Sexual determination and differentiation work in myriad ways across the animal kingdom. In vertebrates, like mammals and fish, sexual determination leads to the development of either ovaries or testis. These organs then secrete hormones that go on to govern the sexual development of the rest of the organism’s body. Insects are a completely different beast.
“The way that insects develop male versus female characteristics is using different kinds of RNA splicing and processing,” said Judy Wexler, who graduated from UC Davis in 2018 with a Ph.D. in Population Biology. “The same genes are used to direct male development or female development; it’s just how those genes and RNA are processed that will tell a cell, ‘You’re a male cell, or you’re a female cell.’”
According to Wexler, insects are the only known animal to perform sexual determination and differentiation via this toggle-like system, which makes use of the biological phenomenon known as “alternative splicing.” Even their closest arthropod relatives, crustaceans, don’t use this sexual development strategy. So it begs the question, how old is the splicing-based mode of sexual differentiation?
“It really is radically different from the way other animal groups manage their sexual differentiation,” said Professor Artyom Kopp, Department of Evolution and Ecology and director of the Center for Population Biology. “The canonical insect sex pathway is in every developmental biology textbook, but nobody has ever looked at how that pathway came about.”
In a study appearing in eLife, Wexler and her colleagues, including Kopp, trace this splicing-based sexual development strategy to hemimetabolous insect orders, or those that develop from larvae to adults without a pupal stage. These types of insects are more ancient than holometabolous insects, which have a pupal stage.
“The main finding from all of the insects we studied is that together, they represent an intermediate state between what we know to be true in crustaceans and then what we know to be true about sexual development in holometabolous insects,” said Wexler. “These three hemimetabolous insects we studied gave us a snapshot of what the evolution of that sexual differentiation pathway could have looked like.”
A pupil without the pupal
Wexler and her colleagues studied three hemimetabolous insect species: the kissing bug (Rhodnius prolixus), the louse (Pediculus humanus) and the German cockroach (Blattella germanica). These insects, like their holometabolous relatives, use a gene regulatory pathway known as transformer (tra)-doublesex (dsx) for sexual determination and differentiation.
“Transformer (tra) and doublesex (dsx) are genes that are present in both males and females,” said Wexler. Tra has two separate versions, called isoforms, which determine the insect’s sex. Dsx, which also has two isoforms, then goes on to influence sexual differentiation, or the development of each sex’s specific physical characteristics. “They have different effects on development depending on the isoforms present and tra controls dsx splicing,” said Wexler.
To look at the underlying genetics of sexual determination and differentiation of hemimetabolous insects, the team extracted RNA from each insect species and used a combination of techniques to characterize their tra and dsx splicing patterns. Like their holometabolous relatives, the German cockroaches boasted two isoforms of the dsx gene. But something was missing in their tra-dsx pathway.
“We were expecting that if we saw male and female forms of dsx, we would also see male and female forms of tra, but we failed to detect any different forms of tra that were male-specific or female-specific,” said Wexler.
“That was mystery from the paper,” she added.
Knockdown genes and sex splicing
Wexler and her colleagues made this discovery in the lab by using a technique called RNA interference (RNAi), which allows them to knockdown genes. The functional experiments of the study were performed on the German cockroach.
“We were really puzzled because we could see that just like in holometabolous insects, tra is controlling dsx splicing,” said Wexler. But “we don’t know how it controls dsx splicing because we could not find a female-specific form of tra.”
In the lab, Wexler and her colleagues interrupted German cockroach sexual development through the RNAi technique.
“We separated them by sex and then we injected males and females with RNAi for either tra or dsx,” said Wexler. “With the females that we injected with RNAi targeting tra, when these females molted into adults, we were like, ‘Oh my god, they look like male cockroaches.’”
The team also found that female cockroaches injected with tra produced broods of solely male cockroaches.
“We have strong reason to believe that when we interfere with tra, all the females die, so it’s female-specific lethal,” said Wexler.
Working out experimental bugs
While no functional experiments were performed in the kissing bug and louse, the team found that splicing patterns seen in the louse were similar to the German cockroach, while the splicing patterns seen in the kissing bug were similar to holometabolous insects.
Wexler, who started a postdoctoral position at the Hebrew University of Jerusalem this month, is excited to investigate the origins of this sexual splicing strategy further.
“What I really want to do when I start a lab is to try and validate some of the RNAi findings using CRISPR,” said Wexler.
But experimenting with CRISPR will prove difficult, as German cockroaches seem to be resistant to the experimental process.
“When they’re using CRISPR, most insect biologists take an insect egg and they inject the CRISPR reagents in the egg and then the egg will develop and some of its cells will have mutations,” said Wexler. “German cockroaches don’t lay eggs and walk away from them.”
Instead, females carry their eggs in an egg case. “If you remove the case from the female, the eggs die,” said Wexler.
“Nobody to my knowledge has done CRISPR in a German cockroach, but I really want to try,” she added.