DOI: 10.15482/usda.adc/26950459.v2
title: Data from: Corazonin responds to nutrient stress and increases flight activity in <i>Apis mellifera</i> workers

description: We asked whether the insect peptide hormone corazonin changes honey bee flight behavior. To address this question, we injected bees with corazonin or a negative control, placed them back in their colony, and measured their daily flight activity.<br>For "concentrationsFlightActivity.xlsx":Fifty 9d old bees were injected in the head with 2µl of a low (25ng/µl), middle (125ng/µl), or high (250ng/µl) dose of CRZ peptide in 1X Ringer’s solution, or 1X Ringer’s solution alone (control). The bees were then marked according to the treatment they received and were returned to their respective hives. For 5 days post-injection (10-15d post-emergence), the entrance of each hive was monitored for 10 minutes each hour for five hours (06:00-11:00 MST, 50 minutes daily per hive). The number of painted bees that left or returned to the hive during this time and their paint color was recorded.Columns in data sheet represent the concentration of peptide injected ("treatment"), whether a flight event was observed ("F vs NF"), hive number ("hive"), age of bee that was observed flying ("age"), and the trial ("round").For "2019FlightActivity.xlsx":Injections and behavioral observations were conducted in June/July 2019 for bees injected at 9d of age. Approximately 700-800 NEBs were obtained, marked, and distributed equally into four separate hives. The marked bees were collected from the hive at 9d and injected with 2µl of CRZ peptide (125ng/µl) or an equivalent concentration of scramble peptide (125ng/µl; negative control) in 1X Ringer’s solution. Each bee was then re-marked according to the treatment they received (CRZ or scramble control) and returned to their hive. Post-treatment flight activity was monitored for five days, for 50 minutes per hive each day. After each daily observation period, we also counted the number of painted bees of both treatments that were found on the frames and hive body or dead bees on the bottom board or at the hive entrance. The data represent whether an individual was observed flying ("F") or was alive but did not fly ("NF").<br>The columns represent whether the individual observed was injected with the control or corazonin peptide ("treatment"), whether the individual observed was flying or alive but not flying ("F vs NF"), the hive number ("hive"), and the age of the observed bee in days post-emergence ("age").For "2020FlightActivity.xlsx":Injections and behavioral observations were conducted in April/May/June 2020 (injected at 8d of age). Approximately 700-800 NEBs were obtained, marked, and distributed equally into four separate hives, as described above. The marked bees were collected from the hive at 8d (Trial 2) and injected with 2µl of CRZ peptide (125ng/µl) or an equivalent concentration of scramble peptide (125ng/µl; negative control) in 1X Ringer’s solution. Each bee was then re-marked according to the treatment they received (CRZ or scramble control) and returned to their hive. Post-treatment flight activity was monitored for five days (10-15d post-emergence) for 50 minutes per hive each day. After each daily observation period, we also counted the number of painted bees of both treatments that were found on the frames and hive body or dead bees on the bottom board or at the hive entrance. The data represent whether an individual was observed flying ("F") or was alive but was not flying ("NF").The columns represent whether the individual observed was injected with the control or corazonin peptide ("treatment"), whether the individual observed was flying or alive but not flying ("F vs NF"), the hive number ("hive"), and the age of the observed bee in days post-emergence ("age").For "injectionSurvivalExperiment.xlsx":Individuals were collected from the entrances of 3 separate colonies and were anesthetized on wet ice for ~15 minutes. The bees were kept separate according to the hive they were collected from. Half of the bees from each hive were handled for ~30-45 seconds and marked with paint on the thorax. The other half of the bees were injected with 2µl of 1X Ringer’s solution (182mM KCl, 46mM NaCl, 3mM CaCl2 dihydrate, 10mM Tris-HCl) in the center of the head on the anterior (face) side, just behind the antennae (as in (Corby-Harris et al., 2019)) and marked with a different color paint. All bees were kept on ice until the injections were complete, warmed at room temperature until they were active, and returned to their source hive. At 24 and 48h post-treatment, we searched the ground near the hives and then opened and inspected the colony for painted bees. The painted bees (live or dead) were counted. This was repeated for three colonies and for three trials (13 February, 26 February, and 18 March 2018). Columns in data sheet represent the colony number, the time of the observation post-treatment ("time of death"), whether the painted bee was observed or not (i.e., censored) at that time point ("censored"), the trial number ("trial"), and whether the bee was injected or simply handled but not injected ("treatment"). [file added January 2025]For "vgCrzExpressionCagesInIncubator.xlsx":The head tissue from 6 bees per cage (10 cages, 5 pollen-fed, 5 pollen-starved) was pooled and crushed in ice-cold TRIzol. RNA was extracted from the head tissue using a “hybrid” TRIzol-column protocol (Untergasser, 2008). The DNased RNA was used as a template for cDNA synthesis using the revertAid First Strand cDNA Synthesis Kit and the supplied random hexamer primer.Gene expression was measured using the synthesized cDNA. In addition to the vg and actin primers used in previous studies (Corby-Harris et al., 2014; Corby-Harris et al., 2016), a crz primer-probe set was developed based on the Apis mellifera crz mRNA sequence (NCBI accession AB201717.1). Expression of vg and actin was assayed using SsoAdvanced Universal SYBR Green Supermix, the cDNA, and primers for vg and actin (the reference gene). Crz gene expression was assayed using the cDNA, the crz primer-probe, and iTaq Universal Probes Supermix. One positive (Apis mellifera gDNA) and one negative control (water) were assayed for each primer pair. All samples were run in triplicate. Expression (Ct) values were averaged across the technical replicates. Gene expression (relative to actin) was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001).The columns represent the cage number ("cage"), the age that the individuals were sampled in days ("age"), whether the individual observed was flying or alive but not flying ("F vs NF"), the hive number ("hive"), expression of crz relative to actin ("crz_expression"), expression of vg relative to actin ("vg_expression"), and whether the bees in the cage were fed pollen or no pollen ("treatment"). [file added January 2025]<br>