Genetic control of moulting and segmentation during post-embryonic development in Lithobius peregrinus (Chilopoda, Lithobiomorpha)
Year: 2010
Type: dissertation
Abstract: The great diversity of arthropod body plans makes this group a remarkable taxon for studying the evolutionary diversification of developmental patterns. In the last decades, developmental genetic studies have been mainly focalized on embryonic development in several arthropod species, whereas post-embryonic development has been investigated to a considerably less extent, and within the limits of very few model species. In many arthropods, post-embryonic development is characterized by an increase in the number of trunk segments. This process is called anamorphosis. In lithobiomorph centipedes juveniles hatch with seven leg pairs and during the first five post-embryonic stages the definitive arrangement with fifteen leg-bearing segments is reached. In these species, despite conspicuous individual variation in growth rates and in temporal molting schedule, each anamorphic stage is characterized by a precise segmental composition of the body. Growth, molting and segmentation are thus intimately correlated developmental processes, and it is easy to hypothesize that variation (and evolvability) in each one of these processes is strictly dependent on the precise relationship it has with the others. The aim of this research was to start an investigation of post-embryonic molting and segmentation process in the centipede Lithobius peregrinus through a genetic approach. In arthropods, molting events driven by oscillations in the titer of the ecdysone, are mediated by the binding of the hormone to a heterodimer of nuclear receptors consisting of the ecdysone receptor (EcR) and the retinoid X receptor (RXR), a homologue of ultraspiracle (USP). In insects, the 20E-receptor complex directly activates a small group of so called ‘early genes’: Broad-Complex (Br-C), E74 and E75, each of which encodes a set of transcriptional factors isoforms. The main role of these genes directly regulated by the hormone is to coordinate the temporal activation in cascade of appropriate sets of ‘late genes’, involved in larval-pupal transformation. In this study we have cloned partial sequences for EcR and RXR gene homologues from L. peregrinus (LpEcR and LpRXR) and we have analyzed their expression profiles during the second larval stage (L1). Sequence comparison of LpEcR and LpRXR with their orthologues in other arthropods shows a maximal degree of identity with chelicerates and hemimetabolous insects (68-81%), and a lesser degree with crustaceans and some holometabolous insects (65-70%). A distinctive character of L. peregrinus EcR and RXR is the presence of several variants: we identified two isoforms for EcR and three isoforms for the RXR receptor. Further studies are necessary to clarify the function of these different variants. The expression profiles shown by LpEcR and LpRXR during the anamorphic stage L1 of L. peregrinus are similar. Expression levels of both receptors increase in the second part of the inter-molt period, suggesting that molting process start 48 hours after the previous molt. Similar expression profiles are found in other arthropods. We failed to isolate an orthologues of BR-C. The lack of homologues in L. peregrinus and crustaceans (B. Konopova, personal communication) suggests that it could be an apomorphic trait of insects. The genetic basis of the developmental mechanisms of body segmentation are well known in the fruit fly Drosophila melanogaster. So-called ‘segmentation genes’ discovered in the fly are classified into gap, pair-rule and segment-polarity genes. These genes occupy different levels in a hierarchical cascade leading from the early gap genes to the later-expressed pair-rule and segment-polarity genes. The latter (like, engrailed, wingless, hedgehog and cubitus interruptus) encode proteins that are expressed in the embryo as stripes with segmental periodicity, and that are required for the formation of the correct pattern of structures within each segment. Homologs of several segment-polarity and pair-rule genes have been studied in other insects, as well as in chelicerates, myriapods, and crustaceans, but we lack basic information about expression of segmentation genes during post-embryonic development. We investigated the expression of two segment-polarity genes, engrailed and wingless, already known to be involved in embryonic segmentation of centipedes, to ascertain their possible role in segmentation during anamorphic stages. We showed that in L. peregrinus, en and wg, are intensively expressed also during all anamorphic stages. Only en seems retain a segment-polarity role in post-embryonic development, although with different modality of expression, whereas wg function seems related to the development of nervous system and leg buds, but with no sign of periodic expression, thus suggesting that it does not retain the same role in segmental patterning that it has during embryonic development. This research also marks a significant methodological advancement, consisting in the protocol set up, in a centipede, for two very powerful techniques of modern molecular biology: Real Time PCR and in situ hybridization on paraffin sections. The results of this work on the anamorphosis of Lithobius peregrinus represent the first molecular data on post-embryonic development in a myriapod species, that add to the very few studies in non-insect arthropods. This work thus represents a basic starting point for the study of development and evolution of molting and segmentation in a poorly investigated phase of arthropod life cycle.
Author: Francesca Bortolin
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W189993634