Dissection of Larval and Pupal Wings of Bicyclus Anynana Butterflies

Protocol Dissection of Larval and Pupal Wings of Bicyclus anynana Butterﬂies

Tirtha Das Banerjee 1,* and Antónia Monteiro 1,2

1 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; [email protected] 2 Yale-NUS College, 10 College Avenue West, Singapore 138609, Singapore * Correspondence: [email protected]

Received: 9 December 2019; Accepted: 6 January 2020; Published: 10 January 2020

Abstract: The colorful wings of butterflies are emerging as model systems for evolutionary and developmental studies. Some of these studies focus on localizing gene transcripts and proteins in wings at the larval and pupal stages using techniques such as immunostaining and in situ hybridization. Other studies quantify mRNA expression levels or identify regions of open chromatin that are bound by proteins at different stages of wing development. All these techniques require dissection of the wings from the animal but a detailed video protocol describing this procedure has not been available until now. Here, we present a written and accompanying video protocol where we describe the tools and the method we use to remove the larval and pupal wings of the African Squinting Bush Brown butterfly Bicyclus anynana. This protocol should be easy to adapt to other species.

Keywords: wing dissection; Bicyclus anynana; butterﬂies

1. Introduction Multiple studies on butterflies are focused on understanding the evolutionary and developmental genetics of their colorful wing patterns. Labs around the world have worked with different species to address a variety of questions at the intersection of these fields. Examples include the discovery of the involvement of the gene optix in wing pattern mimicry of Heliconius butterflies [1,2]; discovery of doublesex as a mimicry supergene in Papilio polytes [3]; involvement of Wnt signaling in wing pattern of butterflies such as Junonia coenia, Heliconius erato and Vanesssa cardui [4–6]; involvement of the genes spalt and BarH-1 in the wing pigmentation of multiple Pieris and Colias species [7–9]; and the involvement of calcium signaling in wing patterning in Junonia orithya [10] to mention a few. Bicyclus anynana has been a popular system to study wing patterning, especially the eyespots, which are novel traits to the nymphalid lineage [11]. Many of the studies have been focused on identifying the local expression of proteins such as Spalt [12], Engrailed/Invected [12], Distal-less [13], Antennapedia [14,15], Notch [16], Cubitus-interruptus [17], Ecdysone Receptor [18], Ultrabithorax [15,17], and the expression of gene transcripts such as hedgehog [14], apterous [19], patched [16], wingless [20], doublesex [21] and decapentaplegic [22] in the developing wing. Gene expression and RNAi studies have proposed that many wound healing network genes are expressed in eyespots [23], that a positional-information mechanism is involved in the formation of the concentric rings [16,20], and that a reaction-diffusion mechanism is involved in setting up the eyespot centers [22] using gene expression and functional analysis via CRISPR-Cas9 [24,25]. All these studies require the removal of wings from the bodies of larvae and/or pupae to examine patterns of gene expression. Protocols describing the dissection of larval and pupal wings have been previously published [26,27].

Methods Protoc. 2020, 3, 5; doi:10.3390/mps3010005 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, 5 2 of 7

However, the protocols are brief and have no accompanying video, making it difficult for newcomers in the field to follow. In this paper, we describe the process of larval and pupal wing removal using a video and explain the process along with all the tools and chemicals needed in the main text below. This protocol can also supplement other similar experiments such as live cell imaging in vivo used to understand cell differentiation and dynamics [28,29].

2. Experimental Design

2.1. Required Materials and Equipment

2.1.1. Materials Curved tweezers (Dumont; Dumont Switzerland, Montignez, Switzerland; Cat. No.: 11274-20); • Methods Protoc. 2020, 3, x FOR PEER REVIEW 2 of 7 Fine straight tweezers (Dumont; Dumont Switzerland; Cat. No.: 11254-20); • previouslyFlat spatula published (Thomas [26,27]. However, Scientific; the prot Thomasocols are brief Scientific, and have no Swedesboro, accompanying video, NJ, USA; Cat. No.: 1208Y75); • making it difficult for newcomers in the field to follow. RegularIn this paper, straight we describe tweezers the process (Dumont; of larval and Dumont pupal wing Switzerland; removal using Cat.a video No.: (see 0203-5-PO); • SupplementarySuperfine Materials) Vannas and scissorsexplain the process 8 cm along (World with all Precision the tools and Instruments; chemicals needed Worldin Precision Instruments, • the main text below. This protocol can also supplement other similar experiments such as live cell imagingSarasota, in vivo used FL, to USA; understand Cat. cell No.: differentiation 501778); and dynamics [28,29]. Blade holder (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0934); • 2. Experimental Design Blades (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0115); • 2.1. Required Materials and Equipment Glass spot plate (PYREXTM; Corning, Corning, NY, USA; Cat. No.: 722085); • 2.1.1.Dissection Materials silicone plate (Dragon Skin 30 Mould Making Silicone Rubber; Cat. No.: 0751635278417. • • PetriCurved plate; tweezers Sigma-Aldrich; (Dumont; Dumont Switzerland, Sigma-Aldrich, Montignez, Singapore; Switzerland; Cat. Cat. No.: No.: 11274-20); P5981-100EA); • Fine straight tweezers (Dumont; Dumont Switzerland; Cat. No.: 11254-20); • InsectFlat spatula pins (Thomas (BioQuip; Scientific; BioQuip, Thomas Scient Ranchoific, Swedesboro, Dominguez, NJ, USA; CA,Cat. No.: USA; 1208Y75); Cat. No.: 1208B2). • • Regular straight tweezers (Dumont; Dumont Switzerland; Cat. No.: 0203-5-PO); • 2.1.2. Superfine Equipment Vannas scissors 8 cm (World Precision Instruments; World Precision Instruments, Sarasota, FL, USA; Cat. No.: 501778); • ZeissBlade holder Dissection (Swann-Morton Microscope No. 4; Swann- (Carl-Zeiss,Morton, Sheffield, Jena, UK; Germany; Cat. No.: 0934); Stemi 305) • • Blades (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0115); • Glass spot plate (PYREXTM; Corning, Corning, NY, USA; Cat. No.: 722085); 2.1.3.• Dissection Reagents silicone plate (Dragon Skin 30 Mould Making Silicone Rubber; Cat. No.: 0751635278417. Petri plate; Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P5981-100EA); • NaClInsect pins (Sigma-Aldrich; (BioQuip; BioQuip, Sigma-Aldrich,Rancho Dominguez, CA, Singapore; USA; Cat. No.: Cat. 1208B2). No.: S9888-500G); • 2.1.2.K EquipmentHPO (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P3786-500G); • 2 4 • KHZeiss PODissection(Sigma-Aldrich; Microscope (Carl-Zeiss, Sigma-Aldrich, Jena, Germany; Stemi Singapore; 305) Cat. No.: 229806-250G); • 2 4 RNAseZap (Themo Fisher Scientific; Thermo Fisher Scientific, Waltham, MA, USA; Cat. No.: • 2.1.3. Reagents • AM9780).NaCl (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: S9888-500G); • K2HPO4 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P3786-500G); • 3. Procedure KH2PO4 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: 229806-250G); • RNAseZap (Themo Fisher Scientific; Thermo Fisher Scientific, Waltham, MA, USA; Cat. No.: 3.1. PreparationAM9780). for Dissection 3. Procedure 1. Transfer 500 µL of 1 PBS into each well of the spot glass plate. × 2. 3.1. PreparationTransfer for around Dissection 100 mL of 1 PBS into the dissection well plate. × 3. 1. WashTransfer the500 µL dissection of 1 × PBS into tools each inwell 70% of the ethanol spot glass plate. prior to dissection. 2. Transfer around 100 mL of 1 × PBS into the dissection well plate. 4. 3. FreezeWash the anaesthetizedissection tools in the70% ethanol larvae prior and to pupaedissection. on ice for 10–20 min. 4. Freeze anaesthetize the larvae and pupae on ice for 10–20 min. CRITICALCRITICAL STEP: If you STEP are performing: If you are experiments performing involving experiments RNA, it is recommended involving RNA, it is recommended that allthat the equipment all the equipment is wiped with RNAseZap. is wiped with RNAseZap. 3.2. Dissection of Larval Wings 3.2.1. DissectionPick one larva of from Larval the ice Wings and carefully secure it in the dissection plate with the help of two pins. One pin should be placed immediately posterior to the head capsule, and the second pin 1. Pickat the end one of larvathe abdomen. from It the is recommended ice and carefully to stretch the secure larva, before it in placing the dissection the second pin, plate with the help of two pins. Oneto make pin the shoulddissection and be placedremoval of immediately wings easier. posterior to the head capsule, and the second pin at the 2. The wings are located around the second and third thoracic legs.

Methods Protoc. 2020, 3, x FOR PEER REVIEW 2 of 7

previously published [26,27]. However, the protocols are brief and have no accompanying video, making it difficult for newcomers in the field to follow. In this paper, we describe the process of larval and pupal wing removal using a video (see Supplementary Materials) and explain the process along with all the tools and chemicals needed in the main text below. This protocol can also supplement other similar experiments such as live cell imaging in vivo used to understand cell differentiation and dynamics [28,29].

2. Experimental Design

2.1. Required Materials and Equipment

2.1.1. Materials • Curved tweezers (Dumont; Dumont Switzerland, Montignez, Switzerland; Cat. No.: 11274-20); • Fine straight tweezers (Dumont; Dumont Switzerland; Cat. No.: 11254-20); • Flat spatula (Thomas Scientific; Thomas Scientific, Swedesboro, NJ, USA; Cat. No.: 1208Y75); Methods• Regular Protoc. straight2020, 3tweezers, 5 (Dumont; Dumont Switzerland; Cat. No.: 0203-5-PO); 3 of 7 • Superfine Vannas scissors 8 cm (World Precision Instruments; World Precision Instruments, Sarasota, FL, USA; Cat. No.: 501778); • Blade holder (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0934); • endBlades of (Swann-Morton the abdomen. No. 4; Swann-Morton, It is recommended Sheffield, UK; to Cat. stretch No.: 0115); the larva, before placing the second pin, to • makeGlass spot the plate dissection (PYREXTM; Corning, and removal Corning, NY, of USA; wings Cat. easier. No.: 722085); • Dissection silicone plate (Dragon Skin 30 Mould Making Silicone Rubber; Cat. No.: 2. The0751635278417. wings arePetri locatedplate; Sigma-Aldrich; around theSigm seconda-Aldrich, Singapore; and third Cat. thoracic No.: P5981-100EA); legs. 3. • HoldInsect pins the (BioQuip; epidermis BioQuip, of Rancho the larva Dominguez, using CA, a straightUSA; Cat. No.: tweezer 1208B2).and using the Vannas scissors make an

2.1.2.incision, MethodsEquipment Protoc. as 2020 indicated, 3, x FOR inPEER Figure REVIEW1A. 3 of 7 4. • AfterZeiss Dissection the incision Microscope try (Carl-Zeiss, to find Jena, the hindwingGermany; Stemi around 305) the third thoracic leg (Figure1C). If you are working3. Hold with the epidermis a young of fifth the instarlarva using larva, a straight the wing tweezer can beand identified using the Vannas adjacent scissors to a whitemake an lump of 2.1.3. Reagents tissueincision, around as the indicated thoracic in Figure leg (Figure 1A. 1D). This white tissue at the base of the wing will become • NaCl4. (Sigma-Aldrich;After the incision Sigma-Aldrich, try to find Singapore; the hindwing Cat. No.: S9888-500G); around the third thoracic leg (Figure 1C). If you are • the trachea that will invade the wing blade and is the preferred spot for handling the wing to K2HPOworking4 (Sigma-Aldrich; with aSigma-Aldrich, young fifth Singapore; instar larva, Cat. No.: the P3786-500G); wing can be identified adjacent to a white lump of • KH2PO4 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: 229806-250G); avoidtissue touching around the the actual thoracic wing leg (Figure tissue attached.1D). This white tissue at the base of the wing will become • RNAseZap (Themo Fisher Scientific; Thermo Fisher Scientific, Waltham, MA, USA; Cat. No.: 5. MakeAM9780).the cuts trachea to the that tissues will invade/trachea the attachedwing blade on and both is the sides preferred of the spot wing for and handling carefully the wing pull to out the wingavoid (touching touching only the the actual white wing tissue) tissue usingattached. a fine tweezer. 3. Procedure 6. Transfer5. Make the cuts hindwing to the tissues/trachea to one of the attached wells ofon the both glass sides plate. of the wing and carefully pull out the wing (touching only the white tissue) using a fine tweezer. 7. 3.1. PreparationThe forewing for Dissection is present just a few millimeters above the third thoracic leg (Figure1A). Perform 6. Transfer the hindwing to one of the wells of the glass plate. 1. theTransfer cuts 500 as µL with of 1 × PBS the into hindwing each well of and the spot pull glass out plate. the wing using a fine tweezer. 2. Transfer7. The around forewing 100 mL is of present1 × PBS into just the a dissection few millimeters well plate. above the third thoracic leg (Figure 1A). Perform 8. 3. TransferWash the dissection cuts the as forewing withtools in the 70% tohindwing ethanol one ofprior theand to dissection. wellspull out of the the wing glass using plate. a fine tweezer. 4. Freeze8. Transferanaesthetize the the forewing larvae and topupae one on of ice the for wells 10–20 min.of the glass plate. CRITICALCRITICAL STEP: If you STEP: are performingBe careful experiments not to involving touch RNA, the wingit is recommended membrane as even a gentle contact that all theCRITICAL equipment isSTEP: wiped Be with careful RNAseZap. not to touch the wing membrane as even a gentle contact with withthe thetweezer tweezer can damage can damage the wing. the wing. 3.2. Dissection of Larval Wings 1. Pick one larva from the ice and carefully secure it in the dissection plate with the help of two pins. One pin should be placed immediately posterior to the head capsule, and the second pin at the end of the abdomen. It is recommended to stretch the larva, before placing the second pin, to make the dissection and removal of wings easier. 2. The wings are located around the second and third thoracic legs.

FigureFigure 1. Dissection 1. Dissection of larvalof larval and and pupal pupal wingswings of Bicyclus anynana anynana. (A.(). ALarval). Larval wings wings are located are located laterallylaterally (black (black arrows), arrows), dorsal dorsal to the to secondthe second and an thirdd third thoracic thoracic legs. legs. The The region region for for incision incision is is marked marked by a dotted line. (B). A larval forewing is located dorsally to the second thoracic leg. (C). A by a dotted line. (B). A larval forewing is located dorsally to the second thoracic leg. (C). A larval larval hindwing is located beside the third thoracic leg. (D). Early larval wings can be identified by hindwing is located beside the third thoracic leg. (D). Early larval wings can be identiﬁed by ﬁnding finding the white tissue (black arrow) around the thoracic legs. Furthermore, to release the pressure the whitedue tissueto the (blackgut it is arrow) recommended around theto make thoracic an initial legs. Furthermore,dorsal incision to(through release which the pressure the gut duecan to the gut itextend) is recommended before the tolateral make incision an initial is made. dorsal (E). incision For the (throughdissection whichof pupal the wings gut canmake extend) incision before as the lateralmarked incision by is the made. dotted (E ).line. For ( theF). Pupal dissection forewing. of pupal (G). wingsPupal hindwing. make incision (H). Early as marked (16–26 byh after the dotted line. (pupation)F). Pupal pupal forewing. forewing. (G). Pupal hindwing. (H). Early (16–26 h after pupation) pupal forewing.

3.3. Dissection3.3. Dissection of Pupal of Pupal Wings Wings 1. Secure1. Secure a pupa a pupa in thein the dissection dissection plate plate withwith the help help of of two two fine ﬁne pins. pins.

2. Make2. Make incisions incisions using using a ﬁnea fine blade blade at at thethe region marked marked in inFigure Figure 1E.1 E. 3. Remove the cuticle using a curved tweezer. The forewing should be visible at the surface of the 3. Removepupa the (Figure cuticle 1F). using If you a are curved working tweezer. with a Thewing forewing that is less should than 26 beh old, visible the forewing at the surface might of the pupabe (Figure still attached1F). If youto the are cuticle working (Figure with 1H). a Using wing a that straight is less tweezer than on 26 one h old, hand, the hold forewing the cuticle might be still attacheddown and tousing the a cuticle curved (Figuretweezer1 onH). the Using other ahand straight, gently tweezer dislodge on the one wing hand, from holdthe cuticle, the cuticle scraping the wing from underneath in gentle nudges, and finally pull out the wing.

CRITICAL STEP: Make sure that the forewing is free from any attachment to the cuticle.

Methods Protoc. 2020, 3, x FOR PEER REVIEW 2 of 7

2. Experimental Design

2.1. Required Materials and Equipment

2.1.1. Materials • Curved tweezers (Dumont; Dumont Switzerland, Montignez, Switzerland; Cat. No.: 11274-20); • Fine straight tweezers (Dumont; Dumont Switzerland; Cat. No.: 11254-20); • Flat spatula (Thomas Scientific; Thomas Scientific, Swedesboro, NJ, USA; Cat. No.: 1208Y75); • Regular straight tweezers (Dumont; Dumont Switzerland; Cat. No.: 0203-5-PO); • Superfine Vannas scissors 8 cm (World Precision Instruments; World Precision Instruments, Sarasota, FL, USA; Cat. No.: 501778); • Blade holder (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0934); • Blades (Swann-Morton No. 4; Swann-Morton, Sheffield, UK; Cat. No.: 0115); • Glass spot plate (PYREXTM; Corning, Corning, NY, USA; Cat. No.: 722085); • Dissection silicone plate (Dragon Skin 30 Mould Making Silicone Rubber; Cat. No.: 0751635278417. Petri plate; Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P5981-100EA); • Insect pins (BioQuip; BioQuip, Rancho Dominguez, CA, USA; Cat. No.: 1208B2).

2.1.2. Equipment • Zeiss Dissection Microscope (Carl-Zeiss, Jena, Germany; Stemi 305)

2.1.3. Reagents • NaCl (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: S9888-500G); • K2HPO4 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: P3786-500G); • KH2PO4 (Sigma-Aldrich; Sigma-Aldrich, Singapore; Cat. No.: 229806-250G); • RNAseZap (Themo Fisher Scientific; Thermo Fisher Scientific, Waltham, MA, USA; Cat. No.: AM9780).

Methods3. Procedure Protoc. 2020, 3, 5 4 of 7

3.1. Preparation for Dissection 1. Transfer 500 µL of 1 × PBS into each well of the spot glass plate. 2. downTransfer andaround using 100 mL a of curved 1 × PBS into tweezer the dissection on thewell plate. other hand, gently dislodge the wing from the cuticle, 3. scrapingWash the dissection the wing tools in from 70% ethanol underneath prior to dissection. in gentle nudges, and finally pull out the wing. 4. Freeze anaesthetize the larvae and pupae on ice for 10–20 min. CRITICALCRITICAL STEP: If you STEP: are performingMake sure experiments that the involving forewing RNA, it is is freerecommended from any attachment to the cuticle. that all the equipment is wiped with RNAseZap. 4. After the forewing is free, transfer the wing to one of the wells of the glass plate using a flat 3.2. Dissection of Larval Wings spatula. Hold the wing by the hinge region, and do not touch the rest of the wing blade with 1. Pick one larva from the ice and carefully secure it in the dissection plate with the help of two thepins.Methods tweezers.One pin Protoc. should 2020 Hold be, 3 ,placed x FOR the immediatelyPEER wing REVIEW with post theerior tweezersto the head capsule, against and the the second spatula pin until the spatula4 breaksof 7 the liquid-airat the end of the surface abdomen. interface. It is recommended You canto stretch also the use larva, the before tweezers placing the to second gently pin, help slide the wing into the to make4. theAfter dissection the forewing and removal is free,of wings transfer easier. the wing to one of the wells of the glass plate using a flat glass wells. 2. The wingsspatula. are located Hold around the thewing second by the and hinge third thoracic region, legs. and do not touch the rest of the wing blade with the 5. To remove the hindwing (Figure1G), make an incision around the wing using a fine blade and tweezers. Hold the wing with the tweezers against the spatula until the spatula breaks the carefullyliquid-air pull out surface a glassy interface. (peripodial) You can also membrane use the tweezers on top to of gently the hindwing. help slide the wing into the 6. After theglass membrane wells. is removed, make a cut at the wing-hinge region and pull out the wing using a curved5. To remove tweezer. the hindwing (Figure 1G), make an incision around the wing using a fine blade and 7. After thecarefully hindwing pull out isa glassy free, (peripodial) transfer the membrane wing to on one top of of the the hindwing. wells of the glass plate using a 6. After the membrane is removed, make a cut at the wing-hinge region and pull out the wing flat spatula.using a curved tweezer. 7. After the hindwing is free, transfer the wing to one of the wells of the glass plate using a flat spatula.CRITICAL STEP: Be careful not to touch the wing membrane as even a gentle contact with the tweezer can damage the wing. CRITICAL STEP: Be careful not to touch the wing membrane as even a gentle contact with 4. Expected Resultsthe tweezer can damage the wing.

Larval Wings4. Expected and Pupal Results Wing LarvalLarval wings Wings and at anPupal early Wing developmental stage are marked by a lack of tracheal invasion in the wing disc andLarval a prominent wings at an white early tissuedevelopmental at the proximal stage are partmarked of theby a wing lack of (Figure tracheal2A,B). invasion Larval in the wings at a later stagewing disc are and larger a prominent and marked white bytissue the at invasionthe proxim ofal trachealpart of the tissuewing (Figure along 2A,B). the veins Larval (Figure wings 2C,D). Pupal wingsat a later around stage are 18–24 larger h and will marked have prominentby the invasion tracheal of tracheal tissue tissue in thealong wing the veins blade (Figure (Figure 2C,D).2E,F). The Pupal wings around 18–24 h will have prominent tracheal tissue in the wing blade (Figure 2E,F). The wings at the pupal stages are much larger and fragile than at the larval stage. Care must be taken to wings at the pupal stages are much larger and fragile than at the larval stage. Care must be taken to preventprevent damage damage to the to wing the wing tissue tissue at thisat this stage. stage.

Figure 2.FigureLarval 2. andLarval Pupal and wingsPupal ofwingsBicyclus of Bicyclus anynana anynana.(A). Early (A) Early larval larval forewing forewing showing showing the prominentthe white tissueprominent that white will ditissueﬀerentiate that will into differentiate the trachea. into Wingsthe trachea. should Wings be should handled be inhandled this regionin this during region during dissections; (B) Early larval hindwing; (C) Late larval forewing; (D) Late larval dissections; (B) Early larval hindwing; (C) Late larval forewing; (D) Late larval hindwing; (E) Pupal hindwing; (E) Pupal forewing; (F) Pupal hindwing. forewing; (F) Pupal hindwing.

Methods Protoc. 2020, 3, 5 5 of 7

5. Discussion Butterflies are becoming a model system to understand the process of color pattern formation in Biology. Over the past three decades numerous research papers have illuminated the processes involved in eyespots development in the wings of butterflies such as Bicyclus anynana and Junonia coenia [11,13,20,22,23,30,31]; color patterning and mimicry in Heliconius and Papilio butterflies [1–3]; and involvement of multiple signaling pathways in wing pigmentation in species belonging to the genus Pieris, Junonia, and Colias [4,7,9,10]. Almost all of these studies involved the process of wing dissections. The dissected wings can be used to localize proteins and gene transcripts involved in color patterning [5,12] and for more advanced techniques such as RNA, FAIRE, and ATAC sequencing [2,23]. Wing dissections, hence, are indispensable for a full understanding of the evolution and development of butterfly wing color patterns. Furthermore, experiments such as in vivo live cell imaging [28,29], used to study cellular dynamics overlap with some the wing dissection steps such as removal of cuticle and might benefit from the protocol mentioned here. To conclude, we have provided a detailed description of the process of wing dissections in a butterfly species which we believe will be helpful for newcomers in the field to adapt to their own species.

6. Reagents Setup

Preparation of 10 PBS Buﬀer × 1. In a 1 L beaker, add 700 mL MilliQ water and reagents mentioned in Table1: 2. Transfer the content to a 1 L measuring cylinder. Raise the volume to one liter using MilliQ water. 3. Mix the solution and transfer the content to a 1 L glass bottle.

4. Autoclave the solution at 121 ◦C for 20 min and store the content at room temperature. Table 1. Reagents for 10 PBS (Phosphate Buffer Saline) buffer preparation. × Reagents Weight/Volume NaCl 81.8 g KH2PO4 5.28 g K2HPO4 10.68 g Note: To prepare 1 PBS, add 10 mL of 10 PBS buffer and 90 mL of MilliQ water. × ×

Author Contributions: T.D.B. and A.M. wrote the manuscript; T.D.B. designed and performed the experiment and developed the associated video article. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded National Research Foundation, Singapore grant R-154-000-B57-281. Acknowledgments: We thank all Monteiro Lab members for their constant support and the comments of three anonymous reviewers that helped in improving the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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