Am. J. Trop. Med. Hyg., 84(1), 2011, pp. 166–173
Copyright © 2011 by The American Society of Tropical Medicine and Hygiene
Mosquitoes belonging to the Anopheles punctulatus group
are sibling species found in the Southwest Pacific region rang-
ing from the Weber Line and Moluccas (former Spice Islands)
to Vanuatu, including New Guinea and islands of the Bismarck
Archipelago, the Solomon Islands and northern Australia. 1
The group includes 13 sibling species ( An. punctulatus , An .
species near punctulatus , morphologically indistinguishable
An. farauti 1–8 [ Farauti complex; former An. farauti 2 and 3,
now An. hinesorum and An. torresiensis , respectively], An.
koliensis , An. clowi , and An. rennellensis ). 1– 5 Studies by Bryan
and Foley and others initially described species diversity
within the Farauti complex by cross-mating, 6, 7 and allozyme
polymorphisms. 5 Beebe and others, and Cooper and others
have extended molecular characterization of the Punctulatus
group by DNA sequence analyses of ribosomal RNA loci. 4, 8– 12
Additional diversity has been suggested among isolates of An.
koliensis between collection sites in East Sepik and Madang
Provinces in Papua New Guinea. 13
Although members of the Punctulatus group have been
characterized as unspecialized in regard to blood feeding
behaviors and breeding habitats, 14 individual species within
the group are not distributed ubiquitously throughout the
region. In previous surveys, An. farauti s.s and An. hinesorum
have been characterized as most widely distributed, through-
out Papua New Guinea, northern Australia, the Solomon
Islands, and Vanuatu. Anopheles farauti s.s. is found most
often within 1–2 km of the coast. In contrast An. hinesorum is
most commonly found in areas between 10 and 100 km from
the coast. Anopheles koliensis and An. punctulatus are also
widely distributed throughout lowlands in Papua New Guinea
and the Solomon Islands. Anopheles torresiensis has been
found in northern Australia and southwestern Papua New
Guinea where climate has been characterized as monsoonal.
Anopheles farauti 4 is found primarily in northern Papua New
Guinea throughout the Sepik and Ramu River plains. Less
widely distributed species include An. farauti 5 and 6, found in
the Papua New Guinea highlands; An. farauti 7, found in the
Solomon Islands and Vanuatu; and An. near species punctula-
tus , found in inland habitats of southern and northern Papua
New Guinea. 4, 14– 16 Anopheles farauti s.l. larvae are found most
frequently in coastal streams and brackish pools or swamps.
Anopheles punctulatus larvae are found in sunlit rainwater
pools made in tire tracks or drainage pools. Anopheles kolien-
sis larvae are found in temporary grassland or forest edge
pools more than 2 km from the coast. 17
In Papua New Guinea, studies have most often implicated
An. farauti s.s . , An. hinesorum , An. farauti 4, An. koliensis , and
An. punctulatus as the primary vectors of malaria and filarial
parasites. 13, 18, 19 However, given the heterogeneous distribution
of An. punctulatus species complex members across Papua
New Guinea, in addition to what is known about the variable
breeding and biting preferences among species 3, 17, 20 it will be
important to understand if all members of the Punctulatus
group transmit parasites, or if there is further heterogeneity
among species in their competence to transmit parasites.
Assessment and continuous monitoring of the vector species
composition in disease-endemic regions is a necessary compo-
nent of vector biology and consistent with the integrated vec-
tor management strategy of the World Health Organization. 21
Although DNA probe hybridization and polymerase chain reac-
tion–restriction fragment length polymorphism (PCR-RFLP)
methods have been developed for performing species-level
classification of Punctulatus group sibling species, 11– 13 currently,
morphology remains the most common method of species
identification. Morphological traits used to distinguish species
(proboscis coloration) are unreliable because individual mos-
quitoes within single species display polymorphism, such that
similar proboscis coloration is observed among species. 1, 4, 13, 22
This finding emphasizes that molecular diagnostic strategies
will be required tools to enhance the capacity of entomologists
High Throughput Multiplex Assay for Species Identification of Papua New Guinea Malaria
Vectors: Members of the Anopheles punctulatus (Diptera: Culicidae) Species Group
Cara N. Henry-Halldin , Lisa Reimer , Edward Thomsen , Gussy Koimbu , Allison Zimmerman , John B. Keven , Henry Dagoro, †
Manuel W. Hetzel , Ivo Mueller , Peter Siba , and Peter A. Zimmerman *
Center for Global Health and Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio; Papua New Guinea Institute of
Medical Research, Madang, Papua New Guinea; Papua New Guinea Institute of Medical Research, Goroka, Eastern Highlands, Papua New Guinea;
School of Population Health, University of Queensland, Brisbane, Queensland, Australia
Abstract . Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling spe-
cies including An. punctulatus , An. koliensis , the An. farauti complex 1–8 (includes An. hinesorum [ An. farauti 2], An. torre-
siensis [ An. farauti 3]). Distinguishing these species from each other requires molecular diagnostic methods. We developed
a multiplex polymerase chain reaction (PCR)–based assay specific for known species-specific nucleotide differences in the
internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease ( An.
punctulatus , An. koliensis , An. farauti 1, An. hinesorum , and An. farauti 4). A set of 340 individual mosquitoes obtained
from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay.
Concordance between molecular and morphological diagnosis was 56.4% for An. punctulatus , 85.3% for An. koliensis , and
88.9% for An. farauti . Among 158 mosquitoes morphologically designated as An. farauti , 33 were re-classified by PCR as
An. punctulatus , 4 as An. koliensis , 26 as An. farauti 1, 49 as An. hinesorum , and 46 as An. farauti 4. Misclassification results
from variable coloration of the proboscis and overlap of An. punctulatus , An. koliensis , the An. farauti 4. This multiplex
technology enables further mosquito strain identification and simultaneous detection of microbial pathogens.
* Address correspondence to Peter A. Zimmerman, Center for Global
Health and Diseases, Case Western Reserve University School of
Medicine, Wolstein Research Building, #4-125, 2103 Cornell Road,
Cleveland, OH 44106. E-mail: firstname.lastname@example.org
MULTIPLEX SPECIES IDENTIFICATION OF PNG MALARIA VECTORS
for assessing vector capacity, insecticide resistance, and distri-
bution patterns of insect species complexes in many infectious
disease scenarios. Significant technical advances can now be
incorporated into new diagnostic strategies to enable more effi-
cient surveillance of these important human disease vectors.
We describe a high-throughput multiplex strategy for
molecular identification of the Punctulatus group sibling spe-
cies that serve as primary vectors of malaria and filarial para-
sites in Papua New Guinea based on species-specific sequence
differences in the internal transcribed spacer 2 (ITS2) region
of ribosomal DNA surveyed in this study and previous stud-
ies. 10 This species identification tool applies technology used
previously to perform multiplex analysis of Plasmodium and
Wuchereria bancrofti infection in human blood samples. 23, 24
MATERIALS AND METHODS
Mosquitoes. Representative mosquitoes were obtained by
the Entomology Unit of the Papua New Guinea Institute of
Medical Research (PNGIMR) through on-going studies and
activities associated with distribution of long-lasting insecticide-
treated bed nets; collection sites from seven provinces in Papua
New Guinea ( Table 1 ). Mosquito capture methods included
landing catches, Centers for Disease Control light traps, 20 and
larval collections. Samples collected at the larval stage were
allowed to mature in the laboratory before morphological
identification could be completed. Sample locations were
defined by global positioning system and represented a range
of altitudes, habitats, and varied distance from the coast to
capture a variety of species ( Table 1 ). Samples collected from
Madang Province (Bilbil), Manus Province (Lorengau), and
Morobe Province (Godowa) represented collections within
< 1 km of the coast. Sampling from West Sepik Province
(Pitapena: 202 km from coast) and Western Highlands Province
(Brimba and Singoropa: 120 km from coast) represented
collections from the most inland locations.
Species morphology. Mosquitoes were morphologically
identified using methods previously described in which mem-
bers of the An. punctulatus group were classified as An.
punctulatus , An. koliensis , or An. farauti s.l. by morphological
characteristics. 4, 22 A set of 340 mosquitoes morphologically
identified as members of the An. punctulatus group were then
kept in coded vials containing silica gel until DNA extraction
could be completed.
DNA extraction. Genomic DNA was extracted from single
whole mosquitoes (n = 340) by using either a QIAamp 96 Kit
or an individual spin blood and tissue kit (QIAGEN, Valencia,
CA, recommended protocol) or a modification of the method
of Bender and others. 25 In the modified method, individual
whole mosquitoes were thoroughly ground by vortexing each
mosquito with a copper pellet (BB) in a 1.5-mL microfuge tube
containing 100 μL of grinding buffer (0.1 M NaCl, 0.2 M sucrose,
0.1 M Tris-HCl, pH 9.1–9.2, 0.05 M EDTA, and 0.5% sodium
dodecyl sulfate). Samples were incubated at 65°C for 30 minutes;
8 M potassium acetate was then added (final concentration =
1 M) to each tube and incubated on ice for 30 minutes.
Samples were centrifuged at 13,500 rpm (15 minutes) and the
supernatant was transferred to a new sterile tube; 100 μL of
100% ethanol was added for precipitation of mosquito DNA.
Tubes were incubated at room temperature (5 minutes) and
then centrifuged at 13,500 rpm (15 minutes). Supernatants
were removed; 100 μL of ice-cold 70% ethanol was added to
each sample, mixed, and centrifuged at 13,500 rpm (5 minutes).
Supernatants were removed and samples were allowed to dry
overnight; precipitated DNA was resuspended in 30–100 μL of
sterile deionized H 2 O or Tris-acetate buffer.
PCR amplification and DNA sequence analysis. PCR
amplifications (25 μL) of the ITS2 locus were performed in a
solution containing 67 mM Tris-HCl, pH 8.8, 6.7 mM MgSO 4 ,
16.6 mM (NH 4 ) 2 SO 4 , 10 mM 2-mercaptoethanol, 100 μM
dATP, dGTP, dCTP, and dTTP, 2.5 units of thermostable DNA
polymerase, and upstream and downstream primers described
by Beebe and Saul. 12 The thermocycling program was 95°C for
2 minutes (1×), 95°C for 30 seconds, 55°C for 30 seconds, 72°C
for 1 minute (35×), 72°C for 4 minutes (1×) and was performed
by using a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-
Rad, Hercules, CA) for all 340 mosquito samples. To evaluate
overall amplification efficiency, PCR products were separated
by electrophoresis on 2% agarose gels (1× Tris-Borate-EDTA
buffer), stained with SYBR ® Gold (Invitrogen, Carlsbad, CA),
and visualized on a Storm 860 using ImageQuant, 5.2 software
(Molecular Dynamics, Sunnyvale, CA). TOPO TA cloning
(Invitrogen) of the amplified ITS2 region was performed on
a subset (n = 90) of the 340 mosquitoes identified, followed
Mosquito collection site data, Papua New Guinea
SiteProvinceAltitude, metersLatitudeLongitude Approximate distance to coast, km
* WHP = Western Highlands Province.
HENRY-HALLDIN AND OTHERS
by single-pass bidirectional plasmid sequencing (Beckman
Coulter Genomics, Danvers, MA) to identify motifs in the
ITS2 sequence for further design and development of species-
specific DNA probes. DNA sequence data was analyzed by
using Geneious version 5.0.3. 26 ClustalW2 27 (default settings:
gap open penalty = 10, gap extend penalty = 6.66) was used
to generate the ITS2 species consensus sequence alignment as
shown in Figure 1 .
Figure 1. Internal transcribed spacer 2 (ITS2) consensus alignment for Anopheles punctulatus (AP), An. farauti s.s. (AF1), An. hinesorum (AH),
An. farauti 4 (AF4), and An. koliensis (AK) from Papua New Guinea. Consensus sequence for each member was created by using ITS2 sequence
from multiple representatives from each species: An. punctulatus (n = 27) (Genbank accession no. HM584428–HM584454), An. farauti s.s. (n = 15)
(HM584365–HM584379), An. hinesorum (n = 12) (HM584380–HM584391), An farauti 4 (n = 16) (HM584392–HM584406, HM584427), and An. koliensis
(n = 20) (HM584407–HM584426). Locations of ligase detection reaction classification and reporter probes are shaded in gray and black, respectively.
MULTIPLEX SPECIES IDENTIFICATION OF PNG MALARIA VECTORS
Molecular species identification. Anopheles punctulatus ,
An. koliensis , An. farauti s.s., An. hinesorum , and An. farauti
4 species were differentiated in a high throughput manner
targeting unique species-specific polymorphisms in the ITS2
ribosomal DNA. After PCR amplification, products were
added to a multiplex ligase detection reaction (LDR) in
which species-specific upstream classification probes (con-
taining anti-TAG sequences specific to Luminex ® micro-
sphere sets) ligate to downstream sequence reporter probes
(3′-biotinylated) when appropriate target template sequences
are available ( Table 2 ). The LDRs were conducted in a solution
(15 μL) containing 20 mM Tris-HCl buffer, pH 7.6, 25 mM
potassium acetate, 10 mM magnesium acetate, 1 mM NAD+,
10mM dithiothrietol, 0.1% Triton X-100, 10 nM (200 pmol) of
each LDR probe, 1 μL of each PCR product, and 2 units of Taq
DNA ligase (New England Biolabs, Beverly, MA). The LDRs
were initially heated at 95°C for 1 minute, followed by 32
thermal cycles at 95°C for 15 seconds and 58°C for 2 minutes.
Labeling and detection of LDR fluorescent microsphere assay
(FMA) products was completed as described 23 by using a
Bio-Plex Array Reader (Bio-Rad).
Individual Luminex FlexMap™ microsphere sets cost
US $25.00/vial. Interrogation of each species-specific target
sequence requires 250 microspheres (1,000 assays/vial); 2.5
A subset (n = 117) of the original 340 mosquitoes was also
characterized by using an ITS2 RFLP assay. 12 This subset was
chosen at random and the PNGIMR Entomology Unit con-
ducted RFLP analysis in a blinded manner. A portion (n = 13)
of this subset of mosquitoes had also been evaluated by DNA
sequence analysis as described above.
Statistical analysis. Specificity, sensitivity, and positive and
negative predictive value calculations were performed by
using standard methods ( http://statpages.org/ctab2x2.html ).
ITS2 sequence analysis and post–PCR-LDR-FMA assay
design. Previous reports of ITS2 polymorphism among
Punctulatus group sibling species 10 demonstrated that there is
significant polymorphism among species. Moreover, previous
analysis showed polymorphism within An. koliensis isolates
in northern Papua New Guinea. 13 Therefore, to identify
conserved regions within ITS2 PCR products specific for LDR-
FMA interrogation, we performed DNA sequence analysis of
multiple alleles for each species of interest. After alignment
of 27 alleles of An. punctulatus , 15 of An. farauti s.s., 12 of An.
hinesorum , 16 of An. farauti 4, and 20 of An. koliensis , conserved
species-specific sequence polymorphisms were identified.
Locations in which species-specific classification and conserved
sequence LDR reporter probes hybridized with template
PCR products are shown in ITS2 consensus alignment for An.
punctulatus , An. farauti s.s., An. hinesorum , An. farauti 4, and
An. koliensis ( Figure 1 ). Consensus sequences for each sibling
species were created from ITS2 sequences after alignment of
multiple representatives from each species: An. punctulatus ,
n = 27 (98.9% similarity within species); An. farauti s.s., n = 15
(99.3% similarity); An. hinesorum , n = 12 (99.6% similarity);
An. farauti 4, n = 16 (99.5% similarity); and An. koliensis ,
n = 20 (98.3% similarity). These consensus sequences were
compared with previously published sequences 10 and found to
be greater than 97.5% similar: AP consensus versus GenBank
accession no. AF033220 = 98.7%, AF1 consensus ( An.farauti
s.s.) versus GenBank accession no. AF055984 = 98.2%, AH
consensus versus GenBank accession no. AF033213 = 98.1%,
AF4 consensus versus GenBank accession no. AF033215 =
97.5%, and AK consensus versus GenBank accession no.
AF033219 = 98.4%.
Specificity of the Punctulatus group post–PCR-LDR-FMA.
The specificity of the assay was then demonstrated by using
probes identified in Figure 1 and Table 2 to differentiate PCR
products amplified from previously sequenced species-specific
controls. Because members of the Punctulatus group are
known to carry Plasmodium 19, 28, 29 and Wuchereria parasites 13
and human blood meals, 3, 28 we included six additional controls
containing Plasmodium falciparum , P. vivax , P. malariae ,
P. ovale , Wuchereria bancrofti , and human genomic DNA.
Results of the specificity test ( Figure 2 ) show by strong
species-specific fluorescent signals ( An. punctulatus median
fluorescence intensity [MFI] > 5,000; An. koliensis > 11,000;
An. farauti s.s. > 20,000; An. hinesorum > 21,000; An. farauti
4 > 5,000) that each LDR-FMA probe set detected only the
species expected, and that all background signals were below
MFIs of 900. This finding demonstrated that the LDR probes
designed to target species-specific polymorphisms described
here and in previously published sequences 10 detected only
the species present and no others. Additionally, fluorescent
signals from the parasite and human samples were below a
Internal transcribed spacer 2 DNA ligation detection reaction primers for Papua New Guinea Anopheles species identification *
Species-specific primers † Primer sequence ‡ FlexMAP™ microsphere §
AF4 and AK Biotin
5′-tacaaatcatcaatcactttaatcGCG CCT GCC CCC AGM GA-3′
5′-Phos-TCT CGC GAC CCA CAT GCA C-3′-Biotin
5′-tcatcaatcaatctttttcacttt TCG CGC GCC AAC CCA TG-3′
5′-Phos-CAC ACT GCC GCG CAA CC/3′-Biotin
5′-ctttaatctcaatcaatacaaatcAGC GCG GGG CGK CCG T-3′
5′-Phos-GTT ACC GGC CCC TGC AC-3′-Biotin
5′-tcataatctcaacaatctttctttCGC ACG CGG CCT CGG CGG GAC TT-3′
5′-ctatctatctaactatctatatcaCTC TGT GTG GGA GGG AGT GCG TT-3′
5′-Phos-CGG TGC GTT YAC YCG ACT AC-3′-Biotin
* AF1 = An. farauti s.s; AH = An. hinesorum ; AP = An. punctulatus ; AF4 = An. farauti 4; AK = An. koliensis .
† Species-specific TAG and Biotin primers are based on GenBank accession nos. AF1 (AF055984, EF042696, and EF619441, HM584365–HM584379), AH (AF033213, HM584380–HM584391),
AP (AF033220, HM584428–HM584454), AF4 (AF033214, HM584392–HM584427), and AK (AF033219, HM584407–HM584426)
‡ Nucleotides in lower case (24 bases) represent TAG sequences added to the 5′ end of each species-specific ligase detection reaction primer. Single letter nucleotide codes: M = A or C; K = G
or T; Y = T or C.
§ Luminex ® microsphere sets are synthesized to exhibit unique fluorescence and each microsphere set is coupled to different anti-TAG sequences, which are complementary to the species-specific
TAG sequence primers.
HENRY-HALLDIN AND OTHERS
background of 900 MFI, demonstrating that the presence of
parasites in a human blood meal would not obscure Anopheles
Evaluation of morphological and molecular identification in
field survey collections. Vector surveys completed previously
in malaria and filariasis endemic communities have shown the
Anopheles mosquito populations to be primarily composed of
An. farauti s.s., An. hinesorum , An. farauti 4, An. koliensis , and
An. punctulatus . 3, 13, 17, 19 Given this background, among the 340
mosquitoes subjected to ITS2 LDR-FMA analysis, morpholog-
ical assessment identified 67 as An. punctulatus , 115 as An. kolien-
sis , and 158 as An. farauti s.l. Amplification of the ITS2 sequence
produced a PCR product of the expected size range (653–783
basepairs) from all 340 insects. ITS2 LDR-FMA observed mono-
specific species identification for each individual mosquito.
The overall comparison of morphology and molecular spe-
cies classification for the 340 mosquitoes sampled are summa-
rized in Table 3 . First, where morphology classifies mosquitoes
into only 3 categories ( An. punctulatus , An. koliensis , and An.
farauti s.l.), the ITS2-based multiplex assay performs species-
level classification for An. punctulatus , An. koliensis , and three
members of the Farauti complex ( An. farauti s.s., An. hineso-
rum , and An. farauti 4).
Comparison of morphology to molecular classification by
ITS2 LDR-FMA shows the following results ( Table 3 ). Of the
67 mosquitoes morphologically identified as An. punctula-
tus , the ITS2 assay identified 57 mosquitoes as An. punctula-
tus (85% concordance). Given the specificity of LDR-FMA
for hybridization to species-specific sequence polymorphisms,
results suggest that 15% of these mosquitoes should be re-
classified as An. koliensis (n = 10). Of the 115 mosquitoes
morphologically identified as An. koliensis , the ITS2 assay
identified 81 as An. koliensis (70% concordance); 34 mos-
quitoes were re-classified (30%) as An. punctulatus (n = 11),
An. farauti s.s. (n = 15), An. hinesorum (n = 3), and An. farauti
4 (n = 5). Of the 158 mosquitoes identified as An. farauti s.l.
by morphology, ITS2 LDR-FMA identified 121 as one of
three Farauti complex sibling species (26 An. farauti s.s., 49
An. hinesorum , and 46 An. farauti 4) (76.5% concordance);
37 mosquitoes were re-classified (23.5%) as An. punctulatus
(n = 33) and An. koliensis (n = 4). Overall concordance between
morphology and molecular identification was 76.2%. The most
common source of discordance (33 of 81 misclassified, 40.7%)
was observed for mosquitoes identified as An. farauti s.l. by
morphology and An. punctulatus by ITS2 LDR-FMA.
Results from comparison of ITS2 LDR-FMA probe clas-
sification to morphology are also shown in Table 3 . Of the
101 mosquitoes identified as An. punctulatus by ITS2 LDR-
FMA, 57 were morphologically identified as An. punctulatus
(56.4% concordance); 11 were identified as An. koliensis and
33 as An. farauti s.l. Ninety-five mosquitoes were identified as
An. koliensis by LDR-FMA, of these 81 had been morphologi-
cally identified as An. koliensis (85% concordance), 10 as An .
punctulatu s, and 4 as An. farauti s.l. Of the 41 mosquitoes iden-
tified as An. farauti s.s. by LDR-FMA, 26 were morphologi-
cally identified as An. farauti s.l. and 15 as An. koliensis . Of the
52 mosquitoes identified as An. hinesorum by LDR-FMA, 49
were morphologically identified as a member of An. farauti s.l.
and 3 as An. koliensis . Of the 51 mosquitoes identified as An.
farauti 4 by LDR-FMA, 46 were morphologically identified as
a member of An. farauti s.l. and 5 as An. koliensis .
Overall sensitivities and specificities of Anopheles spp. iden-
tification in Papua New Guinea are shown in Table 4 . Values
differ based on the direction of these comparisons (morphol-
ogy to molecular, left; molecular to morphology, right).
Comparisons between ITS2 RFLP and ITS2 LDR-FMA
techniques were made for a subset (n = 117) of the 340 mosqui-
toes. ITS2 RFLP species determination was completed by the
PNGIMR Entomology Unit with no prior knowledge of the
ITS2 LDR-FMA results. A 98.3% concordance was observed
between ITS2 RFLP and LDR-FMA species identification meth-
ods ( Table 5 ). Overall concordance between ITS2 RFLP and
morphology was 76%, which was similar to that of ITS2 LDR-
FMA and morphology. Coincidentally, ITS2 DNA sequence
was also obtained for 13 of the 117 mosquitoes evaluated
Figure 2. Detection of species-specific DNAs by the Anopheles
species multiplex polymerase chain reaction–ligase detection
reaction–fluorescent microsphere assay (PCR-LDR-FMA) for mos-
quitoes from Papua New Guinea. Data represent a summary of
12 individual PCR LDR-FMA detecting An. koliensis (AK), An.
farauti s.s. (AF), An. hinesorum (AH), An. farauti 4 (AF4), An. kolien-
sis (AK), and Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae
(Pm), P. ovale (Po), Wuchereria bancrofti (Wb), human (HBS) genomic
DNAs, and negative control (no DNA). Whereas genomic DNAs were
added individually into PCRs for each individual mosquito species,
LDR and FMA included oligonucleotide probes representing all
five Anopheles species. Numbers in parentheses next to species des-
ignations in the legend ((1), (11), (59), (68), (78)) identify Luminex
FlexMap™ microspheres (Luminex Corp., Austin, TX). No DNAs
indicates a blank sample to which no genomic DNAs were added.
Concordance assessment between morphology and internal tran-
scribed spacer 2 classification for Anopheles punctulatus sibling
species, Papua New Guinea *
LDR-FMA Species ID
AP AKAFAH AF4
* LDR-FMA = ligase detection reaction–fluorescent microsphere assay; ID = identi-
fication; AP = An. punctulatus s.s.; AK = An. koliensis ; AF = An. farauti s.s./ An. farauti 1;
AH = An. hinesorum/An. farauti 2; AF4 = An. farauti 4; AF s.l. = An .farauti s.l. (specific
species morphologically unidentifiable).
Sensitivity and specificity of Anopheles spp. identification, Papua New
* LDR-FMA = ligase detection reaction–fluorescent microsphere assay; AP = An. punctu-
latus s.s.; AK = An. koliensis ; AF = An. farauti s.s./ An. farauti 1.
AP AK AFAPAKAF
MULTIPLEX SPECIES IDENTIFICATION OF PNG MALARIA VECTORS
by LDR-FMA and RFLP. All 13 ( An. punctulatus = 5, An.
farauti s.l. = 2, and An. koliensis = 6) of these mosquitoes were
identified by ITS2 RFLP, LDR-FMA and reference sequence
comparison with 100% concordance.
DNA sequence analysis of the ITS2 region of the ribo-
somal RNA genes of An. punctulatus group sibling species
has identified numerous sequence polymorphisms (single
and multiple nucleotide polymorphisms; insertions and dele-
tions; 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, particu-
larly those within the Farauti complex. 10 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 incor-
porating 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 illus-
trating An . punctulatus and An . bancrofti group ITS2 sequence
polymorphisms within individual species, 8, 30 we wanted to
make multiple within and among species sequence compari-
sons before developing species-specific DNA probes for appli-
cation 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
sequences 8 [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 pre-
viously published sequences 10 for An. torresiensis [Genbank
accession no. AF033214], An. farauti 5 [AF033216], An. farauti
6 [AF033217], An. farauti 7 [AF033218], An. near punctu-
latus [AF033221]), further comparisons showed high con-
servation of ITS2 sequence (≥ 99.0% pairwise percent
identity) within species. These comparisons enabled identi-
fication 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 ( Figure 2 ) impli-
cated in transmitting malaria and filarial parasites in Papua
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 com-
plex into three species categories ( An. farauti s.l., An. kolien-
sis , and An. punctulatus ) it was clear that the ITS2 multiplex
assay would provide more definitive identification of individ-
ual 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 gen-
eralized 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 concor-
dant (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. kolien-
sis 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 molec-
ular 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% con-
cordant 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 others 4 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 ob -
served 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. kolien-
sis 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 spe-
cies was present, discordance was observed between DNA
probe and morphology, and in two sites where only one spe-
cies was identified by morphology, different species were iden-
tified by DNA probes. Because morphological classifications
and LDR-FMA probe hybridization discordance was high-
est 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
Concordance assessment of molecular internal transcribed spacer
2 classification methods (RFLP vs. LDR-FMA) for Anopheles
punctulatus sibling species, Papua New Guinea *
LDR-FMA Species ID
AP AKAF AHAF4
* RFLP = restriction fragment length polymorphism; LDR-FMA = ligase detection reac-
tion–fluorescent microsphere assay; ID = identification; AP = An. punctulatus s.s.; AK = An.
koliensis ; AF = An. farauti s.s./ An. farauti 1; AH = An. hinesorum/An. farauti 2; AF4 = An.
HENRY-HALLDIN AND OTHERS
LDR-FMA DNA probes and morphology was observed in col-
lection 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 poly-
morphisms in the primary disease vectors of the Punctulatus
group in Papua New Guinea. This method is based on tech-
nology that facilitates multiplex analysis, automation, and uni-
formity 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 con-
tinuously 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 spe-
cies within the Punctulatus group ( Table 5 ), the MspI RFLP
analysis will not identify strain-specific single nucleotide or
small insertion/deletion polymorphisms (this study and Beebe
and others 10 ) observed within species that appear to be asso-
ciated 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 , 23, 24 it is foresee-
able 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, bit-
ing 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 over-
all complexity of vector-borne disease transmission associated
with the Punctulatus group, it is critical to develop diagnos-
tic surveillance strategies that can monitor markers associated
with infection by efficient methods. In comparison with RFLP
analysis, the LDR-FMA multiplex strategy has greater capac-
ity 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 moni-
tor and evaluate operational control programs to reduce dis-
ease transmission by these important vectors.
Received August 4, 2010. Accepted for publication October 4, 2010.
Acknowledgments: We are grateful to the PNGIMR Entomology
Unit for aid in mosquito collections and morphological identification.
We dedicate this work to our colleague Henry Dagoro who passed
away on August 4, 2010. Over the past 30 years, he contributed care-
ful analysis and significant energy to the study of the An. punctulatus
group sibling species as a senior member of the PNGIMR Entomology
Laboratory. He patiently mentored numerous students and scientists
from around the world on the behaviors and local habitats character-
izing these important human disease vectors.
Financial support: This study was supported by grants from the
National Institutes of Health (AI065717) and the Fogarty International
Center (TW007872, TW007377, and TW007735). Mosquito collection
was also supported by the Global Fund to Fight Aids, Tuberculosis and
Malaria Round 3 malaria grant to Papua New Guinea.
Authors’ addresses: Cara Henry-Halldin, Allison M. Zimmerman, and
Peter A. Zimmerman, Center for Global Health and Diseases, Case
Western Reserve University School of Medicine, Wolstein Research
Building, Cleveland, OH, E-mails: email@example.com, firstname.lastname@example.org,
email@example.com. Lisa Reimer, Edward Thomsen, Gussy Koimbu, John
B. Keven, and Henry Dagoro (deceased), Papua New Guinea Institute
of Medical Research-Madang, Madang, Papua New Guinea, E-mails:
firstname.lastname@example.org, email@example.com, gkoimbu@gmail
.com, firstname.lastname@example.org. Manuel W. Hetzel, Ivo Mueller, and Peter
Siba, Papua New Guinea Institute of Medical Research-Goroka,
Goroka, Papua New Guinea, E-mails: email@example.com,
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