Fig 1.
Experimental animals and their specialised sensory appendages, the pectines.
(A) Mesobuthus eupeus in a natural pose on desert sand. (B) Ventral view of M. eupeus. Pectines (black arrowheads) are located behind the fourth walking legs on the third mesosomal segment, just posterior the genital operculum. (C) Heterometrus petersii in defensive posture. (D) Ventral view of H. petersii. Pectines (white arrowheads) are light in colour and have wide marginal and medial lamellae.
Fig 2.
Pectines of Heterometrus petersii (left) and Mesobuthus eupeus (right). Pectines drawn in natural position, ventral view. Scissors indicate the base of the pecten, where it is connected to the body. Pectines possess marginal lamellae (outer margin of the pectines), medial lamellae (middle section), fulcra (row of more or less dome-shaped mechanosensory pads) and rows of pegs arranged in comb-like fashion. Pegs are studded ventrally with dense fields of peg sensillae (grey areas in diagrams) pointing towards the substrate. All other pecten surfaces are equipped with mechanosensory hair sensillae and trichobothria. (A) and (B) scanning electron microscopic images depicting fulcra and pegs of Heterometrus petersii (left) and Mesobuthus eupeus (right), respectively.
Fig 3.
Experimental set-up for anterograde tracing of peg afferents.
(A) Photo of a cold-anaesthetised Heterometrus petersii fixed to a cork board, ventral side up. Pectines were cleaned, stabilised with plasticine, fixed into position with dental glue and supported with a plastic film. (B) Sketch of the anterograde tracing procedure using Neurobiotin. Pectines are highlighted in beige, examples of incisions at individual pegs in different positions marked in red. Glass capillaries filled with Neurobiotin were put over the incision. Abbreviations: a: anterior; p: posterior.
Fig 4.
Position and morphology of the pecten neuropils in Mesobuthus eupeus (A-C) and Heterometrus petersii (D-F). Maximum projections of cLSM stacks, showing neuropilar regions in grey (synapsin-immunoreactivity). (A) Horizontal section of the posterior part of the synganglion of M. eupeus. The associated neuropils of the pecten extend from the most posterior region anteriorly to the level of the third walking leg neuromere (WLN3). The pecten neuropil is divided into the anterior pecten neuropil (APN) and the posterior pecten neuropils 1–3 (PPN 1–3). (B) Frontal section of the PPN and synganglion located just dorsally. The PPN1 appears to be layered, with “onion peel”-like arches that are smaller in dorsal direction and wider in ventral direction. The area between the hemiganglia is slightly torn (asterisk). (C) Sagittal section of the synganglion of M. eupeus. PPN protrudes ventrally from the synganglion. (D) Horizontal view of PPN of H. petersii. Immunolabelling appears stronger in PPN than in the remaining synganglion. PPN1 has a stronger synapsin-immunoreactivity in the outer cortex, compared to the central region. These subcompartments are not as differentiated as in M. eupeus. APN is ellipsoid and heterogeneously structured, exhibiting a weaker immunoreactivity of synapsin than PPN. (E) Frontal section of the synganglion of H. petersii. The outer cortex of PPN1 appears to be more nodular than the inner layers with stronger synapsin immunoreactivity. (F) Sagittal section of the synganglion of H. petersii. APN is very prominent and positioned ventrally at the level of the WLN3. PPN protrudes ventrally from the synganglion. Abbreviations: a: anterior; APN: anterior pecten neuropil; d: dorsal; p: posterior; PPN1-3: posterior pecten neuropil 1–3; v: ventral; WLN3-4: walking leg neuromere 3–4.
Fig 5.
Detailed view of the posterior pecten neuropils of Mesobuthus eupeus (A) and Heterometrus petersii (B). Maximum projections of horizontal cLSM stacks. Neuropilar tissue in grey and nuclear labelling in magenta. (A) The three major posterior pecten neuropils (PPN1-3) of M. eupeus consist of numerous subcompartments: PPN1 consists of palisade-shaped neuropilar regions (outlined stars). Two large lobules (white dashed lines) are located at the lateral and medial side of the PPN2 with wedge-shaped neuropil subcompartments. An area of arcuate rows (white dashed lines) consisting of smaller glomeruli, is positioned between the lobules. PPN3 is built up of four lobules (asterisks). The anterior pecten neuropil (APN) extends anteriorly. (B) PPN of H. petersii is interspersed with somata and divided in the heterogeneously structured PPN1 and 2 (white dashed line). PPN2 consists of two lobules, located at the lateral and medial sides of the neuropil and numerous glomeruli (white dotted lines). Abbreviations: a: anterior; APN: anterior pecten neuropil; p: posterior; PPN1-3: posterior pecten neuropil 1–3; WLN4: walking leg neuromere 4.
Fig 6.
Axonal pathways of the pectines in Mesobuthus eupeus.
. Maximum projections of horizontal cLSM stacks. Tubulin-immunoreactivity: grey/black. (A) A major tract runs along the posterior pecten neuropil (PPN) laterally (outlined arrowhead). The anterior pecten neuropil (APN) includes a longitudinal tract (outlined arrowhead). Hemiganglia are connected by commissure (outlined star). (B) Higher magnification of PPN2. The glomeruli (outlined arrowheads) and bilateral lobules (outlined arrows) are demarcated by axonal fibres. (C) Higher magnification of PPN1. Afferent bundles are located at the posterior end and are organised in parallel fashion (outlined arrowhead). Abbreviations: a: anterior; APN: anterior pecten neuropil; p: posterior.
Fig 7.
Innervation of the pecten neuropils by sensory axons in Mesobuthus eupeus and Heterometrus petersii.
Maximum projections of horizontal cLSM stacks. Tubulin-immunoreactivity (blue) and anterograde tracing of afferents (green). (A) The entire pecten neuropil exhibits innervation by pecten afferents. Afferent bundles (AB) of the pecten nerve project into the posterior pectines neuropil 1 (PPN1) (white outlined arrow). Adjacent accessory pecten neuropils 1 and 2 (AN1,2) (white arrows), the ascending (AT) and the longitudinal tract (LtT) (white arrowhead) exhibit Neurobiotin labelling. (B) Ventral section of anterior pecten neuropil (APN) and adjacent neuropils. Anterior to PPN3, the ascending tract (arrowhead) proceeds parallel to the APN and innervates the AN1. (C) The longitudinal tract connects the AN1 and AN2 (white arrowhead) dorsally. (D) Anterograde tracing of the entire pecten nerve results in labelling in PPN, APN and AN of Heterometrus petersii. Ascending tract emerges anterior to the walking leg neuromere 4 (WLN4) and innervates the accessory neuropil. Proceeding tract leaves the AN anteriorly. Abbreviations: a: anterior; AB: afferent bundles; AN1,2: accessory neuropil 1, 2; APN: anterior pecten neuropil; AT: ascending tract; LtT: longitudinal tract; p: posterior, PPN1-3: posterior pecten neuropil 1–3; PT: proceeding tract; WLN3-4: walking leg neuromere 3–4.
Fig 8.
Projection pattern of single pegs within the pecten neuropil of Mesobuthus eupeus.
Maximum projections of cLSM stacks. Synapsin-immunoreactivity (grey (not shown in D-F)), anterograde tracing (green) and nuclei staining (magenta). (A-C) Horizontal serial sections arranged from ventral (A) to dorsal (C). (D-F) Insets of A, B and C in a higher magnification. (A) I Innervation is restricted mainly to the lateral part of the posterior pecten neuropil 2 (PPN2) ventrally. (B, C) Gradual dorsal innervation of lateral PPN1 areas. (D) Lateral tract (LT) projects anteriorly from the margin of the neuropil (white arrowhead). (E) Small tract (CT) connects PPN1 and PPN2 (white arrowhead). (F) Lateral tract proceeds anteriorly (white arrowhead). (G) Innervation pattern of distal peg afferents in the right pecten neuropil and a proximal peg in the left pecten neuropil (horizontal view). Proximal pegs project medially into the PPN2, whereas distal pegs project laterally towards PPN1 and PPN2. (H) Sagittal view of the posterior part of the synganglion with focus on PPN which protrudes ventrally. The pecten nerve splits into two tracts. The dorsal tract innervates the postero-ventral synganglion and proceeds anteriorly. The ventral tract innervates PPN dorso-laterally and exits the neuropil anteriorly. (I) Sagittal view of a rather lateral section. Innervation of the PPN is restricted to its dorsal part. Abbreviations: a: anterior; CT: connecting tract; d: dorsal; LT: lateral tract; p: posterior, PPN1-3: posterior pecten neuropil 1–3; v: ventral.
Fig 9.
Median pegs innervate central areas within the pecten neuropil of Mesobuthus eupeus.
Maximum projections of cLSM stacks. Synapsin-immunoreactivity (grey), anterograde tracing (green) and nuclei staining (magenta). (A) Medially situated pegs innervate median parts of the posterior pecten neuropils 1 and 2 (PPN1, 2) (horizontal view). The anteriorly proceeding tract is located near the ganglion midline (arrowhead) and terminates in the anterior pecten neuropil (APN). (B) PPN2 and PPN3 are innervated antero-ventrally by medial peg afferents (white arrow) (sagittal view). (C) In a more lateral section, the innervation is postero-ventral in PPN1 (white arrow). Abbreviations: a: anterior; d: dorsal; p: posterior; PPN1-3: posterior pecten neuropil 1–3; v: ventral.
Fig 10.
Innervation of the posterior pecten neuropil (PPN) of Heterometrus. petersii by distal (A-C) and proximal (D-F) peg afferents. Maximum projections of horizontal cLSM stacks. Synapsin-immunoreactivity (grey (not shown in B, C and E, F)) and anterograde tracing (green). (A) Tract from the pecten nerve enters the synganglion from posterior and proceeds laterally along the margin of the posterior pecten neuropil (PPN). PPN1 and 2 are innervated laterally. Furthermore, the entire anterior area of the anterior pecten neuropil (APN) is innervated by afferents. The innervation pattern reveals conspicuous glomerular-like structures (dotted outlines). (B) Higher magnification of APN from (A). Longitudinal tract splits into distinct branches (white arrowheads), each innervating a glomerular-like structure of APN. (C) Higher magnification of the PPN from (A). The labelled tract is thicker at the posterior end and finer at the anterior end. (D) Projection pattern of proximal peg afferents within the pecten neuropil. Longitudinal tract connects PPN and APN, the former being medially innervated by afferent fibres. (E) Higher magnification of APN from (D). The longitudinal axons terminate in two ellipsoid subcompartments in APN. (F) Higher magnification of the PPN from (D). The main projection area is located laterally and branches into the PPN1 and 2. Abbreviations: a: anterior; APN: anterior pecten neuropil; PPN1,2: posterior pecten neuropil 1, 2; p: posterior.
Fig 11.
Double-labelling of two adjacent distal pegs in Mesobuthus eupeus with lipophilic markers.
Maximum projections of horizontal cLSM stacks. Nuclear labelling in magenta. DiA (yellow) was used for peg 20 (second to last distal peg), DiI (cyan) for peg 18 (fourth to last distal peg). (A) The innervation of peg 18 is distributed medially (dotted line, neuropil outline). (B) Afferents of peg 20 stay on the very lateral side of the posterior pecten neuropil (PPN) and the labelled tract runs along the lateral neuropil margin (dotted line). (C) Merge of (A) and (B). The afferent projections hold a specific spatial position within the pecten nerve but partially overlap in the neuropil (outlined star) (dotted line, neuropil outline). (D) Innervation of the entire anterior pecten neuropil (APN) by afferents of peg 18 (cyan). (E) Innervation of the entire APN by afferents of peg 20 (yellow). (F) Merged image of (D) and (E). White arrows indicate PPN3 and a lateral tract. The afferents of both pegs innervate APN in a similarly homogeneous fashion. (G) Pecten nerve with afferent fibres of peg 18 (cyan) and 20 (yellow) indicated by arrows. Afferents of peg 20 (cyan) of the left pecten indicated by asterisk. Abbreviations: a: anterior; APN: anterior pecten neuropil; PPN3: posterior pecten neuropil 3; p: posterior.
Fig 12.
Overview of variations in the anatomy of the pecten neuropils in Mesobuthus eupeus, Heterometrus petersii, Paruroctonus mesaensis and Vaejovis sp.
Habitats and life styles of the respective scorpion species are listed under the species names. Left column depicts a generelised scheme of the pecten neuropil; relevant subregions are named in the upper left corner and marked in red. Species-specific differences of each subregion are listed under the respective scorpion species. Results in Paruroctonus mesaensis and Vaejovis sp. were taken from neuroanatomical studies of J.M. Melville [74] and H. Wolf [11], respectively. Asterisks refer to the used staining method. Abbreviations: a: anterior; AN: accessory neuropil; APN: anterior pecten neuropil; PPN1-3: posterior pecten neuropil1-3; p: posterior.
Fig 13.
Schematic summary of the pecten neuropils and their innervation in Mesobuthus eupeus (left) and Heterometrus petersii (right). Posterior part of the synganglion in horizontal section. The pecten neuropil of M. eupeus exhibits a compartmentalisation into different subregions: The anterior pecten neuropil (APN) at the level of walking leg neuropil 3 (WLN3), the accessory neuropils 1, 2 (AN1,2) (cyan) and the posterior pecten neuropils1-3 (PPN1-3) in the more posterior part of the synganglion. PPN consists of three conspicuous subregions: The laminar PPN1, the glomerular PPN2 and the lobular PPN3. The pecten neuropil of H. petersii contains an ellipsoid APN, an AN (cyan) and a PPN with probably two main regions (PPN1 and 2). The structures of PPN1 and PPN2 are intertwined, although PPN2 appears more lobular. The PPN of H. petersii is interspersed with somata. The subregions of the PPN are delimited (indicated by dark grey areas in PPN1 and PPN2). PPN and APN are connected by a longitudinal tract (indicated by dark grey area between PPN and APN. The innervation of peg afferents exhibits a somatotopic organisation in the PPN: afferents of distal pegs project into lateral regions (red), afferents of median pegs into the central region (blue) and proximal peg afferents into medial regions (green) of the PPN. Innervation areas overlap, but the exact quantity of afferent overlap is not known. APN is innervated homogenously by all afferent fibres. Abbreviations: a: anterior; AN1,2: accessory neuropil1, 2; APN: anterior pecten neuropil; AT: ascending tract; LtT: longitudinal tract; p: posterior; PPN1-3: posterior pecten neuropil 1–3; PT: proceeding tract; WLN2-4: walking leg neuromere 2–4.