How to sex fruit flies

Drosophila melanogaster, otherwise known as the common fruit fly, is one of the oldest and most powerful model systems used in biology. Fruit flies are cheap to maintain, and have a shorter life cycle and higher fecundity than mammalian models. They also have extraordinary genetic tools with which to investigate many molecular and cellular questions. The diversity and strength of these genetic tools comes from the complex simplicity of Drosophila genetics. Unlike humans, fruit flies have only four chromosomes: three autosomes, denoted 2, 3, and 4, and two possible sex chromosomes, denoted X or Y.

Unlike mammals, however, it is not the absence or presence of the Y chromosome that determines gender. Rather, it is the ratio of female determinants on the X chromosome and male determinants on the autosomes. If there is only one copy of the X chromosome in the fly, the fly is male. If there are two copies, the fly is female. And while the Y chromosome does contain some genes important to spermatogenesis and sperm motility in adult male flies, it does not intrinsically determine gender. The X chromosome, however, encodes genes important to cellular function, and therefore, as you make manipulations on the X chromosome, it becomes important to determine whether you should use males or females.

Superficially, you can tell a male fly from a female fly by comparing the shape and color of their genitalia, as well as the absence or presence of sex combs on their legs. Adult male flies have dark, rounded, complex structures on the lower tip of their abdomen which comprise the genitalia (penis, lateral plate, genital arch). On the other hand, female flies have light, pointed genitalia comprised of very few visible structures. In addition to differences in the color and shape of genitalia, male flies also display a unique set of sex combs on the fourth segment of their front legs. These cells look like a row of thick bristles that appear as a dark mass on their legs and are used by male flies during copulation to grasp the female fly.

While the superficial distinctions between male and female adult flies can be easily seen under a microscope, the difference between male and female larvae is a bit more nuanced. Fruit fly larvae are little tubes of muscle, intestine, fat, heart, brain, and trachea all encased and protected by a sturdy, yet pliable, translucent cuticle. Some fruit fly biologists claim that female larvae are bigger than male larvae, but this distinction is not easily quantified and should not be relied on in the lab. Rather, the best way to determine larval sex is to take advantage of the translucence of the cuticle and look for the absence or presence of gonads. In male larvae, gonads are big, circular, translucent disc organs that can be seen through the cuticle of the lower abdominal segments. In female flies, these translucent discs are not present.

Here is an example of a pinned third instar larva. The head of the larva is denoted with a yellow star and an arrow points to the presence of a gonad. Indeed, it is a translucent, circular disc and we definitely have a male larva on our hands.

Here’s another view of a pinned larva. This time we see both the right and left gonad against the white trachea, proving again that it is a male. When the larva crawls, you’ll see these discs move along the body wall with every contraction but remain in their relative position.

Female larvae will not show the presence of a pronounced disc. Instead, you see predominantly fat bodies and intestines.

Sexing larvae, as it is called in Drosophila larvae, is a highly nuanced skill that takes both time and practice. To double check whether or not you are doing it correctly, you can test your prowess with a mini experiment. Take some larvae, determine their genders, then place each one in their own food vial and allow to eclose into adulthood. Determine the gender of the adult and compare it with your larval determination. Hopefully they match!

Do you have any tips or tricks to sexing larvae? Comment below!

Finding a partner is hard work. When faced with varied mates, we usually consider a range of traits: beauty, personality, wealth, aspirations, kindness, the list goes on. However, few of us would add our partners’ dietary habits to this judgment. But it turns out that, if you are a female fruit fly, you may need to rethink your “perfect partner” criteria and re-prioritize your partner’s diet.

In a recent study published in the journal Scientific Reports, my colleague Stuart Wigby and I showed the negative effects of male diet on female reproduction in fruit flies Drosophila melanogaster. We fed males a variety of diets with different proportions of protein and sugar for four days before males were allowed to mate with virgin females. After mating them for the first time with these males, females were allowed to produce offspring for a day. Next, these same females were mated for a second time with healthy males raised on normal food. After mating with the second males, females were allowed to produce offspring for another three days. Female fruit flies can produce more than 150 offspring in a day and, therefore, four days of offspring production (one day after the first mating + three after the second mating) is a reasonable period of time for assessing female reproduction.

The results are surprising. We found that the diet of the male that copulated with a virgin female can have long-lasting effects on female reproduction. Males that have eaten high protein foods reduced the production of offspring of females over the four days of the experiment even after females had mated again with the healthy male.

Somehow, the diet of female’s first male defines her potential to produce offspring.

But what does this effect mean? It is too early to tell since this is the first study to show this effect. However, we believe that the answer lies in the ejaculate of males. The ejaculate is a cocktail of sperm, sugar, vitamins, proteins and other molecules, and the ejaculates of the majority of species are, to a greater or lesser extent, made of these components. Thus, our results raise the potential for the effects of male diet on female reproduction to be observed elsewhere in animal kingdom, including in humans.

Of course, it is too soon to jump to such conclusions. But if confirmed in humans, these results would suggest that women should choose their first partner not by attractiveness or personality, but perhaps by the number fast food meals her potential partner has eaten lately.

While the list of criteria for finding the perfect match may have just gotten longer, one thing is certain: Choosing the right partner is a very tedious task.

Juliano Morimoto is a Brazilian PhD student at the University of Oxford.

Although both mammals and fruit flies produce XX females and XY males, their chromosomes achieve these ends using very different means. The sex-determining mechanisms in mammals and in insects such as Drosophila are very different. In mammals, the Y chromosome plays a pivotal role in determining the male sex. Thus, XO mammals are females, with ovaries, a uterus, and oviducts (but usually very few, if any, ova). In Drosophila, sex determination is achieved by a balance of female determinants on the X chromosome and male determinants on the autosomes. Normally, flies have either one or two X chromosomes and two sets of autosomes. If there is but one X chromosome in a diploid cell (1X:2A), the fly is male. If there are two X chromosomes in a diploid cell (2X:2A), the fly is female (Bridges 1921, 1925). Thus, XO Drosophila are sterile males. In flies, the Y chromosome is not involved in determining sex. Rather, it contains genes active in forming sperm in adults. Table 17.1 shows the different X-to-autosome ratios and the resulting sex.

In Drosophila, and in insects in general, one can observe gynandromorphs—animals in which certain regions of the body are male and other regions are female (Figure 17.15). This can happen when an X chromosome is lost from one embryonic nucleus. The cells descended from that cell, instead of being XX (female), are XO (male). Because there are no sex hormones in insects to modulate such events, each cell makes its own sexual “decision.” The XO cells display male characteristics, whereas the XX cells display female traits. This situation provides a beautiful example of the association between insect X chromosomes and sex.

Any theory of Drosophila sex determination must explain how the X-to-autosome (X:A) ratio is read and how this information is transmitted to the genes controlling the male or female phenotypes. Although we do not yet know the intimate mechanisms by which the X:A ratio is made known to the cells, research in the past two decades has revolutionized our view of Drosophila sex determination. Much of this research has focused on the identification and analysis of the genes that are necessary for sexual differentiation and the placement of those genes in a developmental sequence. Several genes with roles in sex determination have been found. Loss-of-function mutations in most of these genes—Sex-lethal (Sxl), transformer (tra), and transformer-2 (tra2)—transform XX individuals into males. Such mutations have no effect on sex determination in XY males. Homozygosity of the intersex (ix) gene causes XX flies to develop an intersex phenotype having portions of male and female tissue in the same organ. The doublesex (dsx) gene is important for the sexual differentiation of both sexes. If dsx is absent, both XX and XY flies turn into intersexes (Baker and Ridge 1980; Belote et al. 1985a). The positioning of these genes in a developmental pathway is based on (1) the interpretation of genetic crosses resulting in flies bearing two or more of these mutations and (2) the determination of what happens when there is a complete absence of the products of one of these genes. Such studies have generated the model of the regulatory cascade seen in Figure 17.16.

The first phase of Drosophila sex determination involves reading the X:A ratio. What elements on the X chromosome are “counted,” and how is this information used? It appears that high values of the X:A ratio are responsible for activating the feminizing switch gene Sex-lethal (Sxl). In XY cells, Sxl remains inactive during the early stages of development (Cline 1983; Salz et al. 1987). In XX Drosophila, Sxl is activated during the first 2 hours after fertilization, and this gene transcribes a particular embryonic type of Sxl mRNA that is found for only about 2 hours more (Salz et al. 1989). Once activated, the Sxl gene remains active because its protein product is able to bind to and activate its own promoter (Sánchez and Nöthiger 1983).

This female-specific activation of Sxl is thought to be stimulated by “numerator proteins” encoded by the X chromosome. These constitute the X part of the X:A ratio. Cline (1988) has demonstrated that these numerator proteins include Sisterless-a and Sisterless-b. These proteins bind to the “early” promoter of the Sxl gene to promote its transcription shortly after fertilization.

The “denominator proteins” are autosomally encoded proteins such as Deadpan and Extramacrochaetae. These proteins block the binding or activity of the numerator proteins (Van Doren et al. 1991; Younger-Shepherd et al. 1992). The denominator proteins may actually be able to form inactive heterodimers with the numerator proteins (Figure 17.17). It appears, then, that the X:A ratio is measured by competition between X-encoded activators and autosomally encoded repressors of the promoter of the Sxl gene.

Shortly after Sxl transcription has taken place, a second, “late” promoter on the Sex-lethal gene is activated, and the gene is now transcribed in both males and females. However, analysis of the cDNA from Sxl mRNA shows that the Sxl mRNA of males differs from sxl mRNA of females (Bell et al. 1988). This difference is the result of differential RNA processing. Moreover, the Sxl protein appears to bind to its own mRNA precursor to splice it in the female manner. Since males do not have any available Sxl protein when the late promoter is activated, their new Sxl transcripts are processed in the male manner (Keyes et al. 1992). The male Sxl mRNA is nonfunctional. While the female-specific Sxl message encodes a protein of 354 amino acids, the male-specific Sxl transcript contains a translation termination codon (UGA) after amino acid 48. The differential RNA processing that puts this termination codon into the male-specific mRNA is shown in Figures 17.17B and 17.18. In males, the nuclear transcript is spliced in a manner that yields eight exons, and the termination codon is within exon 3. In females, RNA processing yields only seven exons, and the male-specific exon 3 is now spliced out as a large intron. Thus, the female-specific mRNA lacks the termination codon.

The protein made by the female-specific Sxl transcript contains two regions that are important for binding to RNA. These regions are similar to regions found in nuclear RNA-binding proteins. Bell and colleagues (1988) have shown that there are two targets for the female-specific Sxl protein. One of these targets is the pre-mRNA of Sxl itself. The second is the pre-mRNA of the next gene on the pathway, transformer.

17.9 Other sex determination proteins inDrosophila. Sex-lethal does not work alone, but in concert with several other proteins whose presence is essential for its function. Many of these proteins have other roles during development. http://www.devbio.com/chap17/link1709.shtml

The Sxl gene regulates somatic sex determination by controlling the processing of the transformer (tra) gene transcript. The tra message is alternatively spliced to create a female-specific mRNA as well as a nonspecific mRNA that is found in both females and males. Like the male sxl message, the nonspecific tra mRNA contains a termination codon early in the message, making the protein nonfunctional (Boggs et al. 1987). In tra, the second exon of the nonspecific mRNA has the termination codon. This exon is not utilized in the female-specific message (see Figure 17.18). How is it that the females make a different transcript than the males? The female-specific protein from the Sxl gene activates a female-specific 3´ splice site in the transformer pre-mRNA, causing it to be processed in a way that splices out the second exon. To do this, the Sxl protein blocks the binding of splicing factor U2AF to the nonspecific splice site by specifically binding to the polypyrimidine tract adjacent to it (Figure 17.19; Handa et al. 1999). This causes U2AF to bind to the lower-affinity (female-specific) 3´ splice site and generate a female-specific mRNA (Valcárcel et al. 1993). The female-specific tra product acts in concert with the product of the transformer-2 (tra2) gene to help generate the female phenotype.

The doublesex (dsx) gene is active in both males and females, but its primary transcript is processed in a sex-specific manner (Baker et al. 1987). This alternative RNA processing appears to be the result of the action of the transformer gene products on the dsx gene (see Figure 5.31). If the Tra2 and female-specific Tra proteins are both present, the dsx transcript is processed in a female-specific manner (Ryner and Baker 1991). The female splicing pattern produces a female-specific protein that activates female-specific genes (such as those of the yolk proteins) and inhibits male development. As discussed in Chapter 5, if functional Tra is not produced, a male-specific transcript of dsx is made. This transcript encodes an active protein that inhibits female traits and promotes male traits.

The functions of the Doublesex proteins can be seen in the formation of the Drosophila genitalia. Male and female genitalia in Drosophila are derived from separate cell populations. In male (XY) flies, the female primordium is repressed, and the male primordium differentiates into the adult genital structures. In female (XX) flies, the male primordium is repressed, and the female primordium differentiates. If the dsx gene is absent (and thus neither transcript is made), both the male and the female primordia develop, and intersexual genitalia are produced. Similarly, in the fat body of Drosophila, activation of the genes for egg yolk production is stimulated by the female Dsx protein and is inhibited by the male Dsx protein (Schüpbach et al. 1978; Coschigano and Wensink 1993; Jursnich and Burtis 1993).

According to this model (Baker 1989), the result of the sex determination cascade comes down to what type of mRNA is going to be processed from the dsx transcript. If the X:A ratio is 1, then Sxl makes a female-specific splicing factor that causes the tra gene transcript to be spliced in a female-specific manner. This female-specific protein interacts with the Tra2 splicing factor to cause the doublesex pre-mRNA to be spliced in a female-specific manner. If the doublesex transcript is not acted on in this way, it will be processed in a “default” manner to make the male-specific message.

17.10 Conservation of sex-determining genes. While the pathways of sex determination appear to differ between humans and flies, the discovery of a human gene similar to doublesex suggests that there may be a common end point to the two pathways. http://www.devbio.com/chap17/link1710.shtml

17.11 Hermaphrodites. In C. elegans and many other invertebrates, hermaphroditism is the general rule. These animals are born with both ovaries and testes. In some fishes, sequential hermaphroditism is seen, with an individual fish being female some seasons and male in others. In humans, hermaphrodes are rare and usually sterile. http://www.devbio.com/chap17/link1711.shtml