Dieta e castas: o exemplo das abelhas sociais

Faculdade de Filosofia Ciências e Letras de Ribeirão Preto

Laboratório de Biologia do Desenvolvimento de Abelhas

http://zulu.fmrp.usp.br/beelab

Pré- Genoma

Pós-Genoma

Dieta

O modelo

Um mesmo genótipo

Apis mellifera

Interações gênicas e controle do crescimento

Nijhout, FH, 2003

Uma diferença

Os órgãos reprodutores

Operária Rainha

O terceiro par de pernas

Operária

Rainha

Os títulos de Hormônio Juvenil

Rainha

Operária

HJ

EcD

O genes são diferencialmente expressos - Microarrays

Angel R. Barchuk

Análise da região promotora dos genes superexpressos em rainhas e operárias de Apis mellifera – Reguladores?

Alexandre dos Santos Cristino

Os títulos de Hormônio Juvenil

Rainha

Operária

HJ

EcD

Usando esta informação........ Klaus Hartfelder

Motivos localizados na região up stream dos genes diferencialmente

expressos

JH+ JH+

JH+ JH -

Sítio de ligação ao Ultraspiracle localizado na região up sptream

dos genes diferencialmente expressos – tratamento com HJ

títulos

títulos

Altos

Baixos

Genes do

fisiometabolismo

Genes do

desenvolvimento

Queen

Worker

Aumento de

tamanho

Alteração de

caracteres

morfológicos

Hsc70Cb

Tor

Trap1

usp

Crc

RfaBp

JH

Principalmente nas pernas posteriores, no terceiro par desenvolve a corbícula – ausente nas rainhas

Eles fazem a diferença

Rainha Operária

No metathorax

Ana Bomtorin

A região da corbícula

Worker

Queen

Expressão diferencial de Ubx no desenvolvimento do terceiro par de perna de operárias e rainhas de Apis mellifera. Azul, núcleos ;rosa,

Ubx; vermelho, maior concentração de Ubx.

Um único genótipo

Operária

Rainha

Dois fenótipos

Embryo

Dieta

Diferenciação de castas em Apis mellifera

Barchuk et al. 2007

Maleszka, R (2008)

Resposta a alterações na dieta

Modelo Apis mellifera testado em Drosophila melanogaster

Interações gênicas e controle do crescimento

Nijhout, FH, 2003

Kamakura, 2011

Um trabalho que todos gostariam de ter feito .......

Royalactin induces queen differentiation in honeybees

Drosophila melanogaster

Li et al, 2010

Drosophila melanogaster

Efeito da supressão de proteínas da dieta de Drosophila melanogaster

Níveis do Hormônio Juvenil são controlados por uma esterase

Expressão do gene codificador da esterase do HJ ao longo do desenvolvimento de operárias e rainhas de Apis mellifera

Aline Mackert

Aline Aleixo

Quais os miRNAs afetados pelo silenciamento da JHE?

Sequenciamento dos pequenos RNAs

3 grupos Controle (10)

3 grupos Tratados (10)

14 miRNAs diferencialmente expressos

ame-miR-3728 ame-miR-3727 ame-miR-3759 ame-miR-3793 ame-miR-3720 ame-miR-3477 ame-miR-316

ame-miR-317 ame-miR-3719 ame-miR-996 ame-miR-100 ame-miR-13b ame-miR-306 ame-miR-263b

Silenciamento do gene codificador da esterase do Hormônio Juvenil

0

2000000

4000000

6000000

8000000

10000000

12000000

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 36

N. Reads > 10

JC01

JC03

JC06

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 36

N. Reads > 10

JT01

JT02

JT04

Flávia Freitas

Quais os genes candidatos a alvo desses miRNAs?

Busca pelos genes-alvo dos miRNAs

-RNAhybrid -14 miRNAs X 11062 genes preditos

Resultou em 1011 interações, das quais 562 envolvem genes-alvo com ortólogos em Drosophila melanogaster.

Quais os processos biológicos regulados por esses 14 miRNAs?

Data: 16/04/2012 Ortólogos

Tarefa: miRNAs diferencialmente expressos (tagwise) x Genoma Total = 1011 interações Total = 562 interações

energia =< - 20 kcal/mol ; pvalue < 0.05; seed 2-7 energia =< - 20 kcal/mol ; pvalue < 0.05; seed 2-7

miRNAs selecionados miRNA target mfe pvalue miRNA target ortholog mfe pvalue

ame-miR-3728 ame-miR-3728 GB17247 -30,4 0,001483 ame-miR-3728 GB17247 CG4272 -30,4 0,001483

ame-miR-3727 ame-miR-3728 GB13779 -25,9 0,024596 ame-miR-3728 GB13779 PIP5K59B -25,9 0,024596

ame-miR-3759 ame-miR-3728 GB14496 -25,5 0,031491 ame-miR-3728 GB14496 CG11034 -25,5 0,031491

ame-miR-3793 ame-miR-3728 GB10640 -26,4 0,01804 ame-miR-3728 GB10640 Tollo -26,4 0,01804

ame-miR-3720 ame-miR-3728 GB12348 -24,9 0,045534 ame-miR-3728 GB12348 CG31952 -24,9 0,045534

ame-miR-3477 ame-miR-3728 GB14721 -25 0,042828 ame-miR-3728 GB14326 CG5018 -26,9 0,013219

ame-miR-316 ame-miR-3728 GB30423 -27,9 0,007086 ame-miR-3728 GB11944 G-salpha60A -25,4 0,033493

ame-miR-317 ame-miR-3728 GB30095 -27,3 0,010304 ame-miR-3728 GB17292 CG13467 -24,9 0,045534

ame-miR-3719 ame-miR-3728 GB14326 -26,9 0,013219 ame-miR-3728 GB15316 CG8603 -25,1 0,04028

ame-miR-996 ame-miR-3728 GB18338 -25,5 0,031491 ame-miR-3728 GB14809 endoA -25,8 0,026165

ame-miR-100 ame-miR-3728 GB30112 -25,1 0,04028 ame-miR-3728 GB16081 CG32666 -26,3 0,019195

ame-miR-13b ame-miR-3728 GB11944 -25,4 0,033493 ame-miR-3728 GB11728 CG2972 -25,1 0,04028

ame-miR-306 ame-miR-3728 GB17292 -24,9 0,045534 ame-miR-3728 GB19710 CG4660 -27,4 0,009681

ame-miR-263b ame-miR-3728 GB15316 -25,1 0,04028 ame-miR-3728 GB11325 su(w[a]) -25,6 0,029607

ame-miR-3728 GB15237 -26,8 0,014068 ame-miR-3728 GB19918 CG5036 -25 0,043891

Genes preditos (versão 4.0 do genoma - OGS2) ame-miR-3728 GB17395 -26,8 0,014068 ame-miR-3728 GB17750 CG8320 -26,2 0,020424

11062 genes ame-miR-3728 GB14809 -25,8 0,026165 ame-miR-3728 GB17322 CG7031 -25,7 0,027662

ame-miR-3728 GB12191 -24,8 0,048406 ame-miR-3728 GB19438 CG4342 -25,4 0,033493

ame-miR-3728 GB16081 -26,3 0,019195 ame-miR-3728 GB15084 CG5928 -29,1 0,003326

ame-miR-3728 GB11728 -25,1 0,04028 ame-miR-3728 GB18069 CG3437 -23,3 0,011338

ame-miR-3728 GB19710 -27,4 0,009681 ame-miR-3728 GB18723 CG32052 -25,3 0,03562

ame-miR-3728 GB11306 -27,8 0,007543 ame-miR-3728 GB19944 CG10869 -24,8 0,048406

ame-miR-3728 GB15906 -26,7 0,014971 ame-miR-3728 GB10091 CG7802 -25,1 0,04028

ame-miR-3728 GB11325 -25,6 0,029607 ame-miR-3728 GB10096 slmb -26,6 0,015932

ame-miR-3728 GB19918 -25 0,043891 ame-miR-3728 GB16957 mfr -25,1 0,04028

ame-miR-3728 GB17750 -26,2 0,020424 ame-miR-3728 GB14902 CG4945 -26,7 0,014971

ame-miR-3728 GB11580 -24,8 0,048406 ame-miR-3727 GB11321 CG30437 -27,3 0,03829

ame-miR-3728 GB17322 -25,7 0,027662 ame-miR-3727 GB12933 CG33041 -27,6 0,032413

ame-miR-3728 GB19438 -25,4 0,033493 ame-miR-3727 GB13779 PIP5K59B -32,4 0,002181

ame-miR-3728 GB15084 -29,1 0,003326 ame-miR-3727 GB10640 Tollo -27,2 0,040472

ame-miR-3728 GB17301 -29,9 0,002029 ame-miR-3727 GB14534 CG17758 -29,1 0,014009

ame-miR-3728 GB13233 -26,5 0,011174 ame-miR-3727 GB19937 SoxN -28,3 0,021935

ame-miR-3728 GB14111 -25,8 0,026165 ame-miR-3727 GB10999 CG11132 -30,9 0,005085

ame-miR-3728 GB12697 -25,9 0,024596 ame-miR-3727 GB15897 CG13855 -28,7 0,017534

ame-miR-3728 GB18069 -23,3 0,011338 ame-miR-3727 GB19853 Bgb -27,2 0,040472

ame-miR-3728 GB17136 -26,2 0,020424 ame-miR-3727 GB17100 svp -31,2 0,004293

ame-miR-3728 GB16378 -22,1 0,017946 ame-miR-3727 GB18573 CG8005 -28,8 0,016578

ame-miR-3728 GB18723 -25,3 0,03562 ame-miR-3727 GB19191 Scgdelta -28,7 0,017145

Comparação com os resultados de 2007

títulos

títulos

Altos

Baixos

Genes do

fisiometabolismo

Genes do

desenvolvimento

Queen

Worker

Aumento de

tamanho

Alteração de

caracteres

morfológicos

Hsc70Cb

Tor

Trap1

usp

Crc

RfaBp

JH

GO ID Description p-val corr p-val cluster_freq cluster_freq% total_freq total_freq%

9987 cellular process 7,54E-06 1,03E-04 249/364 68,40% 5484/9550 57,40%

32501 multicellular organismal process 5,01E-15 7,12E-13 175/364 48,00% 2777/9550 29,00%

50794 regulation of cellular process 1,24E-22 7,62E-20 153/364 42,00% 1913/9550 20,00%

32502 developmental process 1,57E-14 1,71E-12 148/364 40,60% 2200/9550 23,00%

7275 multicellular organismal development 2,39E-15 3,68E-13 138/364 37,90% 1941/9550 20,30%

48856 anatomical structure development 1,37E-14 1,68E-12 133/364 36,50% 1876/9550 19,60%

16043 cellular component organization 8,40E-09 2,35E-07 116/364 31,80% 1866/9550 19,50%

48731 system development 1,54E-14 1,71E-12 112/364 30,70% 1452/9550 15,20%

9653 anatomical structure morphogenesis 2,07E-16 9,56E-14 108/364 29,60% 1293/9550 13,50%

51179 localization 1,75E-07 3,64E-06 105/364 28,80% 1723/9550 18,00%

19222 regulation of metabolic process 4,49E-16 1,11E-13 100/364 27,40% 1158/9550 12,10%

48513 organ development 1,46E-13 1,17E-11 94/364 25,80% 1150/9550 12,00%

31323 regulation of cellular metabolic process 2,13E-15 3,58E-13 92/364 25,20% 1039/9550 10,80%

60255 regulation of macromolecule metabolic process 6,60E-16 1,36E-13 92/364 25,20% 1020/9550 10,60%

80090 regulation of primary metabolic process 4,82E-16 1,11E-13 92/364 25,20% 1015/9550 10,60%

48869 cellular developmental process 2,50E-13 1,85E-11 91/364 25,00% 1104/9550 11,50%

30154 cell differentiation 1,34E-12 7,99E-11 87/364 23,90% 1061/9550 11,10%

10468 regulation of gene expression 1,23E-15 2,27E-13 85/364 23,30% 907/9550 9,40%

51171 regulation of nitrogen compound metabolic process 4,82E-16 1,11E-13 83/364 22,80% 859/9550 8,90%

19219 regulation of nucleobase nucleoside nucleotide and nucleic acid metabolic process 4,20E-16 1,11E-13 83/364 22,80% 857/9550 8,90%

23052 signaling 2,72E-08 6,80E-07 78/364 21,40% 1102/9550 11,50%

9889 regulation of biosynthetic process 8,05E-13 5,13E-11 75/364 20,60% 835/9550 8,70%

31326 regulation of cellular biosynthetic process 8,05E-13 5,13E-11 75/364 20,60% 835/9550 8,70%

51234 establishment of localization 3,23E-03 1,67E-02 75/364 20,60% 1457/9550 15,20%

48468 cell development 8,19E-11 3,16E-09 72/364 19,70% 862/9550 9,00%

10556 regulation of macromolecule biosynthetic process 2,34E-12 1,35E-10 72/364 19,70% 800/9550 8,30%

6810 transport 4,66E-03 2,19E-02 72/364 19,70% 1408/9550 14,70%

51252 regulation of RNA metabolic process 1,15E-14 1,51E-12 68/364 18,60% 655/9550 6,80%

50896 response to stimulus 4,79E-04 3,71E-03 68/364 18,60% 1205/9550 12,60%

7399 nervous system development 6,99E-12 3,32E-10 67/364 18,40% 731/9550 7,60%

45449 regulation of transcription 6,17E-12 3,07E-10 67/364 18,40% 729/9550 7,60%

9887 organ morphogenesis 6,34E-13 4,34E-11 65/364 17,80% 661/9550 6,90%

Predição de genes alvos dos micros diferencialmente expressos pelo silenciamento da Esterase do Hormônio Juvenil

Um único genótipo

Operária

Rainha

Dois fenótipos

Embryo

Dieta

Nutrition Reproduction

Royal Jelly

Larval Food

Worker Food

insulina/TOR/IGF pathway

Brain

Corpora allata R

oya

lact

in

insulina/TOR/IGF pathway

Cérebro

Corpora allata R

oya

lact

in

JHE

JH

JHE

JH

Body growth Ovary development Physiometabolic genes

Neurogenesis Worker specific organs Developmental genes

Body Growth Ovary Development Physiometabolic genes

Neurogenesis Worker specific organs Developmental genes

Queen

Worker miR

NA

s m

iRN

As

Modificado de Malezska, 2008

1945 Processos Biológicos

14 miRNAs Diferencialmente

expressos

Alta porcentagem de genes do desenvolvimento

À FAPESP CNPq CAPES

GO ID Description p-val corr p-val cluster_freq cluster_freq% total_freq total_freq%

9987 cellular process 7,54E-06 1,03E-04 249/364 68,40% 5484/9550 57,40%

32501 multicellular organismal process 5,01E-15 7,12E-13 175/364 48,00% 2777/9550 29,00%

50794 regulation of cellular process 1,24E-22 7,62E-20 153/364 42,00% 1913/9550 20,00%

32502 developmental process 1,57E-14 1,71E-12 148/364 40,60% 2200/9550 23,00%

7275 multicellular organismal development 2,39E-15 3,68E-13 138/364 37,90% 1941/9550 20,30%

48856 anatomical structure development 1,37E-14 1,68E-12 133/364 36,50% 1876/9550 19,60%

16043 cellular component organization 8,40E-09 2,35E-07 116/364 31,80% 1866/9550 19,50%

48731 system development 1,54E-14 1,71E-12 112/364 30,70% 1452/9550 15,20%

9653 anatomical structure morphogenesis 2,07E-16 9,56E-14 108/364 29,60% 1293/9550 13,50%

51179 localization 1,75E-07 3,64E-06 105/364 28,80% 1723/9550 18,00%

19222 regulation of metabolic process 4,49E-16 1,11E-13 100/364 27,40% 1158/9550 12,10%

48513 organ development 1,46E-13 1,17E-11 94/364 25,80% 1150/9550 12,00%

31323 regulation of cellular metabolic process 2,13E-15 3,58E-13 92/364 25,20% 1039/9550 10,80%

60255 regulation of macromolecule metabolic process 6,60E-16 1,36E-13 92/364 25,20% 1020/9550 10,60%

80090 regulation of primary metabolic process 4,82E-16 1,11E-13 92/364 25,20% 1015/9550 10,60%

48869 cellular developmental process 2,50E-13 1,85E-11 91/364 25,00% 1104/9550 11,50%

30154 cell differentiation 1,34E-12 7,99E-11 87/364 23,90% 1061/9550 11,10%

10468 regulation of gene expression 1,23E-15 2,27E-13 85/364 23,30% 907/9550 9,40%

51171 regulation of nitrogen compound metabolic process 4,82E-16 1,11E-13 83/364 22,80% 859/9550 8,90%

19219 regulation of nucleobase nucleoside nucleotide and nucleic acid metabolic process 4,20E-16 1,11E-13 83/364 22,80% 857/9550 8,90%

23052 signaling 2,72E-08 6,80E-07 78/364 21,40% 1102/9550 11,50%

9889 regulation of biosynthetic process 8,05E-13 5,13E-11 75/364 20,60% 835/9550 8,70%

31326 regulation of cellular biosynthetic process 8,05E-13 5,13E-11 75/364 20,60% 835/9550 8,70%

51234 establishment of localization 3,23E-03 1,67E-02 75/364 20,60% 1457/9550 15,20%

48468 cell development 8,19E-11 3,16E-09 72/364 19,70% 862/9550 9,00%

10556 regulation of macromolecule biosynthetic process 2,34E-12 1,35E-10 72/364 19,70% 800/9550 8,30%

6810 transport 4,66E-03 2,19E-02 72/364 19,70% 1408/9550 14,70%

51252 regulation of RNA metabolic process 1,15E-14 1,51E-12 68/364 18,60% 655/9550 6,80%

50896 response to stimulus 4,79E-04 3,71E-03 68/364 18,60% 1205/9550 12,60%

7399 nervous system development 6,99E-12 3,32E-10 67/364 18,40% 731/9550 7,60%

45449 regulation of transcription 6,17E-12 3,07E-10 67/364 18,40% 729/9550 7,60%

9887 organ morphogenesis 6,34E-13 4,34E-11 65/364 17,80% 661/9550 6,90%

Predição de genes alvos dos micros diferencialmente expressos pelo silenciamento da Esterase do Hormônio Juvenil