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This review comes from a themed issue on Development and regulation., Proper formation of adult insects requires the integration of spatial and temporal regulatory axes. Whereas spatial information confers identity to each tissue, organ and appendage, temporal information specifies at which stage of development the animal is. Regardless of the type of post-embryonic development, either hemimetabolous or holometabolous, temporal specificity is achieved through interactions between the temporal identity genes Kr-h1, E93 and Br-C, whose sequential expression is controlled by the two major developmental hormones, 20-hydroxyecdysone and Juvenile hormone. Given the intimate regulatory connection between these three factors to specify life stage identity, we dubbed the regulatory axis that comprises these genes as the Metamorphic Gene Network (MGN). In this review, we survey the molecular mechanisms underlying the control by the MGN of stage identity and progression in hemimetabolous and holometabolous insects., Support for this study was provided by the Spanish MINECO (grant PGC2018-098427-B-I00 to DM and XF-M) and by the Catalan Government (2017 SGR 01030 to DM and XF-M). The research has also benefited from FEDER, UE funds.

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., We would like to specially thank Lidia Escoda for technical assistance in the phylogenetic analysis. Support for this research was provided by the Spanish MINECO (Grants CGL2014-55786-P and PGC2018-098427-B-I00 to D.M. and X.F-M.) and by the Catalan Government (2014 SGR 619 to D.M. and X.F-M.). The research has also benefited from FEDER funds. S.C. is a recipient of a pre-doctoral research Grant from the MINECO.

Insects living in the temperate zone enter a physiological state of arrested or slowed development to overcome an adverse season, such as winter. Developmental arrest, called diapause, occurs at a species-specific developmental stage, and embryonic and pupal diapauses have been extensively studied in mostly holometabolous insects. Some other insects overwinter in the nymphal stage with slow growth for which the mechanism is poorly understood. Here, we show that this nymphal period of slow growth is regulated by temperature and photoperiod through separate pathways in the cricket Modicogryllus siamensis. The former regulates the growth rate, at least in part, through the insulin / target of rapamycin (TOR) signaling pathway. Lower temperature down-regulates the expression of insulin-like peptide (Ms’Ilp) and Target of rapamycin (Ms’Tor) genes to slow down the growth rate without affecting the number of molts. The latter regulates the number of molts independent of temperature. Short days increase the number of molts through activation of the juvenile hormone (JH) pathway and down-regulation of myoglianin (Ms’myo), a member of the TGFβ family, which induces adult metamorphosis. In contrast, long days regulate Ms’myo expression to increase during the fifth to sixth instar to initiate adult metamorphosis. When Ms’myo expression is suppressed, juvenile hormone O-methyl transferase (Ms’jhamt) was up-regulated and increased molts to prolong the nymphal period even under long-day conditions. The present findings suggested that the photoperiod regulated Ms’myo, and the JH signaling pathway and the temperature-controlled insulin/TOR pathway cooperated to regulate nymphal development for overwintering to achieve seasonal adaptation of the life cycle in M. siamensis., This study was supported in part by the grant from the Japan Society for the Promotion of Science (23370033) (to K.T.).

In the present study, Chafino et al. identify the upregulation of E93 expression as the trigger for metamorphosis in the holometabolous insect Tribolium castaneum. Depletion of E93 prevents pupa formation, whereas precocious E93 upregulation, by either JH depletion or nutrition restriction, induces premature onset of metamorphosis.Body size in holometabolous insects is determined by the size at which the juvenile larva undergoes metamorphosis to the pupal stage. To undergo larva-pupa transition, larva must reach a critical developmental checkpoint, the threshold size (TS); however, the molecular mechanisms through which the TS cues this transition remain to be fully characterized. Here, we use the flour beetle Tribolium castaneum to characterize the molecular mechanisms underlying entry into metamorphosis. We found that T. castaneum reaches a TS at the beginning of the last larval instar, which is associated with the downregulation of TcKr-h1 and the upregulation of TcE93 and TcBr-C. Unexpectedly, we found that while there is an association between TS and TcE93 upregulation, it is the latter that constitutes the molecular trigger for metamorphosis initiation. In light of our results, we evaluate the interactions that control the larva-pupa transition and suggest alternative models., Support for this research was provided by the Spanish MINECO (CGL2014-55786-P to D.M., E.C., and X.F.-M.) and by the Catalan Government (2014 SGR 619 to D.M. and X.F.-M. and 2017-SGR 1030 to D.M., E.C., and X.F.-M). The research has also benefited from FEDER funds. S.C. is a recipient of a predoctoral research grant from the MINECO.

The adult body size is species-specific and controlled by complex interactions between hormones and the IIS/TOR pathway. To analyze the role of target of rapamycin (TOR) in the growth and development of the insect, expression levels of TOR were silenced in the model and pest insect red flour beetle, Tribolium castaneum. Injection of dsRNA into the last larval instar decreased pupal mass and size, while the amount of food intake by the larvae was not affected. These results place TcTOR downstream of nutrition as a transducer for nutritional signals to increase larval growth. In addition, TcTOR-silencing notably decreased the size of the adult appendages. Analysis of the wings and elytra revealed a decrease in cell size and number of these appendages in the TcTOR-silenced insects. This reduction in size was correlated with a decrease of transcriptional levels of marker genes controlling the cell cycle. Altogether, these results suggest a pivotal role for TcTOR in integrating nutritional signals and regulation of body and appendages growth., This project is supported by the Oversea Study Program of Guangzhou Elite Project (China) to X.L., the Special Research Fund of Ghent University and the Research Foundation-Flanders (FWO-Vlaanderen, Belgium) to G.S., and by the Spanish MINECO (grant CGL2014-55786-P) to D.M., and X.F-M. and by the Catalan Government (2014 SGR 619 and 2017-SGR 1030) to D.M. and X.F-M. The research has also benefited from FEDER funds. S.C. is a recipient of a pre-doctoral research Grant from the MINECO.

Holometaboly is a key evolutionary innovation that has facilitated the spectacular radiation of insects. Despite the undeniable advantage of complete metamorphosis, the female of some holometabolous species have lost the typical holometabolous development through neoteny. In Xenos vesparum Rossi (Strepsiptera: Stylopidae), a derived species of the holometabolous endoparasitic order Strepsiptera, neotenic females reach sexual maturity without the pupal and the imaginal stages, thus retaining their larval morphology (with the exception of the anterior part of the body or cephalothorax), while males undergo normal pupal-based metamorphosis. Expression of the “adult-specifier” E93 factor has been shown to be required for proper metamorphosis in holometabolous insects. Here, we investigated the involvement of E93 in female neoteny by cloning XvE93. Interestingly, while we detected a clear up-regulation of XvE93 expression in pupal and adult stages of males, persistent low levels of XvE93 were detected in X. vesparum females. However, a specific up-regulation of XvE93 was observed in the cephalothorax of late 4th female instar larva, which correlates with the occurrence of neotenic-specific features in the anterior part of the female body. Moreover, the same expression dynamic in the cephalothorax and abdomen was also observed for other two critical metamorphic regulators, the anti-metamorphic XvKr-h1 and the pupal specifier XvBr-C. The specific up-regulation of XvE93 and XvBr-C in the female cephalothorax seems to be the result of an increase in 20-hydroxyecdysone (20E) signaling in this region for we detected higher expression levels of the 20E-dependent nuclear receptors XvHR3 and XvE75 in the cephalothorax. Overall, our results detect a sex-specific expression pattern of critical metamorphic genes in X. vesparum, suggesting that neoteny in Strepsiptera results from the modification of the normal expression of E93, Br-C and Kr-h1 genes.

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes., E.U. was supported by a predoctoral fellowship from Consejo Superior de Investigaciones Cientificas. This work was funded by the Spanish Ministerio de Ciencia e Innovacion (Projects BFU2009-10571 to D.M. and BFU2011-25986 to R.B.). R.B. acknowledges the Consolider program (CSD2007-008-25120), the Departments of Education and Industry of the Basque Government (PI2012/42), and the Bizkaia County. This article is based upon work from COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology). The research has also benefited from FEDER.