The Making of Transgenic Drosophila Guttifera

Article The Making of Transgenic Drosophila guttifera

Mujeeb Shittu 1, Tessa Steenwinkel 1, Shigeyuki Koshikawa 2,3,* and Thomas Werner 1,* 1 Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA; [email protected] (M.S.); [email protected] (T.S.) 2 Faculty of Environmental Earth Science, Hokkaido University, N10W5, Kita-ku, Sapporo, Hokkaido 060-0810, Japan 3 Graduate School of Environmental Science, Hokkaido University, N10W5, Kita-ku, Sapporo, Hokkaido 060-0810, Japan * Correspondence: [email protected] (S.K.); [email protected] (T.W.)

Received: 7 April 2020; Accepted: 26 April 2020; Published: 27 April 2020

Abstract: The complex color patterns on the wings and body of Drosophila guttifera (D. guttifera) are emerging as model systems for studying evolutionary and developmental processes. Studies regarding these processes depend on overexpression and downregulation of developmental genes, which ultimately rely upon an effective transgenic system. Methods describing transgenesis in Drosophila melanogaster (D. melanogaster) have been reported in several studies, but they cannot be applied to D. guttifera due to the low egg production rate and the delicacy of the eggs. In this protocol, we describe extensively a comprehensive method used for generating transgenic D. guttifera. Using the protocol described here, we are able to establish transgenic lines, identifiable by the expression of enhanced green fluorescent protein (EGFP) in the eye disks of D. guttifera larvae. The entire procedure, from injection to screening for transgenic larvae, can be completed in approximately 30 days and should be relatively easy to adapt to other non-model Drosophila species, for which no white-eyed mutants exist.

Keywords: microinjection; Drosophila guttifera; transgenesis; piggyBac

1. Introduction Our ability to genetically manipulate any organism of choice has become a powerful tool for studying gene function. The introduction of a foreign DNA into the genome of an organism to produce germline transformations is known as transgenesis. Until recently, genetic manipulation in Drosophila has been largely achieved by transposase-mediated transgenesis [1,2]. In this technique, transposases catalyze the integration of a transposon into the genome of an organism in an unpredictable manner [3]. However, in recent years, significant improvements in fly genetic techniques have been reported, particularly the integration of transgenes into the genome in a site-specific manner through the use of different integrases and recombinases [4–7]. These techniques have been used to manipulate a variety of species [7–9], but arguably, most of these studies have been conducted in D. melanogaster. Many non-model Drosophila species offer a variety of life history and morphological traits that are absent in D. melanogaster [10]. Thus, our transgenic protocol will enable researchers to study new phenomena beyond the commonly used model organism. Drosophila guttifera is a rare mycophagous species in the quinaria species group, native to the Midwest and Southeast of the USA [10]. D. guttifera has recently become a useful model for pigmentation studies [11]. Most studies carried out on this species are focused on unraveling the functional mechanism for the formation of polka-dotted patterns on the wings of this species. For example, Werner et al. (2010) reported that the wing spots are induced by the Wingless morphogen, and the unique polka-dotted expression pattern of wingless is due to the evolution in cis-regulatory elements

Methods Protoc. 2020, 3, 31; doi:10.3390/mps3020031 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, 31 2 of 14 of the wingless gene [12–14]. Furthermore, Fukutomi et al. (2017) conducted a study measuring the timing of melanin deposition in wings, using a transgenic D. guttifera line [15]. Ongoing and future studies in the field of color pattern development depend on the overexpression and downregulation of genes, which ultimately rely on an effective and efficient transgenesis system. Previously, a review highlighting strategies for making transgenic D. melanogaster was published [16]. However, this protocol cannot be directly applied to D. guttifera transgenesis for the following reasons: (1) The egg production rate of D. guttifera is one to two orders of magnitude lower than that of D. melanogaster, and thus, a large fly population is needed; (2) D. guttifera flies die quickly from the ethanol fumes produced by the yeast required for egg-laying, requiring proper ventilation during the egg collection process; (3) D. guttifera females stick their eggs into the substrate, making them virtually impossible to collect from an agar surface; (4) D. guttifera’s eggs are covered by a thin proteinaceous chorion, which causes rapid embryonic desiccation in this species; (5) there is no white-eyed mutant available for D. guttifera, which is why larvae have to be screened for fluorescent markers; and (6) besides the piggyBac transposon, most other tested transposons do not result in transgenic D. guttifera [12,14,15,17]. For these reasons, critical steps must be deployed to successfully transform D. guttifera. Here, we provide a detailed approach for creating transgenic D. guttifera. This protocol is accompanied by a video (Video S1) and troubleshooting steps (Table1), which makes it easy for researchers in the field to follow. To the best of our knowledge, this protocol is the first to describe how to make transgenic flies in a non-model Drosophila species. It is our expectation that this protocol can be adopted by researchers who might be interested in experiments involving transgenesis in other non-model insect species.

Table 1. Troubleshooting guide (summarizes a variety of problems that we have encountered in the past. The table explains the reasons underlying the problems and oﬀers appropriate actions to work around each problem).

Problem Reason Solution Make sure you use standard needles. Strictly Few larvae hatch. Bad injection needles. follow the instructions under Section 3.6. Add injection oil within 3–5 s after eggs appear Over-desiccation of eggs during lining up. completely dry under the microscope. Use highly pure DNA (no mini-preps). Toxic DNA. Additional isopropanol precipitation after the first extraction of DNA is necessary. The heat shock temperature for D. guttifera transformation is 40 ◦C; however, if this Too-high heat shock temperature. temperature is causing strong reduction in the survival rate, reduce the temperature by 1–2 degrees but never go below 37 ◦C. If the Halocarbon oil mixture is suffocating Use Halocarbon oil 27 only or GEM extra virgin and killing the eggs. This does not normally olive oil. We are using extra virgin olive oil happen, but we have seen this problem in our lab with high success. in our lab. Having only a few injection-surviving Ensure a high injection survival rate. At least Few surviving adults. larvae causes bacterial growth and/or mold 10–20 larvae can keep the food mold-free. formation in the food. Desiccation of pupae due to dry chamber Always keep the chamber and the cotton plugs and/or dry cotton plugs. Always clean the DNA by isopropanol No transgenic larvae. Poor DNA quality. precipitation or prepare new DNA samples. Always ensure the heat shock temperature is not Insufficient heat shock temperature. below 37 ◦C. Check the temperature of your water bath with a calibrated thermometer. Methods Protoc. 2020, 3, 31 3 of 14

2. Experimental Design

2.1. Required Materials and Equipment

2.1.1. Materials Plexiglas egg-laying cage measuring 300 mm 200 mm 200 mm • × × Lamp • Instant yeast (Instaferm) • Sponges • Medium-sized Petri dishes (100 mm 15 mm) • × 1-L plastic beakers for wet chambers • 2-L plastic jar • 5-L beaker • Egg collection filter with Corning Netwell insert (fine-filter, New York, NY, USA) • Egg collection filter with Corning Netwell insert (coarse-filter, New York, NY, USA) • Squeeze bottle • Micro cover glass (18 mm 18 mm) • × Microslide (25 mm 75 mm, 1 mm thick) • × Small brush (to move egg masses) • Very fine brush (to line up individual eggs) • Halocarbon oil 700 (Sigma-Aldrich, cat. no. H8898, Albany, NY, USA) • Halocarbon oil 27 (Sigma-Aldrich, cat. no. H8773, Albany, NY, USA) • 10-mL syringe • 50-mL Falcon tube • FHC capillary tube (Borosil 1.0 mm 0.75 mm ID/Fiber with Omega dot fiber) • × Nitrogen gas • CO gas • 2 Fly food vials • Cornmeal-sucrose-yeast medium (fly food) [10] • Aluminum foil • Two pairs of forceps • Spatula • Cotton plugs • Moist chamber • Anti-fungus paper (see Section 5.1) • 2.1.2. Equipment Flaming/Brown micropipette puller Model P-97 (Sutter Instruments, Novato, CS, USA) • Microinjector (Narishige IM 300, Amityville, NY, USA) • Needle-holder (Narishige, Amityville, NY, USA) • Micromanipulator (Narishige, Amityville, NY, USA) • Inverted microscope (Olympus CKX31, Center Valley, PA, USA) • Dissecting microscope • Water bath (Thermo Scientific, HAAK S3, Waltham, MA, USA) • Mercury burner (Olympus U-RFL-T, Center Valley, PA, USA) • Imaging microscope (Olympus SZX16, Center Valley, PA, USA) with fluorescence filters for GFP • and DsRed Methods Protoc. 2020, 3, x FOR PEER REVIEW 4 of 13

An egg-laying cage can be in operation for up to 10 weeks before the flies must be transferred to a new cage. If fungus grows (pay attention to places where the food comes in contact with the wall), change the cage earlier. Perform the following steps to move the flies to a new cage: Methods Protoc. 2020, 3, 31 4 of 14 (1) Take out all cornmeal-sucrose-yeast medium plates, close them, and continue working at a fly bench with CO2 gas. 3.(2) Methods Cover an area with unfolded paper towels to your right and put a 5-L beaker on it. (3) Have a new cage with a fresh entrance ring plus nylon ready. 3.1.(4) The Put Egg-Laying the populated Cage cage to the left of the new cage (not on the towels), the entrance facing Theupwards egg-laying to prevent cage is CO made2 from of Plexiglasleaking out that of the allows cage good through aeration the back through wall. the nylon entrance at the(5) front Open and the plasticCO2 gas, mesh lead at it the through back of the the nylon cage. to Aeration anesthetize is necessary the flies, to and prevent then the close accumulation the CO2 gas supply. of toxic ethanol fumes produced by the yeast. The cage measures 300 mm 200 mm 200 mm and (6) Remove the ring with the nylon of the old cage and pour the flies into the× 5-L beaker.× holds approximately 10,000 ﬂies from 20 bottles. Before a clean cage can be populated, six small (35 (7) Flies that fall on the towels can now be thrown into the beaker as well. mm 10 mm) plastic Falcon Petri dish bottom halves are taped upside-down (using double-sided (8)× Gas the old cage again to blow loose the remaining living flies and shake the cage over the beaker. tape) onto the bottom of the cage. They will serve as “tables” for the bigger cornmeal-sucrose-yeast (9) Pour the flies from the beaker into the new cage. medium(10) Close plates the andfreshly must filled be cage, spaced bring out it evenly. to its desi Medium-sizedgned place in the empty lab and dishes supply can the be usedflies with to see food. if the spacing(11) Clean is good. the Withoutold cage these and “tables”,the nylon the with cornmeal-sucrose-yeast tap water. Do not use medium soap or plates bleach. would Rinse crush with lots of ﬂiesdistilled on the ground water. with every food exchange (Figure1A,B).

FigureFigure 1. 1.The The egg-laying egg-laying cage cage measuring measuring 300300 mmmm × 200 mm mm × 200200 mm. mm. (A (A) An) An egg-laying egg-laying cage cage holding holding × × ~10,000~10,000 ﬂies, flies, which which contains contains six six plates plates ofof cornmeal-sucrose-yeastcornmeal-sucrose-yeast medium. medium. (B ()B An) An empty empty egg-laying egg-laying cagecage showing showing the the six six small small (35 (35 mm mm x 10 mm) plastic plastic Petri Petri dish dish bottom bottom halves halves taped taped upside-down upside-down onto onto × thethe bottom bottom of of the the cage cage using using double-sided double-sided tape.tape.

3.2.Feed Egg Collection the flies inand the Preparation cage every for Microinjections 2–3 days with six cornmeal-sucrose-yeast medium plates at a time, which are medium-sized (100 mm 15 mm) Petri dish bottom halves, filled to the top with D. guttifera females lay comparably ×few eggs within a given period. For this reason, the cage cornmeal-sucrose-yeastsetup described above medium.is used to Rightobtain before at least feeding 200–300 the freshly flies, wipefertilized off any eggs excess per hour water from from the the platespopulation with a paperof ~10,000 towel mixed-sex and sprinkle flies. with Keep some the cage dry baker’son the yeast.lab bench When at replacingroom temperature old food and plates withnormal new plates,lab humidity follow with the threea 40-W rules lamp below placed to ~30 minimize cm above the it, killing which of is flies: timed to turn on at 6 a.m. and off at 6 p.m. In the cage, females need to be reared on fresh food with dry yeast at all times in (1) Knock the plate gently against the round Petri dish “table” to remove any flies before taking out a order for their ovaries to develop and to achieve a continuous egg production. The age of the females varies,plate. as Usefresh the flies thumb are provided and middle from fingerthe bottle to hold cultures the dish,every whileweek. holdingIf the food the is index in poor finger condition, in place femalesto prevent will retain the food their fromfertilized falling eggs out. inside of their bodies and lay them when the eggs are too old for microinjections. Supply the cage with fresh cornmeal-sucrose-yeast medium 3–4 times a week and (a) If the gap between the food and the Petri dish is large due to excessive drying of the especially one day before injections are carried out. food, then bump (knock the flies) towards the large gap. The food will not move much Eggs that are laid in the morning are often already cellularized and tend to have a thicker chorion, whichdownwards, is disadvantageous and no fly for will injections. be trapped. The egg quality increases later during the day. The best(b) time toIf start the gapthe egg-laying is narrow, is bump 11 a.m. towards Harvest the the side eggs where in 1-hour the foodcycles, still starting sticks at to noon. the plastic.It is This prevents most of the flies in the gap from being squeezed to death. (2) One out, one in! Always take only one plate out at a time and replace immediately with a fresh

plate. If there are ﬂies on the food “table” in the cage, they can be gently pushed away with the front edge of the fresh food plate when putting it down. Then repeat this step with the remaining plates. (3) Freeze and discard the used food as the ﬂies developing from it will be of poor quality and a source of fungus infestations. Methods Protoc. 2020, 3, 31 5 of 14

An egg-laying cage can be in operation for up to 10 weeks before the ﬂies must be transferred to a new cage. If fungus grows (pay attention to places where the food comes in contact with the wall), change the cage earlier. Perform the following steps to move the ﬂies to a new cage:

(1) Take out all cornmeal-sucrose-yeast medium plates, close them, and continue working at a fly bench with CO2 gas. (2) Cover an area with unfolded paper towels to your right and put a 5-L beaker on it. (3) Have a new cage with a fresh entrance ring plus nylon ready. (4) Put the populated cage to the left of the new cage (not on the towels), the entrance facing upwards to prevent CO2 from leaking out of the cage through the back wall. (5) Open the CO2 gas, lead it through the nylon to anesthetize the flies, and then close the CO2 gas supply. (6) Remove the ring with the nylon of the old cage and pour the flies into the 5-L beaker. (7) Flies that fall on the towels can now be thrown into the beaker as well. (8) Gas the old cage again to blow loose the remaining living flies and shake the cage over the beaker. (9) Pour the flies from the beaker into the new cage. (10) Close the freshly filled cage, bring it to its designed place in the lab and supply the flies with food. (11) Clean the old cage and the nylon with tap water. Do not use soap or bleach. Rinse with distilled water.

3.2. Egg Collection and Preparation for Microinjections D. guttifera females lay comparably few eggs within a given period. For this reason, the cage setup described above is used to obtain at least 200–300 freshly fertilized eggs per hour from the population of ~10,000 mixed-sex flies. Keep the cage on the lab bench at room temperature and normal lab humidity with a 40-W lamp placed ~30 cm above it, which is timed to turn on at 6 a.m. and off at 6 p.m. In the cage, females need to be reared on fresh food with dry yeast at all times in order for their ovaries to develop and to achieve a continuous egg production. The age of the females varies, as fresh flies are provided from the bottle cultures every week. If the food is in poor condition, females will retain their fertilized eggs inside of their bodies and lay them when the eggs are too old for microinjections. Supply the cage with fresh cornmeal-sucrose-yeast medium 3–4 times a week and especially one day before injections are carried out. Eggs that are laid in the morning are often already cellularized and tend to have a thicker chorion, whichMethods isProtoc. disadvantageous 2020, 3, x FOR PEER for injections.REVIEW The egg quality increases later during the day. The best5 time of 13 to start the egg-laying is 11 a.m. Harvest the eggs in 1-h cycles, starting at noon. It is advisable that one personadvisable lines that up one two person slides (~200lines up eggs) two during slides a(~ 1-h200 cycle,eggs) whileduring another a 1-hour person cycle, injects while them.anothe Allr person wash stepsinjects of them. the eggs All arewash done steps with of the distilled eggs water.are done with distilled water.

CRITICAL STEPS: STEPS: NeverNever wash wash DD.. guttifera guttifera eggseggs with with ethanol ethanol,, because because it seems it seems to harden to harden the chorionthe chorion and and prevents prevents the the injection injection needles needles from from penetrating penetrating it, it, and and never never leave leave the cage cage without food. Make a starter yeast paste from instant baker’s yeast grains one day before using it, and on the Make a starter yeast paste from instant baker’s yeast grains one day before using it, and on the morning of injection, mix it again with additional dry yeast and water. Keep the yeast paste at room morning of injection, mix it again with additional dry yeast and water. Keep the yeast paste at room temperature during the injections; it should smell a bit like fermented fruit. At 11 a.m., replace all temperature during the injections; it should smell a bit like fermented fruit. At 11 a.m., replace all cornmeal-sucrose-yeast medium plates in the cage with four moist sponges, which are to be placed cornmeal-sucrose-yeast medium plates in the cage with four moist sponges, which are to be placed into medium-sized (100 mm × 15 mm) Petri dish bottom halves and covered with a thin layer of fresh, into medium-sized (100 mm 15 mm) Petri dish bottom halves and covered with a thin layer of fresh, active yeast paste (Figure 2).× A second set of four sponges will be needed for the next round. active yeast paste (Figure2). A second set of four sponges will be needed for the next round. The best way to prepare the sponges is to first make them too wet and then to carefully squeeze off the excess water with two thumbs pressing and moving from the top to the bottom throughout the surface of the sponges, until no drops are running out easily. The sponges should be dry enough that no water runs out when flies are bumped off from them during removal from the cage but wet enough to supply the flies with enough liquid. Sponges that are too dry seem to result in thicker coronas of the eggs (the thickening near the entry site of the injection needle), while sponges that are too wet cause the accumulation of liquid in the cage. The yeast paste is then supplied evenly over the sponge surface. Cover the sponges with lids before placing them into the cage to prevent stray flies from laying eggs on them. Allow the females to lay eggs on the 4 sponges for one hour. The flies prefer to sit on the sponge part that is closest to the cage wall. Sponges that are placed too far away from the wall are often ignored by the flies.

Figure 2. The yeast paste is evenly applied with a flat spatula over the sponge surface.

To collect the eggs, remove the sponges one by one from the cage and immediately replace them with sponges covered with fresh yeast paste. Gently squeeze out the eggs-containing sponges one by one in a 2-L jar filled with 1 L of distilled water (repeatedly bend the sponges from the edge outward to avoid crushing the eggs, and the eggs will drop out of the pores), then allow the eggs to sink to the bottom of the 2-L jar for 2 min. While waiting, wash the sponges and Petri dishes with distilled water. The sponges should be squeezed hard while washing to kill any possible remaining eggs. After the four used sponges are washed, put fresh yeast paste on them and store them in a drawer (away from contaminating flies) until they are used again. At the end of the injection day, wash the sponges thoroughly and place them on a few paper towels on the bench for drying. Assemble—from top to bottom—a funnel, a coarse-filter basket, and a fine-filter basket (Figure 3A). Next, pour the upper half of the egg collection water into the sink (the eggs are at the bottom of the jar). Then swirl and pour the remaining water with the eggs into the funnel/filter system. The coarse-filter traps sponge fibers and flies, while the fine filter collects the eggs

Methods Protoc. 2020, 3, x FOR PEER REVIEW 5 of 13 advisable that one person lines up two slides (~200 eggs) during a 1-hour cycle, while another person injects them. All wash steps of the eggs are done with distilled water. CRITICAL STEPS: Never wash D. guttifera eggs with ethanol, because it seems to harden the chorion and prevents the injection needles from penetrating it, and never leave the cage without food. Make a starter yeast paste from instant baker’s yeast grains one day before using it, and on the morning of injection, mix it again with additional dry yeast and water. Keep the yeast paste at room temperature during the injections; it should smell a bit like fermented fruit. At 11 a.m., replace all cornmeal-sucrose-yeast medium plates in the cage with four moist sponges, which are to be placed into medium-sized (100 mm × 15 mm) Petri dish bottom halves and covered with a thin layer of fresh, active yeast paste (Figure 2). A second set of four sponges will be needed for the next round. The best way to prepare the sponges is to first make them too wet and then to carefully squeeze off the excess water with two thumbs pressing and moving from the top to the bottom throughout the surface of the sponges, until no drops are running out easily. The sponges should be dry enough that no water runs out when flies are bumped off from them during removal from the cage but wet enough to supply the flies with enough liquid. Sponges that are too dry seem to result in thicker coronas of the eggs (the thickening near the entry site of the injection needle), while sponges that are too wet cause the accumulation of liquid in the cage. The yeast paste is then supplied evenly over the sponge surface. Cover the sponges with lids before placing them into the cage to prevent stray flies from laying eggs on them. Allow the females to lay eggs on the 4 sponges for one hour. The flies prefer to sit on the sponge partMethods that Protoc. is closest2020, 3, 31to the cage wall. Sponges that are placed too far away from the wall are often6 of 14 ignored by the flies.

FigureFigure 2. The yeast paste is evenlyevenly appliedapplied withwith aa ﬂatflat spatulaspatula overover thethe spongesponge surface.surface.

ToThe collect best way the eggs, to prepare remove the the sponges sponges is toone first by makeone from them the too cage wet and and immediately then to carefully replace squeeze them withoff the sponges excess watercovered with with two fresh thumbs yeast pressing paste. Gent andly moving squeeze from out thethe topeggs-containing to the bottom sponges throughout one theby onesurface in a of2-L the jar sponges, filled with until 1 L no of dropsdistilled are water running (repeatedly out easily. bend The the sponges sponges should from be the dry edge enough outward that tono avoid water crushing runs out the when eggs, flies and are the bumped eggs will off from drop them out of during the pores), removal then from allow the the cage eggs but to wet sink enough to the bottomto supply of thethe flies2-L jar with for enough 2 min. While liquid. waiting, Sponges wash that arethe toosponges dry seem and Petri to result dishes in thicker with distilled coronas water. of the Theeggs sponges (the thickening should be near squeezed the entry hard site while of the wash injectioning to needle), kill any while possible sponges remaining that are eggs. too wetAfter cause the fourthe accumulation used sponges of are liquid washed, in the put cage. fresh The yeast yeast paste paste on is them then suppliedand store evenly them in over a drawer the sponge (away surface. from contaminatingCover the sponges flies) with until lids they before are placingused again. them At into the the end cage of tothe prevent injection stray day, flies wash from the laying sponges eggs thoroughlyon them. and place them on a few paper towels on the bench for drying. Assemble—fromAllow the females top to layto eggsbottom—a on the 4funnel, sponges a for coar onese-filter hour. Thebasket, flies preferand a to fine-filter sit on the spongebasket (Figurepart that 3A). is closestNext, pour to the the cage upper wall. half Sponges of the egg that collection are placed water too farinto away the sink from (the the eggs wall are are at often the bottomignored of by the the jar). flies. Then swirl and pour the remaining water with the eggs into the funnel/filter system.To collectThe coarse-filter the eggs, remove traps thespon spongesge fibers one byand one flies, from while the cage the andfine immediately filter collects replace the themeggs with sponges covered with fresh yeast paste. Gently squeeze out the eggs-containing sponges one by one in a 2-L jar filled with 1 L of distilled water (repeatedly bend the sponges from the edge outward to avoid crushing the eggs, and the eggs will drop out of the pores), then allow the eggs to sink to the bottom of the 2-L jar for 2 min. While waiting, wash the sponges and Petri dishes with distilled water. The sponges should be squeezed hard while washing to kill any possible remaining eggs. After the four used sponges are washed, put fresh yeast paste on them and store them in a drawer (away from contaminating flies) until they are used again. At the end of the injection day, wash the sponges thoroughly and place them on a few paper towels on the bench for drying. Assemble—from top to bottom—a funnel, a coarse-filter basket, and a fine-filter basket (Figure3A). Next, pour the upper half of the egg collection water into the sink (the eggs are at the bottom of the jar). Then swirl and pour the remaining water with the eggs into the funnel/filter system. The coarse-filter traps sponge fibers and flies, while the fine filter collects the eggs (Figure3B). Rinse the 2-L jar once with 100–200 mL of distilled water, then pour it through the filter system to collect the remaining eggs. With a squeeze bottle of distilled water, wash the eggs off the upper filter and allow them to slip through the coarse mesh into the fine filter below. After obtaining a sufficient number of eggs in the lower filter, remove the baskets from the funnel and wash the eggs thoroughly with distilled water from a squeeze bottle. Then, put the basket with the washed eggs into a bottom half of a small Petri dish, which is halfway filled with distilled water. Line up the eggs under a dissection scope. Methods Protoc. 2020, 3, x FOR PEER REVIEW 6 of 13

(Figure 3B). Rinse the 2-L jar once with 100–200 mL of distilled water, then pour it through the filter system to collect the remaining eggs. With a squeeze bottle of distilled water, wash the eggs off the upper filter and allow them to slip through the coarse mesh into the fine filter below. After obtaining a sufficient number of eggs in the lower filter, remove the baskets from the funnel and wash the eggs thoroughly with distilled water from a squeeze bottle. Then, put the basket with the washed eggs Methodsinto a Protoc. bottom2020 half, 3, 31 of a small Petri dish, which is halfway filled with distilled water. Line up the eggs7 of 14 under a dissection scope.

FigureFigure 3. 3.Egg Egg collection collection with with aa funnelfunnel andand two filters.filters. ( (AA) )The The funnel funnel is is placed placed on on top top of ofthe the coarse coarse filter,filter, and and the the fine fine filter filter is is held held underneath underneath toto collectcollect the eggs. ( (BB) )The The coarse coarse filter filter (left) (left) mainly mainly traps traps thethe flies flies and and sponge sponge fibers, fibers, while while the the fine fine filterfilter (right)(right) retains the the eggs. eggs.

3.3.3.3. Lining Lining up up the the Eggs Eggs for for Microinjections Microinjections PlacePlace a a micro micro cover cover glassglass (18 (18 mm mm × 1818 mm) mm) onto onto a microslide a microslide (25 mm (25 × mm 75 mm,75 1 mm,mm thick) 1 mm with thick) × × witha small a small droplet droplet of water of water so that so it that sticks. itsticks. Dry off Dry the edges off the with edges a piece with of a tissue. piece ofThe tissue. washed The eggs washed are eggsnow are transferred now transferred from the from basket the onto basket the ontocover the glass cover as a glassclump, as using a clump, a small using brush. a small It is brush.essential It is essentialthat thethat eggs the are eggskept wet are at kept all times. wet at During all times. lining During up, always lining pick up, only always the white pick and only structureless- the white and structureless-appearingappearing eggs. It is advisable eggs. It to is put advisable a clump to of put more a clump than 100 of moreeggs into than the 100 upper eggs middle into the of upperthe middlecover ofglass. the coverThen, use glass. a very Then, fine use brown-haired a very fine brown-hairedbrush, make it brush,wet, briefly make dry it wet, it on briefly a tissue dry paper, it on a pull the first egg into position, dry the brush, pull the next egg to extend the line, and so on. tissue paper, pull the first egg into position, dry the brush, pull the next egg to extend the line, and Align the first 10 eggs with a relatively dry (very fine) brush, or otherwise, the surface tension so on. will make it impossible to position them correctly. Then, gently move the clump of ~100 eggs down Align the first 10 eggs with a relatively dry (very fine) brush, or otherwise, the surface tension will while extending the line of selected eggs downwards, otherwise you lose sight of your egg clump, make it impossible to position them correctly. Then, gently move the clump of ~100 eggs down while and the eggs may dry out and die by accident. In the end, the row consists of about 100 eggs, which extendingshall have the their line posterior of selected end eggs towards downwards, the right otherwiseedge of the you cover lose glass. sight The of yourrow of egg eggs clump, should and be the eggspositioned may dry about out and 5 mm die away by accident. from that In theedge. end, Keep the the row eggs consists moist of at about all times 100 eggs,by supplying which shall water have theirwith posterior the brush end to towardsthe clump the of right unaligned edge ofeggs, the as cover well glass. as the The top, row middle, of eggs and should end of bethe positioned row of aboutaligned 5 mm eggs. away from that edge. Keep the eggs moist at all times by supplying water with the brush to the clumpWhen ofthe unaligned row is completed, eggs, as well remove as the all top, unused middle, eggs and and end let of the the row row dry, of aligned while eggs.carefully monitoringWhen the the row eggs is completed, under the removemicroscope. all unused The ro eggsw can and now let the be rowrearranged dry, while slightly carefully U-shaped monitoring to themake eggs them under fit thebetter microscope. to the cornmeal-sucrose-yeast The row can now bemedium rearranged surface slightly later. When U-shaped the egg to line make appears them fit bettercompletely to the cornmeal-sucrose-yeastdry, wait about 3–5 more medium seconds surface(depending later. on When the humidity the egg lineof the appears room) and completely then quickly cover them with the Halocarbon oil mixture (7 parts of type 700 + 1 part of type 27), supplied dry,Methods wait Protoc. about 2020 3–5, 3, xmore FOR PEER seconds REVIEW (depending on the humidity of the room) and then quickly cover5 of 13 themby witha syringe the Halocarbon(Figure 4). The oil mixture Halocarbon (7 parts oil ofstops type further 700 + 1desiccation part of type but 27), allows supplied breathing. by a syringe The (Figureadvisablehalocarbon4). Thethat oil Halocarbonone mix person must belines oil prepared stops up two further at slides least desiccation a(~ day200 beforeeggs but) duringyou allows can a use breathing.1-hour it, so cycle, plan The ahead.while halocarbon another oil person mix mustinjects be CRITICALthem. prepared All wash atSTEPS: least steps aIf day theof the beforeeggs eggs become you are can done too use desiccated with it, so distilled plan (oil ahead. added water. too late), they die and become deformed when the needle pokes against them. If, however, the oil is put on too early, the eggs CRITICAL STEPS: Never wash D. guttifera eggs with ethanol, because it seems to harden the CRITICALwill not stick STEPS: to the Ifglass the and eggs move become away too when desiccated the injection (oil added needle too is poked late), theyagainst die them. and becomeOnly chorion and prevents the injection needles from penetrating it, and never leave the cage deformeda few seconds when lay the between needle pokesboth extremes. against them. Do not If, try however, to inject the a slide oil is with put onswimming too early, or the dead eggs without food. willeggs. not Instead, stick to save the glassyour andneedles move for away a better when slide. the injection needle is poked against them. Only Makea few secondsa starter layyeast between paste from both instant extremes. baker’s Do notyeast try grains to inject one a day slide before with swimmingusing it, and or on dead the morning eggs. of Instead, injection save, mix your it ag needlesain with for additional a better slide. dry yeast and water. Keep the yeast paste at room temperatureCRITICAL during STEPS: the injectionsNote the; following it should importantsmell a bit timelines:like fermented egg-laying fruit. At in the11 a.m. cage, =replace1 h, egg all cornmealcollection-sucrose and-yeast lining medium up = 20 plates min, injectingin the cage one with slide four containing moist sponges, about 100which eggs are= 15to be min. placed It is into mediumimportant-sized to ensure (100 mm that × the15 mm) eggs Petri are in dish the bottom syncytial halves blastoderm and covered stage with during a thin injection, layer of which fresh, activelasts yeast for paste about (Figure 2 h or less2). A at second room temperature.set of four sponges will be needed for the next round. The best way to prepare the sponges is to first make them too wet and then to carefully squeeze off the excess water with two thumbs pressing and moving from the top to the bottom throughout the surface of the sponges, until no drops are running out easily. The sponges should be dry enough that no water runs out when flies are bumped off from them during removal from the cage but wet enough to supply the flies with enough liquid. Sponges that are too dry seem to result in thicker coronas of the eggs (the thickening near the entry site of the injection needle), while sponges that are too wet cause the accumulation of liquid in the cage. The yeast paste is then supplied evenly over the sponge surface. Cover the sponges with lids before placing them into the cage to prevent stray flies from laying eggs on them. Allow the females to lay eggs on the 4 sponges for one hour. The flies prefer to sit on the sponge part that is closest to the cage wall. Sponges that are placed too far away from the wall are often ignored by the flies.

Figure 2. The yeast paste is evenly applied with a flat spatula over the sponge surface.

Methods Protoc. 2020, 3, 31 8 of 14 Methods Protoc. 2020, 3, x FOR PEER REVIEW 7 of 13

FigureFigure 4. 4.(A ()A Adding) Adding the the halocarbon halocarbon oiloil mixturemixture to a line of of eggs. eggs. (B (B) )The The anterior anterior ends ends of ofthe the eggs eggs containcontain the the ﬁlaments filaments (left), (left), while while the the posterior posterior endsends point to the right. right.

3.4.Preparation CRITICAL of TransgeneSTEPS: Note DNA the following important timelines: egg-laying in the cage = 1 h, egg collection and lining up = 20 min, injecting one slide containing about 100 eggs = 15 min. It is (1) Follow the manufacturer’s instructions on the isolation of the plasmid DNA containing the important to ensure that the eggs are in the syncytial blastoderm stage during injection, which transgene of interest from an overnight Escherichia coli culture. In this described experiment, lasts for about 2 h or less at room temperature. we use the Invitrogen HiPure Plasmid Midi-Prep Kit by Thermo Fisher Scientific (Waltham, 3.4.MA, Preparation USA). of Transgene DNA (2) Add 3.5 mL of isopropanol to the eluted DNA in a Corex glass vial. (1) Follow the manufacturer’s instructions on the isolation of the plasmid DNA containing the (3) Close the Corex vial with Parafilm, invert 10 times, take away the Parafilm, and wipe off any transgene of interest from an overnight Escherichia coli culture. In this described experiment, we dropsuse the with Invitrogen Kimwipes. HiPure Plasmid Midi-Prep Kit by Thermo Fisher Scientific (Waltham, (4) CentrifugeMA, USA). at 11,000 rpm for 30 min at 4 ◦C. (5)(2) Mark Addthe 3.5 areamL of of isopropanol the DNA pellet to the with eluted a marker, DNA in decant, a Corex and glass invert vial. the vial on a paper towel. (6)(3) Add Close 350 theµL Corex of sterile vial MQwith water Parafilm, and invert dissolve 10 thetimes, DNA take pellet away by the vortexing. Parafilm, and wipe off any (7) Collectdrops thewith plasmid Kimwipes. DNA in a 1.5-mL Eppendorf tube. (8)(4) Add Centrifuge 35 µL of at 3 11,000 M of sodiumrpm for acetate30 min at (pH 4 °C. 5.5). (5) Mark the area of the DNA pellet with a marker, decant, and invert the vial on a paper towel. (9) Add 875 µL of 200-proof ethanol and invert 10 times. (6) Add 350 µL of sterile MQ water and dissolve the DNA pellet by vortexing. (10) Spin the DNA at 14,000 rpm for 20 min at 4 C on a bench-top centrifuge. (7) Collect the plasmid DNA in a 1.5-mL Eppendorf◦ tube. (11)(8) Remove Add 35 supernatantµL of 3 M of andsodium wash acetate with (pH 300 µ5.5).L of 70% ethanol by inverting the tube 10 times. (12)(9) Centrifuge Add 875 µL again of 200-proof at 14,000 ethanol rpm for and 10 mininvert at 10 room times. temperature. (13)(10)Remove Spin the supernatant DNA at 14,000 and rpm centrifuge for 20 min for at 1 min.4 °C on a bench-top centrifuge. (14)(11)Remove Remove all supernatant ethanol and and dry wash the pelletwith 300 for µL 5 min.of 70% ethanol by inverting the tube 10 times. (15)(12)Dissolve Centrifuge the DNAagain at in 14,000 50–100 rpmµL offor elution 10 min buat ffroomer, measure temperature. the DNA concentration, and add more (13)elution Remove bu supernatantffer if necessary, and centrifuge to a finalconcentration for 1 min. of 1 µg/µL. (14) Remove all ethanol and dry the pellet for 5 min. 3.5.(15) Preparation Dissolve ofthe the DNA Injection in 50–100 Cocktail µL of elution buffer, measure the DNA concentration, and add more elution buffer if necessary, to a final concentration of 1 µg/µL. (1) On ice, add 20 µL of transgene-containing piggyBac construct (1 µg/µL) to a 1.5-mL test tube, 5 µL 3.5.of Preparation the piggyBac of thehelper Injection plasmid Cocktail (phspBac ([1 µg/µL)), and 15 µL of sterile distilled water. Note: We do not add food color, because it is not necessary for the visualization of the injected material. (1) On ice, add 20 µL of transgene-containing piggyBac construct (1 µg/µL) to a 1.5-mL test tube, 5 µL (2) Centrifuge the DNA mixture for 20 min at maximum speed, and transfer the upper 39 µL of it of the piggyBac helper plasmid (phspBac ([1 µg/µL)), and 15 µL of sterile distilled water. Note: We to a fresh tube (do not touch the bottom of the tube with the pipette tip to prevent transferring do not add food color, because it is not necessary for the visualization of the injected material. (2) undissolved Centrifuge the material DNA thatmixture would for 20 clog min the at needle). maximum Spin speed, the DNAand transfer again forthe 20upper min 39 and µL transfer of it µ 38to La fresh of the tube injection (do not mix touch into the another bottom fresh of the tube. tube This with DNA the pipette is now tip clear to prevent of debris transferring and can be usedundissolved to load the material needles. that The wouldpiggyBac clog systemthe needle). pBac Spin (backbone) the DNA/phspBac again for (piggyBac 20 min andhelper) transfer results in38 1 transformationµL of the injection event mix every into another 50 D. guttifera fresh tueggsbe. This with DNA an emptyis now vectorclear of and debris 1 every and can 500 be eggs withused an to 8-kb load insert the needles. [18]. The piggyBac system pBac (backbone)/phspBac (piggyBac helper) results in 1 transformation event every 50 D. guttifera eggs with an empty vector and 1 every 500 eggs with an 8-kb insert [18].

Methods Protoc. 2020, 3, x FOR PEER REVIEW 8 of 13

3.6. Needle Preparation and Microinjections Use a Flaming/Brown micropipette puller Model P-97 and FHC capillary tubing (Borosil 1.0 × 0.75 mm ID/Fiber with Omega dot fiber) to obtain the standard needles. We currently use the followingMethods Protoc. parameters,2020, 3, 31 but these parameters can change based on the filament, machine,9 and of 14 humidity of the room: P = 500, Heat = 496, Pull = 125, Vel = 10, Time = 186 [19]. Do not use gloves when pulling needles, but wash your hands before touching the capillaries! Extreme care should be taken3.6. Needle to avoid Preparation touching and the Microinjections platinum heating filament of the machine with the glass capillaries. Load the needlesUse a at Flaming least one/Brown hour before micropipette using them puller with Model approximately P-97 and 0.5 FHC µL of capillary the DNA tubing mix, and (Borosil store 1.0them in0.75 a moist mm IDchamber/Fiber withat 4 °C Omega for a few dot days. fiber) Use to an obtain automated the standard injection needles. system based We currently on nitrogen use × pressure,the following a micromanipulator parameters, but for these holding parameters the need canle, and change an inverted based on microscope the filament, for viewing. machine, and humidityThe very of the tip room: of every P = needle500, Heat must= first496, gently Pull = be125, broken Vel = off10, before Time =the186 first [19 injection]. Do not can use happen. gloves whenTo do pullingthis, carefully needles, touch but the wash chorion your of hands an egg before with touchingthe needle the tip, capillaries! while continuously Extreme pushing care should the "BALN"be taken or to “INJ” avoid button touching (Figure the platinum S1), until heatingthe DNA filament flows out. of the (Different machine models with the may glass have capillaries. different buttonLoad the names; needles please at least refer one to hour the user before manual.) using them Ensu withre that approximately the needle is 0.5 notµL badly of the broken, DNA mix, as andthis may cause the granular cytoplasmic content to ooze out of the egg (Figure 5A). Inject the eggs with a store them in a moist chamber at 4 ◦C for a few days. Use an automated injection system based on verynitrogen fine pressure,needle tip a micromanipulatorinto the posterior-most for holding part (Figure the needle, 5B) by and hitting an inverted either the microscope “INJ” button for viewing. or the foot pedalThe very (it is tip a matter of every of needlepreference). must first gently be broken off before the first injection can happen. To doIn this, order carefully to prevent touch the the needle chorion from of getting an egg clogge withd, the the needle “BALN” tip, whileor “INJ” continuously button or the pushing foot pedal the should"BALN" be or hit “INJ” frequently, button (Figurewhile the S1), needle until tip the is DNA outside flows of the out. eggs. (Diff Iferent the modelsneedle is may clogged, have carefully different rubbutton the names;tip of the please needle refer against to the an user egg manual.) while holding Ensure the that BALN the needle button is pressed. not badly Additionally, broken, as thismoving may thecause eggs the away granular from cytoplasmic the needle at content high speed to ooze can out help of make the egg the (Figure DNA flow5A). again. Inject the eggs with a very fine needleCRITICAL tip into STEP: the The posterior-most needles must part be (Figurechanged5B) when by hitting the eggs either start the leaking “INJ” out button a lot of or granular the foot pedalcytoplasm, (it is a matter whereas of preference). small clear droplets are acceptable and normal.

Figure 5.5.Needle Needle qualities qualities and and correct correct injection injection site. site. (A) ( TheA) The top needletop needle is a badlyis a badly broken broken needle, needle, while whilethe second the second needle needle is the is standard the standard needle. needle. Note Note that thethat bestthe best needle needle is the is onethe one that that has has a beveled a beveled tip withtip with a small a small opening. opening. (B) ( TheB) The transgene transgene can can be injectedbe injected through through the the posterior posterior part part of the of the egg. egg.

3.7.Methods Post-InjectionIn Protoc. order 20 to20 prevent, 3Treatment, x FOR the PEER needleand REVIEW Heat from Shock getting of the clogged, Eggs the “BALN” or “INJ” button or the foot pedal5 of 13 should be hit frequently, while the needle tip is outside of the eggs. If the needle is clogged, carefully After injecting all eggs on a slide, remove the slide from the microscope stage, remove the cover rubadvisable the tip that of the one needle person against lines up an eggtwo whileslides holding (~200 eggs the) BALNduring button a 1-hour pressed. cycle, Additionally,while another moving person glass from the slide, and briefly let the oil drop off. Quickly wipe off some excess oil against the theinjects eggs them. away All from wash the steps needle of atthe high eggs speed are done can helpwith makedistilled the water. DNA flow again. microscope slide. Stick the cover glass twice into a fresh cornmeal-sucrose-yeast medium vial, thereby carving CRITICAL out a small STEP:STEPS: trenchThe Neverof needlesfood. wash Th musten D place. beguttifera changedthe posterior eggs when with ends the ethanol eggsof the start, eggsbecause leaking with it the seems out cover a lotto slip ofharden granular into thethe trenchchorioncytoplasm, without and pushing whereas prevents the small eggs the clear too injection hard droplets against needles are the acceptable fromfood. Close penetrating and the normal. vial it,with and a cotton never ball leave and thesqueeze cage enoughwithout distilled food. water into the cotton ball to provide immediate moisture (do not make them dripping- 3.7. Post-Injection Treatment and Heat Shock of the Eggs wet butMake sufficiently a starter moist). yeast paste Place fromthe vial instant vertically baker’s into yeast a wet grains chamber one (a day beaker before with using 5-mm-high it, and onwater the onmorning theAfter ground of injecting injection and 2 all ,wet mix eggs paper it onagain a towels slide, with remove stuckadditional against the slidedry the yeast frominner and the wall). water microscope Hold. Keep up stage, theto 8 yeast vials remove pastewith the injectedat coverroom eggsglasstemperature together from the duringwith slide, a rubberthe and injections briefly band (Figure let; it the should oil6). dropAvoid smell o waterff a. bit Quickly likeflowing fermented wipe into o theff somefruit. vials At excessby 11keeping a.m. oil, against replacethe cotton the all microscopecornmeal-sucrose slide. Stick-yeast the medium cover glass plates twice in the into cage a fresh with cornmeal-sucrose-yeast four moist sponges, which medium are vial,to be thereby placed carvinginto medium out a- smallsized (100 trench mm of × food. 15 mm) Then Petri place dish the bottom posterior halves ends and of covered the eggs with with a thethin cover layer slipof fresh, into theactive trench yeast without paste (Figure pushing 2). the A second eggs too set hard of four against sponges the food.will be Close needed the vialfor the with next a cotton round. ball and squeezeThe enoughbest way distilled to prepare water the intosponges the cottonis to first ball make to provide them too immediate wet and then moisture to carefully (do not squeeze make themoff the dripping-wet excess water but with su ffitwociently thumbs moist). pressing Place and the vialmoving vertically from intothe top a wet to chamberthe bottom (a beakerthroughout with the surface of the sponges, until no drops are running out easily. The sponges should be dry enough that no water runs out when flies are bumped off from them during removal from the cage but wet enough to supply the flies with enough liquid. Sponges that are too dry seem to result in thicker coronas of the eggs (the thickening near the entry site of the injection needle), while sponges that are too wet cause the accumulation of liquid in the cage. The yeast paste is then supplied evenly over the sponge surface. Cover the sponges with lids before placing them into the cage to prevent stray flies from laying eggs on them. Allow the females to lay eggs on the 4 sponges for one hour. The flies prefer to sit on the sponge part that is closest to the cage wall. Sponges that are placed too far away from the wall are often ignored by the flies.

Figure 2. The yeast paste is evenly applied with a flat spatula over the sponge surface.

Methods Protoc. 2020, 3, x FOR PEER REVIEW 9 of 13

balls low in the vials, so that they do not touch the wet paper on the side of the wet chamber. Close the Methodschamber Protoc. with2020 aluminum, 3, 31 foil, label the lid, and store it at room temperature. 10 of 14 On the next morning (16–18 h after injection), heat shock the eggs at 40 °C for 90 min in the water bath (use a rubber band to hold the vials together, and submerge the vials as deep as needed to get 5-mm-highthe food level water under on thewater; ground do not and let any 2 wet water paper get towelsinto the stuck vials!). against After the the heat inner shock, wall). put Hold the vials up to 8 vialsback withinto the injected chamber eggs (now together remove with the a rubberwet paper band towels (Figure that6). lined Avoid the water wall). ﬂowing Place the into foil the lid vials back by keepingon, and the store cotton the ballschamber low (with in the some vials, water so that at the they bottom) do not at touch room the temperature wet paper for on theroughly side a of week. the wet chamber.Check daily Close for the problems chamber (mold with or aluminum flooded vial foil,s) labeland the the appearance lid, and store of the it atfirst room pupae. temperature.

FigureFigure 6. (6.A )(A Vials) Vials are bundledare bundled together together with with a rubber a rubber band. band. (B) A wet(B) chamberA wet chamber for the vialsfor the containing vials injectedcontaining eggs. injected eggs.

3.8.On After-Care the next and morning Fly Crosses (16–18 h after injection), heat shock the eggs at 40 ◦C for 90 min in the water bath (useLook a rubber at the bandmoist tochamber hold the every vials day together, to ensure and it submerge remains moistened. the vials as When deep asmost needed pupae to have get the foodformed level and under no more water; wandering do not let larvae any waterare seen, get collect into thethe vials!).pupae from After the the cotton heat plugs shock, with put a thebrush vials backand into two the pairs chamber of forceps (now (Figure remove 7A). the Making wet paper the towelscotton balls that linedvery wet the wall).helps to Place easily the detach foil lid the back on,pupae. and store Do not the leave chamber much (with cotton some left on water the pupae; at the however, bottom) ata few room fibers temperature can remain for attached. roughly Collect a week. Checkthe pupae daily fortemporarily problems on (mold a wet or tissue flooded paper. vials) Take and athe clean appearance and empty of glass the first vial pupae. ("hatching vial"), place a long piece of anti-fungus paper along one side of the wall, and moisten the paper with a squirt 3.8.bottle After-Care (remove and excess Fly Crosses water with a paper towel afterwards so that no free water is in the vial, or else theLook pupae at will the moistdrown). chamber Transfer every the collected day to ensure pupae itwith remains a moist moistened. brush from When the wet most tissue pupae paper have formedonto the and anti-fungus no more wandering paper in the larvae vial. are Then, seen, coll collectect the the remaining pupae from pupae the of cotton the same plugs construct with a brush as anddescribed two pairs above, of forceps and combine (Figure 7themA). Making with the the others cotton in the balls same very hatching wet helps vial. to Close easily the detach hatching thepupae. vial with a fresh cotton ball (push the ball deep enough down to prevent it from picking up water), and Do not leave much cotton left on the pupae; however, a few fibers can remain attached. Collect the store it angled upwards (on a small plastic lid from a pipette box) in a moist chamber (Figure 7B). pupae temporarily on a wet tissue paper. Take a clean and empty glass vial (“hatching vial”), place a long pieceCRITICAL of anti-fungus STEP: Moisten paper along the anti-fungus one side of paper the wall, inside and the moisten glass vials the paperwhenever with needed, a squirt and bottle (removenever excess let the water anti-fungal with a paper paper toweldry out. afterwards Check the sochamber that no twice free daily. water is in the vial, or else the pupae willStart drown). collecting Transfer wild-type the collectedvirgin flies pupae of both with sexes a moist for backcrosses brush from as the soon wet as tissue you papersee the onto first the anti-funguspupae forming paper from in the the vial. injection Then, experiment collect the remainingvials. The collected pupae of virgins the same are constructstored in ascornmeal- described above,sucrose-yeast and combine medium them vials with with the a few others grains in theof dr samey baker’s hatching yeast vial.and must Close be the bumped hatching to fresh vial vials with a freshevery cotton 3–4 days ball (push(or they the will ball get deep stuck enough in slimy down food to). preventThey will it be from sexually picking mature up water), when your and storefirst it angledinjection upwards survivors (on ahatch. small The plastic hatched lidfrom flies from a pipette the injections box) in a (the moist injection chamber survivors) (Figure 7areB). collected once a day in the late afternoon on a CO2 pad and immediately crossed with wild-type virgins of the opposite sex. Use cornmeal-sucrose-yeast medium vials with a few grains of baker’s yeast, and combine 3 males or 10 females of the injection survivors with 15 wild-type virgins of the opposite sex in one vial. The lumping of injection survivors into the same cross is done to produce enough larvae per vial (to keep microorganisms in the culture down). Bump the crosses to fresh food every 3–4 days.

Methods Protoc. 2020, 3, x FOR PEER REVIEW 10 of 13 Methods Protoc. 2020, 3, 31 11 of 14 Look at your crosses and their offspring every day. Screen for transgenic larvae 3 days after the parents have been removed from the vials.

MethodsFigure Protoc. 7. Collection2020, 3, x FORof pupae PEER and REVIEW after-care. (A) Pupae are collected from the cotton plugs with fine 5 of 13 Figureforceps. 7.(B)Collection Pupae collected of pupae on moist and anti-fungus after-care. paper (A in) Pupaea hatching are vial collected and stored from angled the upwards cotton plugs with fine advisableforceps.in a moist that( chamber.B )one Pupae person collected lines on up moist two anti-fungus slides (~200 paper eggs in) during a hatching a 1 vial-hour and cycle, stored while angled anothe upwardsr person injectsin athem. moist All chamber. wash steps of the eggs are done with distilled water. 3.9. Screening for Transgenic Larvae CRITICAL STEPS: Never wash D. guttifera eggs with ethanol, because it seems to harden the TheCRITICAL larval enhanced STEP: greenMoisten fluorescent the anti-fungus protein (EGF paperP) screen inside is carried the out glass in the vials fly wheneverroom with needed, and the mainchorionnever lights let andswitched the anti-fungal prevents off as follows: the paper injection dry out. needles Check fromthe chamber penetrating twice it, daily. and never leave the cage without food. (1) Switch on the UV lamp 10 minutes prior to screening. Start collecting wild-type virgin flies of both sexes for backcrosses as soon as you see the first pupae (2) MicroscopeMake a starter settings: yeast light paste filter from= “GFP”, instant bottom baker’s filter = yeast“Oblique”, grains objective one day = 1× before shutter usingopen, it, and on the forming from the injection experiment vials. The collected virgins are stored in cornmeal-sucrose-yeast morningcamera of tract injection closed,, andmix all it visibleagain microscope with additional lights off dry (check yeast that and the bottom water light. Keep is off the as well!).yeast paste at room medium(3) Repeat vials thewith following a few steps grains until of all dry vials baker’s have been yeast screened: and must be bumped to fresh vials every 3–4 days temperature during the injections; it should smell a bit like fermented fruit. At 11 a.m., replace all (or they(a) Remove will get the stuck larvae in from slimy a food).food vial They by squi willrting be distilled sexually water mature (squeeze when bottle) your and first stirring injection survivors cornmeal-sucrose-yeast medium plates in the cage with four moist sponges, which are to be placed hatch. Thewith hatched a rough flies brush; from then, the pour injections the larva/f (theood injection soup into survivors) a medium-sized are collected Petri dish once a day in the into mediumbottom-sized half. (100 mm × 15 mm) Petri dish bottom halves and covered with a thin layer of fresh, late afternoon on a CO2 pad and immediately crossed with wild-type virgins of the opposite sex. active(b) yeast Collect paste positive (Figure larvae 2). (Figure A second 8) with set aof pair four of spongesforceps into will 2-mL be neededtubes filled for withthe next1-mL round. Use cornmeal-sucrose-yeast medium vials with a few grains of baker’s yeast, and combine 3 males The cornmeal-sucrose-yeastbest way to prepare themedium sponges (briefly is toangle first the make food them by centrifugation, too wet and allowingthen to carefullythe squeeze or 10 females of the injection survivors with 15 wild-type virgins of the opposite sex in one vial. off the excesslarvae waterto be removed with two more thumbs easily from pressing the co llectionand moving forceps). from Wash the and top screen to eachthe bottomvial 3 throughout The lumpingtimes of for injection transgenic survivors larvae. into the same cross is done to produce enough larvae per vial (to the surface of the sponges, until no drops are running out easily. The sponges should be dry enough keep(c) microorganisms Poke two holes into in theeach culture lid (not too down). big that Bump the larvae the crossescan escape) to freshafter putting food every larvae 3–4into days. Look at that no water runs out when flies are bumped off from them during removal from the cage but wet your crossesthe 2-mL and theirfood tubes offspring (only add every up day.to 10 Screenlarvae per for vial). transgenic Label each larvae vial with 3 days the construct after the parents have enough toand supply line information. the flies with enough liquid. Sponges that are too dry seem to result in thicker been removed from the vials. coronas(d) Discard of the eggsscreening (the soup thickening with negative near larvaethe entry and food site debrisof the into injection an empty needle 5-L beaker.), while Rinse sponges that are the Petri dish with distilled water after screening each vial. 3.9.too wet Screening cause forthe Transgenic accumulation Larvae of liquid in the cage. The yeast paste is then supplied evenly over the sponge surface. Cover the sponges with lids before placing them into the cage to prevent stray flies 4. Expected Results fromThe laying larval eggs enhanced on them. green fluorescent protein (EGFP) screen is carried out in the fly room with the mainTheAllow perfect lights the timepoint switchedfemales tofor o layff screeningas eggs follows: on for the transgenic 4 sponges larvae for is one 3 days hour. after The the fliesparents prefer have to been sit on the sponge removed from the vials, as all larvae would have hatched, but the pupae would not have formed yet. (1)partEGFP Switchthat can onlyis closest on be the detected UVto the lamp in cagelarvae 10 minwall.but not prior Sponges in eggs, to screening. pupae,that are or theplaced adult tooflies. far Look away for EGFP from in the the wall are often (2)ignoredlarvalMicroscope eye by disks, the Bolwigflies. settings: organs, light brain, filter and/or= “GFP”, in the bottomanal plates filter (Figure= “Oblique”, 8) [20]. EGFP objective expression= 1is shutter open, × usuallycamera strong tract and clearly closed, visible and allin all visible larval microscope instars. Finding lights multiple off (check positive that larvae the bottomper vial is light not is off as well!). uncommon. Note that many transgenic lines are obtained in later screening sessions, so it is worth (3)the timeRepeat and patience the following bumping steps the crosses until allfor vials2 or 3 haveweeks beenbefore screened: declaring them negative. (a) Remove the larvae from a food vial by squirting distilled water (squeeze bottle) and stirring with a rough brush; then, pour the larva/food soup into a medium-sized Petri dish bottom half. (b) Collect positive larvae (Figure8) with a pair of forceps into 2-mL tubes filled with 1-mL cornmeal-sucrose-yeast medium (briefly angle the food by centrifugation, allowing the larvae to be removed more easily from the collection forceps). Wash and screen each vial 3 times for transgenic larvae. (c) Poke two holes into each lid (not too big that the larvae can escape) after putting larvae into the 2-mL food tubes (only add up to 10 larvae per vial). Label each vial with the construct and line information. Figure 2. The yeast paste is evenly applied with a flat spatula over the sponge surface.

Methods Protoc. 2020, 3, 31 12 of 14

(d) Discard screening soup with negative larvae and food debris into an empty 5-L beaker. Rinse the Petri dish with distilled water after screening each vial. Methods Protoc. 2020, 3, x FOR PEER REVIEW 11 of 13

Figure 8.8.A A transgenic transgenicD. guttiferaD. guttiferalarva larva showing showing enhanced enhanced green ﬂuorescent green fluorescent protein (EGFP) protein expression (EGFP) inexpression the Bolwig in organsthe Bolwig (two organs small glowing(two small dots glowin on theg dots left) on and the the left) eye and disks the/brain eye disks/brain (larger blob (larger to the lowerblob to right). the lower right).

4.5. Troubleshooting Expected Results The perfect timepoint for screening for transgenic larvae is 3 days after the parents have been removedTable from 1. Troubleshooting the vials, as all guide larvae (summarizes would have a variety hatched, of problems but the pupaethat we would have encountered not have formed in the yet. EGFPpast. can The only table be detectedexplains the in reasons larvae butunderlying not in eggs,the problems pupae, and or the offers adult appropriate ﬂies. Look actions for EGFPto work in the larvalaround eye disks, each problem). Bolwig organs, brain, and/or in the anal plates (Figure8)[ 20]. EGFP expression is usuallyProblem strong and clearly visibleReason in all larval instars. Finding multiple positiveSolution larvae per vial is not uncommon.Few larvae Note that many transgenic lines are obtainedMake insure later you screening use standard sessions, needles. so Strictly it is worth follow the Bad injection needles. timehatch. and patience bumping the crosses for 2 or 3 weeks beforethe declaring instructions them under negative. Section 3.6. Over-desiccation of eggs during Add injection oil within 3–5 s after eggs appear

5. Reagents lining up. completely dry under the microscope. Use highly pure DNA (no mini-preps). Additional 5.1. Anti-Fungus Paper Toxic DNA. isopropanol precipitation after the first extraction of DNA is necessary. Dissolve 2 g of sorbic acid and 0.6 g of methyl paraben in 200 mL of 95% ethanol. Roll ~20 paper The heat shock temperature for D. guttifera towels and stick them into a 1-L glass beaker. Slowlytransformation pour the anti-fungus is 40 °C; however, solution if over this temperature the towels to soak them evenly. Too-high Unfold heat the towelsshock temperature. and let them dryis causing completely. strong Anti-fungus reduction in the paper survival does rate, not reduce go bad and can be stored for years. the temperature by 1–2 degrees but never go below 37 °C. 5.2. Halocarbon OilIf Mixture the Halocarbon oil mixture is Use Halocarbon oil 27 only or GEM extra virgin olive suffocating and killing the eggs. This Use a 50-mL Falcon tube, add 35 mL of Halocarbonoil. We are oil using 700 extra and virgin 5 mL olive of Halocarbon oil in our lab with oil 27. does not normally happen, but we Homogenize the mixture on a nutator overnight. high success. have seen this problem in our lab. Few Having only a few injection-surviving 6. Conclusions Ensure a high injection survival rate. At least 10–20 surviving larvae causes bacterial growth and/or larvae can keep the food mold-free. adults.The starting pointmold for formation this protocol in the was food. the standard method that was optimized for D. melanogaster. We quickly learnedDesiccation that “whatever of pupae due works to dry for D. melanogaster does not work for/kills D. guttifera.” Always keep the chamber and the cotton plugs chamber and/or dry cotton plugs. No Always clean the DNA by isopropanol precipitation transgenic Poor DNA quality. or prepare new DNA samples. larvae .

Methods Protoc. 2020, 3, 31 13 of 14

It took therefore several years of innovations and deviations from the original protocol to make D. guttifera survive the procedure from the beginning to the end and to obtain the first transgenic animals. Key innovations are (1) establishing a large cage population (this compensates for the low egg-lay rate), (2) having good cage aeration (this prevents the accumulation of ethanol fumes, which become quickly lethal for ethanol-susceptible species), (3) using sponges as egg-lay surfaces (this allows for easy egg collections, even when females tend to stick their eggs into substrates), (4) no ethanol wash step to visualize the interior of the eggs before injection (this prevents the egg chorion from becoming rock-hard), and (5) washing larvae out of the food for transgenic screening (no need for a white-eyed mutant). We believe that our method, developed for a very delicate species, should be useful to transform most drosophilid species that can be reared in the laboratory on a standard fly-food medium, such as Drosophila tripunctata, Drosophila quinaria, Drosophila subquinaria, and Drosophila deflecta. For recommendations on collecting and rearing many non-model species, please refer to our book [10].

Supplementary Materials: The following are available online at http://www.mdpi.com/2409-9279/3/2/31/s1, Figure S1: The Narishige IM 300 Microinjector, Video S1: The Making of Transgenic Drosophila guttifera. Author Contributions: Conceptualization, S.K. and T.W.; data curation, M.S. and T.W.; formal analysis, S.K. and T.W.; funding acquisition, S.K. and T.W.; investigation, M.S., T.S., S.K., and T.W.; methodology, M.S., T.S., S.K., and T.W.; project administration, T.W.; resources, S.K. and T.W.; supervision, T.W.; validation, M.S., S.K., and T.W.; visualization, M.S., T.S., and T.W.; writing—original draft, M.S. and T.W.; and writing—review and editing, M.S., T.S., S.K., and T.W. All authors have read and agree to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI, grant number 18H02486 to S.K. and the NIH, grant number 1R15GM107801-01A1 to T.W. Acknowledgments: We thank Lucinda Hall, David Trine, Daniel Noren, and Prajakta Kokate for technical assistance. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

References

1. Rubin, G.M.; Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors. Science 1982, 218, 348–353. [CrossRef][PubMed] 2. Santamaria, P.; Roberts, D. Drosophila: A Practical Approach; IRL Press: Oxford, UK, 1986. 3. Handler, A.M.; James, A.A. Insect Transgenesis: Methods and Applications; CRC Press: Boca Raton, FL, USA, 2000. 4. Venken, K.J.; He, Y.; Hoskins, R.A.; Bellen, H.J. P[acman]: A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 2006, 314, 1747–1751. [CrossRef][PubMed] 5. Fish, M.P.; Groth, A.C.; Calos, M.P.; Nusse, R. Creating transgenic Drosophila by microinjecting the site-specific ϕC31 integrase mRNA and a transgene-containing donor plasmid. Nat. Protoc. 2007, 2, 2325. [CrossRef] [PubMed] 6. Venken, K.J.; Bellen, H.J. Transgenesis upgrades for Drosophila melanogaster. Development 2007, 134, 3571–3584. [CrossRef][PubMed] 7. Groth, A.C.; Fish, M.; Nusse, R.; Calos, M.P. Construction of transgenic Drosophila by using the site-specific integrase from phage ϕC31. Genetics 2004, 166, 1775–1782. [CrossRef][PubMed] 8. Allen, B.G.; Weeks, D.L. Transgenic Xenopus laevis embryos can be generated using ϕC31 integrase. Nat. Methods 2005, 2, 975–979. [CrossRef][PubMed] 9. Nimmo, D.; Alphey, L.; Meredith, J.; Eggleston, P. High efficiency site-specific genetic engineering of the mosquito genome. Insect Mol. Boil. 2006, 15, 129–136. [CrossRef][PubMed] 10. Werner, T.; Steenwinkel, T.; Jaenike, J. Drosophilids of the Midwest and Northeast; Michigan Technological University: Houghton, MI, USA, 2018. 11. Koshikawa, S.; Fukutomi, Y.; Matsumoto, K. Drosophila guttifera as a model system for unraveling color pattern formation. In Diversity and Evolution of Butterfly Wing Patterns; Springer: Berlin/Heidelberg, Germany, 2017; pp. 287–301. 12. Werner, T.; Koshikawa, S.; Williams, T.M.; Carroll, S.B. Generation of a novel wing colour pattern by the Wingless morphogen. Nature 2010, 464, 1143–1148. [CrossRef][PubMed] Methods Protoc. 2020, 3, 31 14 of 14

13. Koshikawa, S. Evolution of wing pigmentation in Drosophila: Diversity, physiological regulation, and cis-regulatory evolution. Dev. Growth Differ. 2020.[CrossRef][PubMed] 14. Koshikawa, S.; Giorgianni, M.W.; Vaccaro, K.; Kassner, V.A.; Yoder, J.H.; Werner, T.; Carroll, S.B. Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila. Proc. Natl. Acad. Sci. USA 2015, 112, 7524–7529. [CrossRef][PubMed] 15. Fukutomi, Y.; Matsumoto, K.; Agata, K.; Funayama, N.; Koshikawa, S. Pupal development and pigmentation process of a polka-dotted fruit fly, Drosophila guttifera(Insecta, Diptera). Dev. Genes Evol. 2017, 227, 171–180. [CrossRef][PubMed] 16. Mollinari, C.; González, C. Microinjection of Drosophila Eggs. In Microinjection and Transgenesis; Springer: Berlin/Heidelberg, Germany, 1998; pp. 587–603. 17. Raja, K.K.; Shittu, M.O.; Nouhan, P.M.; Steenwinkel, T.E.; Bachman, E.A.; Kokate, P.P.; McQueeney, A.H.; Mundell, E.A.; Armentrout, A.A.; Peabody, A.M.; et al. The regulation of a pigmentation gene in the formation of complex color patterns in Drosophila abdomens. BioRxiv 2020.[CrossRef] 18. Horn, C.; Wimmer, E.A. A versatile vector set for animal transgenesis. Dev. Genes Evol. 2000, 210, 630–637. [CrossRef][PubMed] 19. Miller, D.F.; Holtzman, S.L.; Kaufman, T.C. Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster. Biotechniques 2002, 33, 366–375. [CrossRef][PubMed] 20. Horn, C.; Jaunich, B.; Wimmer, E.A. Highly sensitive, fluorescent transformation marker for Drosophila transgenesis. Dev. Genes Evol. 2000, 210, 623–629. [CrossRef][PubMed]

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