Characterization of a pseudohemocyanin gene (PtPhc1) and its immunity function in response to Vibrio parahaemolyticus infection in the swimming crab Portunus trituberculatus.

Pseudohemocyanin is a member of the hemocyanin superfamily, but little research is available on its function in immunology. In this study, a Portunus trituberculatus pseudohemocyanin gene, named PtPhc1, was obtained by gene cloning. The PtPhc1 cDNA was 2312 bp in length, encoding 684 amino acids while exhibiting a characteristic hemocyanin structural domain. Tissue expression analysis revealed ubiquitous expression of PtPhc1 across all tissues, with the highest level of expression observed in the hepatopancreas. The expression pattern of PtPhc1 in response to Vibrio parahaemolyticus infection was clarified using RT-qPCR in swimming crabs. Notably, the expression peaked at 24 h, and increased 1435-fold compared to the control group in the hepatopancreas. While the expression level reached the maximum value at 72 h, which was 3.24 times higher than that of the control group in hemocytes. Remarkably, the reduction in PtPhc1 expression led to a noteworthy 30% increase in the mortality rate of P. trituberculatus when exposed to V. parahaemolyticus. In addition, in vitro bacterial inhibition assays exhibited a dose-dependent suppression of bacterial proliferation by recombinant PtPhc1 protein, with a notable inhibition rate of 48.33% against V. parahaemolyticus at a concentration of 0.03 mg/mL. To the best of our knowledge, the results establish the function of pseudohaemocyanin in immunity for the first time, contributing to a deeper comprehension of innate immune regulatory mechanisms in aquatic organisms and advancing strategies for disease-resistant breeding.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp.

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

The Evolution of Hemocyanin Genes in Caenogastropoda: Gene Duplications and Intron Accumulation in Highly Diverse Gastropods.

Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon-intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation.

The krüppel-like factor of Penaeus vannamei negatively regulates transcription of the small subunit hemocyanin gene as part of shrimp immune response.

Hemocyanin is a multifunctional respiratory glycoprotein, which has also been implicated in other biological functions in shrimp. Moreover, recent studies have revealed that hemocyanin is also involved in a broad range of immune-related activities in shrimp. However, in spite of the considerable interest in unraveling the reasons behind the multiple immune-related functions of hemocyanin, little is known about its transcriptional regulation. Here, DNA pull-down and Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) analyses were used to isolate and identify the putative transcription factor(s) that are involved in the transcriptional regulation of the small subunit hemocyanin gene of Penaeus vannamei (PvHMCs). Krüppel-like factor (designated PvKruppel), a zinc finger transcription factor homolog in P. vannamei, was identified among the putative transcription factors, while bioinformatics analysis revealed the presence of Krüppel-like factor binding site (KLF motif) on the core promoter region of PvHMCs. Mutational analysis and electrophoretic mobility shift assay (EMSA) confirmed that PvKruppel could bind to the KLF motif on the core promoter region of PvHMCs. Moreover, in response to lipopolysaccharide (LPS), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge, transcript levels of PvKruppel and PvHMCs were negatively correlated. Furthermore, overexpression of PvKruppel significantly reduced the promoter activity of PvHMCs, while PvKruppel knockdown by RNA interference or lipopolysaccharides (LPS) stimulation resulted in a significant increase in the transcript level of PvHMCs. Taken together, our present study provides mechanistic insights into the transcriptional regulation of PvHMCs by PvKruppel during shrimp immune response to pathogens.

A SNP of the hemocyanin gene (LvHc) is a marker for high growth and ammonia-tolerance in Pacific white shrimp Litopenaeus vannamei.

Expression levels of hemocyanin (LvHc), activating transcription factor 4 (LvAtf4), glutathione S-transferase (LvGst), caspase 2 (LvCasp2) and anti-lipopolysaccharide factor (LvAlf) were examined in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei juveniles exposed to a lethal concentration of ammonia-N (32.15 mg/l). The expression levels of all transcripts except LvAlf were significantly greater (P < 0.05) in tolerant shrimp (Lv-AT; N = 30) that survived up to 72 h post treatment (hpt) than in susceptible shrimp (Lv-AS24 and Lv-AS72; N = 45 and 15), that died within 24 h or between 24 and 72 hpt, respectively. Subsequently, effects of non-lethal concentrations of ammonia-N (control, 10 and 20 mg/l) on the expression of LvHc in juvenile shrimp were examined. Compared to the control, expression levels of LvHc transcripts in hemocytes and the hepatopancreas of tested shrimp changed after exposure to ammonia-N. One SNP (C > T545) was found in the LvHc322 gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was developed for detection of C > T545 genotypes. Juveniles in the lethal exposure test that carried a C/T545 genotype showed a greater average body weight and total length (8.46 ± 0.36 g and 10.05 ± 0.16 cm) than those with a C/C545 genotype (7.48 ± 0.31 g and 9.60 ± 0.13 cm) (P < 0.05). Similar results were found in the second generation (G2) of a growth-improved stock (3 and 4 families of BIOTEC-G2-L1 and BIOTEC-G2-L2) and in commercially farmed shrimp (2 groups). Accordingly, expression levels and SNP of LvHc can serve as markers for selection high growth performance in ammonia-tolerant L. vannamei.

Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Hemocyanin genes as indicators of habitat shifts in Panpulmonata?

Hemocyanin is the primary respiratory protein for the majority of the Mollusca and therefore directly interfaces with the physiological requirements of each species and the environments to which they are adapted. Hemocyanin is therefore likely to have been evolutionarily imprinted by significant habitat shifts. In the gastropod clade Panpulmonata (>30,000 species) major realm transitions have occurred multiple times independently and may have contributed to the diversification of this group. Yet, little is known about the adaptive changes linked to these habitat shifts. In order to gain deeper insight into the evolution of panpulmonate hemocyanins and to infer possible impacts associated with those scenarios, we have assembled and analysed hemocyanin isoforms from 4 panpulmonate species: (i) Helix pomatia, (ii) Cantareus aspersus (both Helicidae, Stylommatophora), (iii) Arion vulgaris (Arionidae, Stylommatophora) and (iv) Lymnaea stagnalis (Lymnaeidae, Hygrophila). Additionally, we describe a new hemocyanin isoform within the genome of the euopisthobranch Aplysia californica. Using these newly acquired hemocyanin data, we performed a phylogenetic analysis that reveals independent duplication events of hemocyanin within lineages that correlate with significant habitat shifts.

Developmental expression and evolution of hexamerin and haemocyanin from Folsomia candida (Collembola).

Haemocyanins constitute a group of copper-containing respiratory proteins, and hexamerins were derived from hexapod haemocyanin but lost the ability to transport oxygen and serve as storage proteins. Although hexamerins have been reported in most insect species, none of them has been identified in Collembola, one of the most primitive hexapod lineages, thereby preventing us from exploring relevant evolutionary scenarios regarding the origin and evolution of hexamerins in hexapods. Here we report on collembolan hexamerins for the first time, and investigated the temporal expression profiles of hexamerin and haemocyanin in the collembolan Folsomia candida. Haemocyanin was expressed over the entire life cycle, with higher expression at the embryonic stage than at other stages, whereas hexamerin expression was restricted to embryos, unlike insect hexamerins, which are generally expressed from larval to adult stages. A phylogenetic analysis and molecular clock estimation suggested that all investigated hexapod hexamerins have a single and ancient origin (~423 Ma), coincident with the rise of atmospheric oxygen levels in the Silurian-Devonian period, indicating a physiological link between molecular evolution and Palaeozoic oxygen changes.

c-Jun regulates the promoter of small subunit hemocyanin gene of Litopenaeus vannamei.

Hemocyanin (HMC) is a respiratory glycoprotein, which also plays multifunctional non-specific innate immune defense functions in shrimp. However, the transcriptional regulatory mechanisms of the hemocyanin gene expression have not been reported. In the present study, we cloned a 4324 bp fragment of small subunit hemocyanin (HMCs) gene of Litopenaeus vannamei including the 5'-flanking region, from upstream 2475 bp to downstream 1849 bp (exon 1-intron 1-exon 2) by genome walking method. Four deletion constructs were then generated and their promoter activity assessed using the luciferase reporter system. Interestingly, we identified an alternative promoter (+1516/+1849 bp) located in exon 2, which has stronger promoter activity than the full-length or the other constructs. Bioinformatics analyses revealed that the alternative promoter region contains two conserved binding sites of the transcription factor c-Jun. Mutational analysis and electrophoretic mobility shift assay showed that Litopenaeus vannamei c-Jun (Lvc-Jun) binds to the region +1582/+1589 bp and +1831/+1837 bp of the alternative promoter. Furthermore, overexpression of Lvc-Jun significantly increased the alternative promoter activity, while co-transfection with dsRNA-Lvc-Jun significantly reduced the alternative promoter activity of HMCs. Taken together, our present data indicate that the transcription factor Lvc-Jun is essential for the transcriptional regulation of the HMCs gene expression.

Functional and structural properties of red color-related pigment-binding protein from the shell of Litopenaeus vannamei.

BACKGROUND: A novel red color-related pigment-binding protein named LvPBP75 isolated from the shell of Litopenaeus vannamei has recently been identified as hemocyanin. However, information on the functional and structural properties of LvPBP75 is insufficient. This study aimed to elucidate the thermal properties and pigment-binding ability of LvPBP75. RESULTS: LvPBP75 showed significant red color change after heat treatment with high concentrations of NaCl (>0.1 mol L-1 ), acidic (<5) or alkaline (>9) pH values and alcohols. LvPBP75 mRNA expression analysis revealed that expression level was highest in hepatopancreas and weakest in muscle. Reconstruction and structural analysis revealed that astaxanthin could bind to hemocyanin derived from the shell of L. vannamei but not to hemocyanins derived from the hepatopancreas or hemolymph of other invertebrates. Three-dimensional models of hemocyanin monomer displayed significant structural differences between native LvPBP75 and hemocyanin derived from shrimp hepatopancreas. CONCLUSION: The results suggest a novel function of hemocyanin as binding with pigment and its involvement in L. vannamei shell color change. The pigment-binding ability of hemocyanins has species and tissue specificity, and their unique structural features play an important role in binding ability. © 2018 Society of Chemical Industry.

Diversity, evolution, and function of myriapod hemocyanins.

BACKGROUND: Hemocyanin transports O2 in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa - Chilopoda, Diplopoda, Symphyla, and Pauropoda - with the aim of identifying hemocyanin-like proteins. RESULTS: We investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O2. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O2 transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O2 binding in the S. dehaani hemolymph, indicating that hemocyanin supports O2 transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult. CONCLUSION: Hemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O2 transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O2 supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.

The spider hemolymph clot proteome reveals high concentrations of hemocyanin and von Willebrand factor-like proteins.

Arthropods include chelicerates, crustaceans, and insects that all have open circulation systems and thus require different properties of their coagulation system than vertebrates. Although the clotting reaction in the chelicerate horseshoe crab (Family: Limulidae) has been described in details, the overall protein composition of the resulting clot has not been analyzed for any of the chelicerates. The largest class among the chelicerates is the arachnids, which includes spiders, ticks, mites, and scorpions. Here, we use a mass spectrometry-based approach to characterize the spider hemolymph clot proteome from the Brazilian whiteknee tarantula, Acanthoscurria geniculata. We focused on the insoluble part of the clot and demonstrated high concentrations of proteins homologous to the hemostasis-related and multimerization-prone von Willebrand factor. These proteins, which include hemolectins and vitellogenin homologous, were previously identified as essential components of the hemolymph clot in crustaceans and insects. Their presence in the spider hemolymph clot suggests that the origin of these proteins' function in coagulation predates the split between chelicerates and mandibulata. The clot proteome reveals that the major proteinaceous component is the oxygen-transporting and phenoloxidase-displaying abundant hemolymph protein hemocyanin, suggesting that this protein also plays a role in clot biology. Furthermore, quantification of the peptidome after coagulation revealed the simultaneous activation of both the innate immune system and the coagulation system. In general, many of the identified clot-proteins are related to the innate immune system, and our results support the previously suggested crosstalk between immunity and coagulation in arthropods.

Distribution and hypoxia-regulation of haemocyanin in springtails (Collembola).

Haemocyanin is the copper-containing respiratory protein present in many arthropods. In the hexapods, respiratory proteins had long been considered unnecessary as sufficient O2 was thought to be obtained via the trachea. Nevertheless, many ametabolous and hemimetabolous hexapod species actually possess haemocyanin. Here we investigated the occurrence of haemocyanin in Collembola (springtails). Haemocyanin was found in 22 collembolan species of the suborders Symphypleona, Tomoceroidea and Entomobryomorpha, demonstrating its widespread occurrence. No haemocyanin was identified in 16 species of these taxa, and it appears to be absent in Poduromorpha. The presence of haemocyanin does not correlate with either the phylogenetic history or lifestyle of the investigated species. We further investigated the function of haemocyanin in Folsomia candida (Entomobryomorpha) by applying different hypoxia regimes. Whereas short-term (1 h) and mild (10% O2 ) hypoxia led to a decrease in haemocyanin mRNA, strong hypoxia (24 h, 1.5% O2 ) resulted in a ∼4300-fold increase in haemocyanin expression. Hypoxia induction of haemocyanin could not be demonstrated in evolutionarily more advanced Hexapoda, where it is restricted to the embryo. The results indicate (1) an important role of haemocyanin in the oxygen supply of F. candida, which may be adaptive in the potentially hypoxic environment in the soil, and (2) a change in haemocyanin function in hexapod evolution.

Molecular cloning and characterization of a hemocyanin from Sepiella maindroni.

Hemocyanins are respiratory proteins occurring freely dissolved in the hemolymph of many arthropods and molluscs. Hemocyanin and hemocyanin-derived peptides have been linked to key aspects of innate immunity. In the present study, the full-length cDNA encoding hemocyanin in Sepiella maindroni (SmHc) was cloned and characterized. Bioinformatic analysis predicted that SmHc contains one open reading frame of 10,032 bp and encodes a polypeptide of 3343 amino acids. Sequence analysis showed that the predicted protein sequence of SmHc contained eight functional units (FUs). Phylogenic analysis revealed that SmHc clustered with the mollusc Hcs. Quantitative real-time PCR assay detected SmHc transcripts were in a wide range of tissues, but mainly distributed in gills. After hypoxia or bacterial challenge, the expression level of SmHc in the gills was significantly higher than that of the control group. These results suggested that SmHc might play important roles in oxygen transport and the modulation of immune response in S. maindroni.

Identification and characterization of an 18.4kDa antimicrobial truncation from shrimp Litopenaeus vannamei hemocyanin upon Vibrio parahaemolyticus infection.

Hemocyanin (HMC) is a multifunctional protein which plays many essential roles in invertebrate organism. Recently more and more immune-related functions have been discovered on this protein. Here the shrimp was infected with Vibrio parahaemolyticus and the shrimp sera were analyzed by two-dimensional gel electrophoresis. Totally 15 spots were identified as significantly up-regulated spots and further analyzed by MALDI-TOF/TOF mass spectrometry (MS). Four of them were identified as HMC derived truncations (HMCS1, HMCS3, HMCS4 and HMCS5). The HMCS4 primary sequence was further determined via Edman N terminal sequencing, MALDI-TOF MS and amino acid sequence alignment. The result indicated that the HMCS4 was a 165aa fragment from shrimp HMC small subunit C-terminal. The HMCS4 immunological activities were further analyzed by agglutination experiment and antibacterial assay in vitro. The results showed that the recombinant HMCS4 (rHMCS4) had strong agglutination and antibacterial activities against pathogenic bacteria at the optimum bacteriostasis concentration. In addition, the HMCS4 immunological activities were explored via mortality assay in vivo. The shrimp was challenged with V. parahaemolyticus and rHMCS4 V. parahaemolyticus mixture separately. The shrimp mortality rate was significantly decreased at 96 h post-infection with rHMCS4 injection. Our data showed that shrimp HMC truncation generation upon infection was an effective immune response against invaded pathogens. Moreover, these findings may have some potential applications in shrimp industry.

High CO2 alters the hypoxia response of the Pacific whiteleg shrimp (Litopenaeus vannamei) transcriptome including known and novel hemocyanin isoforms.

Acclimation to low O2 in many organisms involves changes at the level of the transcriptome. Here we used high-throughput RNA sequencing (RNA-Seq) to explore the global transcriptomic response and specific involvement of a suite of hemocyanin (Hc) subunits to low O2 alone and in combination with high CO2, which naturally co-occurs with low O2. Hepatopancreas mRNA of juvenile L. vannamei exposed to air-saturated water, low O2, or low O2/high CO2 for 4 or 24 h was pooled, sequenced (HiSeq 2500) and assembled (Trinity: 52,190 contigs) to create a deep strand-specific reference transcriptome. Annotation of the assembly revealed sequences encoding the previously described small Hc subunit (HcS), and three full-length isoforms of the large subunit (HcL1-3). In addition to this, a previously unidentified full-length Hc subunit was discovered. Phylogenetic analysis demonstrated the subunit to be a ß-type Hc subunit (denoted HcB), making this the first report of a ß-type hemocyanin subunit in the Penaeoidea. RNAs of individual shrimp were sequenced; regulated genes identified from pairwise comparisons demonstrated a distinct pattern of regulation between prolonged low O2 and low O2/high CO2 treatments by GO term enrichment analysis (Roff-Bentzen, P < 0.0001), showcasing the stabilization of energetically costly translational machinery, mobilization of energy stores, and downregulation of the ubiquitin/proteasomal degradation machinery. Exposure to hypoxia for 24 h resulted in an increase in all of the full-length hemocyanin subunits (HcS, HcL1, HcL2, HcL3, and HcB). The addition of CO2 to hypoxia muted the transcriptomic response of all the Hc subunits to low O2, except for the ß-type subunit.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation.

BACKGROUND: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. RESULTS: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. CONCLUSIONS: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Haemocyanin is essential for embryonic development and survival in the migratory locust.

Haemocyanins are commonly known as copper-containing oxygen carriers within the haemolymph of arthropods, and have been found in many orders of insects. However, it remains unresolved why haemocyanins persist in insects that possess elaborate tracheal systems for oxygen diffusion to cells. Here we identified haemocyanins in the migratory locust Locusta migratoria that consists of two distinct subunits, Hc1 and Hc2. Genomic sequence analysis indicated that Hc1 and Hc2 have four and three gene copies, respectively, which may have evolved via gene duplication followed by divergent evolution of introns. The two subunits exhibit abundant and embryonic-specific expression at the mRNA and protein level; their expression peaks in the mid-term embryo and is not detectable in the late nymphal and adult stages. A larger proportion of the haemocyanins is present in the yolk compared with that in the embryo. Immunostaining shows that haemocyanins in the embryo are mainly expressed in the epidermis. Knockdown of Hc1 and Hc2 results in significant embryonic developmental delay and abnormality as well as reduced egg hatchability, ie the proportion of hatched eggs. These results reveal a previously unappreciated and fundamental role for haemocyanins in embryonic development and survival in insects, probably involving the exchange of molecules (eg O2 ) between the embryo and its environment.

Cloning and characterization of a novel hemocyanin variant LvHMCV4 from shrimp Litopenaeus vannamei.

Recently, we found 3 variants of hemocyanin subunit with higher molecular weight in shrimp Litopenaeus vannamei (Named as LvHMCV1-3). In this study, a novel L. vannamei hemocyanin variant (Named as LvHMCV4) was further cloned and characterized. Bioinformatic analysis predicted that LvHMCV4 contains one open reading frame of 2137 bp and encodes a polypeptide of 678 amino acids. It shares 84-99% cDNA sequences identity to that of the classical form of L. vannamei hemocyanin (LvHMC, AJ250830.1) and LvHMCV1-3. LvHMCV4 possesses a conserved structure characteristic of the hemocyanin family and can be clustered into one branch along with other arthropod hemocyanins in a phylogenetic tree. Further, the full-length DNA of LvHMCV4 contains 2660 bp and two introns, which are located at the 80-538 bp and 2063-2227 bp regions, respectively. In addition, the mRNA transcript of LvHMCV4 was expressed highly in the hepatopancreas, lymphoid, brain and hemocytes, and weakly in the heart, intestine and gill, while no expression was found in the muscle, stomach and gut. Infection by Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes or Staphylococcus aureus up-regulated significantly LvHMCV4 mRNA expression in the hepatopancreas. Furthermore, the recombinant protein of LvHMCV4 (rLvHMCV4) was prepared, which showed agglutination activities against six pathogenic bacteria at concentrations ranging from 15.6 to 125 µg/ml. When co-injected with V. parahaemolyticus in L.vannamei, rLvHMCV4 significantly increased the survival rate after 48 h injection. Together, these studies suggested that hemocyanin variant, LvHMCV4, might be involved in shrimp resistance to pathogenic infection.