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16Russian Journal of Nematology, 2012, 20 (2), 167-170
Short Note
Differential gene expression after osmotic induction in various species of entomopathogenic
nematodes
12 1 3 2 4 2
Xun Yan ' , Richou Han , Shulong Chen , Maurice Moens ' and Patrick De Clercq
1 Guangdong Entomological Institute, 105 Xingang Road West, Guangzhou, 510260, China 2 Ghent University, Coupure Links 653, B-9000, Ghent, Belgium 3 Plant Protection Institute of Hebei Academy of Agricultural and Forestry Sciences, 437 Dongguan Street, Baoding, 071000, China 4 Institute for Agricultural and Fisheries Research, Burg. Van Gansberghelaan 96, 9280 Merelbeke, Belgium;
e-mail: maurice.moens@ilvo.vlaanderen.be
Accepted for publication 18 August 2012
Summary. The expression patterns of fat-2 and sod-2 after osmotic induction were studied in four species of EPN, Steinernema carpocapsae, S. loci, Heterorhabditis bacteriophora and H. indica, which represented a good and a weak desiccation tolerant species from each genus, respectively. The expression of fat-2 was significantly different between the four species. It was down-regulated in H. bacteriophora but was up-regulated in the other three species. The expression of sod-2 was not significantly different between the four species, with an increase in the expression level of 1.6 to 2.0 fold. The possible mechanism of responses of these genes is discussed.
Key words: Differential gene expression, Heterorhabditis, osmotic induction, Steinernema.
Expression patterns of five stress-related genes (fat2, lea, lea5, sod2 and t19d7) have previously been studied in two strains of Steinernema carpocapsae which showed significantly different heat tolerance after osmotic treatment (Yan et al, 2011). It has also been found that different species of entomopathogenic nematodes (EPN) differ in their tolerance to osmotic treatment and subsequent heat exposure (Yan et al., 2010). In the present study, the expression patterns of fat-2 and sod-2 were studied in selected species of EPN. The genus Steinernema was represented by S. carpocapsae All, a strain showing good osmotic tolerance, and S. loci, a strain with week osmotic tolerance. For the genus Heterorhabditis, H. bacteriophora and H. indica were selected because of their good and weak osmotic tolerance, respectively.
Infective juveniles of the four species were cultured in Galleria mellonella larvae at 25°C using
the method of Kaya and Stock (1997). Nematodes were harvested, concentrated to 2*104 IJ ml-1 and treated with osmotic solution for 24 h as described by Yan et al. (2010). qPCR and statistical analysis were as described by Yan et al. (2011). Degenerate primer pairs for actin (ATG TGY GAY GAR GAR GTN GC and ATY TTY TC ATR TCR TCC CA), fat-2 (TTY GTN GTN GGN CAY GAY TGY GG and GGC CAR AAR TGN SWN CCR TCN GG) and sod-2 (CAY CAY CAR AAR CAY CAY GC and CCA NCC CCA NCC NSW NCC YTG NAC NGC) were designed from sequences of these genes from related nematode species and used to clone gene fragments from the different species. The sequences obtained were submitted to GenBank (GenBank Accession Nos. JQ085807-JQ085815). Gene specific primers (Table 1) were designed for qPCR based on the sequences amplified with the degenerate primers.
Fig. 1. Expression of genes (mean fold difference ± SD) in different species of EPN osmotically induced for 24 h. Slo: Steinernema loci; Sca: S. carpocapsae; Hba: Heterorhabditis bacteriophora; Hin: H. indica. Bars with the same letter do not differ significantly according to Tukey's test at P = 0.05 for the same gene.
The primer pairs for qPCR were also used to amplify the gene fragments from the four species. The amplicons were sequenced (Macrogen, Seoul, Korea) to confirm that the target genes had been amplified.
The expression patterns of the two genes in four EPN species after 24 h of osmotic induction are shown in Fig. 1. The expression of fat-2 was significantly different between the four species (F = 139.945, df = 3, 11, P < 0.001). In H. bacteriophora H06, fat-2 was down-regulated while this gene was up-regulated in the other three species. The increase in the expression level offat-2 was the highest (> 6fold) in S. carpocapsae All, followed by H. indica LN2 and S. loci 18. The expression of sod-2 was not significantly different between the four species (F = 0.561, df= 3, 11, P = 0.655), with an increase in the expression level of 1.6 to 2.0 fold.
Fatty acid desaturase 2 (fat-2) may be important in maintaining cell membranes in the liquid crystalline state under water deficit conditions (Peyou-Ndi et al., 2000). Heterorhabditis bacteriophora H06 survived osmotic treatment better than S. loci 18 and H. indica LN2, but the expression of the fat-2 gene decreased. This suggests that there may be other genes that play a more important role in the osmotic response of H. bacteriophora H06. Somvanshi et al. (2008) studied the expression of four other genes related to desiccation-tolerance (Aldehyde dehydrogenase,
Aldh; Glutathione peroxidase, Glp; Nucleosome assembly protein 1, NAP1 and Heat-shock protein 40, Hsp40) in H. bacteriophora TT01 and found that expression peaked at 16 h of desiccation. The expression of these four genes in the desiccation-tolerant species S. riobrave and S. carpocapsae Mex were all down-regulated after 24 h of osmotic treatment, although these four genes were all highly expressed and are biologically important in desiccation stress (Gal et al., 2003). Whether the expression of fat-2 in H. bacteriophora H06 is up-regulated in the early stage of osmotic induction needs to be studied further. The sod-2 gene encodes a protein which detoxifies reactive oxygen species. No significant difference in the expression levels of the sod-2 gene was observed among the different species. It is possible that although SOD is produced as a general response to environmental stress for different organisms, there are other SOD genes and other antioxidant proteins that could be responsible for detoxifying ROS under stress conditions. The sod2 we studied is not involved in the differences in osmotic tolerance among these four species.
Proteins that play important roles in desiccation survival in EPN have been identified from S. carpocapsae and S. feltiae using different molecular methods (Gal et al., 2003; Chen et al., 2005, 2006; Tyson et al., 2007). Although similar gene classes were found to be associated with desiccation stress
Table 1. List of gene-specific primers for qPCR, amplicon length and primer annealing temperature for different species of EPN.
Gene and species GenBank accession Primers Amplicon
No. Forward Reverse length (bp)
Actin
S. loci 18 JQ085807 AGGGAGTCATGGTCGGTATG ATCCCAGTTGGTCACGATTC 119
S. carpocapsae All HQ629785 CATGGGACAAAAGGACTCGT AGGTGTGGTGCCAGATCTTC 131
H. bacteriophora H06 EF571443 GACGCGATCTCACGGATTAT GAGATGACGATGAAGCAGCA 162
H. indica LN2 JQ085809 GAGATGATGCTCCTCGTGCT GGCTTCATCTCCGACGTATG 107
Fatty acid desaturase (fat-2)
S. loci 18 JQ085810 CGGAACGTTCTCCAACTACC CCTTCTCGAGGTGAGAGGTG 134
S. carpocapsae All DR120996 CAGTCGCGGTCCTTGTTGTCGTCT CGCTCCCATCCTTGCCCCTTACT 129
H. bacteriophora H06 JQ085811 TCGGATTATGTGTGGGTCAA TGCGAAGTGTACTGGTGATG 113
H. indica LN2 JQ085812 TCGGAATATGTGTGGCTGAA TGCGAAGTGTACTGATGATG 113
Superoxide dismutase (sod-2)
S. loci 18 JQ085813 ATGGCCACTGGAGATATTCG CTGGGTTCTCCTCCATCCTT 122
S. carpocapsae All DR120999 CACAGCGCATGAAGCACT TCGATCTGGTTGAGGTTGTT 136
H. bacteriophora H06 JQ085814 AGGCAACGTAAAGGAAACGA TGGCAGATGCTAGTTCGACA 134
H. indica LN2 JQ085815 AGGTTCACGATGCTGTTTCG TGCCTCTTGCATCTTCTCAA 197
in these studies, none of the ESTs or proteins detected in these studies was encoded by the same gene. The molecular response to dehydration in EPN is clearly a highly complex process. Further work is needed in this area before the molecular mechanisms underlying differences in stress tolerance in different species of EPN can be elucidated.
This study was supported by the VLIR (Belgium) own initiatives programme ENCHIBE (Entomopathogenic nematodes for sustainable control of chive midge and flea beetle in China, VLIR reference ZEIN2007PR339), Guangdong Provincial Science & Technology Project (2011B040300013), Nonprofit sector project (201003025), National Natural Science Foundation of China (31010103912) and Young Scientists Fund of National Natural Science Foundation of China (31000879, 31101494). The authors thank Prof. J. Jones (JHI) for helpful advice on this MS.
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