Activation of EGFR signaling by Tc-Vein and Tc-Spitz regulates the metamorphic transition in the red flour beetle Tribolium castaneum

Animal development relies on a sequence of specific stages that allow the formation of adult structures with a determined size. In general, juvenile stages are dedicated mainly to growth, whereas last stages are devoted predominantly to the maturation of adult structures. In holometabolous insects, metamorphosis marks the end of the growth period as the animals stops feeding and initiate the final differentiation of the tissues. This transition is controlled by the steroid hormone ecdysone produced in the prothoracic gland. In Drosophila melanogaster different signals have been shown to regulate the production of ecdysone, such as PTTH/Torso, TGFß and Egfr signaling. However, to which extent the roles of these signals are conserved remains unknown. Here, we study the role of Egfr signaling in post-embryonic development of the basal holometabolous beetle Tribolium castaneum. We show that Tc-Egfr and Tc-pointed are required to induced a proper larval-pupal transition through the control of the expression of ecdysone biosynthetic genes. Furthermore, we identified an additional Tc-Egfr ligand in the Tribolium genome, the neuregulin-like protein Tc-Vein (Tc-Vn), which contributes to induce larval-pupal transition together with Tc-Spitz (Tc-Spi). Interestingly, we found that in addition to the redundant role in the control of pupa formation, each ligand possesses different functions in organ morphogenesis. Whereas Tc-Spi acts as the main ligand in urogomphi and gin traps, Tc-Vn is required in wings and elytra. Altogether, our findings show that in Tribolium, post-embryonic Tc-Egfr signaling activation depends on the presence of two ligands and that its role in metamorphic transition is conserved in holometabolous insects.

www.nature.com/scientificreports/ up-regulation of the Halloween genes at the appropriate developmental time to trigger metamorphosis 2 , TGFß/ activin signaling regulates insulin and PTTH/Torso pathways in the PG by controlling the expression of their respective receptors 12 . In addition to these pathways, we have recently shown that the activation of the Epidermal Growth Factor Receptor (Egfr) signaling pathway in PG cells is also critical for ecdysone production at the metamorphic transition 20 . Thus, Egfr pathway activation controls Halloween gene expression and ecdysone vesicle secretion during the last larval stage. Importantly, it has been shown that the control of the metamorphic transition by TGFß/activin, Insulin receptor (InR), and PTTH/Torso pathways is conserved in other non-dipteran holometabolous insects [21][22][23][24][25][26][27][28] . However, the grade of conservation of the Egfr signaling pathway in this process has not been well established.
In Drosophila, Egfr is activated by four ligands with a predicted Egf-like motif: the TGF-like proteins Gurken (Grk), Spitz (Spi) and Keren (krn), and the neuregulin-like protein Vein (Vn). The specific expression and activity of these ligands in different tissues appear to be responsible for different levels of EGF signaling activation in particular developmental contexts 29,30 . For example, Krn acts redundantly with Spi in the embryo, during eye development and in adult gut homeostasis [31][32][33] , whereas Grk is specific of the germline, where it acts to establish egg polarity 34,35 . In contrast, Vn acts as the main ligand in the wing, in muscle attachment sites, during the air sac primordium development and in the patterning of the distal leg region [36][37][38][39][40] . Once Egfr is activated by any of its ligands, the signal is relayed through the sequential activation of the MAPK/ERK kinase pathway to the nucleus, where is finally mediated by the transcription factor Pointed (Pnt) 41,42 .
In the present work, we aim to study the role of Egfr signaling pathway in the control of the metamorphic transition in the more basal holometabolous insect, the red flour beetle Tribolium castaneum, which diverged from Drosophila ∼ 250 million years ago. Despite the early divergence between both species, the role of this pathway in the control of several developmental processes has been revealed to be conserved. For example, it induces the encapsulation of the oocyte by the somatic follicle cell layer and establishes the polarity of the egg chambers and the D-V axis of the embryo during oogenesis 43 . It also controls the formation and patterning of the legs and the proper development of the abdomen and Malpighian tubules during embryogenesis [43][44][45] , and regulates distal development of most appendages such as leg, antenna, maxilla and labium, and promotes axis elongation of the mandibles [46][47][48] . Despite this functional conservation, a remarkable difference between Drosophila and Tribolium is found in the number of Egfr ligands identified, for only a single TGF-EGF ligand, Tc-Spi, has been found in the beetle 44 .
In here, we confirm that Tc-Egfr signaling is required for a normal ecdysone-dependent transition from larval to pupal stages in Tribolium. We found that inactivation of Tc-Egfr signaling in the last larval stage results in the arrest of larval development at the larval-pupal transition, with arrested larvae presenting reduced levels of the Halloween gene Tc-phm, as well as of the ecdysone-dependent transcription factors Tc-Hr3, Tc-E75 and Tc-Broad Complex (Tc-Br-C). Importantly, we provided evidence of the existence of an additional Tc-Egfr ligand in Tribolium, the neuregulin-like protein Tc-Vein (Tc-Vn). We show that Tc-Vn and Tc-Spi act redundantly in the control of larval-pupal transition, while functioning separately during pupal morphogenesis. Taken together, our results strongly suggest that Egfr signaling pathway plays a conserved central role in the control of the ecdysonedependent larval-pupal transition and during the morphogenesis of the pupa in holometabolous insects.

Results
Egfr signaling is required for larval-pupal transition in Tribolium. As a first step towards the characterization of Egfr signaling in post-embryonic Tribolium, we measured mRNA levels of Tc-Egfr and Tc-pnt by RT-qPCR in staged penultimate (L6) and last (L7) instar larvae. Expression of Tc-Egfr and Tc-pnt was low at the onset of both instars, and then steadily increased to reach the maximal level at the final part of each instar, which suggests a role of Egfr signaling during stage transitions (Fig. 1A). To examine this possibility, we analyzed the functions of both factors depleting Tc-Egfr and Tc-pnt by injecting dsRNAs for each transcript in L6 instar larvae (Tc-Egfr RNAi and Tc-pnt RNAi animals). Specimens injected with dsMock were used as negative controls (Control animals). All Tc-Egfr RNAi and Tc-pnt RNAi L6 larvae molted to normal L7 larvae but then failed to pupate at the ensuing molt, as did Control larvae. Instead, they arrested development during the larval-pupal transition ( Fig. 1B and S1 Table). Remarkably, removing the larval cuticle of these animals revealed a larval-like morphology ( Fig. 1B and S1 Table). To discard a possible effect in the nature of the affected transition in the arrested animals, from larva to pupa, we analyzed the expression levels of the juvenile hormone transducer Kruppel homolog 1 Tc-Kr-h1 49 and the metamorphosis-triggering factor Tc-E93 50 . As Fig. 1C shows, arrested Tc-Egfr RNAi and Tcpnt RNAi animals showed the proper downregulation of Tc-Kr-h1 and upregulation of Tc-E93 that is characteristic of larvae being in the last larval instar, thus indicating that lack of Egfr signaling did not affect the nature of the larval-pupal transition (Fig. 1C).
Since Tc-Egfr and Tc-pnt were also expressed in L6, we studied whether Egfr signaling was also required in earlier larval-larval transitions. To this aim, we injected dsRNA of Tc-Egfr in antepenultimate L5 larvae. Under these conditions, all L5-Tc-Egfr RNAi larvae developed normally and underwent two successive molts until reaching L7 (S2 Table), when larvae arrested development before pupation confirming that Egfr signaling is only required for the last larval transition.
Since the larval-pupal transition is ecdysone-dependent, the phenotype presented by Tc-Egfr RNAi and Tcpnt RNAi larvae is consistent with an ecdysone-signaling deficiency. To assess this hypothesis, we injected the active form 20E into Egfr-depleted larvae. Ectopic addition of 20E partially rescued the developmental arrest phenotype as animals underwent pupation although showing morphological defects in elytra, wings and legs, probably due to the pleiotropic effect of the Egfr signaling ( Fig. 1B and S1 Table). To confirm the action of Egfr pathway on ecdysone biosynthesis, we measured mRNA expression levels of the direct ecdysone-dependent genes Tc-Hr3, Tc-E75 and Tc-Br-C in late L7 larvae, which are commonly used as proxies for ecdysone levels 17 www.nature.com/scientificreports/ shows, Tc-Egfr RNAi and Tc-pnt RNAi larvae presented significantly reduced expression levels of these genes compared to Controls. Consistently, the expression of the Halloween gene phm was also decreased in Tc-Egfr RNAi and Tcpnt RNAi larvae (Fig. 1E). However, the expression of three other Halloween genes, Tc-dib, Tc-spo and Tc-shd, was not affected by the absence of Tc-Egfr (Fig. 1E), indicating that the downregulation of Tc-phm is specific and not due to a general transcriptional effect. Interestingly, in addition to the transcriptional regulation of Tc-phm, the gene expression levels of Tc-dib and Tc-spo were also downregulated in Tc-pnt RNAi larvae compared to Controls (Fig. 1E), indicating a stronger inactivation of Tc-Egfr signalling upon depletion of this transcription factor. Taken together, these results indicate that Tc-Egfr signaling is specifically required for a proper ecdysone-dependent larval-pupal transition in Tribolium.

Identification of the Tc-Egfr ligand Tc-Vein in
Tribolium. The next question was to determine which EGF ligand was responsible for the activation of the Tc-Egfr pathway during the larval-pupal transition. As stated before, the TGFα-like protein Tc-Spi is the only Tc-Egfr ligand identified in Tribolium 44 . Expression analysis of Tc-spi during the last two larval instars revealed that it is expressed in a similar pattern to Tc-Egfr ( Fig. 2A), which suggests that Tc-Spi might act as the Tc-Egfr ligand during the transition. We studied this possibility by injecting dsTc-spi into L6 larvae (Tc-spi RNAi animals). Unexpectedly, and in contrast to Tc-Egfr RNAi and Tc-pnt RNAi larvae, all L7-Tc-spi RNAi larvae molted to L7 and then to pupa on a normal schedule ( Fig. 2B and S3 Table), suggesting the occurrence of different Tc-Egfr ligands during the post-embryonic development of Tribolium.
In Drosophila four different Egf ligands have been identified: Dm-Grk, Dm-Ker and Dm-Spi are TGF-like proteins, whereas Dm-Vn belongs to the neuregulin-like family 29 . Tc-Spi is highly similar to the three Drosophila genes Dm-Grk, Dm-Spi and Dm-Ker being the only TGF-like ligand that activates Egfr in Tribolium 31,53 . In contrast, no neuregulin-like protein has been identified in Tribolium. To investigate the presence of a neuregulin-like Ventral views of a Control pupa, Tc-Egfr RNAi and Tc-pnt RNAi larvae with and without cuticle, arrested at the larvalpupal transition and Tc-Egfr RNAi larvae after injection of 20E. Scale bar represents 0.5 mm. (C-E) Transcript levels of Tc-Kr-h1 and Tc-E93 at the indicated stages (C), and Tc-Hr3, Tc-E75 and TcBr-C (D) and biosynthetic ecdysone genes Tc-phm, Tc-dib, Tc-spok and Tc-shd (E) measured by qRT-PCR in 5-day-old L7 Control, Tc-Egfr RNAi , and Tc-pnt RNAi larvae. Transcript abundance values were normalized against the Tc-Rpl32 transcript. Average values of three independent datasets are shown with standard errors (n = 6). Asterisks indicate differences statistically significant at *p < 0.05 and **p < 0.001 (t-test).  3B and S1 Fig). The EGF-like domain is 47 amino acids long and presents the six invariant cysteines and highly conserved glycine and arginine residues characteristic of the motif ( Fig. 3C and S1 Fig. 55,56 ). Finally, Tc-Vn contains a 24 amino acid long PEST region, characteristic of proteins with short half-lives, that localizes between the EGF-like and neuregulin Ig-like domains ( Fig. 3A and S1 Fig).
Phylogenetic analysis of Vn protein sequences showed that Tc-Vn grouped with others coleopteran sequences, confirming that Tc-Vn belongs to neuregulin Ig-like family ( Fig. 4 and S4 Table). Altogether, these data confirmed that Tribolium possesses two Egf ligands, the TGF-like Spitz and the neuregulin-like Vein.

Tc-Vn and Tc-Spi have redundant functions in larval-pupal transition.
Since Tc-Vn is a newly identified protein, we wanted to determine the role of this ligand in Tribolium. Tc-Egfr signaling has been already involved in the regulation of key processes in embryonic, metamorphic and adult stages [43][44][45][46][47][48] . Thus, parental depletion of Tc-Spi, Tc-Egfr, and Tc-Pnt severely reduced egg production, and also resulted in embryos with shorter appendages and problems in the development of thoracic and abdominal segments 43,44 . To study the function of Tc-Vn in adult and embryonic stages, we injected dsTc-vn into adult females (Tc-vn RNAi animals) and found that, in contrast to what is observed in Tc-Egfr-depleted animals, their ovaries developed properly, and eggs were laid normally (S2A- D Fig). Likewise, the resulting embryos developed as normal and eclosed on a regular schedule (data not shown). Consistent with this finding, whereas the relative expression of Tc-spi was strongly detected in ovaries and embryos the levels of Tc-vn were almost absent, supporting the idea that Tc-vn is dispensable for Tc-Egfr activation in adult and embryonic stages (S2E Fig).
To characterize Tc-Vn during post embryonic stages we next analyzed its expression pattern during the last two larval instars. As Fig. 5A shows, Tc-vn is up-regulated at the first day of L6 and its expression is maintained until the beginning of L7 when it declined. Then, Tc-vn is up-regulated again to reach the highest levels in the prepupal stage similarly to Tc-spi expression. To ascertain the role of the ligand, we next injected dsTc-vn in L6 larvae. Similar to the Tc-spi RNAi phenotype, L6-Tc-vn RNAi animals molted to normal L7 larvae and then to pupae although with some morphological defects ( Fig. 5B and S5 Table and Fig. 6). These results demonstrate that Tc-Vn is able to activate the Egfr signaling pathway during the larval-pupal transition.
The fact that the depletion of neither Tc-vn nor Tc-spi prevents larval-pupal transition raises the possibility that both ligands might redundantly activate the Egfr signaling pathway at this stage of development. To address this question, we interfered Tc-vn and Tc-spi simultaneously in penultimate L6 larvae (Tc-vn RNAi + Tc-spi RNAi animals). Interestingly, all Tc-vn RNAi + Tc-spi RNAi animals molted to L7 but then arrested development at the end of the last larval stage, as Tc-Egfr or Tc-pnt larvae (Fig. 5B and S5 Table). This observation was further confirmed by the analysis of Tc-HR3, Tc-E75, Tc-Br-C mRNA levels, which were strongly reduced in Tc-vn RNAi + Tc-spi RNAi larvae compared to the Control (Fig. 5C). Likewise, the expression of Tc-E93 was not significantly affected indicating that depletion of both ligands does not influence to nature of molt as when the Tc-Egf receptor was depleted (Figs. 1C, 5D). Furthermore, the expression of Tc-phm was also decreased in double knockdown animals whereas the expression levels of Tc-dib, Tc-spok and Tc-shd were not affected (Fig. 5E). Altogether, these results demonstrate that Tc-vn and Tc-spi can act redundantly in the control of the metamorphic transition in Tribolium.

Discussion
Egfr signaling in insects is involved in the regulation of multiple processes during organism development such as cell survival, proliferation, and differentiation. In addition to these functions, it has been recently shown in Drosophila that regulates the metamorphic transition by controlling ecdysone biosynthesis in the PG 20 . Interestingly, our study suggests that this role is conserved in the more basal holometabolous insect Tribolium, as the activity of this pathway is specifically required for the larval-pupal transition in this beetle. Furthermore, our study revealed the existence of an additional EGF ligand in Tribolium, the neuroglianin Tc-Vn, which, together with the already described Tc-Spi ligand, activates Egfr in a redundant manner for the induction of the metamorphic transition. In contrast, we report different requirements of both ligands for the formation of pupal structures, such as wings, elytra or gin traps, suggesting a ligand-specific activation of Egfr signaling during metamorphosis.

Egfr signaling regulates larval-pupal transition in Tribolium.
In Drosophila Egfr signaling regulates larval-pupal transition through the control of PG size and the expression of ecdysone biosynthetic genes in this gland 20 . Our results in Tribolium suggest that such regulation might be general feature of holometabolous insects. Several evidences support this possibility: (1) depletion of both Tc-Egfr ligands, Tc-vn and Tc-spi, is It is interesting to note that 20E controls not only the metamorphic switch but also all the previous larvallarval transitions. The fact that Egfr signaling is involved specifically in triggering pupa formation suggests that other factors might control the production of ecdysone in the previous transitions. In this sense, several transcription factors such as Ventral veins lacking (Vvl), Knirps (Kni) and Molting defective (Mld) have been shown to be involved in the control of ecdysone production during early larval stages in Drosophila 57 . Orthologues of Drosophila kni and vvl have been identified in Tribolium, showing conserved functions during embryogenesis 58,59 . Interestingly Tc-vvl has been shown to be a key factor coordinating ecdysteroid biosynthesis as well as molting in larval stages 60 , supporting the idea that Tc-Vvl also exerts similar function in the PG during early larval development. Nevertheless, further studies are required to confirm the role of these genes and to elucidate ecdysone biosynthesis regulation in all larval transitions.
Then, why is Egfr signaling specifically required during the larva-pupa transition? One possibility is that the peak of 20E required to trigger metamorphosis demands the growth of PG cells to increase the biosynthetic enzyme transcription. Interestingly, this process has recently been related to the nutritional state of the animal. In fact, it has been shown in Drosophila that surpassing a critical weight checkpoint that occurs at the onset of the last larval instar is required for the growth of the PG cells and the increase of ecdysone production 1,2,61,62 , which implies that PG cells must reach a certain size to produce enough ecdysone to trigger metamorphosis. Similarly, in Tribolium exists a correlation between the attainment of a threshold size, defined as the mass that determines that the animal is in the last larval instar, and the upregulation of key metamorphic genes that induce the metamorphic transition 63 . Therefore, it is plausible that Egfr signaling is particularly required once the animal reaches the threshold size that determines the initiation of metamorphosis at the ensuing molt. Such activation will ensure the proper increase of ecdysone production to induce the formation of the pupa. In this sense, it is interesting to note that the highest expression of Tc-Egfr is detected at the last larval stage. Similarly, Tribolium possesses two Egfr ligands. To date, a single Tc-Egfr ligand with high sequence similarity to Drosophila Dm-Spi had been identified in the genome of Tribolium probably due to incomplete annotation of the beetle genome and the fact that its depletion in the embryo phenocopied Tc-Egfr knockdown. However, our in silico analysis has revealed the presence of a second Tc-Egf ligand, the neurogulin Tc-Vn, revealing that Tribolium, in contrast to Drosophila, possesses only two EGF ligands. We have confirmed the identity of Tc-Vn in Tribolium by analyzing its protein sequence and its role during development. Phylogenetic analysis clustered Tc-Vn with its coleopteran orthologous proteins. Interestingly, based on the length of the phylogenetic arm, Tc-Vn presents a high range of change, probably due to its minor role on oogenesis and embryogenesis that has reduced the evolutive pressure on Tc-Vn ligand. In contrast, a lower divergence of Tc-Spi is observed, clustering closely to the Drosophila Dm-Egf ligands Dm-Krn and Dm-Spi. This observation is consistent with Tc-Spi exerting a key role during early development as main activator of Tc-Egfr signaling.
Our results indicate that both Egf ligands Tc-Vn and Tc-Spi function redundantly in the control of larvalpupal transition in Tribolium similarly to what occurs in Drosophila. In contrast, the requirement of each ligand is specific for the development of different structures during the pupal stage. Thus, Tc-Vn is required for the formation of wings and elytra, whereas Tc-Spi is responsible of the proper development of gin traps and the urogomphi as well as the mouthpart. In addition, depletion of either Tc-spi or Tc-vn produce distinct defects on the distal part of the leg and the antenna suggesting again a different role on the activation of Egfr signaling in these structures. This different ligand requirement is similar to what has been described in Drosophila. Thus, Dm-Vn is required for wing identity during early imaginal disc development 64 , whereas later on, the combination . Asterisks indicate differences statistically significant at *p < 0.05 and **p < 0.001 (t-test) ***p < 0.0001 (t-test).   www.nature.com/scientificreports/ the most distal part of the leg 40,66 . In addition, our data also show that the activation of the Tc-Egfr pathway in oogenesis relay entirely on Tc-Spi. In this context, the similarity of Tc-Spi to the Dm-Egf Drosophila ligand Dm-grk 35 , a dedicated ligand specific of the ovaries, might explain this exclusive requirement in this process in Tribolium. Therefore, our results confirmed that Egfr signaling is activated by at least two different ligands in most of the holometabolous insects in morphogenetic processes. The different expression and activity of those ligands probably has favored the co-option of Egfr signaling in different processes, from oogenesis to the biosynthesis of ecdysone, contributing to the generation of new organ and shapes along evolution.

Phylogenetic analysis of Tc-Vn.
To understand the phylogenetic relationship of Vein proteins, amino acid sequences from Vein proteins were collected from different insect taxa, including that of Tribolium as well as from four primates species as an outgroup (S3 Table) and aligned using MAFFT 70 v7.130b. Ambiguously aligned positions were trimmed using trimAlv1.2 71 , with the parameters -gt 80 and -cons 20. A maximum-likelihood phylogenetic tree was reconstructed using RAXML v8.0.17 72 with the PROTGAMMAWAG model.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Total
RNA from individual larva of Tribolium was extracted using the GenElute™ Mammalian Total RNA kit (Sigma). cDNA synthesis was carried out as previously described 73,74 . Relative transcript levels were determined by quantitative real-time PCR (qPCR), using Power SYBR Green PCR Mastermix (Applied Biosystems). To standardize the quantitative real-time RT-PCR (qPCR) inputs, a master mix that contained Power SYBR Green PCR Mastermix and forward and reverse primers was prepared to a final concentration of 100 µM for each primer. The qPCR experiments were conducted with the same quantity of tissue equivalent input for all treatments, and each sample was run in duplicate using 2 µl of cDNA per reaction. As a reference, same cDNAs were subjected to qRT-PCR with a primer pair specific Tribolium Ribosomal Tc-Rpl32. www.nature.com/scientificreports/