| Pyrus bretschneideri Rehd.is the most important native commercial pear species in China,with highly self-incompatibility,and honey bees are regarded as its most ideal pollinators.However,the interaction between pollinators and flowering plants in nature is extremely complex,and it has been found in practical application that pears are "problem crops" that honey bees do not like to forage,and the colony state of honey bee colonies and various characteristics of pear flowers may affect the pollination interaction process of bee-flowering plants.In this study,the foraging behavior of Apis mellifera ligustica colonies to pear flowers under three different management strategies:colonies introduced to pear orchard at sequence,colony containing different larvae number and colony size,were evaluated for developing an efficient pollination technology to promote the foraging behavior of honey bees to pear flowers.By observing the foraging behavior of A.m.ligustica and Apis cerana cerana on native P.bretschneideri and exotic European Pyrus communis in the flight cages,and behavior choice in Y-tube olfactory tests,it was determined that honey bees had a significant foraging preference for pear flowers,and it was revealed that pear flower volatiles played a key role in the foraging decision.The flower volatile compounds of two pear species were identified by GC-MS,and the antenatal and behavioral responses of honey bees to different compounds were explored by GC-EAD technology and Y-tube olfactory tests.Transcriptome sequencing was performed on olfactory-related genes in antennae of A.m.and A.c.cerana that response to the volatiles of P.bretschneideri flower,in order to explore the influence mechanism of volatiles of pear flowers on bee foraging behavior at the molecular level.The main results were as follows:(1)The amount of total pollen,the amount proportion of pear pollen collected by honey bee colonies that introduced at sequences[sequence 1 bee colonies were introduced at 20%pear flowering(S1),sequence 2 bee colonies were introduced at 50%pear flowering(S2),and sequence 3 bee colonies were introduced at 80%pear flowering(S3)]were evaluated.The results showed that the foraging efficiency of S2 colonies were the highest among different sequential colonies.The amount of total pollen and pear pollen collected by S2 colonies were significantly higher than those of S1 and S3 colonies(P<0.05),and the amount of pollen collected by all honey bee colonies were increased significantly day by day(P<0.05).The proportions of pear pollen collected by S1 and S2 colonies were significantly higher than that of S3 colonies(P<0.05),and the proportion of pear pollen collected by honey bee colonies were decreased day by day.The results indicated that the flowering stage selection of bee colonies into pear orchards had a significant impact on the foraging efficiency of honey bees.The amount of total pollen,the amount proportion of pear pollen collected by honey bee colonies containing different larvae frames(6-frame colonies containing 0 larvae frame,1 larvae frame,2 larvae frames,and 3 larvae frames,respectively)were evaluated.The results showed that the foraging efficiency of colonies containing 2 frame larvae were the highest among different treatment colonies.The amount of total pollen collected by 2-and 3-frame larvae colonies were significantly higher than those of other two groups(P<0.05),and the amount of pollen collected by all bee colonies increased significantly daily(P<0.05).The proportion and amount of pear pollen of 2-frame larvae colonies were the highest among different groups(P<0.05).These results suggested that increasing the larvae number in the colony can promote the foraging enthusiasm of honey bees,but too many larvae may decrease the foraging specificity to the target crop.The amount of total pollen,the amount proportion of pear pollen collected by honey bee colonies with different sizes(3-frame,4-frame,5-frame,and 6-frame)were evaluated,and the results showed that the foraging efficiency of 5-and 6-frame colonies were the highest among different treatment colonies.The total pollen and pear pollen collected by 5-and 6-frame colonies were significantly higher than those collected by 3-and 4-frame colonies(P<0.05),and the amount of pollen collected by all honey bee colonies were increased significantly day by day(P<0.05).The proportion of pear pollen collected by 3-frame colonies was the highest(P<0.05),and the proportion of pear pollen collected by honey bee colonies was decreased with days.decreased with the increase of colony size,but it was significantly higher than that of other flowering plants(P<0.05).It was concluded that enhancing the colony strength can increase the foraging activity of honey bee for pear,5-frame and 6-frame colonies had the same level foraging efficiency in this area.(2)The behavior observation experiment in the flight cages showed that A.m.and A.c.cerana had significantly foraging preference for P.communis flowers rather than P.bretschneideri flower in the pear orchard(P<0.05),and there was no interspecies-difference in the foraging preference of honey bees(P>0.05).The Y-tube olfactory tests showed that A.m.and A.c.cerana also showed the behavior choice to the flower volatiles of two pear species which was similar to the foraging behavior in the pear orchard(P<0.05).These results indicated that the floral volatiles played a key role in foraging decision of honey bees to forage pear flowers.(3)The identification of flower volatiles of two pear species by GC-MS showed that there were 31 volatile compounds in the P.bretschneideri flower,and 27 volatile compounds in the P.communis flower.methyl L-isoleucine+methyl was the main volatile compounds of P.bretschneideri flower,and 2-hydroxy3-methylpentanoate+linalool was the main volatile compounds of P.communis flower.Totally ten types of compounds were both included in two pear species,and the quantity and relative content of compound types were significantly different between the two pear flowers(P<0.05).Of all the volatile compounds,only 17 compounds were present simultaneously in both pear flowers,and the relative contents of these compounds were significantly different between pear flowers(P<0.05).The compound types and relative contents of volatiles can vary due to genetic differences among subspecies,it was these that caused obvious differences in the whole scents of pear flowers.(4)EAG showed that the antennae response of A.m.and A.c.cerana to the flower volatiles of P.communis was significantly higher than that of P.bretschneideri flower(P<0.05).GC-EAG results showed that among all the volatile components of pear flower,honey bee antennae responded to 16 chemicals,including 5 contained in both pear species:1-nonanol,linalool,methyl 2-hydroxy-3-methylpentanoate,methyl L-isoleucinate,and α-farnesene.In addition,there were 8 electrophysiologically active compounds in P.bretschneideri:methyl L-valine ester,benzaldehyde,6-methyl-5-hepten-2-one,isophorone,2-methyl octane,longicyclene,longifolene,and caryophyllene;and 3 electrophysiologically active compounds in P.communis:β-ocimene,4-oxoisophorone and lilac alcohol D.Y-tube behavior test indicated that honey bees had attracted(linalool)or repelled(β-ocimene and 6-methyl-5-hepten-2-one)behaviors to different compounds,and there were interspecies differences in the behavior responses of bees to benzaldehyde,isophorone and 1-nonanol(P<0.05).(5)The Illumina sequencing platform was used to analyze the antennae transcriptome differences of A.m.and A.c.cerana that responded to pear flower volatiles.A total of 51 DEGs were found in A.m.ligustica antennae compared to the control group;and 44 DEGs were found in A.c.cerana antennae.GO analysis of A.m.ligustica showed that DEGs were mainly related to signal transmission process,such as odor binding and olfactory receptor activation,cell response to stimulus,and response to stimulus response.A significant upregulated gene OR2 and a significantly downregulated gene OR109 were obtained from both A.m.ligustica and A.c.cerana,and these differentially expressed genes may be involved in the recognition process of honey bees to pear flower scents.In conclusion,the foraging efficiency of honey bees at the pear flowering period can be significantly improved by sequential introduction of honey bee colonies,adjusting the number of larvae or the colony size in the bee colony.Moreover,electrophysiological and behavioral studies confirmed that the pear flower volatiles were a key factor in affecting the foraging decision choice during flower visits.RNA-seq revealed that there were significantly upregulated chemoreceptor genes in the antenna of A.m.ligustica and A.c.cerana when they were selecting for P.bretschneideri flowers.The results provide not only potential technical guidance for the pollination strategy of applying honey bees for pear pollination in early spring,but also a theoretical basis for in-depth understanding and exploration of the molecular mechanism of chemical communication between honey bees and pear flowers. |
