DNA sequence analysis of the ITS2 region of the ribosomal RNA genes of An. punctulatus
group sibling species has identified numerous sequence polymorphisms (single and multiple nucleotide polymorphisms; insertions and deletions; repeat variants or types). Beebe and others have used these molecular polymorphisms to perform phylogenetic analyses to demonstrate sequence differences that routinely distinguish morphologically identical sibling species, particularly those within the Farauti
This work has been the platform for developing both post-PCR DNA probe and RFLP strategies for describing species relationships and ancestry, to perform species identification in field surveys, and to begin evaluation of strain distribution between sites within the Southwest Pacific region. New technology incorporating fluorescent microspheres coupled to an array of variable sequence oligonucleotides (Luminex FlexMAP™ microspheres) enables liquid-phase microarray analysis that significantly expands capacity for developing multiplex DNA sequence-based diagnostic strategies. Given further results illustrating An
group ITS2 sequence polymorphisms within individual species,8,30
we wanted to make multiple within and among species sequence comparisons before developing species-specific DNA probes for application through this technology. Our analysis of more than 100 ITS2 alleles from An. punctulatus
(n = 27), An. koliensis
(n = 20), An. farauti
s.s. (n = 28; includes 13 previously published sequences8
[Genbank accession nos. AF104314–AF104326]), An. hinesorum
(n = 12), and An. farauti
4 (n = 16) showed considerable ITS2 sequence divergence between species (from 65.0% to 92.9% pairwise percent identity; includes previously published sequences10
for An. torresiensis
[Genbank accession no. AF033214], An. farauti
5 [AF033216], An. farauti
6 [AF033217], An. farauti
7 [AF033218], An.
[AF033221]), further comparisons showed high conservation of ITS2 sequence (≥ 99.0% pairwise percent identity) within species. These comparisons enabled identification of sequence regions that varied significantly among species that did not vary within species. Probes based on these sequences have now been shown to differentiate the five Punctulatus
group sibling species () implicated in transmitting malaria and filarial parasites in Papua New Guinea.
Using the multiplex molecular diagnostic assay, we analyzed 340 mosquitoes obtained from 18 locations (7 Provinces in Papua New Guinea) that had been morphologically identified as members of the Punctulatus group. Because morphology can only differentiate members of the An. punctulatus complex into three species categories (An. farauti s.l., An. koliensis, and An. punctulatus) it was clear that the ITS2 multiplex assay would provide more definitive identification of individual mosquitoes than classical morphometric methods, and this was borne out in further identification of An. farauti s.s., An. hinesorum, and An. farauti 4. Comparison of the two methods was performed by classifying mosquitoes into the three generalized species. Among the 101 individual insects identified by the An. punctulatus probe, 44 mosquitoes were classified as either An. koliensis or An. farauti s.l. by morphology (DNA sensitivity versus morphology, morphology positive predictive value versus DNA = 0.564). Species identification between the An. koliensis probe and morphology was more highly concordant (DNA sensitivity versus morphology = 0.853). However, 34 of 115 insects identified as An. koliensis by morphology were observed to hybridize with An. punctulatus and An. farauti probes (morphology sensitivity versus DNA = 0.704). Similarly, 37 of 158 insects identified by morphology as An. farauti s.l. were observed to hybridize to An. punctulatus and An. koliensis probes (morphology sensitivity versus DNA = 0.766). When evaluated in aggregate, DNA probes detecting sibling species of the Farauti complex were observed to hybridize with 23 mosquitoes identified by morphology as An. koliensis. However, none of the insects identified by morphology as An. punctulatus hybridized with any of the Farauti complex probes (DNA sensitivity versus morphology = 0.840). To compare this new LDR-FMA method with the currently established molecular method of identification, ITS2 RFLP, we tested a subset of 117 mosquito samples and found concordance rates between ITS2 RFLP and ITS2 LDR-FMA to be above 98%. Similar to ITS2 LDR-FMA, RFLP species differentiation was 76% concordant with morphological species identification.
Given the variability of proboscis coloration and overlapping geographic distribution of morphologically similar Punctulatus
group sibling species previously documented by Cooper and others4
and Beebe and Cooper,14
we anticipated that we would observe discordance between the two classification schemes (ITS2 LDR-FMA and morphological identification). At 10 of the 18 collection sites, 100% concordance was observed between DNA probe and morphologic classification of An. punctulatus
; in four of these sites An. punctulatus
was the only species collected. In the 12 sites where An. koliensis
was identified by morphology, DNA probe classification was 100% concordant in only one site (Hudini). In the 11 sites where An. farauti
s.l. was collected, 100% concordance between DNA probe and morphology was observed in two sites (Sausi and Bilbil). In all 13 sites where more than one species was present, discordance was observed between DNA probe and morphology, and in two sites where only one species was identified by morphology, different species were identified by DNA probes. Because morphological classifications and LDR-FMA probe hybridization discordance was highest between An. koliensis
and An. farauti
s.l., further research is needed to characterize the relationships of these species more clearly. Finally, observed discordance between ITS2 LDR-FMA DNA probes and morphology was observed in collection sites from all 7 of the provinces included in our survey.
We have described and demonstrated a new multiplex DNA-based assay designed to identify DNA sequence polymorphisms in the primary disease vectors of the Punctulatus
group in Papua New Guinea. This method is based on technology that facilitates multiplex analysis, automation, and uniformity of PCR-based mosquito species diagnosis because procedures from sample processing and DNA extraction to entry of results into database files can now be handled continuously in 96-well plate format. Costs associated with the post-PCR fluorescent microsphere assay for the five mosquito species surveyed total to 12.5 cents per individual mosquito. The LDR-FMA multiplex avoids use of ethidium bromide (biohazardous waste) used to visualize RFLP patterns. Also, although the RFLP method reliably differentiates sibling species within the Punctulatus
group (), the MspI
RFLP analysis will not identify strain-specific single nucleotide or small insertion/deletion polymorphisms (this study and Beebe and others10
) observed within species that appear to be associated with different geographic locations. The LDR-FMA assay design allows for modifications and additions, which could identify these polymorphisms. Moreover, because the same LDR-FMA multiplex strategy is used for diagnosis of Plasmodium
species and Wuchereria bancrofti
it is foreseeable that a single post-PCR assay would be able to perform Anopheles
species identification and diagnosis of important human parasites simultaneously from a single tube.
Understanding habitat preference, vector competence, biting habits, and distribution of Punctulatus group sibling species and strains is paramount to understanding mosquito ecology and designing disease and vector control plans. Given the overall complexity of vector-borne disease transmission associated with the Punctulatus group, it is critical to develop diagnostic surveillance strategies that can monitor markers associated with infection by efficient methods. In comparison with RFLP analysis, the LDR-FMA multiplex strategy has greater capacity for high throughput analysis of known genetic targets and expansion to assess additional polymorphism in a single post-PCR assay. Therefore, this new diagnostic approach provides significant potential to improve research studies and to monitor and evaluate operational control programs to reduce disease transmission by these important vectors.