Nano Colonies: Rearing honey bee queens and their offspring in small laboratory arenas

20 Honey bees create complex societies of self-organized individuals in intricate colonies. Studies 21 of honey bees are carried out in both the field and the laboratory. However, field research is 22 encumbered by the difficulties of making reliable observations and environmental confounders. 23 Meanwhile, laboratory trials produce data that are not field realistic as they lack key 24 characteristics of a natural colony. Additionally, advances in honey bee research have been 25 hindered without reliable methodology to rear queens in the laboratory. Here we provide a new 26 system to reliably produce queens and worker brood in the laboratory and describe how this 27 system fits with artificial insemination of queens as a step towards a continuous self-contained 28 source of bees. The process creates a bridge between field research and laboratory trials and 29 provides a secure system for contagious or regulated elements while maintaining many of the 30 intrinsic characteristics of a honey bee colony. 31

32 33 Honey bees are social organism s that are important to our economy by providing key 34 pollination services for many food crops and producing honey and wax1. Ongoing research into 35 honey bee health is integral for understanding factors that impact colony health. However, there 36 is gap in research methodology that impedes advances; there is no reliable method to rear honey 37 bee queens in the laboratory or to raise bee-reared progeny in the laboratory2. 38 Researchers study honey bees in both field and laboratory conditions. Traditional field 39 trials provide real-world results and maintain all colony conditions, while laboratory trials lack 40 key colony characteristics. However, field trials are costly, laborious, and vulnerable to 41 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint environmental variation. Confounding variables are an inherent problem with field trials due to 42 the difficulty in assessing and accounting for landscape effects including microclimate, pesticide 43 exposure, and interactions with bees encountered during flights that span multiple kilometers3. 44 Laboratory trials offer a more uniform environment as the researcher can control all 45 parameters in the experimental design. Arenas are typically made from clear plastic, allowing 46 complete visibility to the researcher4-6. Due to the ease of setup and low cost, high numbers of 47 replicates are more achievable and confounding variables are limited. Laboratory trials have 48 created great advances in honey bee research, especially in regard to novel biossays5,6. However, 49 laboratory trials exclude many components inherent to the colony environment. Specifically, 50 laboratory arenas lack key colony characteristics such as brood, wax, a queen and associated 51 pheromones, communication pathways and social behaviors that are inherent to field colonies. 52 The absence of these key characteristics makes it difficult to extrapolate findings from the 53 laboratory to the real world. 54 Current in vitro methodology allows researchers to rear brood artificially in the 55 laboratory7. The methodology utilizes artificial feedings by a technician rather than brood care 56 from an attending nurse bee. As a result, the hypopharyngeal glands, a gland unique to 57 hymenopterans that generates a processed food source8, are completely bypassed. What is 58 needed is a bridge between the real-world colony environment provided in field trials with the 59 low cost and reliability of cage trials. 60 Here we describe one such bridge. We have developed a method to get a small number of 61 worker bees to both rear queens in the confines of the laboratory and support a laying queen and 62 raise her offspring. These laboratory colonies can be made with as few as 100 bees housed in a 63 typical incubator. The colonies are referred to as queen-rearing or queen-right “nanos” or “nano-64 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint colonies”. They provide a next step in laboratory methodology to help bridge the complexities of 65 a colony with the simplicity of the laboratory environment. 66 In this study we experimentally produced queens in laboratory settings. In several trials 67 these queens were allowed to open mate, and we compared their mating success and morphology 68 to traditionally produced sister queens. In another trial, we produced and instrumentally 69 inseminated queens in the laboratory and then supported in vivo brood rearing without exposing 70 these queens to the field. Finally, we experimentally produced brood through a reliable in vivo 71 laboratory design. 72 73

74 Overview 75 Queen-Right Nanos 76 Queen-right nanos consist of 100-240 worker bees and a queen. The hive is made from a 77 modified plastic cup ventilated with #8 hardware cloth and noseum netting. A portion of empty, 78 dark (seasoned in colonies after multiple rounds of worker bee production) brood comb serves 79 as the brood area for the colony. Young worker bees are installed either soon after emergence or 80 by being scooped from a brood frame. 81 Queen-Rearing Nanos 82 Queen-rearing nanos are queenless colonies of approximately 100 bees in modified plastic cages 83 that will rear their own queen. The colonies are easy to establish and maintain in the laboratory. 84 Establishment is completed over 2 steps. First, a strong queenless colony with ample provisions 85 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint and young worker bees (a ’cell builder’ colony) is made in the field. First instar larvae are 86 grafted into plastic queen-rearing cages and placed into the cell builder for 12-24 hours. Next, 87 nano colonies are made by securing an initiated queen cell into the nano, and then harvesting a 88 small number of bees (approximately 100) from the cell builder and securing them inside the 89 modified plastic cage. The nano colonies are then given feeders, sealed, and placed into an 90 incubator. 91 Making the Modified Cages 92 The cages are modified by removing the lower third of a 16 oz clear plastic cup, and then 93 securing #8 hardware mesh to the bottom opening with hot glue. The lid of the cage is comprised 94 of two materials: 4x4 inch piece of ‘noseeum’ mesh, and a lid to the plastic cup with the center 95 cut out. A piece of paraffin wax honey comb (Betterbee, Inc.) is cut to fit into the center of the 96 cup for the queen-rearing nanos. In contrast, empty, dark brood comb works best for the queen-97 right nanos because it allows for easy observation of eggs and young larva. The comb is placed 98 in a -20 freezer for 10 minutes, and then cut into squares on a table saw to fit inside the modified 99 plastic cups. 100 2 ml Eppendorf tubes are used as feeders for each colony type. For the sucrose and water 101 feeders, a 2 mm hole is made in the bottom of the tube to allow bees to feed on the contents. 102 Holes were made by perforating the tube with either a small brad nail or a 5/64 drill bit. A small 103 cut in the noseeum lid is made with dissecting scissors and the feeder is then squeezed into this 104 slit. The fabric will stretch as the tube is inserted. The lid of the tube is wider than the hole made 105 by the bottom, and as such the feeder remains suspended in the lid. Pollen feeders were made by 106 cutting 5 mm diameter opening in the bottom of an Eppendorf tube with a razor blade, and then 107 packing the tube with either bee bread or pollen substitute (MegaBee) mixed according to the 108 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint manufacturer’s directions. Pollen feeders were changed every other day. Each queen-rearing 109 nano held 1 water feeder, 2 sucrose feeders (1:1 sucrose w/v) and 1 pollen feeder (Fig. 3). These 110 feeders were changed daily. Seven ml of Supplementary diet (see Supplementary Information 111 Table 1-2) is mixed with 43 ml of honey. The honey mixture is placed into feeders which are 112 identical to the pollen feeders, and inserted into the cages from underneath so that the bees may 113 access the honey similar to Shpigler et al5. A step by step guide to cage construction can be 114 found in Supplementary. 115 Establishing queen rearing nano colonies 116 The queen-rearing nano colonies need two components to be established: a started queen cell and 117 nestmate bees to complete queen rearing. First, queen cells are established by grafting 1st instar 118 worker larvae into plastic queen cups (JZ/BZ) and placing these into a traditional cell builder for 119 12-24 hours. During this time the nestmate bees of the cell builder will engorge the larva on a 120 bed of royal jelly. We consider this a started queen cell. The queen cups are inspected 12-24 121 hours post insertion, gently rolled upwards and visually inspected for a small pool of jelly below 122 a c-shaped larva. Empty cups or cups without a larva are not considered viable. 123 The second component is to populate the cages with worker bees who will attend and finish 124 rearing queen cells. Workers can be acquired in one of two ways. A frame of emerging brood 125 can be placed into an incubator and allowed to emerge, after which 100 newly emerged bees are 126 placed inside a nano cup, provided a protein source, sucrose, and water, and then placed back 127 into an incubator at 33oC for three days in order to mature. Conversely, workers can be obtained 128 directly from the cell builder by shaking the nurse bees from a central frame of the cell builder 129 into a plastic tub, and then scooping 1/3rd cup of bees into a nano, and then quickly sealing it. For 130 the first method, the queen cell is inserted into the cover of the nano for the bees to access it. For 131 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint the second method, the started cell cup is directly pressed against the wax comb inside the nano, 132 and then the nurse bees are added. We used a digital scale and measured 100-130 grams of bees 133 to estimate the nurse bee population. We did this in sets of ten without any observable stress or 134 loss to the queen cells. 135 Inspecting the nano colonies 136 Egg laying, brood development, queen cell development and nestmate mortality are all 137 observable in queen-right or queen-rearing nano colonies. Although the plastic cup arena is 138 inexpensive and easily procured, the concave nature of the cup does not allow for quantification 139 of egg laying or brood development. Observers have some ability to view eggs and track larval 140 development by tilting the cup while shining a light into the cage. We made reference cells prior 141 to nano establishment by plunging a white-out applicator into a series of cells. During inspection 142 these marked cells act as convenient reference points for egg laying and brood development in 143 adjacent cells. The observer simply has to record the status of adjacent cells. An example of a 144 typical queenright nano colony can be seen in Figure 1. 145 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint 146 147 Figure 1: A typical queen-right nano 11 days after establishment. Center is one half of the brood area with capped 148 and uncapped brood visible. Center and right, the adult bee population was anesthetized with carbon dioxide . The 149 marked queen, and the entire worker population is presented. 150 151 We used preliminary trials to test an arbitrary range of how many adult worker bees were 152 needed to establish an artificial colony. Colonies with both low and high ranges of adult bees 153 (100 – 240) successfully reared brood. 154 Bees to establish nanos can be collected either from emerging brood frames or directly 155 from the brood frames in a field colony. Frames of emerging brood from healthy colonies 156 exhibiting no covert signs of disease are placed into an incubator at 33oC and 60% humidity in a 157 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint frame holder. Bees are collected within 24 hours of emergence, and placed into a nano setup. For 158 the latter, when collecting bees directly form a colony, bees collected directly from colony brood 159 frames are placed into the nano colony. Queens can be installed prior to or soon after worker 160 addition. Colonies are then maintained in an incubator at 33oC. 161 Comparing mating success of queens reared in traditional cell builders to laboratory 162 reared queens 163 164 In two trials, queens were reared in the laboratory in parallel with sister reared queens in a 165 traditional cell builder. The two groups were then compared for mating success. During the 166 second trial, additional treatments within the nano group were added. We tested if queens could 167 be reared not just on bee bread, but also artificial pollen substitute. We created a third group 168 testing if the amount of time a queen cell was started in a traditional cell builder could be 169 reduced to as little as 12 hours before being transferred into a nano colony for completion. These 170 additional groups had few replicates in each group (N = 6). 171 Instrumental insemination of lab reared queens 172 We produced queens in the laboratory in two different trials, and then instrumentally inseminated 173 the queens. The queens were re-introduced into nano colonies to lay. 174 175 176 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint 177 Figure 2: Step 1: Nurse bees are procured and placed into a cell builder. 1st instar larvae are grafted into queen cups 178 and placed inside the cell builder for 12-24 hours for initiation. Afterwards, queen cells are placed into nano 179 colonies and given nest mate bees to continue queen rearing. Step 2: Mature queen cells (10 days after grafting) can 180 be moved to traditional mating nucs for open mating or they can remain in the lab for instrumental insemination. 181 182 183 184 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint 185 Figure 3 The arena depicted above produced virgin queens using pollen substitute, sucrose and parafilm comb, and 186 approximately 130 worker bees in the arena. (A) A queen-rearing nano colony with open queen cell and larva 187 viewable from the underside of the arena. The queen cell depicted is 24 hours after insertion into the nano colony. 188 (B) A virgin queen newly emerged from her pupal cell. Not seen are the worker bees which were anesthetized with 189 carbon dioxide and removed prior to queen emergence. 190 191

192 Queen-right nanos 193 In two different trials, queen-right nanos were established (N = 24), and maintained for 194 18 days. All colonies supported a laying queen and produced brood during this time period. 195 Brood progression was visually analyzed on day 18 when the nanos were deconstructed. The 196 majority of nanos had developed brood, 23/24 (95.8%). Of these, 13 had capped brood, with an 197 additional 3 having late stage, L5, larvae, but no capped brood. Manual inspection of the worker 198 brood revealed typical worker pupae, which can be seen in figure 1. No experimental variables 199 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint were tested in these trials, but rather the experiments were carried out as a proof of concept if a 200 small number of bees could rear brood in laboratory settings. Queens were overnight mailed to 201 the Queen and Disease Clinic at North Carolina State University Extension for morphometric, 202 sperm viability and sperm quantity analysis. 203 204 Queen-rearing nanos 205 Comparing mating success of queens reared in traditional cell builders to laboratory 206 reared queens 207 There was no difference in mating success between the two groups in the first trial: 6/14 208 (42.9%) of the queens in each group successfully mated. More queens successfully mated in the 209 cell builder group (7/10, 70%) than any of the nano groups in the second trial (37.5% across all 210 nano groups, figure 4). 211 212 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint 213 Figure 4: Mating success was measured between virgin queens produced using a traditional cell builder 214 and virgin queens reared in the laboratory. There was no difference in mating success during the July trial. More 215 queens by a proportion successfully mated in the traditional cell builder trial (70%) than any other laboratory group 216 (17% - 50%) during the August trial. The differences were not statistically significant, X2(1) = 1.83, p = 0.176. 217 218 Morphometrics of queens reared in a traditional cell builder to laboratory reared queens 219 There was no difference between the body weights of queens between the cell builder (M 220 = 192.77, SD = 9.73) or nano groups (M = 184.08, SD = 11.2), t(14.23)=1.78, p = 0.097. Nor 221 was there a difference in sperm viability between the two groups: cell builder ( M = 0.7957, SD 222 = 0.0709) and nano (M = 0.8408, SD = 0.0464), t(9.064)=-1.51, p = 0.166. There was also no 223 difference in total sperm stored in the spermathecae of mated queens regardless of whether they 224 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint were reared in a traditional cell builder (M = 4.24, SD = 2.29) or a nano colony (M = 5.5, SD = 225 1.42), t(8.76)-1.32, p =0.22. (figure 5) 226 227 228 Figure 5 There was no significant difference between groups when analyzed for sperm viability, t(9.064)= -1.51, p = 229 0.166, and total sperm stored in queen spermatheca’s, t(14.23)=1.78, p = 0.097. 230 231 Instrumental insemination of laboratory-reared queens 232 Few queens successfully laid eggs, and fewer reared worker brood. In the first trial, 233 queens did not immediately begin egg laying after initial carbon dioxide (CO2) exposure. Queens 234 were anesthetized with CO2 a second time after insemination. Egg laying began 2 weeks after 235 insemination. Nine queens began laying (9/23, 39.1%), but none of the colonies successfully 236 reared late-stage larva. We did not confirm if the queens were laying fertilized eggs. In trial 2, 237 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint queens were also anesthetized for a second time after insemination with CO2. Eight queens 238 successfully laid eggs (8/12, 75%); Three colonies reared late-stage worker larva, and one nano 239 colony, headed by a lab produced, instrumentally inseminated queen, successfully reared 240 emerging adult worker bees. 241 242

243 Social insects create complex societies which can be difficult to study in field 244 environments. Current laboratory systems simplify these societies into small groups while losing 245 key characteristics. Here we provide a new laboratory method that utilizes a small number of 246 bees to rear queens or worker brood while maintaining many colony characteristics. This method 247 advances honey bee research by building a bridge between the complexities of field trials and the 248 ease of use of laboratory research. 249 Our nano colonies bring more features inherent in honey bee colonies directly into the 250 laboratory. Our colonies have laying queens, brood and honey comb. We hypothesize our setup 251 could be useful studying exposure to disease or chemical stress on worker physiology. The 252 absence of a queen or brood can affect worker bee physiology, limiting the relevance of current 253 arenas. Our methodology provides a bridge between traditional field trials and the laboratory, 254 and could be employed to advance other areas in honey bee research such as studying queen 255 development, modified viruses, transgenic bees, or studying principle drivers of honey bee 256 health. 257 Using a nano colony, researchers can study queen development and colony establishment 258 in a closed environment and under controlled conditions. These queens can then either be 259 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint returned to the field for natural mating or instrumentally inseminated, and then returned to a new 260 nano colony setup to begin egg laying. We were able to produce queens after nominal larval 261 development in the field, with a small number of nurse bees. Queens were successfully produced 262 with either bee bread harvested from colonies or pollen substitute. We successfully reared and 263 instrumentally inseminated queens in the laboratory, representing a closed loop methodology of 264 queen to brood production, and brings a valuable toolset to the researcher. Queen right nano 265 colonies were also used to develop worker brood within the confines of a laboratory incubator. A 266 relatively small number of approximately 100 (100 – 150) bees supported a laying queen and 267 reared her hatched eggs to the final stage of development as healthy pupae 268 Rearing queens and brood in the laboratory advances honey bee research by bridging the 269 gap between field and laboratory trials. The method can be developed so that all components of 270 each nano colony can be sampled and then examined against each other in a simple, closed 271 system. In this way we may be able to parse out how individual drivers of honey bee health are 272 impacting each component of a honey bee colony9. Variation can be further reduced by using 273 pools of homogeneous bees. Researchers could potentially use this methodology to study the 274 impact of one variable at a time on queen development or in a factorial design studying multiple 275 variables at a time. 276 As is, this system can provide colony-like conditions into the laboratory, offering a closed 277 system in which queens, workers, and developing bees can be observed and readily harvested for 278 study. Nevertheless, this method can be further optimized. We did not test the resiliency of these 279 laboratory colonies, and more work can determine the long term fitness of queens and worker 280 bees reared in this way with different microbiome and nutritional provisions. We utilized 281 inexpensive and widely available 16-ounce clear plastic cups to construct our colonies. This 282 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint convex cup limits clear observation of developing brood but a flat design would allow for 283 observation and quantification of brood development. Finally, it would be ideal to initiate queen 284 rearing directly from eggs, so that their entire development could be controlled. We anticipate 285 these are all solvable issues. 286 287

288 Cory Stevens is an independent researcher at Stevens Bee Co., Bloomfield, MO, USA 289 Jason Bragg is an independent researcher at New River Honey Bees in Calvin, WV, USA. Queen 290 morphometrics and sperm viability were provided by the Queen and Disease Clinic through 291 North Carolina State Extension 292 293 Funding 294 Funding was provided by the New Hampshire Beekeepers Association, the Cosmos Club 295 Foundation, the Nansemond Beekeepers, and the Montgomery County Beekeepers. 296 297 298 299 300 301 302 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint

303 304 1 Khalifa, S. A. et al. Overview of bee pollination and its economic value for crop production. 305 Insects 12, 688 (2021). 306 2 McAfee, A., Pettis, J. S., Tarpy, D. R. & Foster, L. J. Feminizer and doublesex knock- outs cause 307 honey bees to switch sexes. PLOS Biology 17, e3000256, doi:10.1371/journal.pbio.3000256 308 (2019). 309 3 Corona, M., Branchiccela, B., Madella, S., Chen, Y. & Evans, J. Decoupling the effects of 310 nutrition, age and behavioral caste on honey bee physiology and immunity. BioRxiv, 667931 311 (2019). 312 4 Evans, J. D., Chen, Y. P., Prisco, G. d., Pettis, J. & Williams, V. Bee cups: single- use cages for 313 honey bee experiments. Journal of Apicultural Research 48, 300-302, 314 doi:10.1080/00218839.2009.11101548 (2009). 315 5 Shpigler, H. Y. & Robinson, G. E. Laboratory Assay of Brood Care for Quantitative Analyses of 316 Individual Differences in Honey Bee (Apis mellifera) Affiliative Behavior. PLoS One 10, 317 e0143183, doi:10.1371/journal.pone.0143183 (2015). 318 6 Fine, J. D. et al. Quantifying the effects of pollen nutrition on honey bee queen egg laying with a 319 new laboratory system. PloS one 13, e0203444-e0203444, doi:10.1371/journal.pone.0203444 320 (2018). 321 7 Schmehl, D. R., Tomé, H. V. V., Mortensen, A. N., Martins, G. F. & Ellis, J. D. Protocol for the 322 in vitro rearing of honey bee (Apis mellifera L.) workers. Journal of Apicultural Research 55, 323 113-129, doi:10.1080/00218839.2016.1203530 (2016). 324 8 Snodgrass, R. E. Anatomy of the honey bee . (Cornell University Press, 1956). 325 9 Steinhauer, N. et al. Drivers of colony losses. Current opinion in insect science 26, 142-148 326 (2018). 327 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint 328 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint Step by step guide to constructing nano cages (1/5) 1. The materials needed to construct nano colonies. 2. Two constructed nano colonies. (Left) Made with real comb and two feeders. This is used for queen-right nanos. (Right) Made with paraffin wax and a single feeder for a queen-rearing nano. 3-4. Preparing the cage. A 16 oz plastic cup is portioned with a razor blade. The cup is pierced with the tip of the blade, and then the cup is rolled until the cup is severed. In picture (4) the final cut can be seen. 5-6. The bottom of the cage is constructed from #8 hardware cloth. “Tin snips” are an excellent tool to cut pieces to length (5). The pieces can then initially be adhered to the cut plastic cups using hot glue (6). 1 2 3 4 5 6 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint Step by step guide to constructing nano cages (2/5) 7-8. Weighted beakers ensure good contact between the plastic cup and the hardware mesh while the glue is drying (7). Afterwards, the beakers are removed and a bead of hot glue is applied directly on the joint of the hardware cloth and plastic cup. Then leave the cup to dry. 9-12. Access holes for the feeders must be added through the bottom. This can be accomplished with a Dremel tool or (9) by using a pair “Tin snips”. The access hole is cut slightly smaller than the diameter of a 2ml Eppendorf tube. A tube is then inserted into the precut hole (10) to finish. Making the feeders can be done by cutting off the bottom of a 2 ml Eppendorf tube with a razor blade against a cutting surface (11-12). 7 8 9 10 11 12 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint Step by step guide to constructing nano cages (3/5) 13. Sheets of paraffin wax can be easily cut to length using a “multi-tool” and pre-cut cardboard templates. 14-15. Frames of wax are cut by first chilling them in a -20c freezer, and then cutting them to length and width on a table saw. 15 Pieces ready to be inserted into prepared cages. 13 14 15 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint 16-18. The lids (16) function to seal the noseeum netting. Lids are prepared by piercing a plastic cup lid with a razor blade so that the tip of the razor is lightly imbedded into the cutting board. Then the plastic lid is turned until the blade has completely severed the inside of the lid, leaving just a ring. To finish construction of the cups, a lid is secured in place (17). Pollen is loosely packed into a prepared feeder (18). Step by step guide to constructing nano cages (4/5) 16 17 18 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint Step by step guide to constructing nano cages (5/5) 26. A nano colony 8 days after being set up. A bee is consuming pollen through the feeder while others linger or attend brood. Developing larvae are visible through the concave structure of the cage. 19 - 21. Bulk bees from a cell builder (19) are scooped into premade nano setups which already have a started queen cell (20). The queen-rearing nanos about one hour after setup in an incubator (21). 22-25. Newly emerged bees are collected in bulk, and then scooped into premade nano colonies. A scale can be used for quality control (23). Queens can be directly introduced to newly emerge bee nanos through the top entrance (24). Replacing feeders and removing dead bees (25). 19 20 21 22 23 24 25 26 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.20.576222doi: bioRxiv preprint