Abstract: Background and Objective: The adaptive immune system depends on the identification and elimination of foreign molecules. The objective of this article is to analyze the antimicrobial peptide of Styela plicata on the molecular level to know what the triggered mechanisms are after stimulation both in vivo and in vitro; perform a comparative analysis between the response of this sea squirt to a living stimulus, bacteria and to an inert stimulus, pathogen-associated molecular patterns. Materials and Methods: Styela plicata was collected from the Arabian Gulf and the Mediterranean in 2019. Parts of the pharyngeal sac, mantle, blood samples, ovary and testis were analyzed using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and Southern blot. Results: Analysis of sea squirts revealed a degree of variability which does not seem to be related to the geographical origin. Genetically related squirts share the same antimicrobial peptide bands. The in vitro experiments showed similar antimicrobial peptide bands before and after the exposure of the blood cells to the stimuli. Conclusion: This is the first time to detect that 5-methyl-hexahydropyrimidine-2,4-dione showed variability in estuarine areas in the Arabian Gulf.
INTRODUCTION
In sea squirts, the innate immune system is capable of recognizing structures present on the surface of microorganisms through recognition receptors1,2. Teichoic acid from actinobacteria, endotoxins from proteobacteria, DNA from bacteria, zymosan, β-glucans and RNA polycytidylic acid are some examples of structures that activate the innate immune system3. The classic methodology to study innate immunity in invertebrates has focused to find out the inflammations and the responses between the host and parasites4. Most of the references focus on phagocytosis studies and the production of nitrogen and oxygen radicals. The genes characterized and related to the immune system in sea squirts code for antimicrobial peptides, lysozymes and heat shock proteins5. Besides, there is a cDNA library identified from un-stimulated sea squirts which code for a protein against lipopolysaccharides, β-glucans, ficolins and lectins of various pathogens6. Different studies have been carried out in marine invertebrates to find primitive molecules that would act as predecessors as those present in the adaptive immune system7,8. Although histocompatibility processes are related to the adaptive response, there are several examples where allorecognition plays a vital role in the defence of invertebrates9. In recent years, the knowledge about the innate immune system has increased considerably due to the studies carried out in the fruit fly, Drosophila melanogaster10. A series of highly variable genes related to this process have been identified in invertebrates as coagulation factors detected in Biomphalaria glabrata, possessing immune gamma globulin domains at their amino terminus11. In the sea squirt, Botryllus schlosseri a highly polymorphic genomic locus called the fusion histocompatibility locus has been detected12. High levels of polymorphism have been found in molecules related to other processes that require recognition, such as fertilization processes. Specifically, highly variable sterility receptors have been described in two species of ascidians Halocynthia roretzi and Halocynthia aurantium which through self-non-self-recognition mechanisms can avoid self-fertilization6. The same occurs with Ciona intestinalis where a transmembrane protein, like the variable complement receptor, has been characterized13. The results presented by many authors revealed the existence of antimicrobial peptides in which high variability was found, either at the genomic level, cDNA, or protein. To understand this variability at the individual level, the electrophoretic technique has been used for separating DNA molecules for the first time in bivalve mollusks. Later, it has been applied in biology to study mutations in genes14 and microbial diversity15. This study is built per the results of Palanisamy et al.16 who identified the antimicrobial peptide 5-methyl hexahydropyrimidine-2,4-dione in Styela plicata.
This article aimed to analyze the antimicrobial peptide of Styela plicata molecular level to know what the triggered mechanisms are after stimulation both in vivo and in vitro; perform a comparative analysis between the response of this sea squirt to a living stimulus, bacteria and to an inert stimulus, pathogen-associated molecular patterns and derive the possible relationships between the molecular and functional responses. Test whether the antimicrobial peptide is inheritable. Results of this study will provide new knowledge about the defence mechanisms and provide new data to the aquaculture sector.
MATERIALS AND METHODS
Sample collection: The collection of Styela plicata was carried out in Arabian Gulf in 2019. Specimens were transferred to aquaria with continuous aeration and fed on phytoplankton to the laboratory at Imam Abdulrahman Bin Faisal University, Saudi Arabia. Experimentations of this article are continued from June, 2019-July, 2020. Seawater was changed every other day. Other specimens of the same species were collected from Ras El-Tin beach and Abu Qir bay of the Mediterranean Sea off Alexandria, Egypt in June-August, 2019 for a comparative study. These specimens experimented at the Faculty of Science, Department of Microbiology, Quality Control Lab, Alexandria University, Egypt.
Denaturing Gradient Gel Electrophoresis (DGGE): Amplification of a part of the cDNA is done through a universal primer PC: 5-T23 (C/G/A)-3 and a reverse primer PS: 5 GGGACCATCAACTT (A/T)TCT(C/T)-T(C/T/A)TGT3 for 5-methyl-hexahydropyrimidine-2,4-dione. The forward oligonucleotide has a sequence attached in GC at its 5' end. The PCRs were carried out in 60 μL of a mixture of 1.35 U of Taq (TaKaRa ExTaq; TaKaRa Japan) applying the instructions of the manufacturer. The cycle to which the samples were subjected was a touchdown cycle with 72°C extensions for 15 min. The outcomes were precipitated using 1/11 volume of Sodium ethanoate (2 M; pH 4.7) and 2 volumes of ethanol. Loading of the products (app. 850 ng) was done on 6% (w/v) acrylamide gel, 0.75 mm thick and with a denaturing gradient of urea-formamide in a range between 30 and 65%. DGGE was carried out in 1X TAE buffer using the DGGE-2001 system (Cleaver Scientific®) at 100V in 70°C for 16 hrs. staining of the gels was done for 55 min in tromethamine, vinegar and Edetic acid supplemented with SYBR® Gold (Invitrogen) and immediately photographed under ultraviolet light using the Gel DocTM XR system and quantity one program (Bio-Rad, Hercules, CA)17.
In vitro and in vivo stimulation: A total of 15 sea squirts were divided into 5 groups of 3 squirts each. Group 1 was inoculated in 100 μL volume of a solution containing 106 cells/mL of a solution of heat-killed M. lysodeikticus, V. splendidus and V. anguillarum (Science Kit®). Group 2 was stimulated with the same volume of the solution containing live bacterium Vibrio splendidus (optical density 0.033). Treatment 3 consisted of adding 100 μL of a Polyinosinic: polycytidylic acid, polycytidylic acid solution Sigma-Aldrich® (1 mg mL–1) to each sea squirt. Group 4 was exposed to a heat shock which consisted of a bath in seawater at 35°C for 100 min. The rest 3 sea squirts acted as a control and were impregnated with 90 μL of filtered seawater. After stimulating the squirts, they were returned to seawater at 15°C for 24 hrs. After that time, the mantle, pharyngeal sac, parts of ovary, testis follicles and blood cells samples were taken from each sea squirt. Total 750 μL Trizol was added to the tissues and the blood cells were previously subjected to centrifugation at 3500 rpm for 20 min in 5°C and the cells were re-suspended in the same volume of Trizol as in the case of the tissues. Cells and tissues were kept at -80°C until the RNA isolation time. In in vitro experiments, 4 sea squirts were used to carry out the inoculation. RNA isolation was carried out using the Trizol reagent and subsequently treated with 2.4 U of DNase (Thermo Fisher Scientific®) to eliminate DNA contamination18,19.
Geographic and tissue variability: To study the variability of 5-methyl-hexahydropyrimidine-2,4-dione in different geographic areas or/and in different tissues within the same squirt, several sea squirts from different areas of the Arabian Gulf and two areas of the Mediterranean Sea were sampled. Besides, the pharyngeal sac, mantle and blood samples were collected and processed20.
Family profiles and generation of variability: To study the heritability of 5-methyl-hexahydropyrimidine-2,4-dione, a family was generated by induction of spawning and cross-fertilization was artificially performed. The larvae were kept in a closed circuit in aquaria at 20°C with continuous aeration and Pavlova salina was provided in the aquaria (10×100 cells/larva). Samples were carried out at 1, 3 and 5 days Post-hatching. The larvae were homogenized in 750 μL Trizol and have frozen at -90°C until treatment. To determine the mechanism by which the different isoforms of cDNA are initiated from DNA, total nucleic acids from two sea squirts were secluded. The Phenol-chloroform technique was applied to obtain the DNA and the isolation of RNA, as well as the synthesis of cDNA, was carried. The total cDNA and the DNA were applied as templates for the performance of PCR using the mentioned primers under the defined conditions. The resulted materials were joined into the vector PCR 2.2 and DH5α and competent bacteria were transformed with this new construction. The RNA of each one was isolated using Trizol and 5 μg of the samples and used to make the passage to cDNA. PCRs were performed to amplify 5-methyl-hexahydropyrimidine-2,4-dione and the housekeeping gene cytoplasmic β-actin. The cycles to which the samples were subjected in each of the PCRs were the touchdown cycle for antimicrobial peptide and β-actin21,22.
Southern blot: To obtain good quality DNA, different isolation methods were tried such as DNAzol (Invitrogen), phenol-chloroform and the methods described by Schmidt et al.17. After determining its quality on an agar gel (1%), independent reactions were carried out with various restriction enzymes such as EcoRI, NdeI, BamHI, XhoI and AgeI as well as combined reactions with several enzymes using 40 μg of DNA in each. of them. The choice of enzymes was carried out taking into account that the restriction target was not present in the gene of interest. The products of the digestions were run on an agar gel (1%) using a molecular weight standard labelled with digoxigenin. Immunological detection was carried out using the DIG Nucleic Acid Detection Kit (Mole®) following Casso et al.23.
RESULTS
To determine whether is there any variability in 5-methyl-hexahydropyrimidine-2,4-dione among sea squirts, the mRNA expression of three sea squirts was analyzed using the DGGE technique where secondary metabolites and β-actin are shown (Fig. 1a).
| Fig. 1(a-f): | DGGE banding profile of different samples (a) Secondary metabolites and β-actin in 3 squirts (1, 2 and 3). (b) Two squirts (4 and 5) were obtained after in vitro stimulation with poly (I: C) (P) and a mixture of dead bacteria (B). (c) Secondary metabolites band profiles corresponding to 3 squirts (9, 10 and 11) in blood cells (B), pharyngeal sac (C) and mantle (M). (d) Blood cells band profile for secondary metabolites obtained for 3 squirts from 3 different areas of the Arabian Gulf. (e) Blood cells band profile obtained for 3 squirts from S. Khobar, Ras el Tin and Abu Qir and (f) Analysis of the expression of secondary metabolites in blood cells of 5 squirts belonging to different areas. (a) Different tissues are represented (M: Mantle, C: Pharyngeal sacs, B: Blood cells). (b) C and Co represents control and absolute control respectively |
Even though this analysis was repeated with more than 100 sea squirts, the pattern of bands observed was different in each one. Furthermore, the number of bands observed in each analyzed sea squirt was variable, being able to detect from a minimum of 5 to a maximum of 24. However, the variability found for a β-actin gene was significantly low after stimulating the process with polycytidylic acid (P) and a mixture of dead bacteria (B) (Fig. 1b). The results obtained after analyzing the expression of an antimicrobial peptide in blood cells (B), pharyngeal sacs (C) and mantle (M) of 3 sea squirts revealed identical profile to the same sea squirt but it was possible to find squirts where this did not occur (Fig. 1c ). In squirts 9 and 11, the blood cell profile was different from that obtained in the mantle and pharyngeal sacs. However, in sea squirt no. 10 the tissue that showed differences from the others was the pharyngeal sac. The result of Fig. 1a also shows different tissues belonging to three other different sea squirts (1, 2 and 3) where squirts 2 and 3 showed the same pattern in pharyngeal sacs and blood cells but differed in that of the mantle. However, sea squirt 1 presented the same pattern of bands in pharyngeal sacs and mantle and different in blood cells.
| Fig. 2(a-c): | DGGE banding profile of different samples (a) Analysis of secondary metabolites from sister larvae, (b) Expression of secondary metabolites and β-actin in mature ovary and testis and (c) A total of 20 genomic clones of sea squirt no. 1 were sequenced. Most of the genomic sequences of DNA were divided into 2 groups. The lower part represents the protein alignments obtained after the translation of the exon and cDNA sequences. The same sequences appear in the same colour. (a) V1-V8 sister larvae, (b) O mature ovary and T testis and (c) S0 before treatment, S24 after 24 hrs treatment, B dead bacteria, PC polycytidylic acid |
To study the level of variability of an antimicrobial peptide in different geographical areas, samples were taken from different areas of the Arabian Gulf. After carrying out the same analysis as in the previous cases, the results again showed a high number of bands in each individually analyzed sea squirt, corresponding to all the bands found with antimicrobial peptide (expected values in the Blastn<10-4). A common pattern of bands was not found between the sea squirts belonging to the same zone or between those belonging to different areas of the Arabian Gulf (Fig. 1d). Analysis of sea squirts of the Arabian Gulf and two areas of the Mediterranean Sea revealed a degree of variability. Therefore, diversity does not seem to be related to the geographical area squirts from as South Khobar, Ras el Tin and Abu Qir (Fig. 1e). To detect whether those sea squirts belonging to the same habitat could share a similar band profile, 5 sea squirts belonging to two different habitats were sampled. The results showed that there is no shared band pattern among sea squirts belonging to the same area, suggesting that each squirt presents a specific band profile. Analysis of the expression of secondary metabolites in blood cells of 5 squirts belonging to different areas is shown (Fig. 1f). To study whether the band profile could be shared between different members of the same family, the spawning was induced experimentally to carry out fertilization. The variability of the antimicrobial peptide was analyzed by DGGE in a total of 82-newly metamorphosed larvae belonging to the same parent (Fig. 2a). This was the first time that a band pattern shared by several larvae was detected, assuming that only offspring of the same patent squirts share identical antimicrobial peptide bands. The number of total bands obtained in each newly metamorphosed squirt was significantly lower than that found in adult ones. To determine whether the band profile of the larvae resulted from a mixture of the bands present in the parent, the same analysis was carried out using DGGE of the ovary and testis samples (note, this squirt is hermaphrodite but has separate ovary and testis entities), observing that interestingly, the testis germ cells did not express antimicrobial peptide, whereas they did express other genes such as β-actin. To confirm the possible fact that antimicrobial peptide was inherited through the maternal route, PCRs were performed using RNAs from mature gonads of more than five squirts. The expression of the antimicrobial peptide was compared with that of β-actin to rule out the fact that the non-detection of the antimicrobial peptide was due to the poor quality of the RNA due to the high enzymatic load of the testis. The expression of the secondary metabolite and β-actin in mature ovary and testis is shown (Fig. 2b). Despite the low quality of β-actin, it is observed how all tissues express antimicrobial peptide, while it is not detected in any of the analyzed testes. To determine the mechanisms by which diversity is generated in antimicrobial peptides, a comparison analysis of genome and cDNA clones of two sea squirts was carried out. The exons obtained in the genomic DNA analysis were aligned to be able to compare them with the cDNA sequences. A total of 20 genomic clones of sea squirt no. 1 were sequenced. Most of the genomic sequences of DNA were divided into 2 groups (Fig. 2c). All the clones belonging to the same group showed 100% homology. The rest of the clones analyzed were unique sequences (a total of 9), each representing 5% of the total clones analyzed and showing 99% homology with the most representative groups. These data suggest that the unique sequences must have formed from point mutations of the two most represented forms. A total of 31 cDNA clones were sequenced and grouped again, in 2 groups with 35 and 29% of the clones in each one of them. The rest of the sequences were divided into 8 groups (a maximum of 3). The equal opportunity between these 8 groups and the 2 majority groups were greater than or equal to 98%. Thus, for sea squirt no. 1, all minor forms of both genome and cDNA clones appear to come from the two major forms. However, none of the minority forms of DNA is capable of generating forms of cDNA. For sea squirt no. 2, a total of 8 genomic clones were sequenced, all of which were grouped into 3 groups with 37.5, 37.5 and 25% of the clones respectively. In this case, no minority form was obtained. In the cDNA, most of the clones were grouped into 2 groups that contained 35.7 (C12) and 28.5% (C22) of the analyzed sequences. The rest of the clones detected were classified as unique sequences, all of them showing 100% homology with the most representative sequences. As can be seen in Fig. 3a, in sea squirt no. 1, only 4 sequences are equal 2-2, coinciding with the majority forms, both genome and cDNA. In Fig. 3b the sea squirt no. 2 there are 3 pairs of identical sequences where the 3 majority genomic forms are found, the 2 majority cDNA forms and, in this case, a minority form of cDNA also appears coinciding with majority genomic forms. The expression profile of antimicrobial peptide was estimated after its exposure time to different external stimuli such as bacteria, polyinosinic-polycytidylic acid and a heat shock, both in vitro (primary culture of blood cells) and in vivo (isolating blood cells). The results of the in vitro experiments are shown 0-24 hrs treatment in Fig. 4a, where it can be seen that the profile of the antimicrobial peptide bands before and after the exposure of the blood cells to the stimuli is the same for the two sea squirts analyzed. As in the last experiment, only the blood cells can be collected following the addition of the stimulus, without causing damage to the squirt. The location and number of the bands in the DGGE gel were the same before and 24 hrs after the administration of the stimulus (polyinosinic-polycytidylic acid and dead bacteria) for the 3 sea squirts investigated. The same squirts with which the experiment was carried out were kept in an aquarium for one week to evaluate if the profile of bands could vary over time. Although not all squirts survived, the same profile was found in the survivors analyzed. Concerning heat shock treatment, the band profile in blood cells was the same in two squirts before and after the stimulus. However, in those sea squirts stimulated with live bacteria, the number of bands at 24 hrs post-infection seems to be reduced. Furthermore, the number of bands detected in the blood cells was slightly higher than in the pharyngeal sacs and the mantle, except in the controls where the distribution of the bands in the three tissues analyzed was more similar to each other than in the other two treated squirts with live bacteria and by heat shock (Fig. 4b). The results obtained in the Southern blot were not conclusive. The experiment has performed a total of 10 times varying the DNA extraction methods, as a positive control of the technique, a probe against histone H3 was used, using the pair of primers, observing a strong hybridization. The best result was obtained by isolating the DNA with phenol-chloroform, digestion with EcoRI, BamHI and XhoI, for one and half hours and with a hybridization temperature of 55°C. The Southern blot hybridized with a probe against 5-methyl-hexahydropyrimidine-2,4-dione is shown (Fig. 4c). The results obtained showed the presence of 3 faint bands after digestion with EcoRI (lane 1) suggesting the existence of 3 gene copies. In lanes 2 and 3, 2 bands are seen that would suppose the existence of hybridization of the probe with 2 different genome fragments, therefore, 2 copies of the gene. However, the digestions with 2 enzymes (lanes 4 and 5) did not show the expected results that would correspond to the sum of results obtained with the squirt enzymes.
| Fig. 3(a-b): | FASTA schematic representation between the genomic and cDNA forms of secondary metabolite found for the two squirts Protein alignments obtained after the translation of the exonic and cDNA sequences. The same sequences appear in the same colour in squirt 1 and 2 |
| Fig. 4(a-c): | DGGE banding profile of different samples (a) Secondary metabolites in blood cells before treatment 0 and 24 hrs after in vivo stimulation with dead bacteria (B) and poly (I: C9 (P). C and Co represent control and absolute control respectively. (b) Secondary metabolites in blood cells, pharyngeal sac (BC) and mantle (M) 24 hrs after in vivo stimulation with live bacteria and by heat shock. (c) Southern Blot hybridized with a probe against 5-methyl-hexahydropyrimidine-2,4-dione. (a) h0 before treatment, 24 after 24 hrs treatment, (b) BC: Branchial Chamber, M: Mantle |
DISCUSSION
In this study, DGGE was used to test the variability of an antimicrobial peptide in Styela plicata collected from different parts of the Arabian Gulf and two areas of the Mediterranean Sea. Our results showed that the band profile observed for each sea squirt was unique and exclusive in each one, regardless of their geographical origin and degree of gonadal development. Despite the high number of sea squirts analyzed in this study, no squirts shared sequences with another, except those belonging to the same parent. This variability could even be linked to the capability of sea squirts to distribute and dwell in different ecological niches. The antimicrobial peptide is expressed from the early stages of development and in all tissues analyzed except the testis, assuming that the function of these metabolites is for the survival of these organisms. Although the sea squirt of this study is not considered a colonial species, the squirts do show a tendency to aggregate.
Marine invertebrates are subjected to various stressors and are susceptible to infectious diseases or/and different opportunistic pathogens24. Several data are suggesting that sea squirts must have resistance mechanisms against all pathogens. Bacterial lipopolysaccharides and other recombinant molecules such as heterologous tumour necrosis factor-alpha activate different signal transduction cascades involved in innate immunity in invertebrate blood cells. After immunostimulation with β-glucan, marine invertebrates do not increase their bactericidal activity as occurs in vertebrates25. The most striking result was the isolation of a great number of antimicrobial peptides from marine invertebrates26. This study observed that innate immunity is present from the first larval stage and in all analyzed tissues. This result has not been previously detected in other sea squirts. Peptides against microbial fauna are the main source of innate immunity in marine invertebrates. The cationic molecules were identified, in Styela clava as Styelin D27 and 5-methyl-hexahydro pyrimidine-2,4-dione in Styela plicata. However, there are hardly any studies where this technique is used for the analysis of genetic polymorphisms and none of them was carried out in sea squirts. In molluscs, specifically in the snail Biomphalaria glabrata, the existence of a high number of different cDNA sequences has been detected in a molecule related to clotting factors28. The fester gene detected in Botryllus schlosseri, possesses a great genetic and somatic diversity which suggests that this gene may be involved in histocompatibility processes29. This study compared the band profile of the antimicrobial peptide with the profile of a gene not related to the immune system β-actin. The lack of variability in this control gene allows concluding that unevenness is not a shared feature in sea squirt genes. Besides, the level of variability of the antimicrobial peptide seems to be universal since it was observed in sea squirts from different areas. The processes where highly polymorphic genes play an important role are allorecognition mechanisms. This gene is involved in the compound sea squirts. When zooids have common genes, a vascular system is formed between them to build the Association30. However, antimicrobial peptides share common characteristics with the fester gene since each squirt has a unique reservoir of shapes, which, at least as in Bottryllus schlosseri are built by optional splicing are built by optional splicing31. The implication of this study guides that living organisms possess an innate immune system which responds automatically to pathogens. The findings of this study can be applied to other marine organisms to study their degree of response against infectious diseases. This study recommends that aquaculture of marine organisms must be improved to increase the productivity. It is difficult to test the response of marine organisms in their natural habitat and to obtain better results the experimentations are limited to be in the laboratory under defined conditions.
CONCLUSION
The antimicrobial peptide 5-methyl-hexahydropyrimidine -2,4-dione represents the innate immunity issues in the pleated sea squirt Styela plicata. Although this species is hermaphrodite, the antimicrobial peptide is transmitted to the offspring through the oocytes and not through the sperm. DGGE showed that the number of bands in sea squirts was variable and the variability found in β-actin was significantly lower. The degree of variability does not seem to be related to geographical origin. Results of the probe against histone H3 showed a strong hybridization.
SIGNIFICANCE STATEMENT
This study concludes that the antimicrobial peptide 5-methyl-hexahydropyrimidine-2,4-dione is important for the survival of the pleated sea squirt in the Arabian Gulf.
This study will help the researcher to uncover the critical areas of innate immunity in the estuarine ecosystem that many researchers were not able to explore. Thus a new theory on antimicrobial protection capability may be arrived at.
ACKNOWLEDGMENT
Authors acknowledge the Deanship of Scientific Research; Ministry of Higher Education at Imam Abdulrahman Bin Faisal University, Saudi Arabia. This research was conducted under project number 2019-352-PYSS.