**3. Discussion**

### *3.1. Jellyfish Mucus Is a Rich Library for the Discovery of Novel Bioactive Compounds*

Mucus provides a unique and multi-functional hydrogel interface between the epithelial cells and their external environment [49]. In most phyla of aquatic and terrestrial metazoans, it has exceptional properties, including elasticity, changeable rheology and an ability to self-repair, and therefore is an ideal medium for trapping and immobilizing pathogens [50]. Moreover, mucus is a rich library of bioactive components functioning against invasion or microorganisms in innate immunity. Sea star integument produces mucus with antioxidant proteins such as peroxiredoxin, catalase and SODs, and anti-microbial proteins such as lysozyme, melanotransferrin and ribosomal proteins [18]. Fish skin mucus serves as the first line of defense against pathogens and external stressors with antioxidant proteins, lectin, calmodulin, histone proteins, Cystatin-B, Apolipoprotein A1, and heat shock proteins [15]. In the human body, the protective mucus widely covers the epithelial cells on the surface of various tissues and organs such as respiratory organs, stomach, intestine, genitourinary organs, etc. [51–53]. Nasal mucus plays crucial roles in preventing microbial infections and protecting the lower airways from the unhealthy conditions of ambient air through fibrinogen, plasminogen, and complement factor C3 [54], whereas self-protective proteins from cervical mucus include serpins, phosphorylated proteins and heat-shock proteins [55].

In recent years, the large-scale outbreak of jellyfish blooms and dramatic increase of patients by jellyfish envenomation has drawn grea<sup>t</sup> attention in both fishery and medicine [56]. Immediate pain, redness and swelling occurs on local skin after being injured by contacting the tentacles of jellyfish, while serious systemic symptoms include multiple organ failures or even death [57]. The venom that is released from nematocyst of jellyfish tentacle possesses a large amount of toxins and other active proteins leading to these various manifestations, and is therefore naturally considered to be a rich library of bioactive compounds. Using the integrated methods of traditional liquid chromatography, amino acid sequencing and cDNA library alignment, the hemolytic proteins were firstly purified and identified from two box jellyfish, *Carybdea rastoni* [58] and *Carybdea alata* [59], in 2000; subsequently, more than ten hemolytic proteins have successfully been identified as a novel cytotoxic protein family [60–62]. By the combination of transcriptomics and proteomics, more than 170 potential toxin proteins, including metalloproteinases, an alpha-macroglobulin domain containing protein, two CRISP proteins, a turripeptide-like protease inhibitor, and particularly, nine novel members of a taxonomically restricted family of cnidarian pore-forming toxins, were successfully identified from the jellyfish *Chironex fleckeri* on the basis of homology to known toxins in public sequence databases [63]. Similarly, 174 potential toxic proteins with 27 homologs to the toxins from venomous animals, including phospholipase A2, zinc metalloproteinase-disintegrin agkistin, serine protease inhibitor, plancitoxin-1, alpha-latrocrustotoxin-Lt1a, etc. were scanned in the jellyfish *Cyanea nozakii* [64].

When an envenomation happens, the skin firstly contacts the jellyfish mucus; this is also an important poisoning source described by fishers indicating that mucus also contains a large number of active components potentially deriving from nematocyst venoms. Because mucus covers the surface of tentacles, including nematocysts, any release of nematocyst venom has a certain portion of debris, thereby displaying a conventional material communication with mucus. Meanwhile, considering the long evolutionary history of jellyfish and the key role of mucus in innate immunity of other aquatic organisms, there should also be a large number of bioactive compounds to function as adhesion and in defense. In this study, we explored the stress-induced mucus secretion and its composition by a combination of proteomics and metabolites using a low-toxicity jellyfish, *A. coerulea*. We successfully scanned 1208 and 2421 proteins in mucus and tissue, respectively. Among them, 225 proteins are exclusively identified in mucus while 183 proteins are up-regulated when compared to that of the tissue. As expected, the mucus-enriched proteins possess dozens of functions as indicated by GO and KEGG analyses. Moreover, 21 self-protective proteins, such as metalloproteinases, serine proteinase inhibitors, SODs and complements were successfully scanned against potential external invasion.

### *3.2. Tryptamine Release Indicates an Elevated Stress of Jellyfish When Stimulated*

Metabolomics is a technique for studying metabolic networks in biological systems by examining the metabolite profiles and their dynamic changes before and after stimulation or disturbance [65,66]. The number of metabolites is much less than those of genes and proteins, and small changes in gene and protein expression can be amplified at the metabolite levels [67]. In this study, we have found three groups of metabolites with lower, equal and higher expressions in mucus when compared to those in tissue by metabolomics. Because metabolites in mucus have the tendency to disperse into the surrounding seawater [68,69] and all are feasibly synthesized and secreted from jellyfish tissue, it is reasonable that metabolites with lower concentrations (L-Glutamate, Succinylacetone, Linoleyl linolenate, Uridine, L-Proline, Inosine, Hypoxanthine, L-Valine and Guanosine) in mucus are in normal conditions while those with equal concentrations (4-Hydroxy-L-proline, Citrulline, L-Leucine, 3-(Phosphoacylase mido)-L-alanine and L-Threonine) should already have been well enriched with the balance between release and diffusion.

Of particular interest, the metabolite Tryptamine displays the maximal FC value of 7.8, which is far higher and indicates much better enrichment than that of other metabolites. Tryptamine is a group of monoamine alkaloids including serotonin (5-hydroxytryptamine, 5-HT) and melatonin, as well as other compounds known for their neurotransmitter properties [70]. It derives from the amino acid-trytophan by tryptophan decarboxylase (EC 4.1.1.105 and EC 4.1.1.28), which is also named aromatic L-amino acid decarboxylase (AADC or AAAD), DOPA decarboxylase (DDC) and 5-hydroxytryptophan decarboxylase, and which plays an important role in the dopaminergic system participating in the uptake and decarboxylation of amine precursors in the peripheral tissues [71,72]. The typical tryptamine, serotonin, is one of the most important and widely studied hormones in humans and other vertebrates involved in regulation and modulation of multiple processes within the central nervous system and behavior [73]. It also plays an important role in gastrointestinal motility, vascular tone and platelet function, and has been related to various pathophysiological processes [74]. However, less research on tryptamine is reported in marine invertebrates. It is reported that cells with serotonin are concentrated at the anterior pole of hydrozoan planulae [75]. Brain serotonin levels in crayfish are reported to greatly increase when exposed to pressure [76]. Because jellyfish have a well-developed peripheral nervous system, and it is reported that tryptamine can be accumulated and released in large quantities under stress in marine invertebrates [76,77], we hypothesize that the increases of tryptamine release and mucus secretion indicate an elevated stressful response when stimulated by taking the jellyfish out of seawater. This will provide a new mechanism for jellyfish to secrete mucus although further validation is needed.

### *3.3. Self-Protective Proteins Play a Key Role in Innate Immunity of Jellyfish*

In mucus-enriched proteins, we have successfully scanned dozens of self-protective proteins including 11 metalloproteinases, four serine protease inhibitors, three SODs, and three complements. According to the activity of metalloproteinase that degrades extracellular matrix proteins and bioactive molecules on the surface of the jellyfish *A. coerulea*, three functions are proposed as an important self-protective factor. The first function is to process the extracellular matrix such as collagen, laminin and THBS to form the main component of the mucus [78]. The second self-protective effect is to directly digest or degrade the invasive toxic component or microorganism, which can further induce the third function, transferring the external stimuli to the body by activating or deactivating the signaling through cutting or digesting the extracellular part of membrane proteins such as Her2 receptors. On the contrary, serine protease inhibitors form stable complexes with their target enzymes to control the activity of serine proteases [79], thereby playing an important role in innate immunity and environmental stability in the body [80]. On the surface of jellyfish, serine protease inhibitors are able to prevent the over-digestion of functional proteins through inhibiting the serine proteases from both jellyfish themselves and the exogenous invading pathogens. As the natural antioxidant function, SODs in jellyfish mucus should catalyze the production of O2 and H2O2 from superoxide

(O2−), which results in less harmful reactants and protects cellular components from being oxidized by ROSs [81,82] that are feasibly from jellyfish tissue, invading microorganisms or environmental conditions such as water pollution, eutrophication, anoxia and radiation. In addition to the above, we also listed three complements. The complement system is a highly complicated defense system in the innate immunity of invertebrates, which directly participates in the lysis of pathogen cell [83]. Most studies on cnidarian complement have focused on C3 [83,84], of which the activation plays a central role and is required for all three pathways of complement activation [85]. By comparison, the complement factor B is a component of the alternative pathway of complement activation to form the pore complexes and lyse the invading micrograms.

### **4. Materials and Methods**

### *4.1. A. coerulea Samples*

*A. coerulea* were collected alive from an artificial aquafarm in Shanghai, China in May 2017. Jellyfish were transported in a 3-L plastic bag fully filled with seawater, to prevent damage from sloshing. In the laboratory all individuals were maintained in buckets of seawater at 18–22 ◦C.

### *4.2. Transcriptome Sequencing and Its Annotation*

Total RNA of jellyfish *A. coerulea* was extracted with TRIzol (Life Technologies) following the manufacturer's procedure. RNA purity, concentration and integrity were evaluated using NanoDrop 2000 UV–Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA 6000 Nano LabChip®Kit (Agilent Technologies, Santa Clara, CA, USA). The mRNA was enriched by polyT oligo-conjugated magnetic beads, then fragmentation buf Santa Clara, America fer was added to break the mRNA into short fragments. A single strand of cDNA was synthesized by using random hexamers as template. The double-stranded cDNA was synthesized by adding buffer, dNTPs, DNA polymerase I and RNase H. The double-stranded cDNA was purified by AMPure XP beads (Beckman, Atlanta, GA, USA). The purified double-stranded cDNA was repaired at the end, A-tail was added and sequencing junction was connected, and AMPure XP beads were used to select the fragment size. Finally, PCR was amplified and purified by AMPure XP beads, and the final cDNA library was obtained. Illumina HiSeq sequencing was carried out after qualification.

The raw data of sequencing were evaluated by FastQC (version: 0.11.2) and filtered by Trimmomatic (version: 0.36) to remove the joints as well as the sequences with low quality. Then the clean data were de novo assembled into transcripts by Trinity, which were further annotated by blast using the databases NR (National Center for Biotechnology Information non-redundant protein sequences) and Swissprot. According to the annotation results of transcripts NR and Swissprot, GO and KEGG analyses were finally performed according to the annotation of transcripts by NR and Swissprot.

### *4.3. Mucus Collection and Tissue Homogenate Preparation*

The jellyfish *A. coerulea* were fasted for 48 h and gently washed thoroughly with sterile filtered artificial seawater before experiments. After measurement of body weight, jellyfish were gently put into a funnel with one layer of medical gauze, which was then sealed with a plastic wrap to avoid liquid evaporation. Jellyfish mucus was collected every 10 min with a 15 mL centrifuge tubes, for a total of 1 h. The debris was removed by centrifugation at 4000 rpm for 10 min and the supernatant was collected and stored at −80 ◦C for further experiments. Jellyfish tissue homogenate was obtained using the method of ultrasonic extraction. In every working cycle, the working time and resting time were 20 s and 1 min with a power of 200 W. The total working time was 2 min. The supernatant of jellyfish tissue homogenate was collected after centrifugation at 4000 rpm for 10 min at 4 ◦C, which was then stored at −80 ◦C for further analysis.

Four replicates of all experiments were performed, using independent batches of *A. coerulea* samples. Protein concentrations of both mucus and tissue homogenate were determined by Bradford's assay. Bovine Serum Albumin (BSA) was used to construct the standard curve.
