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Establishment of a free-mating, long-standing and highly productive laboratory colony of Anopheles darlingi from the Peruvian Amazon.

Villarreal-Treviño C, Vásquez GM, López-Sifuentes VM, Escobedo-Vargas K, Huayanay-Repetto A, Linton YM, Flores-Mendoza C, Lescano AG, Stell FM - Malar. J. (2015)

Bottom Line: Natural copulation was successfully induced in F1 adults under a thermoperiod of 30 ± 1 °C during the day and 25 ± 1 °C at night, and with a 30-min LED light stimulation period at dusk.By March 2015, the An. darlingi colony has been successfully reared to the F(26) generation.This rearing methodology may be a transferable, cost-effective alternative to labour-intensive forced mating practices widely used in maintaining other Anopheles colonies.

View Article: PubMed Central - PubMed

Affiliation: Centro Regional de Investigación en Salud Pública/Instituto Nacional de Salud Pública (CRISP/INSP), Tapachula, Chiapas, Mexico. cvilla@insp.mx.

ABSTRACT

Background: Anopheles darlingi is the main malaria vector in the Amazon region and is among the most efficient malaria vectors worldwide. However, due to the lack of a well-established laboratory colony, key control-relevant aspects of the bionomics, behaviour, genetics, and vector-parasite relationships of An. darlingi remain unknown. Here, biological parameters that had been successful in initiating other Anopheles colonies were optimized and improved for An. darlingi, with the aim of establish a free-mating, stable, and highly productive laboratory colony.

Methods: Wild An. darlingi adult females were field collected from Zungarococha, Loreto Department, Peru (03°49'32.40″S, 73°21'00.08″W), and taken to the NAMRU-6 Insectary in Iquitos where F(1) offspring were produced and reared. Natural copulation was successfully induced in F1 adults under a thermoperiod of 30 ± 1 °C during the day and 25 ± 1 °C at night, and with a 30-min LED light stimulation period at dusk. Oviposition success was enhanced using egg-laying containers with a dark-coloured surface. Larval feeding regimes were standardized for optimal larval development. Optimized copulation induction methods were used to facilitate mating in An. darlingi until the F(10) generation. No copulation induction assistance was needed in subsequent generations.

Results: In 19 generations, the An. darlingi colony produced a total of 763,775 eggs; 441,124 larvae; 248,041 pupae; and 231,591 adults. A mean of 0.56 sexual encounters/female/cage (n = 36 cages) was recorded across the first ten generations (F(1)-F(10)). A mean insemination rate of 54.7 % (n = 5,907 females) ranging from 43.6 % (F(2)) to 66.6 % (F(10)) was recorded across nine generations (F(2)-F(10)). Free-mating was casually observed in the F(8) generation, and subsequently confirmed in the F(9) and F(10) generations; comparable insemination rates and egg laying between stimulated (51.6 %, 12.9 eggs/female), and non-stimulated (52.3 %, 11.2 eggs/female) females were recorded. The time from egg to adult development ranged from 10 to 20 days. Moreover, the colony was relocated to a new laboratory within Iquitos in the F(14) generation without any noted changes in its productivity. By March 2015, the An. darlingi colony has been successfully reared to the F(26) generation.

Conclusions: This constitutes the first report of a free-mating, highly productive, and long-standing An. darlingi laboratory colony established through natural copulation induction, which will support critical malaria research. This rearing methodology may be a transferable, cost-effective alternative to labour-intensive forced mating practices widely used in maintaining other Anopheles colonies.

No MeSH data available.


Related in: MedlinePlus

Copulation induction period and number of natural copulations observed per 100 An. darlingi F1 adult females kept in medium (46 × 46 × 46 cm) and large (61 61 × 61 cm) cages under laboratory conditions in the NAMRU-6 insectary in Iquitos, Peru
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Fig1: Copulation induction period and number of natural copulations observed per 100 An. darlingi F1 adult females kept in medium (46 × 46 × 46 cm) and large (61 61 × 61 cm) cages under laboratory conditions in the NAMRU-6 insectary in Iquitos, Peru

Mentions: During first attempts of applying and optimizing thermoperiod and light exposure regimes used for An. pseudopunctipennis [22] in February 2013, it was observed, and digitally recorded for the first time An. darlingi natural copulation under laboratory conditions. This significant milestone is publicly available in Additional file 1A, and documents the sexual behavior of An. darlingi for the first time. In this first experiment, copulation induction was tested in medium and large screened cages. In both cage sizes, copulation increased from day 1 to day 4, and then decreased as shown by the daily number of natural copulations (Table 1) and the number of copulations per 100 females (Fig. 1). In the medium-sized cage, the highest number of natural copulations was recorded on day 4 (15 total: two per 100 females), and the lowest number on day 6 (four total: 0.5 per 100 females). Copulation was substantially higher in the large cage on days 2–4 (60–100 total: 5–8 per 100 females). The number of copulations per 100 females (mean ± standard deviation) on days 2–4 was significantly higher in the large cage (6.3 ± 1.8) than in the medium cage (1.5 ± 0.5) (t-test: p = 0.0238). Therefore, large cages with a density of approximately 2,500 adult mosquitoes were used since to provide the best conditions for natural copulation in subsequent generations. Copulation induction was conducted only for five days up to the F10 generation when free-mating was confirmed.Table 1


Establishment of a free-mating, long-standing and highly productive laboratory colony of Anopheles darlingi from the Peruvian Amazon.

Villarreal-Treviño C, Vásquez GM, López-Sifuentes VM, Escobedo-Vargas K, Huayanay-Repetto A, Linton YM, Flores-Mendoza C, Lescano AG, Stell FM - Malar. J. (2015)

Copulation induction period and number of natural copulations observed per 100 An. darlingi F1 adult females kept in medium (46 × 46 × 46 cm) and large (61 61 × 61 cm) cages under laboratory conditions in the NAMRU-6 insectary in Iquitos, Peru
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465318&req=5

Fig1: Copulation induction period and number of natural copulations observed per 100 An. darlingi F1 adult females kept in medium (46 × 46 × 46 cm) and large (61 61 × 61 cm) cages under laboratory conditions in the NAMRU-6 insectary in Iquitos, Peru
Mentions: During first attempts of applying and optimizing thermoperiod and light exposure regimes used for An. pseudopunctipennis [22] in February 2013, it was observed, and digitally recorded for the first time An. darlingi natural copulation under laboratory conditions. This significant milestone is publicly available in Additional file 1A, and documents the sexual behavior of An. darlingi for the first time. In this first experiment, copulation induction was tested in medium and large screened cages. In both cage sizes, copulation increased from day 1 to day 4, and then decreased as shown by the daily number of natural copulations (Table 1) and the number of copulations per 100 females (Fig. 1). In the medium-sized cage, the highest number of natural copulations was recorded on day 4 (15 total: two per 100 females), and the lowest number on day 6 (four total: 0.5 per 100 females). Copulation was substantially higher in the large cage on days 2–4 (60–100 total: 5–8 per 100 females). The number of copulations per 100 females (mean ± standard deviation) on days 2–4 was significantly higher in the large cage (6.3 ± 1.8) than in the medium cage (1.5 ± 0.5) (t-test: p = 0.0238). Therefore, large cages with a density of approximately 2,500 adult mosquitoes were used since to provide the best conditions for natural copulation in subsequent generations. Copulation induction was conducted only for five days up to the F10 generation when free-mating was confirmed.Table 1

Bottom Line: Natural copulation was successfully induced in F1 adults under a thermoperiod of 30 ± 1 °C during the day and 25 ± 1 °C at night, and with a 30-min LED light stimulation period at dusk.By March 2015, the An. darlingi colony has been successfully reared to the F(26) generation.This rearing methodology may be a transferable, cost-effective alternative to labour-intensive forced mating practices widely used in maintaining other Anopheles colonies.

View Article: PubMed Central - PubMed

Affiliation: Centro Regional de Investigación en Salud Pública/Instituto Nacional de Salud Pública (CRISP/INSP), Tapachula, Chiapas, Mexico. cvilla@insp.mx.

ABSTRACT

Background: Anopheles darlingi is the main malaria vector in the Amazon region and is among the most efficient malaria vectors worldwide. However, due to the lack of a well-established laboratory colony, key control-relevant aspects of the bionomics, behaviour, genetics, and vector-parasite relationships of An. darlingi remain unknown. Here, biological parameters that had been successful in initiating other Anopheles colonies were optimized and improved for An. darlingi, with the aim of establish a free-mating, stable, and highly productive laboratory colony.

Methods: Wild An. darlingi adult females were field collected from Zungarococha, Loreto Department, Peru (03°49'32.40″S, 73°21'00.08″W), and taken to the NAMRU-6 Insectary in Iquitos where F(1) offspring were produced and reared. Natural copulation was successfully induced in F1 adults under a thermoperiod of 30 ± 1 °C during the day and 25 ± 1 °C at night, and with a 30-min LED light stimulation period at dusk. Oviposition success was enhanced using egg-laying containers with a dark-coloured surface. Larval feeding regimes were standardized for optimal larval development. Optimized copulation induction methods were used to facilitate mating in An. darlingi until the F(10) generation. No copulation induction assistance was needed in subsequent generations.

Results: In 19 generations, the An. darlingi colony produced a total of 763,775 eggs; 441,124 larvae; 248,041 pupae; and 231,591 adults. A mean of 0.56 sexual encounters/female/cage (n = 36 cages) was recorded across the first ten generations (F(1)-F(10)). A mean insemination rate of 54.7 % (n = 5,907 females) ranging from 43.6 % (F(2)) to 66.6 % (F(10)) was recorded across nine generations (F(2)-F(10)). Free-mating was casually observed in the F(8) generation, and subsequently confirmed in the F(9) and F(10) generations; comparable insemination rates and egg laying between stimulated (51.6 %, 12.9 eggs/female), and non-stimulated (52.3 %, 11.2 eggs/female) females were recorded. The time from egg to adult development ranged from 10 to 20 days. Moreover, the colony was relocated to a new laboratory within Iquitos in the F(14) generation without any noted changes in its productivity. By March 2015, the An. darlingi colony has been successfully reared to the F(26) generation.

Conclusions: This constitutes the first report of a free-mating, highly productive, and long-standing An. darlingi laboratory colony established through natural copulation induction, which will support critical malaria research. This rearing methodology may be a transferable, cost-effective alternative to labour-intensive forced mating practices widely used in maintaining other Anopheles colonies.

No MeSH data available.


Related in: MedlinePlus