Effects of In Vitro Exposure to Diarrheic Toxin Producer Prorocentrum lima on Gene Expressions Related to Cell Cycle Regulation and Immune Response in Crassostrea gigas

Abstract

Background Crassostrea gigas accumulates diarrheic shellfish toxins (DSP) associated to Prorocentrum lima of which Okadaic acid (OA) causes specific inhibitions of serine and threonine phosphatases 1 and 2A. Its toxic effects have been extensively reported in bivalve mollusks at cellular and physiological levels, but genomic approaches have been scarcely studied.Methodology/Principal FindingsAcute and sub-chronic exposure effects of P. lima were investigated on farmed juvenile C. gigas (3–5 mm). The Pacific oysters were fed with three dinoflagellate concentrations: 0.3, 3, and 30×103 cells mL−1 along with a nontoxic control diet of Isochrysis galbana. The effects of P. lima on C. gigas were followed by analyzing expression levels of a total of four genes, three involved in cell cycle regulation and one in immune response by polymerase chain reaction and real time quantitative PCR, where changes in time and cell concentration were found. The highest expression levels were found in oysters fed 3×103 cells mL−1 at 168 h for the cycle regulator p21 protein (9 fold), chromatin assembly factor 1 p55 subunit (8 fold), elongation factor 2 (2 fold), and lipopolysaccharide/β-1, 3 glucan binding protein (13 fold above base line). Additionally, the transcript level of all the genes decreased in oysters fed wich the mixed diet 30×103 cells mL−1 of dinoflagellate after 72 h and was lowest in the chromatin assembly factor 1 p55 subunit (0.9 fold below baseline).ConclusionsOn C. gigas the whole cell ingestion of P lima caused a clear mRNA modulation expression of the genes involved in cell cycle regulation and immune system. Over-expression could be related to DNA damage, disturbances in cell cycle continuity, probably a genotoxic effect, as well as an activation of its innate immune system as first line of defense.

Differential expression of immune response genes in Pacific oyster, Crassostrea gigas spat, fed with dinoflagellates Gymnodinium catenatum and Prorocentrum lima

“…The analysis of our results showed that the expression level in the studied genes was affected by cell concentration of the toxic dinoflagellates, as well as exposure time on C. gigas spat. These observations were consistent with those reported by other authors Romero-Geraldo & Hernández-Saavedra, 2014;García-Lagunas et al, 2013Romero-Geraldo et al, 2014, 2016. The expression level of cvt and lgbp gene showed a similar tendency, up-regulated for the three treatments at day three and down-regulated until the end of the experiment, indicating that DSP and PSP toxins inhibited the expression of these genes after five days of feeding.…”

“…Paralytic shellfish toxins (PSTs) are a group of neurotoxic alkaloids, grouped into three structural families: carbamate (saxitoxin-STX, neosaxitoxin-NEO and gonyautoxins-GTX1 to GTX4), decarbamoyl and N-sulfocarbamoyl (Oshima, 1995). These dinoflagellate species have been associated with DSP and PSP episodes in different parts of the world, and in the Gulf of California, they have also been extensively used in aquatic toxicological studies (Pinto-Silva et al, 2005;Wikfors, 2005;Flórez-Barrós et al, 2011;García-Lagunas et al, 2013Romero-Geraldo et al, 2014, 2016.…”

“…comm.). In this context, genes with functions in early immune response were selected, such as toll-interacting protein (tlp), a ubiquitin-binding protein that interacts with TLR signaling components and is highly conserved in evolution from invertebrate to vertebrate (Zhang et al, 2015); cavortin (cvt), which is the major hemolymph protein of the Pacific oyster, characterized as the hemocyte Cu/Zn superoxide dismutase (Lambert et al, 2007;Scotti et al, 2007); lipopolysaccharide and β-1,3glucan-binding protein (lgbp), which is a pattern recognition protein (PRP) that recognizes lipopolysaccharide and β-1,3-glucan (βG) and subsequently triggers innate immunity (Romero-Geraldo et al, 2014). Additionally, interferon-induced protein 44 gene (if44) that may induce a cellular GTP depletion involved in cell cycle arrest (Renault et al, 2011) and immunoglobin domain (Ig) [and Leucine-rich repeat (LRR)] gene (imm) was selected because it is also a competent immune recognition module domain present in some proteins, which could function as immune effector or pro-inflammatory factor in oyster (Wang et al, 2017).…”

“…Gymnodinium catenatum was reared as described in García-Lagunas et al (2013). P. lima and Isochrysis galbana were grown as described in Romero-Geraldo et al (2014). Diploid individuals (n = 180) of Pacific oyster spats (3-4 mm of length) were obtained from the hatchery and acclimated under controlled conditions of temperature, salinity, and oxygen for 10 days.…”

“…Diploid individuals (n = 180) of Pacific oyster spats (3-4 mm of length) were obtained from the hatchery and acclimated under controlled conditions of temperature, salinity, and oxygen for 10 days. The cell concentration of treatments was chosen by previous research by we work group (García-Lagunas et al, 2013Romero-Geraldo et al, 2014). Oysters were exposed to four diets: (T1) combined diet of G. catenatum (3×10 3 cell mL -1 ) and P. lima (3×10 2 cell mL -1 ) and a fixed amount of Isochrysis galbana (7.5×10 5 cell mL -1 ); (T2) combined diet of G. catenatum (1.5×10 3 cell mL -1 ), P. lima (1.5×10 3 cell mL -1 ) and I. galbana (7.5×10 5 cell mL -1 ); (T3) combined diet of G. catenatum (3×10 2 cell mL -1 ) and P. lima (3×10 3 cell mL -1 ), I. galbana (7.5×10 5 cell mL -1 ); and (T4) a non-toxic control diet of I. galbana alone (7.5×10 5 cell mL -1 ).…”