295 ISJ 14: 295-302, 2017 ISSN 1824-307X SHORT COMMUNICATION Immune related genes expression in juveniles of an invasive snail after challenged with Lipopolysaccharide Y-M Xiong 1 , Z-H Yan 1 , J-E Zhang 2,3,4 , H-Y Li 1 1 Department of Aquiculture, College of Animal Science, South China Agricultural University, Guangzhou, China 2 Institute of Tropical and Subtropical Ecology, South China Agricultural University, Guangzhou, China 3 Key Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture P. R. China, Guangzhou, China 4 Guangdong Provincial Engineering Technology Research Center of Modern Eco-agriculture and Circular Agriculture, Guangzhou, China Accepted August 17, 2017 Abstract Invasion by Pomacea canaliculata is an increasing problem worldwide. Its rapid invasion and spread depends on the immune mechanism of efficient treatment of unfavorable factors and pathogenic microorganisms in the environment. Transcriptome technology is widely applied in studying the interaction of molluscs and pathogenic microorganisms and the development of disease. In this study, transcriptome technology is used to detect the expression of immune-related genes after the P. canaliculata juvenile snails were challenged with Lipopolysaccharide (LPS) for 48 h. A total of 402 immune-related differentially expressed genes(DEGs) were identified in comparison with the control. These DEGs involved in antigen recognition gene, immune stress and detoxification. The results showed that genes associated with detoxification metabolism were significantly up-regulated, such as cytochrome c oxidase, cytochrome b oxidase, NADH dehydrogenase, sterol 17-alpha-hydroxylase and cytochrome P450. These data will provide fundamental information for subsequent studies of the immune defense mechanisms against pathogens in the juvenile snail of P. canaliculata. Key Words: Immune response; apple snail; transcriptome; innate immunity; molluscs Introduction Pomacea canaliculata is the only freshwater snail listed among the 100 worst invasive species worldwide (Yang et al., 2017). P. canaliculata originally distributed in South America, which now has invaded in North America, Asia and Europe (Accorsi et al., 2017). Its large size and taste for aquatic plants makes it a threat for causing ecological and economical damage in wetlands and crops fields(Youens and Burks, 2008; Hayes et al., 2012). In addition to the impact on crops, P. canaliculata also is the intermediate host of the nematode Angiostrongylus cantonensis which responsible for potentially lethal encephalitis to human being (Lv et al., 2009; Song et al., 2016). At present there are no accepted and efficient strategies for controlling the spread of P. canaliculata. P. canaliculata lack clear evidence of adaptive ___________________________________________________________________________ Corresponding author: Hai-Yun Li Department of Aquiculture College of Animal Science South China Agricultural University Guangzhou, China E-mail: hyli@scau.edu.cn immunity and yet thrive in freshwater, where is rich in pathogenic bacteria, this means the P. canaliculata should have a perfect and effective immune system to disposal pathogens. In the past, research on bacterial challenge mainly focused on adult molluscs (Liu et al., 2017). Moreover, pathogens infection can also have serious consequences on mollusc’s survival, especially during the juvenile stages (Genard et al., 2013). For instance in oysters, severe juvenile mortality was induced when they were treated with a Vibrio strain at the concentrations from 10 4 to 10 8 CFU individual (Lacoste et al., 2001); while another Vibrio strain, named TNEMF6, was also demonstrated to be responsible for the unexplained mortalities of juvenile oyster C. gigas living in Bay of Morlaix (Le et al., 2002). Although some progresses have been made in the study of the damage caused by pathogens on mollusc juvenile, the mechanism of pathogens that cause great harm to mollusc juvenile remains need further study. In recent years, using transcriptome sequencing can facilitate understanding the immune responsemechanisms of aquatic animals during pathogen stimulation (Dheilly et al., 2014), the technology has been used in Pelodiscus sinensis (Xu et al., 2016), Biomphalaria glabrata (Zhang et al., 2016) mailto:hyli@scau.edu.cn 295 Fig. 1 The length distribution of unigenes. The X axis represents the length of unigene. The Y axis represents the number of unigene. and Crassostrea gigas (Liu et al., 2017). The purpose of the study is to reveal the immune-related DEGs expression profiles of P. canaliculata juvenile snails between LPS challenged group and control, which can help us understand the immune defense mechanisms of P. canaliculata juvenile snails. Materials and Methods The fertilized eggs incubation and juvenile snail collection Eggs were cultured in an incubator at 25 ℃ (Sun et al., 2010). The hatching snails were collected to 100 mm petri dish and washed with sterile water three times. LPS purchased from Sigma (L2630-25MG). Experimental snails were bred in petri dish with 10 mg/ml LPS (LPS dissolve in sterile water), control snails cultured use sterile water. After 48 h cultured, juvenile snails were collected in the frozen tube and placed in liquid nitrogen rapidly. Finally, the samples were stored at -80 ℃ refrigerator for subsequent experiments. Transcriptome sequencing and de novo assembly Total RNA was extracted from juvenile snails in each group according to instruction of the manufacturer (TRIzol Reagent, ThermoFisher). Obtained samples of total RNA and use DNase I to digest DNA, after extracting total RNA and treated with DNase I, Oligo(dT) are used to isolate mRNA. The mRNA will fragment and then as templates to synthesize cDNA. The synthesized cDNA was subjected to end-repair and single nucleotide A (adenine) addition. After that, short fragments are connected with adapters. During the quality control steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. Then the library is sequenced using Illumina HiSeq 4000 platform.The cutadapt used to define raw reads for which containing low-quality, adaptor-polluted and high content of an unknown base reads additionally. Clean reads of each sample were assembled use Trinity software (Haas et al., 2013) and with Tgicl to cluster transcripts to unigenes. The clustered unigenes were aligned to SILVA databases to exclude those matching rRNAs of the score above 60. Unigenes functional annotation The unigenes were Blast (Altschul et al., 1990) to NT, NR, COG, KEGG and SwissProt to get the annotation function, use Blast2GO (Conesa et al., 2005) with NR annotation to get the GO annotation, and use InterProScan5 (Quevillon et al., 2005) to get the InterPro annotation. Software parameters settings are based on references. Identification DEGs and analysis Unigene expression analysis was clean reads mapped to unigenes using Bowtie2 (Langmead and Salzberg, 2012). Gene expression level was calculated with RSEM according to former study (Li and Dewey, 2011). DEGs detected with PossionDis according as Audic S, et al. (Audic and Claverie, 1997). 296 295 Table 1 Summary of functional annotation results of unigene Database Numbers Percentage Annotated in NR 10788 30.86% Annotated in NT 11729 33.55% Annotated in Swissprot 11830 33.84% Annotated in KEGG 23977 68.58% Annotated in COG 12251 35.04% Annotated in Interpro 12544 35.88% Annotated in GO 6925 19.81% Annotated in all databases 2561 7.32% Total sequences number 34963 100% Results and Discussion RNA-seq de novo assembly Next-generation sequencing technology was used in the study to detect immune-related genes expression pattern of LPS challenge, and generated about 17.87 Gb bases in total after Illumina Hiseq4000 sequencing (SRA accession number SRX3029507). Then assemble all samples together. The average length is 1,080 bp and N50 is 2,231 bp. These results demonstrated that the sequencing quality was quite fine and suitable for subsequent transcriptome analysis. Length distribution of all unigenes is shown in Figure 1. Finally, RNA-Seq data had been submitted to the sequence read archive (SRA) database of NCBI with the accession number for SRX3029507. Unigene functional annotation The unigenes were aligned with sequences recorded in the major functional databases (NR, NT, Swissprot, COG, KEGG, GO and Interpro). A total of 34,963 unigenes were been annotated and 2,561 (7.3 %) unigenes were annotated in all databases (Table 1). In this study, the species distribution was statistically analyzed for unigenes which annotated in NR database (Fig. 2), the highest proportion of the best Blastx hit was found for Aplysia californica (33.32 %), Lottia gigantean (22.69 %) and Crassostrea gigas (12.27 %). The result means more than 68.28 % of the unigenes best matched to molluscan species, despite the limited number of molluscan sequences in the public databases. Using GO and KEGG database to analyze the functional classification of annotated sequences. Unigenes were assigned to GO database to determine their functional classifications, a total of 6,925 (19.81 %) unigenes were classified into three categories of molecular function, cellular component and biological process, comprised of 19, 19 and 25 subcategories (Fig. 3). According to the results of the GO analysis, immune-related genes involved in the response to the LPS challenge belong to the GO terms of “immune system process” and “response to stimulus” as well, and the result revealed 133 and 952 unigenes were belonged to the subcategory “immune system process” and “response to stimulus” respectively. Fig. 2 Species distribution of best matched in NR database 297 296 Fig. 3 Functional distribution of GO annotation. X axis represents the number of unigenes. Y axis represents the GO functional category. To classify the biological pathways in the juvenile snail of P. canaliculata, the unigenes were mapped to the KEGG database, which is a bioinformatics database for systematic analysis (Kanehisa and Goto, 2000). A total of 23,977 (68.58 %) unigenes were annotated in KEGG and grouped into 32 level 2 terms (Fig. 4). Among the 32 predicted pathways, immune-related unigenes were grouped into the immune system cluster and divided into 11 immune-related pathways, including platelet activation, leukocyte transendothelial migration, hematopoietic cell lineage, chemokine signaling pathway, complement and coagulation cascades, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, TNF signaling pathway and NF-kappa-B signaling pathway. Immune-related DEGs analysis In order to obtain further details on the immune response of P. canaliculata challenged with LPS, immune-related DEGs were analyzed according to GO and KEGG enrichment result. In the study, a total of 402 immune-related DEGs were obtained between the experimental group and the control group, including 189 genes up-regulated and 213 genes down. 54 immune-related DEGs were enriched in different functional classes of GO, such as the immune system 298 295 Fig. 4 Functional distribution of KEGG annotation. X axis represents the number of unigenes. Y axis represents the KEGG functional category. Table 2 List of immune-related DEGs enrichment in KEGG pathways Item Pathway ID Up-regulated Down-regulated Hematopoietic cell lineage ko04640 48 67 Complement and coagulation cascades ko04610 27 33 Platelet activation ko04611 111 73 Leukocyte transendothelial migration ko04670 65 103 NF-kappa B signaling pathway ko04064 12 46 Toll-like receptor signaling pathway ko04620 31 6 NOD-like receptor signaling pathway ko04621 12 20 TNF signaling pathway ko04668 42 14 RIG-I-like receptor signaling pathway ko04622 3 14 Cytosolic DNA-sensing pathway ko04623 10 5 Chemokine signaling pathway ko04062 19 20 299 296 Table 3 Representative of significantly up-regulated immune related DEGs after challenge with LPS Contig Description Fold change P-value CL4201.Contig1_All Cytochrome c oxidase subunit II 14.24 2.54E-204 Unigene8090_All Cytochrome b 12.23 1.36E-58 Unigene48594_All NADH dehydrogenase subunit 4 10.90 6.46E-10 CL5717.Contig1_All Steroid 17-alpha-hydroxylase 10.45 1.29E-84 CL5310.Contig6_All Cytochrome P450 5.52 6.52E-28 Unigene36519_All Fibrinogen-related molecule 7.61 5.11E-05 CL3301.Contig3_All TNF receptor-associated factor 4 7.61 1.24E-20 Unigene23546_All TNF receptor-associated factor 3 8.18 2.90E-17 CL4150.Contig2_All TNF-inducible gene 6 protein 7.20 4.44E-08 Unigene25880_All Latrophilin-2 4.57 1.18E-06 Unigene24146_All Peptidoglycan recognition protein S1L 4.39 0 CL2173.Contig3_All Cell division cycle protein 16-like protein 7.38 2.19E-08 Unigene15602_All C-type lectin domain family 4 7.38 1.07E-284 Unigene25286_All C-type lectin domain family 4 member E 9.29 1.42E-26 CL705.Contig1_All Complement C1q-like protein 2 5.30 8.52E-21 CL4389.Contig2_All C1q domain protein 3.21 5.71E-54 Unigene14048_All C1q domain profile 4.65 0 CL364.Contig12_All NF-kappa-B inhibitor-like protein 1 6.75 7.45E-07 CL2261.Contig3_All NF-kappa-B inhibitor cactus 3.15 0 Unigene3864_All Neurotrypsin 6.20 0 Unigene14909_All HSP70 6.05 4.28E-19 Unigene23401_All Apoptosis 2 inhibitor 5.73 5.20E-156 CL4290.Contig2_All Inhibitor of apoptosis protein 2.26 3.52E-45 Unigene757_All Baculoviral IAP repeat-containing protein 7 4.63 7.83E-43 Unigene21173_All Baculoviral IAP repeat-containing protein 2 4.22 2.30E-109 Unigene14450_All Dermatopontin 2 5.33 4.76E-11 Unigene5788_All Dermatopontin 3 3.58 2.63E-24 Unigene9600_All Toll-like receptor 2 3.45 1.30E-10 Unigene5098_All Toll-like receptor 4 9.76 2.41E-13 CL7289.Contig3_All Collagen alpha-1 3.89 1.05E-14 CL6048.Contig2_All Protein AMBP 2.95 9.84E-51 CL2116.Contig6_All Kunitz-like protease inhibitor 2.56 0 process(37), immune system development(3), response to stress(19), innate immune response(8), response to lipopolysaccharide(2). In addition, 402 immune-related DEGs were enriched into 11 KEGG database metabolic pathway subcategories, including platelet activation(184), leukocyte transendothelial migration(168), hematopoietic cell lineage(115), chemokine signaling pathway(39), complement and coagulation cascades(60), NOD-like receptor signaling pathway(32), Toll-like receptor signaling pathway(37), cytosolic DNA-sensing pathway(15), RIG-I-like receptor signaling pathway(17), TNF signaling pathway(56) and NF-kappa-B signaling pathway(58) (Table 2). Notably, many immune-related DEGs were involved in more than one function owing to their multiple roles in regulation of immune response. So the sum of each term DEGs is larger than total immune-related DEGs. When P. canaliculata juvenile snails were challenged by LPS, some genes related to the 300 296 immune defense system were significant up-regulated (>2 fold change), these genes including Toll-like receptor, C-type lectin, C1q domain containing proteins, peptidoglycan-recognition proteins, fibrinogen-related proteins (FREPs), cytochrome c oxidase and TNF receptor-associated factor (Table 3). In invertebrates, the innate immune system mainly relies on pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMP) (Akira et al., 2006). Toll-like receptors (TLRs) are very important PRRs, which play a critical role to recognize PAMPs in innate immune (Lu et al., 2016). In this study, the results showed that TLR2 and TLR4 genes were up-regulated after challenged by LPS. LPS is a component of the cell wall of Gram negative bacteria, is a potent activator of innate immunity in mammals, which can recognise by TLR4 and leads to subsequent cell signaling pathway responses. Ren et al. (2014) reported that TLR2 is involved in regulation of antimicrobial peptides (AMP) gene expression of Hyriopsis cumingii, but the mechanism of how TLR2 regulates AMP expression is unclear. Some Lectin-like proteins also have been detected up-regulated in this study. Lectin-like proteins play an important role in the defense of gastropods against pathogen infection (Bulat et al., 2016). Lectin proteins often recognize a specific carbohydrate moieties of the pathogen’s surface (Vasta et al., 2015). These proteins are including Galectin-4, Calnexin, Calreticulin, C-type lectins, I-type lectins and C1q domain containing proteins. Inhibitors of nuclear factor kappa B (IκBs) are major control components of the Rel/NF-κB signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates (Gao et al., 2016; Oyanedel et al., 2016). In Zhikong scallop Chlamys farreri, the mRNA expression of CfIκB and CfNF-κB genes increase expression after LPS stimulation (Wang et al., 2011). The present study revealed that cytochrome c oxidase was the most highly up-regulated DEGs after challenged with LPS. Indeed, increase expression of a number of genes associated with degradation or detoxification was observed, including cytochrome c oxidase subunit II, cytochrome b, NADH dehydrogenase subunit 4, steroid 17-alpha-hydroxylase and cytochrome P450. The immune system of molluscs consists of blood cells (hemocytes), which are the main cellular components of the hemolymph that covers all soft tissue. Hemocytes depends on a multi-enzyme (referred to NADPH and cytochrome oxidase) production of cytotoxic molecules (like nitric oxide and hydrogen peroxide) for killing and elimination pathogen (Humphries and Yoshino, 2008). The production of nitric oxide (NO) by hemocytes of bivalve molluscs has been reported in M. edulis (Ottaviani et al., 1993), V. ater (Franchini et al., 1995), and the carpet shell clam Ruditapes decussatus (Tafalla et al., 2003). The hemocytes of these species are able to produce NO in response to various stimuli, such as LPS, zymosan, and live bacterial challenge by Vibrio tapetis (Tafalla et al., 2003). In conclusion, the study first using high-throughput transcriptome technology analyzed the expression of immune-related genes after the P. canaliculata juvenile snails were challenged with LPS. The work will provide a molecular resource for further functional studies of P. canaliculata. Acknowledgements This research was supported by the National Natural Science Foundation of China (No. U1131006). The authors report no conflicts of interest. The authors themselves are responsible for the content and writing of the paper. References Accorsi A, Benatti S, Ross E, Nasi M, Malagoli D. A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata. Dev. Comp. Immunol. 72: 37-43, 2017. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 124: 783-801, 2006. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J. Mol. Biol. 215: 403-410, 1990. Audic S, Claverie JM. The significance of digital gene expression profiles. Genome Res. 7: 986-995, 1997. Bulat T, Smidak R, Sialana FJ, Jung G, Rattei T, Bilban M, et al. Transcriptomic and proteomic analysis of Arion vulgaris-Proteins for probably successful survival strategies? PLOS ONE 11: e150614, 2016. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674-3676, 2005. Dheilly NM, Adema C, Raftos DA, Gourbal B, Grunau C, Du Pasquier L. No more non-model species: the promise of next generation sequencing for comparative immunology. Dev. Comp. Immunol. 45: 56-66, 2014. Franchini A, Conte A, Ottaviani E. Nitric oxide: an ancestral immunocyte effector molecule. Adv. Neuroimmunol. 5: 463-478, 1995. Gao S, Ren Y, Zhang H, Pan B, Gao H. Identification and expression analysis of IκB and NF-κB genes from Cyclina sinensis. Fish Shellfish Immunol. 56: 427-435, 2016. Genard B, Miner P, Nicolas JL, Moraga D, Boudry P, Pernet F, et al. Integrative study of physiological changes associated with bacterial infection in Pacific oyster larvae. PLOS ONE 8: e64534, 2013. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8: 1494-1512, 2013. Hayes KA, Cowie RH, Thiengo SC, Strong EE. Comparing apples with apples: clarifying the identities of two highly invasive Neotropical Ampullariidae (Caenogastropoda). Zool. J. Linn. Soc.-lon. 166: 723-753, 2012. Humphries JE, Yoshino TP. Regulation of hydrogen peroxide release in circulating hemocytes of the planorbid snail Biomphalaria glabrata. Dev. Comp. Immunol. 32: 554-562, 2008. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28: 27-30, 2000. Lacoste A, De Cian MC, Cueff A, Poulet SA. Noradrenaline and alpha-adrenergic signaling 301 297 induce the hsp70 gene promoter in mollusc immune cells. J. Cell Sci. 114: 3557-3564, 2001. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9: 357-359, 2012. Le Roux F, Gay M, Lambert C, Waechter M, Poubalanne S, Chollet B, et al. Comparative analysis of Vibrio splendidus-related strains isolated during Crassostrea gigas mortality events. Aquatic Living Resour. 15: 251-258, 2002. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12: 323, 2011 Liu Z, Wang L, Zhou Z, Liu Y, Dong M, Wang W, et al. Transcriptomic analysis of oyster Crassostrea gigas larvae illustrates the response patterns regulated by catecholaminergic system upon acute heat and bacterial stress. Dev. Com. Immunol. 73: 52-60, 2017. Lu Y, Zheng H, Zhang H, Yang J, Wang Q. Cloning and differential expression of a novel toll-like receptor gene in noble scallop Chlamys nobilis with different total carotenoid content. Fish Shellfish Immunol. 56: 229-238, 2016. Lv S, Zhang Y, Liu H, Zhang C, Steinmann P, Zhou X, et al. Angiostrongylus cantonensis: morphological and behavioral investigation within the freshwater snail Pomacea canaliculata. Parasitol. Res. 104: 1351-1359, 2009. Ottaviani E, Franchini A, Franceschi C. Presence of several cytokine-like molecules in molluscan hemocytes. Biochem. Biophys. Res. Commun. 195: 984-988, 1993. Oyanedel D, Gonzalez R, Flores-Herrera P, Brokordt K, Rosa RD, Mercado L, et al. Molecular characterization of an inhibitor of NF-kB in the scallop Argopecten purpuratus: First insights into its role on antimicrobial peptide regulation in a mollusk. Fish Shellfish Immunol. 52: 85-93, 2016. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, et al. InterProScan: protein domains identifier. Nucleic Acids Res. 332: W116-W120, 2005. Ren Q, Lan J, Zhong X, Song X, Ma F, Hui K, et al. A novel Toll-like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii. Dev. Com. Immunol. 45: 198-208, 2014. Song L, Wang X, Yang Z, Lv Z, Wu Z. Angiostrongylus cantonensis in the vector snails Pomacea canaliculata and Achatina fulica in China: a meta-analysis. Parasitol. Res. 115: 913-923, 2016. Sun J, Zhang Y, Thiyagarajan V, Qian P, Qiu J. Protein expression during the embryonic development of a gastropod. Proteomics 10: 2701-2711, 2010. Tafalla C, Gomez-Leon J, Novoa B, Figueras A. Nitric oxide production by carpet shell clam (Ruditapes decussatus) hemocytes. Dev. Com. Immunol. 27: 197-205, 2003. Vasta GR, Feng C, Bianchet MA, Bachvaroff TR, Tasumi S. Structural, functional, and evolutionary aspects of galectins in aquatic mollusks: From a sweet tooth to the Trojan horse. Fish Shellfish Immunol. 46: 94-106, 2015. Wang M, Yang J, Zhou Z, Qiu L, Wang L, Zhang H, et al. A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop Chlamys farreri. Dev. Com. Immunol. 35: 511-520, 2011. Xu J, Zhao J, Li Y, Zou Y, Lu B, Chen Y, et al. Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis. Fish Shellfish Immumol. 56: 417-426, 2016. Yang L, Cheng TY, Zhao FY. Comparative profiling of hepatopancreas transcriptomes in satiated and starving Pomacea canaliculata. BMC Genet.18: 18, 2017. Youens AK, Burks RL. Comparing apple snails with oranges: the need to standardize measuring techniques when studying Pomacea. Aquat. Ecol. 42: 679-684, 2008 Zhang S, Loker ES, Sullivan JT. Pathogen-associated molecular patterns activate expression of genes involved in cell proliferation, immunity and detoxification in the amebocyte-producing organ of the snail Biomphalaria glabrata. Dev. Com. Immunol. 56: 25-36, 2016. 302