downregulated. Thus, it appears that the expression of this gene
causes the suppression of wings at T1, a conclusion that is consistent with the wingless T1 phenotype of all modern insects.
At this point, students were directed to transfer this knowledge
to a reconstruction of a Paleozoic insect. These insects bore wings
on the first thoracic segment, in contrast to extant taxa, but similar
to flour beetles exposed to RNAi-mediated downregulation of Scr.
Consequently, students might predict that Scr, which has been
shown to inhibit the development of wings on the first thoracic segment, is not expressed in the first thoracic segment of a Paleozoic
insect with three pairs of wings. An alternative hypothesis is that
the expression pattern of Scr has remained static over insect evolution,
but that the gene evolved a novel function during the Paleozoic,
namely the ability to suppress “wing-building genes,” which ushered
in the transition to winglessness within its expression domain.
Expression patterning images, especially from phylogenetically
basal insect taxa (e.g., through in situ hybridization) can be used
as evidence allowing students to discuss the relative merits of each
Case Vignette 3: Wing Modifications in Diptera &
Background. As outlined above, most insects develop a pair of wings
on both T2 and T3. However, in several insect orders, the forewings and hindwings are dimorphic and thus can be used in a novel
For example, Diptera fully develop only their forewings, while the
hindwings develop into halteres, an important balancing organ for
flies (Grimaldi & Engel, 2005). Through knockdown and knockout
mutations, it has long been known that the expression of the Hox
gene, Ultrabithorax (Ubx), gives identity to the third thoracic segment
and its appendages (Lewis, 1978). Consequently, Ubx promotes haltere development in flies, and reduced expression of Ubx (either
through mutations or RNAi) leads to a transformation of the halteres
into a second pair of forewings, a phenotype that lends the gene its
name. On the other hand, ectopic expression of Ubx in the second
thoracic segment has been shown to give T2 (and its appendages) a
T3 identity (Heffer & Pick, 2013). Thus, flies with such mutations
develop a second pair of halteres in place of their forewings. Thus,
Ubx has been shown to be an important regulator of the morphological forewing and hindwing dimorphism found in flies.
Case vignette. We presented our students with this case vignette
during a discussion of the orders Diptera and Coleoptera. In the
latter, forewings evolved into elytra, while the hindwings retained
the flight function, in contrast to dipterans (Gullan & Cranston,
2014). Using the background presented above, we asked the students to predict the effect of Ubx RNAi on beetles like T. castaneum
(Tomoyasu et al., 2005) or the charismatic dung beetle Onthophagus
(Wasik et al., 2010). As in flies, Ubx is expressed in the third but not
in the second thoracic segment in beetles and thus gives the third
thoracic segment and associated appendages their unique identity.
We presented the students with wild-type pictures of the respective
species and used these to compare and contrast forewing and
hindwing morphology in beetles and flies. After this discussion, we
asked them to predict the phenotype they would expect in Ubx
As in all insects, Ubx serves to regulate T3 identity in beetles and
therefore initiates gene expression associated with building the hind
flight wings in the group. Thus, just as in Drosophila, Ubx RNAi in
Tribolium transforms the third thoracic segment into a second tho-
racic segment. In contrast to flies, this causes the hindwing to
develop into a second pair of heavily sclerotized elytra in beetles
(Tomoyasu et al., 2005).
We further asked the students to predict how ectopic expression
of Ubx in T. castaneum might affect forewing morphology (under
such conditions, elytra are transformed into hindwings). We used
figures from Tomoyasu et al. (2005) to show the students whether
their predictions were supported by experimental evidence.
Case Vignette 4: Evolution of Leg Morphology
Background. In addition to determining the identity of the third thoracic segment, Ubx has also been co-opted as an important regulator
of relative leg length in a variety of insect species (Heffer & Pick,
2013), including grasshoppers (Orthopera: Gryllus spp. and Acheta
spp.), praying mantises (Mantodea: Tenodera spp.), and cockroaches
(“Blattodea”: Periplaneta spp.). Here, specific regions of the hind leg
experience high expression levels of Ubx during development, which
correspond to an elongation of those regions (Heffer & Pick, 2013).
For example, many Orthoptera develop a greatly enlarged femur
and tibia of the hind legs, adapted for jumping. Through in situ
hybridization, it has been shown that these exaggerated regions of
the hind leg show a region-specific increase in Ubx expression during
development (Heffer & Pick, 2013).
Case vignette. This vignette was presented during our discussion
on specific insect orders as outlined above. Here, after introduction
of Orthoptera, “Blattodea,” and Mantodea, we presented the students with preserved specimens of these groups and asked them to
identify the correct order. Afterward, the students were asked to
focus on describing the relative contribution of each hind leg segment (coxa, femur, tibia, tarsi) to overall leg length, when compared
with the foreleg. This also allowed the students to revisit basic insect
leg anatomy and was followed by a discussion on the fitness advantages of hind-leg elongation.
At this point, we presented the students with the background
above and asked them to predict the answers to these questions:
(a) Which of the hind-leg segments have increased Ultrabithorax
(Ubx) expression during insect development?
(b) If an Ubx RNAi experiment was conducted in each of the
species, which of the hind-leg segments would be affected?
(c) Looking at isolated in situ hybridization images from an
Orthopteran embryo, which is the fore-leg and which is
The students were then shown pictures of in situ expression and
asked whether their predictions were confirmed by actual research
(these images can be found in Mahfooz et al., 2004, 2007).
To remind students of the global influence of Ubx in the identity
within T3, and to tie in this vignette with previous material, additional questions might include a hypothetical knockdown experiment and predictions of the collective effects on leg and wing
development in Orthoptera. Forewings and hindwings in Gryllus
are overall very similar in appearance, and therefore, while we would
expect to observe extreme shortening of the T3 femur and tibia in
response to Ubx RNAi, we would expect only minimal effects on