| In social bee,worker and queens develop from diploid fertilized eggs of the same genotype.However,there are significant differences between them,in physiology,development,morphology,work and behavior,what named caste determination.Previous research found two types of female last-instar larvae exist in natural bee colony.The worker larva and queen larva last-instar weight is 165.00(±9.98)mg and 320.75(±13.52)mg respectively,what shows typical dimorphism of body weight.However,what is the relationship between the weight of last-instar larvae and caste determination? There is an urgent need for in-depth study.This study takes Apis mellifera as research objects to investigate the effect of last-instar larvae weight on caste differentiation.This experiment consists of three parts :1.To explore the differences of body nutrient retention between queen and workers from different days,we detected water,crude protein,crude fat,minerals and amino acids contents from 5 to 16 d of queen bees and 5 to 21 d(18 d and 19 d undetected)of worker bees in natural colony.Statistical analysis showed that the moisture content of workers was significantly higher than that of queens on days 5,6,7,10,12,13,and 16(P<0.05);In addition,the accumulation of crude protein in queens was significantly higher(P<0.05)than in workers,except for 6-and7-day-old larvae(P <0.05);The proportion of crude fat in queens was significantly higher than in workers on days 7,9,10,11,and 12(P <0.05);In queen bee samples,the Mn contents of12-16 d,Cu contents of 16 d,K contents of 6 d,10-12 d and 14-16 d,Fe contents of 11 d and13-16 d,Zn contents of 6 d,11 d,12 d and 16 d were significantly higher than those of worker samples(P <0.05);What’s more,sixteen kinds of amino acid were detected from 5 to 16 d,the results showed that amino acid contents of queen samples were significantly higher than that of worker samples at the same period(P <0.05).2.Simulation of space-limiting effects by using Apis cerana cell to cultivate Apis mellifera,And then we detected the weight of female last-instar larvae and emerging bees.The results showed that the weight of female last-instar larvae and emerging bees were significantly lower than those of the control group(P<0.05).Female bee larvae were reared using a sugar-deficient larvae diet,and their larval weight and survival index were measured.And the feeding time of queen larvae in the natural colony were limited,and the queen larvae of 180-200 mg were transferred to the pupal plate.Then the developmental period,morphology,and fecundity indexes of the queen larvae were measured.The effect of nutritional restriction on the growth and development of female larvae was investigated.We found that the female larvae can develop into Queen-like(320-360 mg)with the absence of sugar,but they are unable to complete the entire metamorphic development process and the mortality rate after pupation reached up to 100%.In addition,after the nutritional restriction of the queen larva,its developmental period were significantly higher than those of the control group(P <0.05).The appearance and ovarian morphology of queen larva were also different from those of the control group.We use normal diet to feed 2-7 d female larvae in 24 cell plates to meet their nutritional and space requirements,and then detect the last-instar weight of all larvae to explore the effects of lifting of nutrition and space restrictions on the female last-instar larvae weight.The results showed that female larvae continued to grow to 320 mg within 2-3 d,whose last-instar weight higher than natural workers(P <0.05),but those of 4-7 d can not.3.Female larvae are raised with nutrient and space-adequate and the Worker-like(160-180mg)and Queen-like(320-360 mg)individuals were starvation treated.The fecundity,morphology and behavior of the two type of individuals were measured.There are significant differences between Queen-like and Worker-like individuals in ovarian development level,body length,femur length,tibia length,tarsus length and tarsus width of the metapedes(P<0.05).And there are also significant differences in attack behavior between the Queen-like and Worker-like bees(P<0.05).4.Transcriptional levels of larvae,prepupae,white-eyed pupa and new bees during late metamorphosis were measured.And we selected 10 differentially expressed genes and verified the transcriptome sequencing results by RT-q PCR.Transcriptome-level data showed that 6285 differentially expressed genes(DEGs)in Queen-like individuals were significantly clustered into 8 profiles(profile 12,14,20,21,22,23,24,25)(P <0.05),And there are 6987 DEGs in Worker-like individuals were also significantly clustered into 8 profiles(profile 13,16,17,20,21,22,23,25)(P <0.05).Moreover,the DEGs in the significantly enriched profiles were highly similar between Queen-like samples and Worker-like samples.However,there were conspicuous difference in gene expression between Queen-like and Worker-like individuals at the same developmental stage.Various metabolic-related negative regulatory genes in Workerlike larvae were significantly up-regulated(P <0.05).In addition,the DEGs were enriched in metabolic-related KEGG pathway.RT-q PCR verification results were consistent with sequencing DEGs data.The relative expression levels of apoptosis-related genes and methyltransferases in Worker-like bees were significantly higher than those in Queen-like bees(P <0.05),and the development-related genes in Queen-like bees were significantly higher(P<0.05). |
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