The first field-based evidence of mosquitoes naturally infected with Dirofilaria spp. in Mexico was recently reported from Celestún, a coastal locality in the Yucatan Peninsula with a high prevalence of canine dirofilariasis (Manrique-Saide et al., 2008). Following collections of canine-attracted mosquito species during the dry season of 2007, the black salt marsh mosquito, Aedes taeniorhynchus (Diptera: Culicidae), was tentatively proposed as the principal vector of canine dirofilariasis in the area because it showed the strongest tendency for preferential feeding on dogs and was the only species found by microscopy to have infective third-stage larvae of Dirofilaria sp. in the vicinity of the mouthparts. The incrimination of this species remained provisional because it was not possible to confirm the species of the Dirofilaria found. Additionally, other potential mosquito vectors remained to be investigated because mosquito diversity was expected to vary between seasons. Here, we report for the first time the presence of Dirofilaria immitis in natural populations of Ae. taeniorhynchus in Mexico in the rainy season, as confirmed by polymerase chain reaction (PCR). The feeding and infection rates of mosquitoes collected from dog-baited traps in Celestún during the rainy season of 2007 are presented and contrasted with previous dry season findings, and support the incrimination of Ae. taeniorhynchus as the primary vector of D. immitis in coastal Yucatan, Mexico.
The study was carried out in the town of Celestún, located on the western shore of the Yucatan Peninsula, on the Gulf of Mexico (20°51′39′′ N, 90°23′33′′ W) (Fig. 1). The climate is hot and semi-arid, with monthly maximum temperatures of 35–40 °C, relative humidity of 65–95% and an average annual rainfall of 767 mm. The rainy season runs from June to September, when more than 80% of total annual precipitation occurs, and the dry season runs from October to May (Secretaría de Medio Ambiente y Recursos Naturales, 2000). The study area is located within one of the most important wetland systems in southern Mexico, on a strip of land bordered on one side by the gulf and on the other by a coastal lagoon (Secretaría de Medio Ambiente y Recursos Naturales, 2000).
Mosquitoes were collected according to Manrique-Saide et al. (2008). Briefly, nine dogs identified as infected with Dirofilaria sp. by the thick smear technique and the modified Knott's technique (Knott, 1939; Atkins, 2005) were selected with their owners' consent. Verbal informed consent was obtained from each dog owner. The use of animals complied with the ethical standards required by the Mexican government (Norma Oficial Mexicana NOM-042-SSA2-2006 and Ley Para la Protección de la Fauna del Estado de Yucatán). Each dog was placed in a trap (set at its residence) where mosquito collections were carried out for 18 consecutive nights from 18.00 hours to 06.00 hours during August 2007. Traps consisted of metal cages (1.60 × 0.60 × 1.00 m) covered with wire mesh and partially enclosed within a mosquito net. Mosquitoes were removed each morning and transferred to mosquito cages in a laboratory where they were kept at room temperature (25–27 °C) and provided with cottonwool soaked in 10% sucrose solution until they eventually died. Mosquitoes captured in each trap on each day were counted and identified to species and sex according to morphological characteristics (Carpenter & LaCasse, 1955; Darsie & Ward, 2004).
Immediately after death, all female mosquito specimens were individually dissected. Heads, thoraxes and abdomens were teased apart in independent saline droplets, and preparations were examined at 10–40× magnification for the presence of filarial nematodes [particularly first- (L1) and third-instar (L3) larvae, which represent extracellular stages of the larval cycle]. Filarial larvae found in the mosquitoes were identified as Dirofilaria sp. based on morphological characteristics described by Taylor (1960).
After dissection, each mosquito positive for any larval stage of Dirofilaria sp. was transferred to a 1.5-mL Eppendorf tube by carefully rinsing each slide with 200 µL of 70% ethanol. The ethanol was allowed to evaporate and each tube was stored at −20 °C until DNA extraction. DNA was extracted from the majority of samples within 7 days of dissection. Samples were homogenized with a sterile polypropylene pestle in 500 µL of a lysis buffer (200 mm Tris HCl, pH 7.5; 250 mm NaCl; 25 mm EDTA; 0.5% sodium dodecyl sulphate) (Edwards et al., 1991) and then incubated at room temperature (approximately 25 °C) for at least 60 min. After a 5-min centrifugation at 7000 g, in an Eppendorf 5415c centrifuge (Brinkman Instruments, Inc., Westbury, NY, U.S.A.), 300 µL of supernatant was transferred to a fresh tube, an equal volume of isopropyl alcohol was added and the sample incubated at room temperature for 10 min. After a 15-min centrifugation at 16 500 g, the resulting pellet was vacuum-dried in a Savant vacuum-drying system (ThermoFisher Scientific, Inc., Asheville, CA, U.S.A.) and suspended in 50 µL of double-distilled DNAse-RNAse-free water and stored at −20 °C until use.
Each PCR reaction was carried out using the GoTaq Green Master Mix (Promega Corp., Madison, WI, U.S.A.). The DIDR pan-filarial primers described by Rishniw et al. (2006) were used to amplify the ITS2 region of the ribosomal DNA of both D. immitis and Dirofilaria repens (forward primer: 5′-AGT GCG AAT TGC AGA CGC ATT GAG-3′; reverse primer: 5′-AGC GGG TAA TCA CGA CTG AGT TGA-3′). The PCR reaction mix consisted of 10 µL of Green Master Mix, 5 µL of water (provided in the kit), 1 µL of each primer (10 pmol) and 3 µL of DNA template (containing 10–15 ng of total DNA) for a final reaction volume of 20 µL. The PCR programme consisted of a denaturing step at 94 °C for 2 min, 32 cycles of denaturing (30 s at 94 °C), annealing (30 s at 60 °C) and extension (30 s at 72 °C), a final 7-min extension at 72 °C and a final chill at 10 °C in a Techne TC-312 thermal cycler (Barloworld Scientific Ltd, Stone, U.K.). The amplification products (10 µL) were visualized by electrophoresis on a 1.5% agarose gel stained with ethidium bromide and then photographed under ultraviolet light (330 nm) (Ultra Violet Products, Upland, CA, U.S.A.) with an EDAS 290 gel documentation system (Kodak Corp., Rochester, NY, U.S.A.). DNA for D. immitis-positive controls was extracted from adult worms obtained from infected canines. The PCR master mix without any added template DNA was used as a negative control.
In total, 292 female mosquitoes of 12 species (Diptera: Culicidae) were collected from dog-baited traps in Celestún (Table 1). These included Anopheles albimanus, Anopheles crucians, Anopheles pseudopunctipennis, Culex coronator, Culex interrogator, Culex nigripalpus, Culex quinquefasciatus, Culex salinarius, Aedes aegypti, Aedes scapularis, Aedes sollicitans and Aedes taeniorhynchus. As expected, a greater diversity of mosquito species was collected during the rainy season compared with the dry season (Manrique-Saide et al., 2008), but in concurrence with data previously reported, Ae. taeniorhynchus and Cx. quinquefasciatus were the most common species feeding on dogs (Table 1). Aedes taeniorhynchus represented 77.7% of the total number of females collected and together with Cx. quinquefasciatus represented 91.8% of all female mosquitoes captured. During the rainy season, higher levels of blood feeding (measured by the percentage of the total catch for each species that was blood-fed) were observed for both Ae. taeniorhynchus (43.6%) and Cx. quinquefasciatus (46.3%), and a greater diversity of mosquitoes overall were found to have blood-fed.
|Species||Total captured||Total fed, n (%)||Larval infection by microscopy, n (%)||L3 infection in head (infectious), n (%)||D. immitis infection detected by PCR, n (%)|
|Aedes taeniorhynchus||223||227||71 (31.8)||99 (43.6)||23 (10.3)||76 (33.5)||10 (4.5)||23 (10.1)||14 (6.2)|
|Culex quinquefasciatus||40||41||15 (37.5)||19 (46.3)||3 (7.5)||13 (31.7)||0 (0)||0 (0)||12 (29.3)|
|Aedes sollicitans||6||5||2 (33.3)||2 (40.0)||1 (16.6)||1 (20.0)||0 (0)||0 (0)||1 (20.0)|
|Culex interrogator||2||3||2 (100)||0||1 (50.0)||0 (0)||0 (0)||0 (0)||—|
|Aedes aegypti||1||4||0||2 (50.0)||0 (0)||2 (50.0)||0 (0)||0 (0)||1 (25.0)|
|Anopheles pseudopunctipennis||0||5||—||4 (80.0)||—||3 (60.0)||—||0 (0)||1 (20.0)|
|Culex coronator||0||2||—||2 (100)||—||1 (50.0)||—||0 (0)||1 (50.0)|
|Anopheles crucians||0||1||—||1 (100)||—||1 (100)||—||1 (100)||1 (100)|
|Anopheles albimanus||0||1||—||1 (100)||—||1 (100)||—||—||1 (100)|
|Aedes scapularis||0||1||—||1 (100)||—||1 (100)||—||0 (0)||1 (100)|
|Culex nigripalpus||0||1||—||0||—||0 (0)||—||0 (0)||—|
|Culex salinarius||0||1||—||0||—||0 (0)||—||0 (0)||—|
|Total (%)||272||292||90 (33.1)||131 (44.9)||28 (10.3)||99 (33.9)||10 (3.7)||24 (8.2)|
Filarial nematodes (L1–L3) were observed by microscopy in nine of the mosquito species collected during the rainy season (Table 1). In the present study, Ae. aegypti was found to be infected and Cx. interrogator was not, by contrast with a previous report by Manrique-Saide et al. (2008). In addition, during this rainy season collection, five additional species of mosquitoes were found to be infected with filariae, including An. crucians, An. pseudopunctipennis, An. albimanus, Cx. coronator and Ae. scapularis. However, L3 were only observed in Ae. taeniorhynchus and An. crucians (Table 1).
Dirofilaria sp. infection observed in Ae. taeniorhynchus was higher during the rainy season in comparison with that reported for the preceding dry season. The proportion of mosquitoes with infectious L3 larvae in their heads was also higher during the rainy season (Table 1). The L3 infection rates found in Ae. taeniorhynchus during the rainy season (10.1%) are moderate compared with those in other mosquito populations in the western hemisphere, where higher rates have been observed in Cx. quinquefasciatus (37.5%) and Ae. scapularis (23.1%) in Rio de Janeiro, Brazil (Labarthe et al., 1998). However, they remain far higher than previously reported rates of infection in Ae. taeniorhynchus in the Americas, which included rates of 3.5% in Rio de Janeiro, Brazil (Labarthe et al., 1998), 1.1% in Florida, U.S.A. (Sauerman & Nayar, 1983) and 0.7% in North Carolina, U.S.A. (Parker, 1986).
Of 76 Ae. taeniorhynchus found to be positive for Dirofilaria sp. by dissection, 14 were confirmed to be positive for D. immitis by PCR (Fig. 2). This is the first report of D. immitis diagnosed by PCR in natural populations of Ae. taeniorhynchus or any other mosquito species in Mexico and indicates an infection rate for D. immitis (6.2%) that is higher than any infection rate for Ae. taeniorhynchus previously reported in the Americas. The identity of the nematode larvae from the mosquitoes negative for D. immitis by PCR remains uncertain. The use of the pan-filarial primers should have amplified a 484-bp product if D. repens was present, but no such product was detected. These as yet unclassified infections may still include D. repens or filarioid parasites of this genus from wild animals. We expect to investigate this further in subsequent studies using D. repens-specific primers to detect mixed infections within mosquitoes, as well as in collected rodents, opossums and other mammals from this area.
These results confirm the status of Ae. taeniorhynchus as the principal domestic canine dirofilariasis vector in the area, as previously suggested (Manrique-Saide et al., 2008), and indicate that its importance as a vector is constant throughout the year. The transmission cycle is probably intense throughout the year and is maintained by the presence of a highly infected population of canine reservoirs (59.8%) (Caro-González et al., unpublished data, 2008), and an abundant vector species (Ae. taeniorhynchus), whose wetland habitats produce multiple generations of mosquitoes each year. Aedes taeniorhynchus is expected to be an important vector of dirofilariasis in other coastal areas of the Yucatan Peninsula with similar ecological characteristics. Forthcoming studies will investigate the role of mosquito species involved in the domestic cycle of canine dirofilariasis located further inland, where Ae. taeniorhynchus is rare or non-existent.