Dissections of Larval, Pupal and Adult Butterfly Brains for Immunostaining

Protocol Dissections of Larval, Pupal and Adult Butterﬂy Brains for Immunostaining and Molecular Analysis

Yi Peng Toh 1,†, Emilie Dion 1,*,† and Antónia Monteiro 1,2

1 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; [email protected] (Y.P.T.); [email protected] (A.M.) 2 Yale-NUS College, 10 College Avenue West, Singapore 138609, Singapore * Correspondence: [email protected] † The authors contributed equally.

Abstract: Butterflies possess impressive cognitive abilities, and investigations into the neural mechanisms underlying these abilities are increasingly being conducted. Exploring butterfly neurobiology may require the isolation of larval, pupal, and/or adult brains for further molecular and histological experiments. This procedure has been largely described in the fruit fly, but a detailed description of butterfly brain dissections is still lacking. Here, we provide a detailed written and video protocol for the removal of Bicyclus anynana adult, pupal, and larval brains. This species is gradually becoming a popular model because it uses a large set of sensory modalities, displays plastic and hormonally controlled courtship behaviour, and learns visual mate preference and olfactory preferences that can be passed on to its offspring. The extracted brain can be used for downstream analyses, such as immunostaining, DNA or RNA extraction, and the procedure can be easily adapted to other lepidopteran species and life stages. Keywords: brain dissections; butterflies; caterpillars; pupa; Bicyclus anynana Citation: Toh, Y.P.; Dion, E.; Monteiro, A. Dissections of Larval, Pupal and Adult Butterfly Brains for Immunostaining and Molecular 1. Introduction Analysis. Methods Protoc. 2021, 4, 53. Despite having small brains, insects’ cognitive abilities are impressive. They can process https://doi.org/10.3390/mps4030053 a large set of information from their environment and adjust their behaviour accordingly [1]. Many species possess remarkable learning and memorisation skills [2,3]. Examples of Academic Editor: Fernando Albericio sophisticated behaviours include concept and category learning in honeybees [4], tool use in ants [5], facial pattern recognition in wasps [6,7], and counting ability in bees [8,9]. Received: 8 June 2021 The neural mechanisms responsible for these sophisticated behaviours have interested Accepted: 31 July 2021 Published: 5 August 2021 researchers for decades (e.g., [10–12]). These mechanisms were largely investigated in the fruit fly (e.g., [13–16]), honey bee (e.g., [17–19]), and more recently in other insects, such as

Publisher’s Note: MDPI stays neutral crickets (e.g., [20–22]), mosquitos (e.g., [23]), or cockroaches (e.g., [24,25]), among others. with regard to jurisdictional claims in Recently, studies on butterflies have also increased, motivated by the large diversity of published maps and institutional affil- behaviours that they can perform. iations. Butterflies are becoming a popular system in the fields of evolutionary neurobiology and neuroethology because they use a large set of sensory modalities for their survival and reproduction [26]. They process visual [27], olfactory [28], auditory [29], and gustatory [30] signals to help them forage, search for a host plant, select mates, avoid predators, or migrate. They can also memorise information from their environment and adjust their Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. behaviour accordingly (e.g., [31,32]). Butterflies’ visual and olfactory systems are also This article is an open access article crucial mediators of reproductive isolation and speciation [33,34]. distributed under the terms and The growing exploration of the butterflies’ neurobiology prompted the development conditions of the Creative Commons of experimental techniques for producing relevant molecular and neuroanatomical data. Attribution (CC BY) license (https:// Brain dissections are an essential step in the experimental process (e.g., [35–37]). Protocols creativecommons.org/licenses/by/ describing the dissection of Drosophila brains are currently available [38–42], but a detailed 4.0/). description of butterfly brain dissections at various developmental stages is still lacking.

Methods Protoc. 2021, 4, 53. https://doi.org/10.3390/mps4030053 https://www.mdpi.com/journal/mps Methods Protoc. 2021, 4, 53 2 of 18

Here, we describe the process of larval, pupal, and adult butterfly brain removal using a video. We also provide a complete written description of the processes, with a list of necessary tools and chemicals. The dissection methods can be applied to different butterfly species, and the brains can be used for DNA or RNA extraction, or for immunostaining. We record the dissection process in the Bush brown butterfly Bicyclus anynana, which is especially interesting due to its plastic and sex-role reversed courtship behaviour [43–46]. Individuals are also capable of learning visual and olfactory cues [47–49], and they can transmit the learned odour preferences to their offspring [48,49]. Because the molecular basis of these unique behaviours can begin to be examined at the level of the brain, it is important to extract this organ for downstream analyses. We dissected the brains of two-day-old butterflies, those of pupae at 30% development, and those of 5th instar larvae. These specific ages were chosen because B. anynana adult individuals showed a change in sex-pheromone and wing-pattern preferences on day two after exposure to the newly emerged females [47,48]. The 30% pupal development corre- sponds to the critical stage where variation in hormone levels influences adult courtship behaviour [50,51]. We chose to dissect 5th instar larval brains because they are bigger at this stage, and they provide higher amounts of RNA and DNA. However, B. anynana larvae start preferring novel food odours from the 3rd instar, and keep the same preference until pupation [49]. To show that our methods can be easily applied to other families and individuals of various sizes, we also dissected the brains of adults, pupae, and caterpillars of Pieris canidia (family: Pieridae) at the same stages as B. anynana, and adults of two additional species from the Nymphalidae and Papilionidae.

2. Experimental Design All materials and equipment needed for brain dissections are described in Figure1. Figure2 presents the anatomy of a larval head, adult head and of a pupa.

2.1. Materials • Curved forceps (Dumont; Dumont Switzerland, Montignez, Switzerland; Cat. No.: 11274-20); • Superfine dissecting forceps (Dumont; Dumont Switzerland, Montignez, Switzerland; Cat. No.: 82027-402); • Blade holder (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0934); • Surgical blades (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0115); • Two dissection silicone plates (Dragon Skin 30 Mould Making Silicone Rubber; Cat. No.: 0751635278417, Petri plate; Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P5981-100EA). Use black or red silicon to best see the white brains, or place a black background underneath a transparent silicon plate; • Glass spot plate (PYREXTM; Corning, Corning, NY, USA; Cat. No.: 722085); • Insect pins (BioQuip; BioQuip, Rancho Dominguez, CA, USA; Cat. No.: 1208B2); • Glass slides (Biomedia, Singapore, Cat. No.: BMH.880102); • Plastic pipettes (unbranded, Singapore), 1000 µL; • Filter tips (Maximum Recovery, Axygen, Union City, CA, USA; no. TF-1000-L-R-S); • Microcentrifuge tubes 1.5 mL (Eppendorf, Hamburg, Germany; Cat. no.: T9661-500EA). MethodsMethods Protoc. Protoc. 20212021, 4, ,4 x, 53 FOR PEER REVIEW 3 of 18 3 of 18

Figure 1. 1.Equipment Equipment and and materials materials needed needed for the for dissection the dissec oftion adult, of pupal, adult, and pupal, larval and brains. larval brains. 2.2. Equipment 2.3. Reagents and Solutions • Zeiss Dissection Microscope (Carl-Zeiss, Jena, Germany; Stemi 305); • OrbitalNaCl (Sigma-Aldrich; Shaker (Kangjian, Sigma-Aldrich, Jiangyan City, China). Singapore; Cat. No.: S9888-500G); • KCl (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P9541-500G); 2.3. Reagents and Solutions • CaCl2 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: C1016-100G); • • NaClZnCl2 (Sigma-Aldrich; (Sigma-Aldrich; Sigma-Aldrich, Sigma-Aldrich, Singapore; Singapore; Cat. No.: Cat. S9888-500G); No.: 208086-100G); • KCl (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P9541-500G); • K2HPO4 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P3786-500G); • CaCl2 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: C1016-100G); • ZnCl2Sucrose (Sigma-Aldrich; (Sigma-Aldrich; Sigma-Aldrich, Sigma-Aldrich, Singapore; Singapore; Cat. No.: Cat. 208086-100G); No.: S0389-1KG); • • KHEPES2HPO4 (Sigma-Aldrich;(Sigma-Aldrich; Sigma-Aldrich, Sigma-Aldrich, Singapore; Singapore; Cat. No.: Cat. P3786-500G); No.: H3375-100G); • Sucrose16% Formaldehyde (Sigma-Aldrich; (ThermoFisher Sigma-Aldrich, Scient Singapore;ific, Cat.Singapore; No.: S0389-1KG); Cat. No.: 28908); • HEPESEthanol (Sigma-Aldrich; absolute (ThermoFisher Sigma-Aldrich, Scient Singapore;ific, Singapore, Cat. No.: H3375-100G);Cat. No.: 64175); • 16%RNALater Formaldehyde® RNA (ThermoFisherStabilisation Reagent Scientiﬁc, (Qiage Singapore;n, Singapore, Cat. No.: 28908); Cat. No.: 76104); • • EthanolRNAse absoluteZapTM RNase (ThermoFisher Decontamination Scientiﬁc, Singapore, Solution Cat.(ThermoFisher No.: 64175); Scientific, Singapore, • RNALater® RNA Stabilisation Reagent (Qiagen, Singapore, Cat. No.: 76104); Cat. No.: AM9780);

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Methods Protoc. 2021, 4, x FOR PEER REVIEW 4 of 18 • RNAseZapTM RNase Decontamination Solution (ThermoFisher Scientiﬁc, Singapore, Cat. No.: AM9780); Methods Protoc. 2021, 4, x FOR PEER REVIEW 4 of 18

FigureFigure 2. Anatomy of (A) a B. anynana butterflybutterfly head (front view), (B) a caterpillar head (front view), Figureand (C 2.) of Anatomy a pupa. of (A) a B. anynana butterfly head (front view), (B) a caterpillar head (front view), and (C) of a pupa. and (C) of a pupa. 3.3. Dissection of Adult Brains 3.3.1.3.1. Dissection Preparation of for Adult DissectionDissection Brains ◦ 3.1.•• PreparationFreezeFreeze anaesthetiseanaesthetise for Dissection thethe butterfliesbutterflies inin aa −−2020 °CC freezer freezer for for 3–4 3–4 min; min; •• WashWash thethe dissectiondissection toolstools andand platesplates inin 70%70% ethanolethanol priorprior toto dissection;dissection; • •• FreezePlace a anaesthetise few drops of the HEPES-buffered butterflies in saline a −20 (HBS) °C freezer onto a for dissection 3–4 min; plate using a • Place a few drops of HEPES-buffered saline (HBS) onto a dissection plate using a Washplasticplastic the pipette,pipette, dissection andand set set tools it it aside aside and (see (see plates recipe recipe in for for70% HBS HBS ethanol and and all all other prior other reagents to reagents dissection; at theat the end end of • Placethisof this article); a article); few drops of HEPES-buffered saline (HBS) onto a dissection plate using a •• plasticIfIf thethe brainbrain pipette, tissuestissues and needneed set toto it undergoundergo aside (see immunostaining,immunostaining, recipe for HBS fillfill aaand wellwell all inin theotherthe spotspot reagents plateplate withwith at the end ofZnFAZnFA this solution solutionarticle); using using a a plastic plastic pipette, pipette, and and set se itt it aside aside before before starting starting any any dissection. dissection. If • IftheIf the the brains brainbrains are tissues are used used for need DNAfor toDNA orundergo RNA or RNA extraction, immunostaining, extraction, fill the fill RNAse-free the fill RNAse-free a well Eppendorf in theEppendorf spot tubes plate with with about 300 µL of 100% ethanol or RNAlater, respectively. Place only up to 3 brains ZnFAtubes withsolution about using 300µL a ofplastic 100% ethanolpipette, or and RNAlater, set it aside respectively. before startingPlace only any up dissection.to in the same well (for immunostaining). Number of brains to be pooled in one tube If3 brainsthe brains in the aresame used well (foforr immunostaining).DNA or RNA extraction, Number of brainsfill the to RNAse-free be pooled in oneEppendorf variestube varies with thewith amount the amount of DNA of DNA or RNA or RNA needed needed for downstream for downstream analyses; analyses; tubes with about 300µL of 100% ethanol or RNAlater, respectively. Place only up to 3 brains CRITICAL in the same STEPSTEP IfwellIf DNADNA (fo orror immunostaining). RNARNA workwork isis involved,involved, Number itit isis recommendedrecommended of brains to be toto pooled wipewipe in one thethe equipmentequipmenttube varies withwith with RNAseZapRNAseZap the amount inin stepstep of 2,DNA2, andand useoruse RNA aa micropipettemicropipette needed for andand downstream filterfilter tipstips insteadinstead analyses; inin stepssteps 33 andand 4.4. CRITICAL STEP If DNA or RNA work is involved, it is recommended to wipe the3.2. equipment Dissection Procedure with RNAseZap in step 2, and use a micropipette and filter tips instead in steps3.2.1. 3 Exposure and 4. of Adult Brains • Take the butterfly out of the freezer and place it on a second clean dissection plate. 3.2. DissectionThe first seriesProcedure of steps are performed dry. Detach the head of the butterfly from the 3.2.1. restExposure of the body of Adult using Brains a pair of superfine dissecting forceps. Pull the head away from the body using your dominant hand while pressing down on the wings with the • Take the butterfly out of the freezer and place it on a second clean dissection plate. The first series of steps are performed dry. Detach the head of the butterfly from the rest of the body using a pair of superfine dissecting forceps. Pull the head away from the body using your dominant hand while pressing down on the wings with the

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3.2. Dissection Procedure 3.2.1. Exposure of Adult Brains • Take the butterfly out of the freezer and place it on a second clean dissection plate. The first series of steps are performed dry. Detach the head of the butterfly from the rest of the body using a pair of superfine dissecting forceps. Pull the head away from the body using your dominant hand while pressing down on the wings with the other

hand. Ensure the point of pull is as close to the head as possible, to reduce the amount Figureof 2. thoracic Anatomy tissue of (A) needed a B. anynana to be butterfly removed head (Figure (front3 view),A); (B) a caterpillar head (front view), •and (RemoveC) of a pupa. the rest of the butterﬂy from the dissecting plate, leaving only the head. There will be some remaining thoracic regions left on the back of the head most of 3. Dissectionthe time. of Secure Adult the Brains head from moving with the forceps in your non-dominant hand, 3.1. Preparationwhile pulling for awayDissection the thoracic region with the other forceps in your dominant hand • (FigureFreeze 2anaesthetiseB). Remove thethe thoracicbutterflies tissue in a until−20 °C you freezer can visualise for 3–4 themin; connection point of • theWash labial the palpsdissection to the tools head and (Figures plates2 Ain and70%3 C);ethanol prior to dissection; •• DetachPlace a thefew labial drops palps of HEPES-buffered and proboscis (Figuressaline (HBS)2A and onto3C,D) a dissection by pulling plate them using hard a awayplastic from pipette, the head.and set The it easiestaside (see and recipe fastest for way HBS to completelyand all other remove reagents the palpsat the andend theof this proboscis article); is to grip them from the back of the head and pull them away from the • headIf the usingbrain thetissues forceps need in to your undergo dominant immunostaining, hand (Figure fill3D), a whilewell in gently the spot pressing plate with the partZnFA of solution the head using where a plastic the proximal pipette, ends and ofset theit aside labial before palps starting are located, any dissection. using the otherIf the forcepsbrains inare your used non-dominant for DNA or RNA hand; extraction, fill the RNAse-free Eppendorf • Usingtubes with a pair about of insect 300µL pins, of gently100% ethanol scrape offor RNAlater, the setae that respectively. are attached Place to theonly cuticle up to surrounding3 brains in the the same head. well Gently (for immunostaining). press the head down Number with one of brains insect to pin be while pooled scraping in one thetube setae varies off with with the another amount (Figures of DNA2A andor RNA3E); needed for downstream analyses; CRITICAL STEPSTEP BeIf DNA careful or not RNA to applywork is too involved, much pressure it is recommended when scraping to wipe near thethe eye,equipment as a slight with increase RNAseZap in pressure in step will2, and damage use a micropipette the optic lobe. and This filter step tips will instead reduce in thesteps number 3 and of4. setae ﬂoating on the dissecting solution, and allow for easier exposure of the brain tissue in the later steps; 3.2. Dissection Procedure • Detach the antennae (Figures2A and3F) using a pair of curved forceps. Pull the 3.2.1.antennae Exposure hard of Adult away Brains from the head using the forceps in your dominant hand, while • gentlyTake the pressing butterfly down, out toof thethe extent freezer of and just plac touchinge it on the a headsecond with clean the dissection other forceps plate. in yourThe first non-dominant series of steps hand are (Figure performed3F); dry. Detach the head of the butterfly from the • Gentlyrest of the pick body up theusing head a pair using of superfine a curved di forcepsssecting and forceps. transfer Pull it the into head the previouslyaway from preparedthe body dissectionusing your plate dominant with a fewhand drops while of pressing HBS (enough down to on cover the wings the tissue with fully, the about 180 µL here) for subsequent exposure of the brain tissue (Figure3G); • Secure the head between the curved forceps and fully submerge it into HBS before exposing the tissue;

CRITICAL STEP The head will tend to ﬂoat on the surface of the saline solution at ﬁrst. It is critical to ensure it is fully submerged (by securing the head between the ends of the forceps and then sinking it in the plate so that the saline solution fully covers the head). This ensures that the brain will be exposed within the solution and not outside of it, reducing tissue damage; • Secure the head with the curved forceps facing upwards, to get a grip on the region of the cuticle in between the two eyes (Figure3H); • Make a small tear in the cuticle between the eyes (Figure2I) and use it as an opening point to remove the rest of the cuticle (Figure3J); • Pull out any easily accessible trachea, using curved forceps in your dominant hand (Figure3K). Note: If the brains are used for DNA and RNA extraction, skip Section 3.2.2 (Fixation), and continue the dissection procedure directly as described in Section 3.2.3 below. MethodsMethods Protoc. Protoc. 2021,2021 4, x, FOR4, 53 PEER REVIEW 6 of6 18of 18

FigureFigure 3. Exposure 3. Exposure of an of anadult adult brai brainn and and fixation. fixation. Steps inin (A(A––GG)) are are performed performed dry dry while while (H– L(H) are–L) submerged are submerged in solution. in solution. ((AA)) The The ideal ideal point point of of pull pull when when detaching detaching the head the head of the of adult the fromadult the from rest the of the rest body of the is indicated body is indicated by the white by dashed the white dashedline. line. (B) The(B) The remaining remaining thoracic thoracic regions regions attached atta toched the butterfly to the butterfly head are grabbedhead are with grabbed fine forceps with fine and forceps pulled away. and pulled (C) away.The (C thoracic) The thoracic region isregion removed is removed until the proximaluntil the ends proximal of the palpsends areof the seen palps (circled are in seen white). (circled (D) The in labialwhite). palps (D) andThe the labial palpsproboscis and the areproboscis then pulled are awaythen pulled from the away head. from€ The the setae head. attached € The to setae the front attached and back to the of the front cuticle and are back scraped of the off cuticle gently are scrapedusing off insect gently pins, using to preventinsect pins, setae to from prevent clouding setae the from surface clou ofding the dissectingthe surface solution of the dissecti in the laterng solution steps. (F) in The the antennae later steps. (F) Theare antennae subsequently are removed.subsequently (G) The removed. head is picked (G) The up head gently is using picked curved up gently forceps using and transferred curved forceps to a dissecting and transferred plate with to a dissectingHBS. (Hplate) The with head HBS. is secured (H) The with head curved is secured forceps towith allow curved easier forceps access to to the allow cuticle easier in between access theto the two cuticle eyes (frontal in between view). the two eyes (frontal view). (I) A small tear is made in the piece of cuticle in between the two eyes. (J) The brain is exposed (I) A small tear is made in the piece of cuticle in between the two eyes. (J) The brain is exposed with the region of the cuticle with the region of the cuticle in between the two eyes removed, subsequently exposing the trachea underneath. (K) The in between the two eyes removed, subsequently exposing the trachea underneath. (K) The sticky white trachea just below sticky white trachea just below the surface of the cuticle are removed. (L) The exposed brain is submerged in a ZnFA the surface of the cuticle are removed. (L) The exposed brain is submerged in a ZnFA solution in a spot plate well, and the solution in a spot plate well, and the opening is covered with a glass slide to prevent evaporation of the solution and opening is covered with a glass slide to prevent evaporation of the solution and drying of the tissues. The glass plate is drying of the tissues. The glass plate is subsequently placed on an orbital shaker with a setting of 80–100 rmp for 16–20 h. subsequently placed on an orbital shaker with a setting of 80–100 rmp for 16–20 h. 3.2.2. Fixation of Adult Brains for Immunostaining • Gently lift the head up with the exposed brain with the curved forceps and transfer it into a well filled with ZnFA solution (Figure 3L). Cover the well with a glass slide, then place the well plate on an orbital shaker and agitate it for 16–20 h at 80–100 rates per minute (rmp);

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Figure 2. Anatomy of (A) a B. anynana butterfly head (front view), (B) a caterpillar head (front view), and (C) of a pupa.

3. Dissection of Adult Brains Methods Protoc. 2021, 4, 53 7 of 18 3.1. Preparation for Dissection • Freeze anaesthetise the butterflies in a −20 °C freezer for 3–4 min; • Wash the dissection tools and plates in 70% ethanol prior to dissection; 3.2.2. Fixation of Adult Brains for Immunostaining • Place a few drops of HEPES-buffered saline (HBS) onto a dissection plate using a • Gentlyplastic liftpipette, the head and upset withit aside the (see exposed recipe brain for HBS with and the all curved other forceps reagents and at transferthe end itof into this aarticle); well filled with ZnFA solution (Figure3L). Cover the well with a glass slide, • thenIf the place brain the tissues well need plate to on undergo an orbital immunostaining, shaker and agitate fill ita forwell 16–20 in the h spot at 80–100 plate rateswith perZnFA minute solution (rmp); using a plastic pipette, and set it aside before starting any dissection. • AfterIf the fixingbrains overnight, are used transferfor DNA each or RNA fixed extraction, brain from fill the the well RNAse-free into a dissection Eppendorf plate withtubes a with few dropsabout of300µL HBS of by 100% gently ethanol lifting or it upRNAlater, using the respectively. curved forceps; Place only up to • Again,3 brains the in the brain same might well float (for onimmunostaining). the surface of the Number solution. of brains Grab ato small be pooled part ofin theone braintube varies with the with curved the amount forceps of and DNA fully or submergeRNA needed it into for the downstream solution (Figure analyses;4A). CRITICAL STEPSTEP EnsureIf DNA to or cover RNAthe work well is withinvolved, the glass it is sliderecommended so that the to fixing wipe solutionthe equipment does not with evaporate RNAseZap overnight. in step 2, and use a micropipette and filter tips instead in steps 3 and 4. 3.2.3. Dissection of the Adult Brain •3.2. DissectionGently make Procedure a superficial tear in the cuticle of one eye (Figure4B); •3.2.1.Then Exposure start breakingof Adult offBrains the cuticle into small pieces and remove it gently (Figure4C); • Take the butterfly out of the freezer and place it on a second clean dissection plate. TheCRITICAL first series of STEP stepsDo are not performed pull strongly, dry. orDetach the whole the head optic of lobethe butterfly will be detached from the fromrest the restof the of body the brain. using If a the pair cuticle of superfine cannot bedissecting broken off, forceps. try to Pull dislodge the head it via away a rotating from pull. The region of the cuticle nearest to the central brain sticks very closely to the brain the body using your dominant hand while pressing down on the wings with the tissues, and is hard to remove via normal pulling without damaging the tissues. The easiest way to remove it will be to pull it along the orientation of the eye, going from the dorsal to the ventral point in a circular motion, and breaking it off naturally; • After clearing out the cuticle of one eye, remove the piece of non-brain tissue that is loosely attached to the back of the brain, with small bits of cuticle on it (Figure4D); • Proceed to remove the cuticle of the other eye by repeating steps 3 and 4; • Remove the easily accessible trachea (Figure4E); • Then, scrape away the main body of the ommatidia and the basement membrane of the retina from the optic lobes, using a pair of insect pins (Figure4F). These tissues are darkly coloured;

CRITICAL STEP Do not scrape too hard as the basement membrane is ﬁrmly attached to the lamina of the optic lobe. The lamina gets easily destroyed with just a little pulling pressure; • Remove any remaining trachea by pulling gently with a curved forceps (Figure4G), while pressing the brain down with an insect pin or by using insect pins to scrape them off the brain tissue (Figure4H); • Transfer the fully dissected brain back to the well for downstream immunostaining using a pipette (Figure3I), or into an Eppendorf tube ﬁlled with RNAlater (for RNA extraction) or 100% ethanol (for DNA extraction). MethodsMethods Protoc. Protoc. 2021, 20214, x ,FOR4, 53 PEER REVIEW 8 of 188 of 18

FigureFigure 4. Dissection 4. Dissection of an of adult an adult brain. brain. (A ()A A) Asmall small part part of of the the brain isis grabbedgrabbed to to submerge submerge it fully it fully in HBS. in HBS. (B) While (B) While the the brain brainis submerged, is submerged, a superficial a superﬁcial tear tear in inthe the cuticle cuticle of of one one eye eye is made.made. (C(C)) A A small small piece piece of cuticleof cuticle is removed is removed on the on side the side of theof eye. the ( eye.D) The (D) Thenon-brain non-brain tissue tissue that that is isloosely loosely attached attached to to the backback ofof the the brain brain is is removed, removed, as indicatedas indicated by the by white the white circle.circle. (E) The (E) Thecuticle cuticle of both of both eyes eyes is is removed, removed, exposingexposing some some trachea. trachea. The The trachea trachea is the is white, the stringy,white, stringy, and sticky and substance sticky sub- stanceattached attached to to the the brain brain tissues. tissues. It is It important is important to control to control the pulling the pulling force so fo thatrce the so brainthat the tissues brain are tissues not accidentally are not accidental teared. ly teared.(F )(F The) The ommatidia ommatidia are packedare packed together together into a into reddish-brown a reddish-brown spongy tissue,spongy and tissue, this is and removed this is from removed one side from of the one eye. side of the eye.(G )(G Some) Some trachea trachea still still attached attached is pulled is pu awaylled away with curved with curved forceps. forceps. (H) The ( remainingH) The remaining trachea can trachea also be can scraped also be away scraped away withwith insect insect pins. pins. (I )(I The) The fully fully dissected dissected brain brain can be can used be forused further for further analyses. analyses.

4. Dissection4. Dissection of of Pupal Pupal Brains Brains 4.1.4.1. Preparation Preparation for for Dissection Dissection • Place the pupae on ice for about 15 min prior to dissection; • Place the pupae on ice for about 15 min prior to dissection; • Wash the forceps, scalpel, and dissection plate with 70% ethanol. If the brains are used • Washfor RNAthe forceps, extraction, scalpel, use RNAzap and dissection instead; plate with 70% ethanol. If the brains are • usedPrepare for RNA RNase-free extraction Eppendorf, use RNAzap tubes or well instead; plates ﬁlled with the solution appropriate • Preparefor the RNase-free subsequent Eppendorf experiment, tubes and place or well them plates close filled to the with dissection the solution microscope. appropri- ateFor for DNAthe subsequent and RNA extraction, experiment, prepare and tubes place with them about close 300 toµ theL of dissection 100% ethanol microscope. and ForRNAlater, DNA and respectively. RNA extraction, For immunostaining prepare tubes experiments, with about place 300 uL about of 100% 500 µ Lethanol of the and RNAlater, respectively. For immunostaining experiments, place about 500 uL of the ZnFa fixative solution in the wells. Place a drop of cold PBS on the dissection plate (enough to fully cover the tissue, here about 500 uL).

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ZnFa fixative solution in the wells. Place a drop of cold PBS on the dissection plate Figure(enough 2. Anatomy to fully of (A cover) a B. anynana the tissue, butterfly here head about (front 500 view),µL). (B) a caterpillar head (front view), and (C) of a pupa. 4.2. Dissection Procedure •3. DissectionCut the pupae of Adult into Brains two pieces at the base of the thoracic region using the scalpel blade, 3.1. Preparationand move thefor Dissection head and thoracic regions (Figure2C) into the drop of PBS (Figure5A). • TheFreeze abdominal anaesthetise part the of the butterflies cut pupa in can a − be20 discarded;°C freezer for 3–4 min; •• WithWash one the hand,dissection hold tools the cut and pupae plates inside in 70% the ethanol PBS drop prior using to dissection; forceps. With the other • hand,Place a use few the drops forceps of HEPES-buffered tips to push the thoraxsaline (HBS) cuticle onto down a dissection at the base plate of the using head a (Figureplastic 5pipette,B). The junctionand set it between aside (see the recipe head andfor HBS the thorax and all will other break, reagents making at athe small end openingof this article); in the cuticle (Figure5C); •• HoldIf the thebrain cuticle tissues head need at to the undergo opening immunostaining, just made, and fill with a well the otherin the hand,spot plate grab with the thoraxZnFA solution cuticle. Pull using the a thoracicplastic pipette, region andand all set the it aside tissue before that come starting together any dissection. (antennae, legsIf the for brains example, are Figureused for2C) DNA horizontally or RNA towards extraction, you fill while the firmly RNAse-free holding Eppendorf the head cuticletubes with (Figure about5D). 300µL After itof is 100% fully ethanol detached or from RNAlater, the head respectively. cuticle, discard Place the only thoracic up to tissue.3 brains The in the developing same well butterfly (for immunostaining). head and the dorsal Number side of of brains the brain to be will pooled be exposed in one attube the varies bottom with of thethe pupalamount cuticle of DNA (Figure or RNA5E); needed for downstream analyses; CRITICAL STEP DoIf DNA not pullor RNA the thoracicwork is regioninvolved, upward. it is recommended Pull gently in to a wipe hor- izontalthe equipment direction with to preventRNAseZap the in brain step from2, and tearing use a micropipette and being detached and filter from tips instead the head in capsule;steps 3 and 4. • Hold the pupal head down in the PBS with one hand, and use your dominant hand 3.2. Dissection Procedure to remove remaining thoracic, antennal, and mouthpart tissues still attached to the 3.2.1.cuticle Exposure and of to Adult the developing Brains head (Figure2C). With your dominant hand, grab the • tracheaTake the developing butterfly out around of the each freezer eye andand pullplac ite away;it on a second clean dissection plate. • HoldingThe first theseries pupal of steps head are cuticle performed down dry. with Detach one hand, the head use yourof the dominant butterfly from hand the to removerest of the pieces body of using pupa a cuticlepair of aroundsuperfine the di developingssecting forceps. head Pull (Figure the 5headF), and away gently from detachthe body the using head fromyour thedominant cuticle hand by pushing while itpressing aside gently, down or on scraping the wings it out with of thethe cuticle (Figure5G); • When the developing head is completely detached from the pupa cuticle (Figure5H), remove the non-brain head tissue, such as the ommatidia and the facets around the visual neuropils, and the developing mouth parts on the other side of the brain. Hold the brain in place with the forceps slightly opened, while pulling the non-brain tissue away with the other hand; • If the brain (Figure5I) is used for DNA or RNA extraction, grab it with the forceps and move it to the appropriate reagent (100% ethanol or RNAlater, respectively). If the brains are used for immunostaining, suck the brain up from the PBS with a pipette tip and transfer the brain into the fixative solution. Use a very small pipetting volume, large enough to lift the brain, but small enough to prevent taking an excess of PBS. This method will prevent damage or distortion of the brain tissue. MethodsMethods Protoc. Protoc. 2021,2021 4, x, FOR4, 53 PEER REVIEW 10 of10 18 of 18

FigureFigure 5. Dissection 5. Dissection of a of pupal a pupal brain brain at at 30% 30% pupal pupal development. development. (A(A)) The The anterior anterior part part of theof the pupa pupa is submerged is submerged into 1 into× 1× PBS. PBS.(B) The (B) Thethorax thorax cuticle cuticle is pushed is pushed down down and and ( C) detacheddetached at at the the suture suture at theat the base base of the of headthe head (along (along the dashed the dashed line) to line) to makemake a small a small opening. opening. (D (D) )The The thoracic thoracic tissue andand cuticlecuticle are are pulled pulled away away horizontally horizontally from from the the head head tissue tissue (following (following the directionthe direction of the of thearrow). arrow). (E) ( EThe) The developing developing head head is is visible visible atat thethe bottom bottom of of the the pupal pupal cuticle. cuticle. (F) ( ExtraF) Extra pieces pieces of pupal of pupal cuticlecuticle are removed. are removed. (G) (Then,G) Then, the the brain brain is isgently gently scraped scraped out. out. (H)) WhileWhile detached detached from from the the pupa pupa cuticle, cuticle, extra extra head head tissue tissue can becan removed be removed from from the thebrain brain (e.g., (e.g., cornea, cornea, ante antennae,nnae, trachea trachea (seen(seen inin a a bright bright white white colour)). colour)). (I) The(I) The clean clean brain brain can be can be used usedfor downstream for downstream analyses. analyses.

5. Dissection5. Dissection of of the the Larval Larval Brain Brain and GnathalGnathal Ganglion Ganglion 5.1.5.1. Preparation Preparation for for Dissection Dissection • • PlacePlace the the larvae larvae on on ice ice prior prior to dissection.dissection. After After a fewa few minutes, minutes, the the larvae larvae will will become become immobileimmobile and and can can be be easily easily pickedpicked up up with with flat flat forceps; forceps; • Wash the dissecting forceps, blade, and dissection plate with 70% ethanol. If the brains • Wash the dissecting forceps, blade, and dissection plate with 70% ethanol. If the are used for RNA extraction, use RNAzap instead; • brainsPrepare are Eppendorfused for RNA tubes extr or wellaction, plates use filledRNAzap with instead; the solution appropriate for the • Preparesubsequent Eppendorf experiment tubes conducted or well plates with the filled dissected with the brain solution and place appropriate them close tofor the subsequentthe dissection experiment microscope. conducted For DNA with and RNAthe dissected extraction, brain prepare and tubes place with them about close to the300 dissectionµL of 100% microscope. ethanol and For RNAlater, DNA respectively.and RNA extraction, For immunostaining prepare tubes experiments, with about 300µLplace of about 100% 500 ethanolµL of the and fixative RNAlater, solution inre thespectively. wells; For immunostaining experi- • ments,Place place a drop about of cold 500µL PBS (aboutof the fixative 500 µL, tosolution cover the in tissuethe wells; fully) on the dissection • Placeplate. a drop of cold PBS (about 500 µL, to cover the tissue fully) on the dissection plate.

5.2. Dissection Procedure • Using a scalpel blade, cut the head of the larvae and place it in a drop of PBS on the dissection plate (Figure 6A);

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Figure 2. Anatomy of (A) a B. anynana butterfly head (front view), (B) a caterpillar head (front view), and (C) of a pupa.

3. Dissection of Adult Brains Methods Protoc. 2021, 4, 53 11 of 18 3.1. Preparation for Dissection • Freeze anaesthetise the butterflies in a −20 °C freezer for 3–4 min; • Wash the dissection tools and plates in 70% ethanol prior to dissection; 5.2. Dissection Procedure • Place a few drops of HEPES-buffered saline (HBS) onto a dissection plate using a • Usingplastic a pipette, scalpel and blade, set cut it aside the head (see ofrecipe the larvae for HBS and and place all itother in a dropreagents of PBS at the on end the dissectionof this article); plate (Figure6A); •• PlaceIf the brain the back tissues ofthe need head to undergo towards immunostaining, you and remove fill the a well extra in thoracic the spot tissue plate with still attachedZnFA solution to the hardusing cuticle a plastic (Figure pipette,6B); and set it aside before starting any dissection. • WithIf the one brains hand, are hold used the for head DNA inside or RNA the dropextraction, of PBS fill by the grabbing RNAse-free the cuticle Eppendorf at the opening.tubes with Remove about 300µL small of pieces 100% of ethanol the head or RNAlater, cuticle using respectively. the forceps Place on your only otherup to hand3 brains (Figure in the6C). same The well cuticle (for isimmunostaining). hard, so you can Number break little of piecesbrains withto be thepooled tip ofin theone forceps;tube varies with the amount of DNA or RNA needed for downstream analyses; CRITICAL STEPSTEP DoIf DNA not pull or RNA the mandibles work is involved, away from it is the recommended head yet (Figures to wipe2B andthe 6equipmentD). They are with attached RNAseZap to the in gut, step which 2, and goes use towardsa micropipette the back and of filter the head. tips instead passing in juststeps above 3 and the 4. gnathal ganglion between the connectives (the tissue connecting the lobes and the gnathal ganglion). Pulling the mouthparts would tear the gnathal ganglion and possibly3.2. Dissection the brain Procedure itself, by pulling the connectives away from the brain tissue; •3.2.1.When Exposure most of of Adult the backBrains cuticle is removed, turn the head around and proceed to • removeTake the the butterfly cuticle out on of the the front freezer part and of the plac head.e it on Remove a second the clean antennae, dissection the plate. eyes, andThe otherfirst series head of components steps are performed attached todry. the Detach cuticle the (Figure head2 ofB). the All butterfly head soft from tissue, the includingrest of the thebody brain, using will a pair remain of superfine packed di together.ssecting forceps. When most Pull the cuticle head is away removed, from gentlythe body scrap using the packyour ofdominant head tissue hand (with while the mandiblespressing down still attached) on the wings away fromwith thethe remaining cuticle; • By gently pulling away some head tissue (by grabbing them from the right and from the left), you can see the two brain lobes and the gnathal ganglion sitting in the middle (Figure6E). They can be easily recognised from the other tissue, due to their oval shape, smooth texture, and white colour. After identifying the brain, grab and remove pieces of head tissue with one hand, while holding the mandibles with your other hand until the brain and the gnathal ganglion are well exposed. Alternatively, you can grab the group of soft tissue on the right with the corresponding hand, and the other side of the group of tissue with the left hand, and pull them towards opposite directions, freeing the brain placed in between. Pull gently and pay particular attention not to pull the connectives that link the brain with the gnathal ganglion; • When the brain and the gnathal ganglion are well exposed, remove the other extra tissue still attached to them, by maintaining the brain with one hand through using the forceps slightly opened, and pulling the extra tissue away from it with the other hand. Sometimes, the brain and gnathal ganglion can be extracted directly from the head tissue, by pulling them gently with the forceps (Figure6F). Small trachea (identiﬁed thanks to their shiny white colour) that are strongly attached to each brain lobe should be left in place, as they can tear the brain tissue if pulled away; • Pick up the brain and the gnathal ganglion using a pipette and transfer them to the reagent appropriate for downstream experiments. MethodsMethods Protoc. Protoc. 2021, 20214, x ,FOR4, 53 PEER REVIEW 12 of12 18 of 18

FigureFigure 6. Dissection 6. Dissection of the of larval the larval brain. brain. (A) The (A) head The head is cut is out cut of out the of caterpillar the caterpillar body body with with a scalpel. a scalpel. (B) ( BThe) The head head is isturned face downturned for face an down easier for dissection. an easier dissection. (C) Pieces (C of) Pieces cuticle of are cuticle removed are removed piece pieceby piece by piece around around the the head head tissue. tissue. (D (D) )The The man- diblesmandibles are left attached are left attachedto the head to the tissue head (arro tissuew), (arrow), as removing as removing them them might might damage damage the the brain brain (E ().E ).Gently Gently pulling pulling the the head tissuehead on the tissue right on and theright left will and leftcreate will an create opening, an opening, which which exposes exposes the thebrain brain (arrow). (arrow). (F ()F The) The brain brain (arrow) (arrow) attachedattached to to the gnathalthe ganglion gnathal ganglion (white (whitemass below) mass below) can be can gently be gently pulled pulled ou outt after after removing removing thethe pieces pieces of of head head tissue tissue around around it and it and pickedpicked up with up witha pipette a pipette for forfurther further downstream downstream experiments. experiments.

6. Expected6. Expected Results Results 6.1.6.1. Adult Adult Brain Brain The adult brain is made up of two optic lobes and a central mass, which are clearly The adult brain is made up of two optic lobes and a central mass, which are clearly defined (Figure7A). A more in-depth visualisation of the brain anatomy can be achieved definedthrough (Figure immunostaining, 7A). A more using in-depth an antibody visualis targetingation synapsinof the brain (involved anatomy in the can regulation be achieved throughof neurotransmitter immunostaining, release using at synapses) an antibody and confocal targeting microscopy synapsin techniques. (involved In in brief, the the regula- tionoptic of neurotransmitter lobes consist of five releas pairede at neuropils,synapses) whichand confocal are clearly micr defined:oscopy thetechniques. lamina (La), In brief, themedulla optic lobes (Me), consist accessory of five medulla paired (aMe), neuropils, lobula (Lo),which and are lobula clearly plate defined: (LoP). the The lamina central (La), medullabrain consists(Me), accessory of five paired medulla and one (aMe), unpaired lobula neuropils, (Lo), and which lobula are plate also clearly (LoP). defined: The central brainthe consists antennal of lobes five (AL), paired anterior and one optic unpaired tubercles neuropils, (AOTu), mushroom which are body also lobes clearly (MB-lb) defined: theand antennal calyx (MB-ca), lobes (AL), protocerebral anterior bridgeoptic tubercles (PB), and the(AOTu), central mushroom body (CB) (Figure body7 lobesB–F). (MB-lb) The extraction of a single dissected adult brain using the Rneasy Plus Mini Kit (Qiagen, and calyx (MB-ca), protocerebral bridge (PB), and the central body (CB) (Figure 7B–F). Singapore) yielded an average of 4500 ng of mRNA, which is a sufficiently large amount forThe further extraction experiments of a single (e.g., qPCR, dissected RNAseq). adult brain using the Rneasy Plus Mini Kit (Qi- agen, Singapore) yielded an average of 4500 ng of mRNA, which is a sufficiently large amount6.2. Pupal for further Brain experiments (e.g., qPCR, RNAseq). Similar to the adult brain, the pupal brain also consists of two optic lobes and a central mass which are clearly defined. Visualisation under immunostaining (targeting synapsin) shows three paired neuropils in the optic lobes and five paired and one unpaired neuropil in the central brain. In brief, the neuropils visualised in the optic lobes are the medulla (Me), accessory medulla (aMe), and the lobula (Lo) (Figure8B,C). In the central brain, the paired neuropils are the antennal lobes (AL), anterior optic tubercles (AOTu), mushroom body lobes (MB-lb) and calyx (MB-ca), protocerebral bridge (PB), and the central body (CB) (Figure8A,B,D). However, the AL and the MB-ca are not as well-defined or well-developed in the pupal brain than as in the adult brain.

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FigureFigure 7. (7.A )(A Adult) Adult brain brain after after dissection, dissection, showing showing the th twoe two optic optic lobes lobes and and the the central central brain brain (B –(FB).–F Visualisation). Visualisation of theof the adult adult brain, immunostained with an antibody against synapsin, in different confocal sections from most anterior (B) to brain, immunostained with an antibody against synapsin, in different confocal sections from most anterior (B) to most most posterior (F). (B) Antennal lobes (AL). (C) Accessory medulla (aMe), anterior optic tubercles (AOTu), and mushroom posteriorbody lobes (F). (B(MB-lb).) Antennal (D) lobesLamina (AL). (La), (C medulla) Accessory (Me), medulla the lobula (aMe), (Lo), anterior lobula opticplate tubercles(LoP), and (AOTu), the central and body mushroom (CB). (E body) Methods Protoc. 2021, 4, x FOR PEER REVIEW 14 of 18 lobesMushroom (MB-lb). body (D) Lamina calyx (MB-ca). (La), medulla (F) Protocerebral (Me), the lobulabridge (Lo),(PB). lobula plate (LoP), and the central body (CB). (E) Mushroom body calyx (MB-ca). (F) Protocerebral bridge (PB). 6.2. Pupal Brain Similar to the adult brain, the pupal brain also consists of two optic lobes and a central mass which are clearly defined. Visualisation under immunostaining (targeting synapsin) shows three paired neuropils in the optic lobes and five paired and one unpaired neuropil in the central brain. In brief, the neuropils visualised in the optic lobes are the medulla (Me), accessory medulla (aMe), and the lobula (Lo) (Figure 8B,C). In the central brain, the paired neuropils are the antennal lobes (AL), anterior optic tubercles (AOTu), mushroom body lobes (MB-lb) and calyx (MB-ca), protocerebral bridge (PB), and the central body (CB) (Figure 8A,B,D). However, the AL and the MB-ca are not as well-defined or well-developed in the pupal brain than as in the adult brain. The extraction of a single dissected pupal brain using the Rneasy Plus Mini Kit (Qi- agen, Singapore) yielded an average of 3000 ng of mRNA, a sufficiently large amount that can be used for further experiments (e.g., qPCR, RNAseq).

6.3. Larval Brain The larval brain appears relatively different from the adult and pupal brains. The larval brain consists of two fused lobes connected dorsally to a ventral ganglion by connective tissues. Further analysis of the brain by immunostaining did not reveal any defined neuropils (Figure 9). The extraction of a single dissected larval brain using the Rneasy Plus Mini Kit (Qi- agen, Singapore) yielded an average of 1000 ng of mRNA. This amount is sufficient for subsequent qPCR analysis. Several larval brains can be pooled into one tube, to get a higher RNA yield as required by some sequencing companies.

FigureFigure 8. Visualisation 8. Visualisation of theof the pupal pupal brain brain in in different different confocalconfocal sections from from most most anterior anterior (A) ( Ato) most to most posterior posterior (D) stained (D) stained with synapsin. (A) Mushroom body lobes (MB-lb), anterior optic tubercles (AOTu), and antennal lobes (AL). (B) Central with synapsin. (A) Mushroom body lobes (MB-lb), anterior optic tubercles (AOTu), and antennal lobes (AL). (B) Central body (CB) and accessory medulla (aMe). (C) Medulla (Me) and lobula (Lo). (D) Mushroom body calyx (MB-ca) and pro- bodytocerebral (CB) and bridge accessory (PB). medulla (aMe). (C) Medulla (Me) and lobula (Lo). (D) Mushroom body calyx (MB-ca) and protocerebral bridge (PB).

Figure 9. Visualisation of the larval brain, immunostained with an antibody against synapsin in a single confocal section, showing the lobes, connective tissues, and ganglion.

6.4. Brains of Other Butterfly Species We applied our dissection procedure to brains of other species from different families and of various sizes. We successfully dissected the brain of Pieris canidia at the same stages as in B. anynana: adult, 30% pupal development, and 5th instar larva (Figure 10A–D). We

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The extraction of a single dissected pupal brain using the Rneasy Plus Mini Kit (Qiagen, Singapore) yielded an average of 3000 ng of mRNA, a sufﬁciently large amount that can be used for further experiments (e.g., qPCR, RNAseq).

Figure 8. Visualisation of the6.3. pupal Larval brain Brain in different confocal sections from most anterior (A) to most posterior (D) stained with synapsin. (A) Mushroom bodyThe larval lobes brain (MB-lb), appears relativelyanterior differentoptic tubercles from theadult (AOTu), and pupal and brains.antennal The larvallobes (AL). (B) Central brain consists of two fused lobes connected dorsally to a ventral ganglion by connective body (CB) and accessory medulla (aMe). (C) Medulla (Me) and lobula (Lo). (D) Mushroom body calyx (MB-ca) and pro- tissues. Further analysis of the brain by immunostaining did not reveal any deﬁned tocerebral bridge (PB). neuropils (Figure9).

FigureFigure 9. Visualisation9. Visualisation of the larval of the brain, larval immunostained brain, immunostai with an antibodyned with against an synapsin antibody in a against synapsin in a singlesingle confocal confocal section, section, showing showing the lobes, connectivethe lobes, tissues, connective and ganglion. tissues, and ganglion. The extraction of a single dissected larval brain using the Rneasy Plus Mini Kit (Qiagen,6.4. Brains Singapore) of Other yielded Butterfly an average Species of 1000 ng of mRNA. This amount is sufﬁcient for subsequent qPCR analysis. Several larval brains can be pooled into one tube, to get a higherWe RNA applied yield as required our dissection by some sequencing procedure companies. to brains of other species from different families and of various sizes. We successfully dissected the brain of Pieris canidia at the same stages 6.4. Brains of Other Butterﬂy Species as in B. anynana: adult, 30% pupal development, and 5th instar larva (Figure 10A–D). We We applied our dissection procedure to brains of other species from different families and of various sizes. We successfully dissected the brain of Pieris canidia at the same stages as in B. anynana: adult, 30% pupal development, and 5th instar larva (Figure 10A–D). We also successfully dissected the brains of Malayan eggfly adults (Nymphalidae) (Figure 10E,F) and of Papilio sp butterflies (Papilionidae) (Figure 10G,H).

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Methods Protoc. 2021, 4, 53 15 of 18 also successfully dissected the brains of Malayan eggfly adults (Nymphalidae) (Figure 10E,F) and of Papilio sp butterflies (Papilionidae) (Figure 10G,H).

FigureFigure 10. 10.Brains Brains dissecteddissected from fromPieris Pieris canidia, canidia, Hypolimnas Hypolimnas anomala, anomala,and andPapilio Papilio sp. sp.( A(A––CC) )Pieris Pieris canidia canidia brainbrain at at the the adult adult stage stage (fixed (fixed brain, brain,B B),), at at 30% 30% pupal pupal development development (not (not fixed, fixed,C C),), and and the the 5th 5th instar instar larval stage (not fixed, D). (F) Adult brain from Hypolimnas anomala (Nymphalidae, E). (H) Adult larval stage (not fixed, D). (F) Adult brain from Hypolimnas anomala (Nymphalidae, E). (H) Adult brain from Papilio sp (Papilionidae, G). brain from Papilio sp (Papilionidae, G).

7.7. ReagentsReagents 7.1.7.1. PreparationPreparation of of HEPES-Buffered HEPES-Buffered Saline Saline (HBS) (HBS) •• AddAdd 800 800 mL mL of of MilliQ MilliQ water water and and reagents reagents mentioned mentioned in in Table Table1 to1 to a 1a L1 glassL glass bottle; bottle;

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Table 1. Reagents used to make 1 L of HBS.

Reagents Amount (g) NaCl 8.77 KCl 3.73 CaCl2 5.55 Sucrose 8.56 HEPES 2.38

• Measure the pH of the solution using a pH metre, and adjust accordingly with drops of HCl and NaOH until the solution has a pH of 7.4; • Then, add MilliQ water until the volume in the bottle reaches 1 L; • Autoclave the solution and store it at 4 ◦C thereafter.

7.2. Preparation of 10× Phosphate-Buffered Saline (PBS) • Add 800 mL of MilliQ water and reagents mentioned in Table2 to a 1 L glass bottle;

Table 2. Reagents used to make 1 L of 10× PBS.

Reagents Amount (g) NaCl 81.80 KH2PO4 5.28 K2HPO4 10.68

• Measure the pH of the solution using a pH metre and adjust accordingly with drops of HCl and NaOH until the pH is around 6.8; • Add MilliQ water until the volume in the bottle reaches 1 L; • Autoclave the solution and store it at 4 ◦C thereafter; • For the preparation of 100 mL of 1× PBS, dilute 10 mL of 10× PBS into 90 mL of MilliQ water.

7.3. Preparation of Zinc Formaldehyde (ZnFA) • Add 800 mL of MilliQ water and reagents mentioned in Table3 to a 1 L glass bottle;

Table 3. Reagents used to make 1 L of ZnFA.

Reagents Amount ZnCl 2.51 g NaCl 7.89 g Sucrose 11.98 g 4% Formaldehyde 10 mL

• Measure the pH of the solution using a pH metre and adjust accordingly with drops of HCl and NaOH until the pH is 6.35; • Add MilliQ water until the volume in the bottle reaches 1 L and autoclave the solution for 20 min at 121 ◦C; • Add 4% Formaldehyde and store it at 4 ◦C; • For the preparation of 4% Formaldehyde, dilute 10 mL of 16% Formaldehyde in 30 mL of 1× PBS.

Author Contributions: Conceptualisation, Y.P.T., E.D. and A.M.; methodology, Y.P.T. and E.D.; writing—original draft preparation, Y.P.T. and E.D.; writing—review and editing, E.D. and A.M.; video recording and editing, Y.P.T. and E.D.; funding acquisition, E.D. and A.M.; equal contribution, Y.P.T. and E.D. All authors have read and agreed to the published version of the manuscript. Methods Protoc. 2021, 4, 53 17 of 18

Funding: This research was supported by the Ministry of Education (MOE) Singapore, award MOE2018-T2-1-092 and by the NUS Resilience and Growth Initiative funded by the SG United Traineeship Program, Singapore. Data Availability Statement: All the data collected during this study are available in the article. Acknowledgments: The authors thank T.D. Banerjee for his help in using the microscope and the recording of the butterfly brain dissection videos; L. Guyonvarch for his guidance in editing the caterpillar and pupal brain dissection videos; J. Wee and T.D. Banerjee for providing extra butterfly species; the Firefly Farm for providing the caterpillar food; and anonymous reviewers that helped in improving the manuscript. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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