GC-MS Investigation of Volatiles from Honeybee 2 Worker Larvae and Larval Food at Different Instars 3

: ( E )- β -ocimene was the only found volatile chemical emitted by whole, live worker 13 larvae of Apis mellifera L. by sampling in the vapor phase. While in addition to ( E )- β -ocimene, 14 there is evidence for the existence of other volatiles; but the changes of their composition and 15 contents remain unknown during larval development, as are their differences from larvae to 16 larval food. This is the main purpose of the study. We investigated volatile components of 17 worker larvae and larval food using solid phase dynamic extraction (SPDE) coupled with gas 18 chromatography-mass spectrometry (GC-MS). Nine compounds were identified with certainty 19 and six tentatively, consisting of terpenoids, aldehydes, hydrocarbons, ester and ketone. The 20 contents of volatiles of the second-instar worker larvae differ greatly from larvae of other stages 21 mainly attributable to terpenoids, which made the second-instar worker larvae had 22 significantly higher amounts of overall volatiles. Larval food contained significantly higher 23 amounts of aldehydes and hydrocarbons than the corresponding larvae from the fourth to 24 fifth-instar. We discovered volatiles in worker larvae and their food which were never reported 25 before; we also mastered the change of these volatiles’ contents during larval development.

interpreting their functions.

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Food provided to honeybees could directly or indirectly affect honeybee volatiles 49 production. There is evidence that food shortages might stimulate worker bee larvae into 50 releasing more (E)-β-ocimene [9]; and feeding honeybee worker larvae essential oils via diet 51 supplements may change their volatiles [10]. However, the volatiles extracted from worker larval 52 food have not been reported before. Only volatile carboxylic acids were identified in drone larval 53 food, and other unidentified non-acidic volatiles were noteworthy [11]. If the volatiles of the 54 worker larval food were analyzed combined with the volatiles analysis of the worker larvae, it 55 would provide a deep insight into the relationship between volatiles in larvae and their food 56 during the same larval instar.

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In this paper, we analyzed the volatiles from worker larvae and their food at different 58 instars, using solid phase dynamic extraction (SPDE) combined with high resolution gas 59 chromatography tandem mass spectrometry (GC-MS). Through this research, we discovered 60 some volatiles in worker larvae and their food that have been overlooked before. We believe that 61 these discoveries are a first step towards determining the underlying important functions of 62 volatiles within honey bee nests.

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After the combs were brought back to the laboratory, cells were randomly chosen to provide 73 both the larval food and larvae samples for further analysis. Larvae and larval food was obtained 74 with a small spatula. Larvae were inspected under the microscope, and only live and uninjured 75 larvae were used for the study [9].

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During the equilibration and extraction procedure, the larvae were alive and isolated from food 95 for more than 45 min [9]. The needle was then withdrawn and introduced into the injection port 96 of the gas chromatograph, and pumped with 1 mL nitrogen at 100 µL/s for desorption, at 250°C 97 for 2 min in splitless mode.

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Qualitative and quantitative analysis

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The identification of the compound with authentic standards was performed by comparing 107 the mass spectra (Wiley6 and NIST05) and retention times to those of authentic standards.

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Compounds without standards were identified by comparing the mass spectrum peaks with

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We detected fifteen compounds from the developing larvae and their corresponding food, 128 which could be sorted into seven groups: three aldehydes, one ester, three hydrocarbons, one

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When comparing the content in larvae and food at the same stage as ng/mg, the larvae had 162 significantly higher terpenoid content than food at the 2nd instar, except α-cedrene, β-cedrene 163 and cedrol. At the 2nd instar, the contents of α-cedrene and β-cedrene were insignificant 164 between larva and food; the content of cedrol was significantly lower in larvae than in food. At 165 the 4th instar, the larvae still had significantly higher content of (E)-β-ocimene than food; the 166 content of (Z)-β-ocimene was insignificant between larvae and food. As for other terpenoid 167 compounds, larvae had substantially lower contents than food after the 2nd instar.

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Terpenoids have previously been reported as characteristic products of the Nasanov gland 252 of worker bees, and included nerol, geraniol, (E) and (Z)-citral, nerolic acid, geranic acid and (E, 253 E)-farnesol [14]. In the present study, we found that additional terpenoids are the major 254 constituents of worker bee larvae. Larva and food at the 2nd instar was demonstrated to have 255 greater similarity of dry substance weight per unit volume, compared to other stages (data not 256 shown). Assuming that larvae and food at the 2nd instar had similar densities, larvae would 257 have released significantly higher amount of terpenoids than food, thus, these terpenoids, such

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These sesquiterpene alcohols might be gathered by bees from gum and pollen of Cedrus deodara.

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All aldehydes detected in this study have been previously reported as the volatiles of hives.

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Ethyl 2(E)-decenoate is tentatively identified in honeybees for the first time in the present 294 study. Aliphatic ester is another group released by honeybee which is ubiquitous in colonies.

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Decyl decanoate is secreted by virgin queens from the tergal gland; ethyl oleate is produced by 13 of 15 communication between brood and worker bees [14]. Ethyl 2(E)-decenoate might be a product 299 of incomplete beta oxidation of longer fatty acids.

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(E)-Geranylacetone has not been previously identified as a honeybee volatile.

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Concentrations of this compound in larvae did not show obvious changes with the growth of 302 individual larva, and only displayed a dramatic increase during the capping stage (the 5th 303 instar). Neither did the significant differences of the contents were found in food at different 304 stages. This may suggest that the compound is released by larvae for a specific role in this 305 particular period.

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When tracing the roots of the volatiles of worker larvae and larval food, the factor of the 307 adult worker bee also needs to be considered. Additionally, constancy of volatile components in

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Our results show that more volatiles could be identified from honeybee worker larvae and 315 their food, in addition to (E)-β-ocimene. We provide evidence that these volatiles change and 316 have a set of rules during larvae development.