Evaluation of Larval Development of Dirofilaria immitis in Different Populations of Aedes aegypti and Aedes albopictus

doi:10.4236/ojvm.2013.36045

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Open Journal of Veterinary Medicine, 2013, 3, 277-281

http://dx.doi.org/10.4236/ojvm.2013.36045 Published Online October 2013 (http://www.scirp.org/journal/ojvm)

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: *[email protected]

Received June 27, 2013; revised July 27, 2013; accepted August 17, 2013

bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

ABSTRACT

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.

aegypti.

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.

G. A. DE CARVALHO ET AL.

278

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

Infection

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

Examination

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

G. A. DE CARVALHO ET AL. 279

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 Diroﬁlari 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

[20].

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.

G. A. DE CARVALHO ET AL.

280

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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