Engrailed-like immunoreactivity in the embryonic ventral nerve cord of the Marbled Crayfish (Marmorkrebs).

The homeobox transcription factor Engrailed is involved in controlling segmentation during arthropod germ band formation but also in establishing individual neuronal identities during later embryogenesis. In Crustacea, most studies analysing the expression of Engrailed so far have focussed on its function as segment polarity gene. In continuation to these previous studies, we analysed the neuronal expression of the Engrailed protein by immunohistochemistry in the embryonic nerve cord of a parthenogenetic crustacean, the Marbled Crayfish (Marmorkrebs). We paid particular attention to the individual identification of Engrailed expressing putative neuroblasts in the crayfish embryos. Engrailed positive cells in the neuroectoderm were counted, measured and mapped from 38 to 65% of embryonic development. That way, several Engrailed positive putative neuroblasts and putative neurons were identified. Our findings are compared with earlier studies on Engrailed expression during germ band formation in Crustacea. Recent data on neurogenesis in an amphipod crustacean have provided compelling evidence for the homology of several identified neuroblasts between this amphipod and insects. The present report may serve as a basis to explore the question if during crustacean neurogenesis additional communalities with insects exist.

The engrailed-expressing secondary head spots in the embryonic crayfish brain: examples for a group of homologous neurons in Crustacea and Hexapoda?

Hexapoda have been traditionally seen as the closest relatives of the Myriapoda (Tracheata hypothesis) but molecular studies have challenged this hypothesis and rather have suggested a close relationship of hexapods and crustaceans (Tetraconata hypothesis). In this new debate, data on the structure and development of the arthropod nervous system contribute important new data ("neurophylogeny"). Neurophylogenetic studies have already provided several examples for individually identifiably neurons in the ventral nerve cord that are homologous between insects and crustaceans. In the present report, we have analysed the emergence of Engrailed-expressing cells in the embryonic brain of a parthenogenetic crayfish, the marbled crayfish (Marmorkrebs), and have compared our findings to the pattern previously reported from insects. Our data suggest that a group of six Engrailed-expressing neurons in the optic anlagen, the so-called secondary head spot cells can be homologised between crayfish and the grasshopper. In the grasshopper, these cells are supposed to be involved in establishing the primary axon scaffold of the brain. Our data provide the first example for a cluster of brain neurons that can be homologised between insects and crustaceans and show that even at the level of certain cell groups, brain structures are evolutionary conserved in these two groups.

Evolution of arthropod visual systems: development of the eyes and central visual pathways in the horseshoe crab Limulus polyphemus Linnaeus, 1758 (Chelicerata, Xiphosura).

Despite ongoing interest into the architecture, biochemistry, and physiology of the visual systems of the xiphosuran Limulus polyphemus, their ontogenetic aspects have received little attention. Thus, we explored the development of the lateral eyes and associated neuropils in late embryos and larvae of these animals. The first external evidence of the lateral eyes was the appearance of white pigment spots-guanophores associated with the rudimentary photoreceptors-on the dorsolateral side of the late embryos, suggesting that these embryos can perceive light. The first brown pigment emerges in the eyes during the last (third) embryonic molt to the trilobite stage. However, ommatidia develop from this field of pigment toward the end of the larval trilobite stage so that the young larvae at hatching do not have object recognition. Double staining with the proliferation marker bromodeoxyuridine (BrdU) and an antibody against L. polyphemus myosin III, which is concentrated in photoreceptors of this species, confirmed previous reports that, in the trilobite larvae, new cellular material is added to the eye field from an anteriorly located proliferation zone. Pulse-chase experiments indicated that these new cells differentiate into new ommatidia. Examining larval eyes labeled for opsin showed that the new ommatidia become organized into irregular rows that give the eye field a triangular appearance. Within the eye field, the ommatidia are arranged in an imperfect hexagonal array. Myosin III immunoreactivity in trilobite larvae also revealed the architecture of the central visual pathways associated with the median eye complex and the lateral eyes. Double labeling with myosin III and BrdU showed that neurogenesis persists in the larval brain and suggested that new neurons of both the lamina and the medulla originate from a single common proliferation zone. These data are compared with eye development in Drosophila melanogaster and are discussed with regard to new ideas on eye evolution in the Euarthropoda.

Ontogeny of the Marmorkrebs (marbled crayfish): a parthenogenetic crayfish with unknown origin and phylogenetic position.

Development, growth, and egg production of the Marmorkrebs (marbled crayfish), a crayfish with parthenogenetic reproduction, uncertain geographic origin, and taxonomic position, was studied under laboratory conditions. Length and weight increments strongly depended on temperature being highest at 30 degrees C, and lowest at 15 degrees C. At 25 degrees C, cephalothorax length and weight increased by 17.5 mm and 1700 mg, respectively, in the course of 150 d, whereas at 15 degrees C these parameters increased by only 7 mm and 100 mg during the same period of time. Photoperiod slightly affected growth at 25 degrees C. During growth experiments, mortality was lower at 20 degrees C compared to higher (25 degrees , 30 degrees C) or lower temperatures (15 degrees C), and lower under short-day than under long-day conditions. Females matured early (at an age of 141-255 d, a cephalothorax length of 14-21.5 mm, and a weight of 0.63-2 g) compared to other crayfish species. Reproductive females with a cephalothorax length of between 25-35 mm produced large clutches (up to 416 eggs) and brooding periods varied between 22 and 42 d. In order to establish a staging scheme for Marmorkrebs embryos, embryos were photographed, externally visible ontogenetic events charted, and dissected embryos stained with a nuclear dye. These experiments indicate that their development is virtually identical to that of other crayfish. In conclusion, these results and others show that the Marmorkrebs may be taken as a representative valid model organism for future developmental studies on Crustacea.

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.