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Odorant-binding proteins of the malaria mosquito Anopheles funestus sensu stricto.

Xu W, Cornel AJ, Leal WS - PLoS ONE (2010)

Bottom Line: In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae.Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae.Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America.

ABSTRACT

Background: The mosquito Anopheles funestus is one of the major malaria vector species in sub-Saharan Africa. Olfaction is essential in guiding mosquito behaviors. Odorant-binding proteins (OBPs) are highly expressed in insect olfactory tissues and involved in the first step of odorant reception. An improved understanding of the function of malaria mosquito OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally friendly mosquito controlling strategies.

Methodology: In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae. To compare the absolute efficiency/potency of these chemicals, corrections were made for differences in volatility by determining the exact amount in a stimulus puff. Fourteen AfunOBP genes were cloned and their expression patterns were analyzed. AfunOBP1, 3, 7, 20 and 66 showed olfactory tissue specificity by reverse transcriptase PCR (RT-PCR). Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae. Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

Conclusion: This work presents for the first time a study on the odorants and OBPs of the malaria vector mosquito An. funestus, which may provide insight into the An. funestus olfactory research, assist in a comparative study between major malaria mosquitoes An. gambiae and An. funestus olfactory system, and help developing new mosquito control strategies to reduce malaria transmission.

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Quantitative real-time PCR (qPCR) analysis of An. funestus OBPs from female and male antennae.Normalized by (A) actin gene and (B) AfunOR7. Error bars show standard deviation. Significantly differentially expressed AfunOBP genes for female and male antennae distributions were determined as p-value <0.05 by student t-test.
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pone-0015403-g007: Quantitative real-time PCR (qPCR) analysis of An. funestus OBPs from female and male antennae.Normalized by (A) actin gene and (B) AfunOR7. Error bars show standard deviation. Significantly differentially expressed AfunOBP genes for female and male antennae distributions were determined as p-value <0.05 by student t-test.

Mentions: Then, expression levels of olfactory-specific AfunOBP1, 3, 7, 20 and 66 were compared between female and male antennae by using quantitative real-time PCR (qPCR). Only female adult mosquitoes need a protein-rich blood meal to acquire nutrients necessary for eggs maturation after mating, while males do not feed on blood. Therefore, female specific or female enriched OBPs may be more specifically involved in host-seeking behavior. For such comparison, two different genes were used as endogenous control, actin and the OR83b-like odorant receptor cloned from An. funestus (AfunOR7). When actin was used as control, female antennae over male antennae expression ratios (FA/MA) of all OBP genes ranged from 3.77 to 11.12, indicating a general enrichment in female antennae relatively to male antennae (Fig. 7A). Two genes, AfunOBP1 and AfunOBP3 displayed the highest enrichment in female antennae with FA/MA ratios of 7.89 and 11.12, respectively. AfunOBP7, 20 and 66 ratios displayed comparable but lower enrichment in female antennae, around 4 times. When AfunOR7 was used as control, FA/MA ratios were significantly reduced for all genes tested (Fig. 7B). Again, AfunOBP1 and AfunOBP3 displayed the highest enrichment in female antennae with FA/MA ratios of 3.22 and 4.54, respectively, and other genes (AfunOBP7, 20 and 66) displayed comparable but lower enrichment in female antennae, around 1.5–1.8 times. Expression analysis by qRT-PCR demonstrates that two antennae-specific OBP genes, AfunOBP1 and AfunOBP3, are both enriched in female antennae. Similar study on gender ratios (female/male) was performed on AgamOBP genes by microarray and qPCR [44]. AgamOBP1 and AgamOBP3, the orthologous genes of AfunOBP1 and AfunOBP3, were also detected at higher levels in female antennae than in male antennae [44]. By using microarray, AgamOBP3 showed a 9.2 times higher transcripts level in female antennae and AgamOBP1 showed a 4.2 times enrichment [44]. By using qPCR, AgamOBP3 showed a 8.1 times higher transcripts level in female head than in male and AgamOBP1 showed a 4.4 times enrichment [44]. Both approaches indicate that OBP1 and OBP3 are also female abundant OBPs in An. gambiae [44]. An AfunOBP1 orthologous gene from the Asia malaria vector Anopheles stephensi, AsteOBP1, was also cloned and studied recently [45]. The level of AsteOBP1 transcript was 7-fold higher in female antennae than in male antennae by qRT-PCR, revealing that AsteOBP1 was also abundant in female An. stephensi.


Odorant-binding proteins of the malaria mosquito Anopheles funestus sensu stricto.

Xu W, Cornel AJ, Leal WS - PLoS ONE (2010)

Quantitative real-time PCR (qPCR) analysis of An. funestus OBPs from female and male antennae.Normalized by (A) actin gene and (B) AfunOR7. Error bars show standard deviation. Significantly differentially expressed AfunOBP genes for female and male antennae distributions were determined as p-value <0.05 by student t-test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2962654&req=5

pone-0015403-g007: Quantitative real-time PCR (qPCR) analysis of An. funestus OBPs from female and male antennae.Normalized by (A) actin gene and (B) AfunOR7. Error bars show standard deviation. Significantly differentially expressed AfunOBP genes for female and male antennae distributions were determined as p-value <0.05 by student t-test.
Mentions: Then, expression levels of olfactory-specific AfunOBP1, 3, 7, 20 and 66 were compared between female and male antennae by using quantitative real-time PCR (qPCR). Only female adult mosquitoes need a protein-rich blood meal to acquire nutrients necessary for eggs maturation after mating, while males do not feed on blood. Therefore, female specific or female enriched OBPs may be more specifically involved in host-seeking behavior. For such comparison, two different genes were used as endogenous control, actin and the OR83b-like odorant receptor cloned from An. funestus (AfunOR7). When actin was used as control, female antennae over male antennae expression ratios (FA/MA) of all OBP genes ranged from 3.77 to 11.12, indicating a general enrichment in female antennae relatively to male antennae (Fig. 7A). Two genes, AfunOBP1 and AfunOBP3 displayed the highest enrichment in female antennae with FA/MA ratios of 7.89 and 11.12, respectively. AfunOBP7, 20 and 66 ratios displayed comparable but lower enrichment in female antennae, around 4 times. When AfunOR7 was used as control, FA/MA ratios were significantly reduced for all genes tested (Fig. 7B). Again, AfunOBP1 and AfunOBP3 displayed the highest enrichment in female antennae with FA/MA ratios of 3.22 and 4.54, respectively, and other genes (AfunOBP7, 20 and 66) displayed comparable but lower enrichment in female antennae, around 1.5–1.8 times. Expression analysis by qRT-PCR demonstrates that two antennae-specific OBP genes, AfunOBP1 and AfunOBP3, are both enriched in female antennae. Similar study on gender ratios (female/male) was performed on AgamOBP genes by microarray and qPCR [44]. AgamOBP1 and AgamOBP3, the orthologous genes of AfunOBP1 and AfunOBP3, were also detected at higher levels in female antennae than in male antennae [44]. By using microarray, AgamOBP3 showed a 9.2 times higher transcripts level in female antennae and AgamOBP1 showed a 4.2 times enrichment [44]. By using qPCR, AgamOBP3 showed a 8.1 times higher transcripts level in female head than in male and AgamOBP1 showed a 4.4 times enrichment [44]. Both approaches indicate that OBP1 and OBP3 are also female abundant OBPs in An. gambiae [44]. An AfunOBP1 orthologous gene from the Asia malaria vector Anopheles stephensi, AsteOBP1, was also cloned and studied recently [45]. The level of AsteOBP1 transcript was 7-fold higher in female antennae than in male antennae by qRT-PCR, revealing that AsteOBP1 was also abundant in female An. stephensi.

Bottom Line: In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae.Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae.Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America.

ABSTRACT

Background: The mosquito Anopheles funestus is one of the major malaria vector species in sub-Saharan Africa. Olfaction is essential in guiding mosquito behaviors. Odorant-binding proteins (OBPs) are highly expressed in insect olfactory tissues and involved in the first step of odorant reception. An improved understanding of the function of malaria mosquito OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally friendly mosquito controlling strategies.

Methodology: In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae. To compare the absolute efficiency/potency of these chemicals, corrections were made for differences in volatility by determining the exact amount in a stimulus puff. Fourteen AfunOBP genes were cloned and their expression patterns were analyzed. AfunOBP1, 3, 7, 20 and 66 showed olfactory tissue specificity by reverse transcriptase PCR (RT-PCR). Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae. Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

Conclusion: This work presents for the first time a study on the odorants and OBPs of the malaria vector mosquito An. funestus, which may provide insight into the An. funestus olfactory research, assist in a comparative study between major malaria mosquitoes An. gambiae and An. funestus olfactory system, and help developing new mosquito control strategies to reduce malaria transmission.

Show MeSH
Related in: MedlinePlus