Pycnogonum Litorale Research Articles

The sea spider Pycnogonum litorale overturns the paradigm of the absence of axial regeneration in molting animals

Regenerative abilities and their evolution in the different animal lineages have fascinated generations of biologists. While some taxa are capable of restoring entire individuals from small body fragments, others can regrow only specific structures or lack structural regeneration completely. In contrast to many other protostomes, including the segmented annelids, molting animals (Ecdysozoa) are commonly considered incapable of primary body axis regeneration, which has been hypothesized to be linked to the evolution of their protective cuticular exoskeleton. This holds also for the extraordinarily diverse, segmented arthropods. Contradicting this long-standing paradigm, we here show that immatures of the sea spider Pycnogonum litorale reestablish the posterior body pole after transverse amputation and can regrow almost complete segments and the terminal body region, including the hindgut, anus, and musculature. Depending on the amputation level, normal phenotypes or hypomeric six-legged forms develop. Remarkably, also the hypomeric animals regain reproductive functionality by ectopic formation of gonoducts and gonopores. The discovery of such complex regenerative patterns in an extant arthropod challenges the hitherto widely assumed evolutionary loss of axial regeneration during ecdysozoan evolution. Rather, the branching of sea spiders at the base of Chelicerata and their likely ancestral anamorphic development suggests that the arthropod stem species may have featured similar regenerative capabilities. Accordingly, our results provide an incentive for renewed comparative regeneration studies across ecdysozoans, with the aim to resolve whether this trait was potentially even inherited from the protostome ancestor.

Early lecithotrophic stages of Nymphon grossipes, and the role of larval appendages and glands in different larval types of pycnogonids.

Nymphon grossipes is a common subtidal species belonging to a small and unique group of chelicerates, that is, the sea spiders. These animals have an anamorphic phase during post-embryonic development and often hatch as small, oligomeric and exotrophic larvae (protonymphons) with four postocular segments, cheliphores, and two pairs of larval legs. A common alternative to protonymphons is a large lecithotrophic larval type, where animals hatch at more advanced stages and have a foreshortened anamorphic development. Based on external morphology, N. grossipes was believed to be an intriguing intermediate between these two conditions and its hatchlings were called "lecithotrophic protonymphons." Here, we examine the anatomy and ultrastructure of instars I and II and review the variety of roles of larval appendages and associated glands in other sea spiders in order to correctly place the larva of this species among pycnogonid larval types. Compared to "typical protonymphons," N. grossipes young hatch with an advanced segmental and appendage composition: six postocular segments instead of four, buds of walking legs 1 and hidden buds of walking legs 2. This state corresponds to the instars II/III (rather than larvae) of Nymphon brevirostre and Pycnogonum litorale. Modifications of the larval appendages, chelar, and spinning glands are aligned with ecological needs of different larval types along a few typical dimensions: locomotion and feeding, dispersal, and attachment to the parent. Although the main challenge for N. grossipes young is secure attachment to the egg package while they growth, there are some discrepancies in their anatomy: N. grossipes retains an oyster basket, but an otherwise nonfunctional digestive system, and a strong silken thread for attachment, but no corresponding reduction of the larval legs. Thus, it is likely that the switch to lecithotrophy happened in the recent evolutionary history of this species.

Cover Image

Sea spiders (Pycnogonida) are a small group of basal arthropods, sister to chelicerates. They have an unusual adult bauplan, oligosegmented larvae, and a protracted postembryonic development. In this issue of the Journal of Morphology Alexeeva and Tamberg (pp. 329–354) report about the postembryonic development of Pycnogonum litorale. The cover image shows a light micrograph of a histological midsagittal section of an instar VIII.

Anatomical changes in postembryonic development of Pycnogonum litorale.

Sea spiders (Pycnogonida) are a small group of arthropods, sister to other chelicerates. They have an unusual adult bauplan, oligosegmented larvae, and a protracted postembryonic development. Pycnogonum litorale (Strøm, 1762) is an uncommonly long-lived sea spider with a distinctive protonymphon and adult anatomy. Although it was described ~250 years ago, little is known about its internal organization and development. We examined the anamorphic and early epimorphic development of this species using histology, light microscopy, and SEM, and provide the first comprehensive anatomical study of its many instars. Postembryonic development of P. litorale includes transformations typical of pycnogonids: reorganization of the larval organs (digestive, nervous, secretory), formation of the abdomen, trunk segments (+ appendages), primary body cavity and reproductive system. Specific traits include the accelerated articulation of the walking legs, formation of the subesophageal and posterior synganglia, and the system of twin midgut diverticula. In addition, P. litorale simultaneously lose the spinning apparatus and all larval appendages. We found that developmental changes occur in synchrony with changes in ecology and food sources. The transition from the anamorphic to the epimorphic period in particular is marked by considerable anatomical and lifestyle shifts. HIGHLIGHTS: Postembryonic development of P. litorale includes numerous anamorphic and epimorphic stages. The instars acquire abdomen, trunk segments, body cavity, and gonads, while losing all larval appendages. Developmental changes are synchronized with changes in lifestyle and food sources.

The (not very) typical protonymphons of Pycnogonum litorale.

Sea spiders are unique and poorly known marine chelicerates. Their larvae are even less studied, especially at the ultrastructural level. Here, we examined the hatchlings of Pycnogonum litorale (Strøm, 1,762) using histology, SEM and TEM. Existing classifications place these larvae among "typical" protonymphons, together with Nymphon brevirostre. Our results, however, revealed major differences between the two species. Hatchlings of P. litorale are endotrophic for 1-2 weeks, with yolk deposits in the body wall and a reduced secretory apparatus. They lack a body cavity, demonstrate an unusual modification of the midgut sheath cells and a complex subesophageal ganglion, which includes neuromeres of the prospective walking legs 1. These larvae also possess well-developed glia and complex sensory structures: eyes, V-shaped mechanoreceptive bristles, integrated chemo- and mechanoreceptors, and three types of concealed mechanoreceptors embedded into the body wall and only seen on the sections. In this paper we also propose a new interpretation of the pycnogonid larval types: we present a set of traits useful for diagnosis and a preliminary classification. Finally, we discuss the complexity of glial types in sea spiders and other arthropods.

Comparative study of bisected proboscides of Pycnogonida

To comparatively describe “inner” pharynx armatures in pycnogonids, we bisected proboscides of Achelia langi (Dohrn, 1881), Anoplodactylus californicus Hall, 1912, Ascorhynchus castellioides Stock, 1957, Austrodecus glaciale Hodgson, 1907, Callipallene margarita (Gordon, 1932), Colossendeis macerrima Wilson, 1881, Endeis spinosa (Montagu, 1808), Nymphon macronyx Sars, 1877, Pallenopsis patagonica (Hoek, 1881), Pantopipetta sp., Pigrogromitus timsanus Calman, 1927, and Pycnogonum litorale (Strom, 1762) and analyzed them with the scanning electron microscope (SEM). Moreover, proboscides were stained with actin green to visualize the muscle arrangement with fluorescence and confocal microscopes (A. langi) and were analyzed with microcomputed X-ray tomography (μCT; Ascorhynchus japonicus). As a result of our observations, sets of characters that vary between taxa are established. These traits include length and width of proboscis, shape and structure of the inner mouth opening, borders and armature of the antimeres, shape and position of denticle arrays and rows, relative length of pharyngeal filter apparatus, and arrangement and structure of the filter bristles. Analyses of these characters indicate a substantial variability on the pantopod’s proboscis inner surface probably as an adaptation to different food sources. Finally, we suggest that the presence of the oyster basket represents a ground pattern character of Pycnogonida.

The pattern of a specimen of Pycnogonum litorale (Arthropoda, Pycnogonida) with a supernumerary leg can be explained with the "boundary model" of appendage formation.

A malformed adult female specimen of Pycnogonum litorale (Pycnogonida) with a supernumerary leg in the right body half is described concerning external and internal structures. The specimen was maintained in our laboratory culture after an injury in the right trunk region during a late postembryonic stage. The supernumerary leg is located between the second and third walking legs. The lateral processes connecting to these walking legs are fused to one large structure. Likewise, the coxae 1 of the second and third walking legs and of the supernumerary leg are fused to different degrees. The supernumerary leg is a complete walking leg with mirror image symmetry as evidenced by the position of joints and muscles. It is slightly smaller than the normal legs, but internally, it contains a branch of the ovary and a gut diverticulum as the other legs. The causes for this malformation pattern found in the Pycnogonum individual are reconstructed in the light of extirpation experiments in insects, which led to supernumerary mirror image legs, and the “boundary model” for appendage differentiation.

A scanning electron microscopy study of the embryonic development of Pycnogonum litorale (Arthropoda, Pycnogonida)

The phylogenetic position of the enigmatic Pycnogonida (sea spiders) is still controversial. This is in part due to a lack of detailed data about the morphology and ontogenesis of this, in many aspects, aberrant group. In particular, studies on the embryonic development of pycnogonids are rare and in part contradictory. Here, we present the first embryological study of a pycnogonid species using scanning electron microscopy (SEM). We describe the late embryogenesis of Pycnogonum litorale from the first visible appendage anlagen to the hatchling in 11 embryonic stages. The three pairs of appendage anlagen gain in length by growth, as well as by extension of furrows into the embryo. The opening of the stomodaeum is located far in front of the anlagen of the chelifores and has a Y-shaped lumen from the onset. During further embryogenesis, the position of the mouth shifts ventrally, until it is located between the chelifores. The proboscis anlage grows out as a circumoral wall-like structure, which is initially more pronounced ventrally. Hypotheses about the evolution of the proboscis by fusion of originally separated components are critically discussed, because the proboscis anlage of P. litorale shows no indications of a composite nature. In particular, a participation of post-cheliforal elements in proboscis formation is rejected by our data. Further, no preoral structure and no stage in proboscis formation was found, which could plausibly be homologized with the labrum of othereuarthropods. Thus, our study supports the assumption of a complete lack of a labrum in Pycnogonida.

Cleavage and gastrulation in Pycnogonum litorale (Arthropoda, Pycnogonida): morphological support for the Ecdysozoa?

The early cleavage and gastrulation of the pycnogonid Pycnogonum litorale is investigated in detail by fluorescence microscopy, confocal laser scanning microscopy, and histology. The cleavage is holoblastic with equally sized blastomeres and an irregular radial pattern. There is no stereotypic cell lineage, and timing and spindle directions of individual mitoses vary to a high degree. Gastrulation begins at the 63-cell stage with the retardation and enlargement of a cell which adopts the form of a bottle and fills the interior of the egg, followed by immigration and epiboly of smaller cells surrounding the large bottle-shaped cell. The gastrulation site marks the dorsal side of the embryo and the stomodaeum forms adjacent to the area of gastrulation. The pattern of the early development of Pycnogonum is compared with that of other Pycnogonida resulting in a putative ground pattern of pycnogonid development. Furthermore, our results are discussed in the wider framework of putative arthropod and cycloneuralian relationships. This comparison implies morphological support for the Ecdysozoa.

Larval development and morphogenesis of the sea spider Pycnogonum litorale (Ström, 1762) and the tagmosis of the body of Pantopoda

Aspects of pantopod ontogeny have been known for a long time, but specific information is available for only a few species. Our account of the postembryonic development of Pycnogonum litorale is based on laboratory-reared individuals and SEM studies. We documented particularly all early developmental stages, with emphasis on morphogenetic changes of head structures and appendages. In P. litorale the protonymphal limbs, the chelicerae and two more uniramous legs, degenerate already during the larval phase; only the third one, the ovigers, reappears in male juveniles. Other Pantopoda vary in this aspect from retention of all three protonymphal appendages to their complete reduction, as in P. litorale. Accordingly, the two post-cheliceral larval appendages are separate legs in front of the walking legs in the adults, the ‘parapalps’ and the ‘ovigers’, but they do not occur in all pantopods. The scarcity of studies of the ontogeny of Pantopoda prevents us from a more conclusive picture, but our data are promising to state that additional such studies will increase the usability of ontogenetic data for a phylogenetic analysis of Pantopoda, the crown group of the Pycnogonida. We also discuss the phylogenetic implications of our data in the light of new information from Hox genes and developmental-biological data on body segmentation and tagmosis of the Chelicerata. These suggest the homology of chelicerae and antenn(ul)ae of other euarthropods. Accepting this, we conclude that the adult pycnogonid/pantopod head, the cephalosoma, corresponds to the euarthropod head and that the protonymph with three appendage-bearing segments may represent an even shorter, possibly phylogenetically older larval type than the euarthropod ‘head larva’ bearing four pairs of appendages. In further consequence, the fourth walking legs of Pycnogonida/Pantopoda should correspond to the first opisthosomal appendages, the chilaria, of euchelicerates. This implies that within Pycnogonida the post-prosomal region became compacted during evolution to a single leg-bearing segment plus a tubular end piece. Accordingly, neither the anterior nor the posterior functional boundaries of the walking-leg region correspond to the original tagma borders.

Growth and reproduction of Pycnogonum litorale (Pycnogonida) under laboratory conditions

The complete life cycle of Pycnogonum litorale Strom was observed under laboratory conditions. At 15 °C and a 16 h light:8 h dark photocycle, the duration of embryonic development from oviposition to egg-hatching varied from 32 to 111 d. The onset of egg hatching ranged from 32 to 81 d after oviposition in different egg batches, and the period between the first and the last hatch within a single egg batch varied between 16 and 57 d. The larval period, consisting of five larval instars, lasted between 66 and 113 d. Juvenile development required seven (exceptionally 8 or 9) moults and lasted between 263 and 400 d. The average interval between successive moults in juveniles increased with increasing size from 24 to 82 d. Though females did not moult more often than their male conspecifics, they reached a size from 8.5 to 11.0 mm (average 10.1 mm) while adult males measured only 7.0 to 8.5 mm (average 7.7 mm). At low temperatures (2 °C) moulting was almost completely inhibited. After raising the temperature to 6 °C, the moulting frequency increased to a rate almost as high as at a constant temperature of 15 °C. Moulting was also retarded by starvation and accelerated by subsequent feeding. Adults lived for up to 9 years without further moults, with several periods of mating and oviposition at irregular intervals. In combination with previous long-term field observations, the present results provide a more complete picture of the life cycle of P. litorale in a natural habitat. The great variation in the duration of the different developmental stages, the ability to survive periods of cold and starvation, and the longevity of the adults are important for the survival of pycnogonid populations under changing environmental conditions.

Life cycle and population dynamics of Pycnogonum litorale (Pycnogonida) in a natural habitat

A natural population of Pycnogonum litorale Strom was examined every 4 weeks over a period of 15 months and thereafter at yearly intervals for 15 years. Adult pycnogonids – mating couples and males carrying egg batches – and freshly hatched protonymphon larvae within these egg batches were found throughout the year. The second, third, and fourth instar larvae were only found from April to July, during the vegetation period of their hydroid host Clava multicornis. After metamorphosis to the fifth instar (first juvenile instar) the pycnogonids have a significantly larger proboscis than during the larval period, and they feed on the sea anemone Metridium senile. First juvenile instars were found on M. senile from May to August. Older and larger juvenile stages were found over longer time spans throughout the year, and the maximum number of successive instars shifted slowly from June to December. Freshly moulted adults occurred throughout the year. Males, which on the average are smaller, usually reach the adult stage during late autumn of the first year and females, at the end of the following spring. We conclude that in nature the development from egg to adult stage is completed within one year. Continuous reproduction and asynchronous embryonic development provide offspring throughout most of the year. The annual cycle is synchronized by the vegetation period of C. multicornis, the only host of these pycnogonid larvae in the investigated habitat, and by the arrest of growth during low winter temperatures. The low level of locomotory activity of P. litorale probably requires an environment in which both host species coexist. The abundance of C. multicornis, M. senile, and juvenile pycnogonids decreased from 1990 to 1996, maybe due to hydrographic conditions.

Ecdysteroids fromPycnogonum litorale (Arthropoda, Pantopoda) act as chemical defense againstCarcinus maenas (Crustacea, Decapoda)

Pycnogonum litorale (Ström) is unpalatable to the common shore crabCarcinus maenas, a generalist predator in the pycnogonid's habitat. A feeding bioassay reveals that the crabs are deterred by ecdysteroids that occur in high levels in all developmental stages ofP. litorale. The total ecdysteroids in the pycnogonids reach 5.9×10(-4) M. The 20-hydroxyecdysone 22-acetate (20E22A), which is the predominant ecdysteroid in the pycnogonids, and 20-hydroxyecdysone (20E), the arthropod molting hormone, were tested for their antifeedant effect onC. maenas. When contained in food pellets in homogeneous concentrations, 20E and 20E22A significantly reduced food consumption at 1.25×10(-4) and 5.0×10(-4) molar levels, respectively. The present results demonstrate for the first time chemical defense in arthropods in a marine predator-prey relationship. Furthermore, they provide evidence that ES contained in one animal can act as feeding deterrents on another animal.

Defensive Secretion of Ecdysteroids in Pycnogonum litorale (Arthropoda, Pantopoda)

Abstract In response to disturbance, the pycnogonid Pycnogonum litorale discharges a mixture of eight ecdysteroids (ES). Repeated intensive molestation causes 99% secretion of the endogenous ES present. The concentration of the total ES in the defensive effluent is 1.0 × 10-3 mol · 1-1. 20-Hydroxyecdysone 22-acetate, the predominant ES, reaches 0.8 × 10-3 mol · 1-1. This is sufficient to deter significantly feeding by the common shore crab Carcinus maenas, a generalist predator in the habitat of the pycnogonid. There is evidence that the secreted ES of P. litorale contribute to its unpalatability. The present paper describes for the first time defensive secretion in marine arthropods.

22-deoxy-20-hydroxyecdysone and its phosphoric ester from ovaries of the silkworm, Bombyx mori

A principal unidentified compound in the free ecdysteroid fraction and its conjugated form were purified from c.3 kg of ovaries of the silkworm, Bombyx mori, mainly by means of thin-layer chromatography and high-performance liquid chromatography. The purified compounds were identified as 22-deoxy-20-hydroxyecdysone (22d20E) and 22-deoxy-20-hydroxyecdysone 3-phosphate (22d20E3P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. Although 22d20E had previously been isolated from the leaves and stems of the yew tree, Taxus cuspidata, and from the whole bodies of the sea spider, Pycnogonum litorale, it had not yet been obtained from insect ovaries. 22D20E3P was newly identified in the present study.

Excessive Abundance and Dynamics of Unusual Ecdysteroids in Pycnogonum litorale Ström (Arthropoda, Pantopoda)

In embryos, larvae, and juveniles of Pycnogonum litorale, unusual ecdysteroids have been found at excessively high levels. Concentrations and dynamics of the following eight ecdysteroids were determined by HPLC: 20-hydroxyecdysone, 20-hydroxyecdysone 22-glycolate, (25 R) and (25 S) isomers of 20,26-dihydroxyecdysone 22-acetate, 22-deoxy-20,26-dihydroxyecdysone, 20-hydroxyecdysone 22-acetate, 22-deoxy-20-hydroxyecdysone, and ecdysone 22-glycolate. The excessively high content of ecdysteroids is found in all developmental stages. Juvenile pycnogonids contain the highest total ecdysteroid amounts ever found in arthropods: 0.1% of their body dry weight. 20-Hydroxyecdysone 22-acetate is the prominent ecdysteroid and represents at all stages of both sexes, 66.2-85.8% of the total ecdysteroids. This compound, as well as all other ecdysteroids without a free 22-OH group, are presumed to be inactive as molting hormones. In contrast to these compounds, 20-hydroxyecdysone is present only in amounts similar to those in other arthropods. Furthermore, it is the only ecdysteroid with its peak at apolysis. It is regarded as the molt-promoting hormone. The origin and function of the other ecdysteroids in different developmental stages are discussed.

20-hydroxyecdysone stimulates molting in pycnogonid larvae (arthropoda, pantopoda)

Molting in pycnogonid larvae, Pycnogonum litorale, is accelerated by exposing the animals to 20-hydroxyecdysone at concentrations of 10 to 1000 ppm during Days 5 to 9 after the first ecdysis. Hormone application at the beginning or at the end of the molt cycle, as well as treatment all through the molt cycle, delays or even inhibits the molt by upsetting the sequence of molting processes.

Novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods.

Cross-linked and uncross-linked acrylamide polymers were used to alleviate technical difficulties in cryosectioning and autoradiographic processing of marine arthropods (Pycnogonum litorale), larval insect tissues (Sarcophaga bullata), and amphibian (Xenopus laevis) oocytes. Rapidly polymerized cross-linked acrylamide was used to prepare sections from an animal with a hard thick cuticle, P. litorale. Fragmentation and compression artifacts caused by tissue density differences were avoided and internal tissues were well preserved. Our results indicate that closely applied external support eliminates difficulties typically associated with arthropod cryosectioning. Pre-polymerized, uncross-linked acrylamide provided similar protection when used for sectioning larval tissue from S. bullata. Soft tissues under the cuticle were undamaged in tracheal and epidermal preparations. This polymer was also an excellent embedding material for soft tissues such as salivary glands, quickly penetrating tissue cavities and eliminating air bubbles. Uncross-linked acrylamide was used as an embedding and culturing medium for oocytes from X. laevis. The polymers were non-toxic and allowed the preparation of thin frozen sections containing as many as 50 large oocytes per section.

Ecdysteroids in the pycnogonid Pycnogonum litorale (Ström) (arthropoda, pantopoda)

Two ecdysteroids were isolated from juveniles and adults as well as from eggs of the pycnogonid Pycnogonum litorale Ström and characterized by bioassay, thin layer chromatography, gas liquid chromatography, and high performance liquid chromatography. One of the hormones could be identified as 20-hydroxyecdysone whereas the other behaves in HPLC in a way that is similar to but not identical with ecdysone. This unidentified ecdysone-like hormone exceeded 20-hydroxyecdysone in all developmental stages (1.67:1 to 3:1). Determination of moulting hormone titers by a radioimmunoassay showed highest amounts of hormones in juvenile females (150 ng/mg tissue dry wt). The results are compared with those obtained from other arthropods and are discussed in view of the evolution of hormonal systems.

The influence of exogen and endogen factors on the moulting of Pygnogonum litorale (Ström)

Pycnogonum litorale has a terminal anecdysis. After attaining maturity it does not moult again. Maturity can be attained at different body size, the female usually being larger than the male.The pre-adult instar can also be entered at various body sizes. The duration of the pre-adult instar depends upon body size: At 19°C its mean duration was 86 days, that of the preceding instar 71 days.The freshly moulted animal is transparent. It feeds soon after each moult, which is essential for hardening the integument. Hardening and stretching of the integument continue for about three weeks.Photoperiods differing from those at which the animal was collected, delay moulting. Long day favours adult development.Temperatures of 2-3° C block the moults.The adult moult is not so easily influenced by these factors as the juvenile moults. Possibly a critical phase aster which moulting can no longer be prevented occurs sooner during the pre-adult instar than in earlier instars.After loss of leg parts the rest of the leg is not automomized. The limbs of juvenile animals regenerate at the following moult. Moulting is not accelerated but rather delayed.With Ecdyson and Ecdysterone moults have not yet been evoked.