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Protocol for Initiating and Monitoring Bumble Bee Microcolonies with Bombus impatiens (Hymenoptera: Apidae)
用凤仙花启动和监测大黄蜂微群的方法    

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Abstract

Populations of some bumble bee species are in decline, prompting the need to better understand bumble bee biology and for assessing the effects of environmental stressors on these important pollinators. Microcolonies have been successfully used for investigating a range of endpoints, including behavior, gut microbiome, nutrition, development, pathogens, and the effects of pesticide exposure on bumble bee health. Here, we present a step-by-step protocol for initiating, maintaining, and monitoring microcolonies with Bombus impatiens. This protocol has been successfully used in two pesticide exposure-effects studies and can be easily expanded to investigate other aspects of bumble bee biology.


Disclaimer: The views expressed in this article are those of the author(s) and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency.

Keywords: Bumble bee (大黄蜂), Bombus (熊峰), Microcolony (小菌落), Pollinators (传粉者), Pesticides (农药)

Background

Bumble bees are valuable pollinators in agricultural and natural settings (Kleijn et al., 2015). Disconcertingly, populations of some bumble bee species are in serious decline (Cameron et al., 2011). Many factors are believed to contribute to the reported population declines, including poor nutrition, parasites, pathogens, and pesticides (Brown and Paxton, 2009; Goulson, 2005, 2013, 2015; Meeus et al., 2011; Wood et al., 2019). Recognizing their importance and the number and complexity of factors affecting their populations, there is a need to better understand bumble bee biology and the effects of environmental stressors on bumble bees.


Microcolonies are formed when a small group of bumble bee workers is isolated in a queenless environment. Under these conditions, the workers self-organize to build nest structures and lay unfertilized eggs that produce drones (Free, 1955). The model is versatile, offering the ability to investigate a range of endpoints, including behavior, the gut microbiome, nutrition, development, pathogens, and pesticide exposure (reviewed in Klinger et al., 2019).


Currently, there are no detailed protocols for initiating and monitoring microcolonies published for any bumble bee species, only condensed protocols in the methods sections of research publications (Gradish et al., 2012, 2013; Smagghe et al., 2007). Here, we detail a step-by-step protocol for initiating and monitoring bumble bee microcolonies with the common eastern bumble bee (Bombus impatiens Creson) (Hymenoptera: Apidae). We also provide detailed instructions for preparing microcolony food provisions. An overview of the procedures for initiating and monitoring microcolonies can be found in Figure 1. The protocols presented here were originally described in two peer-reviewed publications (Camp et al., 2020a, 2020c) and a subsequent publication comparing these two studies (Weitekamp et al., 2022). While these protocols were designed for assessing the effects of pesticide exposure on bumble bees, they can be easily expanded to investigate other aspects of bumble bee biology, including behavior, nutrition, development, pathogens, and gut microbiome (reviewed in Klinger et al., 2019).



Figure 1. Overview of procedures for initiating and monitoring microcolonies.

(A and B) Prepare syrup and pollen stocks for provisioning the microcolonies. Although syrup can be prepared in advance and stored at 4°C, pollen patties should be made fresh on the day of use. Transfer pollen to dishes and collect the weight. (C) Use only age-matched, newly emerged B. impatiens workers when using this protocol. To facilitate experimental manipulation, chill workers on ice. Distribute five bees to each microcolony chamber. Provision microcolony chambers with a ~3 g pollen patty for nest building and a syringe feeder filled with 50/50 inverted syrup. Supplement the nest with ~2 g of pollen paste on day 5. (D) Provide microcolonies with pollen patties for feeding (starting on Day 7) and 50/50 inverted syrup every Monday, Wednesday, and Friday for the duration of the experiment (recommend no more than 49 days). Collect the weight of the old syringe feeders and pollen dishes to use when calculating food consumption. (E) Investigators are encouraged to collect data on worker mortality and drone production (i.e., timing to emergence of 1st drone, number of drones emerged, and drone weight). Syrup and pollen consumption values should be corrected for evaporation and worker mortality. The black vertical arrow on the righthand side indicates the order of operations for initiating and monitoring microcolonies.


Part I: Protocol for microcolony food preparation

Materials and Reagents

  1. Fresh or fresh-frozen honey bee-collected corbicular pollen (see Protocol for Microcolony Food Preparation Notes #1) either sourced from investigator-maintained honey bee colonies or a commercial vendor (Swarmbustin’ Honey, catalog number: BP-DKLB).

  2. Sorbic acid (Amresco, catalog number: 0667-500G)

  3. Citric acid anhydrous (Fisher, catalog number: A940-500)

  4. Pure cane sugar (e.g., Domino Sugar)

  5. Distilled water (Gibco, catalog number: 15230)

  6. Potassium Sorbate Solution (see Recipes)

Equipment

  1. Laminar flow hood

  2. 4°C laboratory refrigerator (Thermo Scientific, catalog number: TSV18CPSA)

  3. -20°C laboratory freezer (Thermo Scientific, catalog number: TSX3020FARP)

  4. Basic coffee grinder or (ideally) commercial blender (Waring, catalog number: 7010S)

  5. Vacuum food sealer (FoodSaver, catalog number: FM2100)

  6. Freezer storage bags for vacuum food sealer (FoodSaver, catalog number: FSFSBF0226NP)

  7. Analytical top loading scale/balance (Ohaus, catalog number: AX2202/E)

  8. Analytical balance standards: 200 mg, 500 mg, 1 g, 2 g, 10 g, 20 g, 30 g, 100 g, 200 g, 300 g, 500 g, and 1 kg

  9. Hand-operated, electronic pipet for large volumes (Drummond Pipet-Aid, catalog number: 4-000-101)

  10. Hot plate with stir function (2) (Cimarec, catalog number: SP195025)

  11. pH meter (Orion Star, catalog number: STARA2110)

  12. pH meter calibration standards: pH 4.0 and pH 7.0 (VWR, catalog number: E452-500ML and E459-500ML)

General supplies

  1. N95 disposable respirator (VWR, catalog number: 89201-508)

  2. Mortar and pestle (VWR, catalog number: 470019-978)

  3. Sterile bottletop 0.45 µm filters (VWR, catalog number: 10042-462)

  4. 60 mL Luer slip syringe with tips cut off (Exel International, catalog number: ES60)

  5. 25 mL graduated glass pipet (VWR, catalog number: 76003-570)

  6. Aluminum foil

  7. Disposable paper mats for covering working surfaces (Versi-Dry Lab Table Soakers, catalog number: 62080-00)

  8. 2 L Pyrex bottle (Corning, catalog number: 1395-2L)

  9. Magnetic stir bars (Komet, catalog number: 50087909)

  10. 35 mm × 10 mm disposable Petri dish lids (Falcon, catalog number: 351008)

  11. 43 mm aluminum weigh dishes (QORPAK, catalog number: MET-03105)

  12. 1 L Pyrex beakers (2) (Corning, catalog number: 1000-1L)

Procedure

  1. 50/50 Inverted Syrup: 1 L Bottles (1.5 L produced)

    1. For easier clean-up, cover the hot plate with aluminum foil prior to use.

    2. Combine 1,000 mL distilled water with 850 g pure cane sugar and stir on a hot plate until all sugar granules are dissolved.

    3. Add 0.85 g citric acid anhydrous and continue stirring while heating to a rolling boil.

    4. Cover beaker with aluminum foil and boil for 20 min (see Figure 2).



      Figure 2. Syrup at rolling boil and treated with citric acid.


    5. Allow to cool on a room temperature stir plate, while covered and stirring with a stir bar.

    6. Once cooled, add 7.5 mL (5 mL per 1 L produced) Potassium Sorbate Solution (see Recipes below).

    7. Record the pH and label the container appropriately (see Protocol for Microcolony Food Preparation Notes #2).

    8. Parafilm bottle cap and store 50/50 inverted syrup at 4°C for up to 14 days once opened (30 days unopened).


  2. Preparing Pollen (see Protocol for Microcolony Food Preparation Notes #3 and 4)

    1. Grind frozen pollen to a fine powder with a coffee grinder or (ideally) commercial blender (Figure 3A-C).



      Figure 3. Pollen consistency.

      (A) Frozen fresh collected honey bee corbicular pollen. (B) Honey bee orbicular pollen in a blender cup. (C) Honey bee corbicular pollen ground to a fine powder for making patties and paste.


    2. Calibrate the analytical balance prior to use.

    3. Store ground pollen in vacuum seal freezer bags at a weight of 500 g per bag.

    4. Store ground pollen at -20°C until ready to use.


  3. Preparing Pollen Paste for Nest Initiation and Routine Feeding

    1. Remove one vacuum-sealed bag of freshly collected honey bee pollen from the -20°C freezer when ready to use.

    2. Weigh out the desired amount of frozen pollen in 100 g increments at a time.

    3. Reseal any unused ground pollen in a new vacuum seal bag, label appropriately, and store at -20°C.

    4. Use a commercial blender to blend the pollen to a fine powder consistency (Figure 4A-C).

    5. Add 38.5 mL of 50/50 inverted syrup to 100 g of ground pollen and mix with a spoon to a peanut butter-like consistency (Figure 4A).

    6. Place a damp paper towel over the pollen paste while working to prevent evaporation.

    7. Transfer the pollen paste to disposable 43 mm aluminum weigh dishes. For nest initiation, weigh out 3.0–3.25 g of pollen paste and place offset to one side in the lid of a pre-weighed 35 mm × 10 mm disposable Petri dish (Figure 4B). For routine feeding, fill to the top of the dish, but leave a small space on one side for transfer with forceps to the microcolony chambers (Figure 4C). If using pollen as the dosing vehicle for a pesticide exposure-effects study, either work from the lowest concentration to the highest concentration or use clean forceps when switching to a new dose group.



      Figure 4. Pollen processing.

      (A) Pollen paste with peanut butter-like consistency. (B) Pollen paste for routine feeding in a feeding dish. A small void is left to allow manipulating the feeding dishes with forceps while minimizing the risk of cross-contaminating exposure groups. (C) Nest dish with initiation patty offset to allow space for adding additional pollen paste on day 5.


    8. Pollen patties for nest initiation and paste for routine feeding and nest initiation should be made fresh on the day of initiation or feeding.


    Protocol for Microcolony Food Preparation Notes
    1. Fresh-frozen pollen should be stored in vacuum sealer bags or other air-tight containers at -20°C for up to 2 years. If stored longer, investigators should confirm palatability relative to freshly collected pollen before committing to a large, time-consuming experiment.

    2. The pH of 50/50 inverted syrup should be between 4 and 5.

    3. To maximize continuity within an experiment, all pollen needed for one study should be pooled and blended to produce a single, uniform food stock.

    4. Grinding pollen in advance and distributing it into either single-use bags or bags sufficient to cover experimental needs for one week at a time will save a significant amount of time during the experiment.


    Part II: Protocol for microcolony initiation and monitoring

    Materials and Reagents

    1. Newly emerged B. impatiens workers (Biobest (Romulus, MI), Koppert Biological Systems (Howell, MI) or another commercial vendor; see Protocol for Microcolony Initiation and Monitoring Notes #3).

    2. 50/50 inverted syrup and pollen paste/patties prepared as described in Part I (above)

    3. Data sheets for recording data (see Supporting information figures 1–6)

    4. Head lamp (e.g., Petzl Tactikka) and/or small handheld flashlight with red light filter (e.g., Mini Maglight PRO LED)

    5. Parafilm wrap (Masterflex, catalog number: PM992)

    6. Mortar and pestle (VWR, catalog number: 470019-978)

    7. Disposable paper mats for covering working surfaces (Versi-Dry Lab Table Soakers, catalog number: 62080-00)

    8. 20 mL oral dosing syringes (Medi-dose, catalog number: NAW-2000) with manufacturer-supplied tight-fitting caps (drill a hole in each syringe 1/8” hole located at the 2 mL mark prior to use)

    9. 43 mm aluminum weigh dishes (Qorpak, catalog number: MET-03105)

    10. Specimen forceps (12” length; VWR, catalog number: 82027-382)

    11. General-purpose laboratory tape (VWR, catalog number: 89097-912)

    12. Rectangular ice pan, approximate dimensions 15” L × 10” W × 6” D (VWR, catalog number: 10146-216)

    13. Rectangular plastic container, approximate dimensions 7.5” L × 5” W × 2.5” D (Cambro, catalog number: 42PP190)

    14. 50 mL conical tubes (Corning, catalog number: 352070)

    15. Disposable 35 mm × 10 mm Petri dishes (Falcon, catalog number: 351008)

    16. 43 mm aluminum weigh dishes (QORPAK, catalog number: MET-03105)

    Equipment

    1. 4°C laboratory refrigerator (Thermo Scientific, catalog number: TSV18CPSA)

    2. -20°C laboratory freezer (Thermo Scientific, catalog number: TSX3020FARP)

    3. Analytical top loading scale/balance, 0.1 mg (OHAUS, catalog number: 30100604)

    4. Analytical balance standards: 10 g, 20 g, and 30 g

    5. 5” stainless-steel geology sieve (1.57” depth × 5” diameter), #10 mesh (SciOptic, ASTM 10, catalog number: 305 stainless steel)

    6. Clear observation tops for geology sieves with access lids (see Protocol for Microcolony Initiation and Monitoring Notes #1).

    7. Bottom plates with holes drilled for ventilation for geology sieves to sit on top of (see Protocol for Microcolony Initiation and Monitoring Notes #2).

    8. Environmental chamber with temperature and humidity controls

    Software

    1. Microsoft Excel Spreadsheet Software® (v16.0; Redmond, WA)

    2. GraphPad Prism® (v6; La Jolla, CA)

    Procedure

    1. Prepare nest provisions and feeders (see Protocol for Microcolony Initiation and Monitoring Notes #4)

      1. Prepare syrup feeders using 20 mL oral dosing syringes with a pre-drilled 1/8” hole located at the 2 mL mark. Holes should be parafilmed to facilitate filling syringes with either control 50/50 inverted syrup or, if the experiment calls for it, test article-containing 50/50 inverted syrup.

      2. Fill syringe feeders by submerging the syringe tip into a beaker or conical tube containing 50/50 inverted syrup or test article-containing 50/50 inverted syrup by pulling up on the plunger. Cap syringes after filling with syrup.

      3. Remove the parafilm and record the syrup weight in the datasheet for the experiment (see Supporting information figure 1 for an example data collection sheet).

      4. Prepare pollen patties as described in Part I (above).


    2. Prepare microcolony chambers for nest initiation

      1. Label each microcolony observation top with the date of initiation and assigned microcolony number.

      2. Place stainless steel sieve on the bottom plate with an absorbent paper towel placed in between the pieces, and clear observation lid on top.

      3. Place the lid of a 35 mm × 10 mm disposable Petri dish marked with the microcolony number on the bottom, in the microcolony chamber with a 3–3.25 g nest initiation patty for nest building (Figure 5).



      Figure 5. Microcolony chamber components.

      Stainless steel geology sieve (1.57” depth × 5” diameter) with pass-through floor (#10 mesh), bottom plate with holes drilled for ventilation, see-through top for collecting observations, removable lid for accessing chamber interior, and syringe feeder. Design adopted from Bayer CropSciences.


    3. Weighing and seeding microcolonies with newly emerged B. impatiens workers (Protocol for Microcolony Initiation and Monitoring Notes #5)

      1. Fill the ice pan and place the rectangular plastic container in the center of the ice pan with 50 mL conical tubes positioned around the inside edge of the ice pan for storing collected newly emerged workers.

      2. Transfer enough newly emerged workers to the 4°C refrigerator for 10–15 min to support the experiment (i.e., Number of workers needed = [(5 workers/microcolony × the number of microcolonies desired) + 10% extra bees to account for dead/injured bees]).

      3. Remove bees from 4°C and transfer them to the shallow rectangular plastic container on ice.

      4. Next, transfer five randomly selected bees at a time to the 50 mL conical tubes. Transfer the conical tubes to the lab bench to allow the bees to become active again.

      5. Check the conical tubes for any dead or damaged bees prior to weighing; replace dead or damaged bees as needed.

      6. Weigh the conical tube with the five newly emerged bees for the first microcolony. Add the bees to their microcolony and then weigh the empty conical tube to determine the weight of the bees. Record these numbers in the datasheet for the experiment (see Supporting Information figure 2 for an example data collection sheet).


    4. Supplementing the nest initiation patty

      1. Five days after microcolony initiation, supplement the nest initiation patty with an additional ~2 g of pollen paste.


    5. Microcolony routine feeding (see Protocol for Microcolony Initiation and Monitoring Notes #6–9)

      1. Seven days after microcolony initiation, give each microcolony ~2 g pollen paste for feeding in a disposable 43 mm aluminum weigh dish.

      2. Provide microcolonies with fresh, pre-weighed ~3.5 g pollen paste and either control 50/50 inverted syrup or test article-containing 50/50 inverted syrup every Monday, Wednesday, and Friday starting one-week post initiation.

      3. Record the weight of the new dish with new pollen paste and new syrup feeder into the datasheet for the experiment (see Supporting information figure 1 for an example data collection sheet).

      4. Record the weight of the old pollen paste with the original dish and previous syrup feeder to determine consumption.

      5. To more accurately quantify pollen/syrup consumption levels, include two evaporation controls in the study design. These controls should be set up and processed exactly like all other microcolonies, except they do not contain bees (see Supporting information figures 3 and 4 for an example data collection sheet).


    6. Monitoring microcolonies and data collection (see Protocol for Microcolony Initiation and Monitoring Notes #10–12)

      1. Using an environmental chamber, maintain microcolonies in darkness at 25°C ± 0.5°C and 50% ± 5% relative humidity throughout the duration of the study. Red light may be used when microcolonies are outside of the environmental chamber on the bench. Avoid white light where possible.

      2. Evaluate each microcolony from initiation to study termination (see Supporting information figure 5 for an example data collection sheet).

      3. During each observation, collect the following information: 1) number of dead workers; 2) days to first drone emergence; 3) number of drones emerged; and 4) drone weight.

      4. Optional, additional information can be collected, including 1) time to first uncapped egg chamber; 2) days to first capped egg chamber; 3) days to first larval mass; and 4) days to first pupal cell.

      5. Any drones that emerge should be removed when the microcolonies are fed (i.e., Monday, Wednesday, and Friday). After removal, weigh each drone individually (see Supporting information figure 6 for an example data collection sheet).

      6. Optional but encouraged: randomly select and photographically track at least one microcolony from each experimental group (Wednesdays recommended) to capture microcolony progression and developmental milestones.


    7. Terminating microcolonies

      1. When the experiment is complete, after 42 or 49 days, either proceed to additional assay endpoints or euthanize the workers, drones, and remaining brood by CO2 narcosis followed by transfer to -80°C.


      Protocol for Microcolony Initiation and Monitoring Notes
      1. The observation tops can be made according to the specifications detailed in Figure 6A and 6B. As designed, the tops have a recessed 5” diameter ring that prevents the lids from slipping off the top of the sieve. However, other designs for the top can be used, provided they 1) are large enough to cover the sieve [bees will be housed in the space between the mesh floor of the sieve and the observation top (see Figure 7)], have an opening for the syringe feeder, and have ventilation holes to allow air circulation.



        Figure 6. Materials and measurements for the microcolony observation top, removable lid, and bottom plate.

        Microcolony chamber observation tops (A), removable lids (B), and bottom plates and (C) used previously (Camp et al., 2020a, 2020c; Weitekamp et al., 2022) can be reconstructed according to the specifications shown. dia. = diameter.


      2. Bottom plates can be made according to the specifications detailed in Figure 6C. The bottom plates were designed to prevent bee waste and other debris that pass through the perforated floor of the sieve from contaminating other microcolonies and from fouling the environmental chamber. The design of the bottom plates includes a recessed 5” diameter ring that the sieve drops into. However, investigators can use any bottom plate design, provided the bottoms have ventilation holes to allow air circulation and are large enough for the sieve to sit on top of it.

      3. Microcolonies should not be initiated with randomly aged workers when using this protocol. Using age-matched, newly emerged workers reduces the possibility of confusing age-related deaths for treatment effects and promotes consistency across microcolonies within an experiment and across experiments. When using this protocol, investigators should use fresh or fresh-frozen honey bee-collected corbicular pollen with this protocol. Provisioning microcolonies with old (i.e., >2 years old) or improperly stored pollen may impact microcolony progression and productivity.

      4. Pollen patties for nest initiation and paste for routine feeding should be made fresh on the day of initiation or feeding.

      5. Since worker size can impact microcolony nest development and food consumption rates by workers (Peat and Goulson, 2005; Couvillon and Dornhaus, 2010; Amsalem and Hefetz, 2011; Roger et al., 2017a, 2017b), protocol users are encouraged to seed microcolonies with bees of a similar mass.

      6. Microcolonies must be provided ad libitum access to pollen and syrup for the duration of the experiment. Restricting access to food provisions will disrupt microcolony development, reduce productivity, and complicate the interpretation of experimental results.

      7. The delay in providing pollen specifically for feeding is to reduce the likelihood that the workers will attempt to lay eggs on both the pollen for feeding and nest initiation patty.

      8. Microcolonies consume significantly more pollen when feeding developing larvae. Therefore, it is best to give productive microcolonies ~3.5 g of pollen paste to minimize the risk that they will run out of pollen.

      9. Unless retention is required for additional analysis, dispose of the old pollen dish.

      10. To help keep the bees calm when manipulating the microcolonies, place the chambers on a disposable paper mat that will absorb vibrations.

      11. To promote consistency across microcolonies, rotate the position of individual microcolonies within the environmental chamber.

      12. If a founding worker dies in the first 24 h, replace it with a new newly emerged worker (obtain weight of new worker).

      13. Because the number of worker bees in a microcolony can impact nest productivity (Gradish et al., 2013) and food consumption rates, protocol users are encouraged to track the number of dead workers throughout the experiment.

      14. Production of drones is a key metric of microcolony success. The time to first drone emergence, the number of drones emerged, and drone weight can all be readily quantified. Importantly, all these measures can be affected by experimental treatments providing insights into how a test material impacted the microcolony.

      Data analysis

      This pair of protocols was developed to explore the effects of pesticide exposure on microcolony progression and productivity (see Camp et al., 2020a, 2020c; Eitekamp et al., 2022). While that is the case, these protocols can easily be used to address a variety of research questions, thereby enabling investigators to gain significant insight into other aspects of bumble bee biology. Based on experience using this system, 8–10 microcolonies should be used per experimental group. Below is an overview of how to process and analyze data collected from pesticide exposure-effects studies using this protocol. Detailed methods for processing and analyzing data for these endpoints can be found in Camp et al. (2020a, 2020c) and Weitekamp et al. (2022). To aid new investigators, sample data collection sheets along with guidance on how to record and process microcolony data are provided as supplementary information at the end of this protocol.

      Analysis of microcolony data can be broken down into two interrelated parts. For the first part, microcolonies should be visually inspected and photographed to identify treatment-related effects on nest progression. Microcolonies established according to this protocol will progress according to the timeline shown in Figure 7. When collecting observations weekly, uncapped egg chambers will appear by the end of week 1. Capped egg chambers sometimes appear late in week 1, but most often by the end of week 2. Larval masses can be detected during weeks 2 and 3. Pupal cells will appear during weeks 3 and 4. The first drones appear during week 5 and will continue to emerge for the remainder of the study. If desired, observations can be collected more frequently to tease out subtle effects on microcolony development. However, collecting observations is time-consuming, and frequent disruptions may impact worker behavior and ultimately study outcome.



      Figure 7. Microcolony progression through seven weeks of development.

      Microcolonies were initiated with five newly emerged B. impatiens workers and provisioned with a nest initiation patty of pollen paste (3 g) and 50/50 inverted syrup to stimulate nest building. After 5 days, nests were given an additional 2 g of pollen paste. Starting on day 7, microcolonies were given fresh pollen paste and syrup every Monday, Wednesday, and Friday. Starting from Day 0 (i.e., nest initiation), photos show microcolony progression from uncapped egg chambers to study termination on day 49. Bars with labels above indicate the range when feature typically appears. Numbered circles indicate features of the microcolony, including 1 = nest initiation patty, 2 = pollen supplement given on day 5, 3 = uncapped egg chamber, 4 = larval mass, 5 = pupal cell, and 6 = evacuated pupal cell.


      In addition to qualitatively assessing microcolony status, data from various endpoints should be analyzed statistically, including microcolony development milestones (i.e., time to first uncapped egg chamber, days to first capped egg chamber, days to first larval mass, and days to first pupal cell), syrup and pollen consumption, drone production and weight, and worker survival. These data should be expressed as mean ± standard deviation (STDEV). Using GraphPad Prism® (v6; La Jolla, CA), differences between the control and treatment groups can be assessed with One-way Analysis of Variance (ANOVA) and Dunn’s multiple comparison test. If, according to the Brown-Forsythe or Bartlett’s tests, variances are significantly different, instead use the non-parametric Kruskal-Wallis with Dunnett’s multiple comparisons test.


      Data reporting

      To allow comparisons to be made between studies and to facilitate study replication, investigators are encouraged to report the 1) syrup formulation used for feeding, 2) pollen source and age, 3) age and source of the worker bees, and 4) environmental conditions (i.e., temperature, relative humidity, and light regimen) used during the experiment. In addition, the start, end, and data collection dates should also be recorded and reported. All data should be made available to other investigators either as supplemental information or through a public repository for scientific data.


      Data Analysis Notes

      1. When investigating the effects of pesticide exposure on microcolony development and productivity, investigators should include an untreated control group, a positive control group, and, if applicable, a solvent control group in the study design. Evaporation controls should also be included to correct food consumption estimates when conducting pesticide exposure studies.

      2. When desired, the pesticide can be delivered to the microcolony through the syrup and/or pollen provisions. Dosing pesticides via syrup is generally easier than with pollen. However, not all pesticides are water-soluble, and, for that reason, solvents may be required to solubilize the test material. In that event, investigators will need to empirically identify a suitable solvent concentration for use in their experiment [e.g., 1% acetone (Camp et al., 2020b)].

      3. When evaluating the effects of pesticide exposure on microcolonies, the delivery vehicle (i.e., pollen or syrup) can impact study outcome. Developing bumble bee brood consumes large amounts of pollen and, for that reason, delivering test material through the pollen may target the brood. To that point, acetamiprid delivered in the pollen, but not when delivered in the syrup, reduced average drone weight (Camp et al., 2020a, 2020c; Weitekamp et al., 2022). Consequently, important brood effects could be missed when only dosing through the syrup.

      4. Syrup and pollen consumption values should be corrected for evaporation and, when determining consumption on a per bee basis, worker mortality. Also, if a syrup-filled syringe leaked, leading to an inaccurate consumption value, the value should be replaced with the average syrup consumption value for that treatment group and day.

      5. Published results suggest that the microcolony model may only be appropriate for assessing brood effects for substances with low toxicity to adult workers (Krueger et al., 2021).

      Recipes

      1. Potassium Sorbate Solution

        Prepare a 25% w/v sorbic acid and potassium salt solution by dissolving 25 g of sorbic acid in distilled water to achieve a final volume of 100 mL; sterile filter (0.45 µm) and store at 4°C for up to 90 days.

      Acknowledgments

      The author thanks Drs. J. E. Simmons, C. Weitekamp, and YH. Kim for thoughtful and critical review of this manuscript. The author is also grateful to Drs. D. Schmehl, A. Cabrera, J. Strange, and D. Cox-Foster for their generosity and guidance. This protocol was adapted from previous work published peer-reviewed journals (Camp et al., 2020a, 2020c; Weitekamp et al., 2022).

      Competing interests

      The authors declare no conflict of interest.

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简介

[摘要]一些大黄蜂物种的种群数量正在下降,这促使需要更好地了解大黄蜂生物学并评估环境压力源对这些重要传粉媒介的影响。微菌落已成功用于研究一系列终点,包括行为、肠道微生物组、营养、发育、病原体以及农药暴露对大黄蜂健康的影响。在这里,我们提出了一个分步协议,用于使用Bombus impatiens启动、维护和监控微菌落。该协议已成功用于两项农药暴露效应研究,并可轻松扩展以研究大黄蜂生物学的其他方面。

[背景] 大黄蜂是农业和自然环境中宝贵的传粉媒介(Kleijn等人, 2015 年) 。令人不安的是,一些大黄蜂物种的种群数量正在严重下降(Cameron等人, 2011 年) 。许多因素被认为是导致报告的人口下降的原因,包括营养不良、寄生虫、病原体和杀虫剂(Brown 和 Paxton,2009;Goulson,2005、2013、2015;Meeus等, 2011;Wood等, 2019 ) .认识到它们的重要性以及影响其种群的因素的数量和复杂性,有必要更好地了解大黄蜂生物学和环境压力源对大黄蜂的影响。
当一小群大黄蜂工人在无蜂王的环境中被隔离时,就会形成微菌落。在这些条件下,工人们自我组织建造巢穴结构并产下未受精的卵,从而产生无人机(Free,1955) 。该模型用途广泛,能够研究一系列端点,包括行为、肠道微生物组、营养、发育、病原体和农药暴露(Klinger等人, 2019 年综述) 。
目前,没有针对任何大黄蜂物种发布的启动和监测微菌落的详细方案,只有研究出版物的方法部分中的浓缩方案(Gradish等人, 2012 年,2013 年;Smagghe等人, 2007 年) 。在这里,我们详细介绍了用常见的东部大黄蜂( Bombus impatiens )启动和监控大黄蜂微群落的分步协议。 Creson)(膜翅目:蜜蜂科)。我们还提供有关准备微菌落食品的详细说明。图 1 概述了启动和监测微菌落的程序。此处介绍的协议最初在两份经过同行评审的出版物(Camp等人, 2020a,2020c)和随后的比较这两项研究的出版物(Weitekamp等人, 2022)中进行了描述。虽然这些方案是为评估农药暴露对大黄蜂的影响而设计的,但它们可以很容易地扩展到研究大黄蜂生物学的其他方面,包括行为、营养、发育、病原体和肠道微生物组(Klinger等人综述, 2019) 。


图 1. 启动和监测微菌落的程序概述。
(A 和 B)准备糖浆和花粉储备以供应微菌落。虽然可以提前制备糖浆并在 4 ° C 下储存,但花粉饼应在使用当天新鲜制作。将花粉转移到盘子上并收集重量。 (C)使用此协议时,仅使用年龄匹配、新出现的B.凤仙花工人。为了便于实验操作,冰冷的工人。将五只蜜蜂分配到每个微集落室。提供带有 ~ 3 g 花粉饼的微菌落室,用于筑巢和填充 50/50 倒置糖浆的注射器进料器。在第 5 天用约 2 克花粉糊补充巢穴。 (D)在实验期间每周一、周三和周五提供带有花粉饼的微菌落进行喂养(从第 7 天开始)和 50/50 倒糖浆(建议不超过 49 天)。在计算食物消耗量时,收集旧注射器喂食器和花粉盘的重量。 (E)鼓励调查人员收集有关工人死亡率和无人机生产的数据(即第一架无人机出现的时间、无人机出现的数量和无人机重量)。糖浆和花粉消耗值应根据蒸发和工人死亡率进行校正。右侧的黑色垂直箭头表示启动和监控微菌落的操作顺序。

关键字:大黄蜂, 熊峰, 小菌落, 传粉者, 农药



第一部分:微菌落食物制备规程


材料和试剂


1. 新鲜或新鲜冷冻的蜜蜂采集的球状花粉(参见微生物群食物制备说明#1 的协议),来自研究者维护的蜜蜂群或商业供应商( Swarmbustin ' Honey,目录号: BP-DKLB )。
2. 山梨酸( Amresco ,目录号: 0667-500G)
3. 无水柠檬酸(Fisher,目录号: A940-500)
4. 纯蔗糖(例如,多米诺糖)
5. 蒸馏水(Gibco,目录号: 15230)
6. 山梨酸钾溶液(见食谱)


设备


1. 层流罩
2. 4°C实验室冰箱(Thermo Scientific,目录号:TSV18CPSA)
3. -20°C实验室冰箱(Thermo Scientific,目录号:TSX3020FARP)
4. 基本咖啡研磨机或(理想情况下)商用搅拌机(Waring,目录号:7010S)
5. 真空食品封口机( FoodSaver ,目录号:FM2100)
6. 用于真空食品密封机的冷冻储存袋( FoodSaver ,目录号:FSFSBF0226NP)
7. 分析顶部装载秤/天平(Ohaus,目录号:AX2202/E)
8. 分析天平标准品:200 mg、500 mg、1 g、2 g、10 g、20 g、30 g、100 g、200 g、300 g、500 g和1 kg
9. 用于大容量的手动电子移液器(Drummond Pipet-Aid,目录号:4-000-101)
10. 具有搅拌功能的热板(2)( Cimarec ,目录号:SP195025)
11. pH计(Orion Star,目录号:STARA2110)
12. pH 计校准标准:pH 4.0 和 pH 7.0(VWR,目录号:E452-500ML 和 E459-500ML)


一般用品


1. N95 一次性呼吸器(VWR,目录号:89201-508)
2. 研钵和杵(VWR,目录号:470019-978)
3. 无菌瓶口0.45 µm 过滤器(VWR,目录号:10042-462)
4. 60 mL Luer滑动注射器,尖端被切断( Exel International,目录号:ES60)
5. 25 mL刻度玻璃移液管(VWR,目录号:76003-570)
6. 铝箔
7. 用于覆盖工作表面的一次性纸垫( Versi -Dry Lab Table Soakers,目录号:62080-00)
8. 2升耐热玻璃瓶(康宁,目录号:1395-2L)
9. 磁力搅拌棒(Komet,目录号:50087909)
10. 35 mm × 10 mm 一次性培养皿盖(Falcon,目录号: 351008)
11. 43 mm铝制称重盘( QORPAK,目录号:MET-03105 )
12. 1 L Pyrex 烧杯(2)(Corning,目录号:1000-1L)


程序


A. 50/50 转化糖浆:1 升瓶装(生产 1.5 升)
1. 为了更容易清理,在使用前用铝箔盖住热板。
2. 将 1,000 mL 蒸馏水与 850 g 纯蔗糖混合,在热板上搅拌直至所有糖颗粒溶解。
3. 加入 0.85 g 无水柠檬酸并继续搅拌,同时加热至沸腾。
4. 用铝箔盖住烧杯并煮沸 20 分钟(见图 2)。




图 2. 糖浆滚沸并用柠檬酸处理。


5. 在室温搅拌板上冷却,同时盖上盖子并用搅拌棒搅拌。
6. 冷却后,加入 7.5 mL(每生产 1 L 5 mL)山梨酸钾溶液(参见下面的配方)。
7. 记录 pH 值并适当地标记容器(参见Microcolony食品制备说明 #2 协议)。
8. 封口膜瓶盖并在 4°C 下储存 50/50倒置糖浆,一旦打开,最多可保存 14 天(未开封 30 天)。


B. 准备花粉(参见Microcolony食物准备说明 # 3 和 4 的协议)
1. 用咖啡研磨机或(理想情况下)商用搅拌机将冷冻花粉研磨成细粉(图 3A-C)。




图 3. 花粉一致性。
(A)冷冻新鲜采集的蜜蜂球状花粉。 (B)搅拌杯中的蜜蜂圆形花粉。 (C)蜜蜂球状花粉研磨成细粉,用于制作肉饼和糊状物。


2. 使用前校准分析天平。
3. 将地面花粉储存在真空密封冷冻袋中,每袋重量为 500 克。
4. 将地面花粉储存在 -20°C 直至准备使用。


C. 为筑巢和常规喂养准备花粉糊
1. 准备使用时,从 -20°C 冰箱中取出一袋真空密封的新鲜收集的蜜蜂花粉。
2. 一次以 100 克的增量称出所需数量的冷冻花粉。
3. 将任何未使用的地面花粉重新密封在新的真空密封袋中,适当贴上标签,然后在 -20°C 下储存。
4. 使用商业搅拌机将花粉混合成细粉稠度(图 4A-C)。
5. 将 38.5 mL 的 50/50倒糖浆添加到 100 g 地面花粉中,并用勺子混合成花生酱般的稠度(图 4A)。
6. 在工作时将湿纸巾放在花粉糊上以防止蒸发。
7. 将花粉糊转移到一次性 43 毫米铝制称重盘中。筑巢时,称出 3.0–3.25 g 花粉糊,并将偏移量放在预先称重的 35 mm ×盖子的一侧 10 毫米一次性培养皿(图 4B)。对于日常喂养,填充到盘子的顶部,但在一侧留出一个小空间,以便用镊子将其转移到微菌落室(图 4C )。如果使用花粉作为农药暴露影响研究的给药载体,则要么从最低浓度到最高浓度,要么在切换到新剂量组时使用干净的镊子。




图 4.花粉处理。 
(A)具有类似花生酱稠度的花粉糊。 (B)用于在喂食盘中进行常规喂食的花粉糊。留出一个小空隙,以便用镊子操纵喂食盘,同时将交叉污染暴露组的风险降至最低。 (C)带有初始肉饼偏移的巢菜,以便在第 5 天添加额外的花粉糊。


8. 用于筑巢的花粉饼和用于常规喂食和筑巢的糊状物应在开始或喂食当天新鲜制作。


Microcolony食品制备说明协议
1. 新鲜冷冻的花粉应在 -20°C 下储存在真空密封袋或其他密封容器中长达 2 年。如果储存时间更长,研究人员应在进行大型、耗时的实验之前确认相对于新鲜收集的花粉的适口性。
2. 50/50转化糖浆的 pH 值应在 4 到 5 之间。
3. 为了最大限度地提高实验的连续性,一项研究所需的所有花粉都应汇集和混合,以产生单一、统一的食物储备。
4. 提前研磨花粉并将其分配到一次性袋子或足以满足一次一周实验需求的袋子中,这将在实验过程中节省大量时间。




第二部分:微菌落启动和监测方案


材料和试剂


1. 新出现的B. impatiens工人( Biobest (Romulus, MI)、 Koppert Biological Systems (Howell, MI) 或其他商业供应商;见微生物启动和监测说明 #3协议)。
2. 50/50倒糖浆 和按照第一部分(上图)所述制备的花粉糊/馅饼
3. 用于记录数据的数据表(参见支持信息图 1 – 6 )
4. 头灯(例如, Petzl Tactikka )和/或带红光过滤器的小型手持手电筒(例如,Mini Maglight PRO LED)
5. 封口膜包装( Masterflex ,目录号:PM992)
6. 研钵和杵(VWR,目录号:470019-978)
7. 用于覆盖工作表面的一次性纸垫( Versi -Dry Lab Table Soakers,目录号:62080-00)
8. 20 mL 口服给药注射器(Medi-dose,目录号: NAW-2000),带有制造商提供的紧密盖(使用前在每个注射器上钻孔 1/8 英寸孔位于 2 mL 标记处)
9. 43 mm铝称重盘( Qorpak ,目录号: MET-03105)
10. 标本钳(12 英寸长;VWR,目录号:82027-382)
11. 通用实验室胶带(VWR,目录号: 89097-912 )
12. 矩形冰盘,近似尺寸 15” L × 10” W × 6” D(VWR,目录号:10146-216)
13. 矩形塑料容器,近似尺寸 7.5” L × 5” W × 2.5” D(Cambro,目录号:42PP190)
14. 50 mL锥形管( Corning,目录号:352070)
15. 一次性 35 mm × 10 mm 培养皿(Falcon,目录号: 351008)
16. 43 mm铝制称重盘( QORPAK,目录号:MET-03105 )


设备


1. 4°C实验室冰箱(Thermo Scientific,目录号:TSV18CPSA)
2. -20°C实验室冰箱(Thermo Scientific,目录号:TSX3020FARP)
3. 分析顶部装载秤/天平,0.1 mg(OHAUS,目录号:30100604)
4. 分析天平标准品:10 g、20 g 和 30 g
5. 5” 不锈钢地质筛(1.57” 深度× 5” 直径),#10 目( SciOptic ,ASTM 10,目录号: 305 不锈钢)
6. 清除带盖的地质筛的观察顶部(参见微生物启动和监测说明 #1协议)。
7. 底板上钻有通风孔,用于放置地质筛(参见微生物启动和监测说明 #2的协议)。
8. 带温度和湿度控制的环境室


软件


1. Microsoft Excel 电子表格软件® (v16.0; Redmond, WA)
2. GraphPad Prism® ( v6;加利福尼亚州拉霍亚)


程序


A. 准备巢穴和喂食器(见微生物启动和监测说明#4协议)
1. 使用 20 mL 口服给药注射器准备糖浆进料器,在 2 mL 标记处有一个预先钻孔的 1/8” 孔。应将孔封上封口膜,以方便用对照 50/50倒置糖浆填充注射器 或者,如果实验需要,可以测试含有 50/50转化糖浆的物品。
2. 通过将注射器尖端浸入含有 50/50倒置糖浆的烧杯或锥形管中填充注射器进料器 或含有50/50转化糖浆的试验品 通过拉起柱塞。填充糖浆后盖上注射器。
3. 取下封口膜并在实验数据表中记录糖浆重量(参见支持信息图 1以获取示例数据收集表)。
4. 按照第一部分(上图)所述准备花粉饼。


B. 为巢开始准备微菌落室
1. 用开始日期和分配的微菌落编号标记每个微菌落观察顶部。
2. 将不锈钢筛子放在底板上,将吸水纸巾放在碎片之间,并在顶部清除观察盖。
3. 将底部标有微菌落编号的 35 毫米× 10 毫米一次性培养皿的盖子放在带有 3–3.25 克筑巢起始肉饼的微菌落室中(图 5)。




图 5. 微菌落室组件。
不锈钢地质筛(1.57 英寸深× 5 英寸直径),带穿透底板(#10 目),带钻孔通风孔的底板,用于收集观察结果的透明顶部,用于进入腔室内部的可拆卸盖子和注射器进料器.采用拜耳作物科学的设计。


C. 用新出现的B. impatiens工人称重和播种微菌落(微生物启动和监测说明 #5协议)
1. 填充冰盘并将矩形塑料容器放置在冰盘中心,在冰盘内边缘放置 50 mL 锥形管,用于存放收集到的新出现的工人。
2. 将足够的新出现的工人转移到 4°C 冰箱中 10-15 分钟以支持实验(即,需要的工人数量 = [(5 个工人/微群 x 所需的微群数量)+ 10% 额外的蜜蜂占死/受伤的蜜蜂])。
3. 将蜜蜂从 4°C 中取出,并将它们转移到冰上的浅矩形塑料容器中。
4. 接下来,一次将五只随机选择的蜜蜂转移到 50 mL 锥形管中。将锥形管转移到实验室工作台上,让蜜蜂再次活跃起来。
5. 称重前检查锥形管是否有蜜蜂死亡或损坏;根据需要更换死亡或受损的蜜蜂。
6. 五只新出现的蜜蜂为第一个微群称重锥形管。将蜜蜂添加到它们的微群中,然后称量空锥形管以确定蜜蜂的重量。在实验数据表中记录这些数字(参见支持信息图 2以获取示例数据收集表)。


D. 补充筑巢馅饼
1. 微菌落开始后五天,用额外的 ~ 2 克花粉糊补充巢开始馅饼。


E. 常规喂养(参见微菌落启动和监测说明 #6 - 9协议)
1. 小菌落开始后 7 天,给每个小菌落约 2 克花粉糊,用于在一次性 43 毫米铝称重盘中喂食。
2. 为微菌落提供新鲜的、预先称重的约 3.5 g 花粉糊和对照 50/50倒置糖浆 或含有 50/50转化糖浆的试验品 每周一、周三和周五,从启蒙后一周开始。
3. 将带有新花粉糊和新糖浆进料器的新盘子的重量记录到实验数据表中(有关数据收集表的示例,请参见支持信息图 1 )。
4. 用原来的盘子和以前的糖浆进料器记录旧花粉糊的重量,以确定消耗量。
5. 为了更准确地量化花粉/糖浆消耗水平,在研究设计中包括两个蒸发控制。这些控制应该像所有其他微菌落一样设置和处理,除了它们不包含蜜蜂(参见支持信息图 3 和图 4以获取示例数据收集表)。


F. 监测微菌落和数据收集(参见微生物启动和监测说明 #10 – 12协议)
1. 整个研究期间将微菌落保持在 25°C ± 0.5°C 和 50% ± 5% 相对湿度的黑暗中。当微菌落在工作台上的环境室之外时,可以使用红光。尽可能避免使用白光。
2. 评估从开始到研究终止的每个微菌落(参见支持信息图 5以获取示例数据收集表)。
3. 在每次观察期间,收集以下信息: 1)死亡工人的数量; 2) 距首次无人机出现的天数; 3)出现的无人机数量; 4) 无人机重量。
4. 可选的,可以收集其他信息,包括 1) 首次打开蛋室的时间; 2) 天数到第一次封盖蛋室; 3) 到第一个幼虫质量的天数; 4) 第一个蛹细胞的天数。
5. 即周一、周三和周五),应移除任何出现的无人机。移除后,分别称量每架无人机(参见支持信息图 6以获取示例数据收集表)。
6. 可选但鼓励:从每个实验组中随机选择并拍摄至少一个微菌落(建议在星期三),以捕捉微菌落的进展和发育里程碑。


G. 终止微菌落
1. 2麻醉对工人、无人机和剩余的育雏实施安乐死,然后转移到 -80°C。


微生物启动和监测记录协议
1. 可以根据图 6A 和 6B 中详述的规格制作观察顶部。按照设计,顶部有一个 5 英寸直径的凹槽环,可防止盖子从筛子顶部滑落。但是,可以使用其他顶部设计,只要它们 1) 足够大以覆盖筛子 [蜜蜂将被安置在筛子的网状底板和观察顶部之间的空间中(参见图 7)],具有注射器进料器的开口,并有通风孔以允许空气流通。




图 6. 微菌落观察顶部、可拆卸盖子和底板的材料和测量值。
可以根据所示规格重建先前使用的微菌落室观察顶部(A) 、可拆卸盖子(B)和底板和(C) (Camp等人, 2020a,2020c;Weitekamp等人, 2022) 。直径= 直径。


2. 底板可以根据图 8C 中详述的规格制造。底板的设计是为了防止蜜蜂排泄物和其他通过筛孔底板的碎屑污染其他微菌落并污染环境室。底板的设计包括一个直径为 5 英寸的凹陷环,筛子落入该环。然而,调查人员可以使用任何底板设计,只要底部有通风孔以允许空气流通,并且足够大,筛子可以放在上面。
3. 使用此协议时,不应由随机年龄的工人启动微菌落。使用年龄匹配的新出现的工作人员可以减少将与年龄相关的死亡与治疗效果混淆的可能性,并促进实验中和实验之间微菌落之间的一致性。使用本协议时,调查人员应使用本协议的新鲜或新鲜冷冻的蜜蜂采集的球状花粉。使用旧的(即>2 岁)或不正确储存的花粉提供微菌落可能会影响微菌落的进展和生产力。
4. 用于筑巢的花粉饼和用于常规喂食的糊状物应在开始或喂食当天新鲜制作。
5. 由于工人规模会影响微群落巢的发育和工人的食物消耗率(Peat 和 Goulson,2005 年;Couvillon 和 Dornhaus,2010 年;Amsalem 和 Hefetz,2011 年;Roger等人, 2017a,2017b) ,因此鼓励协议用户播种微群落与质量相似的蜜蜂。
6. 必须向微菌落提供随意接触花粉和糖浆的机会。限制获取食物供应将破坏小菌落的发展,降低生产力,并使实验结果的解释复杂化。
7. 延迟提供专门用于喂食的花粉是为了减少工人试图在花粉喂食和筑巢肉饼上产卵的可能性。
8. 在喂养发育中的幼虫时,小菌落会消耗更多的花粉。因此,最好给生产性微菌落约 3.5 克花粉糊,以尽量减少花粉耗尽的风险。
9. 除非需要保留以进行其他分析,否则请丢弃旧的花粉盘。
10. 为了帮助保持蜜蜂在操纵微菌落时保持冷静,请将腔室放在可吸收振动的一次性纸垫上。
11. 为了促进整个微菌落的一致性,请在环境室内旋转单个微菌落的位置。
12. 如果创始工人在前 24 小时内死亡,则将其替换为新出现的工人(获取新工人的权重)。
13. 由于微群落中工蜂的数量会影响巢穴生产力(Gradish等人, 2013 年)和食物消耗率,因此鼓励协议用户在整个实验过程中跟踪死亡工蜂的数量。
14. 无人机的生产是微殖民地成功的关键指标。首次无人机出现的时间、无人机数量和无人机重量都可以轻松量化。重要的是,所有这些措施都可能受到实验处理的影响,这些处理提供了关于测试材料如何影响微菌落的见解。


数据分析


这对协议旨在探索农药暴露对微菌落进展和生产力的影响(参见 Camp等人,2020a、2020c; Eitekamp 等。 , 2022)。在这种情况下,这些协议可以很容易地用于解决各种研究问题,从而使研究人员能够深入了解大黄蜂生物学的其他方面。根据使用该系统的经验,每个实验组应使用 8-10 个微菌落。下面概述了如何使用该协议处理和分析从农药暴露影响研究中收集的数据。可以在Camp等人中找到处理和分析这些端点数据的详细方法。 (2020a, 2020c) 和 Weitekamp等人。 (2022 年) 。为了帮助新的调查人员,样本数据收集表以及有关如何记录和处理微菌落数据的指南在本协议末尾作为补充信息提供。
微菌落数据的分析可以分为两个相互关联的部分。对于第一部分,应目视检查微菌落并拍照,以确定与处理相关的对巢进展的影响。根据该协议建立的微菌落将根据图 7 所示的时间线进行。每周收集观察时,未封盖的蛋室将在第 1 周结束时出现。封盖的蛋室有时会在第 1 周后期出现,但最常见的是在结束时第 2 周。在第 2 周和第 3 周可以检测到幼虫团块。蛹细胞将在第 3 周和第 4 周出现。第一批雄蜂在第 5 周出现,并将在研究的剩余时间里继续出现。如果需要,可以更频繁地收集观察结果,以梳理出对微菌落发育的微妙影响。但是,收集观察结果非常耗时,并且频繁的中断可能会影响工人的行为并最终影响研究结果。




图 7. 七周发育过程中的微菌落进展。
由五名新出现的B. 凤仙花工人启动微菌落,并提供花粉糊 (3 g) 和 50/50倒置糖浆的巢启动肉饼 刺激筑巢。 5天后,给巢穴额外添加2克花粉糊。从第 7 天开始,每周一、周三和周五给微菌落提供新鲜的花粉糊和糖浆。从第 0 天开始(即巢开始),照片显示了从无盖卵室到第 49 天研究终止的微菌落进展。上面带有标签的条形表示特征通常出现的范围。编号的圆圈表示微菌落的特征,包括 1 = 巢起始肉饼,2 = 第 5 天给予的花粉补充剂,3 = 未封盖的卵室,4 = 幼虫质量,5 = 蛹细胞和 6 = 疏散的蛹细胞。


除了定性评估微菌落状态外,还应统计分析来自不同终点的数据,包括微菌落发育里程碑(即,第一次开盖卵室的时间、第一次加盖卵室的天数、第一次幼虫质量的天数和第一次蛹的天数细胞)、糖浆和花粉的消耗、无人机的生产和重量以及工人的生存。这些数据应表示为平均值±标准偏差 (STDEV)。使用 GraphPad Prism ® (v6; La Jolla, CA),可以通过单向方差分析 (ANOVA) 和 Dunn 的多重比较检验来评估对照组和治疗组之间的差异。如果根据 Brown-Forsythe 或 Bartlett 检验,方差显着不同,则使用具有 Dunnett 多重比较检验的非参数 Kruskal-Wallis。


数据报告
为了在研究之间进行比较并促进研究复制,鼓励研究人员报告 1) 用于喂养的糖浆配方,2) 花粉来源和年龄,3) 工蜂的年龄和来源,以及 4) 环境条件(即,温度、相对湿度和光照方案)在实验期间使用。此外,还应记录和报告开始、结束和数据收集日期。所有数据都应作为补充信息或通过科学数据的公共存储库提供给其他研究人员。


数据分析笔记
1. 在调查农药暴露对微菌落发育和生产力的影响时,研究人员应在研究设计中包括未处理的对照组、阳性对照组和溶剂对照组(如果适用)。在进行农药暴露研究时,还应包括蒸发控制以纠正食物消耗估计。
2. 需要时,可以通过糖浆和/或花粉供应将杀虫剂递送至小菌落。通过糖浆施用杀虫剂通常比花粉更容易。然而,并非所有农药都是水溶性的,因此,可能需要溶剂来溶解测试材料。在这种情况下,研究人员需要凭经验确定用于实验的合适溶剂浓度 [例如,1% 丙酮(Camp等人, 2020b) ]。
3. 在评估农药暴露对微菌落的影响时,传递载体(即花粉或糖浆)会影响研究结果。发育中的大黄蜂育雏会消耗大量花粉,因此,通过花粉传递测试材料可能会针对育雏。至此,啶虫脒在花粉中递送,但在糖浆中递送时,降低了无人机的平均重量(Camp等人, 2020a,2020c;Weitekamp等人, 2022) 。因此,仅通过糖浆给药可能会错过重要的育雏效果。
4. 糖浆和花粉消耗值应根据蒸发进行校正,在确定每只蜜蜂的消耗量时,应根据工人死亡率进行校正。此外,如果装满糖浆的注射器泄漏,导致消耗值不准确,则应将该值替换为该治疗组和当天的平均糖浆消耗值。
5. 已发表的结果表明,微菌落模型可能仅适用于评估对成年工人具有低毒性的物质的育雏效应(Krueger等人, 2021 年) 。


食谱


1. 山梨酸钾溶液
通过将 25 g 山梨酸溶解在蒸馏水中以达到 100 mL 的最终体积,制备 25% w/v 山梨酸和钾盐溶液;无菌过滤器 (0.45 µm) 并在 4°C 下储存长达 90 天。


致谢


作者感谢 Drs。 JE Simmons、C. Weitekamp 和 YH。 Kim 对这份手稿进行了深思熟虑和批判性的审查。作者也感谢 Drs。 D. Schmehl、A. Cabrera、J. Strange 和 D. Cox-Foster 的慷慨和指导。该协议改编自之前发表的同行评审期刊(Camp等人, 2020a,2020c;Weitekamp等人, 2022) 。


利益争夺


作者宣称没有利益冲突。


参考


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引用:Lehmann, D. M. (2022). Protocol for Initiating and Monitoring Bumble Bee Microcolonies with Bombus impatiens (Hymenoptera: Apidae). Bio-protocol 12(12): e4451. DOI: 10.21769/BioProtoc.4451.
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