Hemocyanin Gene Research Articles

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.

Immune response of the Pacific white shrimp, Litopenaeus vannamei, previously reared in biofloc and after an infection assay with Vibrio harveyi

The effectiveness of the immune defense of Litopenaeus vannamei previously reared in biofloc or in a traditional clear seawater rearing system was assessed after a bacterial challenge with a pathogenic strain of Vibrio harveyi. The changes caused by its previous rearing system condition or the challenge were assessed in terms of metabolites (glucose, cholesterol, acylglycerides, protein), hemocyanin, the antioxidant defense system (superoxide dismutase, and catalase), and gene expression related to immune response (superoxide dismutase, alpha2 macroglobulin, prophenoloxidase, hemocyanin, and penaeidin‐3a). The biofloc rearing system was associated with a significant increase in protein, the antioxidant defense system, and the superoxide dismutase, alpha2 macroglobulin, and prophenoloxidase genes. For shrimp previously reared in biofloc, a positive interaction with the presence or absence (control) of V. harveyi significantly affected the hemocyanin concentration, and the interaction between the two rearing systems in shrimp challenged with the bacteria produced a higher transcription of the hemocyanin gene. Likewise, biofloc and clear seawater maintained a higher transcription of prophenoloxidase gene after the bacterial challenge. In the absence of the bacteria, shrimp of the biofloc group produced a higher transcription of the penaeidin‐3a gene. V. harveyi caused hepatopancreatic lesions and mortalities only in shrimp previously reared in clear seawater. These results suggest that biofloc helps to prevent the development of disease by improving the shrimp immune response.

Comparative transcriptome analysis reveals networks of genes activated in the whitefly, Bemisia tabaci when fed on tomato plants infected with Tomato yellow leaf curl virus

The whitefly Bemisia tabaci can transmit hundreds of viruses to numerous agricultural crops in the world. Five genera of viruses, including Begomovirus and Crinivirus, are transmitted by B. tabaci. There is little knowledge about the genes involved in virus acquisition and transmission by whiteflies. Using a comparative transcriptomics approach, we evaluated the gene expression profiles of whiteflies (B. tabaci MEAM1) after feeding on tomato infected by a begomovirus, Tomato yellow leaf curl virus (TYLCV), in comparison to a recent study, in which whiteflies were fed on tomato infected by the crinivirus, Tomato chlorosis virus (ToCV). The data revealed similar temporal trends in gene expression, but large differences in the number of whitefly genes when fed on TYLCV or ToCV-infected tomato. Transcription factors, cathepsins, receptors, and a hemocyanin gene, which is implicated in mediating antiviral immune responses in other insects and possibly virus transmission, were some of the genes identified.

Dietary nano-selenium relieves hypoxia stress and, improves immunity and disease resistance in the Chinese mitten crab (Eriocheir sinensis).

Hypoxia is a relevant physiological challenge for crab culture, and the hemolymph plays a crucial role in response to the hypoxia. In a 60d feeding trial, Chinese mitten crabs (Eriocheir sinensis) fed a diet containing 0.2mg/kg nano-selenium (nanoSe) showed a significantly increased weight gain rate (WGR) and a reduced feed coefficient (FC) compared to those fed diets with 0, 0.1, 0.4, 0.8, and 1.6mg/kg nanoSe. Another 90d feeding trial was conducted to determine the influence of dietary nanoSe on the immune response in juvenile Chinese mitten crabs kept under the condition of hypoxia. The results showed that hypoxia stress resulted in significantly increased hemocyte counts (THC, LGC, SGC, and HC), expression levels of the hemocyanin gene and protein, lactic acid level, and antioxidant capacity (T-AOC activities, SOD activities, GSH-Px and GSH content) in hemolymph supernatant. When these crabs were infected with Aeromonas hydrophila bacteria, hypoxia exposure increased mortality, but it was alleviated by a diet supplemented with 0.2mg/kg nanoSe. The up-regulative effects of nanoSe (0.2mg/kg) on antioxidant capacity, hemocyte counts, and hemocyanin expression under hypoxia exposure were further strengthened throughout, whereas lactic acid levels induced by hypoxia stress were restored. Thus, the observations in this study indicate that the level of dietary nanoSe is important in regulating immunity and disease resistance in crabs kept under hypoxia stress.

EVIDENCE FOR BIOGENIC COPPER (HEMOCYANIN) IN THE MIDDLE CAMBRIAN ARTHROPOD MARRELLA FROM THE BURGESS SHALE

Beginning with the discovery of the middle Cambrian Burgess Shale, the rare examples of exceptional preservation of soft tissues in fine-grained strata have been celebrated as windows into the diversity and complexity of ancient marine animal life. Even so, with the loss of original organic compounds, the physiological attributes of the fossils can be inferred only on the basis of modern descendants or sister groups among extant representatives that can serve as analogs. To test whether soft-bodied fossils from the Burgess Shale and similar Cambrian Lagerstatten could preserve chemical evidence of their original biology, a number of specimens from different groups were analyzed with synchrotron-based X-ray fluorescence imaging to determine the spatial distribution of a range of elements, especially those in exceedingly trace amounts that cannot be detected by more conventional analytical methods. The relative distribution of common elements like Fe, K, Ti, and Ca is related to the composition of the host shale, abundance of pyrite, and presence or absence of preserved exoskeletal calcite. However, the black stain commonly preserved with the arthropod Marrella splendens is preferentially enriched in Cu due to localized residue in the carbon and minute crystals of chalcopyrite. This is interpreted as indicative of the original chemical composition of a primary biotic fluid, likely blood. Since modern-day arthropods generally utilize Cu-containing hemocyanin for oxygen transport, it is hypothesized that hemocyanin was the biogenic source of the observed Cu. This is the first chemical evidence for blood composition in the invertebrate fossil record. These observations reinforce the consensus molecular clock age for the hemocyanin gene family, which traces its origins back to the ‘Cambrian explosion’, thus providing evidence for the early origin of this oxygen carrier among some arthropod groups.

Molecular cloning and characterization of three novel Hemocyanins from Chinese mitten crab, Eriocheir sinensis

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustaceans. However, little is known about the hemocyanin from the Chinese mitten crab, Eriocheir sinensis. In this study, three forms of hemocyanins designated as EsHc1, EsHc2 and EsHc3 were cloned from E. sinensis by using expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The open reading frames (ORFs) of EsHc1, EsHc2 and EsHc3 genes were 2182, 2580 and 2220 bp encoding proteins with 678, 662 and 691 amino acids, respectively, and all contain three tandem hemocyanin domains: 1) hemocyanin N, 2) hemocyanin M or tyrosinase, and 3) hemocyanin C domain. BLASTP and phylogenetic tree analysis showed that EsHc1 was clustered together with cryptocyanin 1 from Portunus pelagicus (PpCc1) and cryptocyanin from Metacarcinus magister (MmCc), EsHc2 and EsHc3 with hemocyanin from Pacifastacus leniusculus (PlHc) and hemocyanin subunit 1 from M. magister (MmHc1) were gathered into one clade. EsHc1 was mainly expressed in hepatopancreas and hemocytes with a lower level of expression in nerves, eyestalk, muscles, intestine, gills and heart; whereas EsHc2 and EsHc3 were mainly expressed in hepatopancreas and were also detected in hemocytes. Quantitative real-time RT-PCR analysis showed that EsHcs mRNA transcription in hepatopancreas was significantly expressed at various time points after infection with Lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, Vibrio parahaemolyticus and Aeromonas hydrophila. In summary, there is evidence that the three isoforms of hemocyanin genes participate in the innate immune response against bacteria infecting the Chinese mitten crab.

Evolutionary implications of dipluran hexamerins

Hexamerin, as a member of the highly conserved arthropod hemocyanin superfamily, has been shown to be a good marker for the phylogenetic study of insects. However, few studies have been conducted on hexamerins in basal hexapods. The first Diplura hexamerin CspHex1 was reported only recently (Pick and Burmester, 2009). Remarkably, CspHex1 was suggested to have evolved from hexapod hemocyanin subunit type 2, which is very different from all insect hexamerins originated from hexapod hemocyanin subunit type 1. Does this finding suggest double or even multiple origins of hexamerins in Hexapoda? To find more evidence on the evolution of dipluran hexamerins, eight putative hexamerin gene sequences were obtained from three dipluran species, as were three hemocyanin genes from two collembolan species. Unexpectedly, after adding the new sequences into the phylogenetic analyses, all dipluran hexamerins including CspHex1 grouped together and as sister to the insect hexamerins, with high likelihood and Bayesian support. Our analysis supports a single origin of the hexamerins in Hexapoda, and suggests the close relationship between Diplura and Insecta. In addition, our study indicates that a relatively comprehensive taxa sampling is essential to solve some problems in phylogenetic reconstruction.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6–48h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab.

Cloning and tissue expression of hemocyanin gene in Cherax quadricarinatus during white spot syndrome virus infection

The role of hemocyanin in terms of immune defense was explored in Cherax quadricarinatus infected by WSSV. The cDNA of a hemocyanin gene (CqHC) was cloned from the hepatopancreas of C. quadricarinatus (crayfish) by the Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of CqHC consisted of 2167bp with a 2043bp open reading frame encoding a 680 amino acid protein with a predicted signal peptide of 19 amino acids. The deduced CqHC protein sequence was found to contain two conserved copper-binding sites and to share 74%, 73%, 72%, 71% and 71% similarity with Penaeus monodon, Marsupenaeus japonicus, Fenneropenaeus chinensis, Metacarcinus magister and Litopenaeus vannamei, respectively. Phylogenetic analysis revealed that CqHC and HC from F. chinensis, M. japonicus and P. monodon clustered in the same phylogenetic branch. Real-time PCR analysis showed that CqHC was widely distributed, with the highest level in hepatopancreas, at lower amounts in hemocytes, gills and antennal gland. HC levels in hepatopancreas, hemocytes and gills from C. quadricarinatus were also compared after white spot syndrome virus (WSSV) infection. After 24h of infection, CqHC levels rose significantly higher than that of the controls (P<0.05) but then sharply declined over the infection period. The HC gene expression in hemocytes and gills (but not hepatopancreas) of crayfish injected with polysaccharides before infection were higher than that of the non-polysaccharide injected infected groups, showing an immunoprotective rate of 51.86% at 7days after exposure, indicating that polysaccharide can act as immunopotentiator and could improve the antiviral ability of C. quadricarinatus against WSSV.

Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4×6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.

Gene expression profile of hepatopancreas from grass shrimp Palaemonetes pugio exposed to cyclic hypoxia

Estuarine organisms often experience periods of cyclic hypoxia characterized by hypoxia in the early morning and normoxia in the afternoon. Here we examine the genomic responses of grass shrimp, Palaemonetes pugio, exposed to cyclic hypoxia in the laboratory. Differentially expressed genes in the hepatopancreas were determined in cyclic hypoxic vs. normoxic control groups after 1, 2, 5 and 10days of exposure to cyclic hypoxia using microarrays printed with 661 annotated transcripts obtained from multiple EST (expressed sequence tag) libraries. Sampling on each day was conducted at two different time series, one in the morning (representing low concentration of dissolved oxygen (DO), designated C-AM) and one in the afternoon (representing high DO concentration, designated C-PM). Distinct differences were observed between the number and identity of specific genes that were significantly down- or up-regulated in shrimp collected at the low DO and high DO points of the cyclic DO cycle. However, cluster analysis showed that the overall response patterns of high (C-PM) and low DO (C-AM) exposures were in the same cluster at 1, 2, and 5days. In contrast, the response patterns on different days were in different clusters. Day 1 was characterized by up-regulation of 17 unknown genes in the morning and a transient down-regulation of several hemocyanin genes, which returned to normoxic control levels in the afternoon. Days 2 and 5 showed significant down-regulation of 10 (C-AM) and 15 (C-PM) unknown genes, respectively. On day 10 the high DO samples showed a dramatic increase in the number of up-regulated genes, including several distinct hemocyanin genes, and this profile did not cluster with any of the other treatment groups. Vitellogenin, cathepsin L, cytochrome c oxidase subunit III, and fatty acid binding protein 10 were the signature down-regulated genes at day 10 (C-AM). According to GO annotation, the most abundant group of genes for both cyclic low (C-AM) and high (C-PM) DO exposure was associated with transport, defense response, and metabolic process. The differentially expressed genes were mapped to KEGG metabolic and regulatory pathways according to the gene distribution in Drosophila pathway database. Cyclic high (C-PM) DO affected a broad range of pathways compared to cyclic low (C-AM) DO.

Molecular phylogeny of South African abalone, its origin and evolution as revealed by two genes

The marine family Haliotidae (Mollusca: Gastropoda) includes approximately 56 extant abalone species found worldwide. None of these species are globally distributed while four areas of endemism (temperate Australia, South Africa, New Zealand and North Pacific) have been recognized. Phylogenetic relationships of 18 abalone species including five South African species were reconstructed using a combined data set containing sequence data of the mitochondrial NADH-dehydrogenase subunit 1 (ND1) gene and the nuclear hemocyanin gene. The molecular topologies confirmed former findings of two major abalone lineages with Northern and Southern Pacific distribution within the Haliotidae family. The phylogeny revealed all five South African species as a monophyletic group with a sister relationship to the Australian endemics clade. It further suggested a relatively recent radiation of the South African species and places it within the same evolutionary context as the Mediterranean/Atlantic and Australasian clades. Molecular phylogeny also revealed a split within the South African group as well as further speciation within one of the two subclades. Possible place of origin for South African abalone are discussed and ecological specialization, e.g. differential adaptation to environmental conditions, is proposed as the most likely scenario describing divergence within the South African clade.

Gene expression profile, protein production, and functions of cryptocyanin during the crustacean molt cycle

Orchestration of the synthesis of a new exoskeleton and degradation of the old exoskeleton during molting requires precise programming. We have studied expression patterns of members of the hemocyanin gene family, hemocyanin, cryptocyanin, and phenoloxidase, in Cancer magister to better understand the molecular basis of the complex regulation and physiological transformations that occur during molting. Results of electrophoresis, immunohistology, in situ hybridization, and real-time qPCR studies on animals sampled across their molt cycles have established that cryptocyanin functions in forming the new exoskeleton. The protein is synthesized in the hepatopancreas, transported in the hemolymph and moved across the epithelium to the new exoskeleton. The presence of cryptocyanin and hemocyanin in oocytes, embryos and zoeas of multiple species indicates that cryptocyanin is available from the earliest onset of cuticle formation in development. Analysis by cDNA microarray supports our studies on hemolymph proteins hemocyanin and cryptocyanin, indicating that these genes are independently regulated. The cryptocyanin transcriptional patterns that change as a function of the molt cycle have also been reported in other species. Cryptocyanin, analogous to vertebrate serum albumin, may also participate as a carrier protein for hormones, divalent cations, or other molecules, as an effector of osmotic balance, and as part of the immune defense during premolt and early postmolt.

Cloning and characterization of the hemocyanin gene of prawn Macrobrachium nipponense

Cloning and characterization of the hemocyanin gene of prawn Macrobrachium nipponense

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

BackgroundCrustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.ResultsA total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.ConclusionsThe expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process.

Identification of upregulated immune-related genes in Vibrio harveyi challenged Penaeus monodon postlarvae

A subtracted cDNA library was constructed and analyzed to elucidate the response of Penaeus monodon postlarvae challenged with Vibrio harveyi. As many as 960 randomly selected cDNA fragments generated through suppression subtractive hybridization were single pass sequenced. Forty five genes and 20 hypothetical proteins were identified, a few being first reports from shrimps. The most abundant immune relevant genes were ferritin, hemocyanin, and TCTP (translationally controlled tumor protein) indicating their upregulation as also confirmed through qPCR. Post-infection qPCR analyses confirmed 2.04, 2.09, 3.28, 5.49, 6.47, and 11.63 fold rise respectively in ferritin, penaeidin, MnSOD, lysozyme, TCTP, and hemocyanin genes. These genes may be involved in the regulation of the host defense against V. harveyi.

Hemocyanin in mollusks—A molecular survey and new data on hemocyanin genes in Solenogastres and Caudofoveata

The most common respiratory protein of mollusks is the blue, copper-containing hemocyanin (van Holde and Miller, 1995). It is not bound to hemocytes but suspended in the hemolymph. Its molecular mass ranges from 3500 10 to 8000 10 Da (dalton) or even more (Herskovits, 1988). These differences in molecular weight are due to the fact that the basic decamers that constitute the barrel-shaped protein may aggregate to didecamers or multidecameric elongated particles (Herskovits, 1988). In cephalopods and chitons (Polyplacophora), there are exclusively decamers, whereas in protobranch bivalves and gastropods the predominantly observed aggregation state is didecamers (Herskovits, 1988; van Holde andMiller, 1995; Lieb andMarkl, 2004; Bergmann et al., 2006, 2007; Gatsogiannis et al., 2007). A typical hemocyanin monomer is composed of eight globular functional units (FU), which are arranged like pearls on a string and termed FU-a to FU-h. An exception are cephalopod hemocyanins that contain only seven FUs. According to molecular clock calculations, the single FUs evolved within the early Precambrian, thus they were present already before the extant molluscan classes derived (Lieb et al., 2000; Lieb and Markl, 2004). Further analyses of hemocyanin-genes from cephalopods, gastropods, a protobranch bivalve, and polyplacophorans (the latter is unpublished data) showed that all hemocyanin genes possess highly conserved phase 1 linker-introns, which separate the individual FU-exons from each other [(FU-a-intron-FU-b-intron-FU-c-intron-FU-d-intron...) Lieb et al., 2001; Altenhein et al., 2002; Bergmann et al., 2006, 2007]. Since these introns are present in all molluscan hemocyanin genes, they seem to be ancient and probably at first used to border a sin-

Characterization of the putative farnesoic acid O-methyltransferase (LvFAMeT) cDNA from white shrimp, Litopenaeus vannamei: Evidence for its role in molting

Methyl farnesoate (MF) is the crustacean homolog of the insect juvenile hormone and is believed to regulate growth and reproduction in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to MF. Here we report the cloning and characterization of two forms of FAMeTs (i.e. LvFAMeT-S and LvFAMeT-L) from the shrimp Litopenaeus vannamei. LvFAMeT transcript has a wide tissue distribution pattern in L. vannamei and is also expressed in nauplius, zoea, mysis, post-larval stages and adults. Unlike FAMeTs reported in other decapods, transcripts of two different sizes were detected in L. vannamei. We postulate that the wide distribution of LvFAMeT expression may be related to its role in growth and regulation of molting. To study the functions of LvFAMeT in molting, the RNA interference (RNAi) technique was used. Injection of double stranded RNA (dsRNA) for LvFAMeT knocked down the expression of LvFAMeT in shrimp for at least 3 days and the shrimp did not advance to the final stage of molt cycle. Furthermore, the expression of the molt-related genes encoding cathepsin-L and the hemocyanin gene was disturbed. Subsequently, 100% mortality of the shrimp was observed in the LvFAMeT dsRNA-injected shrimp. In contrast, control shrimp completed their molt and proceeded to the next molt cycle. We postulate that, as an important enzyme for the conversion of FA to MF, RNAi injection knocked down the expression of LvFAMeT which could potentially result in a decrease in the production of MF and subsequently, could affect the molting process. The newly identified LvFAMeT may be involved in the control of molting in shrimp. The results of this study demonstrate the potential use of the RNA interference technique to study other putative genes identified in crustaceans.

Crustacean hemocyanin gene family and microarray studies of expression change during eco-physiological stress

Proteins in the arthropod hemocyanin gene family are involved in major physiological processes, including aerobic respiration, the innate immune response, and molting. Members of this family, hemocyanin, cryptocyanin, and phenoloxidase, are multisubunit molecules that assemble into hexamers and higher aggregates. The hemocyanin hexamers show species-specific subunit heterogeneity. It is hypothesized that this subunit diversity is maintained as a mechanism of selection for functional diversity under changing developmental and environmental conditions. There is good evidence for a strong relationship between subunit composition and functional diversity in the hemocyanins. We have amplified, cloned, and sequenced the complete cDNAs of the 6 hemocyanin genes, 2 cryptocyanins, and 1 phenoloxidase of Cancer magister. Alignment of the amino acid sequences provides the first opportunity to assess in 1 species of brachyuran crustacean the similarities and differences among all the hemocyanin subunits and compare them with cryptocyanin and phenoloxidase. A phylogeny of sequences of crustacean members of the arthropod hemocyanin gene family is described. Construction of a cDNA library for C. magister microarray studies is in progress. The microarrays will be queried using transcriptional profiles from crabs sampled during developmental, molting, and physiological perturbations. The combination of genomics, proteomics, and gene-by-gene analyses will help us dissect how much a gene sequence in this hemocyanin family can vary while conserving function and which aspects of preservation of shape and structural flexibility are essential for functional stability. Integrating focused gene studies with global-expression profiling can eventually lead to the identification of functional networks at the level of the gene, the multisubunit molecule, and the whole organism.

The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M(r) = approximately 3.5 million), assembled from 10 identical copies of an approximately 350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of approximately 50 kDa each (FUs a-g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5' UTR of 58 bp, a 3' UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M(r) = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin "OdH" from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 +/- 24 million years ago, in close agreement with fossil records from the early Devonian.