Open Journal of Veterinary Medicine, 2013, 3, 277-281 Published Online October 2013 (
Evaluat io n o f Lar va l D ev elo pm ent o f Dirofilaria immitis
in Different Populations of Aedes aegypti
and Aedes albopictus
Gílcia Aparecida de Carvalho1*, Rafael Trindade Maia2, Rafael Antonio Nascimento Ramos3,4,
Carlos Fernando Salgueirosa de Andrade5, Maria Aparecida da Gloria Faustino3,
Leucio Câmara Alves3
1Unidade Acadêmica de Garanhuns, Universidade Federal Rural de Pernambu co, Garanhun s, Brazil
2Centro de Desenvolvimento Sustentável do Semiárido-Unidade Acadêmica de Educação do Campo,
Universidade Federal de Campina Grande, Sumé, Brazil
3Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, Brazil
4Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, Valenzano, Italy
5Universidade Estadual de Campinas, Campinas, Brazil
Email: *
Received June 27, 2013; revised July 27, 2013; accepted August 17, 2013
Copyright © 2013 Gílcia Aparecida de Carvalho et al. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Dirofilaria immitis is an important nematode parasite commonly known as heartworm. This filarioid is transmitted by
culicid vectors and primarily affects dogs, but other animals may also become infected, such as wild carnivores, cats
and humans. The aim of the present study was to assess the development of D. immitis larvae in differen t culicid popu-
lations under laboratory condition s. Adult females of populations of Aedes aegypti from the city of Recife (P1), the city
of Campinas (P2) and the Rockefeller strain from the Centers for Disease Control (P3) and one population of Aedes
albopictus from Recife (P4) were fed for two hours with infected dog blood containing 2,000 microfilariae/ml of D.
immitis. After artificial feeding, the specimens were maintained under controlled conditions. Ten females from each
population were dissected daily over 14 days. The infection ratio and vector efficiency index were calculated and D.
immitis development from L1 to L3 was assessed. The larvae in P1, P2, P3 and P4 reached the third stage in 11, 10, 14
and 9 days, respectively. The vector efficiency index was 53.8%, 20.0%, 7.4% and 25.2% in P1, P2, P3 and P4, respec-
tively. The findings demonstrate that D. immitis larvae develop in all culicid populations stud ied herein. Based on mos-
quito mortality, development time and VEI the A. albopictus population from Recife (P4) demonstrated the best per-
formance as vector. This is the second report of D. immitis development in A. albopictus from Brazil. The present data
reinforce the role of this sp ecies as vector of D. immitis in an area where greater importance has long been given to A.
Keywords: Culicid; Vector; Heartworm; Dog; Experimental Infection
1. Introduction
Dirofilariasis is an important parasitic disease caused by
the nematode Dirofilaria immitis, also known as heart-
worm [1]. This filarioid primarily affects dogs, but infec-
tion has also been reported in cats [2,3], wild carnivores
[4,5] and humans [6]. In this later host, infection is ch ar-
acterized by the presence of pulmonary nodules (the
so-called “coin lesion”) and no apparent symptoms [7].
Transmission occurs through mosquito vectors, which
are the most important vectors that belong to the genera
Culex, Aedes and Anopheles [8,9]. Briefly, first stage
larvae (L1) are taken up by blood-sucking female mos-
quitoes and develop through to the infective final larvae
stage (L3) in approximately 14 days [10]. The vector
competence of a culicid population is the primary factor
for D. immitis transmission in an endemic area. However,
characteristics such as the immune reaction, environ-
mental conditions (e.g., temperature and humidity) and
genetic traits among D. immitis populations may be fac-
tors which affect the vector capacity of culicids [11].
*Corresponding author.
opyright © 2013 SciRes. OJVM
In Brazil, studies on D. immitis vectors are scarce. In
the southeastern region of the country, Aedes scapularis
and Aedes taeniorhynchus have been shown to be the
main vectors, while Culex quinquefasciatus seems to
have secondary importance [2]. Also in this region (state
of Rio de Janeiro), an experimental study demonstrated
Aedes aegypti as a potential vector of D. immitis [12]. In
the northeastern region Cx. quinquefasciatus and A.
taeniorhynchus have been found naturally infected [13],
but little is known regarding the epidemiological impor-
tance of these species, especially the latter. Unfortunately,
the vector competence of two species well established
throughout Brazil (A. aegypti and A. albopictus), has
been poorly studied.
Therefore, the aim of the present study was to assess
the development of D. immitis larvae in three population s
of A. aegypti and one of A. albopictus from different geo-
graphic areas under laboratory conditions.
2. Material and Methods
2.1. D. immitis Microfilariae
Blood containing microfilariae was obtained from a dog
positive for D. immitis in the microscopic analysis (see
below). The dog was a three-year-old male that lived in
the metropolitan region of Recife (7˚45'0"S and
34˚51'0"W), state of the Pernambuco, Brazil.
2.2. Microscopic Diagnosis (Modified Knott Test)
The microscopic analysis was performed using the modi-
fied Knott test [14]. Briefly, 1 ml EDTA blood was
mixed with 9 ml of distilled water and centrifuged for 5
min at 300 × g. The supernatant was discarded, leaving 1
ml of solution to which two drops of methylene blue so-
lution were added. The sediment was transferred to glass
slides, covered with coverslip and examined und er a light
microscope at different magnifications. All filarioid lar-
vae found were morphologically identified as D. immitis.
2.3. Mosquito Populations and Experimental
Four different mosquito populations were used: A. ae-
gypti from the city of Recife, Brazil (P1), A. aegypti from
the city of Campinas, Brazil (P2), A. aegypti Rockfeller
strain from the Centers for Disease Control in the USA
(P3) and A. albopictus from Recife (P4).
A total of 4,800 female mosquitoes (3,600 for the test
populations and 1,200 for the control) aged from three to
seven days were used [15]. The artificial blood meal was
performed as previously described [10]. The test mos-
quitoes were fed for two hours with the infected blood,
which was previously assessed as containing 2,000 mi-
crofilariae/ml. Non-infected blood was offered to the
control group. After feeding, the mosquitoes were main-
tained under controlled temperature (28˚C ± 2˚C) and
relative humidity (>70%).
2.4. Mosquito Dissection and Microscopic
For the detection of D. immitis larvae an d developmental
stages, ten mosquitoes from each population were dis-
sected daily. On Day 14, all remaining living mosquitoes
were also dissected. The mosquitoes were fixed on slides
containing a drop of 0.9% physiological saline solution,
dissected with a sterile scalpel and immediately exam-
ined under a light microscope (Olympus BX41 TF) at
different magnifications. All stages of D. immitis larvae
were morphologically identified [16].
2.5. Data Analysis
The infection ratio (IR) [17] and vector efficiency index
(VEI) were calculated [18]. Linear regression was used
to determine the influence of the ingestion of D. immitis
microfilariae on mosquito mortality. Analysis of variance
was used to compare larval development up to the infec-
tious stage in the female mosquitoes as well as mortality
in the different populations. The BioStat program (ver-
sion 2.0) was employed for the statistical analysis [19].
3. Results
After feeding, the mosquitoes exhibited a similar rate of
engorgement: 97.6%, 91.5%, 97 .6% and 93.3% in P1, P2,
P3 and P4, respectively, and 96.6% in the control group.
Table 1 displays the mean ingestion of microfilariae in
each population. Microfilaria ingestion was statistically
similar among P1, P3 and P4 (P > 0.05), whereas P2 in-
gested a significantly smaller number of microfilariae (P
< 0.01).
Throughout the study period, mosquito mortality was
greater in P1 than P2, P3 and P4 (P < 0.01). However,
this higher mortality rate was not influenced by the
number of microfilariae ingested (F = 0.6899; P > 0.05).
Two peaks in mosquito mortality were found in all po-
pulations, one on the second day post-infection and one
after the detection of L3. At the end of the study, overall
mortality in the control gr oup was 3.4%.
The infection rate among the populations ranged from
11.6% to 17.5% (Table 2). This parameter was not in-
fluenced by the proportion of engorged females (F =
12.8754; P > 0.05), but was affected by the number of
microfilariae ing ested (F = 141.3808; P < 0.01).
D. immitis larval development from L1 to L3 occurred
in all infected populations. In P1, L 1 was found on Day 3
post-infection, L2 on Day 7 and a low number of L3 (n =
7) were retrieved on Day 11. In P2 and P3, L1 on Day 6
post-infection, L2 was found on Day 8 and L3 was found
Copyright © 2013 SciRes. OJVM
Table 1. Mean number of microfilariae ingested, rate of
recovery of L3 and VEI in mosquito females of different
populations (A. aegypti Recife—P1, A. aegypti Campinas—
P2, A. aegypti lineage Rockfeller—P3 and A. albopictus
P4). Specimens were fed with dog blood containing 2,000
microfilaria/ml of D. immi t is.
Pop. Mean of L1 ingested SD* Mean of L3 SD** VEI (%)
P1 13 10.4a 7.0 0B 53.8
P2 5 4b 1 0A 20
P3 13.4 7.7a 1 0A 7.4
P4 11.3 9.4a 2.8 2C 25.2
*Mean number of microfilariae ingested after blood meal; **Mean number of
(L3) recovery 14 days post-infection; different letters in the same column
indicate statistical difference.
Table 2. Total number of dissected and infected mosquito
females of different populations (A. aegypti Recife—P1, A.
aegypti Campinas—P2, A. aegypti lineage Rockfeller—P3
and A. albopictus—P4). Specimens were fed with dog blood
containing 2,000 microfilaria/ml of D. immitis.
Pop. Dissected females Infected females n (%)
P1 849 149 (17.5%)
P2 821 95 (11.5%)
P3 878 149 (16.9%)
P4 838 131(15.6%)
on Days 10 and 14, respectively. Melanized larvae and
few larvae remaining as L1 were detected at the end of
the study in all popu lations. D. immitis larvae exhibited a
comparatively short development time when infecting A.
albopictus (P4), with larvae reaching L1, L2 and L3 at
Days 2, 5 an d 9 , re spectively .
The number of L3 obtained 14 days post-infection (Ta-
ble 1) was not influenced by the number of microfilariae
ingested (F = 0.0415; P > 0.05). The VEI ranged from
7.4% to 53.8% in the different populations (Table 1).
4. Discussion
The present study provides evidence that D. immitis lar-
vae can develop efficiently in all cu licid population s stu-
died herein. Based on mortality, development time and
VEI the A. albopictus population (P4) demonstrated the
best performance as ve c tor of this nematode.
The mean ingestion of microfilariae was similar
among P1, P3 and P4, whereas P2 ingested a signifi-
cantly smaller number. From a biological standpoint,
microfilaria ingestion is an important limiting factor re-
lated to the vector competence o f a mosquito population.
However, the mechanisms that impair the ingestion of D.
immitis microfilariae and lead to mosquito infection re-
main unclear. It has been shown that some mosquito
populations have efficient defense mechanisms against
species of Dirolari a soon after ingestion [20]. Prob ably,
when microfilariae reach the Malpighian tubule, intra-
cellular development is blocked by defense mechanisms
activated by the host, resulting in larval death and lysis
A greater mortality rate was found among the mosqui-
toes in P1. Interestingly, this parameter was not influ-
enced by the number of microfilariae ingested (F =
0.6899; P > 0.05). Both mortality peaks occurred during
critical post-infection times: first during the penetration
of L1 into the Malpighian tubule cells (between Days 1
and 2 post-infection) and soon after the detection of L3.
In both events, Malpighian tubule cells were destroyed,
with the magnitude of the destruction depending on the
quantity of larvae, causing death in P1. As previous
states [21] an efficient mosquito vector should survive
independently of the number of ingested microfilariae
thus, P1 may exhibit a comparatively low degree of vec-
tor competence.
D. immitis development was observed in all popula-
tions studied herein, with the maximum VEI (53.8%)
found for A. aegypti from Recife (P1). However, this
population may not be co nsidered an efficient vector due
to the high mortality rate observed (70.7%). The minimal
differences in development time found among the popu-
lations of A. aegypti were expected, since the same
nematode strain was used for all populations. Previous
studies have been reported different development times
for this filarioid when different strains of nematode and
mosquitoes are us ed [16,22].
Among the four populations studied, one merits par-
ticular attention, A. albopictus from Recife (P4) exhibited
low mortality and a considerable VEI (25.2%). Further-
more, larvae reached the third infectiv e stage in only nin e
days. A. albopictus is rep orted to be th e primary potential
vector of D. immitis in Italy [23]. The importance of this
mosquito as a vector for D. immitis in Brazil has been
assessed in a single study [12]. Nonetheless, its vector
capacity has interesting epidemiological implications. A.
albopictus is considered a secondary vector of human
arboviruses due to its preferential feeding on animals
rather than humans, unlike the highly anthropophilic be-
havior of A. aegypti [24]. Based on feeding preference A.
albopictus may have a major veterinary importance for D.
immitis transmission among animals, which should be
studied better. It is important stress that the A. albop ictus
population evaluated herein proved to be susceptible to
infection and allowed D. immitis larvae development.
However, further studies evaluating the vector capacity
of other populations of this species in different regions of
Brazil should be carried out.
Copyright © 2013 SciRes. OJVM
5. Conclusion
In conclusion, D. immitis larvae developed in all mos-
quito populations studied. Based on factors as mortality,
development time and VEI, the A. albopictus population
from Recife demonstrated the best performance as vector.
This study is the second description of the development
of D. immitis in A. albopictus in Brazil. Moreover, the
findings suggest that this culicid species may perform an
important role as a vector for D. immitis in an area (i.e.,
Brazil) where greater importance has long been attributed
to A. aegypti. Field studies should be carried out to clar-
ify the real importance of this culicid species in the
transmission of D. immitis in Brazil.
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