Expression Differences in the Caste Development of Honeybee Using Solexa Sequencing Method

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1 Expression Differences in the Caste Development of Honeybee Using Solexa Sequencing Method Songkun Su1, Xiangqian Guo2,3, Aung Si4, Fang Liu1, Yi Zhan1, Shuanjin Dai1, Shenglu Chen1, Shaowu Zhang4,1, and Runsheng Chen2 1 College of Animal Sciences, Zhejiang University, Hangzhou, China 2 Bioinformatics Laboratory and National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, 3 Graduate School of the Chinese Academy of Sciences, Beijing, China 4 Centre of Excellence in Vision Science, Research School of Biological Sciences, The Australian National University, Canberra, Australia

2 Introduction The honeybee is becoming an important model organism in many fields of biology for a range of reasons: complex social behaviors, dramatic differences in the caste development of queens and workers, impressive learning ability, and paramount pollination value [HGSC, 2006]. The single queen in each honeybee colony, living 1 to 2 years and laying 2,000 eggs per day during producing season [Page, R. E. & Peng, C. Y, 2001], has ten times lifespan of the sterile workers, which number in the tens of thousands, and display sophisticated cognitive abilities [Menzel, R, 2001; Giurfa, M., Zhang, S., Jenett, A., Menzel, R. & Srinivasan, M. V, 2001].

3 Although queens and workers arise from fertilized diploid eggs, the dramatic distinctions between queens and workers are mainly caused by nutrition input, hormone mediation, and gene expression [Evans, J. D. & Wheeler, D. E., 2001]. The gene expression profiles of queen-caste and worker-caste pupae have been found to be considerably different by several molecular-genetic approaches [Jay D. Evans and Diana E. Wheeler, 1999; Corona, M., Estrada, E. & Zurita, M., 1999; Hepperle, C. & Hartfelder, K, 2001].

4 New-generation sequencing technologies are becoming a powerful means to understand the functioning of genes in the post-genome era [Schuster, 2008; Wold & Myers, 2008].

5 Queen Germ egg Queen cell Worker cell Queen Worker Cells Unfertilized egg Drone The caste development of honeybee

6 Sequencing method profiles

7 Here, we use the new sequencing method of Solexa to determine the expression profiles of 4, 5, 6-day old queen and worker larvae, a time regarded as crucial in the process of caste divergence. We predict new coding genes through this new technique to discover more gene information in honeybee function genome.

8 Material and Methods Honeybee colonies preparation and larvae samples collection RNA extraction and sequencing Sequences mapping and data analysis Predicting the potential functions of gene candidates Comparing the expression profiles of worker and queen larvae Test the functions of different expressed genes in field

9 Results 1. Solexa sequencing results of expression profiles of worker and queen larvae

10 We retrieved 2.86 and 2.34 million distinct tags from the worker larvae and queen larvae libraries respectively. Combining the genes from the two libraries detected by Solexa sequencing and analysis, we obtained 6322 unambiguous sequence tags which stood for 6322 different honeybee genes. Then we took out the upstream 2kb and downstream 2kb sequences of these unique mapped tags to predict their gene coding potency by genscan, and obtained 9258 gene candidates, including 3566 gene candidates greater than 300 nt in length.

11 2. The potential functions of gene candidates Analysis of Gene Ontology (GO) revealed that these gene candidates have the potential to code genes responding to postembryonic development, reproductive development and sexual reproduction.

12 Predict the potential functions of new gene candidates a. Similarity distribution of mapping gene candidates b. E-value distribution of mapping gene candidates

17 Functional classification of gene candidates profiled by Gene ontology

18 3. Differences in expressional profiles of worker larvae and queen larvae

19 Distribution of ratio of distinct tag copy number between two library

20 The Genes Expression Difference

21 The differences of gene expression level were analyzed between worker larvae and queen larvae. We found 1278 genes and 1451 genes which were up-regulated and downregulated respectively in worker larvae at the p<0.05 level. There were 935 genes and 1116 genes which were upregulated and down-regulated in worker larvae at the p<0.01 level.

22 The functional classification of up- and down-regulated genes in queen larvae

28 4. Test the functions of different expressed genes in field

29 Honeybee larvae kept in hive colony with queen cell

30 The queen cell frame taken from honeybee colony

31 The plastic queen cell with honeybee larvae

32 RNA interference Group1: DEPC water 5ul Group2: 50 5ul C27(10ng/ul) Group3: 100 5ul C27(20ng/ul) Group4: 500 5ul C27(100ng/ul) Group5:Nothing Control 3-day 4-day feed small RNA

33 Add srna into plastic queen cell in lab

34 Add srna into plastic queen cell in lab

35 The capped queen cell frame just taken from colony

36 The old capped queen cell just taken from colony

37 The old capped queen cell kept with wiry cage in colony

38 The virgin queen from plastic queen cell

39 The virgin queens stored in eppendorf tubes at -80

40 ng 500ng 500ng 500ng Water Control Water Control Water Control Water Control Control Control Control Control Body Weight Results B b G3 A a G1 A a G2 1% 5% Mean Deal G1: control; G2: Water Control; G3: sirna

41 Discussions 1. Solexa sequencing is a powerful way to discover new genes in the honeybee genome 2. Different gene expressions of caste development

Eusocial species. Eusociality. Phylogeny showing only eusociality Eusocial insects. Eusociality: Cooperation to the extreme

Eusocial species. Eusociality. Phylogeny showing only eusociality Eusocial insects. Eusociality: Cooperation to the extreme Eusociality: Cooperation to the extreme Groups form colonies with reproductive and worker castes. Eusociality has evolved most often in insects: Ants Eusocial species Honeybees Termites Wasps Phylogeny