A Maternal Effect On Queen Development In Honey Bees (Apis Mellifera)

Honeybee keeping has a long history,the queen is the core of the colony.The quality of the queen has strong effects on the fitness of a colony.Artificial breeding of queen have fascinated scientists for decades.Influences from the mother on offspring phenotype,known as maternal effects,are an important cause of adaptive phenotypic plasticity.Eusocial insects show dramatic phenotypic plasticity with morphologically distinct reproductive(queen)and worker castes.The dominant paradigm for honey bees(Apis mellifera)is that castes are epigenetically,rather than genetically determined,with the environment and diet of young larvae causing caste differentiation.A role for maternal effects has not been considered,but here we show that egg size influences queen development also.In this study,Apis mellifera ligustica(Aml)was used as experimental material to investigate the effect of maternal influence on queen egg size and queen development.Eggs sampled from queen cells(QE)and worker cells(WE)were analyzed and compared.Egg width and length was measured with a zoom-stereo microscope system.Eggs laid in queen cells(QE),laid in worker cells(WE),and 2-day old larvae from worker cells(2L)were transferred to artificial queen cells to be reared as queens in a standardized environment.The larvae were subsequently transferred to queen cells and added to the same queenless hive to be reared.Cells from the three groups(QE,WE,2L)were mixed randomly.After 11 days,queens were harvested immediately on emergence.Queen weight,thorax width and length were measured using the methods above.Queens laid significantly bigger eggs in the larger queen cells than in the worker cells.This study was repeated across 3 colonies,in total 152 eggs were measured.Eggs laid in queen cells(QE)were 13.26 %(157.51 ± 12.37 ?g vs 138.93 ± 10.90 ?g)heavier and 2.43 %(1.56 ± 0.04 mm vs 1.52 + 0.05 mm)longer and 4.18 %(0.374 + 0.010 mm vs 0.359 + 0.013 mm)thicker than eggs laid in worker cells(WE).Adult queens from QE were larger and heavier than either of the other treatment groups.Adult queens from QE were(258.65 ± 22.82 mg vs 234.50 ± 36.00 mg)larger than WE in 2 years,indicating that queen QE wasps were the largest individual.Adult queens from QE were heaviest in all five colonies,and queens from QE were significantly heavier than queens from WE(258.65 ± 22.82 mg vs 234.50 ± 36.00,mg)in three colonies out of five.Our haematoxylin-eosin staining(HE)results showed that five day old adult queens from QE had the greatest number of ovarioles in the right ovary,significantly more than queens from 2L(165.50 ± 10.65 vs 145.90 ± 14.89)in all three colonies,but there was no significant difference between queens from QE and WE(165.50 ± 10.65 vs 160.00 ±9.48).This study demonstrates a maternal effect on caste differentiation,where queens that emerged from queen-cell eggs(QE)had higher quality than queens developed from worker cell eggs(WE).To further examine the consequences of egg source on adult queen phenotype we compared the gene expression profiles of newly emerged adult queens from QE,WE and2 L using RNA-Seq.The heads and thoraces of two newly emerged queens from each group were collected,and pooled for RNA-Seq.This experiment was repeated twice using two colonies and both repeats were considered together in our analyses of gene expression differences in 2016.This RNA-Seq experiment was repeated again in 2018 with two extra colonies using same methods.A small number of differentially expressed genes(DEGs)were detected in comparisons between groups from both 2016 and 2018RNA-Seq results.Of the 121 DEGs identified across all comparisons in 2016 RNA-Seq experiment,6 genes with a high expression level were selected and gene expression differences assessed with q RT-PCR to affirm the results of our RNA-Seq analyses.Two years' RNA-Seq results both showed that the greatest differences were detected in comparisons between queens reared from QE and 2L,followed by 2L/WE and WE/QE comparisons.Interestingly,this is of interest because raising queens from 2L rather than WE has already been shown to have a significant impact on queen reproductive development and morphology.Of the DEGs identified in the WE/QE comparison,31(2016)and 19(2018)have been documented previously in comparisons of queen and worker honey bees,or queen honey bees varying in caste development or reproductive condition.Besides,59 of 191 DEGs from three comparisons of the 2016 RNA-Seq results were also identified in the 2018 RNA-Seq results.This suggests the gene expression differences between adult queen from QE and WE are reflective of variation in the caste development process.Our DEGs contained a disproportionately large number of genes such as juvenile hormone methyltransferase,7 abaecin and hexamerin genes involved in hormone synthesis,ovary development,cuticle development and immune functions.The DNA methylation levels of newly-emerged queens were reared from QE,WE,and 2-day larvae(2L).Epigenetic analysis showed that QE/2L comparison had the most different methylated genes(DMGs,614)followed by WE/2L(473),and QE/WE(371),though whole DNA methylation levels were similar for all conditions.Interestingly,a great number of DMGs(42)were in genes belonging to m TOR,MAPK,Wnt,Notch,Hedgehog,Fox O,and Hippo signaling pathways that are involved in regulating caste differentiation,reproduction and longevity.This study demonstrates for the first time that honeybees have epigenetic differences caused by maternal effect,which can influence queen-worker caste differentiation.The transcriptome data of newly-emerged queens were combined with methylation data.The results showed that the correlation between the expression level of QE,WE and2 L queen transcriptome and the methylation level was low,indicating that there was a very weak correlation between DNA methylation and gene expression in the three groups.The relationship between DNA methylation and the expression of different genes involved in immune response and development was analyzed.The results showed that only a few genes showed negative correlation between DNA methylation and gene expression.