Factors regulating metamorphosis in hemimetabolan insects

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad Pompeu Fabra.--2020-09-21.-- ZoologíaThe present thesis deals with the mechanisms regulating metamorphosis, particularly in hemimetabolan insects, by using the cockroach Blattella germanica and the mayfly Cloeon dipterum, as models. We have examined possible candidates to regulate different aspects of metamorphosis, chosen in some cases on the basis of transcriptomic data of B. germanica available in our laboratory. Then, we have functionally tested them with RNAi. Moreover, we have also studied the mayfly Cloeon dipterum, a species belonging to Palaeoptera, an early-branching insect group of which very little is known about metamorphosis and its regulatory mechanisms. In this case, we have examined the expression of key genes that are involved in metamorphosis in neopteran insects, and we have approached the functional studies through treatments with a juvenile hormone mimic. In B. germanica, we found that myoglianin reduces juvenile hormone acid methyl transferase expression in the penultimate nymphal instar, which is a prerequisite for metamorphosis. Myoglianin is also involved in the production of the large ecdysone pulse needed to promote the metamorphic molt. Previous studies have shown that the factor FTZ-F1 induces the death of the prothoracic gland (PG) after the imaginal molt. But we have found that the action of FTZ-F1 is mediated by the adult specifier factor E93. In C. dipterum, we observed that metamorphosis occurs in the transition from last nymphal instar to subimago. In this transition, the expression of the antimetamorphic factor Kr-h1 decreases, and that of E93 increases. We also observed that juvenile hormone inhibits metamorphosis and induces the expression of Kr-h1

Factors regulating metamorphosis in hemimetabolan insects

The project consists in studying different aspects of the regulation of insect metamorphosis, using the cockroach Blattella germanica and the mayfly Cloeon dipterum, as laboratory models. In B. germanica the idea has been to study the possible role of Myoglianin in regulation of the decrease of juvenile hormone production that occurs at the beginning of the last nymphal instar. Also, to study the possible role of the adult specifier factor E93 in the destruction of the PG after the imaginal molt. In C. dipterum the plan has been to study the mechanisms that regulate metamorphosis, particularly during the formation of the subimago, and to compare these mechanisms with those operating in neopteran insects, which are condensed in the so-called MEKRE93 pathway.El projecte consisteix en estudiar diferents aspectes de la regulació de la metamorfosi dels insectes, utilitzant la panerola Blattella germanica i l’efímera Cloeon dipterum com a models de laboratori. A B. germanica, la idea ha estat estudiar el possible paper de la mioglianina en la regulació de la disminució de la producció d’hormona juvenil que es produeix al començament de l’últim instar nimfal. També ha estat previst estudiar el possible paper del factor especificador de l’adult E93 en la destrucció de la glàndula protorácica després de la muda imaginal. A C. dipterum, el pla ha estat estudiar els mecanismes que regulen la metamorfosi, particularment durant la formació del subimago, i comparar aquests mecanismes amb els que operen en insectes neòpters, condensats en l'anomenada via MEKRE9

Factors regulating metamorphosis in hemimetabolan insects

The project consists in studying different aspects of the regulation of insect metamorphosis, using the cockroach Blattella germanica and the mayfly Cloeon dipterum, as laboratory models. In B. germanica the idea has been to study the possible role of Myoglianin in regulation of the decrease of juvenile hormone production that occurs at the beginning of the last nymphal instar. Also, to study the possible role of the adult specifier factor E93 in the destruction of the PG after the imaginal molt. In C. dipterum the plan has been to study the mechanisms that regulate metamorphosis, particularly during the formation of the subimago, and to compare these mechanisms with those operating in neopteran insects, which are condensed in the so-called MEKRE93 pathway.El projecte consisteix en estudiar diferents aspectes de la regulació de la metamorfosi dels insectes, utilitzant la panerola Blattella germanica i l’efímera Cloeon dipterum com a models de laboratori. A B. germanica, la idea ha estat estudiar el possible paper de la mioglianina en la regulació de la disminució de la producció d’hormona juvenil que es produeix al començament de l’últim instar nimfal. També ha estat previst estudiar el possible paper del factor especificador de l’adult E93 en la destrucció de la glàndula protorácica després de la muda imaginal. A C. dipterum, el pla ha estat estudiar els mecanismes que regulen la metamorfosi, particularment durant la formació del subimago, i comparar aquests mecanismes amb els que operen en insectes neòpters, condensats en l'anomenada via MEKRE9

Myoglianin triggers the pre-metamorphosis stage in hemimetabolan insects

Insect metamorphosis is triggered by a decrease in juvenile hormone (JH) in the final juvenile instar. What induces this decline is therefore a very relevant question. Working with the cockroach Blattella germanica, we found that Myoglianin (Myo), a ligand in the TGF-β signaling pathway, is highly expressed in the corpora allata (CA, the JH producing glands) and prothoracic glands (PG, which produce ecdysone) during the penultimate nymphal instar (N5). In the CA, high Myo levels during N5 repress the expression of jhamt, a JH biosynthesis gene. In the PG, decreasing JH levels trigger gland degeneration, mediated by the factors Kr-h1, FTZ-F1, E93 and IAP1. Also in the PG, a peak of myo expression in N5 stimulates the expression of ecdysone biosynthesis genes, such as nvd, thus enhancing the production of the metamorphic ecdysone pulse in N6. The myo expression peak in N5 also represses cell proliferation, which can contribute to enhance ecdysone production. The data indicate that Myo triggers the pre-metamorphic nymphal instar in B. germanica, and possibly in other hemimetabolan insects.This work was supported by the Spanish Ministry of Economy and Competitiveness (grants CGL2012–36251 and CGL2015–64727-P to X.B.) and the Catalan Government (grant 2017 SGR 1030 to X.B.). It also received financial assistance from the European Fund for Economic and Regional Development (FEDER funds). O.K. received a Royal Thai Government Scholarship to complete a PhD thesis in X.B. laboratory, in Barcelona

E93-depleted adult insects preserve the prothoracic gland and molt again

Insect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 is crucial in the emergence of metamorphosis, because it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion towards the end of the final nymphal instar downregulates the expression of E93, whereas E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues about how nymphal epidermal cells definitively become adult cells, and whether it is possible to reverse this process.O.K. received a Royal Thai Government Scholarship to carry out a PhD thesis in X.B.’s laboratory in Barcelona. This work was supported by the Ministerio de Economia y Competitividad (CGL2012–36251, CGL2015–64727-P and PID2019-104483GB-I00 to X.B.), the Consejo Superior de Investigaciones Científicas (2019AEP029), the Generalitat de Catalunya (2017 SGR 1030 to X.B.) and the European Regional Development Fund

Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera)

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.Our work is supported by Spanish Ministry of Economy and Competitiveness (Grants CGL2015-64727‐P and PID2019-104483GB-I00 to X.B.; PGC2018-093704-B-I00 to F.C., and MDM-2016-0687 [“María de Maeztu” Programme for Units of Excellence in R&D] in which F.C. is a consortium participant), by the Catalan Government (Grant 2017 SGR 1030 to X.B.), and by the European Fund for Economic and Regional Development (FEDER funds). O.K. received a Royal Thai Government Scholarship to do a PhD thesis in X.B. laboratory in Barcelona, and A.V.-A. received a predoctoral fellowship of the Spanish Ministry of Economy and Competitiveness, associated with the Grant CGL2015-64727‐P. I.A. was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Grant Agreement 657732.Peer reviewe

Broad complex and wing development in cockroaches

In hemimetabolan insects, the transcription factor Broad complex (Br–C) promotes wing growth and development during the nymphal period. We wondered whether Br–C could trigger the initiation of wing development, using the cockroach Blattella germanica as a model. We show that first instar nymphs have their unique identity of these three thoracic segments specified. During embryogenesis, the expression of Br–C and some wing-related genes show two matching waves. The first takes place before the formation of the germ band, which might be involved in the establishment of various developmental fields including a potential “wing field”, and the second wave around organogenesis, possibly involved in the initiation of wing development. However, the expression of Br–C in early embryogenesis concentrates in the developing central nervous system, thus not co-localizing with the expression of the typical wing-related gene vestigial, which is expressed at the edge of the thoracic and abdominal segments. This suggests that Br–C is not specifically involved in the establishment of a potential “wing field” in early embryogenesis. Moreover, maternal RNAi for Br–C depletes the first wave of Br–C expression but does not affect the early expression of wing-related genes. As maternal Br–C RNAi did not deplete the second expression wave of Br–C, we could not evaluate if Br–C is involved in the initiation of wing development. Alternatively, using nymphal RNAi of Br–C and Sex combs reduced (Scr), we show that Br–C contributes to the formation of ectopic wing structures that develop in the prothorax when Scr is depleted. The gene most clearly influenced by Br–C RNAi is nubbin (nub), which, in nymphs is crucial for wing growth. Together, these results suggest that Br–C does not specifically contribute to the establishment of the “wing field”, but it does seem important later, in the initiation of wing development, enhancing the expression of wing-related genes, especially nub. This supports the hypothesis previously proposed by the authors, whereby Br–C might have facilitated the evolution of holometaboly. However, there is no doubt that other factors have also contributed to this evolution.The work of Xavier Belles was supported by the Spanish Agencia Estatal de Investigación (grant number PID2019-104483 GB-I00/AEI/10.13039/501100011033; CGL2015-64727-P; and CGL2012-36251), including support for the visit of Ana Fernandez-Nicolas to the Tomoyasu lab), the Catalan Government (2017 SGR 1030), and European Fund for Economic and Regional Development (FEDER funds). The work of Yoshinori Tomoyasu was supported by the Miami University Faculty Research Grants Program (CFR), the National Science Foundation (NSF; IOS1557936), and an NSF Graduate Research Fellowship to Courtney Clark-Hachtel.Peer reviewe