Schmid et al.

Parasites & Vectors (2017) 10:6

DOI 10.1186/s13071-016-1939-x

RESEARCH Open Access

Avian malaria on Madagascar: bird hosts

and putative vector mosquitoes of different
Plasmodium lineages
Sandrine Schmid1*, Anke Dinkel1, Ute Mackenstedt1, Michaël Luciano Tantely2, Fano José Randrianambinintsoa3,
Sébastien Boyer2 and Friederike Woog4

Abstract
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.
Keywords: Haemosporida, Uranotaenia, Anopheles mascarensis, Haemoproteus, Ploceidae, Pycnonotidae

Background breeding birds are endemic. This makes Madagascar an

The island of Madagascar is located approximately interesting area for the examination of the ecological and
400 km east of Africa in the Indian Ocean. Due to its evolutionary dynamics of vector-host-parasite associations
isolation from mainland India and Africa it has many of avian malaria.
endemic species and is classified as an important bio- Avian malaria is caused by haemosporidian parasites in-
diversity hotspot [1]. More than half of Madagascar′s cluding the genera Plasmodium, Haemoproteus and Leu-
cocytozoon. In contrast to human malaria, avian malaria is
* Correspondence: Sandrine.Schmid@uni-hohenheim.de
distributed almost worldwide and has been detected in a
1
Universität Hohenheim, Institut für Zoologie, FG Parasitologie, wide range of species [2]. The three genera have similar
Emil-Wolff-Straße 34, 70593 Stuttgart, Germany life-cycles with differences during the asexual phase in the
Full list of author information is available at the end of the article

© 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.
Schmid et al. Parasites & Vectors (2017) 10:6 Page 2 of 7

peripheral blood of their host [3]. The life-cycle involves and small streams, dense and humid evergreen forest
the sexual phase and sporogony both occurring in the in- covers.
vertebrate host (vector) and the merogony and develop- A total of 418 adult mosquitoes of at least 18 species
ment of gametocytes which takes place in avian hosts [4]. (Table 1) were collected using twelve CDC (Center for
For natural transmission of Plasmodium to the vertebrate Disease Control and Prevention) light traps [14] during
host, the parasite undergoes a series of obligatory de- one week (November 17–21, 2014). The sampling period
velopmental, propagative and migrational processes in- was based on the knowledge of high mosquito diversity
side a mosquito vector. These include zygote formation and density in forested areas of the central highland at
and ookinete development in the midgut lumen, oocyst the beginning of the rainy season (i.e. November) [15].
formation and sporogony on the basal side of the mid- The traps were operated for 12 h periods from 6 pm to
gut, sporozoite migration through the haemocoel, and 6 am at each sampling point. To sample a representative
invasion of the salivary glands [5]. Sporozoites, the in- mosquito population, traps were distributed along three
fective stages, are then present in the salivary gland sampling lines, thus, valley-floor, slope and ridge for
(thoracic part) of a mosquito and detectable for several each biotope, with four traps in each sampling point.
weeks [6]. Since the end of the 20th Century, a number Mosquitoes were anesthetized with chloroform vapor
of field and laboratory experimental infection studies and identified using the keys of Ravaonjanahary [16] for
were used to identify dipterans as vectors transmitting Aedes, Grjebine [17] for Anopheles, Doucet [18] for
haemosporidian parasites (e.g. [7, 8]). Recently, most Coquillettidia, Edwards [19] for Culex, Brunhes & Hervy
studies use molecular methods to identify vector feeding [20] for Orthopodomyia and da Cunha Ramos [21] for
preferences [9] and sporogonic stages of the parasites in Uranotaenia. After drying, mosquitoes were stored in
salivary glands to identify potential vectors [10]. ELISA plates to allow identification. DNA extractions
To date, few studies exist on blood parasites of Mala- were performed separately from head, thorax and abdo-
gasy birds. Either blood samples were examined micro- men of each mosquito in order to determine the infection
scopically [11, 12] or just a small number was analyzed rates and diversity of parasite lineages within the thoracic
by PCR [13]. So far, data about vectors in Madagascar (including salivary glands) and abdominal parts, using the
transmitting avian malaria parasites are lacking. To iden- Zymo Research extraction kit (Quick-gDNA™ MiniPrep;
tify mosquitoes as suitable vectors for Plasmodium spe- Zymo Research Europe GmbH, Freiburg, Germany). The
cies, a demonstration of infective sporozoites in insects isolated DNA was stored at -20 °C until used.
is essential. Due to predominant light infections and low A total of 686 blood samples were collected from 45
parasite prevalence (often < 1%) in the majority of vector bird species mist-netted in the Maromizaha rainforest in
populations, the applicability of insect dissection and the years 2006, 2007, 2010, 2012 and 2014. Sampling
microscopic examination methods is limited in wildlife was done by taking a drop of blood from the brachial
[6]. For parasite detection we therefore used a highly vein and stored in buffer [22]. For DNA extraction we
sensitive nested PCR. Positive results allowed us to de- used QIAamp DNA Blood Mini Kit (QIAGEN, Hilden,
termine significant links between mosquitoes and hae- Germany) following the manufacturer's instructions. DNA
mosporidian species. was stored at -20 °C until further use.
The purpose of our study was to assess the presence To prevent contamination with target DNA, a negative
of avian malaria parasites in potential mosquito vectors control was included in each test run as well as a posi-
and avian hosts using molecular techniques and to esti- tive control to ensure PCR was working properly. Target
mate infection rates among mosquito populations in the sequence for amplification was a part of the haemospo-
Maromizaha rainforest located in eastern Madagascar ridian mitochondrial cytochrome b gene. PCR was con-
(Andasibe). In addition, Plasmodium spp. sequences iso- ducted in two steps whereby the primer pair HAEMNF
lated from mosquitoes were compared to those found in and HAEMNR2 [23] was used to amplify a 580 bp frag-
birds within the study area. A complete life-cycle with ment in the first PCR, and the internal primer pair
putative vector, host and parasite is proposed. HAEMF and HAEMR2 [24] used to amplify a 524 bp frag-
ment in the second PCR. The reaction mixture consisted
Methods of 10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 20 pmol
Mosquitoes and birds were caught in the Maromizaha of each primer, 200 μM of each dNTP, 1.25 units Ampli-
rainforest located in the eastern part of Madagascar (18° Taq (Applied Biosystems, Carlsbad, USA) and for the first
56′49″S, 48°27′33″E), 30 km from Moramanga city, PCR approximately 10–100 ng DNA in a total volume of
with an altitude above 943 m and a highest peak of 50 μl. For the second PCR 2 μl of the amplification prod-
1,213 m. The area is protected and part of the uct was used as template. Both PCRs were performed for
Mantadia-Maromizaha-Zahamena rainforest corridor, a 40 cycles with each cycle consisting of denaturation at
terrain that consists of hills with mountain ridges, valleys 94 °C for 30 s, annealing at 50 °C for 60 s and
Schmid et al. Parasites & Vectors (2017) 10:6 Page 3 of 7

Table 1 PCR results of examined mosquito species

Genus Species n H/T A Plasmodium Haemoproteus
Aedes circumluteolus 9
Anopheles lacani 1
mascarensis 2 1 1
gambiae (s.l.) 3
Coquillettidia grandidieri 4
Culex annulioris 5 1 1
antennatus 3
decens 11
giganteus 8
pipiens 164 3 3 2 4
sp. 5
Lutzia tigripes 3
Orthopodomyia milloti 1
Uranotaenia alboabdominalis 45 3 2 1
anopheloides 4
n. sp.* 96 5 2 4 3
neireti 6 1 1
sp. 48 1 1 2
418 14 7 12 (2.87%) 9 (2.15%)
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 exclusively present in the salivary glands of arthropod vectors.
the PCR products stained with GelRed™ (BIOTREND, The resulting haemosporidian DNA sequences of mosquitoes
Köln, Germany) were viewed on a 1.5% agarose gel. with positive thoracic parts therefore include Plasmodium sp.
Amplification products were then purified using the PCR Uan1 (n = 1; from Uranotaenia n. sp.); Plasmodium sp.
Product Purification Kit (Roche, Mannheim, Germany) WA46 (EU810628.1) (n = 3; from Uranotaenia sp., Ur. neireti
and after sequencing (GATC Biotech AG) the resulting and Ur. alboabdominalis); Plasmodium sp. Ual1 (n = 1; from
sequences were compared to sequences published in Ur. alboabdominalis); Plasmodium sp. Cp1 (n = 1; from Culex
GenBank [25]. pipiens); Plasmodium sp. mosquito132 (AB308050.1) (n = 2;
from Culex pipiens, Uranotaenia n. sp.); Plasmodium sp.
Results PV12 (GQ150194.1) (n = 1; from Anopheles mascarensis);
A total of 418 mosquitoes were examined, representing at and Plasmodium sp. P31 (DQ659572.1) (n = 1; from Urano-
least 18 species of seven genera (Table 1). Culex (46.9%) taenia n. sp.). The sequences were compared to those found
and Uranotaenia (47.6%) were the two most frequent in bird blood samples collected in the years 2006, 2007, 2010,
mosquito genera collected in the study. Other mosquito 2012 and 2014.
species found include those of the genera Aedes, Anoph- The Plasmodium sp. mosquito132 lineage was found in
eles, Coquillettidia, Lutzia and Orthopodomyia. Using the 51 bird blood samples from 15 bird species (Foudia omissa,
two step PCR, we found 21 of the 418 mosquitoes (5.02%) Xanthomixis cinereiceps, Nesillas typica, Hypsipetes mada-
to be haemosporidian DNA-positive. gascariensis, Copsychus albospecularis, Zosterops maderas-
These positive samples were of the mosquito species patanus, Monticola sharpei, Ploceus nelicourvi, Foudia
Anopheles mascarensis, Culex annulioris, C. pipiens, Ura- madagascariensis, Xanthomixis zosterops, Saxicola torqua-
notaenia alboabdominalis, Uranotaenia neireti, Uranotae- tus, Tylas eduardi, Philepitta castanea and Pseudobias
nia n. sp. (Tantely, pers. Comm.) and Uranotaenia sp. wardi) belonging to eight bird families (Acrocephalidae,
The haemosporidian DNA detected include five Haemo- Bernieridae, Muscicapidae, Philepittidae, Ploceidae, Pycno-
proteus lineages, isolated from nine mosquitoes and seven notidae, Vangidae and Zosteropidae).
Plasmodium lineages from 12 mosquitoes (Table 2). Plasmodium sp. P31 was also found in 39 blood sam-
Only mosquitoes with positive thoracic parts are consid- ples of weaver birds (Ploceidae), Foudia omissa (n = 35),
ered to be putative vectors since infectious sporozoites are Foudia madagascariensis (n = 3) and Ploceus nelicourvi
Schmid et al. Parasites & Vectors (2017) 10:6 Page 4 of 7

Table 2 Isolated parasite lineages of this study

Parasite lineage Homology (%) [GenBank ID] Mosquito vector Bird host species
Haemoproteus spp.
Ual1 Uranotaenia alboabdominalis
Cp1 99 [AY099042.1] Culex pipiens
Cp2 99 [AY099042.1] Culex pipiens (n = 3); Culex annulioris
Uan1 98 [AY099040.1] Uranotaenia n. sp. Nesillas typica (n = 1)
Haplotype 1252 100 [KJ488706.1] Uranotaenia n. sp. (n = 2)
Plasmodium spp.
Ual1 95 [EU834703.1] Uranotaenia alboabdominalis (T)
Uan1 97 [JN819332.1] Uranotaenia n. sp. (T)
Cp1 99 [AB308050.1] Culex pipiens (T)
WA46 100 [EU810628.1] Uranotaenia sp. (T + A); Ur. alboabdominalis (T); Ur. neireti (T) Hypsipetes madagascariensis (n = 5)
Mosquito 132 100 [AB308050.1] Culex pipiens (T); Uranotaenia n. sp. (T + A) 51 birds of 15 species
PV12 100 [GQ150194.1] Anopheles mascarensis (T) 37 birds of 11 species
P31 100 [DQ839060.1] Uranotaenia n. sp. (T) 39 birds of the family Ploceidae
Abbreviations: T thoracic part, A abdominal part

(n = 1). The lineage Plasmodium sp. PV12 was found in thorax and midgut wall of mosquitoes but undergo abort-
37 blood samples of 11 bird species (Foudia omissa, Sax- ive sporogonic development [6]. Therefore, mosquitoes
icola torquatus, Philepitta castanea, Ploceus nelicourvi, are not competent vectors for this parasite. Haemosporid-
Nesillas typica, Bernieria madagascariensis, Hypsipetes ian parasites can be used to determine links between
madagascariensis, Phedina borbonica, Foudia madagas- blood-sucking insects and their blood-source animals [6].
cariensis, Copsychus albospecularis, Motacilla flaviven- All identified Haemoproteus lineages in the present study
tris) belonging to eight bird families (Acrocephalidae, were similar or highly homologous to sequences previ-
Bernieridae, Hirundinidae, Motacillidae, Muscicapidae, ously isolated from birds. This evidence therefore im-
Philepittidae, Ploceidae and Pycnonotidae). plicates that Uranotaenia alboabdominalis,
Plasmodium sp. WA46 was as well found in five Uranotaenia n. sp., Culex annulioris and Culex pipiens
Madagascar Bulbul (Hypsipetes madagascariensis, Pyc- feed on birds in the study area and could be possible
nonotidae) blood samples. vectors for Plasmodium species.
Seven Plasmodium lineages were detected in 12 mos-
Discussion quito species. Of all examined mosquito species in this
Blood-sucking arthropods are necessary for the life-cycle study containing Plasmodium DNA, only Culex pipiens
of avian malaria parasites. They act as definitive hosts has been previously known to be a suitable vector for
and vectors of avian malaria parasites and are thus respon- avian Plasmodium species [27]. Culex pipiens was the
sible for the transmission. Identification of such vector ar- most abundant mosquito in our study (39%) followed by
thropods is therefore essential to unravel the transmission the newly-described species Uranotaenia n. sp. (22.7%).
cycles of vector borne diseases like avian malaria [26]. For We detected DNA of Plasmodium only in three Culex
many parasite species of the genera Plasmodium, Haemo- pipiens samples (2× thorax, 1× abdomen), indicating that
proteus or Leucocytozoon suitable arthropod vectors have this mosquito species may not play the most important
already been identified [27]. Mosquitoes are the only role for avian Plasmodium transmission in the study
known vectors for Plasmodium species. area.
To identify vectors of avian malaria on Madagascar, The endemic mosquito species Anopheles mascarensis
we examined 418 mosquitoes from at least 18 species is reported to act as a vector of Plasmodium falciparum,
individually using a part of the mitochondrial cyto- the causative agent of human malaria [28]. We found an
chrome b gene. Sequences found in the mosquitoes avian Plasmodium lineage in the thorax of one sampled
were compared to a large dataset of sequences isolated Anopheles mascarensis indicating that this endemic mos-
from 45 bird species (n = 686) of the same area. Twenty- quito species plays an important role for both, human
one mosquitoes were found to contain DNA of avian and avian malaria transmission.
Haemosporida. Ten of the 12 (83.3%) Plasmodium lineages found
We found Haemoproteus DNA in nine mosquitoes. were isolated from mosquitoes belonging to the genus
Haemoproteus species develop oocysts in the head, Uranotaenia. This is the first report of Uranotaenia
Schmid et al. Parasites & Vectors (2017) 10:6 Page 5 of 7

species acting as vectors for avian malaria. Although fur- pipiens as vector for this lineage also on Madagascar
ther studies are needed, the genus Uranotaenia might but with Uranotaenia n. sp. we found a second vector
even be the major vector for avian malaria in this area. mosquito. We isolated DNA of Plasmodium sp. mos-
The isolated Plasmodium lineages were compared to quito132 also from 51 bird blood samples. The samples
previously published sequences in GenBank and thus stem from individuals of 15 bird species belonging to
identified. We isolated the sequence Plasmodium sp. eight bird families. The finding of the lineage in so
Ual1 from the thorax of one Uranotaenia alboabdomina- many diverse bird species leads us to the conclusion
lis. The sequence was 95% homologous to the previously that Plasmodium sp. mosquito132 is a generalist.
described lineage Plasmodium minuoviride haplotype From the endemic mosquito Anopheles mascarensis
CCA0640 (EU834703.1). This lineage was isolated by Per- we isolated the lineage Plasmodium sp. PV12 [31]. The
kins et al. [29] from Prasinohaema prehensicauda, a skink lineage was originally isolated from the mosquito
(Squamata) endemic to Papua New Guinea. Because Coquillettidia aurites in Cameroon. We did not find the
species of the genus Uranotaenia are known to feed on lineage in the four samples of Coquillettidia grandidieri
cold-blooded animals [15] and due to the high homology examined in this study. Because of the small sample size
we conclude that the sequence we found also stems from we cannot exclude this mosquito species as a possible
a Plasmodium species infecting reptiles. The lineages Plas- vector. Our findings suggest that Anopheles mascarensis
modium sp. Uan1 (isolated from thorax of Uranotaenia n. is a vector of Plasmodium sp. PV12 in the study area.
sp.) and Plasmodium sp. Cp1 (isolated from thorax of We also detected this lineage in 37 bird blood samples.
Culex pipiens) were highly homologous to sequences pre- The samples are from eleven 11 bird species belonging
viously isolated from birds. Therefore, we conclude that to eight families. The finding of the lineage in so many
these sequences represent two new Plasmodium lineages diverse bird species leads us to the conclusion that Plas-
infecting birds. The other four sequences isolated were modium sp. PV12 is a generalist.
identical with lineages previously described and were also The Plasmodium lineage isolated from Uranotaenia
found in bird blood samples examined during this study. n. sp. was identical to the previously described Plasmo-
The presence of the identical haemosporidian cytochrome dium sp. P31 [32]. The lineage was originally isolated
b sequence in birds and mosquitoes could be an indica- from Foudia madagascariensis (Ploceidae) captured on
tion of a vector-host relationship between the species Madagascar. In the current study the same sequence
described. was isolated from 39 blood samples of three weaver bird
We isolated the lineage Plasmodium sp. WA46 [30] species, including Foudia omissa, Foudia madagascarien-
from four mosquitoes belonging to the genus Uranotae- sis and Ploceus nelicourvi. Therefore, Plasmodium sp. P31
nia. The parasite sequence was found in the thoracic seems to be specific to weaver birds (Ploceidae). Fodies
parts of Uranotaenia alboabdominalis, Ur. neireti and in were among the most frequently sampled birds. From ob-
one undescribed Uranotaenia species. We conclude that servational studies they are known to move from the for-
all three mosquito species are probably suitable vectors est to the open, more degraded areas, and may therefore
for the Plasmodium lineage WA46. The very same lineage spread blood parasites into more pristine areas. The
was also found in five bird blood samples of the weaver bird lineage was detected in 39 samples from three
Madagascar bulbul (Hypsipetes madagascariensis, Pycno- different years (2006, 2007, 2010, 2012 and 2014). The
notidae). The blood was sampled from different individ- high and apparently stable prevalence in the birds suggests
uals in the years 2007, 2010, 2012 and 2014 suggesting a that there is a stable life-cycle of Plasmodium sp. P31 on
stable occurrence in the study area. The lineage Plasmo- Madagascar with its intermediate host being weaver birds
dium sp. WA46 was originally isolated by Beadell et al. and Uranotaenia n. sp. as the first described vector.
[30] from the common bulbul (Pycnonotus barbatus, Pyc- To confirm the role of the genus Uranotaenia,
nonotidae). Our findings support the hypothesis, that the Anopheles mascarensis and Culex pipiens in the trans-
lineage WA46 is a specialist for the bird family Pycnonoti- mission cycle of the reported avian malaria parasites on
dae. As Hypsipetes madagascariensis is the only bulbul Madagascar, further analyses of blood-fed mosquitoes
species on Madagascar we assume that the life-cycle of from infection experiments are required.
Plasmodium sp. WA46 in our study area includes at least
three vector mosquito species of the genus Uranotaenia Conclusions
and the host bird Hypsipetes madagascariensis. This study provides the first insight into avian malaria
The lineage Plasmodium sp. mosquito132 [26] was transmission in the Maromizaha forest in eastern
isolated from the thoracic parts of Culex pipiens and Madagascar. Five Haemoproteus lineages and seven
Uranotaenia n. sp. in this study. Ejiri et al. [26] originally Plasmodium lineages were detected in the examined
isolated the lineage from Culex pipiens quinquefasciatus mosquitoes. Complete life-cycles for the specialist Plas-
captured in Japan. We supported the role of Culex modium lineages WA46 and P31 and for the generalist
Schmid et al. Parasites & Vectors (2017) 10:6 Page 6 of 7

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