Developmental gene expression as a phylogenetic data class: support for the monophyly of Arachnopulmonata.

Despite application of genome-scale datasets, the phylogenetic placement of scorpions within arachnids remains contentious between two different phylogenetic data classes. Paleontologists continue to recover scorpions in a basally branching position, partly owing to their morphological similarity to extinct marine orders like Eurypterida (sea scorpions). Phylogenomic datasets consistently recover scorpions in a derived position, as the sister group of Tetrapulmonata (a clade of arachnids that includes spiders). To adjudicate between these hypotheses using a rare genomic change (RGC), we leveraged the recent discovery of ancient paralogy in spiders and scorpions to assess phylogenetic placement. We identified homologs of four transcription factors required for appendage patterning (dachshund, homothorax, extradenticle, and optomotor blind) in arthropods that are known to be duplicated in spiders. Using genomic resources for a spider, a scorpion, and a harvestman, we conducted gene tree analyses and assayed expression patterns of scorpion gene duplicates. Here we show that scorpions, like spiders, retain two copies of all four transcription factors, whereas arachnid orders like mites and harvestmen bear a single copy. A survey of embryonic expression patterns of the scorpion paralogs closely matches those of their spider counterparts, with one paralog consistently retaining the putatively ancestral pattern found in the harvestman, as well as the mite, and/or other outgroups. These data comprise a rare genomic change in chelicerate phylogeny supporting the inference of a distal placement of scorpions. Beyond demonstrating the diagnostic power of developmental genetic data as a phylogenetic data class, a derived placement of scorpions within the arachnids, together with an array of stem-group Paleozoic scorpions that occupied marine habitats, effectively rules out a scenario of a single colonization of terrestrial habitat within Chelicerata, even in tree topologies contrived to recover the monophyly of Arachnida.

Formation of the diverticular lumen that enables oocyte fertilization in katoikogenic scorpions, Heterometrus spinifer and Opistophthalmus boehmi (Scorpionidae).

The structure of diverticula of the female gonads was analyzed in two scorpions from the family Scorpionidae by means of standard microscopic techniques (light microscopy, histochemistry, transmission electron microscopy). In scorpions, the female gonad, termed the ovariuterus, participates in two consecutive processes: oogenesis and embryogenesis. In sexually reproducing scorpions, the ovariuterus is also involved in fertilization. Both scorpions under study reproduce sexually. They also represent the katoikogenic type of development, which means that oogenesis, fertilization, and embryogenesis take place in the diverticula, which are sac-like outpocketings of the ovariuterine tubules. Formation of a lumen in the diverticulum is indispensable for sperm entry to enable fertilization and subsequent embryogenesis. The aim of the study was to test our hypothesis that the diverticulum lumen forms due to the engagement of the centrally located stalk cells. In this report, we show that in two species of katoikogenic scorpions, at the final stages of oogenesis, the cytoplasm of the stalk cells contains secretory organelles. In the stages preceding fertilization, secretory activity of the stalk cells and fragmentation of their apical parts lead to formation of the diverticulum lumen by a process similar to "cord hollowing" that commonly occurs for lumen formation in other morphogenesis model systems.

Homeosis in a scorpion supports a telopodal origin of pectines and components of the book lungs.

BACKGROUND: The morphological and functional evolution of appendages has played a key role in the diversification of arthropods. While the ancestral arthropod appendage is held to be polyramous, terrestriality is associated with the reduction or loss of appendage rami, which may obscure the homology of different appendage derivatives. Proxies for appendage homology have included surveys of cross-reactive antibodies for wing markers like Nubbin/PDM, which have suggested that the abdominal appendages of arachnids (e.g., book lungs, tracheal tubules) are derived from ancestral gills (epipods). RESULTS: Here, we discovered a rare case of inferred homeosis in a scorpion in which the bilobed genital opercula and the pectines are transformed to walking legs, and an abnormal sternite shows a book lung close to an everted structure comparable to the morphology of some Palaeozoic scorpion fossils. CONCLUSIONS: The observed morphology is consistent with abnormal expression of homeotic genes during embryonic development. The phenotype of this abnormal specimen suggests that the genital opercula, the pectines, and parts of the book lung may be derived from the telopodite of abdominal appendages rather than from epipods. This interpretation contradicts the "ancestral gill" hypothesis but reconciles features of the Palaeozoic scorpion fossil record with the embryology of modern scorpions.

Scorpions and life-history strategies: from evolutionary dynamics toward the scorpionism problem

This work aims to contribute to the general information on scorpion reproductive patterns in general including species that can be noxious to humans. Scorpions are unusual among terrestrial arthropods in several of their life-history traits since in many aspects their reproductive strategies are more similar to those of superior vertebrates than to those of arthropods in general. This communication focuses mainly on the aspects concerning embryonic and post-embryonic developments since these are quite peculiar in scorpions and can be directly connected to the scorpionism problem. As in previous similar contributions, the content of this communication is addressed mainly to non-specialists whose research embraces scorpions in several fields such as venom toxins and public health. A precise knowledge of reproductive strategies presented by several scorpion groups and, in particular, those of dangerous species may prove to be a useful tool in the interpretation of results dealing with scorpionism, and also lead to a better treatment of the problems caused by infamous scorpions.(AU)

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids.

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.

Hox gene duplications correlate with posterior heteronomy in scorpions.

The evolutionary success of the largest animal phylum, Arthropoda, has been attributed to tagmatization, the coordinated evolution of adjacent metameres to form morphologically and functionally distinct segmental regions called tagmata. Specification of regional identity is regulated by the Hox genes, of which 10 are inferred to be present in the ancestor of arthropods. With six different posterior segmental identities divided into two tagmata, the bauplan of scorpions is the most heteronomous within Chelicerata. Expression domains of the anterior eight Hox genes are conserved in previously surveyed chelicerates, but it is unknown how Hox genes regionalize the three tagmata of scorpions. Here, we show that the scorpion Centruroides sculpturatus has two paralogues of all Hox genes except Hox3, suggesting cluster and/or whole genome duplication in this arachnid order. Embryonic anterior expression domain boundaries of each of the last four pairs of Hox genes (two paralogues each of Antp, Ubx, abd-A and Abd-B) are unique and distinguish segmental groups, such as pectines, book lungs and the characteristic tail, while maintaining spatial collinearity. These distinct expression domains suggest neofunctionalization of Hox gene paralogues subsequent to duplication. Our data reconcile previous understanding of Hox gene function across arthropods with the extreme heteronomy of scorpions.

Reproductive system of female scorpion: a partial review.

The female scorpion ovariuterus was examined in 10 scorpion species belonging to five families: Buthidae, Vaejovidae, Scorpionidae, Urodacidae, and Diplocentridae. Two main patterns of development are known in scorpions: (1) The apoikogenic type with an ovariuterus containing yolk-rich eggs housed in follicles. This type is found in many scorpion taxa (largely buthids). A peculiar case of apoikogenic ovariuterus is a "beaded" ovariuterus where most of the ova's embryogenesis takes place inside the ovariuterus rather than on pedicels situated on the external wall of the ovariuterus as in most buthids. This type is found in a few scorpion species. (2) The katoikogenic type with an ovariuterus where the embryo develops in a diverticulum composed of four parts: a stalk (pedicel), a thickened collar, a conical portion containing the ovum, and an appendix containing the oral feeding apparatus where the embryos' chelicerae grip a "teat"-like structure, described in four families: Hemiscorpiidae, Scorpionidae, Urodacidae, and Diplocentridae. There are three kinds of diverticulae: small rudimentary finger-like diverticulae, embryonic (ED) large projections, and postpartum diverticulae (PPD) empty diverticulae, which are remnants after parturition. The subject is reviewed and its bearing on reproduction in scorpions are discussed.

Observations on the life history of Chaerilus philippinus Lourenço & Ythier, 2008 (Scorpiones, Chaerilidae) from the Philippines.

Biological observations on Chaerilus philippinus were based on specimens from the region of Appari, North of Luzon in the Philippines. The total duration of embryonic development was estimated as being between 110 to 136 days, while the moults between successive juvenile instars and adulthood took place at ages that averaged 7, 39, 73, 190 and 327 days. These developmental periods are shorter and different from those previously observed among species of non-buthid scorpions. They prove to be rather similar to those observed in buthid scorpions, however. Morphometric growth values of the different instars are similar or smaller than those of other species of scorpions that have been studied. Aspects of maternal care and social behaviour are also commented.

Development of respiratory structures in embryos and first and second instars of the bark scorpion, Centruroides gracilis (Scorpiones: Buthidae).

The SEM was used to study the development of respiratory structures in successive stages in relation to the overall changes occurring in the scorpions. Book lung development is a slow process, starting with spiracles and a sac-like atrium in the early embryo and continuing lamellar formation to 150 or more in the adult. In the embryo, the primordial epithelial cells become aligned in a planar pattern as they secrete granules of material that aggregate spontaneously to form the cuticular walls of the lamellae. A blade-like structure is formed consisting of cells sandwiched within the two cuticle walls they secreted. These cells are in the primordial air channel. The adjacent hemolymph channel is nearly devoid of cells, but cross-bridges develop and help stabilize the cuticle walls and maintain the width of the channel. The cells in the primordial air channel undergo cytolysis, leaving it open for air except for cuticular cross-bridges. Development continues in the newborn (first instars); the air channels of some lamellae still contain cells and are not yet functional for gas exchange. The first instars are weak and relatively inactive. They climb up on the mother's dorsum until the first molt (about 8 days). With the cuticular walls of the lamellae in place, cells adhering to the wall in the hemolymph channel produce a thin, new tissue layer (epithelium) on the lamellar wall facing the hemolymph channel. This layer has many discontinuities as though it is slowly developing. Formation of the tissue layer and cytolysis of the cells in the air channels continue through the first molt in which little book lung cuticle is shed as exuvium. The air channels of the second instars (foraging nymphs) are now cell free and open for air passage except for the cross-bridges. The tissue layer is still incomplete and continues to be formed. It may provide the hypodermal primordium for cuticle replacement in later molts, but development was not studied beyond the second instar except for comparison with book lungs in the adult. The blade-like lamellae in the adult are larger and more numerous than in the second instar, but in the anterior book lung the shape of the cuticle wall and cross-bridges and the widths of the air and hemolymph channels are about the same as in the second instar. The air channels in the posterior part of the lamellae have distinctive, vein-like space-holders. The similarity of the adult anterior lamellae with those in the second instar suggests retention of this part through the 4-5 molts to maturation, and/or cell processes like those in the embryo are repeated, but this needs to be examined in further studies of cell and cuticle changes before and during the molts.

Orthodenticle and empty spiracles genes are expressed in a segmental pattern in chelicerates.

Members of the orthodenticle (otd/Otx) and empty spiracles (ems/Emx) gene families are head gap genes that encode homeodomain-containing DNA-binding proteins. Although numerous studies show their central role in developmental processes in brain specification, a surprisingly high number of other developmental processes have been shown to involve their expression. In this paper, we report the identification and expression of ems and otd in two chelicerate species: a scorpion, Euscorpius flavicaudis (Chactidae, Scorpiona, Arachnida, Euchelicerata) and a spider, Tegenaria saeva (Aranea, Arachnida, Euchelicerata). We show that both ems and otd are expressed not only in an anterior head domain but also along the entire anterior-posterior axis during embryonic development. The expression patterns for both genes are typically segmental and concern neurectodermal territories. During patterning of the opisthosoma, ems and otd are expressed in the lateral ectoderm just anterior to the limb bud primordia giving rise to respiratory organs and spinnerets (spider). This common pattern found in two divergent species thus appears to be a conserved character of chelicerates. These results are discussed in terms of evolutionary origin of respiratory organs and/or functional pathway recruitment.

Developmental changes in the embryo, pronymph, and first molt of the scorpion Centruroides vittatus (scorpiones: buthidae).

For the first time the scanning electron microscope was used to compare developmental changes in scorpion embryos and the first and second stadia. In the buthid species of this study, Centruroides vittatus, and all other scorpions, the newborn climb up on their mother's back and remain there without feeding for several days. At this location, they undergo their first molt and in a few days they disperse, fully capable of foraging in the terrestrial environment. The results here support earlier suggestions that the first stadium (pronymph) is a continuation and extension of embryological development. The first molt results in a nymph with exoskeletal features much like those in the adult. In the first molt the metasoma becomes relatively longer, and the sting (aculeus) becomes sharp and functional. The metasomal segments are modified for dorsal flexion and sting use. The embryos and the pronymphs have spiracles that open into an invagination near the posterior margin of flap-like abdominal plates in segments 4-7 of the ventral mesosoma. The second instars have spiracles that lead to book lungs farther anterior in sternites. Tubular legs with cylindrical segments in embryos and pronymphs become more sculptured and oval in the transverse plane. Each leg in the pronymph has a blunt, cup-shaped tip while distal claws (ungues, dactyl) are present in the second instar and subsequent stages. There are some sharp bristles and primordial sensilla in the pronymphs, but the second stadium has adult-like surface features: rows of knobs or granulations (carinae), serrations on the inner surfaces of cheliceral and pedipalpal claws, filtering hairs at the mouthparts, peg sensilla on the pectines, and mechano- and chemoreceptor sensilla on the body and appendages. Scorpion embryos and pronymphs have some structures like fossil scorpions thought to have been aquatic. There is a gradual development of features that appear to be terrestrial adaptations. Evidence is provided for the formation of the sternum from third and fourth leg coxal primordia and possibly from the first abdominal segment. This study is the first to provide evidence for a forward shift of the gonopore along with other structures in the anterior abdomen.

hedgehog is a segment polarity gene in a crustacean and a chelicerate.

The evolution of arthropod segmentation has been studied by comparing expression patterns of pair-rule and segment polarity genes in various species. In Drosophila, the formation and maintenance of the parasegmental boundaries depend on the interactions between the wingless (wg), engrailed (en) and hedgehog (hh) genes. Until now, the expression pattern of hh has not been analysed to such a great extent as en or wg. We report the cloning and expression analysis of hh genes from Euscorpius flavicaudis, a chelicerate, and Artemia franciscana, a branchiopod crustacean. Our data provide evidence that hh, being expressed in the posterior part of every segment, is a segment polarity gene in both organisms. Additional hh expression sites were observed in the rostrum and appendages of Euscorpius and in the gut of Artemia. From the available data on hh expression in various bilaterians, we review the various hypotheses on the evolution of hh function and we suggest an ancestral role of hh in proctodeum specification and gut formation.

Development of segments and appendages in embryos of the desert scorpion Paruroctonus mesaensis (Scorpiones: Vaejovidae).

The scanning electron microscope was used to study the changing features of scorpion embryos from the blastula through early stages in the development of appendages. The earliest scorpion fossils (Silurian period) have structures more advanced than the embryos herein, so the possibility is considered that these embryos still retain and display some features indicative of evolutionary patterns in adult pre-Silurian ancestors. The blastodisc stage is followed by a knob-like germinal center that gives rise to most of the embryo body. The germinal center elongates on the ventral surface of the spherical yolk mass. The broad cephalic lobe is first delineated from the following pedipalpal segment. The limbbuds for the pedipalps and anterior walking legs appear, as additional segments are added at a growth zone at the rear of the embryo body. Initially, in the cephalic lobe there are no limbbuds; then the cheliceral buds emerge from the posterior part of the lobe. The stomodeum appears first in the anterior half of the cephalic lobe, but an oral groove forms and the mouth is displaced posteriorly within the groove. This repositioning allows space anteriorly for invagination (semilunar grooves) of epithelium for the brain and medial eyes. The mouth is directed ventrally in all stages of this study. The widespread chelicerae are initially posterior to the mouth, but later move anterior and dorsal to it. Small limbbud bulges on mesosomal segments disappear later and never become protruding appendages. Metasomal segments are produced free from the yolk surface in a ventral flexure beneath the embryo body. The telson starts as two spherical lobes, but later elongates and tapers distally, not yet developing the sharp sting (aculeus) seen in Silurian and all subsequent scorpions. The walking legs are digitigrade, as in most fossil aquatic scorpions. Segments are delineated in the appendages; the chelicerae and pedipalps are divided distally for chela (claw) formation. Bilateral swellings (limbbuds) on the third abdominal segment become larger than the others, indicating the site of pectine formation. The early fin-like pectines are somewhat posterior in the mesosoma, suggesting ancestral swimming, maneuvering, and balancing for the elongate abdomen. The pectinal surface is initially smooth but later transverse striations increase the surface area as a possible respiratory adaptation. Pectinal teeth (present in Silurian and all subsequent scorpions) and forward movement and merging of anterior abdominal segments are not yet evident in embryos of this study.

Egg maturation and parthenogenetic recovery of diploidy in the scorpion Liocheles australasiae (Fabricius) (Scorpions, ischnuridae).

In the scorpion Liocheles australasiae, egg maturation and parthenogenetic recoveries of chromosome number and nuclear DNA content were examined by histological, karyological observations and quantitative measurements of DNA. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus and a second polar body nucleus, not followed by cytoplasmic fission. The first polar body, in one case, was successively divided into two second polar bodies; in the other case it was not divided. In either case, these polar bodies remained attached to the early embryo. The fate of these polar bodies during further embryogenesis were studied. In the karyological analysis, the chromosome number was divided into two groups, one from 27-32, the other was 54-64. The former was presumably the metaphase chromosome number at the meiotic division; the latter was presumably the metaphase chromosome number at the mitotic division. DNA content in the diploid nucleus of the primary oocyte, doubled before the maturation divisions, was reduced through the maturation divisions by one-half in the nuclei of the secondary oocyte and the first polar body and by one-fourth in the nuclei of the egg and the second polar bodies. The first reduction of DNA content corresponded to halving the number of the chromosomes in the first maturation division and the second to the nuclear division in the secondary oocyte. These reductions represent a common process of egg maturation, except the final production of the mature egg with two haploid nuclei, an egg nucleus, and a second polar body nucleus. These two nuclei, which were formed apart in the mature egg, drew near to fuse into a zygote nucleus. The chromosome number and nuclear DNA content were doubled in the zygote and each blastomere in embryos, supporting the hypothesis that the egg nucleus fuses with the second polar body nucleus and this conjugation initiates subsequent embryonic development.

Conservation and variation in Ubx expression among chelicerates.

Chelicerates are an ancient arthropod group with a distinct body plan composed of an anterior (prosoma) and a posterior portion (opisthosoma). The expression of the Hox gene Ultrabithorax (Ubx) has been examined in a single representative of the chelicerates, the spider Cupiennius salei. In spiders, Ubx expression starts in the second opisthosomal segment (O2). Because the first opisthosomal segment (O1) in spiders is greatly reduced relative to other chelicerates, we hypothesized that the observed Ubx expression pattern might be secondarily modified. Shifts in the anterior boundary of the expression of Ubx have been correlated with functional shifts in morphology within malacostracan crustaceans. Thus, the boundary of Ubx expression between chelicerates with different morphologies in their anterior opisthosoma could also be variable. To test this prediction, we examined the expression patterns of Ubx and abdominal-A (collectively referred to as UbdA) in two basal chelicerate lineages, scorpions and xiphosurans (horseshoe crabs), which exhibit variation in the morphology of their anterior opisthosoma. In the scorpion Paruroctonus mesaensis, the anterior border of early expression of UbdA is in a few cells in the medial, posterior region of the O2 segment, with a predominant expression in O3 and posterior. Expression later spreads to encompass the whole O2 segment and a ventral, posterior portion of the O1 segment. In the xiphosuran Limulus polyphemus, early expression of UbdA has an anterior boundary in the segment. Later in development, the anterior boundary moves forward one segment to the chilarial (O1) segment. Thus, the earliest expression boundary of UbdA lies within the second opisthosomal segment in all the chelicerates examined. These results suggest that rather than being derived, the spider UbdA expression in O2 likely reflects the ancestral expression boundary. Changes in the morphology of the first opisthosomal segment are either not associated with changes in UbdA expression or correlate with late developmental changes in UbdA expression.

Ovariuterus of Pandinus imperator, Koch (Scorpiones; Scorpionidae): comparison of virgin female with mother.

The ovariuterus of the female Pandinus imperator Koch (Scorpiones; Scorpionidae), was compared in a virgin female and a female that had previously given birth at least twice (in the laboratory). The virgin female did not have any embryonic diverticulae (Ed) nor did it have any degenerated, post-partum diverticulae (Dd), whereas in the mother scorpion several Dd were clearly seen on the ovariuterus. This latter female lacked any embryonic diverticulae (Ed). The number of the Dd corresponds well with the number of juveniles in the last brood born to that female during the previous year. Based on the total number of diverticula observed, and the average known litter size, it is suggested that these long-lived scorpions are potentially able to breed at least six times during their lifetime. Since they apparently do not breed in consecutive years (as is evident from the lack of Ed in the female that had bred a year before) and perhaps only every alternate year, they are capable of breeding for 12 years. If a litter amounts to about 25 young, a female is capable of producing 150 young. As it takes about three years to mature from nymph to adult, the life expectancy in this species is therefore about 15 years, by a conservative estimate.