A highly secure method for rearing Aedes aegypti mosquitoes
© The Author(s) 2018
Received: 16 April 2018
Accepted: 14 May 2018
Published: 23 May 2018
Vector-borne infectious diseases are caused by pathogenic microorganisms transmitted mainly by blood-sucking arthropod vectors. In laboratories, the handling of insects carrying human pathogens requires extra caution because of safety concerns over their escape risk. Based on standard insect containment practices, there have been cases where costly enhancements were required to definitely protect laboratory workers and neighbors from potential infection through mosquito bites. Here, we developed a mosquito rearing method that provides a reliable and cost-effective means to securely contain pathogen-infected females of the yellow fever mosquito Aedes aegypti.
To debilitate the motility of A. aegypti females, mosquitoes were rendered completely flightless by ablation of either wing. The “single-winged” mosquitoes exhibited a severe defect in flying ability and were incubated in a container with inside surfaces covered with a net stretched to approximately 1-mm mesh, which helped the mosquitoes hold on and climb up the wall. In this container, flightless females consistently showed similar blood feeding and egg laying activities to intact females. Eighty-five percent of the flightless mosquitoes survived at 1 week after wing ablation, ensuring feasibility of the use of these mosquitoes for studying pathogen dynamics.
This mosquito rearing method, with a detailed protocol, is presented here and can be readily implemented as a highly secure insectary for vectors carrying human pathogens. For researchers in an environment where highly strict containment practices are mandatory, this method could offer appropriate opportunities to perform research on pathogen–mosquito interactions in vivo.
Vector-borne infectious diseases such as dengue fever and malaria account for one sixth of all illnesses and disabilities around the world, leaving more than half the world’s population at risk . Disease-causing microorganisms, virus, bacteria, protozoan parasites, and worms are transmitted from host animals to humans mainly through blood-sucking arthropod vectors. The vectors can perceive multiple sensory cues including emitted odor, carbon dioxide, and body heat to precisely locate their hosts and feed on blood. In particular, highly motile flying insects such as mosquitoes and flies have the ability to disperse over a long range and efficiently transmit pathogens.
Microorganisms originating from host blood are amplified and disseminated inside vectors, thereafter migrating into the mouthparts, settling in the throat, or invading salivary glands for transmission into new hosts. One of the important measures possibly leading to the success of blocking pathogen transmissions is to understand the vector–pathogen interactions underpinning the pathogen burden in vectors. One of the major impediments for research on pathogen–vector interaction is derived from safety concerns regarding the risk of the inadvertent escape of vectors carrying human pathogens when infectious agents are introduced into vectors. In the case of being on the loose, the vectorial capacity of flying insects associated with high motility and excellent host perception could be a serious threat for laboratory workers. Infected workers could unintentionally become a potential source of pathogens in areas where adequate vectors capable of transmission are indigenous in the wild.
The World Health Organization and others have published laboratory biosafety manuals, which provide practical guidance on biosafety techniques [2, 3]. The insectary should be designed in accordance with the biosafety level determined by the risk group of the pathogen. In addition, appropriate precautions are necessary to manage flying vectors (mostly mosquito species) infected with human pathogens, including containment conditions in which the infectious agents should be safely handled (e.g., multiple doors and air shower booth). Moreover, in keeping with institutional guidelines, risk assessments for the safety of laboratory workers and neighbors should be implemented during infection experiments. In any case, comprehensive safety measures to secure any people should be prepared regardless of the technical proficiency of the workers. It is, therefore, necessary to develop additional equipment and/or methods for handling pathogen-infected flying vectors more securely.
Here, we describe an improved method for rearing mosquitoes without the risk of escape, which thereby robustly secures the safety of people in laboratory. Female mosquitoes are rendered flightless by cutting off either wing, resulting in little adverse effect on their fitness. The attenuated mobility of the flightless females makes it less laborious to implement strict containment processes. This procedure proposed here can mitigate the risk of inadvertent infection of laboratory workers and neighbors when applied to research involving mosquitoes carrying human pathogens.
This study was carried out in accordance with the Guideline for Laboratory Animals of The Jikei University School of Medicine and the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions of the jurisdiction of the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The protocol was approved by the Committee on the Animal Experiments of The Jikei University School of Medicine (Permit Number 2016-051).
Rearing of A. aegypti mosquitoes
The A. aegypti strain employed in this study originated from the Liverpool strain and was a gift from Dr. Ryuichiro Maeda (Obihiro University of Agriculture and Veterinary Medicine). Eggs obtained using a standard procedure were hatched in reverse osmosis (RO) water. The first instar larvae were transferred to a plastic container with RO water and daily fed with fish food (Kyorin Co., Ltd., Hikari [#4971618-013378]). The larvae were kept in an insectary room set at 27 °C, and water in the container was refreshed every 2–3 days. Pupae were harvested in a plastic cup and placed within a cage (bottom 27 cm × 27 cm, top 25 cm × 25 cm, height 27 cm), in which a filter paper (Whatman, Grade 4, 1004-125) soaked in 10% sucrose solution was placed in a 50-mL glass flask. The cage was kept in an incubator (Panasonic, MIR254-PJ) set at 27 °C with humidity over 90% in cycles of 12 h light and 12 h darkness. The cup left in the cage was put out 2 days after starting incubation. The sucrose solution was changed every 3–4 days.
Preparation and maintenance of flightless mosquitoes
The customized container was constructed from a cardboard cup (TOKAN, 31-476-001, top diameter 97.2 mm × height 100.0 mm × bottom diameter 74.3 mm), its lid (TOKAN, diameter 97.2 mm), a disposable polyester/polyurethane sink drain net (width 15.5 cm × length 21 cm), and a rubber band. The schematic procedure to fabricate the container is shown in Fig. 1b–d and Additional file 1: Figure S1A. A donut-shaped lid was made by cutting a round hole (approximately 60 mm diameter) in the original lid without the outer frame, and the disposable sink drain net (mesh hole size < 1 mm) was used to cover the hole (Additional file 1: Figure S1B). A cup without a bottom was prepared to insert the net-containing, donut-shaped lid (Additional file 1: Figure S1A and C). The open side of the net was fastened using the outer frame of original lid (Additional file 1: Figure S1D and E). The container was closed with the excess part of the net using a rubber band (Additional file 1: Figure S1F–H).
A cotton pad cut to fit the size of the lid was soaked with 10% sucrose and placed on the container. The cotton pad was avoided from dripping and changed every 3–4 days to fend off fungi contamination. The container was kept inside a plastic box (length 23.8 × width 15 × height 16.4 cm) as a secondary security barrier. The box was kept in an incubator set at 27 °C with humidity over 90% in cycles of 12 h light and 12 h darkness.
Observation of flight ability and posture
To observe the body posture of intact or flightless females in a resting state, about 20–30 mosquitoes were collected in a customized container covered in all inside surfaces with a net. The container was tapped a few times to make all the mosquitoes release hold of the wall. Five minutes later, the mosquitoes resting at the wall (top, side, or bottom) were observed for 10 s.
The mosquitoes 1 day after wing surgery and the controls were fed on blood from mice through an artificial feeding system. Briefly, the blood of male BALB/cAJc1 mice was freshly prepared on the day of feeding by using a syringe loaded with an aliquot of 1 mg/mL heparin in PBS. The equipment for blood feeding was a 25 cm2 cell culture flask (Thermo Fisher, 156367) of which one side was covered with Parafilm M (Bemis, PM996) stretched to about four times its original area. Mice blood (2 mL) was applied into a space between the Parafilm M and the surface of the flask using a syringe. The flask itself was filled with a warmed (42 °C) solution of 100% glycerol (Wako), which was changed every 30 min to maintain the temperature of the blood. A container with ~ 30 mosquitoes was placed in a transparent bucket (top diameter 31 cm × height 27 cm × bottom diameter 22 cm) into which CO2 was supplied through a CO2 pad fixed under the lid to activate the blood foraging of mosquitoes. The blood feeder was placed onto the mosquito container for 1 h, ensuring that the membrane side was in direct contact with the net.
Measurement of the amount of blood ingested by mosquitoes
Immediately after blood feeding, all the mosquitoes were anesthetized using CO2 and the number of the fully engorged mosquitoes was counted. Each blood-fed mosquito was then individually placed in a weighed PCR tube and compared with control unfed mosquitoes. The weight of each tube containing a single mosquito was measured using a microbalance (Sartorius, MSA6-6S-OTR-DM). The amount of ingested blood was estimated by subtracting the mean weight of the control unfed mosquitoes from the weight of engorged ones.
One day after blood feeding, only fully engorged females were collected and placed into a mosquito container. Two days after blood feeding, each mosquito was individually transferred into a polystyrene vial (Chiyoda Science, KBF-1S, diameter: 25 mm × height: 96 mm) in which a cellulose acetate plug (Fisher Scientific, AS271) filled with RO water was squeezed into the bottom and a piece of filter paper (Whatman, grade 1) was then placed on the plug. The vial was closed with a cotton pad soaked with 10% sucrose. Two days after transferring, the number of eggs laid on the filter paper was counted.
Twenty to 30 fully engorged females were transferred into a mosquito container, which was placed in a plastic box. To avoid contamination by fungi and other microbes, a sheet of wrap was seated on the bottom of the box and changed occasionally to keep the inside of the box clean. Sucrose solution (10%) was supplied with a cotton pad placed on the top of the container and changed every 3–4 days. Another cotton pad, which was only filled with RO water, was set on the bottom of the container for mosquitoes to lay egg on. Six to 8 days after blood feeding, the cotton pad on the bottom was replaced with new one with 10% sucrose solution to increase the opportunity for nutrient uptake by aged mosquitoes. The sucrose solution was changed every 3–4 days. The box with a mosquito container was kept in an incubator set at 27 °C with humidity over 90% in cycles of 12 h light and 12 h darkness.
All statistical analyses (chi-square test, Student’s t test, Kaplan–Meier log-rank test) were performed using R version 3.4.1 (www.r-project.org).
Flight ability test of mosquitoes after wing ablation
Dropped inside circle
Blood feeding efficiency of flightless females
Blood feeding from
Survival of flightless females in 8 days after blood feeding
Net covering wall of a container
The secure method for laboratory experiments using pathogen-infected mosquitoes shown in this report provided a reliable and cost-effective way to securely contain pathogen-infected females. Analyzing emerging and re-emerging arboviruses, such as Zika and dengue virus, in Aedes mosquitoes is indispensable for comprehensive understanding of pathogen–vector interactions, which could contribute to establishing effective management strategies of infectious diseases caused by arboviruses. The artificial blood feeding method is a versatile system for introducing a variety of arboviruses into vector mosquitoes. The advantages derived from the blood sucking method can enhance knowledge about arbovirus dynamics in mosquitoes. Histological and cytopathological analyses have shown the specific temporal and spatial distributions of arthropod-borne viruses in mosquitoes [9–15] and detailed evolutional analyses of viral genomic RNAs during the course of traversal across mosquito tissues revealed selective pressures from imposing repeated population bottlenecks [16, 17]. To increase the safety of mosquito handling during and after artificial blood feeding, removing mosquito wings is not a completely novel idea because it is quite obvious that wingless mosquitoes are unable to fly anymore. The fragility of mosquitoes, however, has often been problematic when applying physical manipulations such as wing ablation to mosquitoes [18, 19]. To maintain partially disabled mosquitoes alive for long enough for various experiments, a mesh net was employed to cover one wall of the mosquito reservoir intramurally, providing adequate scaffolding for mosquitoes to hold on to. Although advanced analyses related to mosquito flying, e.g., analysis of courtship behavior, cannot be accomplished, the flightless females are extremely safer even outside the special mosquito reservoir when handled with lab coat and gloves following a standard procedure and a diverse range of pathogen–vector interactions can be explored using this method. Practically, the slightly higher mortality of flightless females observed in our study would not compromise the observation of virus behavior in mosquitoes, including virus proliferation and dissemination, because it takes up to 2 weeks for mosquitoes to become competent to transmit viruses after ingestion of a viremic blood meal [20–22]. Although the egg hatching rate is remained to be examined, the fertility of flightless females was sufficient to lay eggs and should contribute to elucidating the interactions of pathogens with the host reproductive system, e.g., vertical transmissions of flaviviruses [23–25]. The applicability of this method should not be limited to cover only Aedes mosquitoes, rather other mosquito species transmitting pathogens, although modifications of this method might be required to adapt to species-dependent characteristics .
Although further examination regarding the pathogen infection in the flightless mosquitoes remains to be elucidated, the mosquito rearing method for Aedes species presented here was extremely secure, and the protocol was inexpensive and less labor intensive, making it possible to perform intensive studies using mosquitoes carrying human hazardous pathogens, even in counties where highly secure containment systems are mandatory. This protocol could also be applicable to Anopheles and Culex mosquitoes, which are major vectors for other devastating diseases, such as malaria, West Nile fever, and Japanese encephalitis.
We thank all the members in the Kanuka laboratory for their assistance. We are grateful to Chisako Sakuma for the handling of A. aegypti, Kazutomo Ogata for the husbandry of the mosquitoes, and Erisha Saiki for the support with artificial blood feeding methods.
This work was supported by JSPS KAKENHI Grant Number 16K15065 (MO), The Jikei University Graduate Research Fund (HK), and the International Collaborative Research Program for Tackling the NTDs Challenges in African countries from Japan Agency for Medical Research and Development, AMED (JP17jm0510002h0003) (HK).
Availability of data and materials
The datasets supporting the conclusion of this study are included within the article and the supplemental data.
MO and HK conceived the project. MO and HK designed the experiments, which MO performed. MO and HK wrote the manuscript. Both authors approved the final manuscript.
Ethics approval and consent to participate
The authors declare that they have no competing interests.
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- Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet Lond Engl. 2012;380:2095–128. https://doi.org/10.1016/S0140-6736(12)61728-0.View ArticleGoogle Scholar
- WHO|Laboratory Biosafety Manual-Third Edition [Internet]. Available: http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/. Cited 13 Mar 2018.
- American Committee of Medical Entomology, American Society of Tropical Medicine and Hygiene. Arthropod containment guidelines. A project of the American Committee of Medical Entomology and American Society of Tropical Medicine and Hygiene. Vector Borne Zoonotic Dis Larchmt N. 2003;3:61–98. https://doi.org/10.1089/153036603322163448.View ArticleGoogle Scholar
- Lyski ZL, Saredy JJ, Ciano KA, Stem J, Bowers DF. Blood feeding position increases success of recalcitrant mosquitoes. Vector Borne Zoonotic Dis Larchmt N. 2011;11:1165–71. https://doi.org/10.1089/vbz.2010.0164.View ArticleGoogle Scholar
- Volozina NV. The effect of the amount of blood taken and additional carbohydrate nutrition on oogénesis in females of blood-sucking mosquitoes of the genus Aedes (Diptera, Culicidae) of various weights and ages. Entomol Rev. 1967;46:27–32. Available: https://www.cabdirect.org/cabdirect/abstract/19682902392 Google Scholar
- Joy TK, Arik AJ, Corby-Harris V, Johnson AA, Riehle MA. The impact of larval and adult dietary restriction on lifespan, reproduction and growth in the mosquito Aedes aegypti. Exp Gerontol. 2010;45:685–90. https://doi.org/10.1016/j.exger.2010.04.009.View ArticlePubMedPubMed CentralGoogle Scholar
- Xu C, Legros M, Gould F, Lloyd AL. Understanding uncertainties in model-based predictions of Aedes aegypti population dynamics. O’Neill S, editor. PLoS Negl Trop Dis. 2010;4:e830. https://doi.org/10.1371/journal.pntd.0000830.View ArticlePubMedPubMed CentralGoogle Scholar
- Canyon DV, Hii JL, Muller R. Effect of diet on biting, oviposition, and survival of Aedes aegypti (Diptera: Culicidae). J Med Entomol. 1999;36:301–8.View ArticlePubMedGoogle Scholar
- Whitfield SG, Murphy FA, Sudia WD. St. Louis encephalitis virus: an ultrastructural study of infection in a mosquito vector. Virology. 1973;56:70–87.View ArticlePubMedGoogle Scholar
- Gaidamovich SY, Khutoretskaya NV, Lvova AI, Sveshnikova NA. Immunofluorescent staining study of the salivary glands of mosquitoes infected with group A arboviruses. Intervirology. 1973;1:193–200.View ArticlePubMedGoogle Scholar
- Larsen JR, Ashley RF. Demonstration of Venezuelan equine encephalomyelitis virus in tissues of Aedes aegypti. Am J Trop Med Hyg. 1971;20:754–60.View ArticlePubMedGoogle Scholar
- Lamotte LC, Japanese B. Encephalitis virus in the organs of infected mosquitoes. Am J Hyg. 1960;72:73–87.PubMedGoogle Scholar
- Bowers DF, Abell BA, Brown DT. Replication and tissue tropism of the alphavirus Sindbis in the mosquito Aedes albopictus. Virology. 1995;212:1–12. https://doi.org/10.1006/viro.1995.1447.View ArticlePubMedGoogle Scholar
- Weaver SC, Scott TW, Lorenz LH, Lerdthusnee K, Romoser WS. Togavirus-associated pathologic changes in the midgut of a natural mosquito vector. J Virol. 1988;62:2083–90.PubMedPubMed CentralGoogle Scholar
- Weaver SC, Lorenz LH, Scott TW. Pathologic changes in the midgut of Culex tarsalis following infection with western equine encephalomyelitis virus. Am J Trop Med Hyg. 1992;47:691–701.View ArticlePubMedGoogle Scholar
- Grubaugh ND, Weger-Lucarelli J, Murrieta RA, Fauver JR, Garcia-Luna SM, Prasad AN, et al. Genetic drift during systemic arbovirus infection of mosquito vectors leads to decreased relative fitness during host switching. Cell Host Microbe. 2016;19:481–92. https://doi.org/10.1016/j.chom.2016.03.002.View ArticlePubMedPubMed CentralGoogle Scholar
- Lequime S, Fontaine A, Ar Gouilh M, Moltini-Conclois I, Lambrechts L. Genetic drift, purifying selection and vector genotype shape dengue virus intra-host genetic diversity in mosquitoes. PLoS Genet. 2016;12:e1006111. https://doi.org/10.1371/journal.pgen.1006111.View ArticlePubMedPubMed CentralGoogle Scholar
- Helinski MEH, Parker AG, Knols BGJ. Radiation biology of mosquitoes. Malar J. 2009;8(Suppl 2):S6. https://doi.org/10.1186/1475-2875-8-S2-S6.View ArticlePubMedPubMed CentralGoogle Scholar
- Komp WHW. Guide to mosquito identification for field workers engaged in malaria control in the United States. Public Health Rep 1896–1970. 1923;38:1061–80. https://doi.org/10.2307/4576745.View ArticleGoogle Scholar
- Dubrulle M, Mousson L, Moutailler S, Vazeille M, Failloux A-B. Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after oral infection. PLoS One. 2009;4:e5895. https://doi.org/10.1371/journal.pone.0005895.View ArticlePubMedPubMed CentralGoogle Scholar
- Tjaden NB, Thomas SM, Fischer D, Beierkuhnlein C. Extrinsic incubation period of dengue: knowledge, backlog, and applications of temperature dependence. PLoS Negl Trop Dis. 2013;7:e2207. https://doi.org/10.1371/journal.pntd.0002207.View ArticlePubMedPubMed CentralGoogle Scholar
- Chan M, Johansson MA. The incubation periods of dengue viruses. PLoS One. 2012;7:e50972. https://doi.org/10.1371/journal.pone.0050972.View ArticlePubMedPubMed CentralGoogle Scholar
- Rosen L. Mechanism of vertical transmission of the dengue virus in mosquitoes. C R Acad Sci III. 1987;304:347–50.PubMedGoogle Scholar
- Watts DM, Eldridge BF. Transovarial transmission of arboviruses by mosquitoes: a review. Med Biol. 1975;53:271–8.PubMedGoogle Scholar
- Aitken TH, Tesh RB, Beaty BJ, Rosen L. Transovarial transmission of yellow fever virus by mosquitoes (Aedes aegypti). Am J Trop Med Hyg. 1979;28:119–21.View ArticlePubMedGoogle Scholar