Sci Nat (2016) 103: 13 DOI 10.1007/s00114-016-1333-8 ORIGINAL PAPER The pattern of a specimen of Pycnogonum litorale (Arthropoda, Pycnogonida) with a supernumerary leg can be explained with the “boundary model” of appendage formation Gerhard Scholtz1 & Georg Brenneis1,2 Received: 21 July 2015 /Revised: 7 January 2016 /Accepted: 11 January 2016 /Published online: 30 January 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract A malformed adult female specimen of Pycnogonum Introduction litorale (Pycnogonida) with a supernumerary leg in the right body half is described concerning external and internal structures. Animal malformations, or teratologies, and unusual The specimen was maintained in our laboratory culture after an morphologies have always attracted human attention. This is injury in the right trunk region during a late postembryonic stage. reflected in numerous specimens housed in curiosity cabinets The supernumerary leg is located between the second and third and natural history collections. Yet, since Geoffroy Saint- walking legs. The lateral processes connecting to these walking Hilaire’s(1826) pioneer work and, in particular, the ground- legs are fused to one large structure. Likewise, the coxae 1 of the breaking study of Bateson (1894) on the categorization and second and third walking legs and of the supernumerary leg are importance of animal malformations and irregularities, these fusedtodifferentdegrees.Thesupernumerary leg is a complete naturally occurring or experimentally produced deviations walking leg with mirror image symmetry as evidenced by the from the normal patterns of body organizations played a cru- position of joints and muscles. It is slightly smaller than the cial role for the understanding of developmental mechanisms normal legs, but internally, it contains a branch of the ovary and evolutionary processes (see Blumberg 2009). In a recent and a gut diverticulum as the other legs. The causes for this article, Guinard even proposed an “evolutionary teratology” malformation pattern found in the Pycnogonum individual are as a discipline in its own right that “highlights the production reconstructed in the light of extirpation experiments in insects, of developmental anomalies (more or less drastic) over evo- which led to supernumerary mirror image legs, and the “bound- lutionary times, which become integral parts of groups and ary model” for appendage differentiation. taxa” (Guinard 2015,p.20). There are a few reported cases of malformations in the marine Pycnogonida, also known as Pantopoda or sea spiders, Keywords Malformation . Regeneration . Limb an arthropod group that is nowadays most convincingly development . Morphology . Micro-computed tomography . placed within the Chelicerata (for discussion, see Giribet and Sea spider . Pantopoda Edgecombe 2013). Most of these instances are of anecdotal character (Dogiel 1911; Schimkewitsch and Dogiel 1913; Bouvier 1914;Arita1936;Ohshima1942a, b; Gordon 1932; Hedgpeth 1947;Child1979;Stock1987) and the described Communicated by: Sven Thatje structural abnormalities concern the duplication, fusion, or * Gerhard Scholtz absence of limbs and other body parts and have been most [email protected] likely caused by irregular regeneration processes after injury of the animals. Furthermore, exceptional cases of gynandro- morphy have been documented in the otherwise strictly 1 Humboldt-Universität zu Berlin, Institut für Biologie, Vergleichende diecious and mostly sexually dimorphic animals (Child and Zoologie, Philippstr. 13, 10115 Berlin, Germany Nakamura 1982; Krapp and Viquez 2011; Lucena et al. 2015). 2 Neuroscience Program, Wellesley College, 106 Central Street, In general, the observed patterns of these malformations cor- Wellesley, MA 02481, USA respond to those found in other arthropods (Bateson 1894). 13 Page 2 of 9 Sci Nat (2016) 103: 13 However, compared with the remaining chelicerate groups 2009). The living animal was anaesthetized with CO2 and (e.g., Patten 1896;Brauer1917; Ćurčić et al. 1991;Jacuński photographed from dorsal and ventral perspectives with a et al. 2005; David 2012) as well as with crustaceans (e.g., Keyence VHX-1000 microscope by combining stacks of im- Bateson 1894; Shelton et al. 1981; Scholtz 2014; Scholtz ages at various z-levels with the implemented software. et al. 2014), myriapods (e.g., Hubert 1968;Juberthie-Jupeau Afterwards, the animal was fixed in Bouin’ssolution(saturat- 1961;Leśniewska et al. 2009; Janssen 2013), and hexapods ed aqueous picric acid, pure acetic acid, and 10 % formalde- (e.g., Bateson 1894; Cappe de Baillon 1927; Cockayne 1929; hyde solution) for several hours at room temperature and sub- Balazuc 1948, 1958; Puissegur and Benadona 1973; Hesse- sequently washed and stored in 70 % ethanol. Additional pic- Honegger 1998), well-documented instances of pycnogonid tures of the fixed specimen were taken with a Zeiss Lumar malformations remain scarce. Presumably, this is rather due to V12 stereomicroscope equipped with epifluorescence, mak- the lower number of pycnogonid investigations than to a lack ing use of the green autofluorescence of the animal’scuticle of regeneration abilities or a lower frequency of cases. when excited with blue light. In fact, the reported examples of pycnogonid malformations indicate a high potential of wound healing and regeneration in Micro-computed tomography these animals. Yet, in most cases, the actual causes of the specific structural abnormalities are unknown, and the few experiments The sample was dehydrated with a graded ethanol series and that have been conducted are somewhat ambiguous and contra- incubated in a 1 % iodine solution (iodine, resublimated [Carl dictory in their interpretation (Loeb 1895; Morgan 1904). Studies Roth GmbH & Co. KG, Karlsruhe, Germany; cat. #X864.1] in in other chelicerates have shown that malformations are not only 99.8 % ethanol) overnight. After incubation, the sample was caused by regeneration but also by exposure to heavy metals washed several times in 99.8 % ethanol and transferred into a (Itow et al. 1998), X-rays (Seitz 1966), increased temperature vial. Scans were performed with an Xradia MicroXCT-200 (Napiórkowska et al. 2015), and the misexpression of genes Xray imaging system (Carl Zeiss Microscopy GmbH). (Sharma et al. 2015). Hence, similar mechanism can be expected Settings were optimized for the specimen, and objectives were to act with respect to pycnogonid malformations, in addition to chosen according to sample size and region of interest. irregular regeneration. Accordingly, the 0.39× and 4× objectives were used resulting Here, we describe an adult female specimen of in pixel sizes of 34.76 and 5.53 μm/px, respectively. Samples Pycnogonum litorale Strøm, 1762 with a supernumerary limb, were scanned in 99.8 % ethanol. Exposure times were 2 s which is situated between the right second and third walking (0.39× scan) and 15 s (4× scan). Scanning parameters were legs. This individual was unintentionally damaged during its 30 kVand 6 W, resulting in a current of 200 μA. Tomography first juvenile stage (=sixth postembryonic instar) in the trunk projections were reconstructed by using the XMReconstructor region between the right second and third walking legs. For software (Carl Zeiss Microscopy GmbH), resulting in images the first time, we use micro-computed tomography (micro- stacks (TIFF format). All scans were performed by using CT) to study not only the external morphology but also the Binning 2 (summarizing 4 px, resulting in noise reduction) internal organization of a malformed pycnogonid. Except for and subsequently reconstructed by using Binning 1 (full res- its mirror image organization and a somewhat deviating pat- olution) to avoid information loss. tern of gut diverticula, the supernumerary limb shows a high Analyses of the micro-CT data were performed with the 3D degree of external and internal similarity to the other walking reconstruction program “Imaris” (Bitplane AG, Switzerland, legs. version 7.0.0). Within the “surpass mode” of this program, 3D The pattern of the observed malformation is discussed in volumes are generated from the recorded image stacks. the light of extirpation experiments in insects, where the for- Volumes are shown either in the default “maximum intensity mation of additional legs could be induced (Bohn 1974), and projection” (MIP) or alternatively in the “Blend” option, with respect to the “boundary model” for insect appendages which renders scanned structures non-transparent and thus (Meinhardt 1986, 2008). facilitates evaluation of the external shape of an object. To virtually remove “non-target” regions that obstruct the view of more interiorly located substructures of interest, “clipping Material and methods planes” were applied. “Oblique slicers” were used to create virtual sections with specific orientation through different re- Preparation, fixation, and photography gions of the specimen. All midgut components included in the 4× scan were manually segmented using the “surface tool” The specimen of P. litorale is an adult female with a body and upon masking of all surrounding structures added as ad- length of 11 mm measured from the tip of the proboscis to ditional channel into the 4× scan. This enabled to better illus- the end of the anal tubercle. It was raised in our laboratory trate the position, course, and branching pattern of the midgut culture of this species (for details, see Ungerer and Scholtz diverticula within