Hemocyanin Gene Research Articles

Molecular Identification and Hemocyanin Gene (HMC) Characterization of the Shrimp Litopenaeus vannamei Infected by Acute Hepatopancreatic Necrosis Disease (AHPND)

Molecular Identification and Hemocyanin Gene (HMC) Characterization of the Shrimp Litopenaeus vannamei Infected by Acute Hepatopancreatic Necrosis Disease (AHPND)

Identification and Molecular Analysis of Hemocyanin as a Body Resistance Gene in Litopenaeus vannamei

Production of white shrimp (Litopenaeus vannamei) tends to decrease every year caused by diseases, one of which is White Feces Disease (WFD). Genetically, monitoring has been carried out on the parent characters of SPF (Specific Pathogen Free) shrimp imported from Hawaii and Florida with low genetic variation. If there is a threat such as disease caused by bacteria and the gene that codes for disease control or the body's resistance gene is missing, it allows the individual to become susceptible to disease. Gene that codes for body resistance in white shrimp is hemocyanin. Hemocyanin gene studies are needed as a first step in controlling disease in white shrimp. Method used is a descriptive method. Procedures carried out are sample collection, detection of WFD, molecular analysis of hemocyanin and data analysis. Results showed that healthy shrimp had hemocyanin while shrimp with WFD had very little or no amount of hemocyanin. Hemocyanin gene profile in healthy shrimp was also analyzed and there were 4 nucleotide change made changes to 4 amino acid. Changes in amino acids affect the stability of hemocyanin and cause polymorphism of hemocyanin. Phylogenetic analysis showed dendogram split into two clades between L. vannamei and P. monodon.

The Yin Yang 1 of Penaeus vannamei regulates transcription of the small subunit hemocyanin gene during Vibrio parahaemolyticus infection

Hemocyanin is a respiratory protein, it is also a multifunctional immune molecule that plays a vital role against pathogen invasion in shrimp. However, the regulation of hemocyanin gene expression in shrimp hemocytes and the mechanisms involved during pathogen infection remains unclear. Here, we used DNA pull-down followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the Yin Yang 1 transcription factor homolog in Penaeus vannamei (PvYY1) as a key factor that modulates transcription of the small subunit hemocyanin gene of P. vannamei (PvHMCs) in hemocytes during Vibrio parahaemolyticus AHPND (VPAHPND) infection. Bioinformatics analysis revealed that the core promoter region of PvHMCs contains two YY1 motifs. Mutational and oligoprecipitation analyses confirmed that PvYY1 could bind to the YY1 motifs in the PvHMCs core promoter region, while truncation of PvYY1 revealed that the N-terminal domain of PvYY1 is essential for the transactivation of PvHMCs core promoter. Besides, the REPO domain of PvYY1 could repress the activity of the PvHMCs core promoter. Overexpression of PvYY1 significantly activates the promoter activity of PvHMCs core promoter, while PvYY1 knockdown significantly decreases the expression level of PvHMCs in shrimp hemocytes and survival rate of shrimp upon infection with VPAHPND. Our present study provides new insights into the transcriptional regulation of PvHMCs by PvYY1 in shrimp hemocytes during bacteria (VPAHPND) infection.

Sugarcane bagasse improved growth performance, digestive enzyme activity, microbial dynamics, and mRNA transcripts of immune-, growth, and antioxidant -related genes of Litopenaeus vannamei in a zero-water exchange system

Pacific white shrimp (Litopenaeus vannamei) were grown in a biofloc rearing systems with (control treatment) or without sugarcane bagasse (SB treatment), with 3 replicate systems per treatment. SB, is an economic carbohydrate source. Parameters monitored were water-quality, growth indices, digestive enzymes activity, and microbial dynamics. Messenger RNA levels of immune-related, growth-related, and antioxidant genes in hepatopancreas and muscle and gene's single nucleotide polymorphism (SNP) were also considered. The outdoor ponds (44.5 m3), were each stocked with 600 L. vannamei larvae with an initial weight of 0.024 g) to start a three-month experiment. The biofloc development was in two aerated active suspension tanks to which SB (about 700 g/day, adjusted every two weeks) was added to maintain the of the C/N at 16. The water quality parameters, digestive enzyme activity, growth, and survival (P ≤ 0.05) in the SB group was improved compared to the control. SB also increased the total heterotrophic microbiota compared to the control. The mRNA levels of immune- related genes (lipopolysaccharides/b-glucan- binding protein, β-glucan-binding protein, lysozyme, prophenoloxidase, crustin, transglutaminase, and hemocyanin) and antioxidant genes (superoxide dismutase and glutathione peroxidase) significantly increased in SB group (P ≤ 0.05) compared to the control treatment. The SB group was the highest in all growth- related genes such as insulin- like growth factors (I and II), growth hormone, myosin, and paramyosin. Eight SNPs were detected in the hemocyanin gene, and an adenine insertion (ins A-554) was recorded at a high body weight. Our findings implied that using SB, as a carbon source in biofloc system without artificial feeding, could improve water quality, growth indices, digestive enzymes, microbial dynamics and the mRNA levels of all immune-, growth- related and antioxidant genes in L. vannamei.

The transcription factor CSL homolog in Penaeus vannamei positively regulates the transcription of the hemocyanin small subunit gene

Hemocyanin, a copper-containing respiratory protein, is abundantly present in hemolymph of arthropods and mollusks and performs a variety of immunological functions. However, the regulatory mechanisms of hemocyanin gene transcription remain largely unclear. Our previous work showed that knockdown of the transcription factor CSL, a component of the Notch signaling pathway, downregulated the expression of Penaeus vannamei hemocyanin small subunit gene (PvHMCs), indicating the involvement of CSL in regulating the PvHMCs transcription. In this study, we identified a CSL binding motif (“GAATCCCAGA”, +1675/+1684 bp) in the core promoter of PvHMCs (designated as HsP3). Dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA) demonstrated that the CSL homolog in P. vannamei (PvCSL) could directly bind and activate the HsP3 promoter. Moreover, in vivo silencing of PvCSL significantly attenuated the mRNA and protein expression of PvHMCs. Finally, in response to Vibrio parahaemolyticus, Streptococcus iniae and white spot syndrome virus (WSSV) challenge, the transcript of PvCSL and PvHMCs showed a positive correlation, suggesting that PvCSL could also modulate the expression of PvHMCs upon pathogen stimulation. Taken together, our present finding is the first to demonstrate that PvCSL is a crucial factor in transcriptional control of PvHMCs.

Effect of WSSV-Infection on Relative Expression of Hemocyanin Genes in Litopenaeus Vannamei (Boone, 1931)

A Litopenaeus vannamei injected with the serum from the shrimps infected with WSSV-showed the typical symptoms of white spot syndrome at 48hpi. These include lethargy, lack of appetite, reduced and disoriented swimming activity. The negative control and serum from healthy shrimps did not show any band and the test samples contained a band corresponding to 310 bp which was like that of the positive control. Thus, the test samples were confirmed positive for the WSSV-infection. The mean serum protein concentration in healthy shrimp was 86.4 ± 6.3 mg/ml. on the other hand the serum protein concentration of WSSV-injected shrimps declined linearly and significantly to 58.8 ± 3.9, 46.2 ± 5.32, and 38.7 ± 6.7 mg/ml at 12, 24, and 48 hpi, respectively. Relative quantity of hemocyanin gene (I) expression in WSSV-injected shrimps increased gradually from 6 to 48 hpi. Analyses of relative quantification of hemocyanin gene (II) in the experimental groups showed that the relative quantity in WSSV- injected shrimps increased from 6 hpi up to 24 hpi, whereas at 48 hpi the WSSV-injected shrimps showed a decrease in relative quantity of hemocyanin gene (II). Analyses of relative quantification of hemocyanin gene (III) in the experimental groups showed that the relative quantity in WSSV-injected shrimps increased slowly from 6 to 12 hpi and the increase was found to be high at 24 and 48 hpi. The relative quantity increased in sham-injected shrimps. The melt curve analysis showed two peaks corresponding to 18S rRNA and hemocyanin gene I, II and III, respectively. The objective of the present study was directed towards the relative quantification of the hemocyanin genes (I, II and III) in L. vannamei upon WSSV-infection.

Immune gene expression and antioxidant response to vitamin E enriched diets for males Litopenaeus vannamei breeder (Boone, 1931)

The is aim of this study was to evaluate if vitamin E supplementation to diet could improve the spermatic quantity and quality in males of Litopenaeus vannamei. A basal diet with 171.5 mg/kg (included in commercial vitamin premix) and experimental diets with 3 concentrations of vitamin E: 620 (diet A), 1069 (diet B) and 1517 (diet C) mg/kg were tested by 30 days. 500 L tanks were set up for each treatment, and seven males were set in each tank. Circulant cholesterol, triglyceride (TG), glucose, total protein was measured. Sperm quantity and quality were classified as normal, abnormal, or dead. In reproductive tract was measured the activity and differential expression lipid soluble antioxidant system (LSAS) and of immune genes system. The higher sperm quantity was obtained in A diet and decreased in shrimp fed B and C diets (p < 0.05). Males fed diets B and C presented a higher activity and relative expression of lipid system oy suggesting oxidative stress. A significant increment of prophenol oxidase (proFo) and α-2-macroglobulin (α-2 M) transcripts were observed in males fed C diet (p < 0.05). All these variables were present in males with low sperm amount and low hemocyanin gene expression (p < 0.05). Therefore, diet A supplemented with 620 mg/kg vitamin E could benefit males L. vannamei broodstock, taking a count the sperm quantity and all the lipid and immune genes response.

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.

Inhibited expression of seven hemocyanins in hepatopancreatic necrosis syndrome (HPNS) crabs regulated by Kruppel and E75

Hepatopancreatic necrosis syndrome (HPNS) severely threatens the development of Eriocheir sinensis farming. To date, no consensus has been reached about the etiology and pathogenesis of HPNS. In this study, we investigated the effects of HPNS on the copper-containing respiratory protein (hemocyanin), the ecdysone inducible transcription factor (E75), and the zinc finger-containing transcription factor (Kruppel). We also determined the relationship between hemocyanin and the two transcription factors. Seven hemocyanin variants, four E75 isoforms, and a Kruppel-like factor were characterized in E. sinensis. The 3′ and 5′ ends of hemocyanin possessed molecular diversity, which was apparently associated with its multifunctionality. Four E75 isoforms were generated by the alternative splicing of the 5′ exons, and they originated from a single copy gene. Hemocyanins, EsE75, and EsKruppel were mainly expressed in the hepatopancreas, which is the most primary organ for the clinical characterization of HPNS. The expression of the hemocyanins and two transcriptional factors (EsE75 and EsKruppel) were significantly decreased and increased in the crabs with HPNS, respectively. Moreover, silencing of EsE75 and EsKruppel resulted in an evident increase in the synthesis of hemocyanins, indicating that EsE75 and EsKruppel acted as the transcription repressor of the hemocyanin gene. However, the detailed mechanisms by which E75 and Kruppel regulated the transcription of hemocyanin in E. sinensis need to be more thoroughly studied in the future. This study provides new information on hemocyanin, E75, and Kruppel that were influenced by HPNS in E. sinensis. The results will help to enhance the understanding of the causes and pathogenesis of HPNS.

The evolution of hemocyanin genes in Tectipleura: a multitude of conserved introns in highly diverse gastropods

BackgroundHemocyanin is the oxygen transporter of most molluscs. Since the oxygen affinity of hemocyanin is strongly temperature-dependent, this essential protein needs to be well-adapted to the environment. In Tectipleura, a very diverse group of gastropods with > 27,000 species living in all kinds of habitats, several hemocyanin genes have already been analyzed. Multiple independent duplications of this gene have been identified and may represent potential adaptations to different environments and lifestyles. The aim of this study is to further explore the evolution of these genes by analyzing their exon–intron architectures.ResultsWe have reconstructed the gene architectures of ten hemocyanin genes from four Tectipleura species: Aplysia californica, Lymnaea stagnalis, Cornu aspersum and Helix pomatia. Their hemocyanin genes each contain 53 introns, significantly more than in the hemocyanin genes of Cephalopoda (9–11), Vetigastropoda (15) and Caenogastropoda (28–33). The gene structures of Tectipleura hemocyanins are identical in terms of intron number and location, with the exception of one out of two hemocyanin genes of L. stagnalis that comprises one additional intron. We found that gene structures that differ between molluscan lineages most probably evolved more recently through independent intron gains.ConclusionsThe strict conservation of the large number of introns in Tectipleura hemocyanin genes over 200 million years suggests the influence of a selective pressure on this gene structure. While we could not identify conserved sequence motifs within these introns, it may be simply the great number of introns that offers increased possibilities of gene regulation relative to hemocyanin genes with less introns and thus may have facilitated habitat shifts and speciation events. This hypothesis is supported by the relatively high number of introns within the hemocyanin genes of Pomacea canaliculata that has evolved independently of the Tectipleura. Pomacea canaliculata belongs to the Caenogastropoda, the sister group of Heterobranchia (that encompass Tectipleura) which is also very diverse and comprises species living in different habitats. Our findings provide a hint to some of the molecular mechanisms that may have supported the spectacular radiation of one of Metazoa’s most species rich groups.

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal‐selective metallothioneins (MTs) by which they handle metal ions such as Zn2+, Cd2+, and Cu+/Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu‐dependent MT and Hc pathways might be metabolically connected. In fact, the Cu‐specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal‐regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal‐selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH β). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ‐specific transcriptional upregulation in the midgut gland of Cu‐exposed snails. These results are discussed in view of possible interrelationships between the metal‐selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

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.

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.

Nuclear receptor E75 is a transcription suppressor of the Litopenaeus vannamei small subunit hemocyanin gene

Hemocyanin is a respiratory protein that possesses multiple physiological and immunological functions in shrimp. However, the transcriptional regulation of the hemocyanin gene is still poorly understood. Here, the nuclear receptor E75 of Litopenaeus vannamei (LvE75) was identified as one of the transcriptional regulators that modulates the transcription of the small molecular weight hemocyanin gene of L. vannamei (LvHMCs) by inhibiting its core promoter activity in a Dual-luciferase assay. In silico analysis revealed that the core promoter (designated HsP3), which is located at +1517/+1849 bp of LvHMCs contained a putative E75 binding motif (“ACGGAAT”, spanning +1812/+1818 bp). Further, LvE75 was shown to inhibit the core promoter activity by direct binding. Importantly, in vivo silencing of LvE75 resulted in a significant upregulation in the mRNA and protein expression of LvHMCs gene. Taken together, our present results provide direct evidence that LvE75 is a transcriptional suppressor of the LvHMCs gene expression.

Protein-Level Evidence of Novel β-Type Hemocyanin and Heterogeneous Subunit Usage in the Pacific Whiteleg Shrimp, Litopenaeus vannamei

The functional diversity of crustacean hemocyanins is broad, encompassing O2 delivery, innate immune response, metabolite storage, and osmolyte balance, all in a heterogeneous protein structure. As such, the sequence diversity of this class of proteins and its subunit composition are the focus of many studies on crustacean adaptation to environmental challenges. Recent transcriptomic and genomic assemblies on the Pacific whiteleg shrimp Litopenaeus vannamei have identified unique isoforms of hemocyanin including an ancestral β-type subunit thought to be lost in penaeid shrimp. However, it is unknown the degree to which these isoforms are translated as proteins, and their abundances within the hemolymph. The present study uses proteomic approaches to characterize the protein-level abundance and organization of these hemocyanin isoforms within their native oligomeric structures. Fractions of each hemocyanin oligomeric form were purified by size-exclusion high performance liquid chromatography for identification of subunit isoforms using tandem mass spectrometry at <1% protein false discovery rate. Data are available via ProteomeXchange with identifier PXD014575. Relative abundances of hemocyanin oligomers and monomeric subunits from hemolymph and fractions were also quantified by polyacrylamide gel electrophoresis with and without denaturation for comparison of subunit heterogeneity. Hemocyanin subunits were organized primarily as hexamers (95-99% relative abundance) as opposed to dodecamers. Ten unique hemocyanins predicted by transcriptome and genome assemblies were identified by tandem mass spectrometry in both oligomer fractions including the first protein-level evidence of a β-type subunit in penaeid shrimp. Identified hemocyanins mapped to four genomic scaffolds and had two or three exons. A single small (75 kDa) subunit constituted half or more of all isoforms identified in each oligomer. The dodecameric fraction exhibited greater subunit heterogeneity utilizing a significantly lower ratio (1.04:1) of small subunit to large subunit compared to hexamers (2.05:1). One isoform, XP_027232115.1, appeared to be more dominant within dodecamers than hexamers. The ability to distinguish and quantify hemocyanin isoforms within oligomeric structures will aid future studies linking hemocyanin genes to transcripts to function and physiology as well as offer insight into the evolutionary history of crustaceans.

A Simple Genome Walking Strategy to Isolate Unknown Genomic Regions Using Long Primer and RAPD Primer.

BackgroundGenome walking is a DNA-cloning methodology that is used to isolate unknown genomic regions adjacent to known sequences. However, the existing genome-walking methods have their own limitations.ObjectivesOur aim was to provide a simple and efficient genome-walking technology.Material and MethodsIn this paper, we developed a novel PCR strategy (termed SLRA PCR) that uses a single long primer (SLP), a set of gene specific primers (GSP), and a random amplified polymorphic DNA (RAPD) primer for genome walking. SLRA PCR consists of two processes: the first amplification using SLP, and three successive rounds of nested PCR amplified by GSP and RAPD primer. The novelty of the approach lies in the use of long primers (SLP and GSP) and same annealing and extension temperature 68℃ in combination. This method offers higher amplification efficiency, superior versatility, and greater simplicity compared with conventional randomly primed PCR methods for genome walking.ResultsThe promoter regions and the first introns of the insulin-like androgenic gland hormone (IAG) gene and the hemocyanin gene of Macrobrachium nipponense were cloned using SLRA PCR, respectively.ConclusionsThis genome walking strategy can be applied to a wide range of genomes.

Differential expression of immune‐related genes in Pacific white shrimp, Litopenaeus vannamei, previously reared in biofloc and challenged with Vibrio harveyi

This study aimed to evaluate possible modification of some immune-related genes (hemocyanin, alpha2-macroglobulin, prophenoloxidase and penaeidin3a) at the transcriptional level and signs of disease and mortality in Litopenaeus vannamei previously reared in biofloc and challenged with different Vibrio harveyi densities (103–108 CFU per ml). Gene expression was up- or downregulated based on bacterial density, indicating that defence mechanisms were activated to remove the pathogen. The genes analysed were not affected in the control group (without bacteria) or in the 103 CFU per ml group. The prophenoloxidase gene was expressed in the 104 CFU per ml group, whereas transcripts of the alpha2-macroglobulin gene were detected in the 105 and 106 CFU per ml groups. However, the penaeidin3a and hemocyanin genes were upregulated in the 107 and 108 CFU per ml groups, in which signs of disease and mortality in shrimp were evident. These results suggest that the genes involved in the shrimp immune response fluctuate according to pathogen density, and when this density increases (e.g. 107 and 108 CFU per ml), the activated disease tolerance and defence mechanisms are inefficient. For this reason, disease and mortality were observed in the shrimp regardless of the previous condition of the biofloc.

Gene expression and phenoloxidase activities of hemocyanin isoforms in response to pathogen infections in abalone Haliotis diversicolor.

Hemocyanins (Hc), the main protein components of hemolymph in invertebrates, are not only involved in oxygen transport but also linked to non-specific immune responses. In this study, we used abalone (Haliotis diversicolor) Hc to study the basis of its diversified functions through gene, protein, peptides, and phenoloxidase (PO) activity levels. Three complete hemocyanin gene (HdH) sequences were cloned for the first time. By comparing the copies and location of HdH between abalone and other mollusks, we propose that Hc gene duplication and linkage is likely to be common during the evolution of mollusk respiratory proteins. We further demonstrate that all three genes could be expressed in abalone, with expression varying based on the developmental stages, tissue types, and different pathogen infections. However, HdH1 and HdH2 appear to be synthesized by the same cells by fluorescence in situ hybridization. Furthermore, the PO activity of HdH can be induced by trypsin, urea, and SDS in vitro. Viral infection can stimulate its PO activity in vivo by cleaving the protein into fragments. Consequently, we present a comprehensive study of abalone hemocyanin, providing important evidence for an in-depth understanding of the physiological and immune functions of Hc in mollusks.

Discovery of Novel Hemocyanin-Like Genes in Metazoans.

Among animals, two major groups of oxygen-binding proteins are found: proteins that use iron to bind oxygen (hemoglobins and hemerythrins) and two non-homologous hemocyanins that use copper. Although arthropod and mollusc hemocyanins bind oxygen in the same manner, they are distinct in their molecular structures. In order to better understand the range of natural variation in hemocyanins, we searched for them in a diverse array of metazoan transcriptomes by using bioinformatics tools to examine hemocyanin evolutionary history and to consequently revive the discussion about whether all metazoan hemocyanins shared a common origin with frequent losses or whether they originated separately after the divergence of Lophotrochozoa and Ecdysozoa. We confirm that the distribution of hemocyanin-like genes is more widespread than previously reported, including five putative novel mollusc hemocyanin genes in two annelid species from Chaetopteridae. For arthropod hemocyanins, 16 putative novel genes were retained, and the presence of arthropod hemocyanins in 11 annelid species represents a novel observation. Interestingly, Annelida is the lineage that presents the greatest repertoire of oxygen transport proteins reported to date, possessing all the main superfamily proteins, which could be explained partially by the immense variability of lifestyles and habitats. Work presented here contradicts the canonical view that hemocyanins are restricted to molluscs and arthropods, suggesting that the occurrence of copper-based blood pigments in metazoans has been underestimated. Our results also support the idea of the presence of oxygen carrier hemocyanins being widespread across metazoans with an evolutionary history characterized by frequent losses.

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.