The hatching larva of the priapulid worm Halicryptus spinulosus
© Janssen et al; licensee BioMed Central Ltd. 2009
Received: 28 February 2009
Accepted: 26 May 2009
Published: 26 May 2009
Despite their increasing evolutionary importance, basic knowledge about the priapulid worms remains limited. In particular, priapulid development has only been partially documented. Following previous description of hatching and the earliest larval stages of Priapulus caudatus, we here describe the hatching larva of Halicryptus spinulosus. Comparison of the P. caudatus and the H. spinulosus hatching larvae allows us to attempt to reconstruct the ground pattern of priapulid development. These findings may further help unravelling the phylogenetic position of the Priapulida within the Scalidophora and hence contribute to the elucidation of the nature of the ecdysozoan ancestor.
Although many thousands of eggs could be obtained and fertilized in this study, the success rate of development to hatching was extremely low, with the result that only 52 specimens of hatched larvae were available for this study. This is in contrast to a previous study of P. caudatus where many tens of thousands of hatching larvae and large numbers of subsequent larval stage specimens could be obtained under controlled laboratory conditions. This disparity is likely linked to sperm quality. As measured by sperm motility, the quality of H. spinulosus sperm was much lower than for P. caudatus: only very few spermatozoa swam actively and were separated from the gonads and/or sister-spermatozoa; possibly leading to delayed fertilization or polyspermy (if the eggs were fertilized by floating or swimming "groups" of attached spermatozoa). Because of this restriction we were not able to examine all documented features in great numbers. Nevertheless, the stability of the results obtained strongly suggests that the characters we describe are natural features of the H. spinulosus hatching larva.
The first H. spinulosus larva hatched 21 days after fertilization when kept at approximately 4°C. In the following three days the remaining larvae hatched implying that the development is largely synchronized. Most of the obtained larvae (40) were processed for Scanning Electron Microscopy (SEM), 12 were allowed to develop in order to reach subsequent larval stages. Although these latter hatching larvae were alive for several weeks they did not moult and eventually died.
The Halicryptus hatching larva possesses two outgrowths, the neck-flosculi, on opposite sites of the body, at the border between introvert and neck (Fig. 3A–F). They are 1–2 μm long and composed of a short base and circlets of cuticular papillae at the tip that surround a single pore (Fig. 3B, E, F). Interestingly all the neck-flosculi in the H. spinulosus hatching larva are associated with a filiform structure (ca. 3 μm long) emanating from the centre of each flosculus (Fig. 3A–F). The nature and function of these structures is unclear, although the flosculi themselves are clearly sensory organs . In older larval stages of H. spinulosus the number of neck-flosculi increases to at least 15 . For the P. caudatus hatching larva we described two outgrowths in a similar position . These represent tubular structures (neck-tubuli) rather than flosculi. It is unlikely that the ca. 6.5 μm long neck-tubuli in P. caudatus are homologous to the flosculi of H. spinulosus since flosculi in general were never reported for any described P. caudatus larva. One exception is the documentation of a single flosculus-like structure on a putative early larva of P. caudatus , but if this larva actually represents a larva of P. caudatus is unclear (discussed in ).
In a few fixed larvae the rear end formed a tapered structure possibly representing the anal region (Fig. 3G, H). Whether or not a proper anal opening is present is unclear. However its presence is unlikely because of the lack of a mouth and the reported lack of an anus in the P. caudatus hatching larva (cf. ). The hatching larvae of both P. caudatus and H. spinulosus are still yolk-rich and as discussed for the P. caudatus larva, it seems likely that the H. spinulous hatching larva simply digs down into the mud in order to escape from the dangerous surface of the seafloor.
The systematics of the priapulids are not well-characterised. However, Lemburg  places both Priapulus and Halicryptus within the Priapulidae, although distant from each other, a position supported by both Dong et al.  and Wills . Their shared developmental data thus allows a partial reconstruction of the ground plan of the Priapulidae hatching morphology (Fig. 1C): It includes the following features: 1) of light-bulb shape, 2) subdivided into introvert, neck and trunk, 3) lacking a true lorica, 4) without a mouth, 5) without pharyngeal teeth, 6) armed with 7+X scalids and 8) with four posterior tubuli. Further investigation of more basal taxa such as Tubiluchus will be required to extend the investigation of ground plan development to the Priapulida as a whole, an essential prelude to phylogenetic reconstruction of their morphological and developmental evolution .
Halicryptus spinulosus breeding conditions
We fertilized apparently mature eggs of Halicryptus spinulosus during several field seasons from winter 2007 to early spring 2009. Although in all cases a number of eggs were successfully fertilized as recognized by the formation of a fertilization-membrane and the commencement of cleaving, hatched larvae were only been observed once (early winter 2008). In all other cases fertilized eggs underwent irregular cleavage (not shown) and eventually died before reaching the larval stage. Priapulid collection, preparation, fertilisation and breeding were performed in the same way during all of the field seasons. Sexually mature female and male specimens of H. spinulosus were collected from Trosa archipelago close to the Askö marine station on the east coast of Sweden south of Stockholm. Gonads were stripped off and eggs were artificially fertilised as described for Priapulus caudatus [3, 4]. The developing eggs were rinsed every day with fresh filtered see water collected from the same collection site as the adults. Decaying eggs where removed. To maintain water quality throughout the developing phase water was frozen after filtration and thawed the day before use. Embryos and hatching larvae were kept at approximately 4°C in a conventional fridge.
Larvae fixation and preparation
To ensure that the introvert was protruded a needle was used to mechanically push it out from the rear. The hatching larvae were fixed either in 2.5% glutaraldehyde for four hours at room temperature or over night at 4°C in the fridge. Storage and preparation for Scanning Electron Microscopy (SEM) was performed as described in .
For SEM pictures, a Zeiss Supra 35VP scanning electron microscope with field emission gun was used. For light microscopic pictures, a Nikon D70 camera was attached to a Nikon Eclipse E400 light microscope. Further processing of SEM and light microscopy images was carried out in Photoshop CS.
We wish to thank the skippers and administrative staff at Askö Marine Research station for their support during the fieldwork. We are also grateful to Stefan Gunnarsson and Gary Wife for continuous help with the preparations of H. spinulosus larvae. Financial funding was provided by the Swedish Natural Science Council (VR) to GEB and SAW, by the Swedish Royal Academy of Sciences (KVA) to GEB and SAW, Lennanders stiftelse to SAW, and the European Union via the Marie Curie Training network "ZOONET" (MRTN-CT-2004-005624) to GEB and RJ. Christian Lemburg's homepage http://wwwuser.gwdg.de/~clembur/welcome.htm provided a helpful source of priapulid literature.
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