necessary articulation and musculature (Niwa et al., 2010; Clark-Hachtel & Tomoyasu, 2016; Prokop et al., 2017).
Paleozoic fossils indicate that, unlike modern insects, species of
Palaeodictyoptera and Geroptera bore wings on all three thoracic
segments (Figure 1; Kukalová, 1970).
Notably, in these ancient species, the pair of wings on the first
thoracic segment (T1) are markedly smaller than those found on the
second and third thoracic segment (T2 and T3). It has been suggested
that an expansion in size of T2 and T3, which led to a correlated
increase of musculature found in these segments, led to the loss of
the T1 wing and a corresponding increase in flight ability (Ross,
2017). In fact, many modern insects have evolved various mechanisms to couple hindwing and forewing movement, effectively reducing the separate wings to a single, functionally cohesive apparatus,
further increasing flight efficiency (Gullan & Cranston, 2014).
While modern insects do not develop wings on the first thoracic
segment, Tomoyasu et al. (2005) showed that RNAi of the Hox gene,
Sex combs reduced (Scr), in the flour beetle Tribolium castaneum leads
to the development of elytra in T1, demonstrating that Scr is an
important suppressor of wing development in T1. We suggest presenting students with pictures of Scr RNAi individuals (Figure 2),
asking them to describe the wing-specific role of Scr in the first thoracic segment in flour beetles, and finally allowing them to extrapolate this to (modern) insects in general.
Case vignette. We presented this vignette to our students during
a discussion on thoracic anatomy and wing evolution. We provided
them with the background information above, including figures
showing a reconstruction of a hypothetical Paleozoic insect (Figure 1;
Kukalová, 1970) and the phenotypes found by Tomoyasu et al. (2005).
We then asked them to consider and evaluate the following
(a) Sex combs reduced (Scr) is expressed in the first thoracic
(b) Scr is not expressed in the first thoracic segment.
(c) RNAi of Scr would lead to a loss of wings in the first thoracic segments of ancestral insects.
(d) The function of Scr has changed over the course of insect
(e) The expression pattern of Scr has changed over the course
of insect evolution.
In our course, this was the first scenario we used to invite students to apply their understanding of RNAi to a major morphological transition in insect evolution. Many students struggled to find the
correct answer, as it required understanding the “negative logic” of
RNAi. We used this as an opportunity to reiterate the basics of RNAi.
First, the students must understand that RNAi downregulates gene
expression and, thus, traits that appear (or disappear) in RNAi-treated individuals are regulated negatively (or positively) by the specific gene in question. Said another way, if a trait does not develop in
response to RNAi treatment, it can be concluded that the development of this trait is at least partially initiated by the expression of
the RNAi-downregulated gene. On the other hand, if a new trait
appears in RNAi individuals, this is strong evidence that the gene
in question normally acts to suppress the formation of the trait.
In the case of T. castaneum, it was observed that an ectopic
trait (wings at T1) developed in individuals in which Scr was
Figure 1. Reconstruction of Stenodyctia sp. (Palaeodictyoptera)
with wings on all three thoracic segments (courtesy of Kukalová,
Figure 2. Phenotypes of wild type and Scr-RNAi treated
individuals of the flour beetle Tribolium castaneum. Black
arrows indicate elytra; gray arrows indicate membranous
wings. T1–T3 indicate the location of each thoracic segment.
Courtesy of Y. Tomoyasu, University of Miami.