Transcriptome Analysis of Milkfish after Exposure to Aeromonas hydrophila using Next-Generation Sequencing

Jaime Lorenzo N. Dinglasan1, Lorenz Rhuel P. Ragasa1,
Anacleto M. Argayosa1,2, Zubaida U. Basiao1, and Michael C. Velarde1*

1Institute of Biology, College of Science
University of the Philippines Diliman, Quezon City 1101 Philippines
2Department of Biological Sciences, College of Arts Sciences and Education
Trinity University of Asia, Quezon City 1102 Philippines

*Corresponding Author: This email address is being protected from spambots. You need JavaScript enabled to view it.





Milkfish is a major finfish product of the Philippines. But because the whole genome sequence of milkfish is still unknown, it is difficult to study the different molecular pathways involved in milkfish after bacterial exposure. Here, transcriptomic analyses by next-generation sequencing (NGS) were used to identify gene expression in milkfish liver after exposure to bacterial antigens from inactivated A. hydrophila. Obtained reads per individual were assembled de novo and fragments per kilobyte of exon per million mapped reads (FPKM) were measured to identify overall gene expression. Differential expression (DE) was analyzed by Cufflinks-Cuffdiff software. Gene ontology (GO) overrepresentation analysis revealed that milkfish exposed to A. hydrophila altered expression of genes involved in immune response pathways such as T cell and B cell signaling. The most differentially regulated genes include histamine n-methyltransferase (hnmt), nicotinamide phosphoribosyltransferase b (namptb), poliovirus receptor-related 2 like precursor (pvrl2), and the hepcidin antimicrobial peptide 1 – which are all involved in immunity. Overall, the study showed that milkfish liver contains immune-related genes that respond to bacterial antigens.



Southeast Asia’s total aquaculture production is about 9% of the world’s total aquaculture production. Chanos chanos (milkfish) is the top finfish product produced in this region, and the Philippines is one of the major milkfish-producing countries (Martinez et al. 2006). This particular finfish is the most commercially important aquaculture species in the Philippines, being produced at about 0.35 million metric tonnes per year – more than the next most produced finfish, Oreochromis niloticus (tilapia) (BAS 2013). Milkfish are suitable for production in the country due to their environmental adaptability and their suitability to tropical conditions, as their reproductive patterns are reliant on warm temperature and plentiful sunlight (Martinez et al. 2006). However, milkfish are still susceptible to diseases (Chang 2006, Dequito et al. 2015, Virgula et al. 2017, Echem et al. 2018). One such disease that causes high mortality rate is due to the gram-negative bacterium Aeromonas hydrophila (Lio-po and Duremdez-Fernandez 1986, Emata 1994). In contrast to gram-positive bacteria, the cell wall of gram-negative bacteria contains lipopolysaccharides (LPS) as its major component (Anwar and Choi 2014) that contributes to structural integrity, as well as increased pathogenicity of gram-negative bacteria (Turska-Szewczuk et al. 2013). Exposure to inactivated bacteria will allow the stimulation of the host  . . . . read more



ANDERSON L, AMARAL MS, BECKEDORFF F, SILVA LF, DAZZANI B, OLIVEIRA KC, ALMEIDA GT, GOMES MR, PIRES DS, SETUBAL JC, DE MARCO R, VERJOVSKI-ALMEIDA S. 2015. Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq. PLoS Negl Trop Dis 9(12): e0004334.
ANWAR MA, CHOI S. 2014. Gram-negative marine bacteria: Structural features of lipopolysaccharides and their relevance for economically important diseases. Mar Drugs 12(5): 2485–2514.
ARGAYOSA AM, PASCUA CS, SUMERA F, YASON JAL, ESPIGAR ARAE. 2015. Oral Vaccine For Fish. World Intellectual Property Organization 13, A1.
BAIK SC, YOUN HS, CHUNG MH, LEE WK, CHO MJ, KO GH, PARK CK, KASAI H, RHEE KH. 1996. Increased oxidative DNA damage in Helicobacter pylori-infected human gastric mucosa. Cancer Res 56(6): 1279–82.
BARAT A, SAHOO PK, KUMAR R, GOEL C, SINGH AK. 2016. Transcriptional response to heat shock in liver of snow trout (Schizothorax richardsonii)—A vulnerable Himalayan Cyprinid fish. Funct Integr Genomics 16(2): 203–13
BOTTINO C, CASTRICONI R, PENDE D, RIVERA P, NANNI M, CARNEMOLLA B, CANTONI C, GRASSI J, MARCENARO S, REYMOND N, VITALE M, MORETTA L, LOPEZ M, MORETTA A. 2003. Identification of PVR (CD155) and Nectin-2 (CD112) as Cell Surface Ligands for the Human DNAM-1 (CD226) Activating Molecule. J Exp Med 198(4): 557.
[BAS] Bureau of Agricultural Statistics. 2013. Selected Statistics on Agriculture. Quezon City, Philippines: Department of Agriculture. 28p.
CHANG TC, HSIEH CY, CHANG CD, SHEN YL, HUANG KC, TU C, CHEN LC, WU ZB, TSAI SS. 2006. Pathological and molecular studies on mycobacteriosis of milkfish Chanos chanos in Taiwan. Dis Aquat Organ 72(2): 147–51. doi:10.3354/dao072147.
CONESA A, GÖTZ S, GARCÍA-GÓMEZ JM, TEROL J, TALÓN M, ROBLES M. 2005. Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18): 3674–3676.
DE SANTIS C, BARTIE KL, OLSEN RE, TAGGART JB, TOCHER DR. 2015. Nutrigenomic profiling of transcriptional processes affected in liver and distal intestine in response to a soybean meal-induced nutritional stress in Atlantic salmon (Salmo salar). Comp Biochem Physiol Part D Genomics Proteomics 15: 1–11.
DEQUITO AQD, CRUZ-LACIERDA ER, CORRE VL. 2015. A case study on the environmental features associated with Amyloodinium ocellatum (Dinoflagellida) occurrences in a milkfish (Chanos chanos) hatchery. AACL Bioflux 8(3): 390–397.
ECHEM RT, BARBA HM, LI G, PENG F, BUENAVENTURA NJC. 2018. Endoparasites in Chanos chanos (Forsskal, 1775) from the wetlands of Zamboanga City, Western Mindanao, Philippines. J Aquac Res Dev 9(5). doi:10.4172/2155-9546.1000536.
EMATA AC. 1994. Milkfish, rabbitfish and mullet. In: Lacanilao F, Coloso RM, Quinitio GF eds. Proceedings of the Seminar-workshop on Aquaculture Development in Southeast Asia and Prospects for Seafarming and Searanching; 19–23 Aug 1991; Tigbauan, Iloilo, Philippines: SEAFDEC Aquaculture Department. p. 13–23.
FURLAN M, ROSANI U, GAMBATO S, IRATO P, MANFRIN A, MARDIROSSIAN M, VENIER P, PALLAVICINI A, SCOCCHI M. 2018. Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens. J Pept Sci 24(7): e3089.
GALINDO-VILLEGAS J, GARCIA-GARCIA E, MULERO V. 2016. Role of histamine in the regulation of intestinal immunity in fish. Dev Comp Immunol 64: 178–186.
GOFF L, TRAPNELL C, KELLEY D. 2013. cummeRbund: Analysis, exploration, manipulation, and visualization of Cufflinks high-throughput sequencing data. R Package Version 2.26.0.
HAAS BJ, PAPANICOLAOU A, YASSOUR M, GRABHERR M, BLOOD PD, BOWDEN J, REGEV A. 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8(8): 1494–1512.
HU YC, KANG CK, TANG CH, LEE TH. 2015. Transcriptomic analysis of metabolic pathways in milkfish that respond to salinity and temperature changes. PLoS One. 10(8): e0134959
HUANG Y, NIU B, GAO Y, FU L, LI W. 2010. CD-HIT Suite: A web server for clustering and comparing biological sequences. Bioinformatics 26(5): 680–682.
KAMILYA D, BARUAH A, SANGMA T, CHOWDHURY S, PAL P. 2015. Inactivated Probiotic Bacteria Stimulate Cellular Immune Responses of Catla, Catla catla (Hamilton) In Vitro. Probiotics Antimicrob Proteins 7(2): 101–106.
KUMAR R, SAHOO PK, BARAT A. 2017. Transcriptome profiling and expression analysis of immune responsive genes in the liver of Golden mahseer (Tor putitora) challenged with Aeromonas hydrophila. Fish Shellfish Immunol 67: 655–666.
LANGMEAD B, TRAPNELL C, POP M, SALZBERG S. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10(3).
LI W, GODZIK A. 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 22(13): 1658–1659.
LIO-PO G, DUREMDEZ-FERNANDEZ R. 1986. The pathogenicity of bacteria associated with transport-stressed Chanos chanos fingerlings. In: Maclean JL, Dizon LB, Hosillos LV eds. The First Asian Fisheries Forum. Proceedings of the First Asian Fisheries Forum; 26–31 May 1986; Manila, Philippines: Asian Fisheries Society. p. 223–226.
MARCOGLIESE DJ, BRAMBILLA LG, GAGNÉ F, GENDRON AD. 2005. Joint effects of parasitism and pollution on oxidative stress biomarkers in yellow perch Perca flavescens. Dis Aquat Organ 63: 77–84.
MARTINEZ FS, TSENG M, YEH S. 2006. Milkfish (Chanos chanos) Culture: Situations and Trends. Journal-Fisheries Soc Taiwan 33(3): 229–244.
MI H, POUDEL S, MURUGANUJAN A, CASAGRANDE JT, THOMAS PD. 2016. PANTHER version 10: Expanded protein families and functions, and analysis tools. Nucleic Acids Res 44(D1): D336–D342.
MÖLLER AM, KORYTÁŘ T, KÖLLNER B, SCHMIDT-POSTHAUS H, SEGNER H. 2014. The teleostean liver as an immunological organ: Intrahepatic immune cells (IHICs) in healthy and benzo[a]pyrene challenged rainbow trout (Oncorhynchus mykiss). Dev Comp Immunol 46(2): 518–529.
NEAR T, DORNBURG A, FRIEDMAN M. 2014. Phylogenetic relationships and timing of diversification in gonorynchiform fishes inferred using nuclear gene DNA sequences (Teleostei: Ostariophysi). Mol Phylogenet Evol 80(1): 297–307.
NEVES JV, CALDAS C, VIEIRA I, RAMOS MF, RODRIGUES PNS. 2015. Multiple Hepcidins in a Teleost Fish, Dicentrarchus labrax: Different Hepcidins for Different Roles. J Immunol. 195(6): 2696–2709.
PACITTI D, WANG T, MARTIN S, SWEETMAN J, SECOMBES C. 2014. Insights into the fish thioredoxin system: Expression profile of thioredoxin and thioredoxin reductase in rainbow trout (Oncorhynchus mykiss) during infection and in vitro stimulation. Dev Comp Immunol 42(2): 261–277.
RAUTA PR, NAYAK B, DAS S. 2012. Immune system and immune responses in fish and their role in comparative immunity study: A model for higher organisms. Immunol Lett 148(1): 23–33.
RONGVAUX A, GALLI M, GOOL V, DRE PL, SZPIRER C, BUREAU F, ANDRIS F, LEO O. 2008. Nicotinamide phosphoribosyl transferase/pre-B-cell colony-enhancing factor/visfatin is required for lymphocyte development and cellular resistance to genotoxic stress. J Immunol 181(7): 4685–4695.
SHIOZAKI K, NAKANO T, YAMAGUCHI T, SATO M. 2003. Metabolism of exogenous histamine in rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 29(4): 289–295.
STUMBO AD, GOATER CP, HONTELA A. 2012. Parasite-induced oxidative stress in liver tissue of fathead minnows exposed to trematode cercariae. Parasitology. 139(12): 1666–1671.
TAFALLA C, LEAL E, YAMAGUCHI T, FISCHER U. 2016. T cell immunity in the teleost digestive tract. Dev Comp Immunol. 64: 167–177.
TKACHENKO H, KURHALUK N, GRUDNIEWSKA J, ANDRIICHUK A. 2014. Tissue-specific responses of oxidative stress biomarkers and antioxidant defenses in rainbow trout Oncorhynchus mykiss during a vaccination against furunculosis. Fish Physiol Biochem 40(4): 1289–1300.
TORT L, BALASCH JC, MACKENZIE S. 2003. Fish immune system: A crossroads between innate and adaptive responses. Immunologia 22(3): 277–286.
TRAPNELL C, ROBERTS A, GOFF L, PERTEA G, KIM D, KELLEY DR, PIMENTEL H, SALZBERG SL, RINN JL, PACHTER L. 2012. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 7(3): 562–578.
TURSKA-SZEWCZUK A, LINDNER B, KOMANIECKA I, KOZINSKA A, PEKALA A, CHOMA A, HOLST O. 2013. Structural and immunochemical studies of the lipopolysaccharide from the fish pathogen, Aeromonas bestiarum strain K296, serotype O18. Mar Drugs 11(4): 1235–1255.
VIRGULA JC, CRUZ-LACIERDA ER, ESTANTE EG, CORRE VL. 2017. Copper sulfate as treatment for the ectoparasite Amyloodinium ocellatum (Dinoflagellida) on milkfish (Chanos chanos) fry. AACL Bioflux 10(2): 365–371.
WANG Z, GERSTEIN M, SNYDER M. 2009. RNA-Seq: A revolutionary tool for transcriptomics. Nat Rev Genet 10(1): 57–63.
YAN Y-Y, XIA H-Q, YANG H-L, HOSEINIFAR SH, SUN Y-Z. 2016. Effects of dietary live or heat-inactivated autochthonous Bacillus pumilus SE 5 on growth performance, immune responses and immune gene expression in grouper Epinephelus coioides. Aquac Nutr 22(3): 698–707.
YANG S, KIM J, RYU J-H, OH H, CHUN C-H, KIM BJ, MIN BH, CHUN J-S. 2010. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction. Nat Med 16(6): 687–693.