Cláudia Ca olina de Almeida Mendes
Disse a ion p esen ed o ob ain he Ph.D deg ee in De elopmen al Biology
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie | Uni e sidade No a de Lisboa
Oei as,
May 2015
Nu i ional plas ici y and e olu iona y
di e gence in he D osophila o a y
Cláudia Ca olina de Almeida Mendes
Disse a ion p esen ed o ob ain he Ph.D deg ee in De elopmen al Biology
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie | Uni e sidade No a de Lisboa
Oei as,
May 2015
Nu i ional plas ici y and
e olu iona y di e gence in he
D osophila o a y
Resea ch wo k coo dina ed by:
Mendes, C.C.
Nu i ional plas ici y and e olu iona y di e gence in he
D osophila o a y
PhD hesis, Ins i u o Gulbenkian de Ciência, Uni e sidade
No a de Lisboa, 2015
In English, wi h summa y in Po uguese
This hesis has been scanned o plagia ism on Ap il 29 h
2015 and he e was no con lic wi h published wo ks.
Co e image by he au ho © 2015
D awings o adul o a ies o D osophila.
P in ed by 2002 Es údio G á ico, Lda., Odi elas, Po ugal.
Licensed unde c ea i e commons (BY-SA-NC) 3.0

This hesis is dedica ed o
My pa en s, An ónio Mendes and Luciana C uz,
o always encou aging my c ea i i y and cu iosi y.
Decla ação/Decla a ion
Decla o que es a disse ação é o esul ado do meu p óp io abalho
desen ol ido en e Ab il de 2011 e Janei o de 2015 no labo a ó io da D a.
Ch is en Mi h, Ins i u o Gulbenkian de Ciência em Oei as, Po ugal,
com co-o ien ação do D . Élio Sucena do Ins i u o Gulbenkian de
Ciência, Oei as, Po ugal. Es e dou o amen o oi ealizado no âmbi o
do P og ama Gulbenkian de Dou o amen o (edição 2010-2011). Pa e do
capí ulo 1 oi publicado no F on ie s in Physiology como “Mechanisms
egula ing nu i ion-dependen de elopmen al plas ici y h ough o gan-
speci ic e ec s in insec s”, T. Koyama, C.C. Mendes and C.K. Mi h
(2013). O capí ulo 2 e 3 es ão in eg ados num manusc i o subme ido
pa a publicação com au o ia de C.C. Mendes e C.K. Mi h. O capí ulo 4
es á in eg ado num manusc i o em p epa ação com au o ia C.C. Mendes,
E. Sucena e C.K. Mi h.
I decla e ha his disse a ion is a esul o my own esea ch ca ied
ou be ween Ap il 2011 and Janua y 2015 in he labo a o y o D .
Ch is en Mi h, Ins i u o Gulbenkian de Ciência in Oei as, Po ugal,
wi h he co-supe ision o D . Élio Sucena o he Ins i u o Gulbenkian
de Ciência, Oei as, Po ugal. Pa o chap e 1 has been published
in F on ie s in Physiology en i led “Mechanisms egula ing nu i ion-
dependen de elopmen al plas ici y h ough o gan-speci ic e ec s in
insec s”, T. Koyama, C.C. Mendes and C.K. Mi h (2013). Chap e 2 and
3 a e pa o a manusc ip submi ed o publica ion, au ho ed by C.C.
Mendes and C.K. Mi h. Chap e 4 is pa o a manusc ip in p epa a ion,
au ho ed by C.C. Mendes, E. Sucena and C.K. Mi h.
Apoio Financei o/Financial Suppo
Es a disse ação e e o apoio inancei o da Fundação pa a a Ciência
e a Tecnologia, bolsa de dou o amen o #SFRH/BD/51624/2011 e da
Fundação Calous e Gulbenkian.
This disse a ion had he inancial suppo om Fundação pa a a Ciência e
a Tecnologia, doc o al ellowship #SFRH/BD/51624/2011 and Fundação
Calous e Gulbenkian.
This hesis was w i en in L
A
TEX. All images we e made wi h Inkscape,
ImageJ (NIH) and Adobe Pho oshop (Adobe Sys ems).
LIST OF FIGURES
1.1 The elan ionship be ween pheno ype and he en i onmen . 7
1.2 The IIS pa hway in D. melanogas e .............. 11
1.3 The ac i a ion and de ep ession unc ions o ecdysone
signalling............................. 13
1.4 O gans di e in hei sensi i i y o nu i ion. . . . . . . . . 15
2.1 Changes in nu i ion du ing he i s phase o sensi i i y
ha e g ea e e ec s in o a iole numbe han in he second
phase o sensi i i y. . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 O a iole numbe is posi i ely co ela ed wi h ea ly emale
ecundi y. ............................ 31
2.3 O a y de elopmen du ing L3 la al s ages unde op imal
nu i ional condi ions. . . . . . . . . . . . . . . . . . . . . . 32
2.4 Dis inc s age-speci ic de elopmen al p ocesses du ing
o a y de elopmen a e egula ed by nu i ion. . . . . . . . . 34
2.5 TF o ma ion and o a y g ow h espond di e en ly o p e-
and pos -c i ical weigh nu i ion. . . . . . . . . . . . . . . . 36
2.6 C i ical weigh sepa a es wo phases o sensi i i y o
nu i ion in o a iole numbe . . . . . . . . . . . . . . . . . . 37
2.1 Con ol es o En an ibody. . . . . . . . . . . . . . . . . . 40
2.2 La al nu i ion a ec s de elopmen ime and emale weigh . 41
3.1 Expe imen al design o nu i ional manipula ions. . . . . . . 48
3.2 a ic jam-GAL4 is exp essed in o a ian soma ic cells
du ing L3 la al s ages. . . . . . . . . . . . . . . . . . . . . 51

3.3 a ic jam-GAL4 is exp essed in neu oblas s and glial cells
in he la al b ain. . . . . . . . . . . . . . . . . . . . . . . . 52
3.4 Manipula ing IIS o ecdysone signalling in he la al o a y
educes adul o a iole numbe and emale weigh . . . . . . . 54
3.5 Role o IIS du ing o a y de elopmen . . . . . . . . . . . . . 56
3.6 Role o ecdysone signalling du ing o a y de elopmen . . . . 58
3.7 Feeding wild- ype la ae wi h 20E-supplemen ed suc ose-
only ood inc eases TF numbe and o a y olume. . . . . . 60
3.8 The in e play be ween IIS and ecdysone signalling pa hways. 62
4.1 Dis ibu ion o he ou D. moja ensis subspecies. . . . . . 73
4.2 O a iole numbe in emales o D. moja ensis w igleyi is
educed ela i e o o he D. moja ensis subspecies. . . . . . 79
4.3 Daily egg p oduc ion in D. moj. sono ensis and D. moj.
w igleyi.............................. 81
4.4 Adul body size and du a ion o L3 de elopmen in D. moj.
sono ensis and D. moj. w igleyi. ............... 82
4.5 The dynamics o TF o ma ion and o a y g ow h in D. moj.
sono ensis and D. moj. w igleyi. ............... 84
4.6 The e ec s o la al nu i ion in de elopmen al ime, emale
body size and o a iole numbe in D. moj. sono ensis and
D. moj. w igleyi......................... 86
4.7 O a iole numbe and emale body size in F1 and F2 hyb ids. 88
4.8 Changes in dis inc de elopmen al p ocesses unde lie
nu i ional-induced and subspecies-speci ic a ia ion in
o a iolenumbe . ........................ 90
5.1 A hypo he ical h eshold o ecdysone sensi i i y con olled
byIIS...............................101
5.2 Al e na i e mechanisms migh unde lie di e ences in he
onse o TFC di e en ia ion be ween he wo D. moja ensis
subspecies. ...........................103
5.3 The ela ionship be ween IIS ac i i y and nu i ional
sensi i i y.............................105
i
LIST OF TABLES
3.1 Pai wise compa isons o he a e o o a y g ow h in la ae
ed on s anda d ood. . . . . . . . . . . . . . . . . . . . . . . 63
3.2 Pai wise compa isons o he a e o TF o ma ion in la ae
ed on s anda d ood. . . . . . . . . . . . . . . . . . . . . . . 63
3.3 Pai wise compa isons o he a e o TF o ma ion in la ae
ed on suc ose alone. . . . . . . . . . . . . . . . . . . . . . . 63
4.1 Species s ocks used in his s udy. . . . . . . . . . . . . . . . 75
4.2 Gene alized linea model ( amily=Poisson) o
de elopmen al ime. . . . . . . . . . . . . . . . . . . . . . . 86
4.3 Two-away ANOVA model o emale body size. . . . . . . . 86
4.4 Two-away ANOVA model o o a iole numbe . . . . . . . . 87
ii
iii
SUMMARY
The en i onmen can modi y de elopmen al ajec o ies and gene a e
a ange o dis inc pheno ypes wi hou al e ing an o ganism’s genome,
a widesp ead phenomenon called de elopmen al plas ici y. The pas
decades ha e seen a esu gen in e es in unde s anding how de elopmen al
plas ici y con ibu es o e olu iona y p ocesses, as i can p oduce
pheno ypic a ia ion among indi iduals and acili a e di e si ica ion
among popula ions ha inhabi dis inc ecological niches. To be e
unde s and he impo ance o plas ic esponses o e olu iona y change,
we need o explo e how he en i onmen al e s de elopmen o p oduce
pheno ypic a ia ion and hen compa e his o how gene ic a ia ion
in luences hese same de elopmen al p ocesses.
My hesis wo k explo ed he de elopmen al mechanisms unde lying bo h
plas ici y and subspecies-speci ic a ia ion in o a iole numbe , a majo
de e minan o emale ep oduc i e capaci y, in D osophila. O a iole
numbe is de e mined du ing hi d ins a (L3) la al s ages and begins
wi h he di e en ia ion o e minal ilamen cells (TFCs) ha g adually
in e cala e in o s acks called e minal ilamen s (TFs). The numbe o
TFs a pupa ia ion di ec ly de e mines he numbe o o a ioles. The
de elopmen al p ocesses unde lying TF o ma ion a e know o a y bo h
wi h en i onmen al condi ions, like nu i ion, and be ween species.
I i s add essed how nu i ion in luences o a iole numbe in D.
melanogas e . By manipula ing nu i ion a speci ic s ages du ing L3
la al de elopmen , I ound ha o a iole numbe exhibi s wo phases o
sensi i i y o nu i ion (Chap e 2). These wo phases a e sepa a ed by
ix
he nu i ion-dependen de elopmen al ansi ion known as c i ical weigh .
When la ae a e poo ly ed du ing he i s phase o sensi i i y, o a y
g ow h a es s and he onse o TFC di e en ia ion is s ongly delayed,
esul ing in a se e e educ ion in o a iole numbe . On he o he hand,
he e ec s on o a iole numbe in la ae ha a e malnou ished du ing he
second phase a e mo e modes ; o a y g ow h and he o ma ion o new
TFs con inue, al hough a a educed a e ela i e o well- ed la ae.
Secondly, I de e mined he ole o wo ho monal pa hways, he
insulin/insulin-like g ow h ac o signalling (IIS) and ecdysone signalling
pa hways, in egula ing he nu i ional sensi i i y o he o a y (Chap e 3).
My esul s indica e ha bo h pa hways egula e he nu i ional-sensi i e
onse o TFC di e en ia ion, wi h ecdysone signalling playing a pi o al
ole in his p ocess. Con e sely, IIS, and o a lesse ex en , ecdysone
signalling coo dina e he a e o TF o ma ion and o o a y g ow h wi h
nu i ional condi ions.
Las ly, I in es iga ed he de elopmen al changes ha gi e ise o
di e ences in o a iole numbe be ween wo subspecies o D. moja ensis,
he D. moj. sono ensis and D. moj. w igleyi (Chap e 4). As hese
subspecies inhabi geog aphically isola ed a eas and b eed in dis inc
hos cac i, hey p o ide a unique oppo uni y o in es iga e he ea ly
e en s associa ed wi h mo phological di e si ica ion. Based on my de ailed
cha ac e iza ions o o a y de elopmen , I ound ha di e ences in he
a e o o a y g ow h can explain much o he a ia ion in o a iole numbe
be ween D. moj. sono ensis and D. moj. w igleyi. F om hese indings, I
p opose ha e olu iona y changes in he ac i i y o IIS could unde lie he
di e ences in o a y g ow h, and consequen ly o a iole numbe , be ween
hese subspecies (Chap e 5).
In summa y, my esul s unde sco e he impo ance o ho monal pa hways
in coo dina ing s age-speci ic de elopmen al p ocesses wi h en i onmen al
condi ions, and speci ically sugges ha changes in he ac i i y o
ho monal pa hways can accoun o plas ic esponses, and po en ially also
o e olu iona y di e si ica ion.
The powe ul de elopmen al app oach emb aced in his hesis may
be use ul o in es iga e how o he mo phological ai s espond o
en i onmen al a ia ion, and could p o ide signi ican insigh s o un a el
one o he mos cap i a ing mys e ies o biology; ha o he o igin o
di e si y in li ing hings.
x

SUMÁRIO
O ambien e pode al e a o desen ol imen o de um o ganismo e c ia
uma a iedade de enó ipos sem al e a o seu genoma. Es e enómeno,
ex emamen e comum na na u eza, é denominado plas icidade. Nas
úl imas décadas, o in e esse em comp eende como a plas icidade pode
con ibui pa a os p ocessos e olu i os em indo a c esce . É um in e esse
p eenchido ao ac o de a plas icidade se capaz de p oduzi a iação
eno ípica en e indi íduos e acili a a di e si icação en e populações
que habi am di e en es nichos ecológicos. Pa a melho comp eende
a impo ância da plas icidade na e olução, é necessá io explo a de
que o ma o ambien e al e a o desen ol imen o pa a p oduzi di e sos
enó ipos, e iden i ica se al e ações semelhan es no desen ol imen o são
esponsá eis pela a iação eno ípica en e espécies.
Es a ese e e como obje i o in e p e a os mecanismos
de desen ol imen o, que es ão na base de, que de espos as plás icas
no núme o de o a íolos de D osophila, que de di e enças nes e núme o
esul an e de a iação gené ica en e subespécies de D osophila. O núme o
de o a íolos in luencia a capacidade ep odu i a da êmea e é de e minado
du an e o e cei o es ágio la a . O p ocesso de o mação dos o a íolos
começa com a di e enciação de ‘células dos ilamen os e minais’ (TFCs),
que g adualmen e se in e calam, o mando pilhas de células denominadas
‘ ilamen os e minais’ (TFs). O núme o de TFs no momen o da pupa iação
iguala o núme o de o a íolos no adul o. Es e p ocesso pode a ia com as
condições ambien ais da la a, como, po exemplo, com a nu ição, e com
a a iação gené ica en e espécies.
xi
P imei amen e in es iguei como a nu ição in luencia o núme o de
o a íolos em D. melanogas e . Ao manipula a nu ição em di e en es
al u as do e cei o es ágio la al, demons ei que o núme o de o a íolos
exibe dois pe íodos sensí eis à nu ição (Capí ulo 2). Es es são sepa ados
pela ansição de desen ol imen o conhecida como peso c í ico. Quando
as la as são subme idas a um dé ice alimen a du an e o p imei o pe íodo
sensí el, o c escimen o do o á io é ep imido e o início da di e enciação dos
TFCs é ex emamen e a asado, esul ando daí, uma se e a edução no
núme o de o a íolos. Po ou o lado, os e ei os no núme o de o a íolos em,
la as que são mal nu idas, du an e o segundo pe íodo sensí el à nu ição
são mais mode ados; o c escimen o do o á io e a o mação de no os TFs
con inua. No en an o, a sua axa de p og essão é eduzida ela i amen e
a la as bem nu idas. De seguida, explo ei a unção de duas ias de
sinalização ho monal, a ia da insulina e a ia da ecdisona, na egulação
da espos a nu icional do o á io (Capí ulo 3). Es es esul ados indicam
que ambas as ias egulam o início da di e enciação dos TFCs, endo a
ia da ecdisona um papel ulc al nes e p ocesso. Con a iamen e, ambas
as ias, a da insulina, e, em meno g au a da ecdisona, egulam as axas
de o mação de TFs e do c escimen o do o á io em espos a às condições
nu icionais.
Finalmen e, explo ei possí eis al e ações no desen ol imen o que
pudessem explica as di e enças obse adas no núme o de o a íolos en e
duas subespecies de D. moja ensis, a D. moj. sono ensis e a D. moj.
w igleyi (Capí ulo 4). Es as subespecies habi am á eas geog a icamen e
isoladas e desen ol em-se em cac us dis in os, p o idenciando uma
opo unidade única pa a in es iga os p imei os e en os associados com
a di e si icação mo ológica. Baseado nas ca ac e izações de alhadas do
desen ol imen o o á ico que e e uei, demons ei que as di e enças na axa
de c escimen o do o á io podem explica , em g ande pa e, as di e enças
no núme o de o a íolos en e a D. moj. sono ensis e a D. moj. w igleyi.
Tendo em con a es a obse ação, p opus que mudanças e olu i as na
a i idade da ia da insulina pode ão es a na base das di e enças da axa
de c escimen o do o á io, e, consequen emen e, no núme o de o a íolos
en e as duas subespecies (Capí ulo 5).
Resumindo, es es esul ados e elam a impo ância das ias de sinalização
ho monal na egulação de p ocessos que oco em em pe íodos especí icos
do desen ol imen o, e na sua coo denação com as condições ambien ais.
Além disso, suge em ambém que, mudanças na a i idade de ias de
sinalização ho monal são esponsá eis, an o pela espos a plás ica como,
po encialmen e ambém pela di e si icação e olu i a.
xii
A pode osa abo dagem ocada no desen ol imen o, que oi u ilizada nes a
minha ese pode á se ú il pa a in es iga como ou as ca ac e ís icas
mo ológicas espondem à a iação ambien al. Des a o ma, pode á
auxilia a esol e um dos mis é ios mais ca i an es da biologia: o da
o igem da di e sidade de o mas de ida.
xiii
xi
Chap e 1
Figu e 1.1: The elan ionship be ween pheno ype and he en i onmen .
(A) A e age size alues o wo ai s (blue and ed lines) a e di e en due o gene ic
a ia ion. Plas ici y is absen . (B) Bo h ai s espond o en i onmen al a ia ion
and he deg ee o hei plas ic esponse is simila . Plas ici y is p esen , bu gene ic
a ia ion in plas ici y is absen . Fo example, wing and body size in D. melanogas e .
(C) The slopes o he eac ion no ms a e di e en , sugges ing ha he wo ai s espond
di e en ly o he same en i onmen al cue. Plas ici y and gene ic a ia ion in plas ici y
a e p esen . Fo example, male geni alia and wing size in D.melanogas e . (D) A gi en
ai may show dis inc plas ic esponses depending on he en i onmen al cue. Fo
example, o a iole numbe in D.melanogas e . See ex o mo e in o ma ion.
7

Chap e 1
small eyespo s. Tempe a u e shi expe imen s a speci ic de elopmen al
poin s e ealed ha he c i ical pe iod in which empe a u e can induce
changes in wing pa e n occu s la e in la al de elopmen (Kooi and
B ake ield, 1999). Fu he examples ha e been desc ibed in o he
polyphenic bu e lies (Nijhou , 2003b). These s udies a e beginning o
allow us o piece oge he how c i ical pe iods o en i onmen al sensi i i y
a e egula ed.
1.4 Ho monal mechanisms o de elopmen al
plas ici y
Recen esea ch has e ealed ha en i onmen al ac o s o en modula e
de elopmen al ajec o ies o p oduce dis inc pheno ypes by con olling
which, when, and how s ongly genes a e exp essed du ing de elopmen .
The ac ion o ho mones is pe haps one o he bes -unde s ood mechanisms
media ing de elopmen al plas ici y. Ho mones a e known o in eg a e
in o ma ion om he ex e nal en i onmen and egula e mul iple
de elopmen al p ocesses h oughou he en i e o ganism: some bind
di ec ly o ansc ip ion ac o s and ac i a e he exp ession o speci ic
genes (Baniahmad and Tsai, 1993), o he s ac i a e a se ies o in e cellula
signalling cascades ha egula e g ow h (Wu and B own, 2006) and e en
o he s change he DNA me hyla ion p o iles in he genome, egula ing
which genes a e exp essed (Beldade e al., 2011; Snell-Rood e al., 2013).
An in eg a ed pic u e o how ho mones link en i onmen al a ia ion
wi h de elopmen al changes has la gely been d awn om s udies on he
egula ion o body and o gan size in one o he mos di e se animal g oups:
he insec s. Much o he mo phological di e si y seen ac oss insec species
is gene a ed by changes in o gan size and shape ela i e o he whole body
(Shingle on e al., 2007, 2008). In holome abolous insec s, body size is a
unc ion o he la al ea ing en i onmen , in which nu i ional condi ions
play a majo ole. As adul s ha e a scle o ized ou e skele on ha p e en s
u he g ow h, he adul body size is ixed once la ae s ops eeding a he
onse o me amo phosis. Mo eo e , many adul o gans de elop inside he
la al body as imaginal discs and espond o he same cues ha con ol
8
Chap e 1
whole body g ow h. The e o e, like adul body size, he size o many adul
o gans is de e mined by he amoun o g ow h ha imaginal discs achie e
du ing de elopmen .
Al hough signi ican p og ess has been made in unde s anding he
ho monal mechanisms unde lying nu i ional plas ici y o o gan size
in non-model insec s (Beldade e al., 2011), ecen ad ances in D.
melanogas e ha e opened up unique oppo uni ies o gene a e insigh
in o he ho monal mechanisms h ough which nu i ion changes o gan
size and p oduces no el and di e se mo phologies. In D. melanogas e ,
like many holome abolous insec s, h ee de elopmen al ho mones – he
insulin-like pep ides, ju enile ho mone (JH), and he s e oid moul ing
ho mone ecdysone – ansla e signals om he nu i ional en i onmen o
egula e body and o gan g ow h (Mi h and Shingle on, 2012; Nijhou ,
2003a). Al hough JH is a key egula o o g ow h in he obacco
ho nwo m Manduca sex a (Nijhou and Williams, 1974) and he dung
bee les On hophagus au us (Emlen and Nijhou , 1999), i s ole in g ow h
D. melanogas e was, un il ecen ly, con o e sial (Fla , 2005; Mi h
e al., 2014; Riddi o d and Ashbu ne , 1991; Riddi o d e al., 2010). In
he ollowing pages, I will he e o e ocus on wha is known abou he ole
o he D. melanogas e insulin-like pep ides (dILPs), he insulin/insulin-
like g ow h ac o signalling (IIS) pa hway, and he ecdysone signalling
pa hway in egula ing nu i ional plas ici y in body and o gan size.
1.4.1 Nu i ion and he insulin/insulin-like g ow h ac o
signalling (IIS) pa hway
In D. melanogas e , and many o he animals, nu i ion modi ies body and
o gan g ow h h ough he ac ion o he IIS pa hway (Figu e 1.2). In ich
nu i ional en i onmen s, neu osec e o y cells in he b ain, he insulin-
p oducing cells, syn he ize and sec e e high amoun s o dILPs. Only h ee
9
Chap e 1
o he eigh dILPs – dILP2, dILP3, and dILP5 – a e exclusi ely exp essed
in he insulin-p oducing cells (Ikeya e al., 2002; Ruli son e al., 2002).
The exp ession o hese dILPs is nu ien dependen ; s a a ion ep esses
bo h hei syn hesis and sec e ion (B ogiolo e al., 2001; Ikeya e al., 2002).
Fu he , abla ion o he insulin-p oducing cells educes adul body size in a
simila ashion o s a a ion (Ruli son e al., 2002). These indings indica e
ha mos o he nu i ion-dependen g ow h is p esumably egula ed by
he dILP p oduc ion in he insulin-p oducing cells. The addi ional dILPs
a e exp essed in se e al di e en issues, including he imaginal discs, he
mid gu , and he en al ne e co d, and a e hough o ha e sys emic
e ec s on g ow h (B ogiolo e al., 2001; Colombani e al., 2012; Ga elli
e al., 2012).
A e being eleased in o he insec bloods eam, dILPs ac on a ge
issues by binding o he insulin ecep o (InR) (B ogiolo e al.,
2001). Once InR is ac i a ed, a highly conse ed phospho-kinase signal
ansduc ion cascade, he IIS, is induced ul ima ely egula ing cell g ow h
and di ision. This is mainly achie ed by ac i a ing posi i e g ow h
egula o s, such as he p o ein kinase Ak , and supp essing nega i e
g ow h egula o s, such as he ansc ip ion ac o Fo khead Box class
(FOXO) and he Tube ous Scle osis Complex 1 and 2 (TSC1/2) ( e iewed
in (Taniguchi e al., 2006).
The supp ession o TSC1/2 allows an addi ional nu ien -sensi i e
pa hway, he a ge o apamycin (TOR) signalling pa hway, o emain
ac i e. The TOR pa hway esponds di ec ly o in acellula amino acid
concen a ions ia he TOR complex and egula es a numbe o cellula
p ocesses o p omo e g ow h (Gao e al., 2002; Sa basso e al., 2005). In
addi ion, he TOR complex i sel egula es he IIS pa hway by ac i a ing
Ak (Sa basso e al., 2005), which illus a es he ex ensi e c oss alk
be ween he wo nu i ion-sensi i e pa hways. Supp essing any componen
in he IIS pa hway slows g ow h and esul s in smalle adul s in a
simila manne as s a a ion (B i on e al., 2002; B ogiolo e al., 2001).
Combined, hese indings illus a e ha he ci cula ing le els o dILPs and
he IIS pa hway coo dina e g ow h a e wi h nu i ional inpu s.
Addi ionally, he IIS pa hway also con ols he leng h o he g ow h
10
Chap e 1
Figu e 1.2: The IIS pa hway in D. melanogas e The sec e ion o dILPs by he
insulin-p oducing cells in he b ain depends on nu i ion. (A) Unde high nu i ional
condi ions, high le els o dILPs a e exp essed and ac i a e he IIS pa hway, p omo ing
cell g ow h and di ision p ima ly by ac i a ing Ak and sup essing he ac i i i y o
FOXO. (B) In con as , when la ae a e malnou ished, low le els o dILPs a e exp essed,
esul ing in a educed ac i i y o IIS pa hway, and consequen ly, educed g ow h.
11
Chap e 1
pe iod – ano he c ucial de e minan o body and o gan size in insec s.
The IIS pa hway con ols he g ow h pe iod p ima ily by egula ing he
iming o he pulses o he s e oid ho mone ecdysone a speci ic s ages in
de elopmen (Koyama e al., 2014). How does ecdysone, in u n, egula e
he du a ion o he g ow h pe iod and ul ima ely body and o gan size?
1.4.2 Nu i ion and he ecdysone signalling pa hway
Ecdysone is syn he ized and sec e ed by he p o ho acic glands in a
se ies o disc e e peaks h oughou la al and pupal de elopmen . This
pe iodic elease o ecdysone oge he wi h a empo al- and issue-speci ic
exp ession o he ecdysone ecep o complex, a he e odime be ween
Ecdysone Recep o (EcR) and Ul aspi acle (Usp), o ches a e many
aspec s o la al de elopmen : om la al mol s and me amo phosis o
g ow h and di e en ia ion o a ge issues ( e iewed in (Yamanaka e al.,
2013).
Se e al s udies ha e unco e ed ha one small peak o ecdysone ha occu s
ea ly in he hi d and inal ins a (L3) la ae is sensi i e o nu i ional
condi ions (Caldwell e al., 2005; Colombani e al., 2005; Koyama e al.,
2014; Layalle e al., 2008; Mi h, 2005). This small peak o ecdysone
eaches i s maximum a a ound 8 o 10 h a e hi d la al ecdysis (AL3E)
(Koyama e al., 2014; Wa en e al., 2006) and induces a key de elopmen al
ansi ion, c i ical weigh . C i ical weigh de e mines when o end g ow h
and ini ia e me amo phosis, he eby egula ing body and o gan size
(Koyama e al., 2014; Mi h and Riddi o d, 2007; Mi h and Shingle on,
2012). S a ing la ae be o e eaching c i ical weigh signi ican ly delays
he iming o he ecdysone peak, which in u n, delays he onse o
me amo phosis (Beadle e al., 1938; Mi h e al., 2005; Shingle on e al.,
2005; S iepe e al., 2008) and delays pa e ning o he p esump i e adul
issues, he imaginal discs (Mi h e al., 2009). Con e sely, s a a ion
a e c i ical weigh accele a es he onse o me amo phosis and no longe
p e en s con inued pa e ning and g ow h o he imaginal discs in he
absence o nu i ion (Beadle e al., 1938; Mi h e al., 2005; Mi h e al.,
2009; Shingle on e al., 2005).
12

Chap e 1
Figu e 1.3: The ac i a ion and de ep ession unc ions o ecdysone signalling.
Ecdysone binds o i s ecep o , a he e odime be ween EcR and Usp, o induce wo ypes
o unc ions.(A) Ac i a ion unc ion: ecdysone binds o EcR-Usp and di ec ly ac i a es
gene ansc ip ion. (B) De ep ession unc ion: ecdysone binds o EcR-Usp and elie es
he ep essi e ac ion o he EcR-Usp, allowing gene ansc ip ion. Knocking down ei he
EcR o Usp pa ially ac i a es gene ansc ip ion, while o e exp essing a dominan
nega i e o m o EcR wi h a mu a ed ligand-binding domain (EcRDN) p e en s gene
ansc ip ion.
Ecdysone exe s i s e ec s by binding o he EcR/Usp he e odime
complex. This complex ep esses he ansc ip ion o a subse o ecdysone
a ge genes in he absence o ecdysone (Figu e 1.3B) (B own e al.,
2006; Che bas, 2003; Schubige and T uman, 2000; Schubige e al., 2005).
Once ecdysone binds o EcR/Usp, i induces a ge gene ansc ip ion
ei he by di ec ac i a ion ia EcR/Usp (Figu e 1.3A) o by elie ing he
ep essi e ac ion o he EcR/Usp (Figu e 1.3B). Se e al gene ic ools in D.
melanogas e allow us o explo e he speci ic oles o ecdysone signalling
in body and o gan g ow h. Fo ins ance, knocking down ei he EcR o
13
Chap e 1
Usp, using RNAi, elimina es he ep essi e unc ion o EcR/Usp, he eby
pa ially inducing ecdysone unc ion (Figu e 1.3A) (B own e al., 2006;
Che bas, 2003; Mi h e al., 2009; Schubige e al., 2005). Con e sely,
o e exp essing a dominan nega i e o m o EcR wi h a mu a ed ligand-
binding domain abolishes bo h he de ep ession and ac i a ion unc ions
o ecdysone (Figu e 1.3B)(B own e al., 2006; Che bas, 2003; Hu e al.,
2003). In Chap e 3, I ook ad an age o hese wo well-desc ibed gene ic
ools o in es iga e he ole o ecdysone signalling in egula ing nu i ional
plas ici y in o gan size.
1.4.3 O gan-speci ic sensi i i ies o nu i ion
I he le els o ci cula ing dILPs e lec he nu i ional s a us o an insec ,
how do di e en o gans espond di e en ially o nu i ional a ia ion? As
discussed abo e, he size o he male geni alia and he CNS is ela i ely
in a ian ac oss nu i ional condi ions (Cheng e al., 2011; Shingle on
e al., 2005; Tang e al., 2011). This low sensi i i y o nu i ion is achie ed
h ough di e en mechanisms. In he case o he CNS, InR-independen
ac i a ion o he IIS pa hway allows he CNS o main ain i s g ow h a e
e en when ci cula ing dILPs a e low (Cheng e al., 2011). Al e na i ely,
he male geni alia educes i s plas ici y in esponse o nu i ion by
exp essing low le els o oxo mRNA (Tang e al., 2011). When ci cula ing
dILPs and he ac i i y o he IIS pa hway a e educed, FOXO emains
in he nucleus and sup esses g ow h (Jünge e al., 2003). As he male
geni alia exp esses low le els o oxo, i is able o main ain i s size e en
when la ae a e malnou ished (Figu e 1.4A) (Shingle on e al., 2005, 2009;
Tang e al., 2011). O e exp essing FOXO in he male geni alia inc eases
i s sensi i i y o nu i ion and esul s in smalle geni alia (Figu e 1.4A)
(Tang e al., 2011). Despi e he di e ences in mechanisms be ween he
CNS and he male geni alia, ul ima ely hese o gans a e p o ec ed om
he e ec s o poo nu i ion by e aining high le els o ac i i y o he IIS
pa hway i espec i e o nu i ional condi ions.
Changing he le el o ac i i y o he IIS pa hway in o gans ha
scale p opo ionally wi h body size, as he wing discs, can esul in
14
Chap e 1
Figu e 1.4: O gans di e in hei sensi i i y o nu i ion. (A) The male geni al
disc main ains i s size e en when la ae a e poo ly ed. This educ ion in nu i ional
sensi i i y is achie ed by educing he le els o oxo mRNA and e aining high IIS
ac i i y in low nu i ional en i onmen s (g ey line). O e exp essing FOXO in he male
geni alia inc eases i s sensi i i y o nu i ion (blue line). (B, C) Nu i ion a ec s he size
o he wings in p opo ion wi h body size (g ey line). (B) An inc ease o InR exp ession
esul s in an inc ease in nu i ional sensi i i y by enhancing wing size in la ge lies
( ed line). (C) An inc ease o oxo exp ession enhances he nu i ional sensi i i y o
he wing by sup essing wing size in small indi iduals (blue line). (D) The sensi i i y
o nu i ion o he wing discs a ies wi h de elopmen al ime. Be o e c i ical weigh ,
s a a ion se e ely educes he g ow h o he wing disc. On he o he hand, discs
g ow conside ably e en in pos -c i ical weigh la ae ha a e malnou ished.CW:c i ical
weigh . Adap ed om (Shingle on and F ankino, 2013; Shingle on and Tang, 2012;
Shingle on e al., 2008)
.
15
Chap e 1
an exagge a ed esponse o nu i ion (Shingle on and F ankino, 2013;
Shingle on and Tang, 2012). O e exp essing ei he FOXO o InR
speci ically in he wing disc inc eases i s sensi i i y o nu i ion making
i hype allome ic (i.e. disp opo ionally la ge ) in ela ion o body
size (Figu e 1.4B, C). Howe e , his hype allome y is achie ed h ough
di e en ways: inc easing InR exp ession esul ed in an exagge a ed
inc ease in he wing size o la ge indi iduals, bu has li le o no e ec s
in he wing size in smalle indi iduals (Figu e 1.4B) (Shingle on and
Tang, 2012). On he o he hand, an inc ease in oxo exp ession led o
a disp opo ionally small wing size in smalle indi iduals, bu almos no
e ec in la ge indi iduals (Figu e 1.4C) (Shingle on and Tang, 2012).
Thus i appea s ha o gans can display exagge a ed esponses o nu i ion
by modula ing he IIS pa hway a se e al le els o i s ac ion (Shingle on
and F ankino, 2013).
O gans can also change hei sensi i i y o nu i ion wi h de elopmen al
ime. Fo ins ance, s a ing p e-c i ical weigh la ae comp omises wing
disc g ow h and di e en ia ion, bu a e c i ical weigh s a a ion has a
mo e modes e ec on he de elopmen o he wings discs; ha is, discs
g ow conside ably and con inue o di e en ia e e en when pos -c i ical
weigh la ae a e poo ly ed (Figu e 1.4D) (Mi h e al., 2009; Shingle on
e al., 2008). This swi ch in sensi i i y o nu i ion a c i ical weigh
seems o be media ed by changes in he IIS pa hway. Sup essing he IIS
pa hway jus a e c i ical weigh abolishes body g ow h, bu he wing discs
con inue o g ow p esumably un il hei size is app op ia e o he much
educed body size (Figu e 1.4D)(Shingle on e al., 2005, 2008). These
indings ha e led some au ho s o hypo hesize ha an in insic g ow h
a e ha does no equi e nu i ional inpu s may enable u he g ow h o
he de eloping o gans when nu i ion, and acco dingly he IIS, is se e ely
educed (Nijhou e al., 2014; Shingle on e al., 2008).
The ac i i y o ecdysone signalling a c i ical weigh may accoun o he
swi ch in sensi i i y o nu i ion o de eloping o gans. Knocking down
EcR speci ically in he wing discs o s a ed p e-c i ical weigh la ae
allows di e en ia ion o he wing disc o p oceed (Mi h e al., 2009). A
simila obse a ion was epo ed o he op ic lobe o he D osophila CNS
16
Chap e 2
2.1 In oduc ion
De elopmen al plas ici y, he abili y o an o ganism o adjus i s
de elopmen al ajec o y in esponse o en i onmen al a ia ion, is a
seemingly uni e sal p ope y o all mul icellula o ganisms. O en, he
ex en o de elopmen al plas ici y depends no only on he ai s and
en i onmen al condi ions conside ed (Mi h and Shingle on, 2012), bu
also on he exis ence o phases o en i onmen al sensi i i y, commonly
e e ed as c i ical pe iods, du ing which de elopmen al p ocesses can
espond plas ically (Koyama e al., 2013; Nijhou , 2003a). In he mos
ex eme cases, an en i onmen al cue wi hin a c i ical pe iod igge s
a de elopmen al swi ch be ween al e na i e de elopmen al ajec o ies,
gi ing ise o dis inc pheno ypes, such as d ama ic seasonal di e ences
in he pigmen a ion o bu e ly wing pa e ns and di e en body sizes
and shapes in honeybee cas es (B ake ield e al., 1996; Smi h e al.,
2008). Unde s anding how de eloping o gans change hei sensi i i y o
en i onmen al condi ions, and how his in luences hei plas ic esponse,
is an impo an s ep owa ds a comp ehensi e knowledge o how he
en i onmen gene a es new pheno ypic a ian s.
Nu i ional s a us is one o he majo egula o s o body and o gan g ow h
and i s e ec s ha e been ex ensi ely s udied in insec s, in pa icula , in
he ui ly, D osophila melanogas e . In D. melanogas e , like many
insec s, nu i ion egula es g ow h by egula ing a key de elopmen al
ansi ion, c i ical weigh , which occu s a ound 10 h a e moul ing o
he hi d and inal ins a la ae (L3) (Beadle e al., 1938; Koyama e al.,
2014; Mi h e al., 2005; Nijhou and Williams, 1974; Shingle on e al.,
2005; S iepe e al., 2008). S a ing la ae be o e c i ical weigh causes
hem o signi ican ly delay he onse o me amo phosis (Beadle e al.,
1938; Mi h e al., 2005; Shingle on e al., 2005; S iepe e al., 2008),
whe eas s a ing la ae a e c i ical weigh induces ea ly me amo phosis
(Beadle e al., 1938; Mi h e al., 2005; S iepe e al., 2008). As
c i ical weigh de e mines when o end g ow h and ini ia e me amo phosis,
he unde lying mechanisms egula ing i s a ainmen ha e been s udied
ex ensi ely (Beadle e al., 1938; Koyama e al., 2014; Mi h e al., 2005;
Nijhou and Williams, 1974; Shingle on e al., 2005; S iepe e al., 2008).
23

Chap e 2
C i ical weigh also egula es he sensi i i y o de eloping o gans o
nu i ion o e de elopmen al ime. La ae s a ed be o e eaching c i ical
weigh delay he pa e ning o hei p esump i e adul issues, he
imaginal discs (Mi h e al., 2009). Con e sely, s a a ion a e c i ical
weigh allows con inued pa e ning and g ow h o he imaginal discs
(Mi h e al., 2005, 2009; Shingle on e al., 2008). This addi ional ole o
c i ical weigh has been o e looked in cu en esea ch, and impo an ly,
whe he c i ical weigh de e mines pe iods o nu i ional sensi i i y has
no ye been ully in es iga ed. In his chap e , I a emp o elucida e how
de eloping o gans change hei sensi i i y o nu i ion o e de elopmen al
ime, wi h special emphasis on he po en ial ole o c i ical weigh in
media ing nu i ional sensi i i y.
To add ess his issue, I used o a iole numbe in D. melanogas e as a
model. O a ioles a e egg-p oducing s uc u es in he insec o a y ha
di ec ly a ec emale ep oduc i e capaci y (Boulé eau-Me le e al., 1982;
R’ kha e al., 1997; Klepsa el e al., 2013b,a). Al hough li le is known
abou he gene ic cascades in ol ed in o a iole de elopmen (Cheng e al.,
2011; Fo bes e al., 1996; God and Laski, 1995; Pa el e al., 1989; Sahu -
Ba nola e al., 1995; Sa ikaya and Ex a ou , 2015), he cellula e en s
media ing his p ocess a e be e cha ac e ized. O a iole de elopmen
occu s du ing he hi d ins a (L3) la al and ea ly pupal s ages (Ke kis,
1931; King, 1970; King e al., 1968) h ough he in e cala ion o e minal
ilamen cells (TFCs) in o s acks o se en o en la ened cells, called
e minal ilamen s (TFs) (God and Laski, 1995; Sahu -Ba nola e al.,
1995, 1996). Each TF de ines he posi ion o one o a iole and hus, he
numbe o TFs a pupa ia ion is equi alen o he numbe o o a ioles
in he adul (God and Laski, 1995; Hodin and Riddi o d, 1998; Sahu -
Ba nola e al., 1995; Sa ikaya e al., 2012).
Nu i ional condi ions du ing la al s ages egula e o a iole numbe
(Be gland e al., 2008; Hodin and Riddi o d, 2000; Sa ikaya e al., 2012; Tu
and Ta a , 2003). P e ious s udies o he de elopmen al e ec s o nu i ion
on o a iole numbe ha e shown ha dilu ing he ood on which la ae
we e aised al e ed o a iole numbe by changing he o al numbe o TFCs
(Sa ikaya e al., 2012) o he a e o TF o ma ion in la e L3 la ae (Hodin
24
Chap e 2
and Riddi o d, 1998). Howe e , i was unclea whe he o a y de elopmen
exhibi s c i ical pe iods o nu i ional sensi i i y, and impo an ly, how he
de elopmen al p ocesses a e modi ied by nu i ion a di e en pe iods o
sensi i i y. I he e o e examined whe he changes in nu i ion a speci ic
s ages du ing L3 la ae in luence he plas ic esponse o o a iole numbe .
I u he in es iga ed how dis inc s age-speci ic de elopmen al p ocesses
du ing o a y de elopmen espond o changes in nu i ion and accoun o
nu i ional-induced di e ences in o a iole numbe .
2.2 Ma e ials and Me hods
2.2.1 Fly s ock
To assess he e ec s o la al nu i ion on o a iole numbe , I used an
ou b ed popula ion (wild ype) o D osophila melanogas e es ablished
in he labo a o y o D . Élio Sucena in 2007, o igina ing om 160
e ilized emales collec ed in Azei ão, Po ugal (Ma ins e al., 2013).
The popula ion was kep in labo a o y cages wi h high census (>1500
indi iduals) and main ained a cons an empe a u e (25°C) on s anda d
ly ood (4.5% molasses, 7.2% suga , 7% co nmeal, 2% yeas ex ac , 1%
aga and 2.5% Nipagin solu ion).
2.2.2 La al s aging and die a y manipula ions
Adul s we e allowed o lay eggs o wo o six hou s on esh ood
pla es (60 ×15 mm Pe i dish). Egg densi y was con olled o p e en
o e c owding (app oxima ely 200 eggs pe pla e). La ae we e selec ed
0-2 hou s a e ecdysis o L3 (AL3E) and ans e ed on o new ood pla es
(40-60 la ae pe pla e) o eed un il hey eached he app op ia e age. To
de e mine c i ical pe iods o sensi i i y o nu i ion in o a iole numbe ,
20-30 la ae o he app op ia e age we e ans e ed o ials con aining
ei he 20% suc ose on 0.5% aga medium (suc ose-only ood) o s anda d
ly ood (s anda d ood) un il he end o he eeding pe iod (Figu e 2.1A).
On suc ose-only ood, mos la ae su i ed un il pupa ia ion and adul
25
Chap e 2
eclosion. To ob ain L3 o a ies, la ae o he app op ia e age we e dissec ed
and p ocessed o immunocy ochemis y (Figu e 2.1C, 2.3A, 2.5A). All
expe imen s we e pe o med a 25°C.
2.2.3 Measu emen s o li e-his o y ai s: de elopmen al
ime, emale weigh , ea ly emale ecundi y and
o a iole numbe
To de e mine he a e age ime o pupa ia ion, newly ecdysed L3 la ae
we e ans e ed o ials (20-30 la ae pe ial) con aining s anda d
ood. The numbe o la ae pupa ia ing (immobile la ae wi h e agina ed
spi acles) was coun ed in 2 h in e als un il all la ae pupa ia ed. I used
pha a e weigh as a p oxy o adul body size (Mi h e al., 2005). Pha a e
adul s we e collec ed om ood ials and ood esiduals we e ca e ully
cleaned o om he pupal cases using dis illed wa e and a pain b ush.
I dis inguish emales om males by he p esence o absence o male-
speci ic sex combs h ough he pupal case. Female pha a e adul s we e
indi idually weighed on a Sa o ius SE2 ul amic obalance.
To de e mine ea ly ecundi y, newly eclosed emales we e indi idually
main ained in ials on s anda d ood wi h one male o he same ood/ ime
poin . Indi iduals we e ans e ed o esh ials e e y day du ing he i s
h ee days a e eclosion. All eggs we e coun ed daily. To coun adul
o a iole numbe , newly eclosed lies we e main ained in ials ( en emales
and i e males pe ial) on s anda d ood un il he ime o dissec ion
(4-6 days a e eclosion) (Figu e 2.1A). O a ies we e dissec ed in cold
phospha e bu e ed saline con aining 1% T i on X-100 (PBT) and o a ioles
we e eased apa and coun ed unde a dissec ing mic oscope.
2.2.4 Immunocy ochemis y
Female la ae we e selec ed by he small size o hei gonads loca ed in he
pos e io hi d o he a body. La ae we e dissec ed in cold phospha e
bu e ed saline (PBS) and ixed in 4% o maldehyde in PBS o 30 minu es
a oom empe a u e. La ae we e hen washed h ee imes o 20 minu es
26
Chap e 2
wi h PBT and blocked in 2% no mal donkey se um in PBT o 30 minu es.
P ima y an ibody incuba ion in mouse an i-Eng ailed (De elopmen al
S udies Hyb idoma Bank 4D9, 1:40) dilu ed in o 2% no mal donkey
se um in PBT was conduc ed o e nigh a 4°C. A e washing h ee imes
o 20 minu es in PBT, la ae we e incuba ed in he da k wi h goa
an i-mouse Alexa 568 (In i ogen, 1:200) and TRICT-Phalloidin (Sigma,
1:200) dilu ed in o 2% no mal donkey se um in PBT o e nigh a 4°C.
La ae we e insed wi h PBT and o a ies we e moun ed on a poly-L-
lysine-coa ed co e slip using Fluo omoun -G (Sou he nBio ech).
2.2.5 Image Acquisi ion and Analysis
Samples we e imaged using a Zeiss LSM 510 Me a con ocal mic oscope
using a 40x 1.3NA oil objec i e lens. Du ing con ocal image acquisi ion,
he de ec ion pa ame e s we e adjus ed o a oid unde - o o e exposed
pixels, and images we e acqui ed h ough he ull hickness o he o a y
a 1 µm. Images we e p ocessed and analysed using ImageJ (NIH) and
Adobe Pho oshop (Adobe Sys ems). Fo each ime poin /geno ype/ ood
ea men , e minal ilamen cells (TFC) we e iden i ied by Eng ailed
exp ession. Fo ming e minal ilamen s (TFs) we e iden i ied by he
p esence o TFC in s acks wi h he cha ac e is ic la ened cell mo phology,
and o al numbe o o ming TFs we e coun ed. Fo o a y olume, he
ImageJ Volumes plugin was used (Me zin, 2008).
2.2.6 S a is ical Analysis
All expe imen s we e eplica ed a leas wice. The p elimina y
expe imen desc ibed in Figu e 2.1C, D was pe o med one ime wi h small
sample size. The dis ibu ion o esiduals was es ed o no mali y using
Q-Q plo s and he app op ia e s a is ical es was applied. Fo mul iple
compa isons, ANOVAs we e pe o med ollowed by Tukey’s mul iple
compa ison es o e alua e pai wise di e ences. Welch - es (pa ame ic)
and Wilcoxon ank sum es (non-pa ame ic) we e used o es di e ences
in mean alues be ween wo samples. To de e mine di e ences in he a e
o TF o ma ion and o o a y g ow h, slopes we e compa ed using he
27
Chap e 2
unc ion ‘sm.anco a’ unde ‘sm’ lib a y. All da a analyses and s a is ics
we e conduc ed using R 3.1.2 (R De elopmen Co e Team, 2014). Plo s
we e made using G aphPad P ism 6 (G aphPad So wa e). p- alues a e
indica ed in he ex and igu es.
2.3 Resul s
2.3.1 Two phases o sensi i i y o nu i ion egula e he
plas ic esponse o o a iole numbe
To de e mine c i ical pe iods o sensi i i y o nu i ion in o a iole numbe ,
I ed L3 la ae ei he on s anda d ood o on suc ose-only ood a imed
in e als s a ing be ween 0 h o 30 h AL3E un il he end o he eeding
pe iod (Figu e 2.1A). La ae ed on suc ose-only ood a e s a ed o
p o ein, lipids and o he mic onu ien s p esen in yeas , ye show highe
a es o su i al han when s a ed comple ely. O e all, la ae ans e ed
o suc ose-only ood be ween 0 and 25 h AL3E showed a signi ican
educ ion in o a iole numbe when compa ed o he con ols ans e ed
o s anda d ood (Figu e 2.1B). In con as , ans e ing la ae o suc ose-
only ood a 30 h AL3E did no cause a signi ican educ ion in o a iole
numbe (Figu e 2.1B).
In e es ingly, he e ec o he suc ose-only ood in o a iole numbe
depended on he iming a which la ae we e ans e ed o he suc ose-
only ood (Figu e 2.1B). To es o a signi ican change in he esponse
o suc ose-only ood o e ime, I applied a bi-segmen al linea eg ession
model o he da a and es ed o a signi ican change in slope. The
ela ionship be ween o a iole numbe and he age a ans e o suc ose-
only ood (in h AL3E) has a signi ican change in slope a ound a single
b eakpoin (Da ies’ es o a change in slope, p<0.0001) a 11.5 h AL3E
(95% CI: 9.37–13.64 h AL3E) (Da ies, 1987; Muggeo, 2003, 2007). This
es ima ed b eakpoin coincides wi h he a ainmen o c i ical weigh ,
sugges ing ha p e-c i ical weigh o a ies a e mo e sensi i e o changes in
nu i ion han pos -c i ical weigh o a ies.
28

Chap e 2
Figu e 2.1: Changes in nu i ion du ing he i s phase o sensi i i y ha e
g ea e e ec s in o a iole numbe han in he second phase o sensi i i y.(A)
Expe imen al design o de e mine c i ical pe iods o sensi i i y o nu i ion in o a iole
numbe . Only he i s wo ime poin s a e shown (0 h and 5 h AL3E). (B) Adul o a iole
numbe om la ae ans e ed ei he o s anda d ood (yellow ci cles) o o suc ose-only
ood (blue ci cles). Dashed lines show he bes i ed-lines om he segmen al eg ession
analyses. n≥30 o all ea men s. (C) Expe imen al design o de e mine whe he he
leng h o exposu e o suc ose-only ood in luences o a iole numbe . (D) Adul o a iole
numbe om la ae ed on s anda d ood (yellow ci cles); la ae ans e ed o suc ose-
only ood ei he a 5 h AL3E (ligh blue ci cles) o a 20 h AL3E (da k blue ci cles) and
la ae ed on suc ose-only ood o a 20 h in e al ei he be ween 0 h o 20 h AL3E (open
blue ci cles) o be ween 20 h o 40 h AL3E (open da k ci cles). Plo ed alues ep esen
means and e o ba s show 95% con idence in e als o means. Two-way ANOVA using
Tukey’s es : *p<0.05, ***p<0.001, ns non-signi ican . L3: hi d ins a la ae; AL3E:
a e L3 ecdysis.
29
Chap e 2
Ne e heless, he e ec s o he suc ose-only ood in o a iole numbe could
also be a di ec consequence o di e en leng hs o exposu e o he suc ose-
only ood. To es his hypo hesis, I pe o med a p elimina y expe imen
whe e L3 la ae we e ed on suc ose-only ood o 20 h s a ing ei he
a 0 h AL3E o a 20 h AL3E and hen e u ned o s anda d ood un il
he end o he eeding pe iod (Figu e 2.1C). As desc ibed abo e, o a iole
numbe is se e ely educed when la ae we e ans e ed o suc ose-only
ood a 5 h AL3E (Figu e 2.1B, D). Su p isingly, o a iole numbe in la ae
ed on suc ose alone o a sho pe iod be ween 0 h o 20 h AL3E was
simila o s anda d ood con ol (Figu e 2.1D). In con as , when la ae
we e ed on suc ose-only ood om 20 h o 40 h AL3E, o a iole numbe
was signi ican ly educed (Figu e 2.1D). This educ ion in o a iole numbe
was simila when compa ed o la ae ans e ed o suc ose alone a 20 h
AL3E un il he end o de elopmen (Figu e 2.1B, D). These obse a ions
co obo a e a p e ious s udy whe e e- eeding p e-c i ical weigh la ae
a e a b ie pe iod o s a a ion delays pupa ia ion o longe han he
leng h o he s a a ion pe iod, bu does no a ec inal body size. A e
c i ical weigh , when he du a ion o he la al g ow h pe iod is ixed,
sho pe iods o s a a ion ha e no e ec on he iming o pupa ia ion
and hus, la ae a e unable o each hei op imal body size e en a e
e- eeding (Beadle e al., 1938).
O a iole numbe is posi i ely co ela ed wi h egg p oduc ion a e and
he e o e is closely ela ed o i ness (Boulé eau-Me le e al., 1982; R’ kha
e al., 1997; Klepsa el e al., 2013b,a). As expec ed, I ound ha emales
ha we e ed on suc ose-only ood as la ae a imed in e als s a ing
be ween 5 h o 25 h AL3E un il he end o he eeding pe iod laid ewe
eggs in he i s h ee days a e eclosion ela i e o s anda d ood con ol
(Figu e 2.2). When I plo ed o a iole numbe agains daily egg p oduc ion,
I ound ha di e ences in o a iole numbe co ela ed wi h di e ences
in he numbe o eggs laid (Figu e 2.2). Thus, hese esul s con i m
ha o a iole numbe is a good p oxy o ea ly emale ecundi y in D.
melanogas e .
30
Chap e 2
Figu e 2.2: O a iole numbe is posi i ely co ela ed wi h ea ly emale
ecundi y. Numbe o eggs laid was coun ed in he i s h ee days a e eclosion
(diamond: 1s day a e eclosion; squa e: 2nd day a e eclosion; ci cle: 3 d day a e
eclosion) om emales ed on s anda d ood as la ae (yellow symbols) and emales ed
on suc ose-only ood as la ae a imed in e als s a ing be ween 5 h o 25 h AL3E
(symbols wi h di e en shades o blue) un il he end o he eeding pe iod. Plo ed
alues ep esen means and e o ba s show 95% con idence in e als o means. L3:
hi d ins a la ae; AL3E: a e L3 ecdysis.
2.3.2 O a y de elopmen du ing L3 la al s ages
To u he unde s and how nu i ion egula es o a iole numbe , I i s
analysed o a y de elopmen in L3 la ae aised in s anda d ood. When
TFCs di e en ia e om he su ounding o a ian soma ic cells, hey
up egula e exp ession o he ansc ip ion ac o Eng ailed (En) (Pa el
e al., 1989). Thus, I used En as a ma ke o TFC di e en ia ion and TF
o ma ion.
Consis en wi h p e ious s udies, TFCs we e no obse ed in p e-c i ical
weigh o a ies ( om 0-10 h AL3E) (Figu e 2.3A, B) (God and Laski,
1995). A 15h AL3E, TFCs appea ed in he medial side o he o a y
and ew sho TFs we e isible (Figu e 2.3A, B). New TFCs con inued
o eme ge om he su ounding o a ian soma ic cells and g adually
in e cala ed in o o ming TFs. The o ma ion o new TFCs occu s in
a la e al di ec ion (Figu e 2.3A, B) (God and Laski, 1995; Sahu -Ba nola
e al., 1995, 1996). A he end o L3, all o he app oxima ely 18-22 TFs
31
Chap e 2
Figu e 2.3: O a y de elopmen du ing L3 la al s ages unde op imal
nu i ional condi ions.(A) Schema ic d awings ep esen ing o a y de elopmen in
L3 la ae ea ed in s anda d ood. Te minal ilamen s (TFs) a e ep esen ed as da k
g ey symbols. Axis a e p esen ed as A-P, an e io -pos e io ; D-V, do sal- en al; M-L,
medial-la e al. Pic u es show de eloping o a y du ing L3 la al s ages unde s anda d
ood. Eng ailed (g ey) ma ks e minal ilamen cells (TFCs). Scale ba : 20µm. (B)
Numbe o o ming e minal ilamen s (TFs). (C) O a y olume. Plo ed alues
ep esen means and e o ba s show 95% con idence in e als o means. L3: hi d
ins a la ae; AL3E: a e L3 ecdysis.
ha e o med (Figu e 2.3A, B) (God and Laski, 1995; Hodin and Riddi o d,
1998; Sa ikaya e al., 2012). To assess he dynamics o o a y g ow h, I
measu ed o a y olume o e de elopmen ime. O a y olume inc eased
exponen ially h oughou L3 la al de elopmen (Figu e 2.3C), con i ming
esul s p e iously ound in (Ke kis, 1931).
32
Chap e 2
modi ies he g ow h ajec o ies o he wing imaginal discs (Ga cia-Bellido
and Me iam, 1971; Ma in, 1982; B yan and Le inson, 1985). A e
a ainmen o c i ical weigh , wing discs ha e an in insic g ow h a e
ha p omo es conside able g ow h unde poo nu i ional condi ions. This
in insic g ow h is no p esen be o e c i ical weigh , and hus, wing discs
a es g ow h when p e-c i ical weigh la ae a e poo ly ed (Shingle on
e al., 2008). In ligh o hese obse a ions, I p opose ha de eloping
o a ies may also ha e an in insic g ow h a e a e c i ical weigh ha
allows p og ession o g ow h in poo ly- ed la ae.
2.5 Conclusions
The esul s desc ibed in his chap e e ealed ha c i ical weigh plays
a undamen al ole in ep og aming he de eloping o a y’s esponse o
nu i ion. Fu he mo e, his wo k con ibu es o a be e unde s anding
o he de elopmen al p ocesses ha egula e o a iole numbe , and p o ides
he de elopmen al ools ha will be used h oughou his hesis.
Acknowledgemen s
I would like o hank Ví o Fa ia and Nelson Ma ins o aking ca e o he
ou b ed popula ions o D. melanogas e . Ch is en Mi h o eaching me how o
coun o a ioles and o commen ing and p oo eading his chap e . Élio Sucena
o discussions h oughou his p ojec and o commen s on an ea ly e sion
o his chap e . The Uni o Imaging a IGC o all he assis ance du ing image
acquisi ion and analysis. The Fly Facili y, in pa icula Liliana Viei a, o making
ly ood e e y week.
39

Chap e 2
Supplemen a y Figu es
S 2.1: Eng ailed is exp essed in he wing discs o la ae ea ed in suc ose-
only ood. To con i m ha he absence o TFCs (Eng ailed-posi i e cells) was due
o he die manipula ion and no o he immunocy ochemis y p o ocol, I dissec ed
wing discs o cen al ne ous sys em (CNS) and analysed hem oge he wi h he la al
o a ies. Eng ailed is exp essed in he pos e io compa men o he wing disc and in he
neu oblas s in he CNS. Shown is a wing disc o la ae ed on suc ose-only ood om
5 h AL3E exp essing Eng ailed. Phalloidin ma ks F-ac in o ou line cell memb anes.
Scale ba : 20µm. L3: hi d ins a la ae; AL3E: a e L3 ecdysis.
40
Chap e 2
S 2.2: La al nu i ion a ec s de elopmen ime and emale weigh .((A)
De elopmen ime ep esen ed in hou s a e hi d ins a ecdysis (h AL3E) o
pupa ia ion. Wilcoxon ank es using Holm’s p- alue adjus men : ***p<0.001, ns
non-signi ican . (B) Female weigh. Welch Two sample - es using Holm’s p- alue
adjus men : ***p<0.001. La ae ed on s anda d ood (yellow ci cles) and la ae
ans e o suc ose-only ood ei he a 5h AL3E (ligh blue ci cles) o a 15h AL3E
(da k blue ci cles). Plo ed alues ep esen means and e o ba s show 95 con idence
in e als o means. L3: hi d ins a la ae; AL3E: a e L3 ecdysis.
41
Chap e 2
42
3
HORMONAL SIGNALLING
REGULATES PLASTICITY IN
OVARIOLE NUMBER IN
DROSOPHILA MELANOGASTER
“I mean, how could hey know ha because o hei li le dance he
wo ld li es? Bu i does. By simply doing wha hey’ e designed
o do, some hing la ge and magni icen happens.”
– om he ilm Adap a ion (2002)
43
Chap e 3
Abs ac
Ho mones coo dina e body and o gan g ow h wi h en i onmen al
condi ions. Se e al s udies ha e unco e ed ha changes in he iming
and amoun o ho mone p oduc ion a e associa ed o nume ous plas ic
esponses in mo phology, beha iou and physiology. Howe e , how
ho mones ac a speci ic s ages in de elopmen o modi y he sensi i i y
o a de eloping o gan o en i onmen al a ia ion is poo ly unde s ood.
In he p e ious chap e , I ha e shown ha c i ical weigh sepa a es wo
phases o sensi i i y o la al nu i ion in o a iole numbe . He e, I se ou
o es whe he wo ho monal signalling pa hways – he insulin/insulin-
like g ow h ac o signalling (IIS) and ecdysone signalling pa hways –
ac a c i ical weigh o con ol he esponse o he de eloping o a y o
nu i ional condi ions and hus, egula e he plas ic esponse o o a iole
numbe . Indeed, I ound ha bo h IIS and ecdysone signalling pa hways
change he de eloping o a y’s sensi i i y o nu i ion by egula ing dis inc
s age-speci ic de elopmen al p ocesses ha I ha e shown o be modi ied
by nu i ional condi ions. These esul s enhance ou unde s anding o he
s age-speci ic ac ion o ho mones in egula ing plas ic esponses.
Publica ion
Chap e 2 and 3 a e pa o a manusc ip submi ed o publica ion, au ho ed by
C.C. Mendes and C.K. Mi h.
Au ho s’ con ibu ions
Ch is en Mi h and Cláudia Mendes concei ed his s udy. Cláudia Mendes
pe o med and analysed all expe imen s and w o e his chap e .
44

Chap e 3
3.1 In oduc ion
En i onmen ally-induced pheno ypes a e o en induced by ho mone
signals ha in eg a e cues om he ex e nal en i onmen and coo dina e
de elopmen al p ocesses h oughou he whole o ganism. This in eg a ion
o en i onmen al in o ma ion is o en e lec ed in changes in he
iming and/o amoun o ho mone p oduc ion du ing speci ic s ages in
de elopmen . These changes in ho mone p oduc ion can hen a ec
he g ow h and di e en ia ion o de eloping issues and p oduce a wide
ange o dis inc mo phologies (Beldade e al., 2011; Koyama e al.,
2013; Nijhou , 2003a). Mo eo e , changes in he ac i i y o ho monal
pa hways ha e been shown o unde lie di e ences in nu i ional sensi i i y
be ween di e en o gans (Tang e al., 2011). Howe e , he e is s ill a
undamen al gap in ou unde s anding o how ho mones ac a speci ic
s ages in de elopmen o change he sensi i i y o a de eloping o gan o
en i onmen al a ia ion.
In he p e ious chap e , I ha e shown ha a key de elopmen al ansi ion,
c i ical weigh , appea s o ep og am he sensi i i y o he de eloping
o a y o nu i ional condi ions (see Chap e 2). While s a ing la ae
be o e c i ical weigh s ongly comp omises e minal ilamen cell (TFC)
di e en ia ion and o a y g ow h, s a a ion a e c i ical weigh has a
mo e modes e ec on o a y de elopmen ; ha is, o a y g ow h and he
o ma ion o new TFs con inue, al hough a slowe a es, in pos -c i ical
weigh la ae ha a e poo ly ed. Such changes in nu i ional sensi i i y
g ea ly in luence he plas ic esponse o o a iole numbe . In e es ingly,
wing disc de elopmen shows a simila change in nu i ional sensi i i y
a c i ical weigh (Mi h e al., 2009; Shingle on e al., 2008). This
swi ch in he nu i ional sensi i i y o he wing is egula ed by wo
ho monal pa hways, he insulin/insulin-like g ow h ac o signalling (IIS)
and ecdysone signalling pa hways.
A c i ical weigh , he p o ho acic glands p oduce a small peak o ecdysone
(Koyama e al., 2014). Once eleased, ecdysone binds o i s ecep o , a
he e odime be ween Ecdysone Recep o (EcR) and Ul aspi acle (Usp),
in he wing discs and ac i a es he ansc ip ion o a ge genes, allowing
45
Chap e 3
he p og ession o di e en ia ion o con inue e en in he absence o
nu i ion (Mi h e al., 2009). In addi ion, he g ow h o wing discs
becomes less sensi i e o nu i ion a e c i ical weigh (Shingle on e al.,
2008). Because IIS egula es body and o gan g ow h in esponse o
nu i ion, he pulse o ecdysone a c i ical weigh is hough o make he
g ow h o he disc less sensi i e o IIS (Koyama e al., 2013; S iepe e al.,
2008).
In e es ingly, bo h IIS and ecdysone signalling pa hways egula e o a iole
numbe (Gancz and Gilboa, 2013; Gancz e al., 2011; G een and Ex a ou ,
2012, 2014; Hodin and Riddi o d, 1998), and IIS is known o unde lie
he plas ic esponse o o a iole numbe o la al nu i ion (G een and
Ex a ou , 2014). These s udies epo ed ha manipula ing IIS in he
la al o a y a ec s o a y olume and o al TF numbe , bu has no e ec
on he iming o he onse o TFC di e en ia ion (Gancz and Gilboa,
2013; G een and Ex a ou , 2012, 2014). Rep essing ecdysone signalling in
he de eloping o a y delays TFC di e en ia ion and educes o a y olume
(Gancz e al., 2011; Hodin and Riddi o d, 1998). Howe e , hese s udies
ha e no explo ed he e ec s o IIS and ecdysone signalling pa hways wi h
su icien empo al esolu ion o dissocia e hei oles in egula ing TFC
di e en ia ion, he a e a which new TFs a e o med, o he a e o o a y
g ow h. Fu he mo e, he ela i e oles o IIS and ecdysone signalling
pa hways in al e ing de elopmen al p ocesses in esponse o nu i ion ha e
no been explo ed.
He e, I in es iga ed whe he IIS and ecdysone signalling pa hways ac
du ing c i ical weigh o egula e he de eloping o a y’s sensi i i y o
nu i ional condi ions, and ul ima ely, in luence he plas ic esponse
o he o a y. I i s examined he ela i e con ibu ions o each
signalling pa hway in egula ing h ee de elopmen al p ocesses du ing
o a y de elopmen : he onse o TFC di e en ia ion, he a e o TF
o ma ion, and he a e o o a y g ow h in well- ed la ae. I nex explo ed
whe he he nu i ional sensi i i y o hese de elopmen al p ocesses is
media ed by IIS and ecdysone signalling pa hway.
46
Chap e 3
3.2 Ma e ial and Me hods
3.2.1 Fly S ocks
To gene ically manipula e IIS and/o ecdysone signalling pa hways, I used
a ic jam-GAL4 ( j) [yw;PGawBNP1624/CyO; a gi o Lilach Gilboa]
o d i e exp ession in soma ic cells o he la al o a y (Gancz e al.,
2011). This line was c ossed o w1118 (used as a con ol; w1118, j o
j>), w; UAS EcRA.W650A TP3 (>EcRwDN), w; UAS EcR RNAi CA104
(>EcRi), yw;+;UAS PTEN (>PTEN), yw lp; +; UAS InR29.4 (>InR),
w; UAS EcRA.W650A TP3; UAS InR29.4 (>EcRi, InR) o w; UAS
EcRA.W650A TP3; UAS InR29.4 (>EcRDN, InR) . To cha ac e ize he
exp ession pa e ns o a ic jam-GAL4, he ollowing lines we e used: w;
+; UAS GFP (>GFP), w; a ic jam-GAL4; UAS GFP ( j>GFP), w;
ela -GAL4 (d i es exp ession in neu oblas and glial cells in he la al
b ain; ela >) and w; ela -GAL80, a ic jam-GAL4 (inhibi s a ic jam-
GAL4 exp ession in he neu oblas and glial cells in he la al b ain;
ela 80, j>). Fly s ocks we e main ained a 22°C in bo les on s anda d
ly ood (4.5% molasses, 7.2% suga , 7% co nmeal, 2% yeas ex ac , 1%
aga and 2.5% Nipagin solu ion).
Fo ecdysone eeding expe imen s, I used an ou b ed popula ion (wild
ype) o D. melanogas e es ablished in he labo a o y o D . Élio
Sucena in 2007, o igina ing om 160 e ilized emales collec ed in Azei ão,
Po ugal (Ma ins e al., 2013). The popula ion was kep in labo a o y
cages wi h high census (>1500 indi iduals) and main ained a cons an
empe a u e (25°C) on s anda d ly ood (4.5% molasses, 7.2% suga , 7%
co nmeal, 2% yeas ex ac , 1% aga and 2.5% Nipagin solu ion).
3.2.2 La al s aging, die a y manipula ions and ecdysone
eeding expe imen s
Adul s we e allowed o lay eggs o wo o six hou s on esh ood
pla es (60 ×15 mm Pe i dish). Egg densi y was con olled o p e en
o e c owding (app oxima ely 200 eggs pe pla e). To de e mine he e ec s
o supp essing IIS and/o ecdysone signalling pa hways in de eloping
47
Chap e 3
Figu e 3.1: Expe imen al design o nu i ional manipula ions.(A)
Expe imen al design o de e mine he e ec s o sup essing IIS o ecdysone signalling
on o a y de elopmen and o a iole numbe in la ae ed on s anda d ood (yellow line).
(B) A simila expe imen al design was pe o med o examine he e ec s o ac i a ing
IIS and/o pa ially ac i a ing ecdysone signalling on o a y de elopmen . In his se o
expe imen s, la ae we e ans e ed a 5 h AL3E ei he o s anda d ood (yellow line)
o suc ose-only ood (blue line). Dissec ion imes a e ma ked wi h ed c osses.L3: hi d
ins a la ae; AL3E: a e L3 ecdysis.
o a ies, la ae we e selec ed 0-2 hou s a e ecdysis o L3 (AL3E) and
ans e ed on o new pla es (40-60 la ae pe pla e) wi h s anda d ly
ood (s anda d ood) un il he ime o dissec ion (15 h, 29 h and 39 h
AL3E) (Figu e 3.1A). To de e mine he e ec s o ac i a ing IIS and/o
pa ially ac i a ing ecdysone signalling pa hways in de eloping o a ies,
la ae we e collec ed as abo e in 2 h in e als om ecdysis and ed on
s anda d ood. A 5 h AL3E, la ae we e ans e ed o ials (20-30 la ae
pe ial) con aining ei he 20% suc ose on 0.5% aga medium (suc ose-only
ood) o s anda d ood. The suc ose-only ood allowed mos la ae o
su i e un il pupa ia ion and adul eclosion. La al o a ies we e dissec ed
a ou ime poin s (5 h, 15 h, 29 h and 39 h AL3E) (Figu e 3.1B).
Fo ecdysone eeding expe imen s, I supplemen ed 0.15 mg/mL o he
ac i e ecdysone me aboli e 20-hyd oxyecdysone (20E; SciTech Chemicals,
Dej ice-Hanspaulka, Czech Republic) o 1 g o ei he s anda d ood o
suc ose-only ood. The ood was hen well mixed and spun down a day
be o e use. Newly ecdysed L3 la ae we e collec ed as abo e and ed on
48
Chap e 3
I i s analysed o a y de elopmen in j>PTEN la ae ea ed in s anda d
ood condi ions. O a ies om j>PTEN la ae showed a mode a e delay
in he onse o TFC di e en ia ion (Figu e 3.5A, B). Fu he , bo h a e
o TF o ma ion and he a e o o a y g ow h we e g ea ly educed in
j>PTEN o a ies when compa ed o con ol o a ies (w1118; j) (Figu e-
3.5A-A”, B-B”, D, E). Con e sely, ac i a ing IIS in o a ian soma ic cells
by o e exp essing Insulin Recep o ( j>InR) did no a ec he iming o
TFC di e en ia ion (Figu e-3.5A, C), al hough i inc eased he a e o TF
o ma ion and o o a y g ow h (Figu e 3.5A-A”, C-C”, D, E). O e all,
hese esul s sugges ha IIS egula es all h ee de elopmen al p ocesses
in well- ed condi ions.
I nex sough o es whe he ac i a ion o IIS was su icien o o e come
he e ec s o poo nu i ion in la ae ed on suc ose alone. In con ol
la ae ed on suc ose, I ailed o de ec any TFCs be o e 39 h AL3E
(Figu e 3.5F’, F”). In con as , I de ec ed TFCs in j>InR o a ies om
la ae ed on suc ose al eady a 15 h AL3E (Figu e 3.5G’). Such iming
o TFC onse in suc ose ed j>InR la ae was simila o ha o well- ed
con ols (Figu e 3.5A, C, G’, H) and new TFs we e s ill o ming a 39 h
AL3E (Figu e 3.5G”, H).
The a e o TF o ma ion was signi ican ly highe in j>InR la ae ed on
suc ose alone han in simila ly ea ed con ol la ae. Fu he , a he ime
o ans e o suc ose-only ood (5 h AL3E), he o a y olume in j>InR
la ae was signi ican ly bigge han ha o o a ies om con ol la ae
(p<0.001; K uskal-Wallis es ). Howe e , when j>InR la ae we e ed on
suc ose-only ood be ween 5 and 39 h AL3E, o a y g ow h was comple ely
a es ed (Figu e 3.5I). In summa y, ac i a ion o IIS in o a ian soma ic
cells o poo ly- ed la ae is su icien o induce a p ecocious onse o TFC
di e en ia ion and o accele a e he a e o TF o ma ion, bu has no e ec
on o a y g ow h.
3.3.3 Role o ecdysone signalling du ing o a y de elopmen
My p e ious manipula ions o la al nu i ion e ealed ha he onse o
TFC di e en ia ion is highly sensi i e o changes in nu i ion du ing he
55

Chap e 3
Figu e 3.5: Role o IIS du ing o a y de elopmen . (A-G”) Shown is e minal
ilamen s (TFs) ma ked wi h En (g ey).(A-C”, F, G) O a ies om la ae ea ed
on s anda d ood: (A-A”, F) w1118, j (con ol), (B-B”) j>PTEN and (C-C”, G)
j>InR.(D) Numbe o o ming TFs and (E) o a y olume o o a ies om la ae ea ed
on s anda d ood. (F’-F”, G’-G”) O a ies om la ae ans e ed o suc ose-only ood
a 5 h AL3E: (F’-F”) w1118, j (con ol) and j>InR. (F’-F”).(H) Numbe o o ming
TFs and (I) o a y olume o o a ies om la ae ed on suc ose-only ood. n ≥8 o a ies
o all geno ypes. Plo ed alues ep esen means and e o ba s show 95% con idence
in e als o means. In some cases, e o ba s a e oo small o be seen. ANCOVAs:
**p<0.01, ***p<0.001, ns non-signi ican . L3: hi d ins a la ae; AL3E: a e L3
ecdysis. Scale ba : 20µm.
56
Chap e 3
p e-c i ical weigh phase. Once TFC di e en ia ion is ini ia ed a ound
he iming o c i ical weigh , TF o ma ion p oceeds a a educed a e in
poo ly- ed la ae (see Chap e 2). C i ical weigh i sel is egula ed by a
small nu i ion-sensi i e ecdysone peak ha occu s a a ound 8 h AL3E
(Koyama e al., 2014; Mi h e al., 2005; Wa en e al., 2006). Mo eo e ,
bo h EcR and Usp a e exp essed in o a ian soma ic cells du ing L3 la al
s ages (Gancz e al., 2011; Hodin and Riddi o d, 1998). Thus, I easoned
ha ecdysone is likely o induce TFC di e en ia ion.
Unde s anda d ood condi ions, con ol o a ies (w1118, j) showed TFCs
and a ew o ming TFs a 15 h AL3E (Figu e 3.6A-A”, D). Howe e , I
did no de ec any TFCs in j>EcRDN o a ies om well- ed la ae un il
39 h AL3E (Figu e 3.6B-B”, D). Acco dingly, he a e o TF o ma ion
was se e ely educed in j>EcRDN o a ies (Figu e 3.6D). Thus, ecdysone
signalling is necessa y o induce he imely onse o TFC di e en ia ion
and p omo e subsequen o ma ion o new TFs. In addi ion, I ound ha
he a e o o a y g ow h was signi ican ly educed in j>EcRDN o a ies
(Figu e 3.6E), sugges ing ha basal le els o ecdysone a e likely equi ed
o p omo e o a y g ow h.
I hen es ed whe he a pa ial ac i a ion o ecdysone signalling in o a ian
soma ic cells is su icien o induce he onse o TFC di e en ia ion, o
inc ease he a e o TF o ma ion, and p omo e o a y g ow h in p e-
c i ical weigh la ae ed on suc ose-only ood. To do his, I used he
a ic jam-GAL4 d i e line o o e exp ess an RNAi cons uc agains
EcR unde he con ol o UAS ( j>EcRi). In well- ed condi ions, j>EcRi
o a ies showed simila iming in hei onse o TFC di e en ia ion as well
as a e o TF o ma ion ela i e o con ols (Figu e 3.6A, C, D). The
a e o o a y g ow h was educed in j>EcRi o a ies (Figu e 3.6E). When
j>EcRi la ae we e ed on suc ose-only ood be ween 5 and 15 h AL3E,
mos o a ies had TFCs (Figu e 3.6G’, H). In con as , TFCs we e no
de ec able in con ol (w1118, j) la ae ed on suc ose-only ood un il 39 h
AL3E (Figu e 3.6F’, H). Also, he a e o TF o ma ion, bu no o o a y
g ow h, inc eased in j>EcRi la ae ed on suc ose alone (Figu e 3.6H, I).
Toge he , hese esul s indica e ha knocking down EcR in he o a ian
soma ic cells o la ae ed on suc ose alone is su icien o induce p ecocious
57
Chap e 3
Figu e 3.6: Role o ecdysone signalling du ing o a y de elopmen . (A-G”)
Shown is e minal ilamen s (TFs) ma ked wi h En (g ey).(A-C”, F, G) O a ies om
la ae ea ed on s anda d ood: (A-A”, F) w1118 , j (con ol), (B-B”) j>EcRDN and
(C-C”, G) j>EcRi.(D) Numbe o o ming TFs and (E) o a y olume o o a ies om
la ae ea ed on s anda d ood. (F’-F”, G’-G”) O a ies om la ae ans e ed o
suc ose-only ood a 5 h AL3E: (F’-F”) w1118 , j (con ol) and (G’-G”) j>EcRi. (H)
Numbe o o ming TFs and (I) o a y olume o o a ies om la ae ed on suc ose-only
ood. n ≥8 o a ies o all geno ypes. Plo ed alues ep esen means and e o ba s
show 95% con idence in e als o means. In some cases, e o ba s a e oo small o be
seen. ANCOVAs: **p<0.01, ***p<0.001, ns non-signi ican . L3: hi d ins a la ae;
AL3E: a e L3 ecdysis. Scale ba : 20µm.
58
Chap e 3
onse o TFC di e en ia ion and accele a e he a e o TF o ma ion when
compa ed o simila ly ea ed con ols. Howe e , knocking down EcR in
ei he s anda d o suc ose-only condi ions educes o a y g ow h.
Knocking down EcR in he la al o a ies induces he de ep ession, bu
no he ac i a ion unc ion o ecdysone signalling (B own e al., 2006;
Schubige and T uman, 2000; Schubige e al., 2005). To in es iga e he
ull ole o ecdysone signalling in egula ing o a iole numbe plas ici y, I
ed wild- ype la ae om 5 o 29 hou s AL3E on ei he s anda d ood o
suc ose-only ood supplemen ed wi h 0.15 mg/mL o he ac i e ecdysone
me aboli e 20-hyd oxyecdysone (20E). Adding 20E o he s anda d ood
had no e ec on TF numbe (Figu e 3.7A, C, E). Howe e , la ae ed
on 20E-supplemen ed suc ose-only ood had signi ican ly mo e TFs a 29
hou s AL3E han la ae ed on suc ose-only ood plus sol en (e hanol)
(Figu e 3.7B, D, E). Mo eo e , o a y olume signi ican ly inc eased in
la ae ed on bo h s anda d and suc ose-only oods con aining 20E ela i e
o e hanol con ols (Figu e 3.7F). This expe imen con i ms ha ecdysone
is su icien o induce TFC di e en ia ion when p e-c i ical weigh la ae
a e ed on suc ose alone. Mo eo e , i also sugges s ha ecdysone egula es
o a y g ow h h ough i s ac i a ion unc ion.
3.3.4 The in e play be ween IIS and ecdysone signalling
pa hways
Bo h IIS and ecdysone signalling pa hways egula e he onse o TFC
di e en ia ion, he a e o TF o ma ion, and he a e o o a y
g ow h, sugges ing ha nu i ion in luences o a iole numbe h ough bo h
signalling pa hways. Howe e , he ela i e con ibu ion o hese signalling
pa hways in egula ing each de elopmen p ocess appea s o be di e en ,
wi h ecdysone signalling playing a mo e p ominen ole in egula ing TFC
di e en ia ion and IIS con ibu ing mo e o he a es o o a y g ow h.
To es his hypo hesis, I i s up egula ed IIS, using InR, while inhibi ing
ecdysone signalling, ia EcRDN, in o a ian soma ic cells ( j>EcRDN, InR)
o well- ed la ae. O a ies om j>EcRDN, InR la ae delayed he onse o
TFC di e en ia ion and educed he a e o TF o ma ion when compa ed
59
Chap e 3
Figu e 3.7: Feeding wild- ype la ae wi h 20E-supplemen ed suc ose-only
ood inc eases TF numbe and o a y olume. (A-D) Shown is e minal ilamen s
(TFs) ma ked wi h En (g ey). O a ies om la ae ea ed on s anda d ood: (A) plus
e hanol (con ol) o (C) plus 20E (+20E). O a ies om la ae ea ed on suc ose-only
ood: (B) plus e hanol (con ol) o (D) plus 20E (+20E). La ae we e dissec ed a
29 h AL3E. Scale ba : 20µm. (E) Numbe o o ming TFs and (F) o a y olume
o o a ies om la ae ed ei he on s anda d ood plus e hanol (con ol) o on 20E-
supplemen ed s anda d ood (+20E) (yellow poin s) and la ae ed ei he on suc ose
alone plus e hanol (con ol) o on 20E-supplemen ed suc ose-only ood (+20E) (blue
poin s). Plo ed alues ep esen means and e o ba s show 95% con idence in e als
o means. In some cases, e o ba s a e oo small o be seen. Welch Two sample - es :
*p<0.1, ***p<0.001, ns non-signi ican .
60

Chap e 3
o con ol la ae (w1118; j) (Figu e 3.8A-A”, 3.8B-B”, D). Thus, o
induce TFC di e en ia ion, IIS equi es ecdysone signalling o be in ac .
Ne e heless, he a e o o a y g ow h inc eased ela i e o con ol and
j> EcRDN la ae (Figu e 3.8E and Table 3.1). Taken oge he , hese
esul s demons a e ha ecdysone signalling is essen ial in egula ing he
iming o he onse o TFC di e en ia ion. Fu he mo e, hese esul s also
show ha IIS can o e come he g ow h de ec s a ising om dis up ing
ecdysone signalling, sugges ing ha IIS plays a p ima y ole in con olling
o a y g ow h.
I nex es ed he e ec s o up egula ing bo h signalling pa hways on each
de elopmen al p ocess. I pa ially ac i a ed ecdysone signalling, using
EcRi, while up egula ing IIS, wi h InR, in o a ian soma ic cells ( j>EcRi,
InR). In e es ingly, TFCs we e obse ed a 5 h AL3E in o a ies om
j>EcRi, InR ed on s anda d ood (Figu e 3.8G). This onse o TFC
di e en ia ion was no only ea lie han ha o con ol la ae (w1118; j)
(Figu e 3.8), i was also signi ican ly ea lie han he onse o TF o ma ion
in j>InR and j>EcRi o a ies (p<0.0001, χ2= 45, d = 3, Chi-Squa e
Tes ) (Figu e 3.5G and Figu e 3.6G). The a e o TF o ma ion and o
o a y g ow h was as e in o a ies om j>EcRi, InR la ae han o a ies
om ei he con ol o j>EcRi la ae (Figu e 3.8D, E and Table 3.2).
Rema kably, e en hough o a ies g ew a he same a e in bo h j>EcRi,
InR and j>InR la ae, he a e o TF o ma ion was as e in j>EcRi,
InR o a ies (Table 3.2).
In summa y, ac i a ing bo h IIS and ecdysone signalling pa hways in
o a ian soma ic cells o well- ed la ae induced an ea lie onse o TFC
di e en ia ion, and p omo ed a g ea e inc ease in he a e o TF
o ma ion han ac i a ing each signalling pa hway indi idually (Table 3.1
and 3.2). This led me o hypo hesize ha ac i a ing bo h signalling
pa hways may o e come mos o he e ec s o poo nu i ion. When
j>EcRi, InR la ae we e ed on suc ose-only ood be ween 5 and 15
hou s AL3E, he a e o TF o ma ion was signi ican ly as e han he
con ol (w1118; j) o a ies (Figu e 3.8H). In ac , he a e o TF o ma ion
in o a ies om j>EcRi, InR la ae was as e han all p e ious gene ic
manipula ions in poo ly ed la ae (Table 3.3). While a 5 hou s AL3E
61
Chap e 3
Figu e 3.8: The in e play be ween IIS and ecdysone signalling pa hways.
(A-G”) Shown is e minal ilamen s (TFs) ma ked wi h En (g ey).(A-C”, F, G) O a ies
om la ae ea ed on s anda d ood: (A-A”, F) w1118, j (con ol), (B-B”) j>EcRDN,
InR and (C-C”, G) j>EcRi, InR.(D) Numbe o o ming TFs and (E) o a y olume
o o a ies om la ae ea ed on s anda d ood. (F’-F”, G’-G”) O a ies om la ae
ans e ed o suc ose-only ood a 5 h AL3E: (F’-F”) w1118 , j (con ol) and (G’-G”)
j>EcRi, InR. (H) Numbe o o ming TFs and (I) o a y olume o o a ies om la ae
ed on suc ose-only ood. n ≥8 o a ies o all geno ypes. Plo ed alues ep esen
means and e o ba s show 95% con idence in e als o means. In some cases, e o ba s
a e oo small o be seen. ANCOVAs: **p<0.01, ***p<0.001, ns non-signi ican . L3:
hi d ins a la ae; AL3E: a e L3 ecdysis. Scale ba : 20µm.
62
Chap e 3
he o a y olume o j>EcRi, InR o a ies was bigge han he con ol
(p<0.001, K uskal-Wallis es ), I did no obse e any u he inc ease
in o a y olume a e ans e ing j>EcRi, InR la ae o suc ose-only
ood (Figu e 3.8I). Toge he , hese esul s show ha , in poo nu i ional
condi ions, ac i a ing bo h IIS and ecdysone signalling pa hways in
o a ian soma ic cells inc eases he a e o TF o ma ion, bu no o a y
g ow h.
Table 3.1: Pai wise compa isons o he a e o o a y g ow h in la ae ed
on s anda d ood. Shown a e p- alues (** p<0.01, ***p<0.001, ns non-signi ican ;
ANCOVAs using Holm’s p- alue adjus men ) o each pai wise compa ison.
Geno ypes j>EcRDN, InR j>EcRi, InR
w1118, j 0.0001 *** 0.0001 ***
j>EcRDN 0.0014 ** -
j>EcRi - 0.0007 ***
j>InR 0.0014 ** 0.8450 ns
j>EcRi, InR 0.0005 *** -
Table 3.2: Pai wise compa isons o he a e o TF o ma ion in la ae ed
on s anda d ood. Shown a e p- alues (** p<0.01, ***p<0.001, ns non-signi ican ;
ANCOVAs using Holm’s p- alue adjus men ) o each pai wise compa ison.
Geno ypes j>EcRDN, InR j>EcRi, InR
w1118, j 0.0026 ** 0.0029 **
j>EcRDN 0.0055 ** -
j>EcRi - 0.0055 **
j>InR 0.0030 ** 0.0055 **
j>EcRi, InR 0.0029 ** -
Table 3.3: Pai wise compa isons o he a e o TF o ma ion in la ae ed on
suc ose alone. Shown a e p- alues (** p<0.01, ***p<0.001, ANCOVAs using Holm’s
p- alue adjus men ) o each pai wise compa ison.
Geno ypes j>EcRi, InR
w1118, j 0.0001 ***
j>EcRi 0.0017 **
j>InR 0.0017 **
63
Chap e 3
3.4 Discussion
Ho monal egula ion unde lies mos , i no all, well-s udies cases
o de elopmen al plas ici y (Beldade e al., 2011). Howe e , how
ho mones change an o gan’s esponse o en i onmen al condi ions o e
de elopmen al ime and how his in luences hei plas ic esponse a e
poo ly unde s ood. In his chap e , I p o ide e idence ha wo ho monal
signalling pa hways – he IIS and ecdysone signalling pa hways – ac
du ing c i ical weigh o change he sensi i i y o he de eloping o a y o
nu i ional condi ions and hus, egula e he plas ic esponse o o a iole
numbe .
In he p e ious chap e , I ha e shown ha p e-c i ical weigh la ae ed on
suc ose-only ood delay he onse o TFC di e en ia ion (see Chap e 2).
Bo h IIS and ecdysone signalling pa hways egula e he onse o TFC
di e en ia ion; al e ing ei he IIS o ecdysone signalling in he o a y
al e ed he iming o he onse o TFC di e en ia ion. Howe e , he
e ec s o IIS on he onse o TFC di e en ia ion depended on ecdysone
signalling. O a ies in which IIS was up egula ed while ecdysone signalling
was ep essed delayed he onse o TFC di e en ia ion as much as o a ies
in which only ecdysone signalling was ep essed.
E en hough ecdysone signalling was equi ed o induce he onse o
TFC di e en ia ion, he wo pa hways appea o in e ac in a complex
manne o egula e his p ocess. Simul aneously up egula ing bo h IIS
and ecdysone signalling in he o a y esul ed in ea lie onse o TFC
di e en ia ion han up egula ing ei he pa hway on i s own. Po en ially,
nu i ion, ia IIS, may modi y he sensi i i y o he o a y o ecdysone
signalling. Unde high le els o IIS, he o a y may equi e lowe le els o
ecdysone signalling o induce he onse o TFC di e en ia ion, esul ing
in ea lie onse . Fu he s udies will be equi ed o ully unde s and
he na u e o he link be ween IIS and ecdysone signalling in his
de elopmen al p ocess.
Addi ionally, my esul s sugges ha unliganded EcR/Usp ep esses genes
in ol ed in he onse o TFC di e en ia ion. Knocking down EcR in
he o a y elimina es he ep essi e unc ion o he ecep o , and induces
64
Chap e 4
4.1 In oduc ion
In ecen yea s, i has become inc easingly clea ha he en i onmen
no only selec s among pheno ypes, bu i also al e s de elopmen al
ajec o ies, some imes in p o ound ways, o gene a e as onishing a ia ion
in mo phology, beha iou , physiology and li e his o y (Beldade e al.,
2011; P ennig e al., 2010; S ea ns, 1989). Such en i onmen ally-induced
ai s may allow he pe sis ence o a popula ion in a no el and challenging
en i onmen , and can g adually become s abilized and in eg a ed wi h
he es o he pheno ype by means o gene ic accommoda ion (Wes -
Ebe ha d, 2003). A possible ou come om such adap i e e inemen o
en i onmen ally-induced pheno ypes is ha he de elopmen al p ocesses
ha gene a e pheno ypic a ia ion among popula ions should be o some
deg ee simila o he de elopmen al changes ha eme ge as a esponse o
en i onmen al a ia ion (Wund, 2012).
The ema kable plas ic capaci y, pai ed wi h a subs an ial gene ic
a ia ion bo h wi hin and be ween species, makes o a iole numbe in
D osophila an exci ing model o add ess his issue. In p e ious chap e s,
I examined how la al nu i ion shapes o a iole numbe in an ou b ed
popula ion o D. melanogas e . A de ailed cha ac e iza ion o o a y
de elopmen du ing hi d ins a (L3) la al s ages e ealed ha nu i ion
in luences o a iole numbe by al e ing h ee dis inc de elopmen al
p ocesses du ing o a y de elopmen : i) he onse o e minal ilamen cell
(TFC) di e en ia ion, which ma ks he beginning o o a iole de elopmen ,
ii) he a e a which new e minal ilamen s (TF) a e o med, and iii) he
a e o o a y g ow h. Impo an ly, he nu i ion-dependen de elopmen al
ansi ion, c i ical weigh , al e s he ypes o de elopmen al p ocesses
nu i ion can a ec : while s a ing la ae be o e c i ical weigh delays he
onse o TFC di e en ia ion and o a y g ow h, changes in nu i ion a e
c i ical weigh educe he a es o o a y g ow h and o TF o ma ion. He e,
I sough o explo e whe he changes in one o all o he h ee de elopmen al
p ocesses a e esponsible o he di e gence in o a iole numbe among
D osophila species.
The in a/in e speci ic a ia ion in o a iole numbe is co ela ed wi h he
coloniza ion o di e se ecological niches. In gene al, D osophila species
71

Chap e 4
ha eed on a na ow ange o ood sou ces ha e ewe o a ioles han
species wi h a gene alis die (G een and Ex a ou , 2014; Kambysellis
and Heed, 1971). Mo eo e , o a iole numbe shows subs an ial la i udinal
and al i udinal clinal a ia ion on di e en con inen s, p o iding u he
e idence ha o a iole numbe is unde selec ion (Capy e al., 1993; Gibe
e al., 2004; Wayne e al., 2005).
The eple a g oup is one o he la ges species g oup in D osophila genus
comp ising o e 100 species wi h s iking mo phological and beha iou al
di e si y (Du ando e al., 2000; O’G ady and Ma kow, 2012; Smi h e al.,
2012). Pa icula ly in e es ing is he cac ophilic D. moja ensis, which
has eme ged as an impo an sys em o unde s anding he gene ic basis
o local adap a ion, p o iding signi ican insigh s in o he ea ly e en s
associa ed wi h ecological specia ion. D. moja ensis is ound in ou
geog aphically isola ed a eas in he a id egions o sou hwes e n USA
and no hwes e n Mexico and u ilizes nec o ic issues o dis inc local
hos cac us o bo h eeding and b eeding (Figu e 4.1). In he mainland
Sono an Dese , D. moja ensis uses he o gan pipe cac us, S enoce eus
hu be i, and occasionally sha es he columna cina cac us, S. alamosensis,
wi h sis e species D. a izonae. Al hough o gan pipe cac us is abundan in
Baja Cali o nia, D. moja ensis u ilizes almos exclusi ely he pi aya ag ia
cac us, S. gummosus, in his egion. The popula ion in Moja e Dese
uses ed ba el cac us, Fe ocac us cylind aceous, while on San a Ca alina
Island, whe e columna cac i a e absen , he p ickly pea cac us species,
Opun ia demissa and O. li o alis, se e as he hos (Fellows and Heed,
1972a; Ruiz and Heed, 1988).
The di e en cac i used by each popula ion di e in a a ie y o chemical
compounds, including ee a y acids and s e ols, and in he bac e ial and
yeas communi ies ha colonize and decompose he cac us issue in o ood
o hese lies (Fogleman and S a me , 1985; Fogleman e al., 1981; S a me
and Fogleman, 1986). Besides di e ences in chemical composi ion, he
o al size o he plan and hei nec oses also a y among cac us hos s.
La ge cac i, such as he o gan pipe cac us, con ain s able nec oses o
la ge dimensions, whe eas in small cac i, such as Opun ia sp., nec oses
a e small and epheme al (B ei meye and Ma ko w, 1998). Each cac us
72
Chap e 4
Figu e 4.1: Dis ibu ion and cac us hos use o he ou D. moja ensis
subspecies. Map o he sou hwes e n Uni ed S a es and no hwes e n Mexico showing
he geog aphical dis ibu ions o D. moja ensis baja,D. moja ensis w igleyi,D.
moja ensis moja ensis and D. moja ensis sono ensis and hei espec i e hos cac us
(adap ed om (P eile e al., 2009)).
hus o e s a dis inc ecological niche o he esiden lies, which may d i e
gene ic di e gence be ween popula ions o D. moja ensis.
S udies using cy ological and molecula da a ha e clea ly shown s ong
gene ic di e en ia ion be ween he ou popula ions. Ye , he e olu iona y
ela ionships be ween hem, and which popula ion is ances al, ha e
p o en di icul o esol e (Machado e al., 2007; Reed e al., 2007;
Ruiz e al., 1990; Smi h e al., 2012). In addi ion o gene ic a ia ion,
se e al physiological, mo phological, and beha iou al adap a ions ha e
been epo ed be ween popula ions, which has led o hem being ecen ly
desc ibed as subspecies: D. moja ensis baja,D. moja ensis sono ensis,
D. moja ensis moja ensis, and D. moja ensis w igleyi (Figu e 4.1) (Da e
e al., 2013; E ges e al., 2010; P eile e al., 2009; Richmond e al., 2012).
P e ious s udies ha e desc ibed ha o a iole numbe shows mino
a ia ions (i.e. o a iole numbe anges om 25 o 33) among popula ions
73
Chap e 4
o D. moja ensis collec ed om he ield o ea ed in labo a o y cac us
(Ma kow, 1996). I he e o e es ed whe he o a iole numbe a ies
be ween subspecies o D. moja ensis ea ed unde s anda d labo a o y
condi ions. I con i med ha o a iole numbe a ies conside ably be ween
he ou D. moja ensis subspecies. This inding p omp ed me o ocus
on wo D. moja ensis subspecies ha display he g ea es di e gence
in o a iole numbe , D. moj. sono ensis and D. moj. w igleyi. I i s
examined whe he di e ences in o a iole numbe we e associa ed wi h
changes in h ee li e his o y ai s: emale ecundi y, emale body size,
and de elopmen al ime. Nex , I compa ed he o a y de elopmen in each
subspecies o es whe he de elopmen al p ocesses known o egula e
o a iole numbe in esponse o nu i ion also unde lie di e ences in
o a iole numbe be ween he wo subspecies. Finally, I in es iga ed how
en i onmen al and gene ic a ia ion shape o a iole numbe in he wo D.
moja ensis subspecies.
4.2 Ma e ial and Me hods
4.2.1 Species s ocks
The ollowing species we e ob ained om he D osophila Species S ock
Cen e (Uni e si y o Cali o nia, San Diego), wi h he excep ion o one D.
a izonae line which was kindly p o ided by Janelia Fa m Resea ch Campus
(Ashbu n, Vi ginia): D. a izonae,D. moja ensis baja,D. moja ensis
moja ensis,D. moja ensis sono ensis,D. moja ensis w igleyi and D.
mulle i (Table 4.1). Fly s ocks we e main ained a 22°C in bo les on
s anda d ly ood (4.5% molasses, 7.2% suga , 7% co nmeal, 2% yeas
ex ac , 1% aga and 2.5% Nipagin solu ion).
4.2.2 La al s aging
Adul s we e allowed o o iposi o 4-6 hou s on esh pla es (60 ×15
mm Pe i dish) con aining s anda d ly ood. O en, emales would lay
mul iple eggs in e y close p oximi y. When I con olled o o e c owding
74
Chap e 4
Table 4.1: Species s ocks used in his s udy. S ock numbe s om D osophila
S ock Cen e (DSSC) wi h expec ion o one D. a izonae line om Janelia Fa m Resea ch
Campus (JFRC).
Species/Subspecies S ock Numbe
D. a izonae JFRC
D. a izonae 15081-1271.29
D. moj. baja 15081-1351.30
D. moj. baja 15081-1352.34
D. moj. moja ensis 15081-1352.00
D. moj. moja ensis 15081-1352.01
D. moj. sono ensis 15081-1352.26
D. moj. sono ensis 15081-1352.32
D. moj. w igleyi 15081-1352.14
D. mulle i 15081-1371.01
D. mulle i 15081-1379.30
by pe o ming he same p o ocol as desc ibed in Chap e s 2 and 3, his
would a ou bac e ial con amina ion du ing he i s and second la al
ins a , as la ae ended o agg ega e in la ge g oups while o aging and
bu owing in o a small egion o he ood pla e. As la al densi y was
shown o in luence de elopmen al ime and ho ax leng h in di e en
popula ions o D. moja ensis (E ges and Heed, 1987), I con olled la al
densi y a se e al ime poin s h oughou la al de elopmen o a oid
bo h o e c owding and bac e ia con amina ions. This was done by cu ing
he ood con aining la ae in o smalle pieces e e y o he day and gen ly
ans e ing hem o esh ood pla es (60 ×15 mm Pe i dish). A e egg
laying (AEL), pla es we e main ained a 25°C in a 12 h ligh -da k cycle
wi h 70% humidi y. In hese condi ions, bo h D. moj. sono ensis and
D. moj w igleyi exhibi simila gene a ion imes (15-16 days om egg o
adul ). A day 6 AEL, la ae we e selec ed 0-2 hou s a e ecdysis o L3
(AL3E) and ans e ed on o new ood pla es (40-60 la ae pe pla e) un il
he ime o dissec ion [0 h, 20 h, 40 h, 60 h, 80 h AL3E and a pupa ia ion
(D. moj. sono ensis, 110 h AL3E; D. moj. w igleyi, 104 h AL3E)]. Each
ime poin was eplica ed a leas wice.
75
Chap e 4
4.2.3 Immunocy ochemis y, imaging and analysis
Immunocy ochemis y was pe o med as p e iously desc ibed (see
Chap e 2). The p ima y an ibody mouse an i-Eng ailed (De elopmen al
S udies Hyb idoma Bank 4D9, 1:40) was used o iden i y e minal ilamen
cells (TFCs). The ollowing seconda y eagen s we e used: Alexa 568
(In i ogen, 1:200) and TRICT-Phalloidin (Sigma, 1:200). Samples
we e moun ed on a poly-L-lysine-coa ed co e slip using Fluo omoun -
G (Sou he nBio ech) and imaged using a Zeiss LSM 510 Me a con ocal
mic oscope. Measu emen s o o al numbe o o ming e minal ilamen s
(TFs) and o a y olume we e pe o med as p e iously in Chap e 2 and 3
desc ibed using ImageJ (NIH) and Adobe Pho oshop (Adobe Sys ems).
4.2.4 Die a y manipula ions
Well- ed lies we e allowed o deposi eggs o e nigh on pla es con aining
s anda d ly ood. Egg lays we e epea ed in wo independen days
wi h di e en pa en al lies o minimize possible pa en al e ec s on la al
de elopmen . Eggs we e collec ed and g oups o 50 eggs we e andomly
ans e ed o ials con aining one o he ollowing ood concen a ions:
s anda d ly ood (100%), s anda d ly ood dilu ed wi h 1% aga in a
a io o 1:2 (50%), 1:4 (25%) o 1:8 (12.5%). The o al olume o ood
was he same in all ood concen a ions. Fou eplica es we e pe o med
o each ood concen a ion and subspecies. De eloping la ae we e ea ed
a 25°C in a 12 h ligh -da k cycle wi h 70% humidi y un il all indi iduals
eclosed o died. Eclosed lies we e ans e ed o esh ials wi h s anda d
ly ood.
4.2.5 Expe imen al c osses o gene a e F1 and F2 hyb ids
Ten i gin emales om D. moj. w igleyi we e g ouped wi h i e males
om D. moj. sono ensis in ials on s anda d ood (n=5 ials). The
ecip ocal c oss was also made, bu only ou eplica es laid e ile eggs
(n=4 ials). Once emales we e sexually ma u ed (a ound 8-10 days o
age), hey we e le o lay eggs in ood ials o 24 h. O e c owding
76

Chap e 4
was con olled as p e iously men ioned. This p ocedu e was epea ed in
h ee independen days o maximize he numbe o o sp ing. Eclosed F1
hyb ids om each eplica e we e g ouped ( en emales and i e males pe
ial) in esh ials un il sexually ma u e. F1 hyb ids we e hen le o lay
eggs using he same p ocedu e as desc ibed abo e. A subse o F1 hyb id
emales we e ozen a e 8-10 days o o a iole numbe coun s. A simila
p o ocol was implemen ed o ob ain o a ies om F2 hyb id emales.
4.2.6 Measu emen s o li e his o y ai s: de elopmen al
ime, adul body size, o a iole numbe and emale
ecundi y
The a e age ime o pupa ia ion was measu ed by ans e ing newly
ecdysed L3 la ae o ood ials (10-20 la ae pe ial) and coun ing he
numbe o la ae pupa ia ing (immobile la ae wi h e agina ed spi acles)
e e y h ee hou s on day 10-11 AEL. To quan i y adul body size, pha a e
adul s we e indi idually weighed on a Sa o ius SE2 ul amic obalance.
Males o D. moja ensis lack sex combs, so I was unable o dis inguish males
om emales a his s age. A e weighing, I kep each pha a e adul inside
an eppendo un il eclosion and emales and males we e iden i ied based
on hei ex e nal geni alia. O a iole numbe was coun ed o bo h o a ies
om ma ed emales (8-10 days o age) as p e iously desc ibed (see Chap e
2). To de e mine emale ecundi y in each subspecies, I implemen ed
wo expe imen al designs: i) h ee i gin emales we e g ouped wi h
wo i gin males (g ouped emales, n=10 ials/subspecies), and ii) one
i gin emale was g ouped wi h one i gin male (single emales, n=20
ials/subspecies). Flies we e ans e ed o esh ials e e y day du ing
he i s 20 days a e eclosion. The o al numbe o eggs in each ial was
coun ed daily. Va ia ion be ween biological eplica es di e ed signi ican ly
in single emales in bo h subspecies (p<0.0001, D. moj. sono ensis and
D. moj. w igleyi, K uskal-Wallis).
4.2.7 S a is ical Analysis
The dis ibu ion o esiduals was es ed o no mali y using Q-Q plo s
and he app op ia e s a is ical es was applied. Di e ences in o a iole
77
Chap e 4
numbe among pai s o species we e es ed wi h ANOVA ollowed by
Tukey’s mul iple compa ison es . To e alua e di e ences in o al numbe
o eggs laid du ing 20 days a e eclosion, a Poisson eg ession model
(using he R unc ion ‘glm’) was pe o med. Co ela ions be ween emale
body size and o a iole numbe we e es ed using he Pea son’s Co ela ion
coe icien . The dis ibu ion o esiduals was non-no mal o body size
and de elopmen al ime, and hus, a Wilcoxon ank es was used o
pai wise compa isons. Di e ences in he iming o he onse o TFC
di e en ia ion we e es ed wi h a Chi-squa ed es . ANCOVAs we e
used o e alua e di e ences in he a es o TF o ma ion and o o a y
g ow h. To es whe he su i al in ou ood concen a ions was di e en
among subspecies, K uskall-Wallis ank es was pe o med. The same
es was used o e alua e whe he su i al wi hin each subspecies di e s
among ood concen a ions. To compa e he e ec s o nu i ion on
o a iole numbe and emale body size be ween subspecies, ANOVAs we e
pe o med. The dis ibu ion o esiduals o de elopmen al ime was non-
no mal. Fo his da ase , di e ences be ween subspecies we e es ed using
a gene alized line model wi h Poisson dis ibu ion. All da a analyses
and s a is ics we e conduc ed using R 3.1.2 (R De elopmen Co e Team,
2014). Plo s we e made using G aphPad P ism 6 (G aphPad So wa e).
S a is ical es s and p- alues a e indica ed in he ex and igu es.
4.3 Resul s
4.3.1 O a iole numbe di e si y in species o he
D osophila mulle i subg oup
To e alua e he di e si y in o a iole numbe among D. moja ensis
subspecies, I coun ed he numbe o o a ioles in emales o each subspecies
and o wo addi ional cac ophilic species o he eple a g oup, D. a izonae
and D. mulle i. These wo species a e closely ela ed o D. moja ensis, bu
u ilize di e en cac us hos s and ha e b oade geog aphical dis ibu ions
(Oli ei a e al., 2012; Reed e al., 2007). Addi ionally, I collec ed da a
om wo independen lines o each species/subspecies, wi h excep ion
78
Chap e 4
Figu e 4.2: O a iole numbe in emales o D. moja ensis w igleyi is educed
ela i e o o he D. moja ensis subspecies.Adul o a iole numbe in emales
o D. moj. baja ( wo lines, 15081-1351.30 and 15081-1352.34, n=14 and 27), D.
moj. moja ensis ( wo lines, 15081-1352.00 and 15081-1352.01, n=10 and 31), D. moj.
sono ensis ( wo lines, 15081-1352.32 and 15081-1352.26, n=70 and n=69) and D. moj.
w igleyi (one line 15081-1352.14, 14, n=69). D. a izonae ( wo lines, JFRC and 15081-
1271.29, n=17 and 19) and D. mulle i ( wo lines, 15081-1371.01 and 15081-1379.30,
n=21 and 21). The numbe s in he g aph legend ep esen he las digi s om he s ock
numbe o each line used. om he Plo ed alues ep esen means, and e o ba s show
95% con idence in e als o he means. One-way ANOVA using Tukey’s pai wise es
compa ison (ba s wi h same le e s a e no signi ican ly di e en ).
o D. moj. w igleyi, o alida e ha my measu emen s o o a iole
numbe we e species-speci ic (Table 4.1). Indeed, he a iabili y
be ween lines in each species/subspecies was no signi ican ly di e en ,
ensu ing ha o a iole numbe is obus wi hin a gi en species/subspecies
(Figu e 4.2). Con e sely, o a iole numbe di e ed conside ably among
di e en membe s o he eple a g oup. The island subspecies, D. moj.
w igleyi, had he lowes numbe o o a ioles (28.1±3.3) when compa ed
ei he o o he D. moja ensis subspecies (wi h excep ion o D. moj. baja
15081-1351.30) o o o he eple a species, D. a izonae and D. mulle i
(Figu e 4.2). On he o he hand, he subspecies inhabi ing he Sono an
Dese , D. moj. sono ensis, showed he highes numbe o o a ioles among
D. moja ensis subspecies (39.6±2.8 in D. moj. sono ensis 15081-1352.26
and 40.1±3.8 in D. moj. sono ensis 15081-1352.32). Bo h D. a izonae
(only line 15081-1271.29) and D. mulle i showed a simila o a iole numbe
as bo h o he D. moj. sono ensis lines and D. moj. moja ensis line 15081-
1352.00. Based on hese obse a ions, I ocused on wo D. moja ensis
79
Chap e 4
subspecies ha display he g ea es di e gence in o a iole numbe , D.
moj. w igleyi and D. moj. sono ensis 15081-1352.32 (hence o h e e ed
as D. moj. sono ensis and D. moj. w igleyi).
4.3.2 Di e gence o li e his o y ai s in D. moj.
sono ensis and D. moj. w igleyi
Female ecundi y
S udies in se e al species o he melanogas e g oup demons a ed ha
he numbe o o a ioles co ela es posi i ely wi h he numbe o eggs
emales lay (see Chap e 2) (Klepsa el e al., 2013b,a; R’ kha e al., 1997;
Kambysellis and Heed, 1971). I he e o e asked whe he emales o D. moj.
sono ensis, which ha e a highe numbe o o a ioles, lay signi ican ly mo e
eggs han D. moj. w igleyi emales. P elimina y obse a ions e ealed
ha lies o D. moj. sono ensis end o agg ega e in one side o he ood
pla e and lay mul iple eggs in a small egion o he ood pla e. This
o iposi ion beha iou was also obse ed in lies o D. moj. w igleyi,
al hough i was less conspicuous. Hence, o ully in es iga e whe he
emale ecundi y di e s be ween he wo D. moja ensis subspecies, I
coun ed he numbe o eggs laid in each subspecies in wo expe imen al
g oups: g ouped emales ( h ee emales and wo males) and single emales
(one emale and one male).
The dynamics o egg laying in g ouped emales we e e a ic in bo h D.
moja ensis subspecies, ye an in e es ing pa e n eme ged (Figu e 4.3A).
Eggs we e laid in clu ches in gi en days, wi h almos no eggs laid in he
in e ening days. This i egula pa e n o egg laying was mo e no iceable
in single emales (Figu e 4.3B). In wo biological eplica es, in pa icula ,
indi idual emales laid mo e han one hund ed eggs in a single day and e y
ew eggs we e o iposi ed in he emaining days (highe peaks in ligh blue
in Figu e 4.3B). E en hough he dynamics o egg laying was simila in he
wo D. moja ensis subspecies, he o al numbe o eggs laid o e 20 days
was signi ican ly lowe in D. moj. w igleyi ela i e o D. moj. sono ensis,
and his was seen in bo h g ouped and single emales (p<0.0001, Poisson
eg ession model) (Figu e 4.3C, D).
80
Chap e 4
Table 4.4: Two-away ANOVA model o o a iole numbe .
Sou ce o a ia ion d F p- alue
Subspecies 1 335.4036 <0.0001 ***
Food concen a ion 1 29.5408 <0.0001 ***
Subspecies x Food concen a ion 1 0.4847 0.6966 ns
E o 74 - -
species o he melanogas e g oup (Be gland e al., 2008; O gogozo
e al., 2006; Wayne and McIn y e, 2002; Wayne e al., 2001). Howe e ,
only a small numbe o candida e genes ha e been iden i ied and
unc ionally in es iga ed (G een and Ex a ou , 2012, 2014; O gogozo
e al., 2006). G een and Ex a ou (2012) ad oca e ha he iden i ica ion
o ele an candida e genes will bene i om a be e unde s anding o
he de elopmen al mechanisms ha gene a e di e si y in o a iole numbe
(G een and Ex a ou , 2012). Th oughou his hesis, I ex ensi ely
in es iga ed he de elopmen al mechanisms ha con ibu e o a ia ion in
o a iole numbe . In his inal sec ion, I show my p elimina y explo a ion
o he gene ic basis o in aspeci ic a ia ion in o a iole numbe and emale
body size.
Fo his pu pose, I examined o a iole numbe and emale body size in F1
and F2 hyb ids be ween D. moj. sono ensis and D. moj. w igleyi. In
bo h ecip ocal c osses, F1 hyb ids showed in e media e alues in o a iole
numbe , sugges ing ha a ia ion in o a iole numbe in ol es alleles wi h
opposi e dominan e ec s o wi hou any dominance a all (Figu e 4.7A,
B). Female body size in F1 hyb ids was skewed owa ds he smalle
pa en al line, D. moj. w igleyi (Figu e 4.7C, D), which may indica e ha
alleles in ol ed in egula ing smalle body size a e dominan o e hose
ha de e mine la ge body size. Howe e , e en hough la al densi y was
con olled as ca e ully as possible, he numbe o la ae in each bo le
a ied subs an ially, which could con ibu e o a educ ion in emale body
size in F1 hyb ids. Thus, I canno exclude he possibili y ha he smalle
adul size o F1 hyb ids is a consequence o he la al ea ing en i onmen .
In e es ingly, in he F2 hyb ids he a iance o o a iole numbe and emale
body size was simila o ha obse ed in he F1 hyb ids (Figu e 4.7).
87

Chap e 4
Figu e 4.7: O a iole numbe and emale body size in F1 and F2 hyb ids.
Rela i e equency dis ibu ion (in pe cen ages) o o a iole numbe (n=20-45) (A, B)
and emale body size (n=20-90) (C, D) in he pa en al lines, D. moj. sono ensis (ligh
blue line) and D. moj. w igleyi (ligh o ange line), and he F1 (black line) and F2
hyb ids (g ey line) om bo h ecip ocal c osses be ween D. moj. sono ensis and D.
moj. w igleyi.
88
Chap e 4
Thus, i is likely ha ew loci con ibu e o di e gence in o a iole numbe
and emale body size be ween D. moj. sono ensis and D. moj. w igleyi.
Mo e gene a ions would be equi ed o u he b eak up possible linkage
associa ions and ob ain a b oade ange o pheno ypes.
4.4 Discussion
4.4.1 Plas ic esponses and e ol ed a ia ion in o a iole
numbe
By examining he o a y de elopmen in bo h subspecies, I ound ha
all h ee de elopmen al p ocesses di e be ween D. moj. sono ensis
and D. moj. w igleyi (Figu e 4.8A). The onse o TFC di e en ia ion
was delayed in D. moj. w igleyi ela i e o D. moj. sono ensis. Such
delay is no due o di e ences in de elopmen al ime be ween subspecies,
since D. moj. w igleyi la ae a e he ones ha de elop as e . E en
hough he iming o he onse o TFC di e en ia ion was di e en
be ween species, he a e o TF o ma ion was educed only a he end
o L3 de elopmen in D. moj. w igleyi when compa ed wi h D. moj.
sono ensis. This con as s wi h he nu i ional-induced di e ences du ing
o a y de elopmen (Figu e 4.8B) (see Chap e 2). S a ing p e-c i ical
weigh la ae delays he onse o TFC di e en ia ion and educes he a e
o TF o ma ion h oughou he pos -c i ical weigh phase o de elopmen .
This co ela ion is u he obse ed when ei he insulin/insulin-like g ow h
signalling (IIS) o ecdysone signalling pa hways a e supp essed in he
la al o a y o well- ed la ae (see Chap e 3).
How is i ha changes in he onse o TFC di e en ia ion ha e no e ec on
he a e o TF o ma ion be ween subspecies? A possible explana ion is
ha once he i s TFCs di e en ia e om he su ounding soma ic cells in
D. moj. w igleyi, hey apidly in e cala e in o s acks and hence he numbe
o o ming TFs quickly inc eases. Addi ionally, he ela i e numbe o
TFCs ha ini ially eme ged a he onse o TFC di e en ia ion may also
di e be ween subspecies. Pe haps a he onse o TFC di e en ia ion, a
highe ela i e numbe o TFCs is p oduced in D. moj. w igleyi o a ies,
89
Chap e 4
Figu e 4.8: Changes in dis inc de elopmen al p ocesses unde lie
nu i ional-induced and subspecies-speci ic a ia ion in o a iole numbe .
(A) Changes in he a es o TF o ma ion and o o a y g ow h a e esponsible o
o a iole numbe di e gence be ween D. moja ensis subspecies. (B) Be o e c i ical
weigh , s a a ion delays he onse o TFC di e en ia ion and a es s o a y g ow h.
In con as , he a es o TF o ma ion and o o a y g ow h con inue when pos -c i ical
weigh la ae a e poo ly ed. Howe e , hese a es a e conside ably educed, esul ing
in ewe o a ioles han well- ed la ae.
which could con ibu e o a as e o ma ion o new TFs in a sho pe iod
o ime.
E en hough he a e o TF o ma ion is ini ially simila , a he end o
L3 de elopmen he a e o TF o ma ion is educed, con ibu ing o he
di e gence in o a iole numbe be ween D. moj. sono ensis and D. moj.
w igleyi (Figu e 4.8A). Changes in he a e o TF o ma ion we e also
obse ed when pos -c i ical weigh la ae we e poo ly ed (Figu e 4.8B)
(see Chap e 2). In addi ion, Hodin and Riddi o d (2000) ound ha
ea ing D. melanogas e la ae on dilu ed medium only educed he a e
o TF o ma ion a he end o he L3, simila o wha I obse ed in D.
90
Chap e 4
moj. w igleyi.
The a e o o a y g ow h was conside ably educed in D. moj. w igelyi
when compa ed wi h D. moj. sono ensis. The o al numbe o TFs g ea ly
depends on he numbe o TFCs ha di e en ia e om he soma ic cell
pool (Sa ikaya e al., 2012). The e o e, a slowe a e o o a y g ow h
ei he in D. moj. w igleyi o in s a ed pos -c i ical weigh la ae may
g adually cons ain he numbe o cells a ailable o o m TFCs, he eby
educing he a e o TF o ma ion. Taken oge he , I p opose ha
o a y g ow h a es ha e di e ged be ween D. moj. w igleyi and D.
moj. sono ensis, gene a ing di e ences in o a iole numbe by limi ing
he numbe o soma ic cells a ailable o o m TFCs.
In a p e ious s udy by Hodin and Riddi o d (2000), he au ho s concluded
ha nu i ionally-induced a ia ion in o a iole numbe is es ic ed o
al e a ions in he a e o TF o ma ion, while changes in a b oade ange
o de elopmen al p ocesses can accoun o a ia ion in o a iole numbe
bo h be ween popula ions and be ween species (Hodin and Riddi o d,
2000). My esul s di e om his s udy in se e al ways. Fi s , o
in es iga e he de elopmen al p ocesses ha accoun o nu i ional-
induced di e ences, I ea ed L3 la ae in a medium con aining only
suga and aga , which signi ican ly educed bo h o a iole numbe and
body size and delayed de elopmen al ime. Unde hese condi ions, he
onse o TFC di e en ia ion, he a e o TF o ma ion and he a e o
o a y g ow h we e all a ec ed. In con as , Hodin and Riddi o d (2000)
ea ed la ae om egg o adul in 50% dilu ed ood om i s o iginal
concen a ion in o de o a oid any change in body size and de elopmen al
ime (Hodin and Riddi o d, 2000). Such dilu ed ood p o ides a less
s ess ul en i onmen o la ae o de elop, and only al e ed he a e o
TF o ma ion a he end o la al de elopmen . Taken oge he , he ange
o de elopmen al p ocesses al e ed by nu i ion depends pa ly on he
se e i y o he nu i ional condi ions.
Second, my analysis was pe o med wi h signi ican esolu ion h oughou
L3 la al de elopmen , which allowed me o de ec di e ences in he
onse o TFC di e en ia ion, he a e o TF o ma ion, and he a e o
o a y g ow h be ween D. moja ensis subspecies and be ween nu i ional
91
Chap e 4
esponses. Hodin and Riddi o d (2000), by con as , in e ed di e ences
in he onse o TFC di e en ia ion by coun ing he numbe o TFs a 24 h
AL3E wi hou any analysis on p e ious ime poin s (Hodin and Riddi o d,
2000). Mo eo e , he a es o TF o ma ion we e only compa ed du ing
he wande ing s age (i.e. when la ae lea e he ood o ind a pupa ia ion
si e) and no compa isons we e pe o med amongs a es o o a y g ow h
(Hodin and Riddi o d, 2000).
Thi d, Hodin and Riddi o d (2000) compa ed i e species in he
melanogas e subg oup wi h a much g ea e ange o o a y sizes han
hose s udied he e (Hodin and Riddi o d, 2000). D. sechellia has se e ely
educed o a iole numbe ela i e o D. melanogas e (R’ kha e al.,
1997; O gogozo e al., 2006; Hodin and Riddi o d, 2000; G een and
Ex a ou , 2014). These di e ences in o a iole numbe a e due o a
smalle size o o a ian p imo dium and slowe a es o TF o ma ion
in D. sechellia(G een and Ex a ou , 2012; Hodin and Riddi o d, 2000).
Hence, he di e ence in o a iole numbe be ween hese wo species is
de ined by changes in o a y de elopmen since emb iogenesis. In my
s udy, I ocused on wo subspecies o D. moja ensis wi h mo e mode a e
di e ences in o a y size. He e, I ound ha o a y size is indis inguishable
be ween he wo aces a he onse o he L3. Di e ences in o a y
de elopmen , including o a y g ow h a es and he a e o TF o ma ion,
a ise only in he inal la al s age. Thus, i would seem ha much like o
he plas ic esponse, he numbe o de elopmen al p ocesses unde lying
in a/in e speci ic gene ic a ia ion depends on he magni ude o he
di e ence in o a y size.
Al hough di e ences in o a iole numbe bo h ac oss species and ac oss
popula ions o en co ela e wi h obse ed plas ic esponses, hese a e no
he only mechanisms known o gene a e a ia ion in o a iole numbe .
G een and Ex a ou (2012) ound ha di e ences in o a iole numbe
be ween wo s ains o D. melanogas e esul ed no om di e ences in
o a y g ow h bu om di e ences in he alloca ion o soma ic cells, which
ha e he po en ial o become TFC, o di e en soma ic cell a es (G een
and Ex a ou , 2012). In s ains wi h lowe o a iole numbe s, a g ea e
p opo ion o he soma ic cells adop ed a swa m cell a e han in s ains
92

Chap e 4
wi h highe o a iole numbe s. Whe he his de elopmen al di e ence
co esponds o a plas ic esponse o ano he ype o en i onmen al cue,
such as empe a u e o hypoxia, emains o be disco e ed.
4.4.2 The ela ionship be ween o a iole numbe and emale
body size
I ha e shown ha a ia ion in o a iole numbe be ween he wo D.
moja ensis subspecies is posi i ely co ela ed wi h a ia ion in emale
body size. Howe e , he ela ionship be ween o a iole numbe and emale
body size is no always clea (Be gland e al., 2008; G een and Ex a ou ,
2012; Hodin and Riddi o d, 2000; Kambysellis and Heed, 1971; Robe son,
1956; San os e al., 1992; Wayne e al., 1997). The s eng h o his
ela ionship appea s o a y wi h en i onmen al ac o s, including nu ien
a ailabili y and la al compe i ion (Be gland e al., 2008; Kambysellis and
Heed, 1971; San os e al., 1992). Fo ins ance, in some specialis species,
such as he cac ophilic D. buzza ii and he Hawaiian D. mimica, a posi i e
co ela ion be ween ho ax leng h (a p oxy index o body size) and
o a iole numbe is ound in wild caugh lies, bu no in labo a o y- ea ed
lies (Kambysellis and Heed, 1971; San os e al., 1992). Fu he mo e,
la ae o D. melanogas e ea ed in low yeas concen a ions show a s ong
posi i e co ela ion be ween ho ax leng h and o a iole numbe . This
co ela ion is los when la ae a e ed on op imal nu i ional condi ions
(Be gland e al., 2008; Wayne e al., 1997). Pe haps he mos in e es ing
case is ha o empe a u e-induced di e ences in o a iole numbe and
body size in popula ions o D. melanogas e . La ae ea ed a highe
empe a u es p oduce smalle adul s wi h educed o a iole numbe . In
con as , adul om la ae g own a lowe empe a u es ha e la ge body
size, bu simila educ ion in o a iole numbe (Klepsa el e al., 2013a;
Mi h and Shingle on, 2012). Fu u e s udies examining mo e species and
a g ea e ange o en i onmen al condi ions will lead o as imp o emen s
in ou unde s anding o he ela ionship be ween o a iole numbe and
emale body size.
93
Chap e 4
4.4.3 Gene a ing a hypo hesis on egg laying beha iou
O a iole numbe is an impo an de e minan o emale ep oduc i e
capaci y; emales wi h highe numbe o o a ioles can po en ially lay
mo e eggs (see Chap e 2) (Boulé eau-Me le e al., 1982; Klepsa el e al.,
2013b; R’ kha e al., 1997). Indeed, D. moj. sono ensis emales ha e a
g ea e ep oduc i e capaci y han emales o D. moj. w igleyi. Howe e ,
his di e ence in o al numbe o eggs laid among he wo subspecies
o D. moja ensis only became appa en in he las days o he analysis,
sugges ing ha o he ac o s, such as a e o oogenesis o egg e en ion,
in luence emale ep oduc i e ou pu . Mo eo e , subs an ial di e ences in
male ep oduc i e ai s could also exis be ween he wo D. moja ensis
subspecies, which migh con ibu e o he obse ed di e ences in he o al
numbe o eggs laid.
In e es ingly, bo h subspecies exhibi ed an e a ic dynamic o egg laying,
in which nume ous eggs we e laid in gi en days. The a e o egg p oduc ion
may o e a possible explana ion o his egg laying beha iou . O a ies can
be de ined as synch onous when all o a ioles ha e a leas one ma u e egg
(Kambysellis and Heed, 1971). One such example is ound in D. mulle i,
in which ma u e eggs de elop synch onously and a e o iposi ed in la ge
clu ches in a single day (Ma kow and O’G ady, 2008). A close examina ion
o he numbe o eggs pe o a iole may elucida e whe he he obse ed
egg laying beha iou in D. moja ensis subspecies is a di ec esul o a
synch onous o a y.
Al e na i ely, he abundance and dis ibu ion o sui able o iposi ion si es
may in luence he equency o o iposi ion (Kambysellis and Heed, 1971).
Females o D. moja ensis o iposi in nec o ic cac i, which o e s a mois
and nu i ious en i onmen o la al de elopmen (Fellows and Heed,
1972b; Ruiz and Heed, 1988). The pa chy dis ibu ion o cac us hos s and
he epheme al na u e o nec oses equi e ha D. moja ensis e icien ly
dispe se g ea dis ances o ind he app op ia e b eeding si e (B ei meye
and Ma ko w, 1998; P eile and Ma kow, 2011). In hei na u al
se ing, D. moja ensis emales may e ain ma u e eggs un il a po en ial
o iposi ion si e is ound, which migh lead o an i egula egg laying
beha iou . Howe e , i pos poning o iposi ion is ad an ageous when
94
Chap e 4
sui able esou ces a e in equen ly encoun e ed, why is his beha iou
main ained in labo a o y condi ions? The physiological mechanisms
egula ing he a e o egg laying could be gene ically ixed and hus, e en
in he p esence o abundan ood esou ces, he dynamics o egg laying is
unchanged.
Finally, he s anda d ly ood p o ided in labo a o y condi ions migh no
be op imal o D. moja ensis species. Al hough D. moja ensis is ela i ely
easy o main ain unde labo a o y condi ions, he nec o ic cac i p o ides
a complex nu i ious en i onmen wi h speci ic ol ac o y cues (Da e e al.,
2013) ha may in luence he equency o o iposi ion. Fu he s udies
using ex ac s o nec o ic cac us could p o ide new insigh s on egg laying
beha iou in D. moja ensis subspecies.
4.5 Conclusions
The aim o his chap e was o elucida e he de elopmen al mechanisms
unde lying a ia ion in o a iole numbe be ween subspecies o D osophila
moja ensis. Coupled wi h he ex ensi e s udies ega ding hei ecology,
D. moja ensis subspecies ep esen an a ac i e model o add ess he
ea ly e en s leading o e olu iona y di e si ica ion in o a iole numbe .
Impo an ly, he esul s desc ibed in his chap e open up new a enues
o esea ch in di e se disciplines, including ecology (e.g. wha unde lies
i egula egg laying beha iou ?), cellula biology (e.g. wha a e
he unde lying mechanisms egula ing p oli e a ion, di e en ia ion and
in e cala ion o TFCs) and physiology (e.g. how di e en ai s espond
di e en ly o nu i ion?).
Acknowledgmen s
I would like o hank Ch is en Mi h and Nelson Ma ins o ad ice on s a is ical
analyses. Liliana Viei a o making he ou ood concen a ions. Ch is en Mi h
o commen ing and p oo eading his chap e . The Uni o Imaging a IGC o
all he assis ance du ing image acquisi ion and analysis. The Fly Facili y, in
pa icula Liliana Viei a, o making ly ood e e y week.
95
Chap e 4
96
Chap e 5
Figu e 5.2: Al e na i e mechanisms migh unde lie di e ences in he onse
o TFC di e en ia ion be ween he wo D. moja ensis subspecies. (A) The
small ecdysone peak is sec e ed a di e en ime poin s, esul ing in di e ences in he
iming o bo h he a ainmen o c i ical weigh and he onse o TFC di e en ia ion. (B)
C i ical weigh is a ained a he same ime. Howe e , o a ies om D. moj. sono ensis
and D. moj. w igleyi may show subs an ial di e ences in IIS ac i i y, and hus, di e en
h esholds o ecdysone sensi i i y could exis be ween he wo subspecies. As a esul ,
he onse o TFC di e en ia ion would occu a di e en ime poin s.
in ha sh condi ions) in he nema ode Caeno habdi is elegans (Schaedel
e al., 2012). He e, he au ho s desc ibed he exis ence o a esponsi e
pe iod du ing which wo ms can espond o he ho mone da ac onic acid
and induce al e na i e de elopmen al p og ams. Bo h c owding and
he p esence o daue phe omone aises he h eshold o sensi i i y o
da ac onic acid necessa y o induce adul de elopmen (Schaedel e al.,
2012). S udies in he dung bee le On hophagus au us ha e elucida ed
how h esholds o ho mone sensi i i y and he esponsi e pe iod can be
al e ed by social condi ions (Emlen and Nijhou , 1999). The D osophila
o a y now p o ides an oppo uni y o unco e he molecula mechanisms
unde lying hese ypes o phenomena.
5.3 Plas ici y and e olu ion in o a iole numbe
and body size
No all ai s show he same sensi i i y o la al nu i ion (Shingle on
e al., 2009). One mechanism by which o gans can change hei nu i ional
sensi i i y is by changing he le els o IIS ac i i y (Tang e al., 2011;
103

Chap e 5
Shingle on and Tang, 2012; Koyama e al., 2013; Shingle on and F ankino,
2013)). Fo ins ance, G een and Ex a ou (2014) ound ha he o a ies
o D. sechellia and D. melanogas e di e in he le els o phospho yla ed
Ak , and p opose ha his a ia ion in he le els o IIS unde lies he
di e ence in o a iole numbe be ween he wo species. Changes in he
le els o FOXO ac i i y, a nega i e egula o o IIS, a e also known o
media e he nu i ional sensi i i y o de eloping o gans (Tang e al., 2011).
A low le els o oxo mRNA exp ession, g ow h o he wing discs is no
inhibi ed e en in la ae ha a e poo ly ed, allowing lies o main ain a
la ge wing size when nu i ion and IIS is low (Figu e 5.3A, B). Whils
mode a e le els o oxo inc ease he nu i ional sensi i i y o he wing, a
e y high le els o oxo exp ession he wing shows educed sensi i i y o
nu i ion, and hus, lies bea small wings e en when nu i ion and IIS a e
high (Figu e 5.3A, B) (Tang e al., 2011). Thus, plo ing he deg ee o
nu i ional plas ici y agains IIS ac i i y gene a es a bell-shaped cu e.
Using a simila logic o ha used by Tang e al. (2011), I hypo hesize ha
he le els o IIS ac i i y egula e he nu i ional sensi i i y o o a iole
numbe and emale body size in he wo subspecies o D. moja ensis.
When la ae we e ea ed ac oss a ange o nu i ional condi ions, emale
body size in D. moj. sono ensis was mo e sensi i e o nu i ional a ia ion
when compa ed wi h D. moj. w igleyi (i.e. slopes o eac ion no ms
we e signi ican ly di e en ).Mo eo e , emales o D. moj. sono ensis
ha e highe numbe o o a ioles and la ge body size ela i e o D.
moj. w igleyi emales. I he e o e p opose ha whole body o D.
moj. sono ensis emales migh display mode a e le els o IIS signalling,
esul ing in an inc ease in i s sensi i i y o la al nu i ion (Figu e 5.3C,
D). Con e sely, emales o D. moj. w igleyi migh show low le els o
IIS ac i i y h oughou he body, making hem bo h smalle and less
sensi i e o nu i ional a ia ion (Figu e 5.3C, D). In e ms o hei o a iole
numbe , bo h subspecies show simila nu i ional sensi i i y (i.e. slopes
o eac ion no ms we e equal). O a y g ow h a e in D. moj. w igleyi is
signi ican ly educed in compa ison wi h D. moj. sono ensis, and hence,
le els o IIS ac i i y in he de eloping o a y a e likely di e en be ween
he wo subspecies. This could occu i he le els o IIS ac i i y we e a
104
Chap e 5
Figu e 5.3: The ela ionship be ween IIS ac i i y and nu i ional sensi i i y.
(A) Changes in he le els o FOXO ac i i y can accoun o di e ences in nu i ional
sensi i i y. Ei he low o high le els o oxo mRNA in he wing discs educe hei
sensi i i y o nu i ional a ia ion. In con as , wing discs exp essing mode a e le els
o oxo mRNA inc ease hei nu i ional sensi i i y. (B) The e o e, he ela ionship
be ween nu i ional sensi i i y and IIS ac i i y shows a bell-shape cu e. (C) Di e en
plas ic esponses and gene ic a ia ion o body size be ween D. moj. sono ensis and
D. moj. w igleyi. (D) D. moj. sono ensis migh show mode a e le els o IIS ac i i y,
esul ing in highe nu i ional sensi i i y o body size. (E) Simila plas ic esponses,
bu gene ic a ia ion o o a iole numbe be ween he wo subspecies. (F) Le els o IIS
ac i i y may be a opposi e ex emes o he bell-shape cu e, esul ing in simila le els
o nu i ional sensi i i y, bu di e en o a y sizes.
105
Chap e 5
opposi e ex emes o he bell-shaped cu e, esul ing in simila le els o
plas ici y bu di e en o a y sizes (Figu e 5.3E, F).
My wo k has ocussed on he de elopmen al and physiological mechanisms
unde lying a ia ion in o a iole numbe . Howe e , we a e beginning
o unde s and he gene ic basis o o gan and body size. G een and
Ex a ou (2014) u he demons a ed ha changes in he ac i i y o
IIS can accoun o di e ences in o a iole numbe be ween D. sechellia
and D. melanogas e : high IIS ac i i y in he de eloping o a y o D.
melanogas e can p omo e he o ma ion o a highe numbe o o a ioles
(G een and Ex a ou , 2014), p esumably by inc easing he a e o o a y
g ow h. Fu he mo e, in oducing one copy o D. melanogas e InR
in o a D. sechellia backg ound inc eases he numbe o o a ioles in his
species (G een and Ex a ou , 2014). A simila e olu iona y change in
IIS ac i i y be ween D. moj. sono ensis and D. moj. w igleyi could
unde lie di e ences in o a y g ow h, which ul ima ely esul in di e ences
in o a iole numbe .
Toge he wi h he cu en li e a u e on plas ici y and e olu ion o o gan
size, my wo k unde line ha changes in he ac i i y o a majo ho monal
pa hway, he IIS, can accoun o plas ic esponses, and po en ially
acili a e e olu iona y di e si ica ion among popula ions and species.
Unde s anding he gene ic mechanisms unde lying e olu iona y changes
in he ac i i y o IIS could be a ui ul a enue o esea ch.
Acknowledgmen s
I would like o hank Ch is en Mi h o all he b ains o ming un il he las
minu e, which was c ucial o cla i y my andom hypo heses ha we e discussed
in his chap e , and o commen ing and p oo eading his chap e .
106
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