Neuronal control of suppression, initiation and completion of egg deposition in Drosophila melanogaster

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. 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