Abstract

INTRODUCTION:

The lack of highly-productive Nyssorhynchus darlingi laboratory colonies limits some studies. We report the first well-established laboratory colony of Ny. darlingi in Brazil.

METHODS:

Mosquitoes were collected from Porto Velho and were reared at the Laboratory of Fiocruz/RO. After induced mating by light stimulation in the F1 to F6, the subsequent generations were free mating. Larvae were reared in distilled water and fed daily until pupation.

RESULTS:

In 11 generations, the colony produced a high number of pupae after the F5 generation.

CONCLUSIONS:

These results demonstrate the potential for permanently establishing Ny. darlingi colonies for research purposes in Brazil.

Keywords:
Nyssorhynchus darlingi; Colony establishment; Brazil; Malaria vector

Nyssorhynchus darlingi is the most significant malaria vector in the Amazon region and one of the most efficient malaria vectors in the world¹1. Hiwat H, Bretas G. Ecology of Anopheles darlingi Root with respect to vector importance: a review. Parasit Vectors. 2011;4(1):177-90.. However, important entomological aspects of Ny. darlingi remain unknown because continuous propagation of this species is difficult to achieve in the laboratory. The difficulty in developing colonies of some mosquito species is due to challenges in finding the right conditions for successful mating by non-artificial methods and selecting appropriate populations for their ability to mate in a restricted space²2. Benedict MQ, Knols BG, Bossin HC, Howell PI, Mialhe E, Caceres C, et al. Colonisation and mass rearing: learning from others. Malar J. 2009;8(Suppl 2):S4-15.. A free-mating laboratory colony of Ny. darlingi was established in Peru five years ago³3. Moreno M, Tong C, Guzmán M, Chuquiyauri R, Llanos-Cuentas A, Rodriguez H, et al. Infection of laboratory-colonized Anopheles darlingi mosquitoes by Plasmodium vivax. Am J Trop Med Hyg. 2014;90(4):612-6.^,44. Villarreal-Treviño C, Vásquez GM, López-Sifuentes VM, Escobedo-Vargas K, Huayanay-Repetto A, Linton YM, et al. Establishment of a free-mating, long-standing and highly productive laboratory colony of Anopheles darlingi from the Peruvian Amazon. Malar J . 2015;14(1):227-39., but, until now, no free-mating laboratory colonies have been successfully established outside Peru. Previous attempts to establish Ny. darlingi colonies have been beset with difficulties⁵5. Giglioli G. Laboratory colony of Anopheles darlingi. J Natl Malar Soc. 1947;6(3):159-64.^,66. Bates M. The laboratory colonization of Anopheles darlingi. J Natl Malar Soc . 1947;6(3):155-8.^,77. Buralli GM, Bergo ES. Maintenance of Anopheles darlingi root, 1926 colony, in the laboratory. Rev Inst Med Trop Sao Paulo. 1988;30(3):157-64.. In Brazil, no Ny. darlingi colony has ever been permanently established, and currently, no colonies are available for research purposes.

A highly productive laboratory colony of Ny. darlingi is an essential resource for malaria research in the Brazilian Amazon; this is especially true for research into malaria caused by Plasmodium vivax, which has been the predominant species causing malaria in Brazil since the mid-1990s⁸8. Oliveira-Ferreira J, Lacerda MVG, Brasil P, Ladislau JLB, Tauil PL, Daniel-Ribeiro CT. Malaria in Brazil: an overview. Malar J . 2010;9(1):115-30.. In endemic regions of the Brazilian Amazon, experimental studies into the biology of P. vivax-Nyssorhynchus interactions have been limited by the lack of laboratory-reared Ny. darlingi mosquitoes.

In the few studies that have been conducted, P. vivax-Ny. darlingi interactions (in the form of direct feeds or membrane feeding assays) have only been observed in laboratory-reared F1 generations⁹9. Klein TA, Lima JPB, Tada MS, Miller R. Comparative susceptibility of anopheline mosquitoes in Rondonia, Brazil to infection byPlasmodium vivax. Am J Trop Med Hyg . 1991;45(4):463-70.^,1010. Da Silva ANM, Santos CCB, Lacerda RN, Machado RLD, Povoa MM. Susceptibility of Anopheles aquasalis and An.darlingi to Plasmodium vivax VK210 and VK247. Mem Inst Oswaldo Cruz. 2006;101(5):547-50.^,1111. Rios-Velásquez CM, Martins-Campos KM, Simões RC, Izzo T, Dos Santos EV, Pessoa FAC, et al. Experimental Plasmodium vivax infection of key Anopheles species from the Brazilian Amazon. Malar J . 2013;12(1):460-70.. Due to the importance of establishing such a mosquito colony in Brazil, a self-mating Ny. darlingi colony was initiated in March 2018 at the Entomology Laboratory of Fiocruz Rondônia. Colony initiation was achieved using an adapted version of the natural induction technique developed for Ny. darlingi in the Peruvian Amazon³3. Moreno M, Tong C, Guzmán M, Chuquiyauri R, Llanos-Cuentas A, Rodriguez H, et al. Infection of laboratory-colonized Anopheles darlingi mosquitoes by Plasmodium vivax. Am J Trop Med Hyg. 2014;90(4):612-6.^,44. Villarreal-Treviño C, Vásquez GM, López-Sifuentes VM, Escobedo-Vargas K, Huayanay-Repetto A, Linton YM, et al. Establishment of a free-mating, long-standing and highly productive laboratory colony of Anopheles darlingi from the Peruvian Amazon. Malar J . 2015;14(1):227-39..

The colony was initiated using 320 adult females that were collected by BG-Malaria traps and protected human landing in a peridomicile environment (08°39.145’ S/063°56.155’ W), 31 km from Porto Velho City, Rondônia, Brazil. Approximately 800-2,000 three to five-day-old Ny. darlingi males and females (ratio 1:1) of each generation were placed in screened cages (61 × 61 × 61 cm) and fed honey-water solution (15%) ad libitum. Copulation induction was conducted on five to seven consecutive evenings, and each cage was exposed to a light beam (four cycles of 10 min on and 10 min off), the insectary temperature was reduced to 24 ± 1 °C at 6 p.m., and humidity was maintained at 80%. In the initial phases, these cycles were performed manually using a flashlight, but after the F3 generation, an automated system was developed and implemented. Following the final light cycle, female mosquitoes were fed with chicken blood for 15 min during the first 4-6 days of induction. Oviposition was induced by cutting one wing until natural oviposition occurred. Copulation induction was conducted for 5 days (up to the F6 generation), after which free-mating was confirmed and natural oviposition was observed in the F5 generation. Larvae were reared in pans (30.3 × 22.1 × 7.5 cm) containing approximately 200 larvae per pan, and larvae were fed with Tetra Marine Granules^®.

Species identity (F1 to F4 generations, 10 specimens per generations) was confirmed by DNA barcoding¹²12. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Marine Biol. Biotechnol. 1994;3(5):294-9. and the colony exhibited 99-100% identity with Ny. darlingi sequences from Peru (KP193458), Venezuela (KC555065), and Brazil (JF923693) available in GenBank.

Preliminary evaluation using infected human blood in artificial membrane assays confirmed that Ny. darlingi in the colony (F5, F6, F7, F10, F11, F12) are susceptible to infection by P. vivax. Oocysts ranged from 1 to 692 per mosquito (mean ± SD = 69.3 ± 90.2), and sporozoites ranged from 200 to 43,440 per mosquito (mean ± SD = 8,580.5 ± 8,462.1).

At present, our colony has been maintained for 13 generations over the course of 11 months. Pupae production has been high since the F5 generation, and copulation induction and oviposition are no longer necessary (Figure 1), which indicates the development of a stenogamic colony. A drop in pupae production in the F8 and F9 generations occurred as a result of the holiday period in Brazil (December and January). Pupae survival rates range between 83.3 and 97.1%, which indicates that the mosquitoes have adapted to the laboratory conditions.

FIGURE 1
Pupae production of Nyssorhynchus darlingi reared for 11 generations in the Fiocruz Rondônia insectary in Porto Velho, Brazil.

These results demonstrate the potential for permanently establishing Ny. darlingi colonies for research purposes in all parts of Brazil. With colony mosquitoes, experiments will no longer be limited to the Brazilian Amazon, where P. vivax patients are often used for ex vivo infection experiments.

Our Ny. darlingi colony has allowed us to study unknown aspects of this important vector’s biology, such as circadian cycle behavior, the role of P. vivax asymptomatic parasite carriers, the role microbiota play in Ny. darlingi infection, and the ability of potential drugs to block malaria transmission.

Going forward, future generations of the colony will continue to be used for P. vivax infection experiments, and we also hope to analyze genetic variation between each generation and between future colonies.

Our main purpose in creating this colony has been to create a resource that will give the entire scientific community access to a malaria vector that is of great significance in the Amazon.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. Dionicia Gamboa (Universidad Peruana Cayetano Heredia, Lima, Peru), Dr. Manuela Herrera (Universidad Peruana Cayetano Heredia, Lima, Peru), and Dr. Joseph Vinetz (Yale School of Medicine, USA) for their collaboration.

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