Avian malaria on Madagascar: bird hosts and putative vector mosquitoes of different Plasmodium lineages
Schmid et al. Parasites & Vectors
Avian malaria on Madagascar: bird hosts and putative vector mosquitoes of different Plasmodium lineages
Sandrine Schmid 2
Anke Dinkel 2
Ute Mackenstedt 2
Michaël Luciano Tantely 1
Fano José Randrianambinintsoa 0
Sébastien Boyer 1
Friederike Woog 3
0 Université de Reims , 9 Boulevard de la Paix, 51100 Reims , France
1 Institut Pasteur de Madagascar, Unité d'Entomologie Médicale , BP 1274 Avaradoha, Antananarivo 101 , Madagascar
2 Universität Hohenheim, Institut für Zoologie, FG Parasitologie , Emil-Wolff-Straße 34, 70593 Stuttgart , Germany
3 Staatliches Museum für Naturkunde , Ornithology, Rosenstein1, 70191 Stuttgart , Germany
Background: Avian malaria occurs almost worldwide and is caused by Haemosporida parasites (Plasmodium, Haemoproteus and Leucocytozoon). Vectors such as mosquitoes, hippoboscid flies or biting midges are required for the transmission of these parasites. There are few studies about avian malaria parasites on Madagascar but none about suitable vectors. Methods: To identify vectors of avian Plasmodium parasites on Madagascar, we examined head, thorax and abdomen of 418 mosquitoes from at least 18 species using a nested PCR method to amplify a 524 bp fragment of the haemosporidian mitochondrial cytochrome b gene. Sequences obtained were then compared with a large dataset of haemosporidian sequences detected in 45 different bird species (n = 686) from the same area in the Maromizaha rainforest. Results: Twenty-one mosquitoes tested positive for avian malaria parasites. Haemoproteus DNA was found in nine mosquitoes (2.15%) while Plasmodium DNA was found in 12 mosquitoes (2.87%). Seven distinct lineages were identified among the Plasmodium DNA samples. Some lineages were also found in the examined bird samples: Plasmodium sp. WA46 (EU810628.1) in the Madagascar bulbul, Plasmodium sp. mosquito 132 (AB308050.1) in 15 bird species belonging to eight families, Plasmodium sp. PV12 (GQ150194.1) in eleven bird species belonging to eight families and Plasmodium sp. P31 (DQ839060.1) was found in three weaver bird species. Conclusion: This study provides the first insight into avian malaria transmission in the Maromizaha rainforest in eastern Madagascar. Five Haemoproteus lineages and seven Plasmodium lineages were detected in the examined mosquitoes. Complete life-cycles for the specialist lineages WA46 and P31 and for the generalist lineages mosquito132 and PV12 of Plasmodium are proposed. In addition, we have identified for the first time Anopheles mascarensis and Uranotaenia spp. as vectors for avian malaria and offer the first description of vector mosquitoes for avian malaria in Madagascar.
Haemosporida; Uranotaenia; Anopheles mascarensis; Haemoproteus; Ploceidae; Pycnonotidae
The island of Madagascar is located approximately
400 km east of Africa in the Indian Ocean. Due to its
isolation from mainland India and Africa it has many
endemic species and is classified as an important
biodiversity hotspot . More than half of Madagascar′s
1Universität Hohenheim, Institut für Zoologie, FG Parasitologie,
Emil-Wolff-Straße 34, 70593 Stuttgart, Germany
Full list of author information is available at the end of the article
breeding birds are endemic. This makes Madagascar an
interesting area for the examination of the ecological and
evolutionary dynamics of vector-host-parasite associations
of avian malaria.
Avian malaria is caused by haemosporidian parasites
including the genera Plasmodium, Haemoproteus and
Leucocytozoon. In contrast to human malaria, avian malaria is
distributed almost worldwide and has been detected in a
wide range of species . The three genera have similar
life-cycles with differences during the asexual phase in the
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
peripheral blood of their host . The life-cycle involves
the sexual phase and sporogony both occurring in the
invertebrate host (vector) and the merogony and
development of gametocytes which takes place in avian hosts .
For natural transmission of Plasmodium to the vertebrate
host, the parasite undergoes a series of obligatory
developmental, propagative and migrational processes
inside a mosquito vector. These include zygote formation
and ookinete development in the midgut lumen, oocyst
formation and sporogony on the basal side of the
midgut, sporozoite migration through the haemocoel, and
invasion of the salivary glands . Sporozoites, the
infective stages, are then present in the salivary gland
(thoracic part) of a mosquito and detectable for several
weeks . Since the end of the 20th Century, a number
of field and laboratory experimental infection studies
were used to identify dipterans as vectors transmitting
haemosporidian parasites (e.g. [7, 8]). Recently, most
studies use molecular methods to identify vector feeding
preferences  and sporogonic stages of the parasites in
salivary glands to identify potential vectors .
To date, few studies exist on blood parasites of
Malagasy birds. Either blood samples were examined
microscopically [11, 12] or just a small number was analyzed
by PCR . So far, data about vectors in Madagascar
transmitting avian malaria parasites are lacking. To
identify mosquitoes as suitable vectors for Plasmodium
species, a demonstration of infective sporozoites in insects
is essential. Due to predominant light infections and low
parasite prevalence (often < 1%) in the majority of vector
populations, the applicability of insect dissection and
microscopic examination methods is limited in wildlife
. For parasite detection we therefore used a highly
sensitive nested PCR. Positive results allowed us to
determine significant links between mosquitoes and
The purpose of our study was to assess the presence
of avian malaria parasites in potential mosquito vectors
and avian hosts using molecular techniques and to
estimate infection rates among mosquito populations in the
Maromizaha rainforest located in eastern Madagascar
(Andasibe). In addition, Plasmodium spp. sequences
isolated from mosquitoes were compared to those found in
birds within the study area. A complete life-cycle with
putative vector, host and parasite is proposed.
Mosquitoes and birds were caught in the Maromizaha
rainforest located in the eastern part of Madagascar (18°
56′49″S, 48°27′33″E), 30 km from Moramanga city,
with an altitude above 943 m and a highest peak of
1,213 m. The area is protected and part of the
Mantadia-Maromizaha-Zahamena rainforest corridor, a
terrain that consists of hills with mountain ridges, valleys
and small streams, dense and humid evergreen forest
A total of 418 adult mosquitoes of at least 18 species
(Table 1) were collected using twelve CDC (Center for
Disease Control and Prevention) light traps  during
one week (November 17–21, 2014). The sampling period
was based on the knowledge of high mosquito diversity
and density in forested areas of the central highland at
the beginning of the rainy season (i.e. November) .
The traps were operated for 12 h periods from 6 pm to
6 am at each sampling point. To sample a representative
mosquito population, traps were distributed along three
sampling lines, thus, valley-floor, slope and ridge for
each biotope, with four traps in each sampling point.
Mosquitoes were anesthetized with chloroform vapor
and identified using the keys of Ravaonjanahary  for
Aedes, Grjebine  for Anopheles, Doucet  for
Coquillettidia, Edwards  for Culex, Brunhes & Hervy
 for Orthopodomyia and da Cunha Ramos  for
Uranotaenia. After drying, mosquitoes were stored in
ELISA plates to allow identification. DNA extractions
were performed separately from head, thorax and
abdomen of each mosquito in order to determine the infection
rates and diversity of parasite lineages within the thoracic
(including salivary glands) and abdominal parts, using the
Zymo Research extraction kit (Quick-gDNA™ MiniPrep;
Zymo Research Europe GmbH, Freiburg, Germany). The
isolated DNA was stored at -20 °C until used.
A total of 686 blood samples were collected from 45
bird species mist-netted in the Maromizaha rainforest in
the years 2006, 2007, 2010, 2012 and 2014. Sampling
was done by taking a drop of blood from the brachial
vein and stored in buffer . For DNA extraction we
used QIAamp DNA Blood Mini Kit (QIAGEN, Hilden,
Germany) following the manufacturer's instructions. DNA
was stored at -20 °C until further use.
To prevent contamination with target DNA, a negative
control was included in each test run as well as a
positive control to ensure PCR was working properly. Target
sequence for amplification was a part of the
haemosporidian mitochondrial cytochrome b gene. PCR was
conducted in two steps whereby the primer pair HAEMNF
and HAEMNR2  was used to amplify a 580 bp
fragment in the first PCR, and the internal primer pair
HAEMF and HAEMR2  used to amplify a 524 bp
fragment in the second PCR. The reaction mixture consisted
of 10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 20 pmol
of each primer, 200 μM of each dNTP, 1.25 units
AmpliTaq (Applied Biosystems, Carlsbad, USA) and for the first
PCR approximately 10–100 ng DNA in a total volume of
50 μl. For the second PCR 2 μl of the amplification
product was used as template. Both PCRs were performed for
40 cycles with each cycle consisting of denaturation at
94 °C for 30 s, annealing at 50 °C for 60 s and
Table 1 PCR results of examined mosquito species
Abbreviations: n number of different individuals screened using PCR, H/T positive head or thorax samples, A positive abdomen samples
* A new species in process of being described (Tantely, pers. Comm.)
elongation at 72 °C for 45 s. After amplification, 5 μl of
the PCR products stained with GelRed™ (BIOTREND,
Köln, Germany) were viewed on a 1.5% agarose gel.
Amplification products were then purified using the PCR
Product Purification Kit (Roche, Mannheim, Germany)
and after sequencing (GATC Biotech AG) the resulting
sequences were compared to sequences published in
A total of 418 mosquitoes were examined, representing at
least 18 species of seven genera (Table 1). Culex (46.9%)
and Uranotaenia (47.6%) were the two most frequent
mosquito genera collected in the study. Other mosquito
species found include those of the genera Aedes,
Anopheles, Coquillettidia, Lutzia and Orthopodomyia. Using the
two step PCR, we found 21 of the 418 mosquitoes (5.02%)
to be haemosporidian DNA-positive.
These positive samples were of the mosquito species
Anopheles mascarensis, Culex annulioris, C. pipiens,
Uranotaenia alboabdominalis, Uranotaenia neireti,
Uranotaenia n. sp. (Tantely, pers. Comm.) and Uranotaenia sp.
The haemosporidian DNA detected include five
Haemoproteus lineages, isolated from nine mosquitoes and seven
Plasmodium lineages from 12 mosquitoes (Table 2).
Only mosquitoes with positive thoracic parts are
considered to be putative vectors since infectious sporozoites are
exclusively present in the salivary glands of arthropod vectors.
The resulting haemosporidian DNA sequences of mosquitoes
with positive thoracic parts therefore include Plasmodium sp.
Uan1 (n = 1; from Uranotaenia n. sp.); Plasmodium sp.
WA46 (EU810628.1) (n = 3; from Uranotaenia sp., Ur. neireti
and Ur. alboabdominalis); Plasmodium sp. Ual1 (n = 1; from
Ur. alboabdominalis); Plasmodium sp. Cp1 (n = 1; from Culex
pipiens); Plasmodium sp. mosquito132 (AB308050.1) (n = 2;
from Culex pipiens, Uranotaenia n. sp.); Plasmodium sp.
PV12 (GQ150194.1) (n = 1; from Anopheles mascarensis);
and Plasmodium sp. P31 (DQ659572.1) (n = 1; from
Uranotaenia n. sp.). The sequences were compared to those found
in bird blood samples collected in the years 2006, 2007, 2010,
2012 and 2014.
The Plasmodium sp. mosquito132 lineage was found in
51 bird blood samples from 15 bird species (Foudia omissa,
Xanthomixis cinereiceps, Nesillas typica, Hypsipetes
madagascariensis, Copsychus albospecularis, Zosterops
maderaspatanus, Monticola sharpei, Ploceus nelicourvi, Foudia
madagascariensis, Xanthomixis zosterops, Saxicola
torquatus, Tylas eduardi, Philepitta castanea and Pseudobias
wardi) belonging to eight bird families (Acrocephalidae,
Bernieridae, Muscicapidae, Philepittidae, Ploceidae,
Pycnonotidae, Vangidae and Zosteropidae).
Plasmodium sp. P31 was also found in 39 blood
samples of weaver birds (Ploceidae), Foudia omissa (n = 35),
Foudia madagascariensis (n = 3) and Ploceus nelicourvi
Table 2 Isolated parasite lineages of this study
Homology (%) [GenBank ID]
Culex pipiens (n = 3); Culex annulioris
Uranotaenia n. sp. (n = 2)
Uranotaenia alboabdominalis (T)
Uranotaenia n. sp. (T)
Culex pipiens (T); Uranotaenia n. sp. (T + A)
Anopheles mascarensis (T)
Uranotaenia n. sp. (T)
Nesillas typica (n = 1)
51 birds of 15 species
37 birds of 11 species
39 birds of the family Ploceidae
Abbreviations: T thoracic part, A abdominal part
(n = 1). The lineage Plasmodium sp. PV12 was found in
37 blood samples of 11 bird species (Foudia omissa,
Saxicola torquatus, Philepitta castanea, Ploceus nelicourvi,
Nesillas typica, Bernieria madagascariensis, Hypsipetes
madagascariensis, Phedina borbonica, Foudia
madagascariensis, Copsychus albospecularis, Motacilla
flaviventris) belonging to eight bird families (Acrocephalidae,
Bernieridae, Hirundinidae, Motacillidae, Muscicapidae,
Philepittidae, Ploceidae and Pycnonotidae).
Plasmodium sp. WA46 was as well found in five
Madagascar Bulbul (Hypsipetes madagascariensis,
Pycnonotidae) blood samples.
Blood-sucking arthropods are necessary for the life-cycle
of avian malaria parasites. They act as definitive hosts
and vectors of avian malaria parasites and are thus
responsible for the transmission. Identification of such vector
arthropods is therefore essential to unravel the transmission
cycles of vector borne diseases like avian malaria . For
many parasite species of the genera Plasmodium,
Haemoproteus or Leucocytozoon suitable arthropod vectors have
already been identified . Mosquitoes are the only
known vectors for Plasmodium species.
To identify vectors of avian malaria on Madagascar,
we examined 418 mosquitoes from at least 18 species
individually using a part of the mitochondrial
cytochrome b gene. Sequences found in the mosquitoes
were compared to a large dataset of sequences isolated
from 45 bird species (n = 686) of the same area.
Twentyone mosquitoes were found to contain DNA of avian
We found Haemoproteus DNA in nine mosquitoes.
Haemoproteus species develop oocysts in the head,
thorax and midgut wall of mosquitoes but undergo
abortive sporogonic development . Therefore, mosquitoes
are not competent vectors for this parasite.
Haemosporidian parasites can be used to determine links between
blood-sucking insects and their blood-source animals .
All identified Haemoproteus lineages in the present study
were similar or highly homologous to sequences
previously isolated from birds. This evidence therefore
implicates that Uranotaenia alboabdominalis,
Uranotaenia n. sp., Culex annulioris and Culex pipiens
feed on birds in the study area and could be possible
vectors for Plasmodium species.
Seven Plasmodium lineages were detected in 12
mosquito species. Of all examined mosquito species in this
study containing Plasmodium DNA, only Culex pipiens
has been previously known to be a suitable vector for
avian Plasmodium species . Culex pipiens was the
most abundant mosquito in our study (39%) followed by
the newly-described species Uranotaenia n. sp. (22.7%).
We detected DNA of Plasmodium only in three Culex
pipiens samples (2× thorax, 1× abdomen), indicating that
this mosquito species may not play the most important
role for avian Plasmodium transmission in the study
The endemic mosquito species Anopheles mascarensis
is reported to act as a vector of Plasmodium falciparum,
the causative agent of human malaria . We found an
avian Plasmodium lineage in the thorax of one sampled
Anopheles mascarensis indicating that this endemic
mosquito species plays an important role for both, human
and avian malaria transmission.
Ten of the 12 (83.3%) Plasmodium lineages found
were isolated from mosquitoes belonging to the genus
Uranotaenia. This is the first report of Uranotaenia
species acting as vectors for avian malaria. Although
further studies are needed, the genus Uranotaenia might
even be the major vector for avian malaria in this area.
The isolated Plasmodium lineages were compared to
previously published sequences in GenBank and thus
identified. We isolated the sequence Plasmodium sp.
Ual1 from the thorax of one Uranotaenia
alboabdominalis. The sequence was 95% homologous to the previously
described lineage Plasmodium minuoviride haplotype
CCA0640 (EU834703.1). This lineage was isolated by
Perkins et al.  from Prasinohaema prehensicauda, a skink
(Squamata) endemic to Papua New Guinea. Because
species of the genus Uranotaenia are known to feed on
cold-blooded animals  and due to the high homology
we conclude that the sequence we found also stems from
a Plasmodium species infecting reptiles. The lineages
Plasmodium sp. Uan1 (isolated from thorax of Uranotaenia n.
sp.) and Plasmodium sp. Cp1 (isolated from thorax of
Culex pipiens) were highly homologous to sequences
previously isolated from birds. Therefore, we conclude that
these sequences represent two new Plasmodium lineages
infecting birds. The other four sequences isolated were
identical with lineages previously described and were also
found in bird blood samples examined during this study.
The presence of the identical haemosporidian cytochrome
b sequence in birds and mosquitoes could be an
indication of a vector-host relationship between the species
We isolated the lineage Plasmodium sp. WA46 
from four mosquitoes belonging to the genus
Uranotaenia. The parasite sequence was found in the thoracic
parts of Uranotaenia alboabdominalis, Ur. neireti and in
one undescribed Uranotaenia species. We conclude that
all three mosquito species are probably suitable vectors
for the Plasmodium lineage WA46. The very same lineage
was also found in five bird blood samples of the
Madagascar bulbul (Hypsipetes madagascariensis,
Pycnonotidae). The blood was sampled from different
individuals in the years 2007, 2010, 2012 and 2014 suggesting a
stable occurrence in the study area. The lineage
Plasmodium sp. WA46 was originally isolated by Beadell et al.
 from the common bulbul (Pycnonotus barbatus,
Pycnonotidae). Our findings support the hypothesis, that the
lineage WA46 is a specialist for the bird family
Pycnonotidae. As Hypsipetes madagascariensis is the only bulbul
species on Madagascar we assume that the life-cycle of
Plasmodium sp. WA46 in our study area includes at least
three vector mosquito species of the genus Uranotaenia
and the host bird Hypsipetes madagascariensis.
The lineage Plasmodium sp. mosquito132  was
isolated from the thoracic parts of Culex pipiens and
Uranotaenia n. sp. in this study. Ejiri et al.  originally
isolated the lineage from Culex pipiens quinquefasciatus
captured in Japan. We supported the role of Culex
pipiens as vector for this lineage also on Madagascar
but with Uranotaenia n. sp. we found a second vector
mosquito. We isolated DNA of Plasmodium sp.
mosquito132 also from 51 bird blood samples. The samples
stem from individuals of 15 bird species belonging to
eight bird families. The finding of the lineage in so
many diverse bird species leads us to the conclusion
that Plasmodium sp. mosquito132 is a generalist.
From the endemic mosquito Anopheles mascarensis
we isolated the lineage Plasmodium sp. PV12 . The
lineage was originally isolated from the mosquito
Coquillettidia aurites in Cameroon. We did not find the
lineage in the four samples of Coquillettidia grandidieri
examined in this study. Because of the small sample size
we cannot exclude this mosquito species as a possible
vector. Our findings suggest that Anopheles mascarensis
is a vector of Plasmodium sp. PV12 in the study area.
We also detected this lineage in 37 bird blood samples.
The samples are from eleven 11 bird species belonging
to eight families. The finding of the lineage in so many
diverse bird species leads us to the conclusion that
Plasmodium sp. PV12 is a generalist.
The Plasmodium lineage isolated from Uranotaenia
n. sp. was identical to the previously described
Plasmodium sp. P31 . The lineage was originally isolated
from Foudia madagascariensis (Ploceidae) captured on
Madagascar. In the current study the same sequence
was isolated from 39 blood samples of three weaver bird
species, including Foudia omissa, Foudia
madagascariensis and Ploceus nelicourvi. Therefore, Plasmodium sp. P31
seems to be specific to weaver birds (Ploceidae). Fodies
were among the most frequently sampled birds. From
observational studies they are known to move from the
forest to the open, more degraded areas, and may therefore
spread blood parasites into more pristine areas. The
weaver bird lineage was detected in 39 samples from three
different years (2006, 2007, 2010, 2012 and 2014). The
high and apparently stable prevalence in the birds suggests
that there is a stable life-cycle of Plasmodium sp. P31 on
Madagascar with its intermediate host being weaver birds
and Uranotaenia n. sp. as the first described vector.
To confirm the role of the genus Uranotaenia,
Anopheles mascarensis and Culex pipiens in the
transmission cycle of the reported avian malaria parasites on
Madagascar, further analyses of blood-fed mosquitoes
from infection experiments are required.
This study provides the first insight into avian malaria
transmission in the Maromizaha forest in eastern
Madagascar. Five Haemoproteus lineages and seven
Plasmodium lineages were detected in the examined
mosquitoes. Complete life-cycles for the specialist
Plasmodium lineages WA46 and P31 and for the generalist
Plasmodium lineages mosquito132 and PV12 are
proposed. Plasmodium sp. WA46 is transmitted from
mosquitoes of the genus Uranotaenia to birds belonging to
the family Pycnonotidae. Plasmodium sp. P31 is
transmitted from Uranotaenia n. sp. to birds belonging to
the family Ploceidae. Plasmodium sp. mosquito132 is
transmitted from Culex pipiens and Uranotaenia n. sp.
to at least 15 different bird species on Madagascar.
Plasmodium sp. PV12 is transmitted from Anopheles
mascarensis to at least 11 different bird species on
This study identified Anopheles mascarensis and
Uranotaenia spp. as putative vectors for avian malaria for
the first time and offers the first description of vector
mosquitoes for avian malaria on Madagascar.
We are indebted to the Malagasy authorities for granting all the relevant
research and export permits and to GERP (Grouped'Etude et de Recherche
sur les Primates de Madagascar), namely Jonah Ratsimbazafy and Rose Marie
Randrianarison, for letting us work at Maromizaha. The work would not have
been possible without the continued support over many years by Haja
Rakotomanana and Daniel Rakotondravony from the University of Antananarivo,
Department of Animal Biology. We thank Nicola Lillich, Jean-Robert Lekamisi and
all the other Malagasy assistants for their help in the field. We would like to thank
all field assistants, particulary Pia Reufsteck, and also Sonja Dumendiak and Katrin
Fachet for their help in the laboratory.
Availability of data and materials
Data generated or analysed during this study are partially included in this
published article. The whole datasets generated during and/or analysed during
the current study are not publicly available due to the use in further publications
but are available from the corresponding author on reasonable request.
SS carried out the molecular analysis and drafted the manuscript. AD and
UM participated in the coordination of the study and helped drafting the
manuscript. AD also carried out molecular analysis. SB facilitated the field
study that was carried out by MLT and FJR involving capture and morphological
identification of the mosquitoes in the field. FW had the idea for the study,
participated in its design and coordination and helped to write the manuscript.
All authors read and approved the final version of the manuscript.
Consent for publication
1. Myers N , Mittermeier R , Mittermeier CG , da Fonseca G , Kent J. Biodiversity hotspots for conservation priorities . Nature . 2000 ; 403 : 853 - 8 .
2. Atkinson CT , Van Riper C. Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon and Haemoproteus . In: Bird-parasite interactions: ecology, evolution, and behavior. London: Oxford University Press ; 1991 . p. 19 - 48 .
3. Valkiunas G . Avian malaria parasites and other Haemosporidia . Boca Raton: CRC Press ; 2005 .
4. Ishtiaq F , Guillaumot L , Clegg SM , Phillimore AB , Black RA , Owens IPF , et al. Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands . Mol Ecol . 2008 ; 17 ( 20 ): 4545 - 55 .
5. Hillyer JF , Barreau C , Vernick KD . Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel . Int J Parasitol . 2007 ; 37 : 673 - 81 .
6. Valkiunas G , Kazlauskiene R , Bernotiene R , Palinauskas V , Iezhova TA . Abortive long-lasting sporogony of two Haemoproteus species (Haemosporida, Haemoproteidae) in the mosquito Ochlerotatus cantans, with perspectives on haemosporidian vector research . Parasitol Res . 2013 ; 112 ( 6 ): 2159 - 69 .
7. Atkinson T , Greiner EC , Forrester J. Wild vectors in Florida of Haemoproteus meleagridis Levine from turkeys . J Wildl Dis . 1983 ; 19 : 366 - 8 .
8. Huff CG . Susceptibility of mosquitoes to avian malaria . Exp Parasitol . 1965 ; 16 : 107 - 32 .
9. Imura T , Sato Y , Ejiri H , Tamada A , Isawa H , Sawabe K , et al. Molecular identification of blood source animals from black flies (Diptera: Simuliidae) collected in the alpine regions of Japan . Parasitol Res . 2010 ; 106 ( 2 ): 543 - 7 .
10. Valkiūnas G , Santiago-Alarcon D , Levin II , Iezhova TA , Parker PG. A new Haemoproteus species (Haemosporida: Haemoproteidae) from the endemic Galapagos dove Zenaida galapagoensis, with remarks on the parasite distribution, vectors, and molecular diagnostics . J Parasitol . 2010 ; 96 ( 4 ): 783 - 92 .
11. Savage AF , Robert V , Goodman SM , Raharimanga V , Raherilalao MJ , Andrianarimisa A , et al. Blood parasites in birds from Madagascar . J Wildl Dis . 2009 ; 45 ( 4 ): 907 - 20 .
12. Barraclough R , Robert V , Peirce M. New species of haematozoa from the avian families Campephagidae and Apodidae . Parasite. 2008 ; 15 : 105 - 10 .
13. Ishtiaq F , Beadell JS , Warren BH , Fleischer RC . Diversity and distribution of avian haematozoan parasites in the western Indian Ocean region: a molecular survey . Parasitology . 2012 ; 139 ( 2 ): 221 - 31 .
14. Boyer S , Tantely ML , Randriamaherijaona S , Andrianaivolambo L , Cardinale E. Mosquitoes sampling strategy for studying West Nile virus vectors in Madagascar: abundance, distribution and methods of catching in high risk areas . Arch Inst Pasteur Madagascar . 2014 ; 71 ( 1 ): 1 - 8 .
15. Tantely M , Rakotoniaina J , Andrianaivolambo L , Tata E , Razafindrasata F , Fontenille D , Elissa N. Biology of mosquitoes that are potential vectors of Rift Valley fever virus in different biotopes of the central highlands of Madagascar . J Med Entomol . 2013 ; 50 : 603 - 10 .
16. Ravaonjanahary C. Les Aedes de Madagascar (Diptera-Culicidae) . Paris: O.R.S.T.O.M; 1978 .
17. Grjébine A. Insectes Diptères Culicidae Anophelinae . Paris: O.R.S.T.O.M; 1966 .
18. Doucet J. Étude des Culicidae de la région de Vangaindrano (Diptera) . Paris: Mémoires de l'Institut Scientifique de Madagascar; 1951 .
19. Edwards FW . Mosquitoes of the Ethiopian Region. III.- Culicine adults and pupae . London: British Museum (Natural History) ; 1941 .
20. Brunhes J , Hervy J. Insectes Diptères Culicidae Culicinae Genre Orthopodomyia de la sous-région malgacheet de la région afrotropicale . Paris: Muséum National d'Histoire Naturelle ; 1995 .
21. Da Cunha Ramos H, Brunhes J. Insecta , Diptera, Culicidae, Uranotaenia. Paris: IRD-CIRAD ; 2004 .
22. Wink M. Use of DNA markers to study bird migration . J Ornithol . 2006 ; 147 : 234 - 44 .
23. Waldenström AJ , Bensch S , Hasselquist D , Östman Ö . A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood . J Parasitol . 2004 ; 90 ( 1 ): 191 - 4 .
24. Bensch S , Stjernman M , Hasselquist D , Orjan O , Hannson B , Westerdahl H , Pinheiro RT. Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds . Proc Biol Sci . 2000 ; 267 ( 1452 ): 1583 - 9 .
25. Benson DA , Cavanaugh M , Clark K , Karsch-Mizrachi I , Lipman DJ , Ostell J , Sayers EW . GenBank . Nucleic Acids Res . 2013 ; 41 : 36 - 42 .
26. Ejiri H , Sato Y , Sasaki E , Sumiyama D , Tsuda Y , Sawabe K , et al. Detection of avian Plasmodium spp . DNA sequences from mosquitoes captured in Minami Daito Island of Japan . J Vet Med Sci . 2008 ; 70 : 1205 - 10 .
27. Santiago-Alarcon D , Palinauskas V , Schaefer HM . Diptera vectors of avian haemosporidian parasites: Untangling parasite life cycles and their taxonomy . Biol Rev . 2012 ; 87 : 928 - 64 .
28. Fontenille D , Campbell G . Is Anopheles mascarensis a new malaria vector in Madagascar? Am J Trop Med Hyg . 1992 ; 46 : 28 - 30 .
29. Perkins SL , Austin CC . Four new species of Plasmodium from New Guinea Lizards: Integrating morphology and molecules . J Parasitol . 2009 ; 95 ( 2 ): 424 - 33 .
30. Beadell JS , Covas R , Gebhard C , Ishtiaq F , Melo M , Schmidt BK , et al. Host associations and evolutionary relationships of avian blood parasites from West Africa . Int J Parasitol . 2009 ; 39 ( 2 ): 257 - 66 .
31. Njabo KY , Cornel AJ , Sehgal RN , Loiseau C , Buermann W , Harrigan RJ , et al. Coquillettidia (Culicidae, Diptera) mosquitoes are natural vectors of avian malaria in Africa . Malar J . 2009 . doi:10.1186/ 1475 - 2875 - 8 - 193 .
32. Beadell JS , Ishtiaq F , Covas R , Melo M , Warren BH , Atkinson CT , et al. Global phylogeographic limits of Hawaii's avian malaria . Proc Biol Sci . 2006 ; 273 ( 1604 ): 2935 - 44 .