Carlo Brena

From embryo to adult—beyond the conventional periodization of arthropod development

The traditional framework for the description of arthropod development takes the molt-to-molt interval as the fundamental unit of periodization, which is similar to the morphological picture of the main body axis as a series of segments. Developmental time is described as the subdivision into a few major stages of one or more instars each, which is similar to the subdivision of the main body axis into regions of one to many segments each. Parallel to recent criticisms to the segment as the fundamental building block of arthropod anatomy, we argue that, while a firm subdivision of development in stages is useful for describing arthropod ontogeny, this is limiting as a starting point for studying its evolution. Evolutionary change affects the association between different developmental processes, some of which are continuous in time whereas others are linked to the molting cycle. Events occurring but once in life (hatching; first achieving sexual maturity) are traditionally used to establish boundaries between major units of arthropod developmental time, but these boundaries are quite labile. The presence of embryonic molts, the ‘gray zone’ of development accompanying hatching (with the frequent delivery of an immature whose qualification as ‘free-embryo’ or ordinary postembryonic stage is arbitrary), and the frequent decoupling of growth and molting suggest a different view. Beyond the simple comparison of developmental schedules in terms of heterochrony, the flexible canvas we suggest for the analysis of arthropod development opens new vistas into its evolution. Examples are provided as to the origin of holometaboly and hypermetaboly within the insects.

The embryonic development of the centipede Strigamia maritima

The geophilomorph centipede Strigamia maritima is an emerging model for studies of development and evolution among the myriapods. A draft genome sequence has recently been completed, making it also an important reference for comparative genomics, and for studies of myriapod physiology more generally. Here we present the first detailed description of myriapod development using modern techniques. We describe a timeline for embryonic development, with a detailed staging system based on photographs of live eggs and fixed embryos. We show that the early, cleavage and nuclear migration, stages of development are remarkably prolonged, accounting for nearly half of the total developmental period (approx 22 of 48 days at 13 °C). Towards the end of this period, cleavage cells migrate to the egg periphery to generate a uniform blastoderm. Asymmetry quickly becomes apparent as cells in the anterior half of the egg condense ventrally to form the presumptive head. Five anterior segments, the mandibular to the first leg-bearing segment (1st LBS) become clearly visible through the chorion almost simultaneously. Then, after a short pause, the next 35 leg-bearing segments appear at a uniform rate of 1 segment every 3.2 h (at 13 °C). Segment addition then slows to a halt with 40-45 LBS, shortly before the dramatic movements of germ band flexure, when the left and right halves of the embryo separate and the embryo folds deeply into the yolk. After flexure, segment morphogenesis and organogenesis proceed for a further 10 days, before the egg hatches. The last few leg-bearing segments are added during this period, much more slowly, at a rate of 1-2 segments/day. The last leg-bearing segment is fully defined only after apolysis of the embryonic cuticle, so that at hatching the embryo displays the final adult number of leg-bearing segments (typically 47-49 in our population).

An analysis of segmentation dynamics throughout embryogenesis in the centipede Strigamia maritima.

BackgroundMost segmented animals add segments sequentially as the animal grows. In vertebrates, segment patterning depends on oscillations of gene expression coordinated as travelling waves in the posterior, unsegmented mesoderm. Recently, waves of segmentation gene expression have been clearly documented in insects. However, it remains unclear whether cyclic gene activity is widespread across arthropods, and possibly ancestral among segmented animals. Previous studies have suggested that a segmentation oscillator may exist in Strigamia, an arthropod only distantly related to insects, but further evidence is needed to document this.ResultsUsing the genes even skipped and Delta as representative of genes involved in segment patterning in insects and in vertebrates, respectively, we have carried out a detailed analysis of the spatio-temporal dynamics of gene expression throughout the process of segment patterning in Strigamia. We show that a segmentation clock is involved in segment formation: most segments are generated by cycles of dynamic gene activity that generate a pattern of double segment periodicity, which is only later resolved to the definitive single segment pattern. However, not all segments are generated by this process. The most posterior segments are added individually from a localized sub-terminal area of the embryo, without prior pair-rule patterning.ConclusionsOur data suggest that dynamic patterning of gene expression may be widespread among the arthropods, but that a single network of segmentation genes can generate either oscillatory behavior at pair-rule periodicity or direct single segment patterning, at different stages of embryogenesis.

Expression of trunk Hox genes in the centipede Strigamia maritima: sense and anti-sense transcripts

SUMMARY We report the coding sequence and embryonic expression of the four trunk Hox genes Antennapedia (Antp), Ultrabithorax (Ubx), abdominal‐A (abd‐A), and Abdominal‐B (Abd‐B) in the geophilomorph centipede Strigamia maritima. In geophilomorph centipedes, all leg‐bearing segments (LBS) are generated during embryogenesis, allowing us to define expression in relation to the full extent of the forming trunk. Persistent Antp expression characterizes the maxillipedal (poison claw) segment, whereas all LBS express the three Hox genes Antp, Ubx, and abd‐A. Abd‐B is never detectably expressed in segmented tissue, but is restricted to a zone around the proctodaeum that contributes to the hindgut. Expression of all these Hox genes initiates in the unsegmented tissue of the blastodisc, with expression of Antp respecting a sharply defined anterior border before the appearance of morphological segmentation in the trunk. The accumulation of Hox gene transcripts is strongly modulated by the maturing segment pattern, suggesting regulatory interactions with multiple levels of the segment patterning machinery. For one of these genes, Ubx, we detect both sense and anti‐sense transcripts. The anti‐sense transcripts originate 3′ to the Ubx coding sequence and overlap the homeobox exon; they are expressed earlier than the Ubx coding transcripts and persistently, in an axially restricted pattern comparable to but distinct from those of the Hox coding transcripts. The pattern of accumulation of Ubx sense and anti‐sense transcripts is strikingly complementary, suggesting the possibility of anti‐sense regulation of Ubx expression.

Ancestral patterning of tergite formation in a centipede suggests derived mode of trunk segmentation in trilobites.

Trilobites have a rich and abundant fossil record, but little is known about the intrinsic mechanisms that orchestrate their body organization. To date, there is disagreement regarding the correspondence, or lack thereof, of the segmental units that constitute the trilobite trunk and their associated exoskeletal elements. The phylogenetic position of trilobites within total-group Euarthropoda, however, allows inferences about the underlying organization in these extinct taxa to be made, as some of the fundamental genetic processes for constructing the trunk segments are remarkably conserved among living arthropods. One example is the expression of the segment polarity gene engrailed, which at embryonic and early postembryonic stages is expressed in extant panarthropods (i.e. tardigrades, onychophorans, euarthropods) as transverse stripes that define the posteriormost region of each trunk segment. Due to its conservative morphology and allegedly primitive trunk tagmosis, we have utilized the centipede Strigamia maritima to study the correspondence between the expression of engrailed during late embryonic to postembryonic stages, and the development of the dorsal exoskeletal plates (i.e. tergites). The results corroborate the close correlation between the formation of the tergite borders and the dorsal expression of engrailed, and suggest that this association represents a symplesiomorphy within Euarthropoda. This correspondence between the genetic and phenetic levels enables making accurate inferences about the dorsoventral expression domains of engrailed in the trunk of exceptionally preserved trilobites and their close relatives, and is suggestive of the widespread occurrence of a distinct type of genetic segmental mismatch in these extinct arthropods. The metameric organization of the digestive tract in trilobites provides further support to this new interpretation. The wider evolutionary implications of these findings suggest the presence of a derived morphogenetic patterning mechanism responsible for the reiterated occurrence of different types of trunk dorsoventral segmental mismatch in several phylogenetically distant, extinct and extant, arthropod groups.

An early temperature-sensitive period for the plasticity of segment number in the centipede Strigamia maritima.

Geophilomorph centipedes show variation in segment number (a) between closely related species and (b) within and between populations of the same species. We have previously shown for a Scottish population of the coastal centipede Strigamia maritima that the temperature of embryonic development is one of the factors that affects the segment number of hatchlings, and hence of adults, as these animals grow epimorphically—that is, without postembryonic addition of segments. Here, we show, using temperature‐shift experiments, that the main developmental period during which embryos are sensitive to environmental temperature is surprisingly early, during blastoderm formation and before, or very shortly after, the onset of segmentation.

More than one way to produce protein diversity: duplication and limited alternative splicing of an adhesion molecule gene in basal arthropods.

Exon duplication and alternative splicing evolved multiple times in metazoa and are of overall importance in shaping genomes and allowing organisms to produce many fold more proteins than there are genes in the genome. No other example is as striking as the one of the Down syndrome cell adhesion molecule (Dscam) of insects and crustaceans (pancrustaceans) involved in the nervous system differentiation and in the immune system. To elucidate the evolutionary history of this extraordinary gene, we investigated Dscam homologs in two basal arthropods, the myriapod Strigamia maritima and the chelicerate Ixodes scapularis. In both, Dscam diversified extensively by whole gene duplications resulting in multigene expansions. Within some of the S. maritima genes, exons coding for one of the immunoglobulin domains (Ig7) duplicated and are mutually exclusively alternatively spliced. Our results suggest that Dscam diversification was selected independently in chelicerates, myriapods, and pancrustaceans and that the usage of Dscam diversity by immune cells evolved for the first time in basal arthropods. We propose an evolutionary scenario for the appearance of the highly variable Dscam gene of pancrustaceans, adding to the understanding of how alternative splicing, exon, and gene duplication contribute to create molecular diversity associated with potentially new cellular functions.

The embryoid development of Strigamia maritima and its bearing on post-embryonic segmentation of geophilomorph centipedes

BackgroundMany arthropods add body segments post-embryonically, including most of the myriapods. However, geophilomorph and scolopendromorph centipedes are epimorphic, i.e. they form all their segments during embryonic time, although this has never been demonstrated directly. Understanding the similarity between embryonic and post-embryonic segmentation is pivotal to understand the possible evolution from anamorphosis to epimorphosis. We have previously demonstrated that in the geophilomorph centipede Strigamia maritima most segments are produced by an oscillatory mechanism operating through waves of expression at double segment periodicity, but that the last-forming (posteriormost) segments are patterned with a different system which might be more similar to post-embryonic segmentation.ResultsWith a careful analysis of a large number of specimens, I show that the first (“embryoid”) phase of post-embryonic development is clearly distinct from the following ones. It is characterized by more moults than previously reported, allowing me to define and name new stages. I describe these embryoid stages and the first free-leaving stage in detail, providing data on their duration and useful identification characters. At hatching, the prospective last leg-bearing segment is limbless and the genital segments are added in the following stages, indicating a residual anamorphosis in Strigamia segmentation. I demonstrate directly for the first time that at least the leg-bearing segments are in general produced during embryonic life, although in some individuals the external delineation of the last leg-bearing segment may be delayed to post-embryonic time, a possible further residual of anamorphic development. Additionally, I show that the development of the poison claws during this post-embryonic phase may have some element of recapitulation.ConclusionsThe data presented in this paper show that the embryoid phase of post-embryonic development of geophilomorph centipedes may represent an extension of embryonic development, possibly in correlation with the evolution of epimorphic development from an anamorphic ancestor, accomplished without completely losing post-embryonic segmentation activity. This continuity in the segmentation process across the embryonic/postembryonic divide may concur to the evolvability of this developmental process.

Demonstration of a heritable component of the variation in segment number in the centipede Strigamia maritima

SUMMARY Here we address the question of how arthropod segment number may evolve by reporting the results of further work on the model system Strigamia maritima. Recently, we showed that there was a plastic component of the variation in segment number within this species; now we demonstrate that there is also a heritable component. This is important because it enables a connection to be made between the known latitudinal trend among species of geophilomorph centipedes (more segments at lower latitudes) and the parallel trend within them. This latter trend is best documented in S. maritima but is also known in several other species. However, while a general connection between the inter‐ and intraspecific trends can now be made, deciding upon a specific hypothesis of the nature of the selection involved is still problematic. We provide two alternative hypotheses, one based on the temperature‐related plasticity in segment number being adaptive, the other based on it being nonadaptive.

Early embryonic determination of the sexual dimorphism in segment number in geophilomorph centipedes

BackgroundMost geophilomorph centipedes show intraspecific variability in the number of leg-bearing segments. This intraspecific variability generally has a component that is related to sex, with females having on average more segments than males. Neither the developmental basis nor the adaptive role of this dimorphism is known.ResultsTo determine when this sexual dimorphism in segment number is established, we have followed the development of Strigamia maritima embryos from the onset of segmentation to the first post-embryonic stage where we could determine the sex morphologically. We find that males and females differ in segment number by Stage 6.1, a point during embryogenesis when segment addition pauses while the embryo undergoes large-scale movements. We have confirmed this pattern by establishing a molecular method to determine the sex of single embryos, utilising duplex PCR amplification for Y chromosomal and autosomal sequences. This confirms that male embryos have a modal number of 43 segments visible at Stage 6, while females have 45. In our Strigamia population, adult males have a modal number of 47 leg-bearing segments, and females have 49. This implies that the sexual dimorphism in segment number is determined before the addition of the last leg-bearing segments and the terminal genital segments.ConclusionsSexual dimorphism in segment number is not associated with terminal segment differentiation, but must instead be related to some earlier process during segment patterning. The dimorphism may be associated with a difference in the rate and/or duration of segment addition during the main phase of rapid segment addition that precedes embryonic Stage 6. This suggests that the adaptive role, if any, of the dimorphism is likely to be related to segment number per se, and not to sexual differentiation of the terminal region.