Among insects, Orthoptera (crickets, grasshoppers, and katydids) are characterized by two distinguishing features: their greatly enlarged hind legs (adapted for jumping) and modified forewings (adapted for sound production). The former trait is controlled by the homeotic gene Ultrabithorax (Ubx), which was shown to regulate the allometric leg growth in both orthopterans and hemipterans (Mahfooz et al., 2004, 2007; Khila et al., 2014). These studies also suggest that the transcriptional modification of Ubx expression may be a universal mechanism for the evolutionary diversification of insect hind legs. In contrast, very little is known regarding the genetic mechanisms that control the development of specific morphological structures on orthopteran forewings, which in turn, are critical for enabling sound production.

As a part of studies regarding the diversification of insect appendages, we have previously examined the embryonic functions of POU homeodomain gene nubbin (nub) in several insect species, including crickets (Hrycaj et al., 2008; Turchyn et al., 2011). The generated results show that nub can have a variety of roles during embryogenesis, including morphogenesis of mouthparts, joint formation in legs, and the establishment of a limbless abdomen. In adults, however, this gene is recognized for its function in growth and proximal–distal patterning of wings (Ng et al., 1995; Cifuentes and Garcia-Bellido, 1997; Neumann and Cohen, 1998). These observations led us to investigate the roles of nub in the development of wings in Acheta domesticus (house cricket). Our results show that its depletion (via RNAi) results in smaller wings, with each pair displaying an altered vein pattern. Moreover, there is a specific effect in male forewings that lost and/or modified their unique structures involved in sound production.

The above results suggest a dual role of nub in cricket wing development. First, there is a general function in regulating the size and overall shape of both fore- and hindwings. This is further corroborated by recent functional studies in a variety of insects, including Onthophagus taurus (scarab beetle), Blatella germanica (cockroach), and Oncopeltus fasciatus (milkweed bug), which all show that knockdowns of nub cause a reduction in adult wing size (Medved et al., 2015; Hu and Moczek, 2021; Fernandez-Nicolas et al., 2022). Second, nub has a lineage-specific role in developing the singing apparatus in male crickets. While preliminary in nature, this study provides a first step toward identifying a genetic network underlying the evolution of sound production in orthopterans.

Both Acheta nymphs and adults were raised at room temperature of 25°C and relative humidity of about 60% on a diet of fresh lettuce supplemented with dry cat food and water. Under these conditions, they underwent eight nymphal stages to become adults in about three months.

RNA isolation, cDNA synthesis, and cloning were conducted using methods previously outlined in established protocols (Li and Popadic, 2004; Hrycaj et al., 2008). The 387-bp long PCR fragment of Acheta nub, which includes the highly conserved 5’ POU homeodomain, a hypervariable linker region, and the 3’ homeodomain was isolated with the help of degenerated primers: EQFAKT (5´-GGAATTCGARCARTT YGCIA-ARAC-3´) and KEKRINP (5´-GCTCTAGAGGRTTIATICKYTTYCYTT-3´). It was then subsequently inserted into a pCR4-TOPO vector and confirmed through sequencing. This fragment was utilized not only to induce specific nub RNAi phenotypes in embryos (Turchyn et al., 2011), but also in our current analysis.

To investigate the functional importance of nub during postembryonic development, four to six μl of a 2.5 μg/μl nub double-stranded RNA (dsRNA) were injected into Acheta dorsal midline between the first and second abdominal (A1 and A2) segments using a Hamilton syringe with a pulled glass capillary needle. More specifically, multiple rounds of nub RNAi treatments were administered at different juvenile stages, starting from the 4^th nymphal stage (n=51) up to the 5^th (n=7) and 6^th (n=53). Each injection was followed by an additional dose of nub dsRNA every ten days until these nymphs emerged as adults or died. Among the total of 111 injected nymphs, 37 died, 12 displayed wild-type characteristics, and 62 exhibited specific nub RNAi phenotypes ranging in severity from moderate to strong. As described previously (Hrycaj et al., 2010), positive control was performed by using universal 18S (QuantumRNA™ 18S Internal Standards, Ambion, Austin, TX, USA) primers with competimer (3:7 ratio) in both wild type and nub-depleted Acheta. The PCR conditions were as follows: denaturation at 95°C for 2 minutes, followed by annealing at 58.3°C and extension at 72°C for 30 seconds, repeated for 33 cycles.

The classical research in Drosophila established that nub plays a key role in the proximal–distal patterning of wings (Ng et al., 1995; Cifuentes and Garcia-Bellido, 1997; Neumann and Cohen, 1998; Zirin and Mann, 2007). The complete knockdown results in a vestigial tissue with a small bump-like appearance, highlighting its essential function during wing primordia development. However, studies in coleopterans (beetles) and hemipterans (milkweed bugs) show that nub depletion via RNAi at later nymphal stages (after the primordia are formed) cause a moderate reduction in wing size (Medved et al., 2015; Hu and Moczek, 2021). These observations reveal that nub is continuously required during wing development. Early on, it is essential for formation of wing primordia, while its later expression is necessary for wings to reach their full size. As illustrated in Figures 1 and 2 , wings in Acheta nub RNAi adults are visibly smaller compared to wild type, consistent with the latter function. In addition, the patterning and melanization of veins are also affected (especially in hindwings). Aside from these general roles in both pairs of wings in both sexes, nub has a distinct function in Acheta males only ( Figures 3 , 4A ), where it regulates the development of sound amplification structures on forewings (mainly the harp, anal area, mirror, and chord). More specifically, in the harp, nub controls the formation of the three large S-shaped annulated veins as well as the overall size of this structure ( Figure 3C2 ). Similar function is observed in the adjacent region (anal area), where nub promotes the development of two L-shaped thick veins: A1 and Cu2 ( Figure 3C1 ). In the mirror, though, nub suppresses vein formation, resulting in a vein-free region ( Figure 3C3 ). Similarly, in the chord, it prevents the growth of cross-connections between the three longitudinal veins ( Figure 3C4 ). Therefore, nub can either activate (harp and anal area) or suppress (mirror and chord) vein and cross-connection development in the distinct regions of the forewings. In addition, it is involved in regulating the size and shape of these regions.

The above described alterations in the morphology of resonators are functionally significant, as Acheta nub RNAi males do not stridulate. Intriguingly, twenty years ago a similar silencing event was observed in natural populations of the Hawaiian field cricket Teleogryllus oceanicus (Zuk et al., 2006). In that instance, a mutant wing morph (flatwing) evolved spontaneously to render males silent as a protection against predatory parasitoid ( Figure 4B ). Extensive follow up research in Teleogryllus provided a wealth of knowledge regarding the evolution of “flatwing” males, including the identification of the gene doublesex (dsx) as potentially responsible for male silencing (Desutter-Grandcolas, 1997; Montealegre et al., 2011; Gu et al., 2012; Pascoal et al., 2014; Bailey et al., 2019; Zhang et al., 2021). As illustrated in Figure 4B , though, the path towards male cricket silencing in Teleogryllus is quite different from the present observations in Acheta. First, the mirror and the chord do not develop in flatwing males. Second, the plectrum (scraper) is absent in some populations (Kauai), but present in others (Oahu). Third, the forewings in these individuals display a trend toward feminization by which they acquire the overall vein patterning very similar to the one present in female crickets. Overall, the insights from Teleogryllus and Acheta are complementary, and provide a better understanding of the level of integration between different components involved in sound production. In Teleogryllus, it is the large perturbations of the system (the absence of the mirror and chord, and the change of the wing identity) that cause the loss of the function. On the other hand, changes in Acheta are more nuanced and encompass changes in the size and morphology of the resonators.

In nature, crickets display a surprisingly large variation in the design of their sound production apparatus, some of which is depicted in Figure 4C . For example, the size of the mirror can range from small (as in Gryllotalpa monaka) to large (as in Oecanthus fultoni). There are species that do not have a mirror at all (such as Riatina pilkena), while in others a harp is absent (as in Pholidoptera griseoaptera). These observations suggest that the formation of sound resonators is regulated by distinct gene regulatory networks (GRNs) that can be activated either independently or in a coordinated fashion. Studies in Teleogryllus point to dsx as the obvious candidate as the common regulator of such GRNs. Similarly, results in Acheta suggest that nub may be co-opted for a role in the development of distinct resonator morphologies. For both genes, it is necessary to extend functional studies to other species in order to determine whether their roles are general or lineage-specific. Moreover, as resonators are spatially precisely defined, their respective GRNs should have matching spatially localized expression patterns. For example, in scarab beetles, dsx is preferentially expressed in the horn imaginal disks in large males causing the subsequent differential enlargement of these appendages in adults (Kijimoto et al., 2012). Combining the functional analyses with the expression pattern studies of genes such as dsx and nub and identifying their downstream targets, may provide critical new insights into the genetic architecture of sound-producing structures in insects.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, HQ543076.

The manuscript presents research on animals that do not require ethical approval for their study.

NT: Writing – review & editing, Formal analysis, Investigation, Methodology, Validation, Visualization. AP: Writing – review & editing, Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft.