Evolution of Strigamia centipedes (Chilopoda): a first molecular assessment of phylogeny and divergence times

We present a first phylogenetic and temporal framework, with biogeographical insights, for the centipedes of the genus Strigamia, which are widespread predators in the forest soils of the Northern Hemisphere and comprise the evo-devo model species Strigamia maritima. The phylogeny was estimated by different methods of maximum likelihood and Bayesian inference from sequences of two mitochondrial (16S, COI) and two nuclear (18S, 28S) genes, obtained from 16 species from all major areas of the global range of the genus and encompassing most of the overall morphological and ecological diversity. Divergence times were estimated after calibration upon the fossil record of centipedes. We found that major lineages of extant species of Strigamia separated most probably around 60 million years (Ma) ago. The two most diverse lineages diversified during the last 30 Ma and are today segregated geographically, one in Europe and another in Eastern Asia. This latter region hosts a hitherto underestimated richness and anatomical diversity of species, including three still unknown, yet morphologically well distinct species, which are here described as new: Strigamia inthanoni sp. n. from Thailand, Strigamia korsosi sp. n. from the Ryukyu Islands and Strigamia nana sp. n. from Taiwan. The northern European model species S. maritima is more strictly related to the Eastern Asian lineage, from which it most probably separated around 35 Ma ago before the major diversification of the latter

Underground radiobiology: a perspective at Gran Sasso National Laboratory

Scientific community and institutions (e. g., ICRP) consider that the Linear No-Threshold (LNT) model, which extrapolates stochastic risk at low dose/low dose rate from the risk at moderate/high doses, provides a prudent basis for practical purposes of radiological protection. However, biological low dose/dose rate responses that challenge the LNT model have been highlighted and important dowels came from radiobiology studies conducted in Deep Underground Laboratories (DULs). These extreme ultra-low radiation environments are ideal locations to conduct below-background radiobiology experiments, interesting from basic and applied science. The INFN Gran Sasso National Laboratory (LNGS) (Italy) is the site where most of the underground radiobiological data has been collected so far and where the first in vivo underground experiment was carried out using Drosophila melanogaster as model organism. Presently, many DULs around the world have implemented dedicated programs, meetings and proposals. The general message coming from studies conducted in DULs using protozoan, bacteria, mammalian cells and organisms (flies, worms, fishes) is that environmental radiation may trigger biological mechanisms that can increase the capability to cope against stress. However, several issues are still open, among them: the role of the quality of the radiation spectrum in modulating the biological response, the dependence on the biological endpoint and on the model system considered, the overall effect at organism level (detrimental or beneficial). At LNGS, we recently launched the RENOIR experiment aimed at improving knowledge on the environmental radiation spectrum and to investigate the specific role of the gamma component on the biological response of Drosophila melanogaster

Genetic control of moulting and segmentation during post-embryonic development in Lithobius peregrinus (Chilopoda, Lithobiomorpha)

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.La grande diversificazione nell’organizzazione del piano corporeo degli artropodi fa di questo gruppo un taxon molto interessante per lo studio dell’ evoluzione dei modelli di sviluppo. Negli ultimi decenni, gli studi di genetica dello sviluppo si sono focalizzati principalmente sulla fase embrionale in diverse specie di artropodi, mentre lo sviluppo post-embrionale è stato raramente studiato solo in alcune specie modello. In molti artropodi lo sviluppo postembrionale è caratterizzato da un aumento del numero dei segmenti del tronco. Nei centopiedi litobiomorfi, l’animale emerge dall’uovo con soli sette segmenti del tronco perfettamente formati raggiungendo poi, al termine delle prime cinque mute, l’assetto definitivo con quindici segmenti pediferi. Questo tipo di sviluppo viene detto emianamorfico. In queste specie, nonostante ci siano ampie variazioni intraspecifiche nel tasso di crescita e nella scansione delle mute, ogni stadio anamorfico è caratterizzato da un precisa composizione segmentale del corpo. Crescita, muta e segmentazione sono quindi processi di sviluppo strettamente correlati ed è facilmente ipotizzabile che la variazione (e la capacità di evolvere) di ciascun processo sia strettamente dipendente dalla precisa relazione con gli altri. L’ obiettivo di questa ricerca è stato lo studio dei processi di muta e di segmentazione durante lo sviluppo postembrionale nel centopiedi Lithobius peregrinus, utilizzando un approccio molecolare. Negli artropodi, la muta è controllata dall’oscillazione dei livelli dell’ecdisone. A livello molecolare, questo ormone svolge la sua funzione regolativa legandosi ad un complesso eterodimerico formato da due recettori nucleari: il recettore dell’ecdisone (EcR) e il recettore X retinoico (RXR), omologo del recettore ultraspiracle (USP). Negli insetti, il complesso EcR/USP regola direttamente i geni target primari dell’ecdisone, tra i quali Broad-Complex (BR-C), E74 ed E75. Questi geni codificano fattori di trascrizione che mediano ed amplificano il segnale ormonale regolando un ampio assortimento di geni target secondari coinvolti nel passaggio larva-pupa.(review in Thummel, 1995; King-Jones & Thummel 2005). In questo studio sono state clonate sequenze parziali dei geni omologhi EcR e RXR in L. peregrinus (LpEcR and LpRXR) ed è stato analizzato il loro profilo d’espressione durante il secondo stadio larvale (L1). Un confronto delle sequenze di LpEcR and LpRXR con sequenze ortologhe in altri artropodi ha evidenziato un’elevato livello d’identità con i chelicerati e gli insetti etero metaboli (68-81%), mentre con i crostacei e gli insetti olometaboli meno derivati (65-70%) l’identità è leggermente inferiore. Una caratteristica di L. peregrinus è la presenza di più varianti di sequenza per entrambi i recettori: sono state identificate due isoforme per EcR e tre isoforme per RXR. Ulteriori analisi saranno necessarie per chiarire un eventuale diversa funzione di queste varie isoforme. Il profilo d’espressione osservato per LpEcR e LpRXR durante lo stadio L1 è simile. I livelli d’espressione di entrambi i geni aumentano nella seconda parte dello stadio, e ciò suggerisce che il processo di muta in questo stadio inizi dopo circa 48 ore dalla precedente ecdisi. I risultati ottenuti sono in linea con quanto è stato osservato in altri artropodi. Il mancato riscontro di un ortologo del gene Broad-Complex in L. peregrinus e nei crostacei (B. Konopova, comunicazione personale) fa supporre che si tratti di un’apomorfia degli insetti. Le basi genetiche dei meccanismi di sviluppo della segmentazione sono stati ampiamente studiati nel moscerino della frutta Drosophila melanogaster. I cosiddetti ‘geni della segmentazione’ in Drosofila sono stati classificati in geni ‘gap’, ‘pair-rule’ e ‘segment-polarity’. Questi geni occupano diversi livelli gerarchici nella cascata di regolazione genica della segmentazione: a monte ci sono i geni ‘gap’, successivamente i geni ‘pair-rule’ ed i geni ‘segment-polarity’. Questi ultimi (come engrailed, wingless, hedgehog and cubitus interruptus) codificano proteine che vengono espresse nell’embrione con periodicità segmentale e che sono richieste per la corretta formazione del pattern di strutture entro ciascun segmento. Geni omologhi dei geni ‘pair-rule’ e ‘segment-polarity’ sono stati studiati in diversi insetti, nei chelicerati, nei crostacei e nei miriapodi, ma mancano informazioni basilari sulla loro espressione durante la segmentazione postembrionale. In questa ricerca è stata studiata l’espressione di due geni ‘segment-polarity’, engrailed e wingless, già coinvolti nella segmentazione embrionale dei centopiedi, per stabilire il loro ruolo nella segmentazione durante la fase anamorfica dello sviluppo. E’ stato così dimostrato che in L. peregrinus, en e wg sono intensamente espressi durante l’anamorfosi. Solamente en sembra conservare un ruolo nella segmentazione postembrionale, sebbene con modalità d’espressione diverse, mentre wg sembra legato allo sviluppo del sistema nervoso e degli abbozzi degli arti, e non presenta espressione periodica. Questo suggerisce che questo gene non svolga più nel processo di segmentazione il ruolo che aveva durante lo sviluppo embronale. Questa ricerca segna inoltre un avanzamento metodologico significativo: per la prima volta in un centopiedi sono stati messi a punto i protocolli di due tecniche di biologia molecolare molto importanti: la Real Time PCR e l’ibridazione in situ su sezioni di paraffina. I risultati di questo studio sull’anamorfosi di Lithobius peregrinus rappresentano i primi dati molecolari disponibili sullo sviluppo postembrionale di un miriapode e rappresentano un importante punto di partenza per lo studio dello sviluppo e dell’evoluzione dei processi di muta e segmentazione in una fase dello sviluppo ancora poco conosciuta

Gene expression during postembryonic segmentation in the centipede Lithobius peregrinus (Chilopoda, Lithobiomorpha)

Postembryonic segmentation (anamorphosis) is widespread among arthropods, but only partially known as for its developmental mechanics and control. Studies on developmental genetics of segmentation in anamorphic arthropods are mostly limited to the germ band stage, during early phases of embryonic development. This work presents the first data on the postembryonic expression of a segmentation gene in a myriapod. Using real-time PCR, we analyzed engrailed expression patterns during the anamorphic stages of the centipede Lithobius peregrinus. A variation pattern in en RNA level during anamorphosis suggests that gene expression is precisely modulated during this period of development and that engrailed is mainly expressed in the posterior part of the body, in the newly differentiating segments of each stage. As anamorphosis is possibly the primitive segmentation mode in arthropods, the postembryonic en expression pattern documented here provides evidence for a conservation of en role in ontogeny, across the embryonic/postembryonic boundary, as well as in phylogeny, across the same boundary, but in the opposite direction, from primitive postembryonic expression to the more derived expression in clades with exclusively embryonic segmentation

Cloning and expression pattern of the ecdysone receptor and retinoid X receptor from the centipede Lithobius peregrinus (Chilopoda, Lithobiomorpha)

In arthropods, molting events are mediated by the binding of the ecdysone hormone to a heterodimer of two nuclear receptors: the ecdysone receptor (EcR) and the retinoid X receptor (RXR), a homolog of ultraspiracle (USP). We have cloned partial sequences of several isoforms for EcR and RXR genes from the centipede Lithobius peregrinus, and studied their expression profile during the second post-embryonic stage. LpEcR and LpRXR inferred amino acid sequences are very similar to other arthropod orthologs, especially to those of chelicerates and hemimetabolous insects, and their expression levels are significantly higher during the 48 h that precede the molt. Results obtained in this study represent the first data on the genetic basis of the ecdysone signal pathway for a myriapod, and in particular for an animal that, through a stereotyped developmental schedule paced by the molt cycle, completes trunk segmentation during post-embryonic life