Insights Into Larval Identification By Honey Bee Workers And Stronger Olfactory Sense Of Accepting Larvae In Queen Cells By Royal Jelly Bees

Feeding young larvae is a nursing behavior achieved by nurse bees(Apis mellifera)once their peripheral olfactory system stimulated by larval pheromone,which is vital for secretion of proteinaceous larval food,royal jelly(RJ).And that is one important aspect of honey bee reproductive investment consists of raising female-destined larvae into new queens by alloparental care of nurse bees in form of royal jelly provisioning.Artificial selection for commercial royal jelly production over 40 years in China has increased reproductive investment and royal jelly production by an order of magnitude.However,mechanism how nurse bees sense the existence of larvae by olfaction and the difference of larval acceptance of queen cells by royal jelly bees(RJBs)and Italian bees(ITBs)are largely unknown.Here,molecular biology,behavior,functional proteomic and other approaches are used to dissect the mechanism of larval identification by nurse bees and compare the olfactory difference between nurse bees of ITBs and RJBs.First,we use cross-feeding experiments to study the acceptance rate of queen cells and rolay jelly production of ITBs and RJBs.The acceptance rate of queen cells of ITBs and RJBs using their respective larvae was 36.45 % ± 3.09 %(significantly lower than that of RJBs),92.59 % ± 1.46 %,respectively.In cross-fostering assays,91.84 % ± 1.35 % of the ITB larvae were accepted by RJBs,and 34.54 % ± 3.88 % of the RJB larvae were accepted by ITBs.However,there was no statistical difference that the RJBs accepted the larvae from ITBs and RJBs(p = 0.935,n = 9),and vice versa in ITBs(p = 0.925,n = 9).This suggests that the enhanced olfaction sense of larval identification by RJB NBs is to match with the acceptance of larvae in queen cells.We analyze the larval volatiles by solid phase microextraction(SPME)allying with gas chromatography-Mass spectrometer(GC-MS).And the measurement of volatile chemicals produced by larvae also conform this result.β-ocimene and allo-ocimene are detected in the larvae,and the abundance levels of β-ocimene and allo-ocimene are not statistically different between both stains of bees.These also suggest that the identity of the grafted larvae did not significantly influence the probability of their acceptance.Then,we use Electorantennography(EAG)and behavior observation experiment to analysis the difference of larval pheromone to the NBs’ antennae in both ITBs and RJBs.Of all the compounds,only β-ocimene and allo-ocimene were responded to the NBs’ antennae in both ITBs and RJBs in dose-dependent manner.The NBs’ antennae of both bee strains are all responded to two chemicals within 3 ms.To be noted,the time responding to β-ocimene and allo-ocimene between the NB antennae of ITBs and RJBs was not significantly different.The individual of the BEP and a mixture of them are not elicited conspicuous antennal nerve depolarization.Only a few of BEP are produced very feeble electrical signals(E < 0.1 mV)at high concentration(C = 0.1),such as methyl oleate and ethyl oleate.But the antennae of RJB NBs strongly responded to β-ocimene and allo-ocimene at four concentrations relative to those in ITBs,particularly,at lower concentrations demonstrates that they have shaped a sensitive peripheral olfactory system for detecting larval signals than the ITBs.In the behavior observation experiment,when the device is divided into three parts,more numbers of NBs are attracted by β-ocimene(23.10 % of ITBs and 26.25 % of RJBs)and allo-ocimene(22.27 % of ITBs and 22.67 % of RJBs)than those by mineral oil.Notably,a few of NBs were enticed by the mixture of 10 aliphatic esters(5.72 % of ITBs and 8.95 % of RJBs)similar to the number bees lured by mineral oil.These evidences demonstrate that 10 aliphatic esters do not play key role in the olfactory system for distant sense but have an aggregation effects in the process of random walk.Specifically,the highest attractant rate of NBs of both stocks was the mixture(24.5 % of ITBs and 26.33 % of RJBs).Third,both bee lines have tailored a distinct proteome landscape and molecular diversity to underpin the divergent olfaction of accepting the larvae in queen cells.In RJBs,the enhanced activity of proteins related to energy supplement and membrane depolarization relative to ITBs are suggest of the activity of signal transduction being induced to cement the larval recognition.This is accomplished by stronger expression of olfactory proteins.And the result of qRT-PCR also suggested that genes related to olfactory proteins,energy metabolism,and signal transduction,are highly expressed in RJBs,most prominently OBP16 and CSP4(more than 6 times).In fluorescent competitive binding assays,most OBPs and CSPs were bound with β-ocimene and allo-ocimene with varied affinity.OBP8 is observed the strongest binding affinity to β-ocimene(Kd = 4.17 μ mol/L),and CSP4 is recorded the strongest binding affinity to allo-ocimene(Kd = 2.87 μ mol/L).Contrasting to above two volatiles,10 brood pheromones were exhibited very weak or no binding affinity to all OBPs and CSPs.Finally,the quenching constant Ksv decreased with temperature increase,and the interaction distance was 2.73 nm and 2.43 nm(< 10 nm),indicating that β-ocimene and allo-ocimene could form stable complexes with CSP4.The observed △H < 0 and △S > 0 of thermodynamics suggest the main driving forces are electrostatic or hydrophobic force.All above thermodynamics findings are in line with the results of ITC experiments.Furthermore,molecular docking,MD simulation and site-directed mutagenesis indicate the binding cavities are located at cavity 1 in C-terminal of CSP4,where Tyr98 and Asp67 are vital amino acids in maintaining the stable form of protein and larval pheromones,and electrostatic energies are the main driving forces.Our findings gain novel insight into the binding mechanism of chemosensory protein with volatile larval pheromones and are important for understanding olfactory interaction of honeybees.These findings reveal novel aspects of pheromonal communication,gain a cognize of nurse bees to recognize and accept larvae in honey bees and explain how sensory changes affect communication and lead to a drastic shift in larva acceptance and colony-level resource allocation to sexual reproduction.