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Abstract Background Hagfishes are fascinating creatures that typically inhabit the deep sea. The deep sea is characterized by its lack of sunlight, primary productivity, and diminishing biomass with increasing ocean depth. Therefore, hagfishes living in this environment must develop effective survival strategies to adapt to the limited food supply. Deep-sea hagfishes have been observed to survive without food intake for up to one year. In this study, we have assembled a high-quality somatic genome of the deep-sea hagfish (Eptatretus okinoseanus) captured below 1,000 m. We compared the genome of E. okinoseanus with the genomes of inshore hagfish, lampreys, and other related species to investigate the genetic factors underlying the deep-sea hagfish adaptations to the environment. Results The E. okinoseanus somatic genome was estimated to be 1.89 Gb and assembled into 17 pseudochromosomes. Phylogenetic analysis showed that shallow-sea and deep-sea hagfishes diverged approximately 58.8 million years ago. We found Perilipin gene family was significantly expanded in deep sea E. okinoseanus, which promotes triacylglycerol storage. Furthermore, a series of genes involved in fatty acid synthesis and metabolism, blood glucose regulation, and metabolic rate regulation were also expanded, rapid evolution or positive selection, and these changes contribute to their efficiency in energy utilization. Among these genes, the positively selected gene JNK may play an important role in energy metabolism. In addition, the olfactory receptors of the deep-sea hagfish were significantly expanded to 86, and three conserved motifs present only in hagfishes olfactory receptors were identified, which may facilitate the rapid localization of carrion. Conclusions This study provides valuable genomic resources for insights into the survival strategies of deep-sea hagfishes in oligotrophic environments.

As the deepest vertebrate in the ocean, the hadal snailfish (Pseudoliparis swirei), which lives at a depth of 6,000–8,000 m, is a representative case for studying adaptation to extreme environments. Despite some preliminary studies on this species in recent years, including their loss of pigmentation, visual and skeletal calcification genes, and the role of trimethylamine N-oxide in adaptation to high-hydrostatic pressure, it is still unknown how they evolved and why they are among the few vertebrate species that have successfully adapted to the deep-sea environment. Using genomic data from different trenches, we found that the hadal snailfish may have entered and fully adapted to such extreme environments only in the last few million years. Meanwhile, phylogenetic relationships show that they spread into different trenches in the Pacific Ocean within a million years. Comparative genomic analysis has also revealed that the genes associated with perception, circadian rhythms, and metabolism have been extensively modified in the hadal snailfish to adapt to its unique environment. More importantly, the tandem duplication of a gene encoding ferritin significantly increased their tolerance to reactive oxygen species, which may be one of the important factors in their adaptation to high-hydrostatic pressure.

The genus Rimicaris is the dominant organism living in hydrothermal vents. However, little research has been done on the functions of their intestinal flora. Here, we investigated the potential functions of Deferribacterota, which is dominant in the intestine of Rimicaris kairei from the Central Indian Ridge. In total, six metagenome-assembled genomes (MAGs) of Deferribacterota were obtained using the metagenomic approach. The six Deferribacterota MAGs (Def-MAGs) were clustered into a new branch in the phylogenetic tree. The six Def-MAGs were further classified into three species, including one new order and two new genera, based on the results of phylogenetic analysis, relative evolutionary divergence (RED), average nucleotide identity (ANI), average amino acid identity (AAI) and DNA–DNA hybridization (DDH) values. The results of the energy metabolism study showed that these bacteria can use a variety of carbon sources, such as glycogen, sucrose, salicin, arbutin, glucose, cellobiose, and maltose. These bacteria have a type II secretion system and effector proteins that can transport some intracellular toxins to the extracellular compartment and a type V CRISPR–Cas system that can defend against various invasions. In addition, cofactors such as biotin, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) synthesized by R. kairei gut Deferribacterota may also assist their host in surviving under extreme conditions. Taken together, the potential function of Deferribacterota in the hydrothermal R. kairei gut suggests its long-term coevolution with the host.

Sponges are widely distributed in the global ocean and harbor diverse symbiotic microbes with mutualistic relationships. However, sponge symbionts in the deep sea remain poorly studied at the genome level. Here, we report a new glass sponge species of the genus Bathydorus and provide a genome-centric view of its microbiome. We obtained 14 high-quality prokaryotic metagenome-assembled genomes (MAGs) affiliated with the phyla Nitrososphaerota, Pseudomonadota, Nitrospirota, Bdellovibrionota, SAR324, Bacteroidota, and Patescibacteria. In total, 13 of these MAGs probably represent new species, suggesting the high novelty of the deep-sea glass sponge microbiome. An ammonia-oxidizing Nitrososphaerota MAG B01, which accounted for up to 70% of the metagenome reads, dominated the sponge microbiomes. The B01 genome had a highly complex CRISPR array, which likely represents an advantageous evolution toward a symbiotic lifestyle and forceful ability to defend against phages. A sulfur-oxidizing Gammaproteobacteria species was the second most dominant symbiont, and a nitrite-oxidizing Nitrospirota species could also be detected, but with lower relative abundance. Bdellovibrio species represented by two MAGs, B11 and B12, were first reported as potential predatory symbionts in deep-sea glass sponges and have undergone dramatic genome reduction. Comprehensive functional analysis indicated that most of the sponge symbionts encoded CRISPR–Cas systems and eukaryotic-like proteins for symbiotic interactions with the host. Metabolic reconstruction further illustrated their essential roles in carbon, nitrogen, and sulfur cycles. In addition, diverse putative phages were identified from the sponge metagenomes. Our study expands the knowledge of microbial diversity, evolutionary adaption, and metabolic complementarity in deep-sea glass sponges.

The hadal zone, which represents the deepest marine habitat on Earth (6,000–11,000 m), is a harsh environment mainly characterized by extremely high hydrostatic pressure, and this habitat is believed to have a high degree of endemism. The deep-sea anemone family Galatheanthemidae comprises two valid species exclusively from the hadal; however, no other information about this family is currently available. In the present study, a sea anemone was collected from a depth of 9,462 m at the Mariana Trench and was defined as Galatheanthemum sp. MT-2020 (Actiniaria Galatheanthemidae). The mitochondrial genome of Galatheanthemum sp. MT-2020 was circular, was 16,633 bp in length, and contained two ribosomal RNA genes, 13 protein-coding genes and two transfer RNA genes. The order of the genes of Galatheanthemum sp. MT-2020 was identical to that of the majority of the species of the order Actiniaria. The value of the AT-skew was the lowest in the whole mitochondrial genome, with a positive GC skew value for the atp8 gene, while other species, except Antholoba achates, had the negative values of the GC skew. Galatheanthemum sp. MT-2020 was clustered with another abyssal species, Paraphelliactis xishaensis, in the phylogenetic tree, and these species diverged in the early Jurassic approximately 200 Mya from the shallow-sea species. The usage ratio of valine, which is one of the five amino acids with the strongest barophilic properties, in the mitochondrial genomes of the two abyssal species was significantly higher than that in other species with habitats above the depth of 3,000 m. The ω (dN/dS) ratio of the genomes was 2.45-fold higher than that of the shallow-sea species, indicating a slower evolutionary rate. Overall, the present study is the first to provide a complete mitogenome of sea anemones from the hadal and reveal some characteristics that may be associated with adaptation to an extreme environment.

Abstract Background The metabolic capacity, stress response and evolution of uncultured environmental Tenericutes have remained elusive, since previous studies have been largely focused on pathogenic species. In this study, we expanded analyses on Tenericutes lineages that inhabit various environments using a collection of 840 genomes. Results Several environmental lineages were discovered inhabiting the human gut, ground water, bioreactors and hypersaline lake and spanning the Haloplasmatales and Mycoplasmatales orders. A phylogenomics analysis of Bacilli and Tenericutes genomes revealed that some uncultured Tenericutes are affiliated with novel clades in Bacilli, such as RF39, RFN20 and ML615. Erysipelotrichales and two major gut lineages, RF39 and RFN20, were found to be neighboring clades of Mycoplasmatales. We detected habitat-specific functional patterns between the pathogenic, gut and the environmental Tenericutes, where genes involved in carbohydrate storage, carbon fixation, mutation repair, environmental response and amino acid cleavage are overrepresented in the genomes of environmental lineages, perhaps as a result of environmental adaptation. We hypothesize that the two major gut lineages, namely RF39 and RFN20, are probably acetate and hydrogen producers. Furthermore, deteriorating capacity of bactoprenol synthesis for cell wall peptidoglycan precursors secretion is a potential adaptive strategy employed by these lineages in response to the gut environment. Conclusions This study uncovers the characteristic functions of environmental Tenericutes and their relationships with Bacilli, which sheds new light onto the pathogenicity and evolutionary processes of Mycoplasmatales.

Despite extreme physical and chemical characteristics, deep-sea hydrothermal vents provide a place for fauna survival and reproduction. The symbiotic relationship of chemotrophic microorganisms has been investigated in the gill of Rimicaris exoculata, which are endemic to the hydrothermal vents of the Mid-Atlantic Ridge. However, only a few studies have examined intestinal symbiosis. Here, we studied the intestinal fauna in juvenile and adult Rimicaris kairei, another species in the Rimicaris genus that was originally discovered at the Kairei and Edmond hydrothermal vent fields in the Central Indian Ridge. The results showed that there were significant differences between juvenile and adult gut microbiota in terms of species richness, diversity, and evenness. The values of Chao1, observed species, and ASV rarefaction curves indicated almost four times the number of species in adults compared to juveniles. In juveniles, the most abundant phylum was Deferribacterota, at 80%, while in adults, Campilobacterota was the most abundant, at 49%. Beta diversity showed that the intestinal communities of juveniles and adults were clearly classified into two clusters based on the evaluations of Bray–Curtis and weighted UniFrac distance matrices. Deferribacteraceae and Sulfurovum were the main featured bacteria contributing to the difference. Moreover, functional prediction for all of the intestinal microbiota showed that the pathways related to ansamycin synthesis, branched-chain amino acid biosynthesis, lipid metabolism, and cell motility appeared highly abundant in juveniles. However, for adults, the most abundant pathways were those of sulfur transfer, carbohydrate, and biotin metabolism. Taken together, these results indicated large differences in intestinal microbial composition and potential functions between juvenile and adult vent shrimp (R. kairei), which may be related to their physiological needs at different stages of development.

ObjectiveThe present study aims to increase the concentration of genetically modified bone marrow mesenchymal stem cells (BMSCs) in the distraction osteogenesis (DO) interstitial space and induce the conversion of BMSCs to osteoblasts to improve the osteogenic efficiency in DO and shorten the treatment period.MethodsBone morphogenetic protein 1 (BMP-1) and green fluorescent protein (GFP) gene-modified cell sheets of BMSCs were constructed by tissue engineering. Thirty-six New Zealand white rabbits were randomly divided into three groups: group A (the blank control group), group B (the GFP group) with the injection of GFP gene-modified BMSC sheets into the DO gap, and group C (the BMP-1 group) with the injection of BMP-1 gene-modified BMSC sheets into the DO gap. Rabbits in all three groups were distracted for 5 days at a distraction rate of 2.0 mm/d, once/day. After distraction, the above-mentioned cell sheet suspension was injected into the distraction gap to observe osteogenesis, which was observed by gross specimen observation, micro-computed tomography (Micro-CT) scanning, and histomorphology.ResultsThe gross specimen observation showed that all animals had smooth and continuous bone cortex in the distraction region with relatively high hardness. The osteogenesis quality or hardness was ranked from the highest to the lowest, as Group C > Group B > Group A. Micro-CT and histomorphological observation revealed that group C had better maturation and bone volume of the new bone in the DO region at weeks 3 and 6 than groups B and A.ConclusionBMP-1 gene-modified BMSC sheets could effectively promote the formation of new bone during rapid DO in the mandible, compensating for the poor osteogenesis caused by rapid distraction and providing a new approach to shorten the DO treatment period in clinical practice.

Citreamicin ε is a group of antitumor compounds produced by Streptomyces species. Cytotoxicology study of two diastereomers, citreamicin ε A and B, showed different apoptotic effects on PtK2 cells with IC50 (half-maximal inhibitory concentration) values of 0.086 and 0.025 μM, respectively. Thus, we performed an iTRAQ (isobaric tags for relative and absolute quantitation)–based quantitative proteomic analysis to reveal the mechanism of cytotoxicity of citreamicin ε A and B in PtK2 cells. A total of 1,079 proteins were identified and quantified, among which 103 and 94 proteins displayed significant changes in expression levels after the treatment of citreamicin ε A and B, respectively. These significantly differentially expressed proteins (DEPs) were further annotated by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein–protein interaction analysis, which revealed the involvement of eight molecular pathways. Among them, expression trends of proteins involved in the nuclear transcription factor κB (NF-κB) pathway displayed the opposite between the two diastereomer treatments, indicating different modes of action of these two compounds. Citreamicin ε A treatment induced rapid activation of the NF-κB pathway, which might promote cell survival and resulted in lower toxicity. Our comparative proteomic analysis provided molecular evidence on the toxicity of two diastereomers compounds to cells, which may shed new light on future mechanism study of these antitumor compounds.

To obtain an accurate picture of microbial processes is essential for in-depth understanding of the dynamics of microbial communities and metabolic potentials. In this study, an apparatus for automatic multiple in situ nucleic acid collections (MISNAC) loaded and controlled by a lander was designed and applied for in situ microbial filtration, cell lysis and nucleic acid collection at different time points during three cruises in the South China Sea. The MISNAC apparatus completed 390 L in situ water filtration and collected a total of 1,176 ng DNA with six working units in four deployments at ∼1,000 m depth. Microbial community structures in water samples obtained by the MISNAC and in situ microbial filtration and fixation (ISMIFF) apparatus, respectively, were compared by analyzing sequences of 16S rRNA amplicons. The result showed that these communities were largely consistent, regardless of the differences in the communities in the samples from different cruises and time points. In addition, the transcriptomes of the samples collected by the MISANC and a Niskin bottle dramatically differed in abundance and diversity of 16S rRNA gene reads, indicating that more in situ RNA molecules were preserved by the MISNAC, compared to the Niskin. The application of the MISNAC apparatus paved the way to time-series changes of deep-sea microorganisms and their response to various environmental factors.

The complete mitochondrial genome of the deep sea amphipod Eurythenes magellanicus was determined in this paper. This molecular was 14,988 bp in length, and contained the typical 13 protein coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and one control region (CR). The gene order of E. magellanicus was identical to that from E. maldoror, a deep sea amphipod inhabiting in a deeper habitat than E. magellanicus. A maximum-likelihood tree based on the 13 PCGs from 25 amphipods indicated that E. magellanicus and E. maldoror were closely related and the origin of deep sea amphipods was not monophyletic.

Crustins are widely distributed among different crustacean groups. They are characterized by a whey acidic protein (WAP) domain, and most examined Crustins show activity against Gram-positive bacteria. This study reports two Crustins, Al-crus 3 and Al-crus 7, from hydrothermal vent shrimp, Alvinocaris longirostris. Al-crus 3 and Al-crus 7 belong to Crustin Type IIa, with a similarity of about 51% at amino acid level. Antibacterial assays showed that Al-crus 3 mainly displayed activity against Gram-positive bacteria with MIC50 values of 10–25 μM. However, Al-crus 7 not only displayed activity against Gram-positive bacteria but also against Gram-negative bacteria Imipenem-resistant Acinetobacter baumannii, in a sensitive manner. Notably, in the effective antibacterial spectrum, Methicillin-sensitive Staphylococcus aureus, Escherichia coli (ESBLs) and Imipenem-resistant A. baumannii were drug-resistant pathogens. Narrowing down the sequence to the WAP domain, Al-crusWAP 3 and Al-crusWAP 7 demonstrated antibacterial activities but were weak. Additionally, the effects on bacteria did not significantly change after they were maintained at room temperature for 48 h. This indicated that Al-crus 3 and Al-crus 7 were relatively stable and convenient for transportation. Altogether, this study reported two new Crustins with specific characteristics. In particular, Al-crus 7 inhibited Gram-negative imipenem-resistant A. baumannii.

Diffusing fluid at a deep-sea hydrothermal vent creates rapid, acute physico-chemical gradients that correlate strongly with the distribution of the vent fauna. Two alvinocaridid shrimps, Alvinocaris longirostris and Shinkaicaris leurokolos occupy distinct microhabitats around these vents and exhibit different thermal preferences. S. leurokolos inhabits the central area closer to the active chimney, while A. longirostris inhabits the peripheral area. In this study, we screened candidate genes that might be involved in niche separation and microhabitat adaptation through comparative transcriptomics. The results showed that among the top 20% of overexpressed genes, gene families related to protein synthesis and structural components were much more abundant in S. leurokolos compared to A. longirostris. Moreover, 15 out of 25 genes involved in cellular carbohydrate metabolism were related to trehalose biosynthesis, versus 1 out of 5 in A. longirostris. Trehalose, a non-reducing disaccharide, is a multifunctional molecule and has been proven to act as a protectant responsible for thermotolerance in Saccharomyces cerevisiae. Putative positively selected genes involved in chitin metabolism and the immune system (lectin, serine protease and antimicrobial peptide) were enriched in S. leurokolos. In particular, one collagen and two serine proteases were found to have experienced strong positive selection. In addition, sulfotransferase-related genes were both overexpressed and positively selected in S. leurokolos. Finally, genes related to structural proteins, immune proteins and protectants were overexpressed or positively selected. These characteristics could represent adaptations of S. leurokolos to its microhabitat, which need to be confirmed by more evidence, such as data from large samples and different development stages of these alvinocaridid shrimps.

Hadal trenches are characterized by not only high hydrostatic pressure but also scarcity of nutrients and high diversity of viruses. Snailfishes, as the dominant vertebrates, play an important role in hadal ecology. Although studies have suggested possible reasons for the tolerance of hadal snailfish to high hydrostatic pressure, little is known about the strategies employed by hadal snailfish to cope with low-nutrient and virus-rich conditions. In this study, the gut microbiota of hadal snailfish was investigated. A novel bacterium named “Candidatus Mycoplasma liparidae” was dominant in the guts of three snailfish individuals from both the Mariana and Yap trenches. A draft genome of “Ca. Mycoplasma liparidae” was successfully assembled with 97.8% completeness by hybrid sequencing. A set of genes encoding riboflavin biosynthesis proteins and a clustered regularly interspaced short palindromic repeats (CRISPR) system was present in the genome of “Ca. Mycoplasma liparidae,” which was unusual for Mycoplasma. The functional repertoire of the “Ca. Mycoplasma liparidae” genome is likely set to help the host in riboflavin supplementation and to provide protection against viruses via a super CRISPR system. Remarkably, genes encoding common virulence factors usually exist in Tenericutes pathogens but were lacking in the genome of “Ca. Mycoplasma liparidae.” All of these characteristics supported an essential role of “Ca. Mycoplasma liparidae” in snailfish living in the hadal zone. Our findings provide further insights into symbiotic associations in the hadal biosphere.

Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and immunity of animals, but little is known about the composition and function of intestinal microbiota in deep-sea invertebrates. In this study, the intestinal microbiota of six holothurian Molpadia musculus were investigated, showing that their midguts were predominantly occupied by Izemoplasmatales bacteria. Using metagenomic sequencing, a draft genome of 1,822,181 bp was successfully recovered. After comparison with phylogenetically related bacteria, genes involved in saccharide usage and de novo nucleotide biosynthesis were reduced. However, a set of genes responsible for extracellular nucleoside utilization and 14 of 20 amino acid synthesis pathways were completely retained. Under oligotrophic condition, the gut-associated bacterium may make use of extracellular DNA for carbon and energy supplement, and may provide essential amino acids to the host. The clustered regularly interspaced short palindromic repeat (CRISPR) and restriction–modification (RM) systems presented in the genome may provide protection against invading viruses. A linear azol(in)e-containing peptide gene cluster for bacteriocin synthesize was also identified, which may inhibit the colonization and growth of harmful bacteria. Known virulence factors were not found by database searching. On the basis of its phylogenetic position and metabolic characteristics, we proposed that the bacterium represented a novel genus and a novel family within the Izemoplasmatales order and suggested it be named “Candidatus Bathyoplasma sp. NZ”. This was the first time describing host-associated Izemoplasmatales.

In this study, the complete mitochondrial DNA sequence of a hagfish Eptatretus sp. Nan-Hai from a depth of 1000 m is presented. The complete sequence was determined using next-generation sequencing and long PCRs. The mitochondrial genome of Eptatretus sp. Nan-Hai is 17,538 bps in size and composed of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one control region (D-loop). The base composition of mitochondrial genome is biased toward A + T content, at 67.21%, with GC skew of −0.35 and AT skew of −0.03. A phylogenetic tree revealed that within the genus Eptatretus, Eptatretus sp. Nan-Hai is closely related to Eptatretus atami.

Ontogeny reversal, as seen in some cnidarians, is an unprecedented phenomenon in the animal kingdom involving reversal of the ordinary life cycle. Three species of Turritopsis have been shown to be capable of inverted metamorphosis, a process in which the pelagic medusa transforms back into a juvenile benthic polyp stage when faced with adverse conditions. Turritopsis sp.5 is a species of Turritopsis collected from Xiamen, China which presents a similar ability, being able to reverse its life cycle if injured by mechanical stress. Phylogenetic analysis based on both 16S rDNA and cytochrome c oxidase subunit I (COI) genetic barcodes shows that Turritopsis sp.5 is phylogenetically clustered in a clade separate from other species of Turritopsis. The genetic distance between T. sp.5 and the Japanese species T. sp.2 is the shortest, when measured by the Kimura 2-Parameter metric, and the distance to the New Zealand species T. rubra is the largest. An experimental assay on the induction of reverse development in this species was initiated by cutting medusae into upper and lower parts. We show, for the first time, that the two dissected parts have significantly different potentials to transform into polyps. Also, a series of morphological changes of the reversed life cycle can be recognised, including medusa stage, contraction stage I, contraction stage II, cyst, cyst with stolons, and polyp. The discovery of species capable of reverse ontogeny caused by unfavorable conditions adds to the available systems with which to study the cell types that contribute to the developmental reversal and the molecular mechanisms of the directional determination of ontogeny.

N-acetylneuraminic acid (Neu5Ac) based novel pharmaceutical agents and diagnostic reagents are highly required in medical fields. However, N-acetylneuraminate lyase（NAL）for Neu5Ac synthesis is not applicable for industry due to its low catalytic efficiency. In this study, we biochemically characterized a deep-sea NAL enzyme (abbreviated form: MyNal) from a symbiotic Mycoplasma inhabiting the stomach of a deep-sea isopod, Bathynomus jamesi. Enzyme kinetic studies of MyNal showed that it exhibited a very low Km for both cleavage and synthesis activities compared to previously described NALs. Though it favors the cleavage process, MyNal out-competes the known NALs with respect to the efficiency of Neu5Ac synthesis and exhibits the highest kcat/Km values. High expression levels of recombinant MyNal could be achieved (9.56 mol L−1 culture) with a stable activity in a wide pH (5.0–9.0) and temperature (40–60 °C) range. All these features indicated that the deep-sea NAL has potential in the industrial production of Neu5Ac. Furthermore, we found that the amino acid 189 of MyNal (equivalent to Phe190 in Escherichia coli NAL), located in the sugar-binding domain, GX189DE, was also involved in conferring its enzymatic features. Therefore, the results of this study improved our understanding of the NALs from different environments and provided a model for protein engineering of NAL for biosynthesis of Neu5Ac.

The bryozoan Bugula neritina has a biphasic life cycle that consists of a planktonic larval stage and a sessile juvenile/adult stage. The transition between these two stages is crucial for the development and recruitment of B. neritina. Metamorphosis in B. neritina is mediated by both the nervous system and the release of developmental signals. However, no research has been conducted to investigate the expression of neuropeptides (NP)/peptide hormones in B. neritina larvae. Here, we report a comprehensive study of the NP/peptide hormones in the marine bryozoan B. neritina based on in silico identification methods. We recovered 22 transcripts encompassing 11 NP/peptide hormone precursor transcript sequences. The transcript sequences of the 11 isolated NP precursors were validated by cDNA cloning using gene-specific primers. We also examined the expression of three peptide hormone precursor transcripts (BnFDSIG, BnILP1, BnGPB) in the coronate larvae of B. neritina, demonstrating their distinct expression patterns in the larvae. Overall, our findings serve as an important foundation for subsequent investigations of the peptidergic control of bryozoan larval behavior and settlement.

As typical biofoulers, barnacles possess hard shells and cause serious biofouling problems. In this study, we analyzed the protein component of the barnacle Amphibalanus (= Balanus) amphitrite shell using gel-based proteomics. The results revealed 52 proteins in the A. Amphitrite shell. Among them, 40 proteins were categorized into 11 functional groups based on KOG database, and the remaining 12 proteins were unknown. Besides the known proteins in barnacle shell (SIPC, carbonic anhydrase and acidic acid matrix protein), we also identified chorion peroxidase, C-type lectin-like domains, serine proteases and proteinase inhibitor proteins in the A. Amphitrite shell. The sequences of these proteins were characterized and their potential functions were discussed. Histology and DAPI staining revealed living cells in the shell, which might secrete the shell proteins identified in this study.

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing.

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.

The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts.

Dental teams treated patients with facial injuries after the May 12, 2008 earthquake in Sichuan province, China. Creating oral and maxillofacial surgeon and dentist reserves can help improve disaster response especially since facial injuries make up a significant portion of earthquake-related injuries.