Molecular Biology Reports

, Volume 46, Issue 1, pp 1023–1031 | Cite as

Multiplex PCR for the rapid detection of insulin-like growth factor in the Pacific oyster, Crassostrea gigas: a useful indicator for growth assessment

  • Ji-sung Moon
  • Youn Hee ChoiEmail author
Original Article


Insulin-like growth factor (IGF) expression plays a critical role in the endocrine regulation of proliferation, differentiation, and growth in shellfish as well as in fish. The Pacific oyster, Crassostrea gigas, is a significant aquaculture species that comprises a large percentage of the Korean shellfish industry; moreover, its growth is economically important in aquaculture. However, when measuring the growth rate in shellfish, the soft tissue weight is difficult to determine because of the shell weight. In the present study, we describe an indirect method of measuring the growth rate using multiplex polymerase chain reaction (PCR) and analyzing levels of molluscan insulin-related peptide (MIP), the acid labile subunit of the IGF-binding protein complex (IGFBP ALS), and insulin-related peptide receptor (CIR) in Pacific oysters. The predicted sizes of amplicons were 776, 537, 380, and 198 bp, and the detection limit of the annealing temperatures was confirmed to be 65 °C. The annual expression of MIP and IGFBP ALS in tissues reached high levels in the winter following the condition index (CI). MIP and IGFBP ALS in male gonads and CIR in female gonads were related to the CI. This newly improved multiplex PCR provides an indirect measure of the growth rate; thus, it can be used to rapidly assess the growth rate. In addition, this method can supplement traditional growth data from oyster farms.


Pacific oyster Insulin-like growth factors Multiplex PCR assay Annual growth rate Molecular biology 



This work was supported by Pukyong National University Research Fund in 2016 (CD 20170051).

Author contributions

YHC conceptualized and acquired funding for this study. JSM carried out the experiments and wrote draft manuscript. YHC revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interests.


  1. 1.
    Statistics Korea (2018) Preliminary results of the fishery production survey in 2017. Daejeon. Accessed 15 Mar 2018
  2. 2.
    Hur YB, Jeon CY, Cho KC, Hur SB (2011) Digestion indices of 12 species of microalgae by the oyster Crassostrea gigas larval development stages. Korean J Malacol 27(4):359–369CrossRefGoogle Scholar
  3. 3.
    Bayne BL (1999) Physiological components of growth differences between individual oysters (Crassostrea gigas) and a comparison with Saccostrea commercialis. Physiol Biochem Zool 72(6):705–713CrossRefGoogle Scholar
  4. 4.
    Rossi F, Herman PMJ, Middelburg JJ (2004) Interspecific and intraspecific variation of δC and δN in deposit-and suspension-feeding bivalves (Macoma balthica and Cerastoderma edule): evidence of ontogenetic changes in feeding mode of Macoma balthica. Limnol Oceanogr 49(2):408–414CrossRefGoogle Scholar
  5. 5.
    Dégremont L, Ernande B, Bédier E, Boudry P (2007) Summer mortality of hatchery-produced Pacific oyster spat (Crassostrea gigas). I. Estimation of genetic parameters for survival and growth. Aquaculture 262(1):41–53CrossRefGoogle Scholar
  6. 6.
    Andrews KS, Beckman BR, Beaudreau AH, Larsen DA, Williams GD, Levin PS (2011) Suitability of insulin-like growth factor 1 (IGF1) as a measure of relative growth rates in lingcod. Mar Coast Fish 3(1):250–260CrossRefGoogle Scholar
  7. 7.
    Wood AW, Duan C, Bern HA (2005) Insulin-like growth factor signaling in fish. Int Rev Cytol 243:215–285CrossRefGoogle Scholar
  8. 8.
    Liu Z, Han T, Fishman S, Butler J, Zimmermann T, Tremblay F, Harbison C, Agrawal N, Kopchick JJ, Schaffler MB, Yakar S (2017) Ablation of hepatic production of the acid-labile subunit in bovine-GH transgenic mice: effects on organ and skeletal growth. Endocrinology 158(8):2556–2571CrossRefGoogle Scholar
  9. 9.
    Gricourt L, Bonnec G, Boujard D, Mathieu M, Kellner K (2003) Insulin-like system and growth regulation in the Pacific oyster Crassostrea gigas: hrIGF-1 effect on protein synthesis of mantle edge cells and expression of an homologous insulin receptor-related receptor. Gen Comp Endocrinol 134.1:44–56CrossRefGoogle Scholar
  10. 10.
    Feng L, Li X, Yu Q, Ning X, Dou J, Zou J, Zhang L, Wang S, Hu X, Bao Z (2014) A scallop IGF binding protein gene: molecular characterization and association of variants with growth traits. PLoS ONE 9(2):e89039CrossRefGoogle Scholar
  11. 11.
    Choi YH, Nam TJ (2014) Influences of the toxicity of cryoprotective agents on the involvement of insulin-like growth factor-I receptor in surf clam (Spisula sachalinensis) larvae. CryoLetters 35:537–543Google Scholar
  12. 12.
    Choi YH, Nam TJ (2015) Toxicity of cryoprotective agents and signaling of insulin-like growth factor in hen clam (Mactra chinensis) embryos. CryoLetters 36:158–164Google Scholar
  13. 13.
    Niu D, Wang F, Zhao H, Wang Z, Xie S, Li J (2016) Identification, expression, and innate immune responses of two insulin-like peptide genes in the razor clam Sinonovacula constricta. Fish Shellfish Immunol 51:401–404CrossRefGoogle Scholar
  14. 14.
    Canesi L, Ciacci C, Betti M, Malatesta M, Gazzanelli G, Gallo G (1999) Growth factors stimulate the activity of key glycolytic enzymes in isolated digestive gland cells from mussels (Mytilus galloprovincialis, Lam.) through tyrosine kinase mediated signal transduction. Gen Comp Endocrinol 116(2):241–248CrossRefGoogle Scholar
  15. 15.
    Shi Y, Guan Y, He M (2013) Molecular identification of insulin-related peptide receptor and its potential role in regulating development in Pinctada fucata. Aquaculture 408:118–127CrossRefGoogle Scholar
  16. 16.
    Gricourt L, Mathieu M, Kellner K (2006) An insulin-like system involved in the control of Pacific oyster Crassostrea gigas reproduction: hrIGF-1 effect on germinal cell proliferation and maturation associated with expression of an homologous insulin receptor-related receptor. Aquaculture 251(1):85–98CrossRefGoogle Scholar
  17. 17.
    Hamano K, Awaji M, Usuki H (2005) cDNA structure of an insulin-related peptide in the Pacific oyster and seasonal changes in the gene expression. J Endocrinol 187(1):55–67CrossRefGoogle Scholar
  18. 18.
    Jouaux A, Franco A, Heude-Berthelin C, Sourdaine P, Blin JL, Mathieu M, Kellner K (2012) Identification of Ras, Pten and p70S6K homologs in the Pacific oyster Crassostrea gigas and diet control of insulin pathway. Gen Comp Endocrinol 176(1):28–38CrossRefGoogle Scholar
  19. 19.
    Zhang G, Fang X, Guo X et al (2012) The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490:49–54CrossRefGoogle Scholar
  20. 20.
    Cong R, Kong L, Yu H, Li Q (2014) Association between polymorphism in the insulin receptor-related receptor gene and growth traits in the Pacific oyster Crassostrea gigas. Biochem Syst Ecol 54:144–149CrossRefGoogle Scholar
  21. 21.
    Choi YH, Kim EY, Nam TJ (2018) Involvement of insulin-like growth factor in intraspecific variation in growth of Pacific oyster Crassostrea gigas during winter. Fish Sci 84:1017–1024CrossRefGoogle Scholar
  22. 22.
    Goldberg AL (2003) Protein degradation and protection against misfolded or damaged proteins. Nature 426(6968):895CrossRefGoogle Scholar
  23. 23.
    Brasher CW, DePaola A, Jones DD, Bej AK (1998) Detection of microbial pathogens in shellfish with multiplex PCR. Curr Microbiol 37.2:101–107CrossRefGoogle Scholar
  24. 24.
    Jeong YS, Jung HK, Jeon WB, Seo HJ, Hong JH (2010) Simultaneous detection of Staphylococcus aureus Salmonella enterica subsp., Vibrio parahaemolyticus by multiplex polymerase chain reaction. J Korean Soc Food Sci Nutr 39(4):595–601CrossRefGoogle Scholar
  25. 25.
    Hossain MT, Kim YO, Kong IS (2013) Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL, tdh and trh genes. Mol Cell Probes 27(5-6):171–175CrossRefGoogle Scholar
  26. 26.
    Zhang DF, Zhang QQ, Li AH (2014) Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria. Lett Appl Microbiol 59(5):471–478CrossRefGoogle Scholar
  27. 27.
    Choi YH, Chang YJ (2003) Gametogenic cycle of the transplanted-cultured pearl oyster, Pinctada fucata martensii (Bivalvia: Pteriidae) in Korea. Aquaculture 220(1-4):781–790CrossRefGoogle Scholar
  28. 28.
    Evseev GA, Yakovlev YM, Li X (1996) The anatomy of the Pacific oyster, Crassostrea gigas (Thurnberg) (Bivalvia: Ostreidae). Publ Seto Mar Biol Lab 37:239–255CrossRefGoogle Scholar
  29. 29.
    Du Y, Zhang L, Xu F, Huang B, Zhang G, Li L (2013) Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR. Fish Shellfish Immunol 34(3):939–945CrossRefGoogle Scholar
  30. 30.
    Markoulatos P, Siafakas N, Moncany M (2002) Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 16(1):47–51CrossRefGoogle Scholar
  31. 31.
    Hecker KH, Roux KH (1996) High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques 20(3):478–485CrossRefGoogle Scholar
  32. 32.
    Barillé L, Lerouxel A, Dutertre M, Haure J, Barillé AL, Pouvreau S, Alunno-Bruscia M (2011) Growth of the Pacific oyster (Crassostrea gigas) in a high-turbidity environment: comparison of model simulations based on scope for growth and dynamic energy budgets. J Sea Res 66.4:392–402CrossRefGoogle Scholar
  33. 33.
    Normand J, Le Pennec M, Boudry P (2008) Comparative histological study of gametogenesis in diploid and triploid Pacific oysters (Crassostrea gigas) reared in an estuarine farming site in France during the 2003 heatwave. Aquaculture 282(1–4):124–129CrossRefGoogle Scholar
  34. 34.
    Zheng K, Liang M, Yao H, Wang J, Chang Q (2012) Effect of dietary fish protein hydrolysate on growth, feed utilization and IGF-I levels of Japanese flounder (Paralichthys olivaceus). Aquac Nutr 18.3:297–303CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Marine Bio-Materials & AquaculturePukyong National UniversityBusanSouth Korea

Personalised recommendations