Microplastics exposure as an emerging threat to ancient lineage: A contaminant of concern for abnormal bending of amphioxus via neurotoxicity.

Growing inputs of microplastics into marine sediment have increased significantly the needs for assessment of their potential risks to the marine benthos. A knowledge gap remains with regard to the effect of microplastics on benthos, such as cephalochordates. By employing amphioxus as a model benthic chordate, here we show that exposure to microplastics for 96 h at doses of 1 mg/L and 100 mg/L results in evident accumulation of the polyethylene microplastics. The accumulated microplastics are as much as 0.027% of body weight upon high-dose exposure, causing an abnormal body-bending phenotype that limits the locomotion capability of amphioxus. Mechanistic insight reveals that microplastics can bring about histological damages in gill, intestine and hepatic cecum; In-depth assay of relevant biomarkers including superoxide dismutase, catalase, glutathione, pyruvic acid and total cholesterol indicates the occurrence of oxidative damage and metabolic disorder; Further, microplastics exposure depresses the activity of acetylcholinesterase while allowing the level of acetylcholine to rise in muscle, suggesting the emergence of neurotoxicity. These consequences eventually contribute to the muscle dysfunction of amphioxus. This study rationalizes the abnormal response of the vulnerable notochord to microplastics, signifying the dilemma suffered by the ancient lineage under the emerging threat. Given the enrichment of microplastics through marine food chains, this study also raises significant concerns on the impact of microplastics to other marine organisms, and eventually human beings.

Genome-wide gene expression analysis reveals novel insights into the response to nitrite stress in gills of Branchiostoma belcheri.

Amphioxus has been widely used as a model for the comparative immunology of vertebrates. Studies have reported that gene expression changes in the amphioxus gill in response to biotic stress, such as microbial and their mimic challenge, but little is known about how gene expression is affected by abiotic stress in the marine environment, such as nitrite. A lack of information regarding gene expression response to abiotic stress hinders a comprehensive understanding of gill defense response in amphioxus. Here, RNA sequencing was used to carry out gene expression profiling analyses of Branchiostoma belcheri gills under nitrite stress. Six libraries were created for the control and treatment groups, including three biological replicates. In total, 2416 differently expressed genes (DEGs) were detected in response to nitrite stress, of which 1522 DEGs were up-regulated in the treatment group in comparison to the control, while the remaining 894 DEGs were down-regulated genes. Functional enrichment revealed that these DEGs are primarily involved in disease, innate immunity, xenobiotic biodegradation and metabolism, and biomolecular processes and apoptosis. We screened 11 key nitrite-responsive DEGs to detect their expression responses to nitrite stress at different time points, and validate the sequencing data using real time quantitative PCR. The results indicated that the expression of gene encoding CYP3A, POD, CASPR1, GST, MAO, DDH, and XDH/XO were induced, while those encoding MRC, GT, DNASE1L, and RIPK5 were reduced, to participate in the anti-nitrite response. This study provides a useful resource for research of molecular toxicology in amphioxus under environmental stress.

Nodal and Hedgehog synergize in gill slit formation during development of the cephalochordate Branchiostoma floridae.

The larval pharynx of the cephalochordate Branchiostoma (amphioxus) is asymmetrical. The mouth is on the left, and endostyle and gill slits are on the right. At the neurula, Nodal and Hedgehog (Hh) expression becomes restricted to the left. To dissect their respective roles in gill slit formation, we inhibited each pathway separately for 20 min at intervals during the neurula stage, before gill slits penetrate, and monitored the effects on morphology and expression of pharyngeal markers. The results pinpoint the short interval spanning the gastrula/neurula transition as the critical period for specification and positioning of future gill slits. Thus, reduced Nodal signaling shifts the gill slits ventrally, skews the pharyngeal domains of Hh, Pax1/9, Pax2/5/8, Six1/2 and IrxC towards the left, and reduces Hh and Tbx1/10 expression in endoderm and mesoderm, respectively. Nodal auto-regulates. Decreased Hh signaling does not affect gill slit positions or Hh or Nodal expression, but it does reduce the domain of Gli, the Hh target, in the pharyngeal endoderm. Thus, during the neurula stage, Nodal and Hh cooperate in gill slit development - Hh mediates gill slit formation and Nodal establishes their left-right position.

Identification of a novel antimicrobial peptide from amphioxus Branchiostoma japonicum by in silico and functional analyses.

The emergence of multi-drug resistant (MDR) microbes leads to urgent demands for novel antibiotics exploration. We demonstrated a cDNA from amphioxus Branchiostoma japonicum, designated Bjamp1, encoded a protein with features typical of antimicrobial peptides (AMPs), which is not homologous to any AMPs currently discovered. It was found that Bjamp1 was expressed in distinct tissues, and its expression was remarkably up-regulated following challenge with LPS and LTA. Moreover, the synthesized putative mature AMP, mBjAMP1, underwent a coil-to-helix transition in the presence of TFE or SDS, agreeing well with the expectation that BjAMP1 was a potential AMP. Functional assays showed that mBjAMP1 inhibited the growth of all the bacteria tested, and induced membrane/cytoplasmic damage. ELISA indicated that mBjAMP1 was a pattern recognition molecule capable of identifying LPS and LTA. Importantly, mBjAMP1 disrupted the bacterial membranes by a membranolytic mechanism. Additionally, mBjAMP1 was non-cytotoxic to mammalian cells. Collectively, these data indicate that mBjAMP1 is a new AMP with a high bacterial membrane selectivity, rendering it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows for the first time that use of signal conserved sequence of AMPs is effective identifying potential AMPs across different animal classes.

Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.