Larval Habitats Diversity and Distribution of the Mosquito
(Diptera: Culicidae) Species in the Republic of Moldova
Author(s): Tatiana M. Sulesco, Lidia G. Toderas, Inga G. Uspenskaia and I. K.
Toderas
Source: Journal of Medical Entomology, 52(6):1299-1308.
Published By: Entomological Society of America
URL: http://www.bioone.org/doi/full/10.1093/jme/tjv142
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SAMPLING, DISTRIBUTION, DISPERSAL
Larval Habitats Diversity and Distribution of the Mosquito
(Diptera: Culicidae) Species in the Republic of Moldova
TATIANA M. SULESCO,1 LIDIA G. TODERAS, INGA G. USPENSKAIA, AND I. K. TODERAS
Institute of Zoology, Academy of Sciences of Moldova, st. Academiei 1, Chisinau, MD-2028, Republic of Moldova.
J. Med. Entomol. 52(6): 1299–1308 (2015); DOI: 10.1093/jme/tjv142
ABSTRACT A countrywide field survey of immature mosquitoes was conducted in Moldova with the
aim to evaluate the Culicidae species composition in different larval habitats and their distribution in the
country. In total, 259 potential larval habitats were sampled in the 53 localities, resulting in 9,456 specimens. Twenty species belonging to the genera Anopheles, Aedes, Culex, Culiseta, and Uranotaenia were
collected. Mean species richness in aquatic habitats ranged from 1.00 to 4.00, and, for example, was
higher in swamps, flood plains, ditches, and large ground pools and lower in rivers, streams, tree-holes,
and containers. Six mosquito species were identified only in a single type of aquatic habitat. Anopheles
maculipennis s.l., Culex pipiens pipiens L., and Culex modestus Ficalbi were the most abundant and
distributed species representing over 80% of the identified specimens. Three, four, and five associated
species were recorded from 23.5% of mosquito-positive aquatic habitats. Our findings demonstrate the
co-occurrence of Cx. p. pipiens and Culex torrentium Martini in natural and rural environments. It is
concluded that the study area has undergone a dramatic ecological change since the previous studies in
the 1950s, causing the near extinction of Culex theileri Theobald from Moldova. An. maculipennis s.l. larval abundance, reduced by the DDT control of the adults in the 1950s, had returned to those of the
1940s. Restoration of An. maculipennis s.l. abundance in combination with imported malaria cases constitute a risk of the reintroduction of malaria transmission in Moldova.
KEY WORDS mosquito ecology, aquatic habitat, spatial distribution, Moldova
A detailed survey of mosquito larval habitats in Moldova last time was carried out when the cases of autochthonous malaria peaked in the 1940s (Markovich
et al. 1949, Prendel et al. 1949). Until the mid-1948,
national malaria control efforts, focused on treatment
of malaria cases and Anopheles maculipennis s.l. larval
control by insecticides, did not give a rapid reduction
of malaria morbidity. Since the advent of dichlorodiphenyltrichloroethane (DDT), most efforts on vector
control in Moldova were focused on mosquito adults
and research on the larval ecology was largely neglected (Markovich et al. 1949). From 1948 to 1956,
the treatments with DDT area around water bodies
and indoor spraying with DDT and hexachlorocyclohexane decreased the An. maculipennis s.l. densities
and malaria morbidity. During these years about 1,145
ha of ground water pools were drained to remove mosquito breeding sites (Miliutina et al. 1957). In the
1960s and 1970s, 39,500 and 23,541 ha of floodplains
of the Dniester and Prut rivers were drained to protect
the soil from water erosion, waterlogging, and reduce
Anopheles larval habitats (Miliutina et al. 1957, Bespalov 1999). Although malaria was eliminated in Moldova
by 1960, introduced cases are reported from the
1
Corresponding author, e-mail: tatiana_sulesco@yahoo.com.
country every year (WHO 2002, 2005). Systematic research on the larval ecology of the potential mosquitoborne disease vectors in Moldova is still limited and often represents short data collection periods in some
regions.
In the 1980s several surveys have been conducted to
characterize mosquito aquatic habitats and seasonal
abundance of some mosquito species in Moldova.
Culex pipiens pipiens L. and An. maculipennis s.l. have
been sampled from the wells and canals of the irrigation systems, which are not in use currently (Tihon
1981). Another paper presented limited information
about the distribution of Aedes cinereus Meigen,
Ae. communis (De Geer), Ae. cantans (Meigen), and
Ae. punctor (Kirby) in temporary ground pools of the
Codri reserve in springtime (Tihon 1984). Finally, the
presence and seasonal abundance of Cx. territans
Walker were reported in permanent ground pools of
the Codri reserve (Uspensky 1989).
However, detailed information on the ecology of the
immature stages of Culicidae species in Moldova
does not exist. As a result, little is known about the habitats, species colonization, and distribution of the Culicidae species with most information in relation mainly
to An. maculipennis s.l. larval control dating back to
publications in the mid of the twentieth century (Markovich et al. 1949, Prendel et al. 1949, Miliutina et al.
1957).
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com
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JOURNAL OF MEDICAL ENTOMOLOGY
As different mosquitoes distributed in Moldova are
known vectors of human and zoonotic diseases, the
obtained faunistic and ecologic data provide important
insight from a public health perspective (Markovich
et al. 1949, Sergeyeva 1953, Gratz 2004, Hubálek
2008). Therefore, our field survey covered numerous
urban and rural localities and natural ecosystems of
Moldova. To control mosquitoes, it is crucial to understand not only the fluctuations of the adult populations
but also the factors affecting larval abundance and distribution. This study was conducted to determine the
spatial distribution of immature Culicidae, species composition, and habitat preferences.
Materials and Methods
Study Area. A broad survey of immature mosquito
breeding sites was conducted in Moldova, situated in
southeast Europe. The study area is mostly agricultural
and cultivated lands make up 75.6% of the Moldovan
territory (Gan 1990). Natural areas represent 15% of
the country total area. Urban and rural areas represent
9 and 76% of the country total area. To the west, Moldova is bordered by the river Prut and to the east by
the river Dniester. The country is divided into five
main landscape regions: the Northern Forest Steppe
Upland, the Balti Meadow Steppe Plain, the Codri Forest Upland, the Lower Dniester Steppe Plain, and the
Bugeac Steppe Plain (Fig. 1). The central part of Moldova contains the steep forested slopes (locally called
Codri), ranging from 350 to 430 m a.s.l., which are
interlaced by deep, flat valleys and ravines. This area is
rich in small lakes and ditches. Originally forested, the
Balti Meadow Steppe Plain was extensively deforested
for agriculture. To the west, the Balti Meadow Steppe
Plain includes the Middle Prut Valley which is dominated by numerous lakes, floodplains and wetlands.
The northern part of Moldova contains the uplands of
the Dniester Hills (240–320 m a.s.l.), which to the east
form the right bank of the Dniester River. The Lower
Dniester Steppe Plain (100–170 m a.s.l.) includes the
southern part of Transnistria, the right bank of the river
Dniester and adjacent areas (Fig. 1). This section of
the Dniester River is characterized by extreme and
irregular floods, due to severe hydrological and hydrographic changes associated with hydroconstruction and
other forms of human impact. The Bugeac Steppe
Plain, located in southern Moldova, is almost entirely
cultivated and characterized by more arid climate. The
Lower Prut Valley, located in the western part of the
Bugeac Steppe, is characterized by swamps, wetlands,
ponds, and floodplain lakes. The area is flooded in
springtime.
Mean annual temperature and rainfall between 2008
and 2013 ranged from 9.3 C and 634 mm in the northwest to 11.4 C and 503 mm in southeast of the country,
respectively (NBS 2014).
Mosquito Sampling. This study is a part of the
countrywide field survey of the mosquito species composition and its geographical distribution in Moldova.
To study the spatial distribution of mosquito breeding
sites and achieve a greater diversity of larval habitats
Vol. 52, no. 6
and species, 53 randomly selected localities and natural
areas (scientific and landscape reserves) were sampled
in urban (n ¼ 7; 13%), rural (n ¼ 37; 70%) and natural
(n ¼ 9; 17%) habitats (Table 1). The sampled site locations are given in Fig. 1. The global positioning system
(GPS) coordinates of each site were recorded. Each
locality was supported by its own identification number
to express the species composition in each habitat type,
locality, and breeding site (Supp Appendix 1 [online
only]). Mosquito immature stages were collected in the
five landscape regions to cover a variety of conditions
specific for the country (Fig. 1). Larval habitats were
checked for the presence or absence of mosquito species between April and October in 2008 and from 2010
to 2013. To increase the coverage area and the mosquito
species records, combination of previously sampled and
new localities were surveyed from 2010 to 2013. In total,
66 (n ¼ 35) and 19% (n ¼ 10) of the localities were
sampled once or twice during the study period, respectively. Thirteen percent (n ¼ 7) of the localities from
urban, rural, and natural habitats were sampled for
more than two sampling years, three times per sampling
season (once in the spring, summer, and fall), to identify
additional mosquito breeding sites and species. Permanent ground pools in the city of Chisinau were inspected
once every 2 wk from April to September 2012. To identify species composition of container breeding mosquitoes, short-term field surveys of artificial containers were
carried out in July and September 2008, September–
October 2010, August 2011, and July 2013. The surveys
of tree-holes in deciduous forests on the presence of
tree-hole breeding mosquitoes were carried out in Hirbovat forest and Codri reserve in April, June, and
August 2010, and July 2013, in Peresecena in late June
2010 and Chisinau forest park in mid-April 2010.
Sampling was done only once for 69% (n ¼ 38) of
permanent ground pools and for each semipermanent
and temporary breeding site identified. Larvae were
sampled using standard dipping techniques with a 600ml quart-sized dipper or pipettes according to the size
and the type of larval habitat (Service 1993, O’Malley
1995, Silver 2008). The number of dips per aquatic
habitat ranged from 3 to 10 for small water bodies, and
from 20 to 50 for large water bodies. The water samples were taken at intervals along the edge of large
aquatic habitats. Larval abundance was expressed as
the number of larvae (all instars) per dip.
No special techniques were used for collecting
Coquillettidia larvae (O’Malley 1995, Silver 2008).
Tree-holes of deciduous trees were verified for the
presence of water and larvae. The larvae and a sample
of water were collected from the tree-hole by siphon. A
sample of scratched detritus from the walls and the
bottom was taken from each dry tree-hole found and
incubated during 1 mo with distilled water under laboratory conditions for egg hatching.
All larval stages, pupae, and a sample of water from
each breeding site were placed in plastic containers
and transported to the laboratory, where larvae were
sorted and separated by genus and instar, and counted
(Table 2). The late (3rd and 4th) instars from each
aquatic habitat were identified to species or reared to
November 2015
SULESCO ET AL.: LARVAL HABITATS OF MOSQUITOES IN MOLDOVA
1301
Fig. 1. A map of Moldova with the localization of the 53 study areas. Square dots: urban sampling sites; round dots: rural
sampling sites; triangular dots: natural sampling sites.
Table 1. Characterization of immature mosquito collections in
breeding sites by habitat type sampled in 2008 and from 2010 to
2013 in Moldova
Habitats
Urban
Rural
Natural
No. (%) sampled localities
7 (13)
37 (70)
9 (17)
No. (%) sampled breeding sites
53 (20.4) 119 (45.9)
87 (33.6)
No. (%) breeding sites positive
21 (15.9)
71 (53.8)
40 (30.3)
No (%). specimens collected
2052 (21.7) 5511 (58.3) 1893 (20.0)
No (%). specimens identified
1696 (82.7) 4779 (86.7) 1533 (81.0)
Total no. species identified
12
14
15
the adult stage and identified. The younger (1st and
2nd) instars were reared in plastic trays until 4th larval
instar or pupation and emergence of the adult stage.
Pupae were identified only after the emergence of the
adult stage. All mosquitoes were stored in 70% ethanol.
Mosquito
Identification
to
Species
Level. Fourth instars and adults were identified to
species or species complex according to taxonomic keys
of Gutsevich et al. (1970) and Becker et al. (2010). Cx.
p. pipiens and Culex torrentium Martini cannot be
accurately distinguished morphologically as adult
females and larvae; therefore, morphological identification was based on the structure of male hypopygium.
Mixed mosquito populations contained both species
were expressed as Cpp./torrentium. The arrangement
of the mosquito species takes into account the systematic classification used in the “Systematic Catalog of
Culicidae” in the website of the Walter Reed Biosystematics Unit (WRBU 2001).
DNA was isolated from individual An. maculipennis
s.l. larvae or adults collected from 20 localities for identification by rDNA polymerase chain reaction (PCR)
using species specific primers (Proft et al. 1999).
Description of the Larval Habitats. During the
field surveys we defined 15 larval habitat types, which
were grouped as follows.
Natural containers of vegetal origin. Water-filled or
dry tree holes (1) of oak (Fagaceae, genus Quercus)
and hornbeam (Betulaceae, genus Carpinus) trees
were the only natural containers found positive for
mosquito larvae or eggs.
Artificial human-made containers that hold water.
Among sampled artificial containers (2) filled with rainwater were found plastic barrels used for irrigation and
enamel old utensil found in the backyards. In addition,
fountains (3) and water supplies (4) were filled with tap
water, and wells (5) with groundwater were sampled.
Ground pools of water of different size and origin.
These included large ground pools of natural (small
lakes from rural and natural areas) and artificial (water
reservoirs from urban areas) origin and small ground
pools created by groundwater discharge from the
springs (6); artificial drainage ditches alongside roadways or fields (7); hoof prints (8) near lakes left by livestock; car tracks (9) left on the road and filled with
rainwater. Some larval habitats were associated with
natural water bodies such as rain pools (10) in open
areas and forests; flooded meadows (11) after heavy
rainfall; slow-moving sections of rivers (12); landward
edges of the extensive floodwater in the floodplains
(13) of the river Prut and shallow inundated areas fed
by groundwater and located close to the Dniester River
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JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 52, no. 6
Table 2. Total number of mosquito larval habitats surveyed, proportion of habitats positive and negative, total number of immature
mosquitoes collected, and proportion of Anophelinae and Culicinae immature stages collected in 2008 and from 2010 to 2013 in the
countrywide survey
Habitats
Total no. Proportion Proportion Total no. of Proportion of Proportion
habitats of habitats of habitats immatures immatures
of pupae
surveyed positive (%) negative (%) sampled identified (%) sampled (%)
Proportion of early
instars sampled (%)
Proportion of late
instars sampled (%)
Anophelinae Culicinae Anophelinae Culicinae
Ground pool
Ditch
River section
Stream
Floodplain
Flooded meadow
Swamp
Rain pool
Tree-hole
Hoof print
Car track
Container
Well
Fountain
Water supply
55
26
14
6
3
4
2
33
35
30
14
14
10
5
8
80.0
73.1
78.6
16.7
100.0
50.0
100.0
39.4
28.6
40.0
28.6
57.1
10.0
20.0
12.5
20.0
26.9
21.4
83.3
—
50.0
—
60.6
71.4
60.0
71.4
42.9
90.0
80.0
87.5
3830
1606
412
77
273
78
66
363
328
855
204
1153
202
2
7
edge; swamp forests (14) along the river Prut and inundated permanently or seasonally; and low-level small
streams (15).
For each breeding site the following characteristics
were recorded: water permanence (permanent, semipermanent, or temporary), type of aquatic vegetation
(emergent, floating, or submerged), presence or
absence of grass, sunlight exposure (sunny, part shade,
or shade), water surface (large, > 15 m2; medium, 5–15
m2; small < 5 m2), nature of water body (natural or artificial) and mean water temperature (three or six readings) registered with digital thermometer. The majority
of aquatic habitats could not be observed over time,
permanency was determined based on the source of
water, aquatic habitat size. and observations made from
repeated visits of some localities.
Data Analysis. In order to characterize the mosquito communities that inhabited different aquatic habitats, mean species richness (arithmetic mean of all
mosquito species identified at a given habitat) was calculated for each type of aquatic habitat (Le Goff et al.
2014). One-way analysis of variance (ANOVA) test was
used for analysis of variation in larval abundance
among months and type of larval habitats. Descriptive
statistics was used to summarize the data for each habitat. Correlation analysis was used to assess the relationship between larval abundance and habitat
characteristics. Results were considered significant at
P < 0.05. Statistical analyses were performed using program package STATISTICA 7.0 (StatSoft 2004).
Results
Larval
Habitat
Diversity;
Species
Composition. In total, 362 sampling visits were made
to 259 potential larval habitats sampled in the 53 study
sites from urban, rural, and natural habitats. Fifty one
percent of the breeding sites (n ¼ 132) and 56%
(n ¼ 204) of visits yielded mosquito collections. Among
the water bodies inspected 66.7 (n ¼ 88) and 77.3%
83.8
77.9
88.6
97.4
92.3
93.6
71.2
85.4
82.6
88.7
84.3
80.6
86.1
100
57.1
4.3
3.1
2.4
15.6
12.8
1.3
37.9
10.7
—
8.0
2.9
7.7
5.4
—
42.9
27.8
35.5
33.7
13.0
4.0
10.3
13.6
18.5
—
1.1
0.5
—
5.9
50.0
—
32.5
36.3
3.4
—
19.0
30.8
16.7
38.0
82.0
48.2
64.7
37.1
65.8
—
—
16.3
15.6
57.3
71.4
11.7
3.8
6.1
4.1
—
0.9
—
—
0.5
50.0
—
19.2
9.5
3.2
—
52.4
53.8
25.8
31.7
18.0
41.9
31.9
55.2
22.3
—
57.1
(n ¼ 102) were found to be breeding habitats for Anophelinae and Culicinae species, respectively (Supp
Appendix 1 [online only]). Of the positive breeding
habitats, 59% (n ¼ 77) were permanent (41 lakes, 19
ditches, two swamps, one stream, one well, one fountain, and one water supply), 20% (n ¼ 26) were semipermanent (three ground pools, three floodplains, two
flooded meadows, 10 tree-holes, and eight containers)
and 22% (n ¼ 29) were temporary (13 rain pools, 12
hoof prints, and four car tracks) (Table 2).
In total, 9,456 Culicidae immatures were sampled, of
which 5,340 (56.5%) were categorized as early instars,
3,604 (38.1%) as late instars, and 512 (5.4%) as pupae.
Larval instars were classified separately for Anophelinae and Culicinae, except for pupae, which were identified only after the emergence of the adults. Among
sampled larvae 3,130 (35%) were identified as Anophelinae (61% early and 39% late instars) and 5,814 (65%)
as Culicinae (59% early and 41% late instars). Due to
mortality of early instars and some field-collected
pupae, 8,008 (84.7%) immatures were identified to species level (Table 2). Twenty species of the genera
Anopheles, Aedes, Culex, Culiseta, and Uranotaenia
were identified using morphological criteria or PCR
technique (Tables 3, 4).
PCR Identification of An. maculipennis
s.l. From a total of 2,682 An. maculipennis s.l. larvae
collected, 238 (9%) randomly selected specimens from
35 aquatic habitats were identified to species level by
PCR technique (Proft et al. 1999). Results showed the
presence of the following four species: An. atroparvus
Van Thiel (5.9%), An. maculipennis s.s. Meigen
(45.4%), An. melanoon Hackett (7.6%), and An. messeae Falleroni (41.1%) (Table 4). Among selected specimens An. labranchiae Falleroni and An. sacharovi
Favre mosquitoes were not identified by PCR.
Mosquito Species Richness. The total number of
species and species composition in urban, rural, and
natural habitats were different. Species richness in natural habitats was higher (n ¼ 15, three species
22 (0.7)
1431 (44.6)
—
4 (0.1)
—
2 (0.1)
—
65 (2.0)
5 (0.2)
881 (27.4)
698 (21.7)
—
—
—
85 (2.6)
—
3 (0.1)
15 (0.5)
3211
2.14
An. claviger
An. maculipennis s.l.
An. plumbeus
An. pseudopictus
Ae. geminus
Ae. vexans
Ae. geniculatus
Ae. caspius
Ae. dorsalis
Cx. modestus
Cx. p. pipiens
Cpp./torrentium
Cx. theileri
Cx. torrentium
Cx. territans
Cs. longiareolata
Cs. annulata
Ur. unguiculata
Total no. immatures
Mean species richness
—
676 (54.0)
—
—
—
4 (0.3)
—
91 (7.3)
51 (4.1)
249 (19.9)
151 (12.1)
8 (0.6)
—
14 (1.1)
6 (0.5)
—
—
1 (0.1)
1251
2.21
Ditch
—
341 (93.4)
—
—
—
—
—
—
—
20 (5.5)
4 (1.1)
—
—
—
—
—
—
—
365
1.46
River
section
—
75 (100)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
75
1.00
Stream
52 (20.6)
—
78 (31.0)
1 (0.4)
—
—
—
—
2 (0.8)
252
2.40
—
39 (15.5)
—
—
—
80 (31.7)
—
—
Flood plain
—
10 (13.7)
—
—
—
63 (86.3)
—
—
—
—
—
—
—
—
—
—
—
—
73
1.00
Flooded
meadow
The values do not provide density comparisons of mosquito immatures among larval habitat types.
Ground pool
Species
—
9 (19.2)
—
1 (2.1)
—
—
—
—
—
18 (38.3)
19 (40.4)
—
—
—
—
—
—
—
47
3.40
Swamp
—
70 (22.6)
—
—
10 (3.2)
30 (9.7)
—
25 (8.1)
—
20 (6.4)
58 (18.7)
81 (26.1)
—
—
—
—
—
16 (5.2)
310
2.08
Rain pool
—
—
31 (8.1)
—
—
—
—
385
1.30
—
—
4 (1.0)
—
—
—
350(90.9)
—
—
—
Tree hole
—
15 (2.0)
—
—
—
3 (0.4)
—
7 (0.9)
3 (0.4)
4 (0.5)
579 (76.4)
—
—
112 (14.8)
—
—
—
35 (4.6)
758
1.90
Hoof print
—
1 (0.6)
—
—
—
—
—
—
—
—
—
108 (62.8)
—
63 (36.6)
—
—
—
—
172
2.00
Car track
—
—
—
—
—
—
—
—
—
—
894 (96.2)
—
—
—
—
35 (3.8)
—
—
929
1.37
Container
—
13 (7.5)
—
—
—
—
—
—
—
—
76 (43.7)
—
—
—
24 (13.8)
61 (35.1)
—
—
174
4.00
Well
Number (proportion in %) of immatures sampled and identified to species/species complex in each type of larval habitat
Table 3. Species composition and mean species richness of Culicidae immatures collected from different types of larval habitats in Moldova in 2008 and from 2010 to 2013
—
2 (100)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2
1.00
Fountain
—
—
—
—
—
—
—
—
—
1 (25.0)
3 (75.0)
—
—
—
—
—
—
—
4
2.00
Water supply
November 2015
SULESCO ET AL.: LARVAL HABITATS OF MOSQUITOES IN MOLDOVA
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JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 52, no. 6
Table 4. Number of An. maculipennis s.l. specimens collected from different aquatic habitats of Moldova and identified to species
level
Type of aquatic
habitat
Ground pool
Ditch
River section
Stream
Rain pool
Flood plain
Swamp
Hoof print
Total
No. aquatic
habitats selected
No. An. maculipennis
s.l. larvae collected
No. An. maculipennis
s.l. processed by PCR
An.
atroparvus
An.
maculipennis s.s
An.
melanoon
An.
messeae
22
6
2
1
1
1
1
1
35
591
269
127
65
11
28
4
3
1098
158
34
21
12
4
4
2
3
238
4
9
0
0
0
0
0
1
14
88
2
0
12
4
0
0
2
108
18
0
0
0
0
0
0
0
18
48
23
21
0
0
4
2
0
98
restricted to natural sampling sites) than in rural
(n ¼ 14, two species restricted to rural sampling sites)
and urban habitats (n ¼ 12) (Supp Appendix 2 [online
only]). In total, nine species were collected in all three
habitats and included the most widespread species
(species complex) An. maculipennis s.l., Cx. p. pipiens,
and Culex modestus. The number of species varied
among different aquatic habitats. The maximum number of species at a single breeding site was five (ditches,
flood plains). Mean species richness in different aquatic
habitats ranged from 1.00 to 4.00 and, for example, was
higher in swamps, flood plains, ditches, and large
ground pools and lower in rivers, streams, tree-holes,
and containers (Table 3).
Six mosquito species were identified only in a single
type of aquatic habitat. Aedes geniculatus Oliver and
An. plumbeus Stephens were sampled in tree-holes,
Ae. geminus Peus in one rain pool, An. claviger (Meigen) in one ground pool and Culex theileri Theobald in
flood plain. For the first time larvae of Culiseta longiareolata (Macq.) were sampled from artificial enamel and
plastic containers (sampling sites C10 and C37) and
recently have been collected along with An. maculipennis s.l., Cx. p. pipiens, and Cx. territans from one well
in Chisinau (sampling site W12) (Supp Appendix 2
[online only]). Despite the recent finding of Cs. longiareolata in Moldova for the first time, this species was
found in southern Ukraine in 1954 not far from Moldova (Naidich 1957).
Abundance and Spatial Distribution. Results of
the spatial distribution survey showed that An. maculipennis s.l., Cx. p. pipiens, and Cx. modestus Ficalbi
were the most abundant and distributed species representing over 80% (n ¼ 6,409) of the identified specimens and occurred in 42.1% (n ¼ 109) of the total
aquatic habitats surveyed (Table 5). An. maculipennis
s.l. made up 33.5% of the mosquitoes collected and
was the most abundant and widespread species collected in 83% (n ¼ 44) of sampled localities (Table 3,
Supp Appendix 2 [online only]). Anopheles maculipennis s.l. inhabited a wide variety of breeding sites,
whereas immatures were sampled predominantly from
edges of large ground pools (72.7% of the total ground
pools surveyed), ditches (57.7%) and slow-moving sections of rivers (78.6%), which supported larval development during most of the sampling period. Collections
from these aquatic habitats comprised 91.3% of the
total An. maculipennis s.l. larvae collected. There was
significant positive correlation between the abundance
of An. maculipennis s.l. and larval habitat temperature
(r ¼ 0.32, P ¼ 0.03). Larvae predominantly were collected from open sunlit habitats with stagnant warm
water and considerable aquatic vegetation.
Culex p. pipiens was the second most distributed and
abundant species in aquatic habitats, representing
31.0% (n ¼ 2,482) of the total mosquito specimens
identified from 51% of the sampled localities. Overall
19.2% (n ¼ 51) of the total number of larval habitats
sampled contained Cx. p. pipiens. Most of the Cx. p.
pipiens specimens (87.5%) were collected from artificial
containers (57.1% of the total containers inspected),
large ground pools (29.1%), and hoof prints (16.7%).
Permanent ground pools supported Cx. p. pipiens larval
production during most of the sampling period and
comprised 31.4% of the total habitats positive for Cx. p.
pipiens, although larval abundance did not vary significantly among months (F ¼ 0.81, df ¼ 5, 105, P ¼ 0.41).
Culex modestus was the third most distributed species, present in 51% (n ¼ 27) of all sampled localities
and recorded mainly in permanent ground pools and
ditches where 90.8% of the total individuals were collected. Cx. modestus adult females reared in the laboratory from larval population collected in Padurea
Domneasca reserve deposited their first batch of viable
eggs after the sugar feeding without prior blood meal.
An autogenous deposition of viable eggs was observed
for the next generation as well.
Tree-Hole Breeding Mosquitoes. During the
survey, 35 tree-holes from three woodland areas and
one urban forest park were identified and examined,
from which 20.0% (n ¼ 7) were filled with water. Overall, 28.6% (n ¼ 10) of the tree-holes contained mosquito
larvae and / or eggs. Three mosquito species were
found breeding in tree-holes (An. plumbeus, Ae. geniculatus and Cx. torrentium). First two species are usually restricted to breeding in tree holes and Cx.
torrentium was occasionally observed to breed in treehole in our surveys. The most abundant species was Ae.
geniculatus found in all examined water-filled tree holes
and in 7.1% (n ¼ 2) of the dry holes sampled in April.
Some 31% (n ¼ 110) of Ae. geniculatus larvae hatched
in laboratory from eggs collected in dry holes. Few eggs
of An. plumbeus along with Ae. geniculatus larvae from
the water-filled hole sampled in late June and a single
—
—
—
—
—
—
—
—
—
1 (12.5)
1 (12.5)
—
—
—
—
—
—
—
—
1 (20.0)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1 (10.0)
—
—
—
—
—
—
—
—
1 (10.0)
—
—
—
1 (10.0)
1 (10.0)
—
—
—
—
—
—
—
—
—
—
—
—
8 (57.1)
—
—
—
—
3 (21.4)
—
—
—
1 (7.1)
—
—
—
—
—
—
—
—
—
3 (21.4)
—
1 (7.1)
—
—
—
—
—
4 (13.3)
—
—
—
1 (3.3)
—
2 (6.7)
1 (3.3)
1 (3.3)
5 (16.7)
—
—
2 (6.7)
—
—
—
3 (10.0)
—
—
3 (8.6)
—
—
—
9 (25.7)
—
—
—
—
—
—
1 (2.9)
—
—
—
—
—
6 (18.2)
—
—
1 (3.0)
1 (3.0)
—
4 (12.1)
—
2 (6.1)
3 (9.1)
3 (9.1)
—
—
—
—
—
2 (6.1)
—
2 (100)
—
1 (50.0)
—
—
—
—
—
2 (100)
2 (100)
—
—
—
—
—
—
—
2 (66.7)
—
1 (33.3)
1 (33.3)
—
—
—
—
1 (33.3)
—
1 (25.0)
—
—
—
1 (25.0)
—
—
—
—
—
—
—
—
—
—
—
—
—
2 (66.7)
—
—
—
1 (33.3)
—
—
—
1 (16.7)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
11 (78.6)
—
—
—
—
—
—
—
2 (14.3)
3 (21.4)
—
—
—
—
—
—
—
1 (1.8)
40 (72.7)
—
2 (3.6)
—
2 (3.6)
—
2 (3.6)
2 (3.6)
21 (38.2)
16 (29.1)
—
—
—
6 (10.9)
—
1 (1.8)
4 (7.3)
An. claviger
An. maculipennis s.l.
An. plumbeus
An. pseudopictus
Ae. geminus
Ae. vexans
Ae. geniculatus
Ae. caspius
Ae. dorsalis
Cx. modestus
Cx. p. pipiens
Cpp./torrentium
Cx. theileri
Cx. torrentium
Cx. territans
Cs. longiareolata
Cs. annulata
Ur. unguiculata
——
15 (57.7)
—
—
—
1 (3.8)
—
4 (15.4)
2 (7.7)
11 (42.3)
4 (15.4)
1 (3.8)
—
1 (3.8)
1 (3.8)
—
—
1 (3.8)
Stream
(n ¼ 6)
Ground
pool (n ¼ 55)
Ditch
(n ¼ 26)
River section
(n ¼ 14)
Flood plain Flooded meadow Swamp Rain pool Tree hole Hoof print Car track Container
Well
Fountain Water supply
(n ¼ 3)
(n ¼ 4)
(n ¼ 2) (n ¼ 33)
(n ¼ 35)
(n ¼ 30)
(n ¼ 14)
(n ¼ 14) (n ¼ 10) (n ¼ 5)
(n ¼ 8)
SULESCO ET AL.: LARVAL HABITATS OF MOSQUITOES IN MOLDOVA
Species
Number (proportion in %) of positive breeding sites
Table 5. Number and proportion of mosquito-positive breeding sites sampled in 2008 and from 2010 to 2013 in Moldova
November 2015
1305
egg sampled from one dry hole in middle April. A
single egg raft and early instar larvae of Cx. torrentium,
and a single egg of An. plumbeus together with
Ae. geniculatus larvae were found in late July in the
same tree-hole filled with water (site 15). No larvae of
An. plumbeus were sampled in the current study.
Co-occurrences of Culicidae Species in
Mosquito Breeding Habitats. At 43% (n ¼ 88) of
positive sampling visits made to 51 different larval habitats, only one species was collected, while in 36
(n ¼ 74) and 13% (n ¼ 26) of positive visits, two and
three associated species were sampled from 50 and 19
aquatic habitats, respectively. Four and five associated
species were found in 7 (n ¼ 14) and 1% (n ¼ 2) of
occasions in 10 and two larval habitats, respectively.
An. maculipennis s.l. was the most widely distributed
species detected in 64.4% (n ¼ 85) of mosquito-positive
habitats. In 25.8% (n ¼ 34) of mosquito- positive water
bodies and 27.0% (n ¼ 55) of positive visits it was
found as the only species. An. maculipennis s.l. most
frequently co-occurred with Cx. modestus and
Cx. p. pipiens in 29.5 (n ¼ 39) and 22.7% (n ¼ 30) of
positive larval habitats, or 28.4 and 15.5 % of positive
sampling visits, respectively (Table 6). The presence of
Cx. modestus in the ground pools and ditches was constantly associated with larvae of An. maculipennis s.l.,
where they both co-occurred together in 46% (n ¼ 20)
of the ground pools and 58% (n ¼ 11) of ditches. However, in 46% of ground pools and 10.5% of ditches An.
maculipennis s.l. was found as the only species. An.
maculipennis s.l. was present in higher abundance than
Cx. modestus in permanent ground pools and ditches
with rich floating and submerged aquatic vegetation
where both species co–occurred together (F ¼ 17.96,
df ¼ 1, 48, P < 0.001).
Morphologically similar larvae of Cx. p. pipiens and
Cx. torrentium Martini were found associated in eight
breeding sites most of which were temporary or semipermanent aquatic habitats. From 56 natural or artificial water bodies positive for Cx. p. pipiens and/or
Cx. torrentium 23.2% (n ¼ 13) of aquatic habitats contained larvae of Cx. torrentium and 91.1% (n ¼ 51) contained larvae of Cx. p. pipiens.
Discussion
This research represents the first recent countrywide
study of immature Culicidae distribution, their habitat
preferences and species richness in Moldova. Of the
36 mosquito species recently identified in Moldova,
20 species were recorded from 132 mosquito breeding
habitats (Sulesco et al. 2013). Since our surveys were
conducted using only one sampling technique and sampling efforts were not equally distributed over all types
of larval habitats it was expected that not all species
recorded in Moldova would be detected.
If our sampling efforts had included more floodplain
areas, Ae. vexans Meig. immatures would have been
more abundant in our collections and probably another
floodwater species like Ae. cinereus, Ae. rossicus Dolb.,
Gor., Mitrof., and Ae. sticticus (Meigen) would have
been detected. The low number of Ae. vexans and the
—
0
0.5
0
0
2.1
0
0
2.1
a
Collections (n ¼ 10) of the species of tree-hole breeding mosquitoes were excluded from analysis, as they restricted to breeding in tree-holes only.
1.5
0
0
0
1.5
28.4
0.5
0
0
0.5
0.5
15.5
0.5
0.5
1.5
3.1
1.0
11.3
0.5
—
—
—
—
—
0.5
0.5
2.6
—
—
—
0.5
—
1.5
4.1
—
6.7
—
—
0.5
—
—
5.2
3.6
—
0.5
—
—
—
—
—
—
—
2.1
—
—
—
—
—
—
—
—
—
0.5
0.5
—
—
—
—
4.6
—
—
0.5
1.0
1.5
2.1
3.6
—
0.5
0.5
—
—
An. maculipennis s.l.
An. pseudopictus
Ae. geminus
Ae. vexans
Ae. caspius
Ae. dorsalis
Cx. modestus
Cx. p. pipiens
Cx. theileri
Cx. torrentium
Cx. territans
Cs. longiareolata
Cs. annulata
Ae.
geminus
Ae.
vexans
Ae.
caspius
Ae.
dorsalis
Cx.
modestus
Cx. p.
pipiens
Cx.
theileri
Cx.
torrentium
Ur.
unguiculata
Cs.
annulata
Cs.
longiareolata
Cx.
territans
Percent of visits positive for mosquito immatures (n ¼ 194)a
Species
Table 6. Co-occurrence of immature mosquito species in aquatic habitats of Moldova
2.1
JOURNAL OF MEDICAL ENTOMOLOGY
An.
pseudopictus
1306
Vol. 52, no. 6
absence of another floodwater species that could occur
in high numbers in inspected floodplains, indicated our
late start of sampling of floodwater mosquitoes after
flooding, when the adults of these species already
emerged and were replaced in floodplains by An. maculipennis s.l. and Cx. modestus detected in high
numbers.
The absence of immatures of some Aedes species
like Ae. annulipes (Meigen), Ae. communis, Ae. cantans
and Ae. punctor, which produce one generation per
year and develop in spring in a variety of permanent
and semi-permanent meadow pools and inside deciduous forests, may be explained by conducting of our
field surveys in such breeding sites from the end of
April, and rare frequency of detection of colonized
aquatic habitats by snow-melt mosquito species. Aedes
caspius (Pallas) and Ae. dorsalis (Meigen) are polycyclic
species in the study area, and were regularly found in
our collections. Tihon (1984) described the presence of
Aedes cinereus larvae both in meadow and woodland
temporary pools in spring. Our recent collections in the
Codri reserve (site 15) detected the presence of
Ae. geminus in one woodland temporary pool.
Anopheles claviger has been found previously in a
stagnant groundwater flowed from the spring (Tihon
1984). Our recent collections of adult mosquitoes also
identified An. claviger females resting in stables within
the Codri reserve. In this study An. claviger larvae
were collected in mid-June from the north-western part
of Moldova among growth of reeds at the edge of Lake
La Fontal (site 14) with highly mineralized cold water
(16.1 C) derived from the springs. In the 1940s during
the malaria outbreaks this species along with An. maculipennis s.l. was common in the villages from central
Moldova, but was less abundant than the latter (Markovich et al. 1949). Currently, it seems that the distribution of An. claviger is restricted only to the forest zones
of the central and northwestern parts of Moldova.
Culex theileri was found singleton in our collections
from Bugeac Steppe Plain in one semi-permanent
pool, formed after flooding from the river Prut. This is
in agreement with previous findings in this area by Prendel (1951, 1956), who additionally described the
abundance of the species in the floodplains of the
Dniester River in Transnistria and its presence in
Bugeac Steppe Plain. Our recent immature and mature
collections in these areas did not reveal the presence of
Cx. theileri. Perhaps distribution and abundance of the
species have been adversely affected by the wide-scale
land reclamation in the past. This is supported by
Tihon’s (1981) observations that Culicidae species richness and abundance were greatly reduced in drained
floodplains of the rivers Reut and Prut due to elimination of large areas of favorable mosquito breeding sites.
Our results show that An. maculipennis s.l.,
Cx. p. pipiens, and Cx. modestus are widely distributed
and the most abundant species in Moldova, which are
well-known vectors of mosquito-borne diseases in
Europe (Hubálek 2008). These observations generally
are in agreement with previous studies, made in the
1940s and 1970s (Prendel et al. 1949, 1956, 1965;
Tihon 1981). We found only one published reference
November 2015
SULESCO ET AL.: LARVAL HABITATS OF MOSQUITOES IN MOLDOVA
on the abundance of An. maculipennis s.l. in control
and experimental mosquito habitats from central Moldova (Markovich et al. 1949). Based on this publication
and our own data, we suggest that An. maculipennis s.l.
larval abundance increased after mosquito control in
the 1950s. Restoration of An. maculipennis s.l. abundance after DDT control operations in 1947–1956, in
combination with imported malaria cases constitute a
risk of the reintroduction of malaria transmission in
Moldova (Gratz 2004, WHO 2005). Prendel et al.
(1949) previously revealed the presence and distribution of An. maculipennis s.s., An. messeae, and An.
atroparvus throughout Moldova based on egg structure. The authors described the distribution and absolute dominance of An. messeae in floodplains of the
rivers Prut and Dniester and villages located in southern Moldova. Prendel (1938, 1941) also found that elevation is an important landscape determinant for An.
maculipennis s.s. distribution and abundance in Moldova. An. maculipennis s.s. has been found predominantly at higher altitudes in hilly forested areas of the
Codri Forest Upland. Anopheles atroparvus has been
shown to prevail in Black Sea coast region and its
occurrence generally increased with the increasing of
salinity of the natural breeding sites from north to
south of the region (Prendel 1938, 1941). In Moldova
An. atroparvus has been regularly found in different
localities and was a common species in Balti Meadow
Steppe Plain where solonetzic soils and brackish waters
occur (Prendel et al. 1949, 1965). At that time, the
malaria outbreaks in some villages from central Moldova were associated mostly with An. maculipennis s.s.
and An. messeae due to their abundance in the daytime
resting sites. Anopheles atroparvus was the less abundant species in these villages (Markovich et al. 1949).
Our preliminary results on the An. maculipennis complex distribution in Moldova generally are in agreement
with previous results, obtained in the 1940s (Prendel
1941, Prendel et al. 1949). Eighty percent of An. maculipennis s.s. specimens identified by PCR were from
larval habitats located in central hilly forested area of
Moldova. Anopheles messeae has been shown to be
associated with river systems where it was the dominant species. Amongst the 98 specimens identified as
An. messeae there might have been as well some of An.
daciae Linton et al. not recovered from the samples as
PCR assay developed by Proft et al. (1999) identifies
both species as An. messeae (Kronefeld et al. 2012).
Anopheles atroparvus and newly found in our region
An. melanoon are the less abundant species and seems
to occur sporadically in Moldova. Anopheles labranchiae has not been detected in our surveys, but larvae
of this species recently have been detected in Iasi,
Romania, located close to the north-western border of
Moldova (Ivanescu et al. 2015). Taking into account
this finding, we may suggest the possible occurrence of
An. labranchiae in Moldova.
Cx. p. pipiens has been reported previously as the
species found throughout Moldova, occurring in natural environments, urban and rural areas, and has not
been distinguished from Cx. torrentium (Prendel 1956,
Prendel et al. 1965; Tihon 1981, 1984). Our results
1307
show the co-occurrence of Cx. p. pipiens and Cx. torrentium in natural and rural environments from northwestern, central and southern Moldova. Small,
temporary water pools without vegetation (rain pools
and car tracks) were the main habitats where larvae of
both species co-occurred.
Cx. modestus has been shown in the 1950s to be
abundant in the floodplains of the rivers Prut and
Dniester and distributed in central, eastern, and southern Moldova (Prendel 1951; Prendel et al. 1965).
Recent studies have demonstrated that Cx. modestus is
widely distributed throughout the country, including
northern Moldova. This species has been shown to be
more abundant and distributed than Cx. p. pipiens in
permanent aquatic habitats located within towns, indicating an adaptation of the species to the urban environment. Its occurrence in a wide array of habitat types
and conditions, and the ability of some Cx. modestus
females to lay eggs without a previous blood meal demonstrate the wide ecological flexibility of this mosquito.
Anopheles hyrcanus (Pallas) characterized by mostly
dark tarsomere IV of the hind legs has not been presented in our larval or adult mosquito collections,
although it has been previously recorded and morphologically distinguished from the pseudopictus form in
Transnistria and southern Ukraine (Prendel 1951). In
our surveys, all specimens morphologically identified as
An. pseudopictus according to WRBU systematic catalog, carried entirely white tarsomere IV of the hind
legs. No specimens with intermediate hind tarsomere 4
were collected in our studies (Ponçon et al. 2008).
Nevertheless, Becker et al. (2010) considered An. pseudopictus as a western Palaearctic form of An. hyrcanus
due to individual morphological variations within each
of these two taxa precluding reliable separation, the
presence of intermediate forms and the lack of information on cross-mating experiments, behavioral, phenological, and ecological differences (Ramsdale 2001,
Ponçon et al. 2008).
Finally, we present species co-occurrence data from
different breeding sites which suggest that larval control of An. maculipennis s.l. will have an impact especially on Cx. modestus and Cx. p. pipiens as these
species frequently co-occurred with larvae of the An.
maculipennis complex.
Variations in Culicidae species composition and
abundance along with habitat preferences play a major
role in the spatial and temporal heterogeneity of mosquito-borne disease risks. A good knowledge of the
geographical localization and ecological characterization
of larval habitats of the abundant mosquito vectors will
help to implement targeted larval control in Moldova.
Supplementary Data
Supplementary data are available at Journal of Medical
Entomology online.
Acknowledgments
Special thanks we extend to Prof. Dušan Petrić and Prof.
Bulent Alten for their valuable comments and suggestions to
1308
JOURNAL OF MEDICAL ENTOMOLOGY
improve the manuscript. We thank the directors of the scientific reserves for giving us permission for sampling on the protected natural areas. We are grateful to Prof. Liviu Miron and
Dr. Maria-Larisa Ivanescu for help in molecular analysis. This
research was supported by funds from the Institute of Zoology ASM project 11.817.08.13 F.
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Received 17 July 2015; accepted 28 August 2015.