquid nitrogen , and stored at 80c until rna extraction .
a total of 1  ml hemolymph was collected from the ventral sinus using a sterile syringe and diluted using half volume of anticoagulant solution , then centrifuged at 8000  g for 10  min at 4c to collect the hemocyte , and stored at 80c immediately until rna extraction .
prior to the challenge experiment , the adult prawns were acclimatized in the laboratory for 2 weeks .
a total of 300 healthy prawns were randomly divided into two groups with five replicates . according to the preliminary experiment ,
the prawn in the bacterial challenge trial was injected with 100  l a. hydrophila in saline suspension ( 1  10  cfu / ml ) obtained from shanghai ocean university , while each prawn in the control group received the same volume of saline injection .
after injection , prawns were put back to the rearing tanks , and hemocyte samples were collected at 0 , 3 , 6 , 12 , 24 , and 48  h after injection , centrifuged , and stored at 80c for rna extraction .
the juvenile m. nipponense were obtained from the same farm as the adults and acclimated for two weeks in the laboratory conditions prior to the feeding trial .
the basal diet was supplemented with copper sulphate ( analytical reagent , shanghai chemical co. , shanghai , china ) at 0 , 10 , 20 , 30 , 40 , 80 , and 160  mg  cu  kg diet , respectively .
the actual copper concentrations in the feeds were analyzed to be 2.8 , 12.2 , 20.9 , 29.8 , 43.1 , 78.9 , and 157.1  mg  kg , respectively , by the flame atomic absorption photometry .
prawn juveniles ( 0.101  0.001  g ) were randomly placed in 21 of 300-l tanks with 30 prawns per tank in triplicate .
prawns were fed to apparent satiation twice daily ( 8:00 and 17:00  h ) for 56 days . to maintain water quality ,
one - third of the tank water was exchanged daily . during the feeding period ,
l , and total ammonia nitrogen < 0.1  mg  l. the cu concentration in rearing water was 1.41.7  g  cu  l. at the end of the feeding trial , all prawns were counted and gained survival rate ( survival rate = 100  ( final prawn number)/(initial prawn number ) ) .
total rna was isolated using rna extraction kit ( aidlab biotech , beijing , china ) following manufacture protocol . in reverse transcription reaction
, 3  g of total rna was used for synthesis of first - strand cdna by the primescript rt - pcr kit ( takara , dalian , china ) .
acquired cdna was stored at 20c for subsequent quantitative real - time pcr ( qrt - pcr ) . to get the full length of mnhc-1 cdna sequence , smart race cdna amplification kit ( clontech , usa )
gene - specific primers of mnhc-1 were designed based on the known fragments initially identified from the est cdna library of m. nipponense .
reaction volume was 50  l containing 5.0  l 10x ex taq buffer ( mg plus ) , 5.0  l 10x upm , 4.0  l cdna template , 4.0  l dntps mix ( 2.5  mm each ) , 1.0  l gsp ( 10  m ) , 0.26  l ex taq hs ( takara , dalian , china ) , and 30.74  l of sterile deionized water . the pcr conditions were set as follows : 5 cycles of 94c for 30  s and 72c for 3  min , then 5 cycles of 94c for 30  s and 70c for 30  s , and 25 cycles of 94c for 30  s , 68c for 30  s , and 72c for 3  min .
primers of mnhc - s1 and mnhc - a1 were used to confirm the mnhc-1 fragment from the est cdna library .
5-cds primer a ( as the rt primer ) , gene - specific primer of mnhc-5gsp , and the upm ( universal primer a mix ) were used for the 5-race .
3-cds primer a ( as the rt primer ) , gene - specific primer of mnhc-3gsp , and the upm were used for the 3-race .
the pcr products were purified by the uniq-10 gel extraction kit ( sangon , shanghai , china ) and cloned into the pucmt vector ( sangon , shanghai , china ) .
the transformed bacteria were identified , confirmed by blue / white screening , and validated by pcr .
more than two recombinant plasmids were sequenced using bigdye terminator v3.1 cycle sequencing kit operating on an automatic dna sequencer ( abi 3730xl dna analyzer ) .
the online orf finder program ( http://www.ncbi.nlm.nih.gov/gorf/ ) was used to predict the gene 's putative open reading frame .
the blast algorithm at the national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/blast/ ) was applied to search for sequence homology .
smart ( simple modular architecture research tool , http://smart.embl-heidelberg.de/ ) was used to predict conserved motifs .
the deduced amino acid sequence of mnhc-1 from m. nipponense and other invertebrate hemocyanin sequences acquired from ncbi database were aligned by software clustalx .
a neighbor - joining ( nj ) phylogenetic relationship was established based on amino acid sequences of the hemocyanin using the mega 5.1 program ( http://www.megasoftware.net/ ) .
the mrna expression of mnhc-1 in the hepatopancreas , muscle , gill , ovary , intestine , heart , and stomach was detected by qrt - pcr with -actin as internal control .
the synthesis of the first - strand cdna was the same as that described above .
gene - specific primers of mnhc - s2 , mnhc - a2 , -actin - s , and -actin - a were used in qrt - pcr ( table 2 ) .
the qrt - pcr liquid compositions and conditions were according to the manufacturer instructions of sybr premix ex taq ( takara , dalian , china ) . the qrt - pcr was conducted on the cfx96 real - time system ( bio - rad , usa ) according to the manufacturer 's protocols . during the detection ,
the melt curve of the amplification products was analyzed to ensure that only one pcr product was amplified and detected at the end of each pcr .
the mrna expression of mnhc-1 in the hemocytes of m. nipponense injected with a. hydrophila or saline water was , respectively , detected at 0 , 3 , 6 , 12 , 24 , and 48  h after injection by a quantitative real - time rt - pcr . at the feeding experiment , mrna expression of mnhc-1 in hepatopancreas of m. nipponense from the feeding treatment at seven copper levels was analyzed by qrt - pcr .
the results of relative mrna expression in challenge test were analyzed by t - test , while other results were subjected to one - way anova and post hoc duncan multiple range tests .
the full length of mnhc-1 cdna from the m. nipponense was 2,163  bp ( genbank accession number jx456149.1 ) , containing a 20  bp 5-untranslated region , a 115  bp 3-untranslated region with poly a tail , and a 2,028  bp open reading frame ( orf ) ( figure 1 ) .
the orf encoded a polypeptide of 675 amino acids with a calculated molecular weight of 78.060  kda and an isoelectric point of 5.45 .
a putative signal peptide of 21 amino acids was found in the n - terminus by using signalp 4.1 program .
smart program predicted that the mnhc-1 belongs to the hemocyanin family , including n terminal domain ( ser24-val150 ) , typical copper - containing domain ( pro154-glu411 ) , and c terminal domain ( pro417-his667 amino acid ) .
six histidine residues ( h212 , h216 , h242 , h362 , h366 , and h402 ) of copper - binding sites were identified .
homology analysis with blast algorithm showed that mnhc-1 amino acid sequences had 75% , 73% , 70% , and 68% similarity to that of the a. moluccensis gamma subunit , p. monodon , c. quadricarinatus , and e. sinensis , respectively .
the mnhc-1 exhibited 64% identity with another subunit of hemocyanin in m. nipponense ( mnhc-2 , genbank accession number jf683437.1 ) .
a phylogenetic tree was constructed with the 24 full - length hemocyanin sequences from arthropod based on the neighbor - joining method ( figure 2 ) .
the result of phylogenetic analysis revealed that mnhc-1 was more closely related to the hemocyanin gamma subunit 1 of freshwater shrimps a. moluccensis and c. multidentata .
the mrna transcripts of mnhc-1 were analyzed in the tissues of hepatopancreas , gill , muscle , hemocytes , intestine , ovary , and stomach . in prawn ,
the highest expression was found in hepatopancreas ( p < 0.05 ) ; the expression value is 20.32 , which is 20 times higher or more compared with other tissues .
the value is as low as 0 so that the gene is not expressed in muscle ( figure 3 ) .
figure 4 exhibits the expression profile of mnhc-1 in the hemocytes challenged by a. hydrophila from 0 to 48  h. the expression of mnhc-1 showed a distinct time - dependent pattern .
the mrna expression level significantly dropped at 3  h after injection ( 0.6 times ) ( p < 0.05 ) and then started to significantly increase and reached the peak in 12  h ( 6.2 times ) ( p < 0.05 ) , followed by a recovery to the initial level in 24  h and 48  h. survival rate ( 7081% ) of prawns was not affected by the dietary copper levels . as shown in figure 5 , the response of mnhc-1 expression in the hepatopancreas was affected by the dietary copper level . with the dietary copper level increasing from 43.1 to 157.1  mg  cu  kg diet ,
the level of mnhc-1 mrna in hepatopancreas of prawns significantly increased ( 1.9-fold to 5.8-fold ) ( p < 0.05 ) and was significantly higher compared to that fed 2.829.8  mg  cu  kg diet ( p < 0.05 ) , but differences in 2.829.8 groups were not significant ( p > 0.05 ) .
those subunits differ considerably in their primary structures and are encoded by distinct genes . in this study , we cloned and characterized the expression pattern of one hemocyanin subunit from m. nipponense ( mnhc-1 ) .
mnhc-1 was a polypeptide of 675 amino acids with a 21-amino acid putative signal peptide .
the signal peptide ends ala - x - ala motif , which is a frequent accordance prior to the cleavage site of signal peptides , suggesting that a cleavage site is located at the 21 - 22 amino acids [ 32 , 35 ] .
structurally , mnhc-1 has conservative copper - binding domains including six histidine residues ( h212 , h216 , h242 , h362 , h366 , and h402 ) of the copper - binding sites ; this domain agrees with other crustaceans [ 4 , 14 , 15 ] .
based on immunological methods , the crustacean hemocyanin subunits are classified into three distinct subunit types : alpha , beta , and gamma .
the phylogenetic analysis showed that mnhc-1 and mnhc-2 belong to separate clade ; mnhc-1 belongs to the gamma subunit which has evolved at a later time compared to alpha and beta subunits in freshwater shrimps .
the present study showed that the highest level of mnhc-1 mrna expression occurred in the hepatopancreas .
the result is the same as the findings in other crustaceans , such as h. americanus , fenneropenaeus chinensis , and e. sinensis , and consistent with the report that hemocyanin synthesis occurs mainly in the hepatopancreas .
mnhc-1 expression was detected in all the examined tissues except for muscle , which is different from another subunit of hemocyanin in m. nipponense ( mnhc-2 ) expressed in the muscle .
we also found that mnhc-1 was expressed in ovary , whereas mnhc-2 was hardly expressed in ovary .
the discrepant expression patterns of these two subunits of hemocyanin may be owing to their functional specialization in different tissues .
hemocyanin is an important multifunctional protein in mollusks and arthropods . besides its role as an oxygen carrier ,
its immune functions including antibacterial activities , agglutination property , and po activity have become hot topics of immunological research [ 2 , 4 , 5 , 7 ] .
zhang et al .   found that the main protein directly bound to the vibrio alginolyticus , vibrio harveyi , a. hydrophila , and staphylococcus aureus in l. vannamei serum was hemocyanin , suggesting that hemocyanin possesses antibacterial functions .
the c - terminus of l. vannamei hemocyanin is possibly related to the immunity in shrimp to different pathogens .
gram - negative bacteria a. hydrophila are a common species of the aeromonas genus in water and water habitants .
the infection of a. hydrophila in fish and prawn including m. nipponense has been one of the major diseases under farming conditions [ 25 , 41 ] .
sun et al .   showed that the hemocyanin gene expression of e. sinensis was significantly upregulated by a. hydrophila infection . in our study ,
temporal and spatial expressions of mnhc-1 in the hemocytes of prawn infected with a. hydrophila showed a clear time - dependent pattern .
the level of mnhc-1 mrna expression significantly decreased at 3  h after injection , then started to significantly increase after 6  h and 12  h , and then reached the peak at 12  h , implying that the hemocyanin is involved in the antibacterial defense of prawn . in another study , the level of mnhc-2 mrna expression in prawn significantly increased over time and peaked at 3  h after the a. hydrophila challenge .
lei et al .   found a similar result in p. japonicus that pjhcl transcriptional upregulation occurred after 4  h of injection of the active wssv .
it is possible that pjhcl may be triggered by the fast expressed proteins in virus .
it is also likely that mnhc-1 may be induced by fast expressed protein in bacteria , but the detailed defensing mechanism of hemocyanin against bacterial infection needs further study .
the expression of hemocyanin subunits varies with environmental or nutritional changes [ 42 , 43 ] .
our present study showed that the level of dietary copper affected the hemocyanin gene expression in prawn .
the level of mnhc-1 mrna in hepatopancreas of the prawn fed 43.1157.1  mg  cu  kg diet was significantly higher compared to that fed 2.829.8  mg  cu  kg .
the increase of dietary cu concentration also increased the cu content in hepatopancreas , especially in the 43.1157.1  mg  cu  kg groups .
therefore , we suggest that the high level of copper content in the hepatopancreas can trigger mnhc-1 mrna expression , whereas the optimal level of dietary copper ( 2040  mg  kg diet ) can increase hemocyanin mrna expression in the hepatopancreas and hemocytes of crab .
these studies suggest that the effect of dietary copper level on hemocyanin mrna expression is species - specific .
it seems that the two subunits of hemocyanin may have structural functions in their own hemocyanin and show different response to the level of dietary copper .
dietary copper may be first used for hemocyanin synthesis as low dietary copper did not reduce the level of mnhc-1 mrna . in conclusion , we cloned the hemocyanin subunit gene ( mnhc-1 ) from the hepatopancreas of m. nipponense .
our results suggest that mnhc-1 may play a critical role in antibacterial defense in prawn .
these results provide the foundation for further studies in biological function and regulation of hemocyanin in crustacean .
 hemocyanin is a copper - containing protein with immune function against disease . in this study , 
 a hemocyanin subunit named mnhc-1 was cloned from macrobrachium nipponense . 
 the full - length cdna of mnhc-1 was 2,163  bp with a 2,028-bp open reading frame ( orf ) encoding a polypeptide of 675 amino acids . 
 the mnhc-1 mrna was expressed in the hepatopancreas , gill , hemocytes , intestine , ovary , and stomach , with the highest level in the hepatopancreas . in the infection trial 
 , the mnhc-1 mrna transcripts in the hemocytes were significantly downregulated at 3  h after injection of aeromonas hydrophila and then upregulated at 6  h and 12  h , followed by a gradual recovery from 24 to 48  h. the mnhc-1 transcriptional expression in the hepatopancreas was measured after m. nipponense were fed seven diets with 2.8 , 12.2 , 20.9 , 29.8 , 43.1 , 78.9 , and 157.1  mg  cu  kg1 for 8 weeks , respectively . 
 the level of mnhc-1 mrna was significantly higher in the prawns fed 43.1157.1  mg  cu  kg1 diet than in that fed 2.829.8  mg  cu  kg1 diet . 
 this study indicated that the mnhc-1 expression can be affected by dietary copper and the hemocyanin may potentially participate in the antibacterial defense of m. nipponense . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion