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A de novo expression profiling of Anopheles funestus, malaria vector in Africa, using 454 pyrosequencing.

Gregory R, Darby AC, Irving H, Coulibaly MB, Hughes M, Koekemoer LL, Coetzee M, Ranson H, Hemingway J, Hall N, Wondji CS - PLoS ONE (2011)

Bottom Line: In total 20.8% of all reads were novel when compared to reference databases.Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

View Article: PubMed Central - PubMed

Affiliation: University of Liverpool, School of Biological Sciences, Cornwall House, United Kingdom.

ABSTRACT

Background: Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.

Results: Using a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.

Conclusion: This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

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Related in: MedlinePlus

qPCR gene expression profiles of six contigs between the susceptible strain [Kela (field, Mali) and a laboratory pyrethroid-resistant strain (FUMOZ-R).The normalised expression ratio of each contig against the RSP7 gene is represented on the primary vertical axis while the secondary vertical axis represents FUMOZ-R/Kela fold change of each contig.
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pone-0017418-g006: qPCR gene expression profiles of six contigs between the susceptible strain [Kela (field, Mali) and a laboratory pyrethroid-resistant strain (FUMOZ-R).The normalised expression ratio of each contig against the RSP7 gene is represented on the primary vertical axis while the secondary vertical axis represents FUMOZ-R/Kela fold change of each contig.

Mentions: The expression levels of the 6 contigs were generally comparable between FUMOZ and Kela [for 20 out of the 24 comparisons (females, males, larvae and pupae for the 6 contigs)]. However, a differential expression between the strains was observed for the contig corresponding to GSTe2 with a 2.5-fold change (P<0.05) for females and 2-fold change in pupae (Figure 6). GSTe2, which belongs to the glutathione transferase family, has been found to be involved in detoxification and in insecticide resistance in An. gambiae, the other malaria vector [5]. However, although there is a significant difference in expression level for GSTe2, between resistant and susceptible, the fold change of the expression is significantly lower than the 18–25 fold change observed for previously identified genes conferring pyrethroid resistance in An. funestus, such as the duplicated P450 genes CYP6P9 and CYP6P4 [12]. This suggests that if GSTe2 is playing a role in this resistance, it is probably a minor role. GSTe2 over-expression was higher in females than in males, a pattern previously observed for other genes related to pyrethroid resistance (CYP6P9 and CYP6P4) in An. funestus, where the resistance level is higher in females than in males [12], [15]. A 2.2-fold change over-expression of GSTd1-3 was also observed in Larvae for FUMOZ compared to Kela while a 4.1-fold change overexpression of the cuticular gene 76 was observed in Kela pupae compared to FUMOZ. The contigs corresponding to the P450s CYP6AH1 and CYP9J14 and the cuticular gene 125 did not show a significant difference.


A de novo expression profiling of Anopheles funestus, malaria vector in Africa, using 454 pyrosequencing.

Gregory R, Darby AC, Irving H, Coulibaly MB, Hughes M, Koekemoer LL, Coetzee M, Ranson H, Hemingway J, Hall N, Wondji CS - PLoS ONE (2011)

qPCR gene expression profiles of six contigs between the susceptible strain [Kela (field, Mali) and a laboratory pyrethroid-resistant strain (FUMOZ-R).The normalised expression ratio of each contig against the RSP7 gene is represented on the primary vertical axis while the secondary vertical axis represents FUMOZ-R/Kela fold change of each contig.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045460&req=5

pone-0017418-g006: qPCR gene expression profiles of six contigs between the susceptible strain [Kela (field, Mali) and a laboratory pyrethroid-resistant strain (FUMOZ-R).The normalised expression ratio of each contig against the RSP7 gene is represented on the primary vertical axis while the secondary vertical axis represents FUMOZ-R/Kela fold change of each contig.
Mentions: The expression levels of the 6 contigs were generally comparable between FUMOZ and Kela [for 20 out of the 24 comparisons (females, males, larvae and pupae for the 6 contigs)]. However, a differential expression between the strains was observed for the contig corresponding to GSTe2 with a 2.5-fold change (P<0.05) for females and 2-fold change in pupae (Figure 6). GSTe2, which belongs to the glutathione transferase family, has been found to be involved in detoxification and in insecticide resistance in An. gambiae, the other malaria vector [5]. However, although there is a significant difference in expression level for GSTe2, between resistant and susceptible, the fold change of the expression is significantly lower than the 18–25 fold change observed for previously identified genes conferring pyrethroid resistance in An. funestus, such as the duplicated P450 genes CYP6P9 and CYP6P4 [12]. This suggests that if GSTe2 is playing a role in this resistance, it is probably a minor role. GSTe2 over-expression was higher in females than in males, a pattern previously observed for other genes related to pyrethroid resistance (CYP6P9 and CYP6P4) in An. funestus, where the resistance level is higher in females than in males [12], [15]. A 2.2-fold change over-expression of GSTd1-3 was also observed in Larvae for FUMOZ compared to Kela while a 4.1-fold change overexpression of the cuticular gene 76 was observed in Kela pupae compared to FUMOZ. The contigs corresponding to the P450s CYP6AH1 and CYP9J14 and the cuticular gene 125 did not show a significant difference.

Bottom Line: In total 20.8% of all reads were novel when compared to reference databases.Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

View Article: PubMed Central - PubMed

Affiliation: University of Liverpool, School of Biological Sciences, Cornwall House, United Kingdom.

ABSTRACT

Background: Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.

Results: Using a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.

Conclusion: This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

Show MeSH
Related in: MedlinePlus