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Rearing of Culex spp. and Aedes spp. Mosquitoes
库蚊和伊蚊的饲养   

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Abstract

Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements. Here, we present a standard protocol for the rearing of Culex spp. and Aedes spp. mosquitoes and maintenance of the mosquito colony.

Keywords: Mosquito(蚊子), Egg(卵), Larva(幼虫), Pupa(蛹), Adult(成蚊), Colony(集落), Blood-feeding(吸血), Aedes(伊蚊), Culex(库蚊)

Background

Mosquitoes undergo complete metamorphosis with four life stages: egg, larva, pupa, and adult. The immature stages are always aquatic. Successful maintenance of mosquitoes in the laboratory depends on providing conditions that are optimal for each developmental stage. These requirements will vary with species, and in fact, many mosquito species, such as Cx. restuans, have not been colonized successfully in the lab. Colony maintenance is a labor-intensive process requiring time and attention to detail in handling and record keeping. When standard protocols are successful, the fitness of the mosquitoes is maintained and their suitability for repeatable experimentation is optimal. This protocol is designed for colonization and maintenance of fresh water Culex species and container-breeding Aedes species. It has been used successfully with Cx. pipiens, Cx, quinquifasciatus, Cx. tarsalis, Ae. aegypti, Ae. albopictus, Ae. triseriatus, and Ae. japonicus, among others.

Since most diseases transmitted by mosquitoes are BSL-2 and BSL-3 biological agents, experimental research with these vectors requires containment based on established guidelines (Benedict et al., 2004) as well as cooperation with institutional biosafety committees. Work with non-indigenous species also requires containment that assures no escape will occur. The Arbovirus Insectary Facility, Wadsworth Center, New York State Department of Health in Albany, NY consists of connecting arthropod BSL-2 (ABSL-2) and ABSL-3 labs. Mosquito rearing is carried out in the ABSL-2 facility under ABSL-2 guidelines. These containment guidelines are important to prevent escape of mosquitoes into the surrounding environment, preventing introduction of new species. Mosquitoes that will be infected with virus for experimental purposes are transferred into the ABSL-3 facility and handled following arthropod ABSL-3 guidelines. The mosquitoes are transferred into the ABSL-3 lab via a pass-through chamber that can be accessed from only one side at a time.

See references (Gerberg et al., 1994; Higgs and Beaty, 1996; Higgs, 2005; Imam et al., 2014) for additional information on mosquito rearing and containment.

Materials and Reagents

  1. Clear polystyrene cups, disposable, capacity 250 ml, height 7.5 cm, bottom diameter 5 cm, top diameter 8.5 cm (e.g., Solo, clear plastic cup, 9 oz, https://www.solocup.com/products/clear-plastic-cup/)
  2. Wooden applicator sticks
  3. Oviposition dish for Aedes spp.: black plastic dish (ramekin), approximately 10 cm width x 5 cm height (e.g., Portion cup, black plastic soufflé, WebstaurantStore Food Service and Supply, catalog number: 127P400B )
    Note: The dish is half-filled with distilled water, and brown seed germination paper (Anchor Paper Company, 38-lb regular weight creped seed germination paper) or a fluted coffee filter with bottom removed is placed partially submerged around the edges.
  4. Plastic bag
  5. Damp sponge
  6. Transfer pipets, 1 ml, polyethylene, disposable (Biologix, catalog number: 30-0135 )
  7. Paper towel
  8. Mosquito emergence jars (Mosquito Breeder) (BioQuip, catalog number: 1425 )
    Note: The apparatus consists of two plastic 1-liter (L) jars that can be screwed together. Water and mosquito larvae are placed in the bottom jar, and emerging adults fly into the upper portion. A mesh hole is provided on top for respiration and food. The BioQuip breeder is equipped with a funnel between the upper and lower units, but in our insectary, better viability of the adults has been achieved by removing the funnel.
  9. Mosquito-holding cartons, created from 0.5- or 1-L ice cream cartons or white food containers with lids (Solo, catalog number: KH16A-J8000 , https://www.solocup.com/; available also from Amazon.com). See Note 1 for details on how to modify for mosquito containers
  10. Fish net, 10 cm, with fine-mesh netting
  11. T-175 flasks (e.g., NuncTM Non-treated Flasks, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178883 )
  12. Cotton coil (e.g., Fantasea 100 % Cotton Coil/12 M per Bag (FSC501), available at Amazon.com)
  13. Petri dish cover
  14. 15-ml conical centrifuge tube
  15. Natural hog sausage casing, available in coils of approximately 32-35 mm diameter, in brine, available at local butcher shops or on-line
    Note: Store in air tight container at 4 °C for up 6 months. Do not freeze.
  16. Gloves
  17. 0.22 µm Nalgene vacuum filter unit
  18. Tulle fabric
  19. Duct tape
  20. Gorilla glue (Gorilla Glue Company, Cincinnati, Ohio), or other polyurethane adhesive that expands slightly as it dries
  21. Culex spp. and Aedes spp. Mosquitoes
  22. Larval food: Kaytee Koi’s Choice Premium Fish Food (available at pet supply stores such as PetSmart or Petco, or at Amazon.com)
    Note: Prepare by grinding in coffee grinder or blender, store in aliquots at -20 °C for up to 6 months. Another brand of koi premium fish food could be substituted.
  23. Sugar cubes (e.g., Domino Dots sugar cubes)
  24. Defibrinated chicken blood for Culex rearing (100 ml, Colorado Serum, catalog number: 31143 ); defibrinated sheep blood for Aedes rearing (100 ml, Colorado Serum, catalog number: 31123 )
    Note: Store at 4 °C and use within 2 weeks.
  25. Bleach
  26. Ethyl alcohol 190 proof (PHARMCO-AAPER, UN1170; http://www.pharmcoaaper.com)
  27. Sucrose (Avantor Performance Materials, J.T. Baker®, catalog number: 4072-07 )
  28. 70% ethanol (see Recipes)
  29. 10% sucrose solution (see Recipes)
  30. 50% sucrose solution (see Recipes)

Equipment

  1. Grinder for preparation of larval food (coffee grinder or blender)
  2. Flask, side-arm filtering, 2 L capacity
  3. Mosquito cages, collapsible, aluminum frame, aluminum 20 x 20 mesh, nylon feeding hammock, access through knitted polyester stockinette sleeve; sizes 30 cm3, 46 cm3, or 60 cm3 (BioQuip, catalog number: 1450 )
  4. Larval flats, 35.6 cm length x 27.9 cm width x 8.3 cm height (Sterilite, catalog number: 1963 )
    Note: Maximum liquid capacity 1 L to prevent spillage during transport.
  5. Glove box, side entry, acrylic, size 30 x 24 x 24 inches (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: H50026-0000 )
  6. Spray bottle
  7. Water bath
  8. Freezer
  9. 1-L glass bottle
  10. Magnetic stir plate
  11. Dissecting microscope (e.g., Carl Zeiss, model: Stemi 2000 )
  12. Battery-powered aspirator (Clarke, catalog number: 13500 )
    Note: It consists of a handheld ‘flashlight’ aspirator body operating off two D-cell batteries, a 16-cm length x 1.25 cm diameter inlet tube, which connects via a stopper into a collecting tube (5 cm height x 2.5 cm diameter) screened on one end.

Procedure

  1. Collection and hatching of eggs
    1. Culex spp. lay their eggs in rafts directly on the water surface, ideally 250-300 eggs per raft. Females settle carefully on still water and lay eggs one by one, arranging them into a head-down array that sticks together to form the raft, which must remain on the water surface to hatch. In the laboratory mosquito colony, collect Culex spp. egg rafts 3-7 days (d) after blood feeding, by placing a clear polystyrene 250 ml cup (oviposition dish) partially filled with distilled water in the mosquito cage overnight (Figure 1A). The egg rafts (Figure 2) are transferred to a larval flat filled with 1 L of distilled water and a pinch (approximately 0.2 g) of ground larval food. Transfer the intact egg rafts by gently picking them up with a wooden applicator stick and placing them on the water surface of the larval flat in the same orientation as in the oviposition cup. If the eggs have already hatched, pour the contents of the oviposition dish directly into the larval flat. Most eggs hatch into larvae within 48 h.


      Figure 1. Oviposition containers for Culex (A) and Aedes (B)


      Figure 2. Culex egg raft and enlarged view of eggs

    2. Aedes spp. lay their eggs singly on damp substrates just above the water line. Their eggs can withstand long periods of desiccation and remain viable. In the laboratory colony, collect Aedes spp. eggs 3-5 d after blood feeding by placing an oviposition dish containing distilled water and a partially submerged fluted coffee filter, or seed paper in the mosquito cage (Figure 1B). After eggs are deposited, remove the paper with attached eggs from the oviposition dish, allow it to partially dry, place it into a plastic bag, and place the bag in a sealed air-tight plastic container for at least three days to allow the eggs to embryonate. Place a damp sponge in the container to maintain humidity. Under these conditions, the Aedes eggs will remain viable for 3-6 months (Ae. aegypti) or 1-2 months (Ae. albopictus). Ae. albopictus, unlike Ae. aegypti, will not tolerate extreme desiccation. Thus, regularly test viability by hatching a small section of the paper containing eggs. Once a month, check eggs for desiccation by observing under a microscope (Figure 3). If they are shrunken and deflated, they may have desiccated, or may not be embryonated.


      Figure 3. Aedes eggs on paper (A) and enlarged view of eggs (B)

      To hatch Ae. albopictus eggs, place egg papers into a larval flat with 1 L of distilled water and a pinch (0.2 g) of ground larval food. After the papers are thoroughly wet, they will sink into the water.
      To hatch Ae. aegypti eggs, place egg papers in a 2-L side-arm flask containing 1 L of distilled water and place under laboratory house vacuum for 1 h to deoxygenate the water. After the deoxygenation step, pour the flask contents into an empty larval flat and add a pinch (0.2 g) of ground larval food.
      Note: For both species, examine the flats daily for hatched larvae, which are 1-1.5 mm long and tend to accumulate in corners of the flat, but will ‘wiggle’ and disperse if the flat is disturbed.
  2. Larval rearing
    1. During this aquatic stage, the Culex and Aedes species larvae will feed voraciously. They often suspend head down, from the surface of the water breathing through siphon tubes. Larvae shed (molt) their exoskeleton four times (4 instar stages), growing larger after each molt. After the fourth instar, larvae metamorphose into pupae. The entire larval stage lasts from 6 to 8 days, depending on temperature, crowding, and nutrition. Larval body lengths range from approximately 1-1.5 mm for first instar, 1.5-3 mm for second instar, 3-5 mm for third instar, and 3.5-7 mm for fourth instar (Video 1 shows fourth instar larvae swimming and suspended at the water surface).

      Video 1. Culex larvae and pupae

    2. After eggs have hatched, use 1-ml disposable transfer pipets to transfer the first instar larvae into flats containing 1 L of deionized water. Aim for a density of 250-300 larvae per flat (approximately 2-3 pipet squirts if removing larvae from a concentrated area of the hatching flat). The larval density is important because crowding leads to stress, competition for food, longer pupation times and smaller adults. Approximately six larval flats will be needed to fill a 30 cm3 mosquito colony cage and one flat with approximately 200 larvae for experimental use (Figure 4).


      Figure 4. Covered larval flats

    3. Feed the larvae daily with ground larval food. Always make sure that there is a small amount of food at the bottom of the pan. Food density should be assessed daily. The following approximations can be used as a guideline: first and second instar, 0.2 g per flat; third and fourth instar, 0.4 g per flat. A scum on the surface of the flat indicates overfeeding, which will affect larval fitness. The scum should be removed by skimming the surface with a paper towel. Do not change the water in the flats because larvae feed best in water conditioned during breakdown of food. If the larvae become too dense, they can be divided into new flats (resulting in slower development) or added to less dense flats from the same batch of eggs. The rearing process proceeds much more efficiently, if egg hatching, larval rearing, pupae, and adult development are as closely synchronized as possible. Keep flats covered with plastic lids to prevent emerging mosquitoes from escaping from the flats.
  3. Pupae
    After the fourth instar, larvae begin to pupate, approximately 6-8 days after hatching and rearing at 26-28 °C. Pupae have short curved bodies with a large head and flippers for swimming at the other end. They are lighter than water and rest at the surface, breathing through a pair of respiratory trumpets. When disturbed, they swim in jerky movements and then float back to the surface (see Video 1 that shows both fourth instar larvae and pupae). Pupation continues for 3-4 days, and the pupal stage lasts for 1-2 days, depending on species and temperature. During daily larval feeding, check the flats for pupae, which float at the surface and appear darker in color than the larvae. Since the pupal stage lasts only days, the pupae should be moved from the larval flats (Figure 5) to mosquito emergence jars as soon as detected to avoid emergence of adults into the larval flats. If mosquitoes are allowed to emerge in the flats, the covered flats must be transferred to a glove box, and the adult mosquitoes collected with an aspirator, as described in step 4b and Video 2. Separate pupae from larvae using one of the following methods and place them into an emergence jar with the bottom chamber half-full of deionized water (Figure 6). Label emergence jars with hatch date and mosquito species/strain. Place a sugar cube or other sugar source on the mesh top of the emergence jar to be available to adults after they emerge, but do not add food to the water, as pupae do not feed.


    Figure 5. Picking pupae from a larval flat

    Video 2. Transferring adult mosquitoes from emergence jar


    Figure 6. Mosquito emergence jar containing pupae and adults

    1. Picking pupae manually.
      Cut off the tip of a 1 ml transfer pipet and draw in pupae one by one from the water surface and transfer to an emergence jar (Figure 7). This is the quickest method when there are < 20 pupae in the flat
    2. Separating pupae from larvae by the ice-water method.
      Pour the contents of the larval flat through a fish net with the drained water going into a clean flat. Using a small amount of distilled water, wash contents of fish net into a clear plastic cup. Fill the cup with icy cold water, swirl gently, which will cause larvae to sink to the bottom and pupae to rise to the top. Use a 1 ml transfer pipette to transfer pupae to the emergence jar. Pour the remaining larvae into the flat that contains strained rearing water. This is a good method to use when there are many pupae but still a substantial number of larvae in the flat. This method also allows one to easily combine larval flats to maintain an optimal density. Pupal separators also can be purchased or made, but because male and female pupae vary in size, the separators may be difficult to adjust so they work well.


      Figure 7. Transfer pipets with tip cut off (A) and containing pupa (B)

  4. Adults
    1. Adults will emerge from pupae in approximately 2 days. After emergence, the adult will sit on the water surface until its body dries and hardens. Most mosquitoes mate shortly after emergence from the pupal stage. The spermatozoa are passed by the male into the spermatheca of the female and usually serve to fertilize all eggs laid throughout the female’s lifetime. Males live for only 3-5 days after emergence, while females can live up to one or two months under laboratory conditions, typically laying as many as three sets of eggs before dying.
    2. Transfer the adults from the emergence jars into a mosquito colony cage (Figure 8) or into mosquito holding cartons for experimental use. Open the emergence jar within a glove box and use the battery-powered aspirator to collect adults (Figure 10 and Video 2). After aspirating the adults into the collecting tube, stopper the inlet tube, detach the collecting tube from the aspirator, and remove collection tube with attached inlet tube through the sleeve of the glove box. Expel mosquitoes into a colony cage or mosquito holding carton by inserting the inlet tube through the sleeve of the cage (Figure 8A) or the port of the holding carton (Figure 9) and gently blowing through the collection tube screen (Video 2). For the mosquito colony, all adults, both female and male, are transferred. For experimental use, one male is included for every ten females in the holding carton. Collecting the adults within the glove box provides an opportunity to sort female and males. Males are easily identified because they are smaller than females and have branched and feathery antennae (Figure 11). Before beginning transfer of adults within the glove box, clean the box with 70% ethanol in a spray bottle, wipe down, and allow the fumes to clear. After transfer is complete, check the glove box for loose mosquitoes and clean the box again. The 70% ethanol will also kill mosquitoes that may have hidden in corners and crevices. 


      Figure 8. Mosquito cage front (A) and top (B) views


      Figure 9. Mosquito holding cartons


      Figure 10. Hand-held mosquito aspirator


      Figure 11. Male and female mosquitoes

    3. Healthy mosquito colonies are maintained by regulating the population size and feeding larvae appropriately. For a 30-cm3 cage, a population of 2,000-3,000 is ideal; low populations result in bottlenecks, and overcrowding can decrease fitness. The population can be estimated by the number of newly emerged adults that are introduced. Place a sugar cube or other source of carbohydrate, such as cotton wad soaked in 10% sucrose, in the nylon feeding hammock on top of the cage and provide water by placing water-soaked white paper towels on the screened top of the cage, keeping them moist with a cotton coil stuffed into a T175 flask filled with deionized water (Figure 8B). Check the water daily (paper towels should be moist), and replace paper towels and cotton coils once every 2 weeks, or sooner if mold develops. Cover the cage with clear plastic to maintain moisture. Clean cages twice per week by removing the paper cage liner, aspirating dead mosquitoes, and replacing with a new liner. 
    4. Mosquitoes placed in holding cartons are maintained by placing cotton pads soaked in 10% sucrose on the mesh top and covering with a Petri dish cover. The entire mesh top is then covered with plastic wrap and secured with a rubber band. Check the sucrose pads and moisten them daily.
  5. Blood feeding
    1. Only female mosquitoes feed on blood, and all female mosquitoes, except those that are autogenous (such as Cx. pipiens form molestus), must take a blood meal in order to obtain nutrients necessary to develop eggs.
    2. For routine maintenance, mosquito colonies should be fed with blood every other week to select for longer-lived mosquitoes. Before feeding, remove the sugar cube and pre-warm a water bath to 45 °C. Prepare the blood meal by mixing 9.5 ml of defibrinated chicken blood (Culex spp.) or sheep blood (Aedes spp.) with 0.5 ml of 50% sucrose in a 15-ml conical centrifuge tube. Cut a 5-7 inch piece of sausage casing. Open the casing and rinse well inside and out. Knot one end and fill with water 3 times to check for leaks. Pour 5-10 ml of prepared blood meal into the casing and knot or tie off the other end (Figure 12). Warm the blood meal by placing the filled casing in a beaker with warm deionized water, and place the beaker in the 45 °C water bath. After 5 min, remove the blood meal, dab excess water on a paper towel, and place it in the feeding hammock of the cage for 1-2 h. After removing the blood meal, clean the hammock and replace the sugar cube. Collect eggs 2-7 days after blood feeding (see step 1).


      Figure 12. Sausage casing before and after filling with blood meal

  6. Insectary maintenance
    1. The temperature should be maintained at 24-28 °C and humidity should be 70-80%, or as high as can be achieved if that is not possible. Floor humidifiers can assist in maintaining humidity; they should be filled daily using deionized water.
    2. Room lights should be set to a 16-h light, 8-h dark cycle with 0.5-h crepuscular periods at dawn and dusk. Dawn: 6:00-6:30, Light: 6:30-21:30, Dusk: 21:30-22:00, or set to whatever environment you want to replicate.
    3. Clean all flats & lids, emergence jars, aspirator collection tubes, etc. by soaking for at least 15 min in 10% Bleach. Rinse well. Do not use soap. Make fresh 10% Bleach once per month.
    4. Wear gloves when working with mosquito colonies; wash hands.
    5. Mosquito cages should be emptied, cleaned with 70% ethanol and autoclaved 1-2 times/year.
    6. Complete weekly logs recording egg laying, blood feeding, flats, temperature range, and mosquito fitness.
    7. Do not discard live mosquitoes! Freeze adults overnight in mosquito cartons (kill cups: see Note 1) before disposal. Pour contents of containers with larvae or pupae through a fish net into a bucket so that larvae/pupae are trapped on the mesh. Rinse the net with very hot water, then wipe the mesh with paper towel and discard in stock pot. Check the bucket for larvae before discarding in the sink. Run hot water in the sink for several minutes.

Data analysis

This protocol does not generate information that needs extensive data analysis. However, experimental use of mosquitoes reared using this protocol for vector competence studies, life table studies, and experimental protocols, such as generation of mosquito salivary gland extract for intradermal inoculation of mice, will produce data requiring analysis. See Bio-protocol Schmid et al. (2017), which also includes information on example data and data analysis.

Notes

Modify ice cream cartons for use as mosquito holding cartons. Cut out the inside top of the lid. Stretch tulle screening over the top of the cup and secure the edges with Gorilla wood glue. Replace the lid. The lid then will be sealed and cannot be opened to introduce or remove mosquitoes. Cut a 2-cm diameter hole (port) in the lower part of the carton and cover it with duct tape. This tape will be used to open and close the container to insert mosquitoes from the aspirator collection tube, or remove mosquitoes for experimental use or disposal. To keep mosquitoes from sticking to the tape at the port, place a port-size piece of tape with sticky side facing the tape. A rubber band around the tape is often used to keep the port closed. At least one of these cartons is used as a ‘kill cup’, which is used to kill excess mosquitoes by inserting mosquitoes through the side port, then closing and placing the carton in the freezer overnight (see Figure 9).

Recipes

  1. 70% ethanol
    1. Combine 74 ml of ethyl alcohol with 26 ml of distilled water
    2. Label as flammable and store at room temperature for up to 3 months
  2. 10% sucrose solution
    1. Add 100 g sucrose to a 1-L glass bottle and fill to 1-L mark with distilled water
    2. Stir until dissolved using a magnetic stir plate
    3. Filter-sterilize with a 0.22 µm Nalgene vacuum filter unit
    4. Store at 4 °C for up to 6 months
  3. 50% sucrose solution
    1. Add 500 g sucrose to a 1-L glass bottle and fill to 1-L mark with distilled water
    2. Stir until dissolved using a magnetic stir plate and gentle heating
    3. Filter-sterilize with a 0.22 µm Nalgene vacuum filter unit
    4. Store at 4 °C for up to 6 months

Acknowledgments

Establishment of the high-containment insectary facilities at the Wadsworth Center, New York State Department of Health, was made possible through funding grant from NIH (1 C06 RR 17715-01) and matching funds from Wadsworth Center. We thank all the insectary staff who over the years have assisted in rearing mosquitoes to establish colonies and supply mosquitoes for use in laboratory studies, and for their help in developing protocols.

References

  1. Benedict, M. Q., Tabachnick, W., Higgs, S. and Wesson, E. D. (2004). Arthropod containment guidelines (Version 3.1). A project of the American Committee of Medical Entomology and American Society of Tropical Medicine and Hygiene. Vector Borne Zoonotic Dis 3(2):61-98.
  2. Gerberg, E. J., Barnard, D. R. and Ward, R. A. (1994). Manual for mosquito rearing and experimental techniques. American Mosquito Control Association.
  3. Higgs, S. and Beaty, B. J. (1996). Rearing and containment of mosquito vectors. In: Beaty, B. J. and Marquardt, W. C. (Ed). Biology of Disease Vectors. 1st edition. University of Colorado PRess 595-605.
  4. Higgs, S. (2005). Care, maintenance, and experimental infection of mosquitoes. In: Marquardt, W. C. (Ed). Biology of Disease Vectors. 2nd edition. Elsevier, 727-39.
  5. Imam, H., Zarnigar, Sofi, G. and Seikh, A. (2014). The basic rules and methods of mosquito rearing (Aedes aegypti). Trop Parasitol 4(1): 53-55.
  6. Schmid, M. A., Kauffman, E., Payne, A., Harris, E. and Kramer, L. D. (2017). Preparation of mosquito salivary gland extract and intradermal inoculation of mice. Bio Protoc 7(14): e2407.

简介

蚊子传播的病原体引起重大的公共卫生问题,并对全球疾病负担作出重大贡献。 蚊子蚊子传播登革热,紫草,黄热病和基孔肯雅病毒; 库蚊的蚊子传播西尼罗河,日本脑炎和圣路易脑炎病毒等。 利用实验室饲养的殖民蚊子的实验可以解决许多问题,如载体生物学,载体能力,载体 - 病原体相互作用和载体控制。 建立健康规范的蚊子殖民地需要制定和实施议定书,注重细节,了解影响蚊子健康的因素,如温湿度,营养素质量和可获得性,人口密度,供血和交配行为, 和产蛋要求。 在这里,我们提出了Culex spp饲养的标准协议。 和伊蚊属 蚊子和养殖蚊子殖民地。
【背景】蚊子经历了四个生命阶段的完全变态:鸡蛋,幼虫,蛹和成年人。不成熟的阶段总是水生的。在实验室中成功维护蚊子取决于为每个发育阶段提供最佳条件。这些要求将随物种而变化,事实上,许多蚊子物种,如Cx。在实验室没有成功地殖民。殖民地维护是一个劳动密集型的过程,需要时间和注意处理和记录保存的细节。当标准协议成功时,保持蚊子的适应性,并且它们对可重复实验的适用性是最佳的。该协议是为淡水库莱克种和殖民养殖的白纹伊蚊种群而定殖和维护的。它已经成功应用于Cx。皮肤科,Cx,quinquifasciatus,Cx。 t alis埃及伊蚊白纹伊蚊triseriatus和Ae。日本等。
  由于蚊子传播的大多数疾病是BSL-2和BSL-3生物制剂,因此这些载体的实验研究需要基于既定的指导方针进行遏制(Benedict et al。,2004)以及与机构生物安全委员会的合作。与非土着物种的工作也需要遏制,不会发生逃脱。纽约州奥尔巴尼州纽约州卫生局Wadsworth中心的Arbovirus昆虫学设施包括连接节肢动物BSL-2(ABSL-2)和ABSL-3实验室。根据ABSL-2指南,ABSL-2设施进行蚊子饲养。这些遏制指南对于防止蚊子进入周围环境是重要的,防止引入新物种。将被感染病毒用于实验目的的蚊子转移到ABSL-3设施中,并处理节肢动物ABSL-3指导方针。蚊子通过一个通过室转移到ABSL-3实验室,一次只能从一侧进入。
  有关蚊子饲养和遏制的更多信息,参见参考文献(Gerberg等,1994; Higgs和Beaty,1996; Higgs,2005; Imam等,2014)。

关键字:蚊子, 卵, 幼虫, 蛹, 成蚊, 集落, 吸血, 伊蚊, 库蚊

材料和试剂

  1. 透明聚苯乙烯杯,一次性,容量250毫升,高度7.5厘米,底部直径5厘米,顶部直径8.5厘米(例如,独奏,透明塑料杯,9盎司, https://www.solocup.com/products/clear-plastic-cup/
  2. 木applicator棒
  3. 大小10厘米x 5厘米高的黑色塑料盘(ramekin),部分杯子,黑色塑料蛋奶油,WebstaurantStore食品服务和供应,目录号:127P400B)
    注意:该盘用蒸馏水半充满,将棕色种子发芽纸(Anchor Paper Company,38磅常规重量起皱种子发芽纸)或带有底部去除的槽形咖啡过滤器部分浸没在边缘周围
  4. 塑料袋
  5. 潮湿海绵
  6. 转移移液管,1 ml,聚乙烯,一次性(Biologix,目录号:30-0135)
  7. 纸巾
  8. 蚊子出血罐(蚊子饲养员)(BioQuip,目录号:1425)
    注意:该设备由两个塑料1升(L)罐组成,可以拧紧在一起。水和蚊子被放置在底部的罐子里,新兴的成年人飞入上部。顶部设有网孔,用于呼吸和食物。 BioQuip育种者在上下单位之间配备漏斗,但在我们的昆虫中,通过去除漏斗可以获得更大的成年人的生存能力。
  9. 由0.5升或1升冰淇淋纸盒或带盖的白色食品容器(Solo,目录号:KH16A-J8000, https://www.solocup.com/ ;也可从Amazon.com获得)。有关蚊子容器如何修改的详细信息,请参见注1
  10. 鱼网10厘米,细网网
  11. T-175烧瓶(例如,未经处理的烧瓶,Thermo Fisher Scientific,Thermo Scientific,目录号:178883)的Nunc TM br />
  12. 棉花线圈(例如,/ />,Fantasea 100%棉线/ 12M /袋(FSC501),可从Amazon.com获得)
  13. 培养皿盖
  14. 15 ml锥形离心管
  15. 天然猪肠衣,直径约32-35毫米的线圈,盐水,可在当地肉店或在线上
    注意:在4℃的气密容器中储存6个月。不要冻结。
  16. 手套
  17. 0.22μmNalgene真空过滤器单元
  18. 薄纱织物
  19. 胶带
  20. 大猩猩胶(Gorilla Glue Company,辛辛那提,俄亥俄州)或其他聚氨酯胶粘剂,随着干燥而稍微膨胀
  21. culex spp。和 Aedes spp。蚊子
  22. 幼鱼食品:Kaytee Koi's Choice Premium鱼食(可在PetSmart或Petco或Amazon.com等宠物用品商店购买)
    注意:通过在咖啡研磨机或搅拌机中研磨制备,以等分试样在-20°C下储存长达6个月。另外品牌的锦鲤鱼饲料可以替代。
  23. 糖立方体(例如,,Domino Dots糖立方体)
  24. 用于Culex饲养的去纤维化鸡血(100ml,Colorado Serum,目录号:31143);去纤维蛋白羊血液用于养殖(100毫升,科罗拉多血清,目录号:31123)
    注意:请于4°C储存,并在2周内使用。
  25. 漂白剂
  26. 乙醇190证明(PHARMCO-AAPER,UN1170; http://www.pharmcoaaper.com
  27. 蔗糖(Avantor Performance Materials,J.T.Baker ®,目录号:4072-07)
  28. 70%乙醇(见食谱)
  29. 10%蔗糖溶液(参见食谱)
  30. 50%蔗糖溶液(见食谱)

设备

  1. 研磨机用于准备幼体食物(咖啡研磨机或搅拌机)
  2. 烧瓶,侧臂过滤,2升容量
  3. 蚊帐,可折叠,铝框架,铝合金20 x 20目,尼龙喂料吊床,通过针织涤纶丝袜套;尺寸30厘米,46厘米或60厘米3(BioQuip,目录号:1450)
  4. 幼虫平坦,35.6厘米长x 27.9厘米宽x 8.3厘米高(Sterilite,目录号:1963)
    注意:最大液体容积为1L,以防止运输过程中溢出。
  5. 手套箱,侧入口,亚克力,尺寸30 x 24 x 24英寸(SP科学软件 - 贝尔艺术产品 - H-B仪器,目录号:H50026-0000)
  6. 喷瓶
  7. 水浴
  8. 冰柜
  9. 1升玻璃瓶
  10. 磁力搅拌板
  11. 解剖显微镜(例如,卡尔·蔡司,型号:Stemi 2000)
  12. 电池供电器(Clarke,目录号:13500)
    注意:它包括一个手持式"手电筒"吸气体,两个D型电池,一个16厘米长x 1.25厘米直径的入口管,通过塞子连接到收集管(5厘米高x 2.5厘米直径)在一端屏蔽。

程序

  1. 收集和孵化鸡蛋
    1. culex spp。直接在水面上放置筏子,最好每筏250-300个鸡蛋。女性小心翼翼地定居在静止的水面上,一个个放下鸡蛋,将它们排列成一个固定在一起形成筏子的头顶阵列,这些筏必须留在水面上才能孵化。在实验室的蚊子殖民地,收集库里克斯 spp。卵筏3-7天(d)通过在蚊帐中放置一部分充满蒸馏水的透明聚苯乙烯250毫升杯(产卵盘)过夜(图1A)。将鸡蛋筏(图2)转移到装满1升蒸馏水和一份(约0.2克)地面幼体食物的幼虫盘中。转移完整的蛋筏通过轻轻地拿起它们用木制的涂抹棒,并将它们放置在幼体平面的水面上,方向与产卵杯相同。如果鸡蛋已孵出,将产卵盘的内容物直接倒入幼虫盘中。大多数鸡蛋在48小时内孵化成幼虫。


      图1. <库> Culex (A)和 Aedes (B)
      的Oviposition容器

      图2.鸡尾酒蛋筏和鸡蛋放大视图

    2. 阿黛斯 spp。将它们的鸡蛋单独放在刚刚在水线上方的潮湿底物上。他们的鸡蛋可以承受长时间的干燥并保持活力。在实验室的殖民地,收集 Aedes spp。通过将含有蒸馏水和部分浸没的有槽咖啡过滤器或种子纸的产卵盘放置在蚊帐中(图1B)将3-5天后的鸡蛋喂入。蛋沉积后,从产卵盘中取出附有蛋的纸,将其部分干燥,放入塑料袋中,将袋放入密封的气密塑料容器中至少三天,以使鸡蛋来胚胎将潮湿的海绵放在容器中以保持湿度。在这些条件下,鸡蛋将持续3-6个月(埃及伊朗伊蚊)或1-2个月( Ae。albopictus )。 阂。白纹伊蚊,不像Ae。埃及伊朗人不会容忍极度干燥。因此,通过孵化含有蛋的纸的一小部分来定期测试活力。每月一次,通过在显微镜下观察,检查鸡蛋的干燥(图3)。如果他们萎缩和收缩,他们可能已经干燥,或者可能不会被淹没。


      图3.纸张上的Aedes鸡蛋(A)和鸡蛋放大视图(B)

      要孵化啊白纹伊蚊鸡蛋,将鸡蛋纸放入1升蒸馏水和一份(0.2克)地面幼虫食物的幼虫平底锅中。纸张彻底湿透后,会沉入水中。
      要孵化啊埃及鸡蛋,将蛋纸置于含有1升蒸馏水的2L侧臂烧瓶中,并在实验室放置1小时以使水脱氧。脱氧步骤后,将瓶子内容物倒入一个空的幼虫平底锅,并加入一些夹心(0.2g)的幼虫食物。
      注意:对于这两种物种,每天检查一些孵化幼虫的单位,长度为1-1.5毫米,倾向于积聚在平面的拐角处,但如果平面受到干扰,则会"晃动"并分散。
  2. 幼虫养殖
    1. 在这个水生阶段,ule x x x>> ae ae ae ae ae ae。。。。。。。。。。。。。他们经常悬挂头,从呼吸的表面通过虹吸管。幼虫脱落(蜕皮)他们的外骨骼四次(4阶段),每次蜕皮后增长更大。四龄后,幼虫变形成蛹。整个幼虫阶段持续6至8天,取决于温度,拥挤和营养。幼虫体长度范围从第一龄约1-1.5毫米,第二龄期为1.5-3毫米,三龄期为3-5毫米,第四龄为3.5-7毫米(视频1显示第四龄幼虫游泳并悬挂在水面)
      Video 1. Culex larvae and pupae

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. 鸡蛋孵出后,使用1毫升一次性转移吸管将第一龄幼虫转入含有1升去离子水的平板。瞄准250-300个幼虫的平均密度(如果从孵化平台的集中区域移除幼虫,大约2-3个移液器喷射)。幼虫密度很重要,因为拥挤导致压力,食物竞争,幼蛹时间和小型成年人。需要大约6个幼虫平面,以填充30厘米3厘米的蚊子殖民地笼子和1个具有大约200个幼虫的平地用于实验用途(图4)。


      图4.覆盖的幼儿单位

    3. 每天用幼虫食物喂养幼虫。始终确保锅底部有少量食物。食物密度应每天进行评估。以下近似值可作为指导原则:第一和第二龄,每平面0.2克;第三和第四龄,每单位0.4克。扁平表面的浮渣表示过量供给,这将影响幼体适应性。应该用纸巾撇去表面来清除浮渣。不要更换单位的水,因为幼虫在食物分解过程中最适合于水。如果幼虫变得太密集,那么它们可以分为新的单位(导致发育较慢)或者加入同一批鸡蛋中较不稠密的单位。如果卵孵化,幼虫饲养,蛹和成年发育尽可能地同步,饲养过程进行得更加有效。保持盖上塑料盖的单位,以防止新兴的蚊子从单位逃脱。

  3. 在第四龄后,幼虫开始蛹,大约6-8天后孵化和饲养在26-28°C。蛹有短的弯曲的身体,大头和鳍状物在另一端游泳。它们比水轻,在表面休息,呼吸一个呼吸号。当他们受到干扰时,他们以动静的方式游泳,然后漂浮回地面(参见视频1,显示四龄幼虫和蛹)。幼虫持续3-4天,根据物种和温度,蛹期持续1-2天。在日常喂养期间,检查蛹的平面,漂浮在表面,并显示出比幼虫更深的颜色。由于蛹阶段只持续了几天,因此,一旦发现蛹就要从幼虫(图5)移到蚊子emerg ence j to to to to to to to to to to to to to to。。。。。。。。。。。。。。。如果蚊子被允许出现在单位中,则被覆盖的公寓必须被转移到手套箱,并且用吸气器收集成年蚊子,如步骤4b和视频2所述。使用以下方法之一将蛹与幼虫分开,并且将它们放入具有半满的去离子水的底部室的出口罐(图6)。标签出口罐与孵化日期和蚊子物种/应变。在出口罐的网格顶部放置糖立方体或其他糖源,才能在成年人出现之后才能使用,但不要向水中添加食物,因为蛹不喂食。


    图5.从幼虫平原采摘蛹

    Video 2. Transferring adult mosquitoes from emergence jar

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player



    图6.包含蛹和成虫的蚊子出现罐

    1. 手动采摘蛹。
      切断1毫升转移吸管的尖端,并从水面逐一吸取蛹,并转移到出口罐(图7)。这是最快的方法,平 20蛹
    2. 用冰水法将幼虫与幼虫分开。
      将幼鱼的内容物倒入鱼网中,排干的水进入干净的公寓。使用少量的蒸馏水,将鱼网的内容物清洗成透明的塑料杯。用冰冷的水填满杯子,轻轻旋转,这将使幼虫沉入底部,蛹上升到顶部。使用1 ml转移移液器将蛹转移到出苗罐。将剩余的幼虫倒入包含紧张养殖水的平台。这是一种很好的方法,当有很多蛹,但仍然有相当数量的幼虫在平面。这种方法还可以容易地结合幼体平面以保持最佳的密度。蛹分离器也可以购买或制造,但是因为雄蛹和雌蛹的大小不同,所以分离器可能难以调整,使其工作良好。


      图7.带有末梢切断(A)并包含蛹(B)
      的移液管
  4. 成人
    1. 大约2天内,大人将从蛹中出现。出现后,成年人将坐在水面上,直到身体干燥并变硬。大多数蚊子从蛹阶段出现后不久就交配。精子通过男性进入女性的精子,通常用于对整个女性一生中所有的卵进行施肥。男性在出现后只有3-5天生活,而女性在实验室条件下可以活到一至两个月,通常在死亡之前放置多达三套鸡蛋。
    2. 将成年人从出血罐转移到蚊子殖民地笼(图8)或用于实验用途的蚊帐中。打开手套箱内的出水瓶,并使用电池供电的吸气器收集成年人(图10和视频2)。将成人吸入收集管后,塞住入口管,将吸管从吸气器中取出,并通过手套箱的套筒拆下带有入口管的收集管。通过将入口管插入保持架(图8A)或保持纸箱(图9)的端口并轻轻吹过收集管屏幕(视频2),将蚊子排入殖民地笼子或蚊子托盘。对于蚊子殖民地,所有成年女性和男性都被转移。对于实验用途,每10名女性在包装箱中包括一名男性。收集手套箱内的成年人提供了分类女性和男性的机会。男性很容易识别,因为它们比雌性小,具有分枝和羽状天线(图11)。在手套箱内开始成年人转移之前,用喷雾瓶中的70%乙醇清洁盒子,擦拭,并将烟雾清除。转移完成后,检查手套箱是否有松动的蚊子,并再次清洁盒子。 70%的乙醇也会杀死可能隐藏在角落和裂缝中的蚊子

      图8.蚊帐前面(A)和顶部(B)视图


      图9.蚊子装箱


      图10.手持蚊子吸引器


      图11.男性和女性蚊子

    3. 通过适当调整人口规模和喂养幼体来维持健康的蚊子殖民地。对于30厘米3厘米的笼子,人口2000-3,000是理想的;低人口导致瓶颈,过度拥挤可能会降低适应度。人口可以通过引进的新出现的成年人的数量来估计。将糖立方体或其他来源的碳水化合物(例如浸在10%蔗糖中的棉絮)放在笼子顶部的尼龙喂养吊床上,并将水浸泡的白纸毛巾放在笼子的筛选顶部,并保持它们用填充有去离子水的T175烧瓶中的棉线圈湿润(图8B)。每天检查水(纸巾应该是潮湿的),并且每2周更换纸巾和棉线圈,或者如果模具发展就更快。用透明塑料盖住笼子以保持水分。清洁笼子每周两次,取下纸笼笼,吸入死蚊子,并更换新的班轮。&nbsp;
    4. 通过将铺在10%蔗糖上的棉垫放置在网状物顶部并用培养皿盖覆盖的方式来保持装在纸箱中的蚊子。然后用塑料包裹物覆盖整个网眼,并用橡皮筋固定。检查蔗糖垫并每天润湿。
  5. 喂血
    1. 只有雌蚊服用血液,所有雌性蚊子,除了那些自发性的蚊子(如鸵鸟),必须服用血粉才能获得发育蛋所需的营养。 br />
    2. 为了日常维护,蚊子殖民地每隔一周都要喂血,选择更长寿的蚊子。喂食前,取出糖立方体,预热水浴至45°C。通过混合9.5ml脱纤维蛋白鸡血清(Culex spp。)或绵羊血液(Eedes spp。)与0.5ml 50%蔗糖在15- ml锥形离心管。切一个5-7英寸的香肠肠衣。打开外壳,内外冲洗干净。结一端,充满水3次,检查是否有泄漏。将5-10毫升准备好的血粉倒入套管中,打结另一端(图12)。通过将装满的外壳放入烧杯中,用温和的去离子水来加热血粉,并将烧杯置于45℃水浴中。 5分钟后,取出血粉,用纸巾擦去多余的水分,放在笼子的喂养吊床上1-2小时。取出血粉后,清理吊床,更换糖立方体。喂血后2-7天收集鸡蛋(见步骤1)。


      图12.填充血粉之前和之后的香肠肠衣

  6. 昆虫维护
    1. 温度应保持在24-28℃,湿度应为70-80%,或者如果不可能,可以达到高度。地板加湿器可以帮助保持湿度;每天应使用去离子水填充。
    2. 房间灯应在黎明和黄昏时段设置为16小时光线,8小时黑暗循环,0.5小时闪烁时间。黎明:6:00-6:30,光:6:30-21:30,黄昏:21:30-22:00,或设置为您要复制的任何环境。
    3. 清理所有单位及盖子,出口罐,吸气管收集管,等。通过在10%漂白剂中浸泡至少15分钟。冲洗干净。不要使用肥皂。每月新鲜10%漂白一次。
    4. 使用蚊子殖民地时戴手套;洗手。
    5. 蚊子笼应清空,用70%乙醇清洗,高压灭菌1-2次/年
    6. 完成每周记录产蛋,喂血,单位,温度范围和蚊子健身的日志。
    7. 不要丢弃活的蚊子!在灭蚊之前,将蚊子过夜冻结(杀死杯子:见注1)。将含有幼虫或蛹的容器的内容物通过鱼网倒入桶中,以使幼虫/蛹被捕获在网状物上。用非常热的水冲洗网,然后用纸巾擦拭网,并放在储罐中。检查桶中的幼虫,然后丢弃在水槽中。在水槽里运行热水几分钟。

数据分析

此协议不生成需要广泛数据分析的信息。然而,使用该方案进行的用于载体能力研究,生命表研究和实验方案的蚊子的实验性使用,例如产生用于皮内接种小鼠的蚊子唾液腺提取物将产生需要分析的数据。参见Bio-protocol Schmid等人。 (2017),其中还包括有关示例数据和数据分析的信息。

笔记

修改冰淇淋纸箱,用作蚊子托盘。切出盖子的内部顶部。拉伸薄纱筛选在杯子的顶部,并用大猩猩木胶粘固边缘。更换盖子。盖子将被密封,不能打开以引入或除去蚊子。在纸箱的下部切割一个2厘米直径的孔(端口),并用胶带盖住。该胶带将用于打开和关闭容器,从吸液管收集管插入蚊子,或者清除蚊子进行实验使用或处置。为了防止蚊子粘在端口的胶带上,请将带有粘性面的胶带的端口尺寸的胶带放在胶带上。磁带周围的橡皮筋经常用于保持端口关闭。这些纸箱中至少有一个被用作"杀手杯",用于通过将蚊子插入侧端口,然后关闭并将纸箱放置在冷冻室中过夜来杀死多余的蚊子(见图9)。

食谱

  1. 70%乙醇
    1. 将74ml乙醇与26ml蒸馏水结合使用
    2. 标记为易燃并在室温下储存长达3个月
  2. 10%蔗糖溶液
    1. 将100克蔗糖加入1升玻璃瓶中,并用蒸馏水填充至1升标记
    2. 使用磁力搅拌板搅拌至溶解
    3. 用0.22μmNalgene真空过滤器单元进行过滤消毒
    4. 在4°C下储存长达6个月
  3. 50%蔗糖溶液
    1. 向1升玻璃瓶中加入500克蔗糖,并用蒸馏水
      填充至1升标记
    2. 使用磁力搅拌板搅拌直至溶解并轻轻加热
    3. 用0.22μmNalgene真空过滤器单元进行过滤消毒
    4. 在4°C下储存长达6个月

致谢

在纽约州卫生部Wadsworth中心建立高遏制的昆虫设施,可以通过NIH资助(1 C06 RR 17715-01)和Wadsworth中心的配套资金。我们感谢所有多年来一直帮助养殖蚊子建立殖民地并提供蚊子用于实验室研究的昆虫工作人员,并协助制定协议。

参考

  1. 本尼迪克特,M. Q.,Tabachnick,W.,Higgs,S。和Wesson,E.D。(2004)。 节肢动物控制指南(版本3.1)。美国医学昆虫学委员会和美国热带医学和卫生学会的一个项目。&nbsp; 矢量生物人畜共患病 3(2):61-98。
  2. Gerberg,E.J.,Barnard,D.R。和Ward,R.A。(1994)。 蚊虫抚养和实验技术手册。&nbsp;
  3. Higgs,S.和Beaty,B.J。(1996)。饲养和遏制蚊子载体。在:Beaty,B.J。和Marquardt,W.C。(Ed)。疾病载体的生物学。第一版。科罗拉多大学PRESS 595-605。
  4. Higgs,S。(2005)。蚊子的护理,维护和实验感染。在:Marquardt,W.C。(Ed)。 疾病载体生物学。第二版。 Elsevier ,727-39。
  5. Imam,H.,Zarnigar,Sofi,G。和Seikh,A。(2014)。 蚊子养殖的基本规则和方法(埃及伊蚊)。&nbsp; Trop Parasitol 4(1):53-55。
  6. Schmid,M.A.,考夫曼,E.,Payne,A.,Harris,E.和Kramer,L.D。(2017)。 制备蚊子唾液腺提取物和皮内接种小鼠。 Bio Protoc 7(14):e2407。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kauffman, E., Payne, A., Franke, M. A., Schmid, M. A., Harris, E. and Kramer, L. D. (2017). Rearing of Culex spp. and Aedes spp. Mosquitoes. Bio-protocol 7(17): e2542. DOI: 10.21769/BioProtoc.2542.
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