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

  • ️Sun Jan 01 2023

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

Georg Brenneis et al. Proc Natl Acad Sci U S A. 2023.

Abstract

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.

Keywords: Arthropoda; Pycnogonida; development; evolution; segmentation.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

Anamorphic development and hindgut differentiation in P. litorale. (A) Copulating male and female in dorsal view. (B) Selected instars (ventral view) illustrate the anteroposterior developmental gradient during the anamorphic post-embryonic development. (C and C’) First bundles of the hindgut dilatator musculature (arrow) and the anlage of the slit-shaped anus (asterisk) are recognizable in late instar IV (ventral view). (D and D’) Proctodeal dilatator muscles (arrow), ring musculature of the through-gut (arrowhead) and the open slit-shaped anus (asterisk) are well-developed in instar V (ventral view). AF, cuticular autofluorescence; ELM, extrinsic leg muscles; G1-4, ventral ganglia 1 to 4; ILM, intrinsic leg muscles; NUC, nuclear staining; PHA, phalloidin labeling; 1 to 4, leg pairs 1 to 4.

Fig. 2.
Fig. 2.

Survival and molting of experimental animals after posterior amputation. Specimens in four immature developmental stages and adults were included in the amputation experiments (left side). The horizontal bars represent single individuals. At the border between differently colored sections of a single bar, the specimen underwent a molt. The stippled outlines of bar sections at the far right indicate that experimental animals reached maturity.

Fig. 3.
Fig. 3.

Axial regeneration patterns in anamorphic instar V. (A) Ventral detail of instar V depicting the different section planes (colored arrows, colors matching with details shown in B–D) in the posterior amputation experiments. The small inset in the Left Bottom corner shows instar V in dorsal view. (B) After partial removal of the primordial fourth TS and the AT, an eight-legged phenotype with vestigial AT (arrowhead) is formed (

SI Appendix, Fig. S4 E and F

). (C) Oblique amputation affecting the AT, the TS4 primordium and the right limb bud of TS3 resulted in the transient regeneration of an AT (asterisk) and an aberrant bifurcating leg 4 (“4”,

SI Appendix, Fig. S5 A–F

). After shedding of both regenerated structures with the next molt, a hypomeric, six-legged phenotype with vestigial AT (arrowhead) is formed. (D) Transverse amputation at the TS2 to TS3 border results in no regeneration (right column;

SI Appendix, Fig. S4 A and B

), or a hypomeric, six-legged phenotype (left column;

SI Appendix, Fig. S4 C and D

) with anus-bearing AT (asterisk). AF, cuticular autofluorescence; G1-4, ventral ganglia 1 to 4; L, leg; NUC, nuclear staining; r, right; 1 to 4, leg pairs 1 to 4.

Fig. 4.
Fig. 4.

Axial regeneration patterns in epimorphic juvenile instars. (A) Sagittal extended optical section through a juvenile (anterior to the left) illustrating two of the three different section planes (colored arrows, colors matching with details shown in B and D) in the amputation experiments. The small inset in the Right Bottom corner shows a juvenile in dorsal view. (B) After removal of the ultimate leg-bearing TS and the AT (asterisk), a normal eight-legged phenotype is formed after two molts. (C) After oblique amputation of the AT, TS4 and part of the right TS3 half, a normal phenotype is likewise restored. Note coagulating hemolymph at the wound site after the amputation procedure (white arrowhead). The stippled blue arrow indicates an additional molt that is not shown. (D) Transverse amputation at the TS2 to TS3 border resulted in a hypomeric, six-legged phenotype with anus-bearing AT (left column) and a seven-legged specimen with normal AT. AF, cuticular autofluorescence; BR, brain; G1-4, ventral ganglia 1 to 4; L, leg; MG, midgut; NUC, nuclear staining; r, right; TUB, tubulin immunolabeling; 1 to 4, leg pairs 1 to 4.

Fig. 5.
Fig. 5.

Internal anatomy of experimental animals after reaching maturity. (A and B) Adult male specimen (Juv_4), four molts after amputation at the TS3 to TS4 border, ventral views. (A) The normal phenotype has regenerated, with midgut diverticula (1 to 4) in all four leg pairs, the hindgut with slit-shaped anal opening (asterisk), and a complete ventral nerve cord (arrowheads mark ganglia 3 and 4). (B) The regenerated leg pair 4 displays regular ventral gonopores (arrowheads), targeted by vasa deferentia and equipped with gonopore muscles (GM). The slit-shaped anus (asterisk) is opened by regular proctodeal dilatator musculature (PM). (C–F) Adult female specimen (Juv_3), five molts after amputation at the TS2 to TS3 border, dorsal (C and E) and ventral (D and F) views. (C) A hypomeric, seven-legged phenotype has formed. (D) All legs are penetrated by midgut diverticula (1 to 4) and the hindgut with slit-shaped anal opening (asterisk) is regenerated, but ventral ganglion 4 is missing (arrowhead highlights ganglion 3). (E) While a gonopore (arrowhead) connected with a regular oviduct (OD) and gonopore musculature (GM) is present on the two regenerated legs in the left body half, it is lacking on the right leg 3 (stippled oval). (F) The nerves targeting the unpaired leg 4 and the proctodeum (LN4 and PN, respectively) emanate from the posterior side of ventral ganglion 3 (G3). The midgut diverticula of the right leg 3 and of the unpaired leg 4 branch off at untypical locations (arrows). AM, distal gut ampulla; CX2, coxa 2; LD, leg diverticulum; LN, leg nerve; OV, ovary; VD, vas deferens.

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