The evolution and functional characterization of CXC chemokines and receptors in lamprey.

Chemokines are a large family of soluble peptides guiding cell migration in development and immune defense. They interact with chemokine receptors and are essential for the coordination of cell migration in diverse physiological processes. The CXC subfamily is one of the largest groups in the chemokine family and consists of multiple members. In this study, we identified homologues of three chemokine ligands (CXCL8, CXCL_F5 and CXCL12) and two CXC receptor like molecules (CXCR_L1 and CXCR_L2) in lamprey. Sequence analysis revealed that they share the same genomic organization with their counterparts in jawed vertebrates but synteny was not conserved. Lamprey CXCL8 and CXCL12 have four conserved cysteine residues whilst the CXCL_F5 has two additional cysteine residues. In addition, CXCL_F5 is evolutionarily related to the fish specific CXC chemokine groups previously identified and contains multiple cationic aa residues in the extended C- terminal region. The two CXCRs possess seven transmembrane domains and conserved structural elements for receptor activation and signaling, including the DRYXXI(V)Y motif in TM2, the disulphide bond connecting ECL2 and TM3, the WXP motif in TM6 and NPXXY motif in TM7. The identified CXC chemokines and receptors were constitutively expressed in tissues including the liver, kidney, intestine, heart, gills, supraneural body and primary leukocytes, but exhibited distinct expression patterns. Relatively high expression was detected in the gills for CXCL8, CXCL_F5 and CXCR_L1 and in the supraneural body for CXCL12 and CXCR_L2. All the genes except CXCL12 were upregulated by stimulation with LPS, pokeweed and bacterial infection, and the CXCL8 and CXCL_F5 was induced by poly (I:C). Functional analysis showed that the CXCL8 and CXCL_F5 specifically interacted with CXCR_L1 and CXCR_L2, respectively. Our results demonstrate that the CXC chemokine system had diversified in jawless fish.

Molecular Characterization and Chemotactic Function of CXCL8 in Northeast Chinese Lamprey (Lethenteron morii).

Chemokine-induced chemotaxis of leukocytes is an important part of the innate immunity and has been shown to mediate inflammation in all groups of jawed vertebrates. For jawless vertebrates, hagfish leukocytes are known to show chemotaxis toward mammalian complement anaphylotoxin and Gram-negative bacteria lipopolysaccharide. However, whether chemokines mediate chemotaxis of leukocytes in jawless vertebrates has not been conclusively examined. Here, we show C-X-C motif chemokine ligand 8 (CXCL8, also named interleukin 8) of the Northeast Chinese lamprey (Lethenteron morii) (designated as LmCXCL8) induces chemotaxis in its leukocytes. We identified LmCXCL8 and found it possesses the characteristic N-terminal cysteine residues and GGR (Gly-Gly-Arg) motif. The Lmcxcl8 gene was found to be expressed in all examined tissues, and its expression was inducible in the lamprey challenged by an infectious bacterium, Pseudomonas aeruginosa. A recombinant LmCXCL8 protein elicited concentration-dependent chemotaxis in peripheral blood leukocytes isolated from the Northeast Chinese lamprey. Based on these results, we conclude that LmCXCL8 is a constitutive and inducible acute-phase cytokine that mediates immune defense and trace the chemotactic function of chemokine to basal vertebrates.

Molecular Evolution of Apolipoprotein Multigene Family and the Original Functional Properties of Serum Apolipoprotein (LAL2) in Lampetra japonica.

Apolipoprotein (APO) genes represent a large family of genes encoding various binding proteins associated with plasma lipid transport. Due to the long divergence history, it remains to be confirmed whether these genes evolved from a common ancestor through gene duplication and original function, and how this evolution occurred. In this study, based on the phylogenetic tree, sequence alignment, motifs, and evolutionary analysis of gene synteny and collinearity, APOA, APOC, and APOE in higher vertebrates may have a common ancestor, lamprey serum apolipoprotein LAL1 or LAL2, which traces back to 360 million years ago. Moreover, the results of immunofluorescence, immunohistochemistry, and flow cytometry show that LAL2 is primarily distributed in the liver, kidney, and blood leukocytes of lampreys, and specifically localized in the cytoplasm of liver cells and leukocytes, as well as secreted into sera. Surface plasmon resonance technology demonstrates that LAL2 colocalizes to breast adenocarcinoma cells (MCF-7) or chronic myeloid leukemia cells (K562) associated with lamprey immune protein (LIP) and further enhances the killing effect of LIP on tumor cells. In addition, using quantitative real-time PCR (Q-PCR) and western blot methods, we found that the relative mRNA and protein expression of lal2 in lamprey leukocytes and sera increased significantly at different times after stimulating with Staphylococcus aureus, Vibrio anguillarum, and Polyinosinic-polycytidylic acid (Poly I:C). Moreover, LAL2 was found to recognize and bind to gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Escherichia coli) and play an important role in the antibacterial process. All in all, our data reveals a long, complex evolutionary history for apolipoprotein genes under different selection pressures, confirms the immune effect of LAL2 in lamprey sera against pathogens, and lays the foundation for further research regarding biological functions of lamprey immune systems.

Label-Free Quantitative Proteomic Reveals Differentially Expressed Proteins in Aeromonas-Immunostimulated Leukocytes of Lampetra japonica.

Lamprey was considered to be one of the most basal jawless vertebrate representatives for studying vertebrate evolution, embryo development, and the origin of adaptive immunity. Here we investigated the effect of the gut-derived Aeromonas on the lamprey leukocytes proteome using the label-free liquid chromatography-tandem mass spectrometry for quantitative proteomics analysis. Significant difference was observed in the regulation of 34 out of 755 proteins in Aeromonas-immunized lamprey. 31 proteins were only identified in saline solution-immunized lamprey and 47 proteins were only identified in Aeromonas-immunized lamprey. Quantitative real-time polymerase chain reaction was used to validate the results of the proteomic analysis. The differentially expressed proteins were found to be associated with several different biological processes. The identification of leukocytes proteins essential for lamprey adaptive immune response induced by gut-derived Aeromonas strain could supply important information on lamprey-Aeromonas interactions and VLR-based adaptive immune signal pathways.

Bacterial community composition in the gut content of Lampetra japonica revealed by 16S rRNA gene pyrosequencing.

The composition of the bacterial communities in the hindgut contents of Lampetrs japonica was surveyed by Illumina MiSeq of the 16S rRNA gene. An average of 32385 optimized reads was obtained from three samples. The rarefaction curve based on the operational taxonomic units tended to approach the asymptote. The rank abundance curve representing the species richness and evenness was calculated. The composition of microbe in six classification levels was also analyzed. Top 20 members in genera level were displayed as the classification tree. The abundance of microorganisms in different individuals was displayed as the pie charts at the branch nodes in the classification tree. The differences of top 50 genera in abundance between individuals of lamprey are displayed as a heatmap. The pairwise comparison of bacterial taxa abundance revealed that there are no significant differences of gut microbiota between three individuals of lamprey at a given rarefied depth. Also, the gut microbiota derived from L. japonica displays little similarity with other aquatic organism of Vertebrata after UPGMA analysis. The metabolic function of the bacterial communities was predicted through KEGG analysis. This study represents the first analysis of the bacterial community composition in the gut content of L. japonica. The investigation of the gut microbiota associated with L. japonica will broaden our understanding of this unique organism.

Characterisation of the bacterial community structures in the intestine of Lampetra morii.

The metagenomic analysis and 16S rDNA sequencing method were used to investigate the bacterial community in the intestines of Lampetra morii. The bacterial community structure in L. morii intestine was relatively simple. Eight different operational taxonomic units were observed. Chitinophagaceae_unclassified (26.5 %) and Aeromonas spp. (69.6 %) were detected as dominant members at the genus level. The non-dominant genera were as follows: Acinetobacter spp. (1.4 %), Candidatus Bacilloplasma (2.5 %), Enterobacteria spp. (1.5 %), Shewanella spp. (0.04 %), Vibrio spp. (0.09 %), and Yersinia spp. (1.8 %). The Shannon-Wiener (H) and Simpson (1-D) indexes were 0.782339 and 0.5546, respectively. The rarefaction curve representing the bacterial community richness and Shannon-Wiener curve representing the bacterial community diversity reached asymptote, which indicated that the sequence depth were sufficient to represent the majority of species richness and bacterial community diversity. The number of Aeromonas in lamprey intestine was two times higher after stimulation by lipopolysaccharide than PBS. This study provides data for understanding the bacterial community harboured in lamprey intestines and exploring potential key intestinal symbiotic bacteria essential for the L. morii immune response.

Identification and characterization of a cathepsin D homologue from lampreys (Lampetra japonica).

Cathepsin D (EC 3.4.23.5) is a lysosomal aspartic proteinase of the pepsin superfamily which participates in various digestive processes within the cell. In the present study, the full length cDNA of a novel cathepsin D homologue was cloned from the buccal glands of lampreys (Lampetra japonica) for the first time, including a 124-bp 5' terminal untranslated region (5'-UTR), a 1194-bp open reading frame encoding 397 amino acids, and a 472-bp 3'-UTR. Lamprey cathepsin D is composed of a signal peptide (Met 1-Ala 20), a propeptide domain (Leu 21-Ala 48) and a mature domain (Glu 76-Val 397), and has a conserved bilobal structure. Cathepsin D was widely distributed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of lampreys. After challenging with Escherichia coli or Staphylococcus aureus, the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with Escherichia coli was also up-regulated in the hearts, kidneys, and intestines. As for the Staphylococcus aureus challenged group, the expression level of lamprey cathepsin D was found increased in the intestines. The above results revealed that lamprey cathepsin D may play key roles in immune response to exogenous pathogen and could serve as a potential antibacterial agent in the near future. In addition, lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed in the human embryonic kidney 293 cells. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin which are the major components in the blood, suggested that lamprey cathepsin D may also act as a digestive enzyme during the adaptation to a blood-feeding lifestyle.

A sensitive analytical method for quantifying petromyzonol sulfate in water as a potential tool for population monitoring of the southern pouched lamprey, Geotria australis, in New Zealand streams.

The migratory southern pouched lamprey, Geotria australis, is a culturally important fish native to New Zealand. Anecdotal evidence suggests that populations of G. australis have declined from historic levels, and presently, this species is rare in many New Zealand rivers and streams. Migratory sea lamprey (Petromyzon marinus) use a pheromone mixture to locate suitable spawning sites. This mixture is comprised of three steroids: petromyzonol sulfate (PS), petromyzonamine disulfate (PADS), and petromyzosterol disulfate (PSDS). We examined the migratory pheromone mixture released by G. australis ammocetes and found that they excrete predominantly PS. PADS has been detected on some occasions in low concentrations, and PSDS either is not released, or is released in extremely low concentrations. By using a recently developed sensitive mass spectrometry method, we compared passive sampling techniques against more traditional active water sampling as methods for estimating lamprey populations in local streams. Passive sampling provided quantitative data for PS from all sites surveyed, with uptake rates of 0.3 to 45.7 pg/day observed. Conversely, active sampling returned only one positive result out of 19 samples, and with a method detection limit of 2.5 × 10(-14) M, this suggests that concentrations of PS in these streams are either extremely low or variable. The combination of passive sampling and triple quadrupole mass spectrometry is a promising tool for monitoring of G. australis in New Zealand streams.

[Parasite fauna of the European river lamprey Lampetra fluviatilis (L.) from Lake Onega].

Data on the parasite fauna of the adult European river lamprey Lampetra fluviatilis (L.) from Lake Onega are reported. Ten parasite species are found, including trematodes Diplostomum petromyzifluviatilis and D. spathaceum (metacercariae), cestode Proteocephalus longicollis, nematodes Cucullanus truttae and Raphidascaris acus, acanthocephalan Echinorhynchus salmonis, ectoparasitic infusoria Chilodonella hexastica, Trichodina tenuidens, and Trichodinella epizootica, and fungus Saprolegnia parasitica. Three species are found to be dominate, namely D. petromyzifluviatilis, Cucullanus truttae, and P. longicollis. Comparative analysis of the parasite faunas of the lampreys from other basins is carried out. Some similarity in the parasite faunas of lampreys and salmonids is discovered.

The prevalence of Clostridium botulinum in European river lamprey (Lampetra fluviatilis) in Finland.

The prevalence of Clostridium botulinum types A, B, E and F in river lampreys caught in Finnish rivers was determined for the first time using a quantitative PCR-MPN (most probable number) analysis. One of 67 raw whole lampreys (1.5%) was positive for the botulinum neurotoxin type E gene, with the estimated C. botulinum count being 100spores/kg. Two type E strains were isolated from the positive sample and confirmed as different genotypes by pulsed-field gel electrophoresis. Although the current procedure of bringing the charcoal-broiled lampreys to market has been without any further packaging or extended storage, interest towards increasing the shelf life of the product by vacuum-packaging is increasing. Our results demonstrate that C. botulinum type E may constitute a safety hazard in processed lampreys from the Baltic Sea area if packaging and extended shelf lives are to be used. To control the potential risk, a storage temperature of 3 degrees C or below should be recommended for these products.

Developing microbial spoilage population in vacuum-packaged charcoal-broiled European river lamprey (Lampetra fluviatilis).

Microbiological and sensory changes in vacuum-packaged charcoal-broiled river lampreys from three lamprey processing plants were monitored as a function of time at 8 degrees C. The lampreys were examined every 7 days up to 8 weeks for aerobic plate count (APC) and lactic acid bacteria (LAB). The highest mean APC and LAB were 6.01 log CFU/g and 4.86 log CFU/g, respectively. Only 6 out of 15 lots reached an APC value of 7.0 log CFU/g during storage. The sensory scores remained at the baseline levels after 8 weeks' storage. Twenty-seven isolates were randomly picked from MRS agar and identified to species level using a 16S and 23S rDNA HindIII RFLP (ribotyping) database and sequencing of the 16S rRNA gene if no database match was obtained. Twelve of the 27 isolates were identified as Lactobacillus curvatus subsp. curvatus, and two Leuconostoc mesenteroides and one Weissella halotolerans strain were also detected. Twelve isolates were not identified by the LAB database. However, they possessed very high (99.9%) 16S gene sequence similarity with either Staphylococcus warneri or Staphylococcus pasteuri type strains. The LAB detected, with the exception of W. halotolerans, have commonly been associated with spoilage of fishery products, but in these vacuum-packaged lampreys, they were not the dominant organisms within the developing spoilage population.

Microbiology of charcoal-broiled European river lampreys (Lampetra fluviatilis) stored at 3 and 22 degrees C.

The microbiological quality of 30 production lots of charcoal-broiled river lampreys was studied at three lamprey processing plants (plants A, B, and C). Samples were taken directly after charcoal broiling and stored at 22 and 3 degrees C. Lampreys were examined on the day of manufacture, and those kept at 22 degrees C were examined every second day for 6 days. Samples kept at 3 degrees C were examined every fourth day for up to 24 days. On the production day, the mean aerobic plate counts (APCs) for broiled lampreys from plants A, B, and C were 2.29 log CFU/g, 1.88 log CFU/g, and undetectable (1.67 log CFU/g), respectively. At 22 degrees C, the mean APCs for samples from plants A, B, and C increased markedly within 4 days, and after 6 days the counts for samples from these plants were 8.56, 5.04, and 6.23 log CFU/g, respectively. Chilling and storage at 3 degrees C remarkably improved the shelf life of the product. The levels of bacteria in charcoal-broiled river lampreys from plant A were higher than those in lampreys from plants B and C. No significant increases in APCs were observed during storage at 3 degrees C for 24 days; mean APCs did not exceed 2.80 log CFU/g for samples from any plant. Staphylococcus aureus was found in two samples. No lactic acid bacteria, thermotolerant coliforms, enterococci, Clostridium perfringens, or Listeria monocytogenes was detected. Microbiological data from this study will be used for the development of a hazard analysis for the determination of critical control points.