Disse a ion p esen ed o ob ain he Ph.D deg ee in Neu oscience
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie | Uni e sidade No a de Lisboa
Oei as,
Decembe , 2021
Neu onal con ol o supp ession, ini ia ion and
comple ion o egg deposi ion in D osophila
melanogas e
C is ina Oli ei a Fe ei a
C is ina Oli ei a Fe ei a
Disse a ion p esen ed o ob ain he Ph.D deg ee in Neu oscience
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie | Uni e sidade No a de Lisboa
Oei as, Decembe , 2021
Neu onal con ol o supp ession, ini ia ion
and comple ion o egg deposi ion in
D osophila melanogas e
Resea ch wo k coo dina ed by:
Neu onal con ol o supp ession,
ini ia ion and comple ion o egg
deposi ion in D osophila melanogas e
C is ina Oli ei a Fe ei a
A Disse a ion
P esen ed o he Facul y
o Uni e sidade No a de Lisboa
in Candidacy o he Deg ee
o Doc o o Philosophy
Supe ised by: Ma ia Luisa Vasconcelos
In e na ional Neu oscience Doc o al P og amme
Champalimaud Resea ch
Lisbon, Po ugal
2021
To he u u e
iii

Acknowledgmen s
This 7-yea jou ney was de ini ely no a smoo h one wi h a lo o di icul
momen s in he cou se and, i I each he end o his pa h, ha was only made
possible because I am o una e o be su ounded by people ha a e good
lis ene s, pa ien and ca ing.
Thank you o he Inna e Beha iou lab (p esen and alumni membe s):
Luisa Vasconcelos, Cecilia Mezze a, Miguel Gaspa , Eliane Ca alho, Má cia
A anha and Sophie Dias. I has been a pleasu e o be pa o his g oup
whe e he willingness o help was always he p ime ounda ion. Fo all he
expe ise and aluable sugges ions ha helped me o g ow as a be e scien is
and o build a be e wo k. In special, a wa m hank you o my supe iso ,
Luisa Vasconcelos, o being an amazing PI: wise, pa ien , who od he whole
way nex o me always looking o a be e ail. Also, a big hank you o
Miguel Gaspa o his willingness o con ibu e o my p ojec , e en hough he
was simul aneously wo king ha d o inish his PhD. To my hesis commi ee,
Eugenia Chiappe and Michael O ge , o he wise sugges ions ha shape his
wo k o be e .
To he ly oom people −a big hank you o being a bu e on he bad
days. Wo king on such a nice and amilia en i onmen made e e y hing wo h
i .
My li e would no be he same wi hou he p ecious −Gab iela Fio eze and
Inês Fe ei a −who I me du ing his jou ney and became e y special iends
o mine. F iends o e e y occasion.
To Susana Lima − hank you o you ca e and, no he leas , companion-
ship on he dance loo .
To my sis e o being he bes . The c ea i e mind in he amily − hanks
o making he exe cise o hinking abou di e en al e na i e ca ee s o me.
I´ e los he coun and i is eassu ing o know ha I´ll ne e be unemployed
wi h all he ideas popping ou you mind.
i
The e a e h ee people which, wi hou hei uncondi ional lo e and suppo ,
I would ha e ne e inished his PhD. This hesis is specially dedica ed o: my
pa en s, Emilia and Ca los, and o my companion, Rica do Ne o.
I would like o hank my pa en s o being an un eplaceable suppo in my
li e. Wi hou hei pe sis ence and lo e, I would no ha e passed om he i s
yea o PhD. This is a ac . Lo e you.
To Rica do Ne o −I will ne e be able o pu in wo lds how much I hank
you. Thanks o you wise ad ices, endless science- ela ed con e sa ions and
lis ening my p esen a ions o e and o e . Bu also, hanks o sha ing wi h
me he good momen s a wo k and in li e. You smile and ca ing make happiness.
Tí ulo
Con olo neu onal da sup essão, iniciação e e minação da deposição do o o em
D osophila melanogas e
Resumo
O compo amen o de o iposição é um aspe o cen al na biologia ep odu-
i a dos inse os endo um impac o p o undo na sob e i ência e i ness das es-
pécies. Em semelhança à maio ia das espécies de inse os, as êmeas D osophila
e oluí am pa a in es i a sua ene gia na p odução de mui a p ogenia em de i-
men o de cuidado ma e nal. A elação mãe-p ogenia e mina com a seleção de
um local ap op iado pa a a deposição dos o os que de e á alimen a e p o e-
ge a p ogenia de e en os ambien ais ex emos e p edado es. Enquan o g ande
p og esso em sido ei o na comp eensão das bases neu onais da seleção de sub-
s a o, exis e pouco conhecimen o sob e a o ganização e unção de ci cui os
mo o es que con olem a deposição do o o. Es a ese, a a és da combinação
de análise compo amen al com a ca ac e ização de ci cui os neu onais, o nece
conhecimen o sob e a a qui e u a de ci cui os mo o es in e io es que execu am
e egulam a deposição do o o.
No segundo capí ulo, desc e emos as ases e os elemen os compo amen ais
associados com o iposição em êmeas D. melanogas e o iginá ias de uma es i pe
sel agem. Ca ac e izamos ês ases dis in as des e compo amen o complexo:
deposição do o o, con o ções abdominais e explo ação. Es abelecemos que a
ase de deposição do o o −quando o o o é pos o −é semp e seguida pela ase
de con o ções abdominais, enquan o que a ase de explo ação −onde a mosca
p ocu a po um local ideal de o iposição −é opcional. Cada ase em um p o-
g ama compo amen al mo o especí ico que pode p og edi numa sequência de
compo amen os ou não. Todos es es elemen os compo amen ais são execu a-
dos pa a p omo e a degus ação do subs a o, a deposição do o o e a o ulação.
Es a desc ição de alhada do compo amen o de o iposição em moscas sel agens
o e ece uma es u u a pa a ques iona os subs a os neu onais adjacen es.
No e cei o capí ulo, o necemos no a e idência sob e um ci cui o mo o no
gânglio abdominal −os neu ónios O Abg −que são su icien es e necessá ios
pa a o compo amen o de o iposição. Usando uma abo dagem in e seccional
baseada no pe il de neu o ansmisso es pa a ob e subg upos uncionais, de-
i
scob imos que os neu ónios O Abg êm ês populações neu onais com con-
ibuições di e en es pa a a o iposição. Expe iências de a i ação op ogené ica
mos am que os subg upos glu ama é gicos (O Abg/VGlu ) e coliné gicos (O -
Abg/Cha) es ão en ol idos na iniciação e e minação da ase da deposição do
o o. Além disso, mos amos que o silenciamen o gené ico de um subg upo
dis in o de neu ónios GABAé gicos (O Abg/Gad1) não em e ei o no núme o
de o os deposi ados, no en an o, a sua a i ação op ogené ica inibe o iposição.
Es es esul ados o emen e suge em que, du an e o iposição, os neu ónios O -
Abg/Gad1 es ão silenciados e, quando a i os, podem bloquea a execução de
o iposição.
Es e abalho o nece in o mações impo an es sob e a iden idade e a qui e-
u a de subs a os mo o es in e io es que con olam um compo amen o com-
plexo a a és da descobe a do papel impo an e do ci cui o O Abg no con olo
de o iposição. Es e abalho se e de base pa a a descobe a de como di e en es
ci cui os es ão conec ados pa a coo dena compo amen o.
ii
Con en s
Acknowledgmen s ............................ i
Tí ulo e Resumo ............................. i
Abs ac .................................. iii
Au ho Con ibu ions and Financial Suppo .......... x
Lis o Abb e ia ions .......................... xi
1 Gene al In oduc ion 1
1.1 Egg-Laying: Inna e and Rep oduc i e Beha iou . . . . . . . . . 2
1.2 When olayanegg.......................... 3
1.2.1 Female Pos -Ma ing Response . . . . . . . . . . . . . . . . 3
1.2.2 Ac i a ing he Pos -Ma ing Response . . . . . . . . . . . 4
1.2.3 Ci cadian Modula ion . . . . . . . . . . . . . . . . . . . . 4
1.2.4 Egg-laying Si e Value . . . . . . . . . . . . . . . . . . . . 5
1.3 Whe e olayanegg ......................... 6
1.3.1 How o Sense he En i onmen . . . . . . . . . . . . . . . 6
1.4 How olayanegg .......................... 10
1.4.1 Female Rep oduc i e Ana omy . . . . . . . . . . . . . . . 10
1.4.2 Beha iou al Mo o P og ammes . . . . . . . . . . . . . . 11
1.5 Egg-laying Ci cui s . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.5.1 When and Whe e: F om ma ing o he decision o laying
anegg............................. 12
1.5.2 How: om he decision o he execu ion o laying an egg . 14
1.6 Analogous Lowe Mo o Ci cui s . . . . . . . . . . . . . . . . . . 18
1.6.1 D osophila Male Sexual and Locus Egg-laying Beha iou s 18
2 Egg-laying beha iou in wild ype lies 21
2.1 Cha ac e iza ion o egg-laying beha iou . . . . . . . . . . . . . . 22
xi

3 Mo o con ol o egg-laying beha iou 26
3.1 Ac i i y o O Abg neu ons p omo es egg deposi ion . . . . . . . 27
3.2 Silencing O Abg neu ons dis up s all mo o elemen s associa ed
wi h egg-laying beha iou . . . . . . . . . . . . . . . . . . . . . . 31
3.3 Ac i i y o GABAe gic O Abg neu ons blocks egg-laying . . . . . 35
3.4 Choline gic O Abg neu ons a e necessa y and su icien o egg
deposi ion............................... 37
3.5 Glu ama e gic O Abg neu ons con ibu e o egg deposi ion ini-
ia ion................................. 40
4 Supplemen a y In o ma ion 45
4.1 Supplemen a y Figu e 4.1 . . . . . . . . . . . . . . . . . . . . . . 46
4.2 Supplemen a y Figu e 4.2 . . . . . . . . . . . . . . . . . . . . . . 48
4.3 Supplemen a y Videos . . . . . . . . . . . . . . . . . . . . . . . . 49
4.3.1 Supplemen a y Video 1 . . . . . . . . . . . . . . . . . . . 49
4.3.2 Supplemen a y Video 2 . . . . . . . . . . . . . . . . . . . 49
4.3.3 Supplemen a y Video 3 . . . . . . . . . . . . . . . . . . . 49
4.3.4 Supplemen a y Video 4 . . . . . . . . . . . . . . . . . . . 49
4.3.5 Supplemen a y Video 5 . . . . . . . . . . . . . . . . . . . 49
4.3.6 Supplemen a y Video 6 . . . . . . . . . . . . . . . . . . . 49
4.3.7 Supplemen a y Video 7 . . . . . . . . . . . . . . . . . . . 49
4.4 Supplemen a y Table 1. Fly s ocks . . . . . . . . . . . . . . . . . 50
4.5 Supplemen a y Table 2. Full geno ypes used in expe imen s . . . 51
5 Expe imen al P ocedu es 52
5.1 Fly s ocks and husband y . . . . . . . . . . . . . . . . . . . . . . 53
5.2 Immunohis ochemis y . . . . . . . . . . . . . . . . . . . . . . . . 53
5.3 P epa a ion o lies o be assayed . . . . . . . . . . . . . . . . . . 54
5.4 Beha iou al assays . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.4.1 24h egg-laying assay . . . . . . . . . . . . . . . . . . . . . 54
5.4.2 Egg-laying a ena and subs a e . . . . . . . . . . . . . . . 54
5.4.3 De ailed beha iou . . . . . . . . . . . . . . . . . . . . . . 55
5.4.4 Op ogene ics ......................... 55
5.4.5 Imagecap u e ........................ 56
5.5 Quan i ica ion and s a is ical analysis . . . . . . . . . . . . . . . 56
5.5.1 Da a p ocessing . . . . . . . . . . . . . . . . . . . . . . . . 56
x
5.5.2 Quan i ica ion o beha iou s . . . . . . . . . . . . . . . . . 56
5.5.3 S a is ical analysis . . . . . . . . . . . . . . . . . . . . . . 58
6 Gene al Discussion and Conclusion 60
6.1 Discussion............................... 61
6.2 Egg-laying beha iou in wild- ype lies . . . . . . . . . . . . . . . 62
6.3 O Abg neu ons: lowe mo o con ol o egg-laying beha iou . . 63
6.4 Execu ion o Egg Deposi ion: Glu ama e gic and Choline gic O -
Abgci cui s.............................. 64
6.5 Regula ion o Egg-laying: GABAe gic O Abg ci cui . . . . . . . 66
6.6 Conclusion .............................. 67
Re e ences 68
x i
Chap e 1
Gene al In oduc ion
1
How he Cen al Ne ous Sys em (CNS) gene a es a sequence o mo o
ac ions o p oduce beha iou , once a decision is signalled by he b ain, is a
long-s anding ques ion in beha iou al neu oscience. I is a complex ask as
his mo o ou pu needs o be: (1) igh ly coo dina ed in ime so ha he
app op ia e sequence o beha iou al elemen s is accomplished; (2) synch onised
wi h he animal in e nal s a e (e.g. ma ing, eeding s a us); and (3) lexible
enough o cope wi h unexpec ed changes in he en i onmen .
The ui ly has a ela i ely simple ne ous sys em (∼200,000 neu ons)
ha manages o pe o m complex beha iou s. As such, he ly has been a
p ime model in beha iou al neu obiology esea ch o many yea s. The cu en
a ailable gene ic oolki , combined wi h minimal cul u ing equi emen s and a
sho li e cycle allow lexible and di e se app oaches o scien i ic ques ions. The
s udy p esen ed he e explo es he ui ly’s po en ial, along wi h he s udy o
egg-laying beha iou , in o de o s eng hen ou knowledge abou he gene al
p inciples go e ning animal mo o beha iou .
1.1 Egg-Laying: Inna e and Rep oduc i e Beha iou
Animals a e bo n wi h a se o p e-de ined beha iou al p og ammes −inna e
beha iou s − ha a e eliably execu ed in esponse o speci ic s imuli (Tinbe -
gen, 1991). These beha iou s a e suppo ed by gene ically ha dwi ed neu al
ci cui s and do no ely on lea ning (Kim e al., 2017). Rep oduc i e beha iou s
a e included in he epe oi e o inna e beha iou s. Fly sexual beha iou s ha e
been highly signi ican o he s udy o ele an ques ions such as: di e ences in
he gene ic a chi ec u e o gende -speci ic beha iou s, molecula and neu onal
basis o social in e ac ions, mul isenso y in eg a ion, neu omodula ion and he
mo o execu ion o beha iou al p og ammes (A anha and Vasconcelos, 2018;
Kim e al., 2017; La u ney and Bille e , 2014).
Egg-Laying beha iou is a sexually dimo phic beha iou essen ial o
he su i al and i ness o o ipa ous species, displayed by emales in he pos -
copula o y phase. I s co ec implemen a ion equi es he coo dina ion o phys-
iological and beha iou al changes ha occu o mee he me abolic and nu-
i ional demands o egg p oduc ion and ma u a ion. Gi en ha D osophila
emales do no display ma e nal beha iou s, i is c ucial ha he beha iou al
changes include decisions ega ding he si es whe e o lay he eggs o ensu e
2
he p ope la ae nu i ion, and p o ec ion om en i onmen al h ea ening ac-
o s. Thus, egg-laying is a complex beha iou and i s egula ion implies he
coo dina ion be ween he pe iphe ic sys em and he CNS in o de o p oduce
he co ec ou pu in space and ime. Speci ically, as pos ula ed by Cu y e al.
(2019), h ee a iables should be conside ed:
1. When (ma ing, ci cadian egula ion and he alue o he egg-laying si e);
2. Whe e (egg-laying si e selec ion);
3. How o lay an egg (beha iou al mo o p og ammes and unde lying
neu onal subs a es).
In he nex sec ions o his in oduc o y chap e , I will e iew he cu en
knowledge on he neu oe hology o egg-laying, ollowing he h ee a o emen-
ioned poin s.
1.2 When o lay an egg
1.2.1 Female Pos -Ma ing Response
A e copula ion, emale lies ini ia e a se ies o changes in di e en compo-
nen s o hei beha iou known as he emale Pos -Ma ing Response (PMR).
The gene ic, physiological and neu onal basis o his esponse in D osophila
melanogas e ha e been ho oughly s udied o e he pas wen y yea s (Ca -
alho e al., 2006; Chen e al., 1988; Hei e z e al., 2014, 2000; Hussain e al.,
2016b; Liu and Kubli, 2003; Mack e al., 2006; McG aw e al., 2008; Peng e al.,
2005; Rezá al e al., 2014; Solle e al., 1999). The PMR is ini ia ed soon a e
ma ing (Liu and Kubli, 2003; McG aw e al., 2008; Shao e al., 2019) and i can
pe sis o se e al days (Ga be e al., 2016; Peng e al., 2005; Ra i Ram and
Wol ne , 2005). The h ee beha iou al hallma ks o he PMR a e: a dec ease
in emale sexual ecep i i y o new cou ing males (Chen e al., 1988; Connolly
and Cook, 1973), an inc ease in he a e o egg laying (Chen e al., 1988; Hei e z
e al., 2000) and a change in he eeding habi s ha co ela es wi h he nu i-
ional demand o egg p oduc ion, which is cha ac e ized by inc eased p e e ence
o yeas , sal and polyamines-en iched ood (Ba nes e al., 2008; Ca alho e al.,
2006; Hussain e al., 2016b; Ribei o and Dickson, 2010; Walke e al., 2015).
3

1.2.2 Ac i a ing he Pos -Ma ing Response
The ac i a ion o he PMR was shown o be mos ly dependen on he ac ion
o a male-speci ic pep ide −Sex Pep ide (SP) (Ga be e al., 2016; Peng e al.,
2005; Ribei o and Dickson, 2010; Walke e al., 2015). Du ing copula ion, SP
is ans e ed oge he wi h he male spe m in o he emale ep oduc i e ac
(Chen e al., 1988; Liu and Kubli, 2003; Peng e al., 2005). The neu onal basis
o SP-dependen modula ion in he emale ly is well dissec ed (Feng e al., 2014;
Häsemeye e al., 2009; Jang e al., 2017; Rezá al e al., 2014, 2012; Yang e al.,
2009; Yapici e al., 2008). SP binds o Sex Pep ide Recep o (SPR), which
is exp essed in a g oup o in e nal SP Senso y Neu ons (SPSNs) loca ed in
he u e us (Häsemeye e al., 2009; Yang e al., 2009). SPR ac i a ion educes
SPSNs’ exci abili y, which in u n dampens he ac i a ion o hei pos synap ic
pa ne s loca ed in he ly Ven al Ne e Co d (VNC) − he SP Abdomi-
nal Ganglion (SAG) Neu ons. SAG neu ons p ojec o he b ain whe e hey
elay he in o ma ion o ini ia e he PMR (Feng e al., 2014). The SAG down-
s eam a ge s in he cen al b ain we e ecen ly iden i ied, he eby un eiling
mo e de ails abou he PMR implemen a ion by he CNS (Wang e al., 2020,
2021). Al hough SP plays a key ole in inducing he PMR, e idence is g owing
on SP-independen PMR mechanisms. In pa icula , ch onic exposu e o he
sex phe omone 11-cis-Vaccenyl Ace a e (cVA) (Leb e on e al., 2015) and he
physical ac o copula ion (Shao e al., 2019) con ibu e o he emale PMR.
Mos o he pos -ma ing beha iou s a e he ou pu o neu onal ci cui s de-
elopmen ally con olled by wo ansc ip ion ac o s, ui less ( u) and dou-
blesex (dsx) (Demi and Dickson, 2005; Feng e al., 2014; Häsemeye e al.,
2009; Rezá al e al., 2014, 2012; Rideou e al., 2010; Walke e al., 2015; Wang
e al., 2021; Yang e al., 2009).
1.2.3 Ci cadian Modula ion
The iming o laying an egg is also egula ed by he ci cadian hy hm. Mon-
i o ing he egg-laying ac i i y o D. melanogas e in a 12h Ligh :Da k (L:D)
cycle shows a 24h pe iodici y ha is cha ac e ized by a peak in egg deposi ion
a he onse o he da k phase (Allemand, 1976; Asbu ne e al., 2005; Sheeba
e al., 2001). The egg deposi ion hy hm is main ained when lies a e subjec ed
o di e en pho ope iod and empe a u e condi ions, as well as a e changes in
he p o ein die quali y (Howlade e al., 2006; Sheeba e al., 2001). The pe sis-
4
ence o he egg-laying hy hm unde a ypical condi ions sugges s he exis ence
o a ci cadian egula o o egg-laying. A e copula ion, he PMR needs o be
in eg a ed wi h he ci cadian clock in o de o p ecisely con ol he iming o
egg-laying. How he CNS o ches a es his in eg a ion is s ill unknown.
1.2.4 Egg-laying Si e Value
The ‘when’ o lay an egg is no only dependen on he pos -copula o y e-
sponses and he ci cadian hy hm. The con ol o e he si e whe e he eggs
a e laid is o majo impo ance o species pe pe ua ion. D. melanogas e does
no display ma e nal beha iou s and he la ae show low mobili y, he eby i is
c ucial ha emales ind a good loca ion o lay hei eggs. Tha loca ion should
p o ide p o ec ion om p eda o s and pa hogenic mic oo ganisms, en i onmen-
al s esso s, and should also be able o sus ain la ae eeding. The e o e, in
spi e o he d i e o lay eggs a e ma ing, i he subs a e condi ions a e no
ideal o egg-laying, p egnan lies will pause egg deposi ion and sea ch o a be -
e egg-laying si e. Indeed, he e is e idence ha emales wi hhold eggs in he
ep oduc i e sys em un il ci cums ances a ou egg deposi ion (Allemand, 1976;
Asbu ne e al., 2005; D ummond-Ba bosa and Sp adling, 2001; Kacsoh e al.,
2015; Ku z e al., 2017; Le è e e al., 2012; Yang e al., 2008, 2015). Di e en
en i onmen al cues can igge a pause in egg-laying a dis inc s ages o he
p ocess, which can mani es as: 1) a dec ease in he a e o egg p oduc ion; 2)
a delay in he o ula ion o ma u e eggs (passing an egg om he o a ies o he
u e us); and 3) apop osis o egg p ecu so cells, which can be induced in ex eme
en i onmen al condi ions, he eby hal ing egg p oduc ion. Dep i ing lies o an
app op ia e subs a e o egg-laying (Asbu ne e al., 2005; D ummond-Ba bosa
and Sp adling, 2001; Yang e al., 2008, 2015), as well as ex eme empe a u es
(Asbu ne e al., 2005) a e su icien o educe egg deposi ion in ma ed emales.
D ummond-Ba bosa e al. (2001) obse ed ha ma ed emales main ained in a
p o ein-poo die educe he egg p oduc ion a e. Exposu e o p eda o s o o
pa hogenic bac e ia ha e also been shown o dec ease egg deposi ion and induce
egg e en ion in he o a ies (Kacsoh e al., 2015; Ku z e al., 2017; Le è e e al.,
2012). A s udy conduc ed by Kacsoh e al. (2015) ound ha apop osis o he
egg p ecu so cells is obse ed when ma ed D osophila a e exposed o emale
pa asi oid wasps. In e es ingly hough, a di e en s a egy is obse ed when
lies a e exposed o pa hogenic bac e ia (ex. Esche ichia coli) cha ac e ized by
5
he accumula ion o ma u e eggs in he o a ies (delay in o ula ion) (Ku z e al.,
2017).
1.3 Whe e o lay an egg
1.3.1 How o Sense he En i onmen
As al eady men ioned, ui lies a e e y selec i e abou he si e whe e
hey lay he eggs and, consequen ly, hey may spend a a iable amoun o ime
sea ching o a p ope egg-laying spo (Cu y and Axel, 2021; Vijayan e al.,
2021; Yang e al., 2008, 2015). I is widely accep ed ha D. melanogas e has a
s ong p e e ence o laying eggs in o ing ui s, which p o ide a so subs a e.
Flies use mul iple senso y modali ies o sea ch o possible subs a es: ol ac ion,
gus a ion, ision and mechanosensa ion a e all impo an o p o ide accu a e
in o ma ion abou he subs a e quali ies. The senso y cues ha s imula e o
inhibi egg-laying in D. melanogas e a e well-known ( e iewed in (Cu y e al.,
2019) and (A anha and Vasconcelos, 2018) (Fig. 1.1).
Figu e 1.1: Summa y o he senso y sys ems and some o he a ac i e (⊕)
and a e si e (⊖) egg-laying cues in ol ed in egg-laying si e selec ion.
Female D. melanogas e igu e adap ed om he collec ion o D osophila species images
a ailable in Nicolas Gompel´s labo a o y websi e (h p://gompel.o g/images-2).
Fo mos o he senso y cues, he i s -o de senso y neu ons a e iden i ied.
Howe e , much less is known abou how hese pe iphe ic senso y ci cui s com-
6
munica e wi h highe -b ain cen e s o accomplish egg-laying si e selec ion. In
he nex sec ions, I will e iew some o he senso y cues iden i ied o posi i ely
(a ac i e) o nega i ely (a e si e) modula e egg-laying.
Ol ac o y cues
Flies smell odou s om a dis ance o in si e by using he an ennae and he
maxilla y palps. The in en o y o ol ac o y cues e alua ed by D. melanogas e
du ing he choice o an egg-laying si e is as .
Egg-laying is s imula ed by ola ile limonene and alencene e penes
p esen in Ci us ui s, bo h de ec ed by Ol ac o y Senso y Neu ons (OSNs)
exp essing he ecep o O 19a loca ed in he an ennae. The a ac ion o Ci us
ui s p o ec s he la ae om pa asi oid wasps, which a e epelled by he smell
o alencene (Dweck e al., 2013). E hanol, one o he main me aboli es o
e men a ion p esen in o ing ui s, is simul aneously an a ac i e odou o
egg-laying (Azanchi e al., 2013) and a po en ial oxic compound o he la ae
(Schumann e al., 2021). Consequen ly, e hanol p e e ence is dose and con ex -
dependen . Female lies p e e o deposi eggs in ood subs a es con aining he
mos ecologically bene icial concen a ions o e hanol (3-5%) (Azanchi e al.,
2013). Ye , hey lay eggs in high-e hanol concen a ion subs a es (10-15%)
when acing p eda o s, as a way o p o ec he la ae om in ec ion (Kacsoh
e al., 2013; Milan e al., 2012). E idence sugges s ha bo h he ol ac o y and
gus a o y sys ems can con ibu e o e hanol disc imina ion (Azanchi e al.,
2013). E hylphenols de i ed om ui an ioxidan s p omo e egg-laying, and
i s de ec ion in adul lies is done by maxilla y palp neu ons exp essing he e-
cep o O 71a (Dweck e al., 2015). Egg-laying si e selec ion is also modula ed
socially h ough ol ac o y cues eleased by o he emales and by males. The
phe omone 9- icosene deposi ed by males in ood si es was shown o wo k
as an agg ega ion and an egg-laying a ac i e cue o emales. I is de ec ed
by O 7a-exp essing neu ons in he an ennae, he same ol ac o y senso y neu-
ons ha also de ec he lea ola ile E2-hexenal, ano he egg-laying en icing
compound (Lin e al., 2015). Ma ed emales also p o ide in o ma ion abou
high-quali y egg-laying si es o o he emales by ma king he ood spo s wi h a
combina ion o emale and male chemicals, including species-speci ic cu icula
hyd oca bons (CHC) and he phe omone cVA (p esen in he male spe m)
(Duménil e al., 2016).
7
inpu . Silencing aDNs abolishes he p e e ence o ma ed emales o lay eggs in
subs a es en iched in male-phe omones wi hou a ec ing he numbe o eggs
laid, sugges ing a ole o aDNs in egg-laying si e selec ion a he han on he
mo o execu ion o his beha iou (Nojima e al., 2021). These indings es ab-
lished a ci cui model o how he b ain coo dina es ma ing and egg-laying.
In he nex sec ion, I will expand hese ci cui s o hei downs eam pa ne s
elaying mo o commands o he VNC.
Figu e 1.4: Diag am o neu onal ci cui s in ol ed in egg-laying and hei
modula ion by ma ing and si e selec ion cues.
In e ac ions be ween di e en neu ons in ol ed in he PMR ( ed), egg-laying si e selec-
ion (yellow) and execu ion (ligh g een). In he b ain, ma ing s a us is p ocessed by
pC1-o iIN neu ons and subs a e cues a e in eg a ed by o iEN and aDN neu ons. In
ma ed emales, pC1-o iIN pa hway is silen and, hus, he ac i i y o o iENs and aDNs
p omo es egg-laying when a sui able subs a e is encoun e ed. o iIN connec ions de-
pic ed by dashed lines ep esen he absence o inhibi o y inpu on o o iEN, o iDN and
aDN neu ons in ma ed emales. Colou scheme in acco dance wi h Fig. 1.3. Adap ed
om (Nojima e al., 2021) and (Wang e al., 2020).
1.5.2 How: om he decision o he execu ion o laying an egg
Descending Ou pu s
The decision o laying an egg is execu ed due o he ac i i y o Descending
Neu ons (DNs) ha elay in o ma ion om he b ain o he VNC. The VNC,
he unc ional analogous o he e eb a e´s spinal co d, is connec ed wi h he
body ho acic appendages (legs and wings) and abdominal segmen s/o gans
o egula e physiology and execu e mo o p og ammes. I ecei es descending
signals om he b ain o ini ia e mo o ac ions, bu also sends ascending signals
ca ying mo o and senso y eedback (Fig. 1.5b) (Cou e al., 2020; Hsu and
Bhandawa , 2016). The VNC is loca ed pos e io ly o he b ain and comp ises
14

by h ee pai s o ho acic uni s (T1, T2 and T3) used wi h a egion called he
Abdominal ganglion (Abg) (Fig. 1.5a) (Cou e al., 2020).
Up o now, h ee DNs −pMN2,DNp42 and o iDNs −we e ound o be
in ol ed in egg-laying beha iou , e en hough, only DNp42 and o iDNs we e
cha ac e ized in a mo e in eg a i e way wi hin egg-laying ci cui s (Huo iala
e al., 2018; Kimu a e al., 2015; Vijayan e al., 2021; Wang e al., 2020).
Huo iala e al. iden i ied ha he geosmin a oidance LHAV1a1 neu ons ou -
pu o he descending neu on DNp42 (Fig. 1.4). Silencing DNp42 abolishes
geosmin a oidance du ing egg-laying, as obse ed upon LHAV1a1 inac i a ion.
The emale-speci ic dsx+ pMN2 was he i s DN desc ibed o elici he mo o
elemen s linked wi h egg deposi ion (Fig. 1.4) (Kimu a e al., 2015). Mo e
ecen ly, a se o dimo phic u+ o iposi ion descending neu ons (o iDNs) we e
cha ac e ized as necessa y and su icien o egg-laying. Silencing o iDNs abol-
ishes egg-laying in ma ed emales, whe eas hei ac i a ion igge s he mo o
elemen s o egg deposi ion: abdomen bending o con ac he subs a e, o ipos-
i o ex usion and egg expulsion. Imaging o iDNs ac i i y du ing spon aneous
egg-laying e ealed ha i s signal ises du ing he sea ch pe iod and peaks du -
ing egg deposi ion. In e es ingly, he a e o ise in o iDNs ac i i y du ing ex-
plo a ion is modula ed by subs a e quali y, which s ongly a ec s how quickly
o iDNs hi he h eshold o ini ia e he mo o p og amme o egg expulsion
(Vijayan e al., 2021). The ac i i y o o iDNs is no a ec ed by ma ing, as
abdomen bending and o iposi o ex usion a e simila ly igge ed in i gin and
ma ed emales, which sugges s a ma ing-dependen modula ion in b ain ci cui s
ups eam o O iDNs (Wang e al., 2020). Impo an ly, o iDNs a e in eg a ed
in he egg-laying ci cui composed by o iIN, o iEN and aDN neu ons (Fig. 1.4)
(Wang e al., 2020). Fu he s udies a e equi ed o cha ac e ize he down-
s eam pa ne s o o iDN neu ons in o de o d aw a mo e comple e pic u e o
a senso y-mo o pa hway dedica ed o egg-laying.
Mo o Execu o s
How is egg-laying execu ion, downs eam o DNs, implemen ed? The mo-
o ci cui s in ol ed in he execu ion o he egg-laying beha iou al p og amme
(sea ch, egg deposi ion and clean and es ) a e poo ly explo ed.
The neu al subs a es ha in eg a e commands om he b ain o gene a e
mo o ou pu s a e loca ed in he VNC and a e e e ed o as lowe mo o
15
Figu e 1.5: Logic o mo o con ol by he D osophila CNS.
(a) S uc u al and unc ional o ganiza ion o he VNC.
(b) In o de o gene a e mo o beha iou , he ne ous sys em in eg a es a a ie y
o s imuli; hose a e i s de ec ed by he pe iphe ic senso y sys ems (e.g. p obosics,
an ennae, legs, o iposi o ) and associa ed senso y ci cui s, which elay he senso y in o -
ma ion o he b ain. In he b ain, he in o ma ion is in eg a ed in highe b ain ci cui s
in ol ed in mo i a ion, lea ning, choice and in e nal s a e, which selec a beha iou al
ou come. The beha iou comes o ac ion ia b ain descending neu ons ha ac i a e
lowe mo o ci cui s in he VNC in ol ed in he execu ion and coo dina ion o mo o
beha iou . Mechanosenso y, p op iocep i e and chemical eedback om he pe iphe y
in o he VNC, as well as ascending inpu s in o he b ain, a e an impo an sou ce o
senso y eedback o he sys em; such eedback allows o ine une beha iou acco dingly
wi h he ex e nal condi ions and, also, ensu es a coo dina ed sequence o mo o e en s.
16
ci cui s. These ci cui s comp ise local neu ons and mo o neu ons. While
mo o neu ons gene a e mo o ou pu by di ec ly inne a ing muscle ibe s,
local neu ons ac on downs eam mo o neu ons o con ol beha iou (Fig. 1.5b)
(Pu es, 2001; Venka asub amanian and Mann, 2019).
The mo o execu ion o egg-laying implies he coo dina ion among be-
ha iou s ha p omo e o ula ion, e iliza ion, sea ch and, ul ima ely, egg
deposi ion in o he subs a e. The e is suppo ing e idence ha dsx-exp essing
neu ons a e key in he con ol o egg-laying beha iou . Silencing ei he
he ac i i y o all dsx+ neu ons, o a es ic ed subse o ∼9 VNC
dsx+/oc opamine gic neu ons, is su icien o educe, o e en abolish
egg-laying in ma ed emales; in u n, hei ac i a ion igge s an inc ease in
he numbe o eggs laid by i gin emales (Rezá al e al., 2014; Rideou e al.,
2010). Ye , how and which sub-popula ions o he dsx+ VNC ne wo k con ol
he egg-laying mo o p og amme is s ill o be unco e ed.
The mechanisms by which he VNC con ols he o iduc muscles o p o-
mo e o ula ion a e ai ly unde s ood. The emale ep oduc i e ac is a mus-
cula sys em which dis ends when he egg passes h ough, being inne a ed by
mo o and senso y p ocesses. The e is moun ing e idence ha o ula ion in
D osophila is modula ed by glu ama e and OA (Lee e al., 2003; Lim e al.,
2014; Rod íguez-Valen ín e al., 2006). While OA p omo es o iduc elaxa ion
by binding o OAMB and Oc β2R ecep o s in he o iduc wall, glu ama e in-
duces o iduc con ac ions (Lee e al., 2003; Lim e al., 2014; Rod íguez-Valen ín
e al., 2006). Impai ing oc opamine gic signalling esul s in s e ile emales wi h
enla ged o a ies ull o ma u e eggs ha a e no o ula ed and, he e o e, no
laid (Lee e al., 2003; Li e al., 2015; Lim e al., 2014; Monas i io i, 2003; Mona-
s i io i and Linn, 1996). In e es ingly, he e-es ablishmen o OA p oduc ion in
a popula ion o Abg neu ons inne a ing he o a ies and o iduc s is su icien o
escue he s e ile pheno ype (Monas i io i, 2003). Mo eo e , s imula ing a clus-
e o Abg glu ama e gic neu ons inne a ing he o iduc igge s ep oduc i e
ac con ac ions (Gou e al., 2014) while i s silencing causes eggs jammed in
he o iduc s (Cas ellanos e al., 2013; Gou e al., 2014; Yang e al., 2008).
17
1.6 Analogous Lowe Mo o Ci cui s
1.6.1 D osophila Male Sexual and Locus Egg-laying Be-
ha iou s
Insec s a e p ime models o s udy he neu onal basis o mo o beha iou s.
Wo ks on D. melanogas e and locus species (Locus a mig a o ia mig a o ioides
and Schis oce ca ame icana) ha e been undamen al o ou comp ehension o
he iden i y and unc ion o lowe mo o ci cui s in a wide ange o beha iou s,
om locomo ion o ep oduc ion (Ayali and Lange, 2010; Feng e al., 2020;
Howa d e al., 2019). He e, I will b ie ly e iew wo s udies, which p o ide
gene al p inciples o how mo o ci cui s a e o ganized o coo dina e ep oduc ion
in insec s. I will i s in oduce a ci cui in D osophila males in ol ed in he
success ul execu ion o geni al coupling du ing cou ship (Pa lou e al., 2016).
Nex , I will e iew wo k on ci cui s con olling di e en beha iou s du ing egg
deposi ion in locus emales (Ayali and Lange, 2010; Lange, 2009a,b).
The s udy unde aken by Pa lou e al. showed ha he ini ia ion and e mi-
na ion o geni al coupling in males is coo dina ed by an Abg ci cui composed
by wo dsx+ popula ions: a glu ama e gic subse (dsx+/VGlu +) ha inne -
a es he male geni alia, and a local GABAe gic subse (dsx+/Gad1+). The
dsx+/VGlu + neu ons ini ia e copula ion by allowing geni al coupling wi h he
emale, whe eas dsx+/Gad1+ neu ons media e geni al uncoupling o e mina e
copula ion, likely by ga ing he ac i i y o he dsx+/VGlu + popula ion. This
ci cui is modula ed by senso y eedback om dsx+ mechanosenso y neu-
ons loca ed in he geni alia, and is poised o connec wi h local dopamine gic
neu ons o modula e copula ion pe sis ence (Fig. 1.6a).
The wo k de eloped o e he pas yea s in locus has been ins umen al o
unde s and how VNC ci cui s coo dina e a wide ange o beha iou s. He e, I
will ocus on he mo o con ol o egg-laying beha iou . The knowledge p e-
sen ed he e is based on h ee exhaus i e e iews on he ield (Ayali and Lange,
2010; Lange, 2009a,b). The locus ep oduc i e sys em has iden ical ana om-
ical s uc u e o he one in D osophila: wo o a ies linked by la e al o iduc s
con e ging on a cen al o iduc connec ed wi h he geni al chambe o u e us.
The e is one spe ma heca o s o e spe m and a pai o accesso y glands. Du ing
egg-laying, emales dig a deep hole in he subs a e wi h he o iposi o al es
o bu ow he eggs. While digging he subs a e, emales e ain he o ula ed
18
eggs in he la e al o iduc s and, only once he digging p ocess is comple ed,
he eggs descend o he u e us o e iliza ion and expulsion. The e o e, he
coo dina ion be ween digging, egg e en ion and e iliza ion beha iou s is c u-
cial o success ul egg-laying. How is his coo dina ion accomplished o p oduce
an o de ly sequence o beha iou s? Th ee mo o ci cui s loca ed in he ab-
dominal ganglion we e shown o con ol digging, egg e en ion and e iliza ion
(Fig. 1.6b). The digging ci cui inne a es he o iposi o al es o induce
con ac ions o subs a e digging. An egg- e en ion ci cui whose ac i i y
s imula es o iduc con ac ions ensu es ha eggs a e no expelled du ing he
digging p ocess. The coo dina ed ac i i y o digging and egg- e en ion neu ons
ensu es ha eggs a e only expelled once he hole is comple ed. The digging
ci cui ecei es senso y eedback om mechanosenso y neu ons in he o iposi-
o al es o main ain o s op digging beha iou . The egg hen descends o he
u e us whe e e iliza ion mus occu be o e expulsion. A e iliza ion ci cui
p omo es con ac ions o he spe ma heca o spe m elease and e iliza ion.
The e iliza ion module likely ecei es eedback om senso y neu ons sensing
he egg in he u e us o ensu e e iliza ion a he app op ia e iming (Ayali and
Lange, 2010; Lange, 2009a,b).
Taken oge he , hese s udies allow us o specula e ha he same ci cui
logic may be applied in he con ol o egg-laying beha iou in D. melanogas e .
In my hesis wo k, I d ew inspi a ion om he wo k ou lined in he p e i-
ous sec ions, and used D. melanogas e egg-laying beha iou as a pa adigm o
u he ou unde s anding o he mechanisms unde lying mo o beha iou .
19

Figu e 1.6: VNC mo o ci cui s con olling ep oduc i e beha iou s in in-
sec s.
(a) Model o he ipa i e ci cui con olling geni al coupling in D osophila male.
Adap ed om (Pa lou e al., 2016).
(b) The h ee mo o ci cui s loca ed in he Abg ha con ol egg- e en ion, digging
and e iliza ion beha iou s in locus . These ci cui s a e loca ed in he VII and VIII h
segmen s o he Abg. Egg-laying is accomplished by he elonga ion o he abdomen
in o he subs a e, while digging a hole by using he o iposi o al es. Adap ed om
(Lange, 2009a).
20
Chap e 2
Egg-laying beha iou in wild
ype lies
21
2.1 Cha ac e iza ion o egg-laying beha iou
In o de o unde s and how he execu ion o he egg-laying mo o p og amme
is coo dina ed, we analysed wild- ype egg-laying beha iou in de ail. Fig. 2.1a
shows a schema ic ep esen a ion o he beha iou al se up. Single ma ed e-
males we e placed in an a ena lined wi h 1% aga ose on h ee walls. Videos
we e eco ded o 45 minu es. We i s analysed he empo al s uc u e o egg
expulsion (Fig. 2.1b). Fo he du a ion o he ideo, each emale laid om 4 o
17 eggs, wi h a median o 11.5 (Fig. 2.1c). All eggs we e laid on he aga ose
walls and 94% o eggs we e bu ied in he aga ose. The median in e -egg ex-
pulsion in e al calcula ed o each emale anges om 2 o 3 minu es wi h he
excep ion o one ly ( ly#7) ha showed a median o 5 minu es (Fig. 2.1d).
These da a indica e a low in e -indi idual a iabili y when ea ing condi ions
and en i onmen a e con olled. To ob ain a de ailed desc ip ion o egg-laying
beha iou , we analysed egg-laying mo o elemen s which we associa ed wi h
di e en egg-laying phases. Egg-laying beha iou in ol es: 1) an explo a ion
phase whe e he ly p esumably sea ches o app op ia e egg-laying si es, 2) he
egg deposi ion phase in which he o iposi o mo o p og amme is ac i a ed o
lay an egg, and 3) he es phase. We chose o call he es phase, ‘abdominal
con o ions’, because we and o he s (B äcke e al., 2019; Cu y and Axel, 2021)
obse ed ha his phase is accompanied by s ong and p olonged abdominal
con o ions, as desc ibed below. Fig. 2.1e shows ideo ames cap u ing each
o he di e en egg-laying mo o elemen s. Some beha iou al denomina ions
we e based on o he desc ip ions o egg-laying (B äcke e al., 2019; Cu y and
Axel, 2021). Egg deposi ion mo o elemen s all p og ess in a sequence culmi-
na ing in egg expulsion and ending wi h g ooming o he e minalia. We show
mo e han one ideo ame o egg pushing as i has wo di e en pos u es. Ab-
dominal con o ions, as men ioned ea lie , a e he single mo o elemen o i s
phase. We de ined explo a ion mo o elemen s as elemen s whe e emales p obe
he subs a e ei he wi h he o iposi o o he p oboscis wi hou p og essing in
con inuous sequence o egg expulsion.
To unde s and he empo al sequence o he di e en beha iou al elemen s,
we plo ed he p obabili y o each mo o elemen a ound he momen o egg ex-
pulsion (Fig. 2.2a-d). Du ing egg deposi ion phase, he mo o elemen s leading
o egg expulsion a e pe o med e e y ime an egg is deposi ed (Fig. 2.2a). A e
an egg is expelled, he emale cu ls he abdomen while walking away, which can
22
Figu e 2.1: Egg-laying beha iou in wild ype lies.
(a) Schema ic o se up and a enas used o eco d egg-laying beha iou . Each a ena is
composed by wo chambe s allowing o eco d wo lies simul aneously. The egg-laying
subs a e used in his s udy was 1% aga ose. In a ed (IR) LEDs we e used o illu-
mina ion and IR came as we e used o eco d ly beha iou . (b) Ras e plo showing
he empo al p o ile o egg expulsion bou s. Each black do ma ks he momen o egg
expulsion. n = 10 lies. (c) Numbe o eggs laid by ma ed Can on S (CS) lies. Median
= 11.5; o al numbe o lies = 10; o al numbe o eggs = 112. (d) Time in e als be-
ween egg expulsion bou s pe ly. Each do ep esen s he in e al be ween consecu i e
egg deposi ion bou s. Median alues o he dis ibu ion o each ly a e shown on he
igh side. n = 10 lies. (e) Video snapsho s illus a ing he di e en egg-laying mo-
o pa e ns analysed wi hin each egg-laying phase. Open iangles be ween snapsho s
deno e sequen ial beha iou s. O iposi o con ac (ma ked by he black a owhead) is
de ined by abdomen bending accompanied by ex usion o he o iposi o and con ac
wi h unde lying subs a e. I can culmina e in egg expulsion o no . Bu owing is
cha ac e ized by sc a ching he su ace e en ually leading o digging he subs a e wi h
he o iposi o . Egg pushing is cha ac e ized by a igid pos u e ha ini ia es a he end
o bu owing beha iou and accompanies egg expulsion. No e ha his beha iou can
be displayed in wo di e en pos u es: 1. e ec body pos u e ( ly head is ele a ed in
ela ion o he abdomen, o 2. leaning body pos u e ( ly leans owa ds he subs a e).
Abdomen cu ling is cha ac e ized by cu ling he abdomen ollowed by walking o wa d,
ei he li ing he cu led abdomen, o by d agging i h ough he subs a e. G ooming
e minalia is sel -explana o y. Abdominal con o ions a e undula ed abdominal mo e-
men s ( ep esen ed by he shape o he black line) accompanied by ex usion o he
o iposi o . P oboscis ex ension is cha ac e ized by he p oboscis being ex ended o
con ac he subs a e (ma ked by he black a ow).
23
Figu e 3.3: Ch onic silencing o O Abg neu ons blocks egg-laying and p o-
mo es egg-jamming.
(a) Numbe o eggs laid pe emale in he 24h a e ma ing du ing inhibi ion o O Abg
neu ons. n = 45 (con ol) and 55 (O Abg) emales. Mean ±s.d. is shown on op he
sca e plo . Mann-Whi ney es , ****p < 0.0001. (b) Pe cen age o emales wi h eggs
jammed in he la e al o iduc s du ing inhibi ion o O Abg neu ons. n = 32 (con ol)
and 36 (O Abg) emales. Fishe ´s exac es , ****p < 0.0001. (c) Image ep esen ing
an O Abg emale ep oduc i e sys em ( igh ) wi h wo eggs jammed (whi e a ows)
in he la e al o iduc s and a con ol (le ) ep oduc i e sys em wi h no eggs in he
o iduc s. No e ha , besides he egg a es ing, O Abg silenced emales also ha e en-
la ged o a ies con aining mo e ma u e eggs when compa ed wi h con ol o a ies.
This esul oge he wi h he obse a ion ha lies su i e and appea
heal hy wi h cons i u i e silencing o O Abg neu ons indica es ha hey a e
speci ically in ol ed in egg-laying. To asce ain ha egg-laying de ec does no
esul om a de ec in egg p oduc ion, we dissec ed he ep oduc i e sys em.
We obse ed eggs jammed in he la e al o iduc s in all es lies oge he
wi h an excess o ma u e eggs in he o a ies (Fig. 3.3b-c), indica ing ha egg
p oduc ion is no a ec ed.
In his se ies o expe imen s, we ound a g oup o neu ons ha a e pa o
he egg-laying mo o ci cui s as hey a e di ec ly in ol ed in he execu ion o egg
pushing and egg expulsion, and ha a e necessa y o egg-laying. These Abg
neu ons p o ide a g ea en y poin o add ess how di e en ci cui s coo dina e
o execu e egg deposi ion.
30

3.2 Silencing O Abg neu ons dis up s all mo o ele-
men s associa ed wi h egg-laying beha iou
We ha e shown ha upon ac i a ion o O Abg neu ons a single egg deposi-
ion mo o elemen - egg pushing - is induced and ha O Abg silenced emales
do no lay eggs. How do O Abg silenced emales beha e? Do hey pe o m all
he beha iou elemen s wi h he excep ion o egg pushing, o a e o he mo o
elemen s a e a ec ed?
To answe hese ques ions we used he anion channel hodopsin G ACR1
(Mohammad e al., 2017) o acu e op ogene ic silencing o O Abg neu ons.
We analysed 15 minu es o ligh s imula ion as well as 10 minu es p e- and 5
minu es pos -s imula ion (Fig. 3.4a). Acu e silencing o O Abg neu ons blocked
egg-laying; he numbe o eggs laid du ing he s imula ion was se e ely educed
and pa ially eco e ed in he pos -s imula ion pe iod (Fig. 3.4a and b).
Figu e 3.4: Acu e silencing o O Abg neu ons blocks egg-laying
(a) ( op) Neu onal G ACR1 silencing p o ocol scheme. G een shaded a ea ep esen s
he s imula ion pe iod. (bo om) Ras e plo shows he egg expulsion e en s. n = 19
(O Abg) and 8 (con ol) lies. (b) Quan i ica ion o he numbe o eggs laid pe ly by
O Abg and con ol emales du ing 5 min pe iods. n = 19 (O Abg) and 8 (con ol) lies.
P e: Mann-Whi ney es , ns p≥0.05; Silencing: Mann-Whi ney es , ****p < 0.0001;
Pos : Mann-Whi ney es , *p < 0.05.
Analysis o he beha iou al elemen s showed ha , wi h he excep ion o
g ooming, all egg deposi ion mo o elemen s a e abolished du ing s imula ion
and pa ially eco e ed pos -s imula ion (Fig. 3.5a-e). The expulsion o ou
eggs du ing he silencing pe iod was done wi hou using mos o he egg de-
posi ion mo o p og amme (Supplemen a y Video 4). The numbe o e mi-
nalia g ooming bou s does no di e om con ol du ing silencing (Fig. 3.5e).
31
Howe e , he ime he emale spen g ooming he e minalia is much la ge in
he es condi ion du ing silencing (Fig. 3.5 ). In e es ingly, bo h measu es o
g ooming a e educed compa ed o con ol in he pos -s imula ion pe iod, sug-
ges ing a ebound e ec on ci cui s modula ing g ooming beha iou . The da a,
so a , shows a wide e ec o silencing O Abg neu ons in all he egg deposi ion
elemen s.
Figu e 3.5: Silencing O Abg neu ons dis up s all mo o elemen s associa ed
wi h egg deposi ion phase.
Quan i ica ion o he numbe o beha iou bou s du ing 5 min pe iods. n = 19 (O Abg)
and 8 (con ol) lies. P e: Mann-Whi ney es in (a),(c) and - es in (b),(d),(e)
ns ≥0.05; Silencing: Mann-Whi ney es in (a),(e), ****p < 0.0001 and ns ≥0.05
and - es in (b),(c),(d), **p < 0.01; Pos : Mann-Whi ney es in (a),(b),(c),
(d),(e), *p < 0.05. ( ) Quan i ica ion o he mean du a ion o g ooming e minalia
bou s. n = 19 (O Abg) and 8 (con ol) lies. P e: Mann-Whi ney es , ns p≥0.05;
Silencing: - es , ****p < 0.0001; Pos : Mann-Whi ney es , **p < 0.01.
We nex analysed he o he egg-laying phases. Abdominal con o ions a e
educed compa ed o con ol, bo h in numbe o bou s (Fig. 3.6a) and beha iou
du a ion (Fig. 3.6b). Addi ionally, he in ensi y o he con o ions and he ex-
en o he o iposi o ex usion a e educed in es lies compa ed o con ols,
as exempli ied in Fig. 3.6c. The beha iou elemen s o he explo a ion mo o
p og amme a e educed du ing silencing (Fig. 3.6d- ). In e es ingly, bo h he
32
explo a ion and abdominal mo o p og ammes du ing he pos -s imula ion pe-
iod a e no di e en om he espec i e con ols (Fig. 3.6a-b, d- ), indica ing
ha he egula ion o hese phases is simple han ha o he egg deposi ion
p og amme whe e he inhibi o y e ec s o G ACR1 s imula ion pe sis .
Figu e 3.6: Silencing O Abg neu ons dis up s all mo o elemen s associa ed
wi h abdominal con o ions and explo a ion phases
(a) and (b) Quan i ica ion o he numbe o abdominal con o ions bou s du ing 5 min
pe iods and he co esponding bou mean du a ion. n = 19 (O Abg) and 8 (con ol)
lies. P e: - es in (a) and Mann-Whi ney es in (b), ns p ≥0.05; Silencing: Mann-
Whi ney es in (a) and (b) *p < 0.05; Pos : Mann-Whi ney es in (a) and - es in
(b), ns p ≥0.05. (c) Video snapsho s o es (bo om) and con ol ( op) lies displaying
abdominal con o ions du ing he silencing pe iod. Silenced lies also display less ex-
ended o iposi o ex usions du ing abdominal con o ions (a owheads). (d),(e) and
( ) Explo a ion phase-associa ed mo o elemen s and co esponding quan i ica ion o
he numbe o beha iou bou s du ing 5 min pe iods. n = 19 (O Abg) and 8 (con ol)
lies. P e: - es in (d) and Mann-Whi ney es in (e) and ( ), ns p ≥0.05; Silencing:
Mann-Whi ney es in (d),(e), and ( ), **p < 0.01, ***p < 0.001, ****p < 0.0001;
Pos : Mann-Whi ney es in (d),(e), and ( ), ns p ≥0.05.
Ou indings show ha silencing O Abg neu ons a ec s all phases o egg-
laying beha iou . This d ama ic esul could e lec a di ec in ol emen o
O Abg neu ons in all egg-laying phases. Al e na i ely, hey could e lec an
33
a es on he egg-laying cycle (Fig. 2.2e) induced by he loss o egg pushing
beha iou and inabili y o comple e egg deposi ion.
34
3.3 Ac i i y o GABAe gic O Abg neu ons blocks
egg-laying
To add ess how di e en neu ons wi hin he O Abg popula ion con ibu e o
he execu ion o egg-laying, we used an in e sec ional app oach o ob ain unc-
ional subg oups (Diao e al., 2015). The GABAe gic O Abg (O Abg/Gad1)
neu ons will be discussed he e while choline gic and glu ama e gic O Abg neu-
ons will be discussed in he ensuing sec ions. O Abg/Gad1 neu ons (∼96
neu ons, n=7 lies) a e local in e neu ons wi h spa se and ain p ojec ions o
o he VNC ganglia (Fig. 3.7a). No p ojec ions o O Abg/Gad1 neu ons we e
obse ed in he b ain (Fig. 3.7b) o he ep oduc i e sys em (Fig. 3.7c).
Figu e 3.7: GABAe gic O Abg ana omy.
(a),(b), and (c) Con ocal images o emale VNC (a), b ain (b) and ep oduc i e
sys em (c) o O Abg/Gad1 neu ons and co esponding inne a ions s ained wi h an i-
GFP (g een) o e eal he ana omy and nc82 o neu opil. An i-F-ac in was used in
(c) o isualize he muscle ibe s. o a : o a y; o : o iduc s; s : seminal ecep acle; u :
u e us. An i-GFP is a ge ing he luo escen p o ein Venus om O Abg/Gad1-LexA
> CsCh imson-mVenus exp essing lies. Scale ba s a), b) 50 µm and c) 200 µm.
Silencing O Abg/Gad1 had no e ec on he numbe o eggs laid in 24h
(Fig. 3.8a) and i s op ogene ic ac i a ion did no elici any beha iou associ-
a ed wi h he egg-laying mo o p og amme (da a no shown). The e o e, i
O Abg/Gad1 neu ons con ibu e o egg-laying, hey may do so by inhibi ing
egg-laying. To es his, we ac i a ed O Abg/Gad1 neu ons o e nigh (16h ac-
i a ion) and measu ed he numbe o eggs laid. We obse ed ha ac i a ion o
O Abg/Gad1 abolishes egg-laying (Fig. 3.8b) and, he dissec ion o he o a ies
a he end o he expe imen , e ealed ha he eggs a e jammed a he la e al
o iduc (Fig. 3.8c-d).
In summa y, he esul s show ha , du ing egg-laying, O Abg/Gad1 neu ons
a e silen , and ha ac i i y in O Abg/Gad1 neu ons p e en s egg-laying. This
subse o O Abg neu ons con ibu es o opposing ou comes compa ed o he
35

gene al line and, hus, ha e he po en ial o ga e egg-laying execu ion by o he
neu ons in he O Abg popula ion.
Figu e 3.8: Ac i a ion o O Abg GABAe gic neu ons is su icien o block
egg-laying.
(a) Numbe o eggs laid pe emale in he 24h a e ma ing du ing inhibi ion o O -
Abg/Gad1 neu ons. n = 46 (con ol) and 49 (O Abg/Gad1) emales. Mean ±s.d. is
shown on op o he sca e plo . Mann-Whi ney es , ns p ≥0.05. (b) Numbe o
eggs laid pe emale du ing he 16h pho oac i a ion wi h CsCh imson o O Abg/Gad1
neu ons. n = 29 (con ol) and 40 (O Abg/Gad1) emales. Mean ±s.d. is shown on
op o he sca e plo . Mann-Whi ney es , ****p < 0.0001. (c) Pe cen age o emales
wi h eggs jammed in he la e al o iduc s a e 16h pho oac i a ion o O Abg/Gad1
neu ons. n = 25 (con ol) and 35 (O Abg/Gad1) emales. Fishe ´s exac es , ****p
< 0.0001. (d) (bo om) Image ep esen ing a ep oduc i e sys em o an O Abg/Gad1
emale a e 16h pho oac i a ion wi h one egg jammed (yellow a ow) in he la e al
o iduc and a con ol ( op) ep oduc i e sys em wi h clea o iduc s. No e ha O -
Abg/Gad1 ac i a ed emales also ha e enla ged o a ies con aining mo e ma u e eggs
when compa ed wi h con ol o a ies (simila o O Abg silencing egg jamming pheno-
ype, see igu e 3.3c).
36
3.4 Choline gic O Abg neu ons a e necessa y and
su icien o egg deposi ion
Choline gic neu ons (O Abg/Cha) a e a la ge ac ion o O Abg neu ons
(Fig. 3.9a) ha include p ojec ions o he b ain (Fig. 3.9b) and he ep oduc i e
sys em (Fig. 3.9c). Silencing O Abg/Cha neu ons leads o a se e e educ ion
in he numbe o eggs laid (Fig. 3.9d) and a la ge ac ion o he emales display
egg jamming in he la e al o iduc (Fig. 3.9e). These esul s show a e y simila
pheno ype o ha obse ed when silencing all O Abg neu ons (Fig. 3.3a-b).
Figu e 3.9: Silencing Choline gic O Abg neu ons blocks egg-laying.
(a),(b), and (c) Con ocal images o emale VNC (a), b ain (b) and ep oduc i e
sys em (c) showing O Abg/Cha neu ons and co esponding inne a ions s ained wi h
an i-GFP (g een) o e eal he memb anes and nc82 o synapses. An i-F-ac in was
used in (c) o isualize he muscle ibe s. o a : o a y; o : o iduc ; u : u e us. An i-
GFP is a ge ing he luo escen p o ein Venus om O Abg/Cha-LexA > CsCh imson-
mVenus exp essing lies. Scale ba s a), b) 50 µm and c) 200 µm. (d) Numbe o eggs
laid pe emale in he 24h a e ma ing du ing inhibi ion o O Abg/Cha neu ons. n =
45 (con ol) and 25 (O Abg/Cha) emales. Mean ±s.d. is shown on op o he sca e
plo . Mann-Whi ney es , ****p < 0.0001. (e) Pe cen age o emales wi h eggs jammed
in he la e al o iduc s du ing inhibi ion o O Abg/Cha neu ons. n = 28 (con ol) and
19 (O Abg/Cha) emales. Fishe ´s exac es , ****p < 0.0001.
Likewise, ac i a ion o O Abg/Cha neu ons using he p o ocol shown in
Fig. 3.2a leads o bo h i gin and ma ed emales assuming an egg pushing
37
pos u e each ime he ligh is ON (Fig. 3.10a-b, Supplemen a y Video 5), as
obse ed when all O Abg neu ons a e ac i a ed (Fig. 3.2b-c). In e es ingly,
quan i ica ion o he numbe o emales laying eggs du ing he s imula ion p o-
ocol e ealed ha all O Abg/Cha emales laid one egg (Fig. 3.10c), in con as
o less han hal o O Abg emales (Fig. 3.2 ).
Figu e 3.10: O Abg Choline gic neu ons a e in ol ed in egg pushing and
expulsion.
(a) Pe cen age o s imula ion e en s in which O Abg/Cha lies displayed an egg
pushing-like pos u e du ing pho oac i a ion wi h CsCh imson. n = 150 (con ol), 144
(M, O Abg/Cha) and 72 (V, O Abg/Cha) s imula ions. (b) Video snapsho (la e al
iew) o an O Abg/Cha emale displaying an egg pushing-like pos u e in esponse o
he s imula ion wi h CsCh imson. (c) Pe cen age o O Abg/Cha emales ha lay eggs
du ing pho oac i a ion wi h CsCh imson. n = 25 (con ol) and 24 (O Abg/Cha) e-
males. Fishe ´s exac es , ****p < 0.0001. (d) Pe cen age o eggs laid by O Abg/Cha
emales du ing s imula ions and ISI. n = 24 eggs. (e) La ency (seconds) o egg expul-
sion (pe iod o ime o egg expulsion du ing s imula ion) o O Abg and O Abg/Cha
pho oac i a ed emales. n = 9 (O Abg) and 24 (O Abg/Cha) emales. Mean ±s.d. is
shown on op o he sca e plo . Mann-Whi ney es , ****p < 0.0001.
Addi ionally, all eggs laid du ing he s imula ion p o ocol by O Abg/Cha e-
males we e laid du ing he i s s imulus (Fig. 3.10d), whe eas egg-laying iming
by O Abg emales du ing he s imula ion p o ocol was a iable, wi h emales
38
laying eggs in he hi d and i h s imulus as well as in he in e s imulus in e -
als (Fig. 3.2g). The esul s show ha , upon ac i a ion, i and when an egg
is laid is a iable o O Abg, bu no o O Abg/Cha emales. We also quan-
i ied, wi hin he 10 second s imula ion bou , when he emales expelled he
egg. We ound a s iking di e ence be ween O Abg and O Abg/Cha emales
(Fig. 3.10e), wi h O Abg emales aking a longe ime o expel he egg. The
inc eased a iabili y ega ding when he egg is expelled du ing he s imula ion
p o ocol, and he inc eased la ency o lay an egg upon ligh ON o he O Abg
emales compa ed o he O Abg/Cha emales, likely esul s om inhibi ion by
he GABAe gic neu ons in he O Abg popula ion. Ac i a ion o O Abg neu-
ons encompasses simul aneous ac i a ion o inhibi o y O Abg/Gad oge he
wi h egg-laying p omo ing O Abg/Cha, which esul s in con lic ing in o ma-
ion leading o a iabili y and delay o he beha iou al execu ion.
O e all, he esul s indica e ha O Abg/Cha neu ons unde lie he execu ion
o egg pushing leading o egg expulsion. Unlike egg-laying beha iou o wild-
ype lies, eggs expelled by op ogene ically ac i a ed O Abg/Cha emales we e
ne e bu ied and we e equally dis ibu ed be ween he ac ylic and he aga ose
su aces, highligh ing he impo ance o o he beha iou al componen s o egg-
laying si e selec ion and egg bu ial.
39
4.1 Supplemen a y Figu e 4.1
46

Supplemen a y Figu e 4.1
(a) and (b) Con ocal images o emale abdominal muscles in he nsyb-Gal4 (a) and
O Abg line (b). Neu onal inne a ions a e s ained wi h an i-GFP (g een) and muscle
ibe s wi h an i-F-ac in (magen a). A7 and A8 indica e he posi ion o he abdomi-
nal segmen s. nsyb-Gal4 line exp ession is shown o compa ison wi h he O Abg line
exp ession. An i-GFP is a ge ing he luo escen p o ein GFP and Venus om nsyb-
Gal4 > mCD8::GFP and O Abg > CsCh imson-mVenus lies. Scale ba s a) and b)
50 µm. (c-k) Con ocal images o O Abg neu onal pola i y in he emale VNC (c-e),
b ain ( -h) and ep oduc i e sys em (i-k). Dend i es (inpu s) a e labelled using he
soma odend i ic ma ke , DenMa k, and axons (ou pu s) a e labelled using he synap ic
esicle ma ke , Synap o agmin. An i-GFP is a ge ing EGFP- agged Synap o agmin
and an i-DsRed is a ge ing mChe y- agged DenMa k. Scale ba s c-k) 50 µm. (l-n)
Con ocal images o male b ain (l), as well as male (m) and emale (n) do sal iew o
VNC showing O Abg neu ons and co esponding inne a ions s ained wi h an i-GFP
(g een) o e eal he ana omy and nc82 o synapses. An i-GFP is a ge ing he luo-
escen p o ein Venus om O Abg > CsCh imson-mVenus exp essing lies. Scale ba s
l), m) and n) 50 µm. (o) Pe cen age o s imula ion e en s in which male O Abg lies
displayed abdomen cu ling and aedeagus ex usion beha iou s du ing pho oac i a ion
wi h CsCh imson. n = 48 (con ol) and 48 (O Abg) s imula ions. (p) Video snapsho
(la e al iew) o O Abg male displaying abdomen cu ling and aedeagus ex usion (as-
e isk) beha iou s in esponse o he s imula ion wi h CsCh imson ( op). A snapsho
o he same male du ing a ligh o pe iod (below) is also shown o compa ison. (q)
Pe cen age o ecep i e emales du ing inhibi ion o O Abg neu ons. n = 60 (con ol)
and 62 (O Abg) emales. Fishe ´s exac es , ns p ≥0.05.
47
4.2 Supplemen a y Figu e 4.2
aand bP obabili ies o g ooming e minalia beha iou du ing a 1-min ime window
a ound egg expulsion o (a) con ol and (b) O Abg/VGlu silenced lies. Time = 0
minu es ma ks he momen o egg expulsion ( ep esen ed by he g ey e ical line). n
= 126 (con ol) and n = 105 (O Abg/VGlu ) egg expulsions.
48
4.3 Supplemen a y Videos
Videos can be accessed ough his link: www.bio xi .o g/con en /10.1101/2021.
08.23.457359 1.supplemen a y-ma e ial
4.3.1 Supplemen a y Video 1
Mo o elemen s displayed by Can on S ma ed emales du ing he explo a ion and egg
deposi ion phases.
4.3.2 Supplemen a y Video 2
Abominal con o ions displayed by Can on S ma ed emales du ing he abdominal con-
o ions phase.
4.3.3 Supplemen a y Video 3
CsCh imson op ogene ic s imnula ion o O Abg neu ons in ma ed emales. The i s
pa o he ideo shows egg pushing accompained by egg expulsion. The second pa o
he ideo shows egg pushing wi hou egg expulsion. The ed do on he op igh side
o he ideo ma ks he s imula ion pe iod.
4.3.4 Supplemen a y Video 4
Egg deposi ion bou du ing G ACR1 inhibi ion o O Abg neu ons in ma ed emales.
The g een do on he op igh side o he ideo ma ks he s imula ion pe iod.
4.3.5 Supplemen a y Video 5
CsCh imson op ogene ic s imnula ion o O Abg/Cha neu ons in ma ed emales. The
i s pa o he ideo shows egg pushing accompained by egg expulsion. The second
pa o he ideo shows egg pushing wi hou egg expulsion. The ed do on he op
igh side o he ideo ma ks he s imula ion pe iod.
4.3.6 Supplemen a y Video 6
CsCh imson op ogene ic s imnula ion o O Abg/VGlu neu ons in ma ed emales elic-
i ing o iposi o con ac beha iou . The ed do on he op igh side o he ideo ma ks
he s imula ion pe iod.
4.3.7 Supplemen a y Video 7
Egg deposi ion bou du ing Ki 2.1 inhibi ion o O Abg/VGlu neu ons in ma ed e-
males.
49
4.4 Supplemen a y Table 1. Fly s ocks
50
4.5 Supplemen a y Table 2. Full geno ypes used in
expe imen s
51

Chap e 5
Expe imen al P ocedu es
52
5.1 Fly s ocks and husband y
See Supplemen a y Table 1 and 2 o geno ypes o D osophila used in
his s udy. F ui lies D. melanogas e we e aised in s anda d co nmeal-aga
medium, using Vienna ood ecipe (in 1 Li e o wa e : 80 g molasses-ba ley
mal , 22 g bee sy up, 80 g co n lou , 18 g g anula ed yeas , 10 g soy lou , 8 g
aga -aga , 8 mL p opionic acid, 12 mL 15% nipagin, 35 mL Ba is in), a 25ºC
and 70% ela i e humidi y in a 12h da k:12h ligh cycle. De ailed in o ma ion
on ly housing and age o each expe imen a e indica ed in he ele an sec ion.
5.2 Immunohis ochemis y
Adul b ains, VNCs, ep oduc i e sys ems and abdomen cu icles we e dis-
sec ed in cold Phospha e-Bu e ed Saline (PBS) and immedia ely ans e ed o
cold Pa a o maldehyde (PFA) 4% in PBL (PBS wi h 0.12 M Lysine) and ixed
o 30 min a Room Tempe a u e (RT), washed h ee imes o 5 min in PBT
(PBS wi h 0.5% T i on X-100) and blocked o 30 min a RT in 10% no mal goa
se um in PBT (Sigma, ca # G9023). Samples we e incuba ed wi h he p ima y
an ibodies in blocking solu ion, o 72h a 4°C. The ollowing p ima y an ibodies
we e used: abbi an i-GFP 1:1000 (Molecula P obes, ca #A11122), chicken
an i-GFP 1:1000 (abcam, ab13970), mouse an i-nc82 1:10 (De elopmen al S ud-
ies Hyb idoma Bank, ca # AB2314866), abbi an i-DsRed 1:1000 (Taka a,
ca # 632496). Samples we e washed h ee imes o 5 min in PBT and incu-
ba ed in Alexa Fluo 488 o 594 seconda y an ibodies 1:500 (In i ogen) o 72h
a 4ºC. To coun e s ain he emale ep oduc i e sys em, Alexa 594-conjuga ed
phalloidin (Molecula P obes, ca # A12381) was used. Samples we e washed
h ee imes o 5 min in PBT and moun ed in Vec aShield medium (Vec o
Labo a o ies, ca #H-1000). Images we e acqui ed on a Zeiss LSM 710 con ocal
mic oscope using a 25X imme sion objec i e (Zeiss) o he b ains/VNCs and a
10X objec i e (Zeiss) o he ep oduc i e sys ems and abdomen cu icles. A e
acquisi ion, colou le els we e adjus ed using Fiji (Schindelin e al., 2012) o
op imal display.
53
5.3 P epa a ion o lies o be assayed
Low ly densi y c osses (10-15 i gin emales x 5 males pe bo le) we e used
in all expe imen s o ea ing lies wi h he app op ia e geno ype. In o de o
maximize he occu ence o egg deposi ion e en s du ing beha iou al expe i-
men s, we ollowed he egg-laying dep i a ion p o ocol desc ibed by Yang e al.
(2015) in all expe imen s, excep in he 24h egg-laying assays. B ie ly, g oups
o 5-7 i gin emales pe ial o he app op ia e geno ypes and 2-3 Can on S
males ( o ma ing) we e collec ed in o no mal ood ials wi h he excep ion
o op ogene ic expe imen s in which lies we e housed in no mal ood con ain-
ing all- ans-Re inal (Sigma, R2500) (all- ans-Re inal concen a ions used: 0.2
mM o CsCh imson ac i a ion and 0.4 mM o G ACR1 silencing). In con as
wi h he o iginal p o ocol (Yang e al., 2015), we yeas pas e was no supplied
o he ood. Flies we e le in he ials o 4 o 7 days a 25ºC and 70% el-
a i e humidi y. Beha iou al assays we e pe o med wi hin ha 4–7 days’ ime
window.
5.4 Beha iou al assays
5.4.1 24h egg-laying assay
Single i gin emales we e gen ly aspi a ed and ans e ed o 35 mm Pe i
dishes o 10 mm o heigh (The mo Fishe Scien i ic) coa ed wi h apple aga (750
mL wa e , 250 mL apple juice, 19,5 g aga , 20 g suga , 10 mL 10% nipagin) and
incuba ed wi h a nai e CS male o 2h unde cons an obse a ion o check o
ma ing occu ence. Pla es whe e ma ing did no happen we e disca ded. Flies
we e kep in he pla e o 24h be o e eggs we e coun ed. A e egg coun ing, he
emale´s ep oduc i e sys em was dissec ed o measu e egg jamming.
5.4.2 Egg-laying a ena and subs a e
Cus om made small ec angula -shaped a enas wi h 2 chambe s we e de-
signed o allow eco ding o 2 lies simul aneously. Each chambe measu es 1.8
(H) x 1.2 (L) x 0.3 (D) cm. Du ing beha iou al assays, he chambe s we e
pa ially illed wi h he egg-laying subs a e, which in his s udy was always 1%
aga ose (SeaKam®LE Aga ose, ca # 50004) dilu ed in dis illed wa e (Milli-
Q®Wa e Pu i ica ion Sys ems Me k). Flies had a ee walking space o 1.5 x
54
0.7 x 0.3 cm in he chambe o egg-laying. The base and he lid o he a ena
we e made o whi e opaque and anspa en ac ylic, espec i ely.
5.4.3 De ailed beha iou
To analyse he mo o elemen s associa ed wi h egg-laying beha iou , emales
we e collec ed soon a e eclosion and housed in g oups ollowing he egg-laying
dep i a ion p o ocol desc ibed abo e. Aged 4-7 days emales we e es ed. Flies
we e gen ly aspi a ed in o he egg-laying a ena and beha iou was eco ded a
20 ames pe second du ing 45 min o CS (Fig. 2.1 and 2.2) and du ing 15
min o O Abg/VGlu silenced emales (Fig. 3.11 , 3.12, 3.13). The same ly
handling p ocedu e was pe o med o op ogene ic expe imen s in which egg-
laying mo o elemen s we e analysed ( o mo e de ailed in o ma ion, see he
op ogene ic s imula ion sec ion).
5.4.4 Op ogene ics
Fo all expe imen s using CsCh imson, excep in he 16h egg-laying assay
(Fig. 3.8b-d), he s imula ion p o ocol included 1 min baseline pe iod ollowed
by 6 epe i ions o 10 s ed-ligh s imuli wi h a powe o 4.40 mW/cm2and
20 s in e al be ween s imuli. Fly beha iou was eco ded a 20 ames pe
second, excep in he O Abg line ac i a ion expe imen s (Fig. 3.2b-g) in which
we used 15 ames pe second. In he O Abg headless emales’ pho oac i a ion
(Fig. 3.2d), he head was gen ly cu using dissec ion o ceps (Dumon #55
Fo ceps, 11295-51) unde CO2 anaes hesia. Flies we e ans e ed o he egg-
laying a ena and allowed o eco e om his p ocedu e o 5-10 min be o e
pho oac i a ion. In he 16h pho oac i a ion egg-laying assay (Fig. 3.8b-d),
ma ed emales we e ans e ed o he apple aga pla es (desc ibed in he 24h
egg-laying assay sec ion). The s imula ion p o ocol included cons an ed-ligh
wi h a powe anging 4.19-4.85 mW/cm2du ing 16h. A he end o his pe iod,
he eggs we e coun ed and he ep oduc i e sys em was dissec ed o measu e
egg jamming. Fo he silencing expe imen using G ACR1 (Fig. 3.4-3.6), he
s imula ion p o ocol included a p e-s imula ion pe iod ha las ed o 10 min,
ollowed by cons an g een-ligh s imula ion wi h a powe o 5-6.23 mW/cm2
du ing 15 min and a pos -s imula ion pe iod o 5 min. Videos we e eco ded a
20 ames pe second.
55
6.2 Egg-laying beha iou in wild- ype lies
Complemen a y o Yang e al. (2008), we showed ha egg-laying beha iou
is s uc u ed in h ee phases - egg deposi ion, abdominal con o ions and explo-
a ion. Each phase includes a speci ic and s e eo yped epe oi e o beha iou al
elemen s conse ed ac oss D osophila species (B äcke e al., 2019) and execu ed
o p omo e subs a e p obing, egg deposi ion and o ula ion. Di e en g oups
simul aneously wo king on his opic eached complemen a y esul s ha we e
ecen ly published (Cu y and Axel, 2021; Vijayan e al., 2021), highligh ing
he need in he ield o a mo e comp ehensi e cha ac e iza ion o his complex
beha iou .
We show ha he egg deposi ion phase ollows a eliable beha iou al se-
quence in which g ooming e minalia is he only op ional beha iou , as also
shown by Cu y and Axel (2021). We es ablished ha egg deposi ion always
ansi s o abdominal con o ions, he sole beha iou o his phase, which is
in line wi h Vijayan e al. showing ha abdominal con o ions ep esen he
o ula ion pe iod and, hus, a e c i ical o he posi ioning o a new egg o be
laid (Vijayan e al., 2021). The explo a o y phase sha es common beha iou al
elemen s wi h he egg-deposi ion phase bu , in his case, hey a e used by lies o
sample he subs a e and do no culmina e in egg expulsion. The explo a o y be-
ha iou s a e no pe o med in a beha iou al sequence and can be displayed wi h
di e en imings, in con as wi h he egg deposi ion mo o p og amme. The
a iabili y in he sequence and iming o explo a o y beha iou s, which was also
epo ed by Cu y and Axel (2021), may o e o he o ganism a beha iou al lex-
ibili y o cope wi h changes in he en i onmen du ing egg-laying si e selec ion.
On his analysis, we did no dis inguish p oboscis ex ension bou s pe o med o
eeding o explo a ion in he con ex o egg-laying. Howe e , since he subs a e
we used lacks nu i i e cues and he lies es ed a e well ed, we hypo hesize ha
mos o he p oboscis ex ension bou s obse ed in ou expe imen s a e speci ic
o egg-laying beha iou . In e es ingly, emales show educed explo a ion in ou
beha iou al assays and his phase is op ional. Two ac o s may explain hese
obse a ions: 1) he e may be explo a ion ha does no include he mo o ele-
men s we conside ed and, ins ead, lies use mechanosenso y and chemosenso y
in o ma ion om he legs and an ennae; 2) we use e y small a enas wi h a
es ic ed space o explo a ion and wi hou complex senso y cues. This ea u e
may allow a quick spa ial and senso y ecogni ion o he en i onmen making
62

explo a ion less equen . Fu u e wo k on he ea u es o explo a ion and si e
selec ion should use mo e complex a enas and en i onmen s.
In his wo k, momen s o dis inc beha iou s we e anno a ed manually,
which ep esen s an impo an in es men o ime and limi s he use o his
beha iou al assay in high- h oughpu expe imen s. The implemen a ion o au-
oma ed anno a ion sys ems (e.g. DeepLabCu , JAABA) o ack ly kinema ics
and classi y egg-laying mo o elemen s will ce ainly o e an ad an age o u-
u e s udies (Kab a e al., 2013; Ma his e al., 2018).
O e all, his de ailed desc ip ion o egg-laying beha iou p o ided a ame-
wo k o in e oga e he unde lying neu onal subs a es.
6.3 O Abg neu ons: lowe mo o con ol o egg-
laying beha iou
This s udy o e s unp eceden ed insigh s in o he lowe mo o con ol o egg-
laying h ough he cha ac e iza ion o a no el popula ion loca ed in he Abg
− he O Abg neu ons. The analysis o hei ana omy and unc ion showed
ha O Abg neu ons a e c i ical o egg-laying beha iou and belong o he
egg-laying mo o ci cui s. We show ha O Abg is a dimo phic popula ion
in ol ed in he con ol o ep oduc i e beha iou s, as shown by he dis inc CNS
ana omy and unc ion be ween emales and males. The di e en ial egula ion
o dsx exp ession suppo s sex-speci ic beha iou s (Kohl e al., 2013; Nojima
e al., 2021; Pa lou e al., 2016; Rideou e al., 2010). We assume ha we a e
a ge ing pa o he dsx+ popula ion a he Abg based on ou in e sec ional
app oach using spli Gal4 lines con olled by dsx egula o y egions, and gi en
he ana omy epo ed o dsx+ neu ons in he VNC (Pa lou e al., 2016; Rideou
e al., 2010).
The Abg egg-laying ci cui is poised o ecei e commands om he b ain
o he execu ion o egg deposi ion. Which a e he ups eam a ge s o O Abg
neu ons? O iDNs inne a ions a he VNC a e ana omically poised o connec
wi h O Abg neu ons (Vijayan e al., 2021; Wang e al., 2020). Tools o s udy
connec i i y be ween neu ons, such as GRASP (Feinbe g e al., 2008) and op-
ogene ic s imula ion in eg a ed wi h calcium imaging, awai he de elopmen
o new gene ic d i e s labelling O iDNs and O Abg popula ions, espec i ely.
63
The O Abg neu ons p o ided a g ea en y poin o add ess how di e en
ci cui s coo dina e egg deposi ion. A de ailed discussion o he h ee abdominal
ganglion popula ions cha ac e ized in his s udy ollows below.
6.4 Execu ion o Egg Deposi ion: Glu ama e gic and
Choline gic O Abg ci cui s
We cha ac e ized wo sub-popula ions wi hin he O Abg ci cui −a glu-
ama e gic and a choline gic − ha implemen he egg deposi ion mo o p o-
g amme.
The O Abg/VGlu ci cui ep esen s a es ic ed ac ion o he dsx+
glu ama e gic-exp essing neu ons in he Abg o D osophila emales. Gi en he
o al numbe o dsx+ neu ons exp essing glu ama e a he emale Abg (∼100
neu ons) epo ed by Pa lou e al (2006), we easoned ha O Abg/VGlu
popula ion ep esen s only 10-18% o all Abg glu ama e gic dsx+ neu ons. The
neu onal manipula ions o O Abg/VGlu popula ion unco e ed a speci ic ole
in he ini ia ion o he egg deposi ion mo o sequence h ough he execu ion
o o iposi o con ac and bu owing beha iou s. In e es ingly, hese mo o
elemen s o egg deposi ion, ha a e also displayed du ing he explo a ion
phase, we e no a ec ed in he explo a ion phase when hese neu ons we e
silenced, sugges ing a di e en con ol o he same mo o elemen s du ing
di e en phases.
Cu iously, he ac ha O Abg/VGlu neu ons a e in ol ed in he ini ia ion
o he egg deposi ion sequence led us o specula e ha hey could ep esen he
analogous neu ons on he emale Abg o he male-speci ic glu ama e gic neu-
ons con olling he ini ia ion o geni al coupling du ing male cou ship (Pa lou
e al., 2016). Addi ionally, he O Abg/VGlu popula ion could be he equi alen
ci cui in he ly o he digging mo o ci cui ound in locus emales (see in o-
duc ion sec ion 1.6). Bu owing beha iou is one o he mo o elemen s elici ed
by O Abg/VGlu ac i a ion and, complemen a y o ou wo k, his beha iou
was ecen ly associa ed wi h digging he subs a e o allow sub e aneous egg
deposi ion (Cu y and Axel, 2021). Based on he a chi ec u e o he locus egg
deposi ion ci cui s, we hypo hesized ha he O Abg/VGlu popula ion could
locally in e ac wi h an egg- e en ion ci cui o block egg expulsion while digging
is s ill occu ing.
64
Egg-laying is mos ly pe o med by ma ed emales, al hough i gin emales
may deposi un e ilized eggs esidually. Ac i a ion o O Abg/VGlu neu ons
leads o ewe e en s o egg deposi ion ini ia ion in i gin emales when com-
pa ed o ma ed emales, hus sugges ing ha ma ing s a us modula ion o
egg-laying is occu ing locally a he Abg, in addi ion o he modula ion in he
b ain (Feng e al., 2014; Shao e al., 2019; Wang e al., 2020). Local modula ion
may esul om di ec oc opamine gic modula ion o in downs eam a ge s.
The O Abg/Cha ci cui ep esen s a b oad popula ion ha is necessa y and
su icien o egg deposi ion. Op ogene ic ac i a ion o O Abg/Cha neu ons
igge s he comple ion o egg deposi ion h ough he execu ion o egg pushing
and egg expulsion, while hei silencing abolishes egg-laying and p omo es egg-
jamming. The egg-jamming pheno ype sugges s a ole o O Abg/Cha neu ons
in o iduc con ac ions. Such modula ion is mos likely indi ec , because we did
no ind any p ojec ions be ween his popula ion and he la e al o iduc s. As
discussed in he in oduc o y sec ion, a ole o OA in he o iduc ac i i y du -
ing egg-laying has been in ensely s udied in insec s. Do O Abg/Cha neu ons
belong o he oc opamine gic ne wo k in ol ed in his p ocess? We p opose ha
he O Abg popula ion is unlikely o be oc opamine gic, gi en he di e ences
in he Abg cell body ana omy be ween O Abg and he oc opamine gic ep e-
sen a i e dTdc2-GAL4 line (da a no shown). None heless, choline gic O Abg
neu ons may s ill modula e ep oduc i e unc ion by esponding o oc opamin-
e gic signalling (e.g. h ough he exp ession OAMB and/o Oc β2R ecep o s).
An RNAi sc eening a ge ed o oc opamine gic ecep o s and unc ional connec-
i i y expe imen s should help cla i y a he ole o OA on he O Abg ci cui
unc ion.
Ou indings hin a a possible in e ac ion be ween he O Abg glu ama e -
gic and choline gic popula ions in he execu ion o egg deposi ion. How is his
in e ac ion se so ha lies pe o m a sequen ial o de o beha iou al elemen s
leading o egg expulsion? A e O Abg/VGlu p esynap ic o O Abg/Cha neu-
ons? I glu ama e gic O Abg we e ups eam pa ne s o choline gic O Abg
neu ons in a linea sequen ial mo o ci cui , we would expec ha ac i a ion
o O Abg/VGlu popula ion elici ed egg pushing and egg expulsion, which a e
ne e obse ed upon his manipula ion. Howe e , we canno ule ou he possi-
bili y o such a sequen ial pa hway because O Abg/VGlu neu ons may wo k
65
oge he wi h o he exci a o y descending inpu o ac i a e O Abg/Cha popu-
la ion (see model schema ic, Fig. 3.13e).
I is also in e es ing o specula e ha bo h popula ions may ecei e senso y
eedback om he ep oduc i e sys em o shape he egg deposi ion mo o p o-
g amme. Fo example, Pos e io U e ine (PU) senso y neu ons loca ed in he
u e us (Cu y and Axel, 2021) could elay eedback o O Abg/VGlu neu ons
abou he p esence o he egg in he o iposi o du ing bu owing beha iou and,
he e o e, modula e he ac i i y o his mo o ci cui o ine une he p og ession
along he beha iou al sequence o comple e egg deposi ion.
6.5 Regula ion o Egg-laying: GABAe gic O Abg
ci cui
The GABAe gic O Abg ci cui ep esen s abou hal (∼46%) o all dsx+
GABAe gic-exp essing neu ons in he Abg acco ding wi h Pa lou e al (2006).
While he glu ama e gic and choline gic O Abg popula ions ep esen mo o
ci cui s in ol ed in he implemen a ion o he egg deposi ion mo o p o-
g amme, he O Abg/Gad1 popula ion seems o play a mo e egula o y ole
on egg-laying. This e idence comes om he ac ha (1) he numbe o
eggs laid is no a ec ed when O Abg/Gad1 neu ons a e silenced and (2)
op ogene ic ac i a ion does no elici any beha iou al elemen associa ed wi h
he egg-laying mo o p og amme (da a no shown) bu , ins ead, is su icien
o block egg-laying. Taken oge he , ou da a indica e ha O Abg/Gad1
ac i i y nega i ely egula es egg-laying whe eby hey need o be silen du ing
egg-laying, likely h ough inhibi o y descending inpu . Local supp ession o
egg-laying may be equi ed when nega i e egg-laying cues a ise o in i gin
emales (ma ing s a us modula ion), which could be media ed by an inc ease in
ac i i y in he GABAe gic popula ion. O Abg/Gad1 neu ons could supp ess
egg-laying by ac ing on he glu ama e gic and choline gic popula ions (see
model schema ic, Fig. 3.13e). We may hypo hesize ha O Abg/VGlu and
O Abg/Gad1 popula ions o m a ci cui o coo dina e egg deposi ion ini ia ion
and supp ession wi h analogous a chi ec u e o he one p oposed by Pa lou e
al. (2016). I O Abg/Gad1 neu ons indeed sup ess egg-laying in un a ou able
en i onmen al condi ions, o which nega i e cues do hey espond? Who is
elaying he in o ma ion abou he en i onmen o O Abg/Gad1? We en ision
66
ha i could be ei he ansmi ed om senso y sys ems (e.g. legs, ep oduc i e
sys em) o om descending inpu coding nega i e cues (e.g. DNp42 pa hway
o geosmin a oidance).
As a conclusion, we iden i ied di e en popula ions o O Abg neu ons in-
ol ed in he con ol o egg-laying mo o ou pu . The undamen al ques ions
on he connec i i y be ween hese O Abg popula ions equi e u he in es iga-
ion, which is cu en ly limi ed o he gene ic d i e s a ailable o label di e en
O Abg neu ons. Fu u e sc eenings on addi ional enhance lines (e.g. enhance
bashing) and di e en exp ession sys ems (e.g. LexA d i e lines) would allow
o independen ly label and manipula e each o he ele an clus e s. This will
open new possibili ies o dissec ing he ci cui . Mo e ecen ly, he EM-based
connec i i y o he T1, T2 and T3 VNC segmen s was ecen ly published high-
ligh ing he senso y and mo o ne wo ks con olling limb mo emen (Phelps e
al. 2021). Mapping he s uc u al connec i i y o he Abg awai s u u e wo k
and would ce ainly con ibu e o ou unde s anding on how he O Abg ci cui
coo dina es and egula es egg-laying beha iou .
6.6 Conclusion
1. Ou indings p o ide a de ailed desc ip ion o egg-laying. We desc ibed
he di e en mo o elemen s, hei pa icipa ion in di e en egg-laying phases
and how lies ansi ion om one phase o he nex . This desc ip ion acili a es
he goal o linking a complex beha iou −egg-laying −wi h i s neu onal
unde pinnings.
2. We p esen insigh s in o he logic o egg deposi ion mo o ci cui s. This
wo k se es as a s epping s one o dissec ascending and descending communica-
ion wi h he b ain, o ex end neu onal dissec ion and connec i i y o egg-laying
popula ions and add ess mechanisms o local ma ing s a us modula ion o egg-
laying.
67

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