Cloning and Real-Time PCR Testing of 14 Potential Biomarkers in Eisenia fetida Following Cadmium Exposure

Abstract

Important biological activities could be affected in metal exposed species, and amongthe main physiological functions, immunity may provide one (or more) effector(s) which expression can be directly affected by a metal exposure in various macroinvertebrates. As many proteinic effectors showed a high degree of homology between species, we have developed a PCR approach to characterize partial mRNA sequences of selected effectors in the laboratory model, Eisenia fetida. After cloning, levels of expression of each gene were analyzed following exposures (80 and 800 mg/kg) to cadmium spiked soils using real-time PCR. An implemented approach was allowed to test quickly potential biomarkers in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, trancriptionally controlled tumor protein, protein kinase C, and ubiquitin. Most of the selected effectors did not show variations of expression level after exposure. Others expressed weak changes of expression as heat shock proteins. At lastfor catalase and metallothionein, early suitable variations of expression were observed.

Pesticides and earthworms. A review

“…In this case, no response may be seen if it is simply delayed (Zhang et al 2013). For this reason, ideal biomarkers exhibit time and dose-dependent variations (Brulle et al 2006). Another point is that exposure durations used under laboratory conditions are not representative of field conditions, where earthworms are continually exposed.…”

Growth Inhibition and Altered Gene Transcript Levels in Earthworms (Eisenia fetida) Exposed to 2,2′,4,4′-Tetrabromodiphenyl Ether

“…Primers of b-actin, SOD, CAT, and Hsp70 used for earthworms (E. fetida) were selected from references (Brulle et al 2006;Chen et al 2011b). The primers of GST were designed based on known sequences (HQ693699).…”

“…They ranged from 0.98 to 1 (Pearson correlation coefficient r 2 [ 0.99), and a strong linearity was observed. The gene transcript levels (R) were calculated according to the formula R = (E Tg ) CPTg /(E act ) CPact , target gene (Tg), and b-actin gene(act) (Pfaffl 2001;Brulle et al 2006). …”

Responses of growth inhibition and antioxidant gene expression in earthworms ( Eisenia fetida ) exposed to tetrabromobisphenol A, hexabromocyclododecane and decabromodiphenyl ether

“…Sequence of primers used for RT-qPCR is shown in Table 1. Primers of β-actin, SOD, CAT and Hsp70 used for earthworms (E. fetida) were selected from references (Brulle et al, 2006;Chen et al, 2011b). Table 1 Sequence of primers used for RT-qPCR.…”

“…They were in the ranged from 0.98 to 1 (Pearson correlation coefficient r 2 N 0.99), and a strong linearity was observed. The relative quantification of target genes with reference gene (R) was calculated according to the formula R = (E Tg ) CPTg / (E act ) CPact , target gene (Tg), β-actin gene(act) (Pfaffl, 2001;Brulle et al, 2006).…”