Purpose

The main objective of this study was to develop DNA-based methods for identifying malaria mosquitoes in a region of Bolivia where there is epidemic malaria and a great diversity of mosquito species and breeding habitats. My goal was to develop a method using multiplex PCR (multiple species specific primers) that was easy, efficient, accurate and could identify any life stage (egg, larva, pupa, or adult) (Figure 1).

  Figure 1.

Methods

Larval Collection. Larvae were collected from fifty-six sites in the Carrasco/Chapare Valley, Bolivia in May 2003.  Of these larvae, about ten were reared to adults as voucher specimens from each site for identification with standard morphological keys.

Sequencing.  Sections of DNA that separate genes, but do not code for anything (known as “spacers”), evolve rapidly through time and, therefore, are good candidates for finding DNA differences between species. I chose a spacer known as the Internal Transcribed Spacer Two (ITS2) to develop species-specific primers.  PCR of the ITS2 was done using primers that anneal to the flanking 5.8s and 28s rDNA genes (Porter and Collins 1991).  Direct sequencing of the PCR product was done using a Beckman CEQ 2000 sequencer following the manufacturer's instructions.

Primer Choice.  Appropriate species specific primer sites were located by ITS2 alignment with CLUSTALW of all targeted species (Figure 2 and 3) along with those available on GenBank.

The species-specific primers were then combined in a multiplex PCR and tested with samples of DNA from the following species: A. trinkae, A. triannulatus, A. rangeli, A. strodei, A. aquasalis, A. albimanus, A. darlingi, A. evansae, A. oswaldoi, A. marajoara, A. albitarsis, A. nuneztovari, A. galveoi, A. deanorum, A. bennarochi, A. konderi, A. braziliensis, A. argrytarsis, and species C.

Figure 2.  Sequence alignment of A. oswaldoi and species C. Black letters indicate that the sequences are identical, and red indicates differences. Highlighted sections indicate species-specific primer sites.

Figure 3. Sequence alignment Albitarsis complex. Black letters indicate that the sequences are identical, and red indicates differences.

Highlighted section indicates Albitarsis complex unique primer and red rectangle represents Bfa-I (C/TAG) restriction enzyme cut site.

Results

Two multiplex PCR were developed to identify two sets of morphologically similar species of malaria mosquitoes incorporating species-specific primers derived from direct sequencing of the ITS2 (Table 1).  One PCR reaction identifies A. oswaldoi and an undescribed species (Species C) that we believe to be new to science and abundant in certain areas of Bolivia (Figure 4).

Table 1.  Ribosomal DNA ITS2 species-specific primers and their melting temperatures (Tm)

A second PCR reaction identifies mosquitoes as belonging to a complex of four species  (Albitarsis Complex), which includes A.deanorum, A. albitarsis A, A. albitarsis B, and A. marajoara. The resulting PCR product is then digested with a restriction enzyme that cuts the DNA at a specific site to distinguish A. marajoara (Figure 4).

Figure 4. DNA bands produced by ribosomal DNA-polymerase chain reaction (PCR) of samples of A. oswaldoi and Species C, and restriction enzyme digest of A. marajoara.

Conclusions

The two multiplex PCR developed can provide an unambiguous and relatively rapid identification of morphologically similar species. In addition, the multiplex PCR developed have proven to be highly specific, not only permitting the identification of A. marajoara (Figure 5), species C (Figure 6), and A. oswaldoi (Figure 7), but also differentiating them from other sympatric anopheline species in the subgenus Nyssorhynchus.

Figure 5. A. marajoara

Figure 6. Species C.

Figure 7. A. oswaldoi

The construction of two multiplex PCR that can diagnose three putative vectors of human Plasmodium in the Neotropics should make it more feasible to initiate studies on anopheline mosquito ecology and the dynamics of transmission of malaria, particularly since the primers can be used with a minute amount of sample material for species identification.