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Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Müller P, Pflüger V, Wittwer M, Ziegler D, Chandre F, Simard F, Lengeler C - PLoS ONE (2013)

Bottom Line: The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies.While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time.As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Services and Diagnostic, Swiss Tropical and Public Health Institute, Basel, Switzerland. pie.mueller@unibas.ch

ABSTRACT
Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

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Related in: MedlinePlus

Total number of peaks versus number of diagnostic peaks present in average spectra from the Anopheles gambiae species complex.The number of diagnostic peaks present is associated with the number of total peaks present in an average peak list. The diagnostic peaks refer to the 68 selected peaks to distinguish within the A. gambiae species complex (Model 1). The plot suggests that the field specimens collected by aspiration (green crosses) were generally of lower quality (i.e. showing fewer peaks) than the specimens that were raised from the larvae; regardless whether from laboratory (black circles) or field caught larvae (red triangles).
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pone-0057486-g004: Total number of peaks versus number of diagnostic peaks present in average spectra from the Anopheles gambiae species complex.The number of diagnostic peaks present is associated with the number of total peaks present in an average peak list. The diagnostic peaks refer to the 68 selected peaks to distinguish within the A. gambiae species complex (Model 1). The plot suggests that the field specimens collected by aspiration (green crosses) were generally of lower quality (i.e. showing fewer peaks) than the specimens that were raised from the larvae; regardless whether from laboratory (black circles) or field caught larvae (red triangles).

Mentions: There might be many – biological and technical – reasons why the specimens from the resting collections performed less well in the MALDI-TOF MS analysis. The specimens – unlike the adults raised from the larval collections – were either blood fed or gravid females or perhaps even carrying pathogens. Therefore, it is expected that a subset of genes would be differentially expressed due to the physiological state of the mosquito (e.g.[48]–[50]). Such differential expression would potentially also affect the number and types of masses detected in the MALDI-TOF spectra. If this was the prime cause of poor performance of Model 1 on the specimens from the resting collections we would predict different masses to come up in the MS spectra. The observation in our data set was, however, a different one. The number of peaks obtained from these specimens was lower than from those raised from larvae. More importantly, the number of diagnostic peaks present in an average spectrum showed the same relationship for all types of samples (Figure 4). It is, therefore, concluded that a major impact on the poorer performance of Model 1 on these specimens is a quality rather than a biological phenomenon. Indeed, some of the spectra even showed zero peaks (Figure 4). Including the spectra from field specimens in the model did also not improve its performance (data not shown). Perhaps the blood in the abdomens somehow negatively influenced the preservation of the specimens from those resting collections. Although somewhat unfortunate for the current study this is an aspect that can be addressed by optimising and standardising sampling, storage and processing procedures in future studies. A preliminary recommendation would be to separate the abdomens from the head and thoraces prior storage.


Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Müller P, Pflüger V, Wittwer M, Ziegler D, Chandre F, Simard F, Lengeler C - PLoS ONE (2013)

Total number of peaks versus number of diagnostic peaks present in average spectra from the Anopheles gambiae species complex.The number of diagnostic peaks present is associated with the number of total peaks present in an average peak list. The diagnostic peaks refer to the 68 selected peaks to distinguish within the A. gambiae species complex (Model 1). The plot suggests that the field specimens collected by aspiration (green crosses) were generally of lower quality (i.e. showing fewer peaks) than the specimens that were raised from the larvae; regardless whether from laboratory (black circles) or field caught larvae (red triangles).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585343&req=5

pone-0057486-g004: Total number of peaks versus number of diagnostic peaks present in average spectra from the Anopheles gambiae species complex.The number of diagnostic peaks present is associated with the number of total peaks present in an average peak list. The diagnostic peaks refer to the 68 selected peaks to distinguish within the A. gambiae species complex (Model 1). The plot suggests that the field specimens collected by aspiration (green crosses) were generally of lower quality (i.e. showing fewer peaks) than the specimens that were raised from the larvae; regardless whether from laboratory (black circles) or field caught larvae (red triangles).
Mentions: There might be many – biological and technical – reasons why the specimens from the resting collections performed less well in the MALDI-TOF MS analysis. The specimens – unlike the adults raised from the larval collections – were either blood fed or gravid females or perhaps even carrying pathogens. Therefore, it is expected that a subset of genes would be differentially expressed due to the physiological state of the mosquito (e.g.[48]–[50]). Such differential expression would potentially also affect the number and types of masses detected in the MALDI-TOF spectra. If this was the prime cause of poor performance of Model 1 on the specimens from the resting collections we would predict different masses to come up in the MS spectra. The observation in our data set was, however, a different one. The number of peaks obtained from these specimens was lower than from those raised from larvae. More importantly, the number of diagnostic peaks present in an average spectrum showed the same relationship for all types of samples (Figure 4). It is, therefore, concluded that a major impact on the poorer performance of Model 1 on these specimens is a quality rather than a biological phenomenon. Indeed, some of the spectra even showed zero peaks (Figure 4). Including the spectra from field specimens in the model did also not improve its performance (data not shown). Perhaps the blood in the abdomens somehow negatively influenced the preservation of the specimens from those resting collections. Although somewhat unfortunate for the current study this is an aspect that can be addressed by optimising and standardising sampling, storage and processing procedures in future studies. A preliminary recommendation would be to separate the abdomens from the head and thoraces prior storage.

Bottom Line: The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies.While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time.As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Services and Diagnostic, Swiss Tropical and Public Health Institute, Basel, Switzerland. pie.mueller@unibas.ch

ABSTRACT
Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Show MeSH
Related in: MedlinePlus