Special thanks to previous B. coprophila stock keepers (Jacob E. Bliss, Paula Bonazinga, Anne W. Kerrebrock, Ingrid M. Mercer, Heidi S. Smith) and research personnel (especially Robert Baird, Michael S. Foulk, Donna Kubai, John M. Urban, Yutaka Yamamoto) for fine-tuning the husbandry protocols. Initial instructions on care of B. coprophila were provided by Helen V. Crouse, Natalia Gabrusewycz-Garcia, Reba M. Goodman, Charles W. Metz, and Ellen Rasch. With gratitude to Yukiko Yamashita and Anne W. Kerrebrock for taking on the Bradysia (Sciara) stock center. Much appreciation to the following people for their helpful preparation of figures: Brian Wiegmann (Figure 1), John M. Urban (Figure 4 upper panel), Laura Ross (Figure 4 lower panel), Yutaka Yamamoto (Figure 5 left panel), Leo Kadota (Figure 7 and Figure 8). Many thanks to Ava Filiss and the Brown University Multidisciplinary Laboratory for their help with photography and filming. Thanks to Robert Baird for comments on this manuscript. Our research and maintenance of B. coprophila has been supported by NIH and NSF, including the most recent support from NIH GM121455 to S.A.G. Further details about B. coprophila are available on the Bradysia (Sciara) Stock Center websites (https://sites.brown.edu/sciara/ and https://sciara.wi.mit.edu) that are currently being constructed.

&lt;i&gt;Bradysia coprophila&lt;/i&gt; Lab Protocols and Chromosomal Research

The author has no conflicts of interest to declare.

The protocols presented here for the husbandry of B. coprophila will be useful for scientists who wish to raise this organism in their laboratories for experiments to delve into its unique biological features. The initial description of the feeding method using yeast and mushroom powder sprinkled onto an agar base to maintain B. coprophila29 was used in the Metz laboratory to rear 14 different species of Sciarid flies5. Subsequently, it was observed that the addition of nettle and/or spinach powder further increased the vitality of B. coprophila (Gabrusewycz-Garcia, personal communication). These methods have been successful for the maintenance of related species within the Sciaridae family, including Bradysia impatiens and Lycoriella ingenua that are currently in culture&#160;(Robert Baird, personal communication).
Other methods (such as the alternative feeding methods described below) have been tried to raise B. coprophila, but the protocols described here have been optimized to have the most favorable ratio of larvae per surface area of agar to obtain the most fertile fat adults and minimize the growth of mold. To scale up, mass mating can be done in glass vials as described in protocol 2 above. Alternatively, a few (2-4) adult females can be placed together with twice as many adult males in a flask such as that used to raise Drosophila (disposable 6 oz. = 177.4 mL square bottom Drosophila polypropylene bottle). In both cases, the researcher must be fully confident that the flask contains only all female-producer or all male-producer mothers.
Only feed the larvae since the pupae and adults do not eat. Do not feed the vial if larvae have turned into pupae (a sign of this is when the first early-to-emerge adult flies appear). Once the adults eclose, put the vials into a cooler incubator (e.g., 16 &#176;C) if available as this will allow the adults to live longer. Feed three times per week (e.g., Monday, Wednesday, Friday), increasing the amount of food given per vial as the larvae grow older. Feed generously, and you will be rewarded with fat fertile adults. However, if you feed too much, white mold will appear and that is a sign to reduce the amount of food you are depositing in a vial. Further, if you feed too much, a thick pad of food will develop on top of the agar and make it harder for the adults to emerge (you can remove the pad with a forceps, but be careful to not take larvae out with the pad-it is best to not have to do this at all). Vials with few larvae (marked &#34;few&#34;) need less food. If you feed too little, the larvae will climb the walls of the vial in search of food. Underfed larvae result in small adults that are less fertile.
Alternative feeding methods 
A variety of methods have been attempted to feed larvae just once during the larval stage rather than 3 times per week.&#160;B. coprophila will not grow on Drosophila style food. John Urban (personal communication) tried mixing B. coprophila food in with the agar, but too much mold grew. He found that adding two mold inhibitors (tegosept and propionic acid) in combination and separately, trying several different concentrations, were all toxic to B. coprophila at levels that inhibit mold. The agar should be pH 6-7 (neutral) as B. coprophila gets sick at an acidic pH (as with propianic acid). Alternatively, to obviate thrice weekly feeding, he tried using a spatula or syringe without a needle to dispense a thick yeast paste (Red Star active dry yeast mixed with a little distilled water to moisten it) as a dollop on top of the agar in each vial one week after mating (i.e., about the time the larvae will begin to emerge).
Another method to avoid thrice weekly feeding is to add a living culture of fungi to each vial.&#160;Bath and Sponsler37 reported that&#160;a slanted agar surface with Sabouraud&#39;s medium should be streaked with a fungal culture of the genera Chaetoconidia (best) or else Baplosporangia or Xllescheria. The fungus was grown several days to one week before B. coprophila was introduced. No feeding was needed after this. A variant of this method was also employed by Ellen Rasch (personal communication). In our hands, the vials were too wet with this method and the larvae drowned, but it could be tried again to optimize the number of larvae relative to the vials with live fungi.
Arthur Forer (personal communication) has had some success in rearing B. coprophila the same way as crane flies38. With this approach, pupae were reared on moist papier m&#226;ch&#233;. Subsequently, the adults were mated and the eggs were deposited on fresh moist papier m&#226;ch&#233;. The resulting larvae were kept on papier m&#226;ch&#233; in petri dishes and fed with powdered nettle leaves twice a week. Pupae were put into a cage to repeat the cycle.
Yukiko Yamashita (personal communication) has tried without success to rear B. coprophila on soil, mimicking the conditions where they are found in nature in potted plants and greenhouses with high humidity. However, mold can become a problem when the humidity level is raised. Nonetheless, moist soil has been used with success to raise Pseudolycoriella (previously Bradysia) hygida larvae in plastic boxes with moist soil; they are fed decomposed Ilex paraguariensis leaves, supplemented in late larval life with 1.2% yeast extract, 1.4% cornstarch, 0.8% oatmeal flour, 1.2% agar12. Similarly, the moist soil can be replaced by moist peat moss with crushed kidney beans to raise Sciarid flies39,40.
Still other methods have been employed to maintain laboratory cultures of Bradysia: (i) autoclaved potato to which yeast and dried blood fertilizer are added41; (ii) manure42,43,44 to which dried blood can be added45; (iii) plastic containers with cotton pads and dampened paper towels with ground soybeans46.
Mites 
Mites can be transferred from Drosophila to B. coprophila. To minimize this, it is best to keep B. coprophila in a separate incubator or room, not close to Drosophila stocks. Moreover, do any B. coprophila maintenance work early in the day before handling Drosophila. Mites can also be transferred to B. coprophila from houseplants, so do not keep plants in the same room as B. coprophila. If mites invade the vials, they can be seen as small white spherical organisms crawling on the body of B. coprophila. Chemical treatments that work to destroy mites in Drosophila cannot be used for B. coprophila as the chemicals kill B. coprophila (B. coprophila is also sensitive to organic fumes such as phenol). The only treatment to rid B. coprophila stocks of mites is to manually collect the embryos on an agar plate, examine each for the absence of mites, and then transfer them to fresh agar vials using a fine paintbrush. Cotton-filled gauze plugs and cellulose acetate foam flugs (as used for Drosophila polypropylene vials) both help to prevent the entry of mites into the vials.
Usefulness of the husbandry protocols 
The protocols described here will enable the growing community of scientists to rear B. coprophila as a new/old emerging model organism to study its unique biological features. New laboratory groups are encouraged to join the growing community to maintain and investigate the unique biological features of Bradysia (Sciara).

A complete understanding of biological principles requires the study of many diverse organisms spanning the Tree of Life. Although a broad range of organisms were described through the end of the 19th century, by the middle of the 20th century experimental studies became restricted to a handful of less than a dozen model organisms. With the advent of the genomic era and the goal to sequence genomes from all species in the Tree of Life1, we are now in a position to expand the types of organisms being used for laboratory experiments and to reap the advantage of their diversity. Such expansion of emerging model organisms for experiments has a prerequisite of being able to maintain them in the laboratory. Here, protocols are described for rearing one such emerging new/old model organism.
The majority of animal life on Earth is accounted for by four super-radiations of Insects2. Within the Insects, there are about 158,000 species of Diptera (true flies)3, with about 3000 species in the family Sciaridae (black fungus gnats)4. The fruitfly Drosophila is the most thoroughly studied of the Dipteran flies. The lower Dipteran fly (Nematocera), Bradysia (previously called Sciara) coprophila, diverged 200 million years ago from Drosophila, which is a &#34;higher Dipteran&#34; fly (Brachycera). Therefore, B. coprophila is in a favorable taxonomic position for comparative studies with D. melanogaster (Figure 1). Moreover, B. coprophila has many unique biological features that are worthy of study in their own right5,6,7. Many of these features disobey the rule of DNA constancy in which all cells of an organism have the same DNA content. In B. coprophila, (i) the paternal genome is eliminated on a monopolar spindle in male meiosis I; (ii) there is non-disjunction of the X dyad in male meiosis II; (iii) germ line-limited (L) chromosomes are eliminated from the soma; and (iv) one or two X chromosomes are eliminated in the early embryo depending on the sex of the individual. Chromosome imprinting to distinguish maternal from paternal homologs was first discovered in B. coprophila and is at play for many of these chromosome elimination events. In addition to chromosome elimination, another bypass of DNA constancy occurs via developmentally regulated, locus-specific DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. Studies of these unique features require laboratory maintenance of B.coprophila; details of its husbandry are presented here to facilitate such studies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66751/66751fig01v3.jpg" /> 
Figure 1: Phylogeny of Bradysia (Sciara) coprophila. Popular model organisms are indicated in blue font and their taxonomic order in red font. Bradysia and other Sciarid fungus gnats as well as mosquitoes are lower dipteran flies (formerly, suborder Nematocera), whereas Drosophila species are higher dipteran flies (suborder: Brachysera). Information on the left side of the figure is from Misof et al.33; information on the right side is from Bertone et al.34 and Wiegmann et al.2. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Previously, the genus Sciara had the largest number (700) of species for any eukaryote, prompting Steffan to subdivide them8. Subsequently, Shin proposed that the Sciaridae family be subdivided into the subfamily Sciarinae (with six genera including Sciara, Trichosia, and Leptosciarella), the subfamiliy Megalosphyinae (including the genus Bradysia) and three other groups (including Pseudolycoriella)9. The phylogeny of the Sciaridae has been studied further by several groups in recent years9,10,11. Over the past several decades, the names of many organisms in the Sciaridae family have changed12. Although most of the literature spanning more than a century refers to the organism that we study as Sciara coprophila, its current taxonomic name is now Bradysia coprophila (syn. Bradysia tilicola and other synonyms)10. They are found worldwide and are commonly known as fungus gnats since they eat mushrooms and other fungi. They were first described in 1804 by Meigen13 in Europe and subsequently by Johannsen14,15 in North America. B. coprophila was collected at Cold Spring Harbor Laboratory and laboratory stocks were established by Charles Metz in the early 1900s when he was a graduate student at Columbia University with Thomas Hunt Morgan. Thus, the current stocks reflect a century of inbreeding. Similarly, the biology of B. coprophila was further elucidated by decades of cytogenetic studies by Helen Crouse (who did her Ph.D. work with Barbara McClintock).
In the 1930s, Bradysia (Sciara) competed with Drosophila melanogaster as a model system for genetic studies. Despite its many unique biological features, B. coprophila was eclipsed by D. melanogaster as a popular model organism since radiation-induced phenotypic mutations were needed for genetic studies and were easier to achieve in the latter, even though B. coprophila is only slightly more resistant to gamma irradiation than D. melanogaster16. In the modern era of genomics, this is no longer a concern. Since the genome sequence17,18,19 (Urban, Gerbi, and Spradling, data not shown) and methods for transformation20,21 (Yamamoto and Gerbi, data not shown) for B. coprophila have recently become available, the time is now ripe to utilize it as a new/old emerging model system, as seen by the growing community of scientists that have adopted it for their research. This article describes procedures for its laboratory maintenance.
B. coprophila lacks a Y chromosome, and the sex of the offspring is determined by the mother. Females that have the X&#39; (&#34;X-prime&#34;) chromosome with a long paracentric inversion will have only daughters, whereas females that are homozygous for the standard (non-inverted) X chromosome will have only sons5 (Figure 2). Sequence information is available for the X&#39; chromosome19, but the molecular mechanism remains to be elucidated on how the X&#39; chromosome determines that offspring will be females. Males never have the X&#39; chromosome, and after fertilization, females are X&#39;X (heterozygous for the X&#39;) or XX. Adult X&#39;X females can be distinguished from XX females by phenotypic markers on the wing (Figure 3). X&#39;X females (who will have only daughters) can be recognized by the dominant Wavy (W) wing marker on the X&#39; (as in the HoLo2 stock)22. Alternatively, XX females (who will have only sons) can be recognized by the recessive petite (p) wing marker on the X as in the 91S stock23. In this case, X&#39;Xp females will have full-length (not petite) wings and will have only daughters. The stock 6980 carries a recessive marker on the X chromosome for swollen (sw) veins24, as well as the dominant marker Wavy on the X&#39;, allowing two markers for selection for crosses. The degree of expression of Wavy can vary and seems weaker in overcrowded vials where food is limiting or if the temperature becomes too warm. The Wavy wing phenotype is exceptionally strong if larvae are kept in the cold room (4&#176;-8 &#176;C) instead of the usual 21 &#176;C. Although the recessive petite wing marker is not variable and is very easy to identify, 91S stocks are used less frequently since they are less healthy than the HoLo2 stock. The B. coprophila mating schemes are presented here (Figure 2), and described in detail for the HoLo2, 7298, and W14 stocks (Supplemental File 1), the 91S stock (Supplemental File 1), the 6980 stock (Supplemental File 1), and the translocation stocks (Supplemental File 1). The translocation stocks are no longer extant; they were reciprocal translocations of heterochromomeres (H1, H2, and H3) on the short arm of the X that contains the ribosomal RNA genes25,26,27.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66751/66751fig02.jpg" /> 
Figure 2: Mating scheme for B. coprophila. This organism has no Y chromosome (the male soma has a single X); mothers determine the sex of their offspring. XX mothers have only sons and X&#39;X females have only daughters. The X&#39; chromosome has a long paracentric inversion when compared to the X chromosome. The paternal or maternal lineage of the X (or X&#39;) chromosome is denoted by blue or red, respectively, in this figure. Sperm are haploid for the autosomes but have two copies of the X chromosome due to non-disjunction in meiosis II. The somatic lineage of early embryos eliminates one or two copies of the paternally derived X if they will be female or male, respectively. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66751/66751fig03.jpg" /> 
Figure 3: Wing phenotypes of B. coprophila. Adult female flies are shown with various wing phenotypes: (A) straight-wing (XX), (B) Wavy wing (X&#39;WX), (C) extreme Wavy wing (X&#39;WX) phenotype that has a shriveled appearance after storing larvae in the cold room, (D) petite wing (XpXp) that is vestigial-like, (E) straight wing with wildtype (XX) and not swollen veins, (F) straight wing with swollen veins (XswXsw) where small bubbles (black arrows) appear on the upper edge of the wing and/or near the tip of both wings, (G) extreme example of swollen where a blister (white arrow) occurs on one or both wings. Males lack the X&#39; chromosome and therefore will never have Wavy wings, but they have petite or swollen wings in the 91S stock or 6980 stock, respectively. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The goal in stock maintenance is to perform crosses where half the crosses are from female-producing mothers and half the crosses from male-producing mothers to have equal numbers of female and male adults in the next generation for subsequent crosses. However, this also involves planning since the life cycle for males is shorter than for females and adult males emerge up to a week before adult females. Nature accommodates for this asynchrony between the sexes by having the male embryos emerge as larvae 1-2 days after the female larvae from a cross on the same date. However, to ensure that male and female adults are available at the same time for laboratory crosses, female development can be somewhat speeded up by leaving vials with female larvae at room temperature rather than at 21 &#176;C or placing vials with male larvae at slightly cooler temperatures (e.g., 16 &#176;C). Another route that is more foolproof is to do crosses with female-producer mothers on Monday and crosses with male-producer mothers on Friday of the same week. The easiest route, which is what we employ, is to perform crosses with female-producer and male-producer mothers on the same day each week and perform crosses on that day in each consecutive week. In that approach, adult females from a cross on week 1 can be mated with adult males that emerged from a cross on week 2.
The life cycle for female B. coprophila is 5 weeks when raised at 21 &#176;C (Table 1). The length of their life cycle is somewhat longer at cooler temperatures or if they are underfed. The life cycle of male B. coprophila is ~4-4.5 weeks since they pupate 0.5-1 week before females. The end of each larval instar is marked by the shedding of the cuticle, which is triggered by a burst in the level of the steroid hormone ecdysone. Unlike D. melanogaster, which has three larval instars, B. coprophila has four larval instars.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Developmental Stage</td>
			<td>Days post mating (dpm)</td>
			<td>Length of stage (days)</td>
		</tr>
		<tr>
			<td>Egg laid</td>
			<td>1-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo</td>
			<td>1-2 to 7-8</td>
			<td>~7 days</td>
		</tr>
		<tr>
			<td>Larva&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Larval instars 1, 2, and 3</td>
			<td>7-8 to 16-19</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>4th larval instar pre-eyespot</td>
			<td>16-19 to 21-24</td>
			<td>5</td>
		</tr>
		<tr>
			<td>4th larval instar eye-spot stage</td>
			<td>21-24 to 25-28</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Pupa&#160;</td>
			<td>25-28 to 30-33</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Adult</td>
			<td colspan="2">lives 1-2 days at 21 &#176;C if mated or lives 2-3 weeks at 16 &#176;C if not mated.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Life cycle of female B. coprophila at 21 &#176;C.
B. coprophila can be kept anywhere in the range of 15 &#176;C-25 &#176;C, with development progressing more slowly at cooler temperatures. This insect prefers a humid environment (being found in the soil of houseplants or mushroom beds), so we keep a beaker with deionized water in the incubator. B. coprophila can be kept at room temperature in a metal bread box with a loosely fitting lid and containing a beaker of water, but they go into heat shock at 37 &#176;C28, which is a danger in hot climates. Michael Ashburner and others have tried with little success to store D. melanogaster in the cold to reduce the time needed for stockkeeping. In contrast, a major advantage of B. coprophila is that vials with mid-stage larvae can be stored for up to 3 months on an open shelf in the cold room (4-8 &#176;C) with minimal care of only feeding once a month. They develop extremely slowly in the cold up to the pupal stage and will emerge as fertile adults when the vials are brought back to 21 &#176;C. Presumably, this mimics their overwintering in the wild. This cold-induced developmental stalling might be comparable to that seen after gamma irradiation of mid-stage B. coprophila larvae16, but developmental stalling is not seen in late-stage larvae that have passed the point of no return for their normal developmental progression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar (bacteriological)</td>
			<td>U.S. Biological</td>
			<td>A0930</td>
			<td>https://www.usbio.net;&#160;</td>
		</tr>
		<tr>
			<td>CO2 FlyStuff Foot Pedal</td>
			<td>Genesee Scientific</td>
			<td>59-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 FlyStuff Blowgun</td>
			<td>Genesee Scientific</td>
			<td>54-104</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 FlyStuff UltimaterFlypad</td>
			<td>Genesee Scientific</td>
			<td>59-172</td>
			<td>https://www.geneseesci.com</td>
		</tr>
		<tr>
			<td>Ether fume hood</td>
			<td>Labconco&#160;</td>
			<td>3955220</td>
			<td>Sits on top of lab bench</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Filter replacement&#160; cat # 6961300</td>
		</tr>
		<tr>
			<td>Food: Brewer&#8217;s Yeast Powder</td>
			<td>Solgar</td>
			<td>&#160; &#160;</td>
			<td>Obtain from Amazon or health food store</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>https://www.solgar.com;</td>
		</tr>
		<tr>
			<td>Food: Nettle Powder&#160; (pesticide free)</td>
			<td>Starwest Botanicals</td>
			<td>209460-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Food: Shitake Mushrooms (pesticide free)</td>
			<td>Starwest Botanicals</td>
			<td>202127-5</td>
			<td>https://www.starwest-botanicals.com;</td>
		</tr>
		<tr>
			<td>Food: Spinach Powder ( pesticide free)</td>
			<td>Starwest Botanicals</td>
			<td>209583-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Food: Straw (pesticide free )</td>
			<td>Starwest Botanicals</td>
			<td>209465-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Jar: clear glass, polypropylene lid</td>
			<td>Fisher Scientific:</td>
			<td>FB02911765</td>
			<td>73 mm dia, 89 mm ht (240 ml) 
			https://www.fishersci.com;</td>
		</tr>
		<tr>
			<td>Needle Probe, wooden handle</td>
			<td>US Geo Supply Inc</td>
			<td>SKU: 4190</td>
			<td>5.75&#8221; long probe, 
			stainless steel needle 
			https://usgeosupply.com; (970)-434-3708</td>
		</tr>
		<tr>
			<td>Vials: glass, preferred:</td>
			<td>Wilmad LabGlass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wilmad-glass custom vials</td>
			<td></td>
			<td></td>
			<td>28-33 mm inner dia, 33 mm outer dia, 9.5 cm ht 
			Wilmad: https://www.SP-WilmadLabglass.com</td>
		</tr>
		<tr>
			<td>Vials: glass (cheaper and ok)&#160;</td>
			<td>Fisher Scientific</td>
			<td>03-339-26H</td>
			<td>29 mm outer dia, 9.5 cm h 
			&#160;https://www.fishersci.com;&#160;</td>
		</tr>
		<tr>
			<td>Vials: glass (a bit narrow)&#160;</td>
			<td>Genesee Scientific&#160;</td>
			<td>32-201</td>
			<td>24.5 mm outer dia,9.5 cm h 
			thttps://www.geneseesci.com</td>
		</tr>
		<tr>
			<td>Vials: polypropylene&#160;</td>
			<td>Genesee Scientific</td>
			<td>32-114</td>
			<td>28.5 mm outer dia,9.5 cm ht</td>
		</tr>
		<tr>
			<td>Vial Plugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>roll of non-absorbent cotton</td>
			<td>Fisher Scientific</td>
			<td>22-456881</td>
			<td></td>
		</tr>
		<tr>
			<td>cheesecloth</td>
			<td>Fisher Scientific</td>
			<td>22-055053</td>
			<td>https://www.fishersci.com;</td>
		</tr>
	</tbody>
</table>

laboratory maintenance of the lower dipteran fly bradysia (sciara) coprophila: a new/old emerging model organism

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. 
It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Author Spotlight: Exploring &lt;i&gt;Bradysia coprophila&#039;s&lt;/i&gt; Unique Biology &amp;#8211; A Guide to Laboratory Maintenance

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

The protocols described here have led to proven success in raising B. coprophila. When recently eclosed fat adults are chosen for mating (Figure 4), over 90% of the crosses can be fertile and yield offspring. The fertility success does vary with different stocks (Table 2). Stock 7298 (X&#39; chromosome with Wavy marker) was the healthiest of the stocks but went through a period of decline, apparently due to activation of DNA mobile elements creating genome rearrangements32. The HoLo2 stock represents a healthy strain derived from 7298, where apparently the genome rearrangements have stabilized, and it has replaced the parental 7298 stock in the stock center. The HoLo2 stock is the one that was used to sequence the B. coprophila genome and is the one used most widely by various laboratory groups. Recently, CRISPR mutagenesis of HoLo2 flies has been used to create the W14 stock with a white eye phenotype to be used for transformation with fluorescent eye markers (Yamamoto and Gerbi, data not shown). The W14 strain is exceptionally robust. The 6980 stock (Wavy wing and swollen vein markers) is somewhat less robust and the 91S stock (petite wing marker) is even less robust.
Successful crosses result in embryos (Figure 5). Embryos undergo elimination of the imprinted paternal X chromosomes at the 7th to 9th cleavage division. In addition, germ-line limited L chromosomes are eliminated from the somatic lineage in embryos at the 5th to 6th cleavage division. The embryos emerge as larvae, which should not be confused with mold that also may be present (Figure 6). Eyespots (anlage to the adult eyes) appear at the second half of the 4th larval instar (Figure 7). The size of the eyespots provides a convenient phenotypic marker for the onset and progression of DNA puff amplification, which is one of only two known examples of naturally occurring developmentally regulated site-specific intrachromosomal DNA (gene) amplification. Subsequently, pupae develop, and the amount of pigment filling their eyes can serve as a developmental marker for meiosis I and II in spermatogenesis (Figure 8) with its unique chromosome behaviors in these divisions.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66751/66751fig04.jpg" /> 
Figure 4: Mating of adult B. coprophila flies. The upper panel shows the three types of adult flies in the HoLo2 stock: female-producing mothers with Wavy wings (X&#39;WX adult females), male-producing mothers with straight wings (XX adult females), and males with straight wings (X0 adult males). Note the pointed ovipositor at the posterior end of the female flies and the hook-shaped clasper at the posterior end of the male flies. The lower panel shows a male and female mating, where the male clasper has grabbed the female&#39;s ovipositor. The sperm will be stored in the spermatheca of the female and will fertilize the eggs as they are discharged to the outside. The length of adults is 2.0 mm (males), 2.5 mm (females). <a href="https://www.jove.com/files/ftp_upload/66751/66751fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66751/66751fig05v2.jpg" /> 
Figure 5: B. coprophila embryos. The upper left panel is a view of embryos using standard light in a dissecting microscope; the nuclei in the syncytial cytoplasm appear as black dots. The panel on the right uses fluorescence microscopy to visualize the propidium iodide-stained nuclei of embryos. The embryos have an average length of 200 microns and an average width of 150 microns. The nuclei for the germ cells cluster at the posterior pole of the embryo as seen for cycles 6 (embryo tilted forward), and 7-9, after which they are interspersed with somatic nuclei. L chromosome elimination in the somatic lineage occurs in cleavage division 5 or 6; X chromosome elimination in the somatic lineage occurs in the 7th, 8th, or 9th cleavage division. Cellularization occurs during interphase of cycle 11. The table at the left shows the average length of time for each division cycle at 22 &#176;C. The table on the left and panel on the right are adapted with permission from de Saint Phalle and Sullivan35. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66751/66751fig06.jpg" /> 
Figure 6: B. coprophila embryos emerge as larvae. A cluster of embryos (blue thick arrow) is seen near the ovipositor of the adult female who died after egg-laying. A week after egg-laying, the embryos become young larvae, several of whom are indicated by the black arrows. The newly emerged larvae have a black jaw at the anterior end and a translucent body. They move on top of the agar surface and should not be confused with mold that does not move. The inset shows some mold, with a white filament (red arrow) and a black spore at its tip (green arrow), and is very slightly smaller than the newly emerged larvae. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66751/66751fig07.jpg" /> 
Figure 7: Eyespot stages of B. coprophila larvae. Eyespots form at the anterior of the larva, just behind the jaw, and are composed of pigment granules that increase in number. The eyespots are the anlage to the adult eye. The upper panel shows larvae that were visualized with a dissecting microscope; the lower panel is a magnified view of the eyespots using a phase contrast microscope to visualize a larva on a microscope slide with a drop of distilled water and a coverslip lightly floating on top. The nomenclature of the eyespot stages is according to Gabrusewycz-Garcia30 where the number of granules is counted in the longest row (e.g., 12) and the number of additional rows excluding the longest row is noted (e.g., 6 for eyespot stage 12x6). Initiation of site-specific DNA amplification in the salivary gland polytene chromosomes begins at eyespot stage 10x5 and is completed at 14x7 when there is a burst of transcription at the locus and expansion of the DNA puffs31. At the subsequent edge-eye/dropped jaw stage, the eyespot granules begin to merge, and they move laterally away from the midline; the length of the larval body shortens. Further, the DNA puffs condense at this stage. It takes about one day at 21 &#176;C to traverse each eyespot stage. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66751/66751fig08.jpg" /> 
Figure 8: Development of B. coprophila pupae. During pupation, all larval tissues histolyze except for the nervous system and are replaced by adult tissues that arise by cell divisions of the imaginal discs. The body color changes from white to tan to brown to black. Pigment gradually fills in the pupal eye; meiosis I and II occur in male pupae with eyes that are 1/4 to 1/2 filled with pigment36. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/66751/66751fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stock name</td>
			<td>Markers&#160;</td>
			<td>Fertility rate&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>7298</td>
			<td>Wavy (W) wing</td>
			<td>~75%</td>
			<td></td>
		</tr>
		<tr>
			<td>HoLo2</td>
			<td>Wavy (W) wing</td>
			<td>~90%</td>
			<td>derived from 7298</td>
		</tr>
		<tr>
			<td>W14</td>
			<td>Wavy (W) wing; White eyes</td>
			<td>~95%</td>
			<td>derived from HoLo2</td>
		</tr>
		<tr>
			<td>6980</td>
			<td>Wavy (W) wing; swollen (sw) veins</td>
			<td>~65%</td>
			<td></td>
		</tr>
		<tr>
			<td>91S</td>
			<td>weak Wavy (W) wing; petite (p) wings</td>
			<td>~50%</td>
			<td>the Wavy marker was introduced in a cross to rescue 91S</td>
		</tr>
	</tbody>
</table>
Table 2: Stocks of Bradysia (Sciara) coprophila. Table 1 of Gerbi6 lists these markers and others that are no longer extant. Five translocations (T1, T23, T29, T32, T70) at the centromere end of the X27 are summarized in Figure 8 of Gerbi6 but are no longer extant.
Supplemental File 1: HoLo2 (and 7298 and W14) crosses, 91S cross and rescue, 6980 cross, translocation crosses. <a href="https://www.jove.com/files/ftp_upload/66751/Supplemental Materials.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Author Spotlight: Exploring Bradysia coprophila's Unique Biology: A Guide to Laboratory Maintenance at JoVE.com

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

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Maintenance and Rearing of Bradysia (Sciara) coprophila Larvae in a Laboratory Setup

To set up the mating crosses, place the Bradysia coprophila adult male flies on a white fly pad. Use fine tipped forceps to gently pick up the fattest adult by its middle or hind leg. Then place the anesthetized adult female flies on a white fly pad.Using fine tipped forceps, pick up an adult female by its hind leg. One day after mating, push the wings of the anesthetized adult females into 2.2%agar in a Petri plate. Once all the female flies are impaled on the agar, gently squeeze the thorax with forceps.The female will lay a cluster of fertilized eggs within 30 to 60 minutes. Use a dissecting microscope to score the newly emerged larvae. Observe the inch worm-like motion of the newly emerged larvae as they crawl on the surface of the 2.2%agar.After removing the vial plug, examine the vial for the age and number of larvae. Pick up some straw between the second and third fingers or between the thumb and second fingers, and rotate the fingers against each other to deposit the appropriate amount of straw into the vial. On the same day that the larvae emerged, or up to two days later, sprinkle in a tiny bit of food that has been poured into a small bowl for easier access, and replace the gauze plug.For one week old larvae, add more food to just cover the surface of the agar and replace the gauze plug. For the two week old larvae, sprinkle in a generous amount of food to cover the agar surface and replace the gauze plug. Once the larvae attain the late fourth instar stage and begin to pupate, add a generous amount of food on the agar surface.Stop feeding once adults begin to emerge. Do not feed vials with adult flies and do not remove the gauze plug. If there is white mold in the vial, use 70%ethanol on a laboratory wipe to clean a long metal probe.Insert the clean probe into the vial to tap down the mold on the surface of the agar. Then clean the probe with a laboratory wipe dampened with 70%ethanol before storing or using it on another vial. Add only a small amount of food without disturbing the top of the agar, and replace the plug.