TY - JOUR
AU - , van de Kamp, Thomas
AB - Abstract Thickened femora of insects are correlated to enlarged muscle masses and serve two basic purposes: jumping/kicking and grasping/holding. Modifications on the ventral femoral wall and the tibial flexor tendon that are possibly involved in catch mechanisms have been described in multiple insect taxa with both jumping and grasping legs. Our comparative study aims to explore the functional and structural similarities of these modification in jumping and grasping leg types from Coleoptera, Hymenoptera, Diptera, and Orthoptera with the combination of cutting edge, noninvasive imaging methods, and classical dissections techniques. Our data indicate that locking mechanisms are present in the jumping and grasping legs of insects. We describe three femoro-tibial lock types based on the location of the interacting sclerites relative to the site of origin of the tibial flexor tendon. All of the three types can be found in jumping insect legs, whereas only one type is present in grasping legs. The locking mechanism might aid in keeping the femoro-tibial joint in a flexed position for an extended period of time. Our data indicate that morphologically similar modifications in the femoro-tibial joint are involved in energy-saving mechanisms both in jumping and grasping legs in insects. The insect femoro-tibial joint is a relatively simple mechanical system as it is composed of two cylindrical leg segments (tibia and femur) that are movably connected to each other with a ring-like conjunctiva, two antagonistic muscles—tibial flexor and tibial extensor—and with a dicondylic joint composed of a pair of lateral articulations (Fig. 1). Straightening (extension) and bending (flexion) of the leg is achieved by the alternate movement of the two tibial muscles which arise from the femur with wide, fan-shaped site of attachments and inserts at the base of the tibia with elongate tendons (Fig. 1A–C). The ratio of the size of the tibial flexors and extensors is highly variable and corresponds to the adequate function of the leg that serves a broad variety of motion or grasping related behaviors (Furth and Suzuki 1990a). The muscles are equivalent in normal walking legs while the flexor muscle is enlarged in grasping and the extensor in jumping/kicking legs (Figs 1A–C, Furth and Suzuki 1990a). Fig. 1. Open in new tabDownload slide Generalized representation of an insect leg and modifications in the tibio-femoral joint for jumping and grasping. Two muscles connect the tibia and the femur. The tibial flexor (blue) bends (flexes) while the tibial extensor (brown) straightens (extends) the femoro-tibial joint. The tibial extensor is enlarged in a jumping legs (A), the two muscles are similar in their mass in walking legs (B), and the tibial flexor is enlarged in grasping legs (C). The locking mechanism between the tibial flexor muscle tendon and the ventral wall of the tibia is composed of the Heitler's lump (HL) and the genuflexor sclerite (GFS) in orthopterans (flexed (D) and extended (E) positions, modified after Gronenberg 1996; cx = coxa, tr = trochanter, tib = tibia, fem = femur, tar = tarsus; distal to the left). Fig. 1. Open in new tabDownload slide Generalized representation of an insect leg and modifications in the tibio-femoral joint for jumping and grasping. Two muscles connect the tibia and the femur. The tibial flexor (blue) bends (flexes) while the tibial extensor (brown) straightens (extends) the femoro-tibial joint. The tibial extensor is enlarged in a jumping legs (A), the two muscles are similar in their mass in walking legs (B), and the tibial flexor is enlarged in grasping legs (C). The locking mechanism between the tibial flexor muscle tendon and the ventral wall of the tibia is composed of the Heitler's lump (HL) and the genuflexor sclerite (GFS) in orthopterans (flexed (D) and extended (E) positions, modified after Gronenberg 1996; cx = coxa, tr = trochanter, tib = tibia, fem = femur, tar = tarsus; distal to the left). Changes in the muscle mass ratio is often accompanied with tendonal modifications, which have been reported from the enlarged fore, middle, and hind legs of numerous insect taxa. The tibial flexor sclerite, an atrophied basal sclerotization at the tibial flexor tendon, is perhaps the most common tendonal modification in jumping and grasping legs. In locusts, the tibial flexor sclerite is involved in a lock (catch) and plays an important role in the energy-releasing catapult mechanism (Heitler 1974, Gronenberg 1996). Although the tibial flexor muscle contracts and bends (flexes) the tibia, the tibial flexor sclerite is pulled over an internal projection (invagination) of the ventral femoral wall (Heitler's lump) into a locked position (Figs. 1D and F, and 2A–G). When the tibial extensor muscle starts to contract, the lock prevents the flexor tendon to move and straightens (extends) the femoro-tibial joint and it requires an extra power from the extensor muscle to eventually overcome the lock. During the time of release, energy from the contracting extensor muscle is stored in an apical, resilin rich structure of the femur, the semilunar process (SLP: Figs. 2A–C, and 10A and B). The stored energy from the semilunar processes allows the locusts to jump multiple times of their body length. Fig. 2. Open in new tabDownload slide The femoro-tibial joint of the hind leg of the locust, Omocestus haemorrhoidalis (Charpentier, 1825) (Orthoptera: Acrididae); (A–C) closing of the joint, (D) open joint, (E) closed joint (conjunctiva is located between Heitler's lump and GFS), (F) open joint (CLSM), (G) same at greater magnification (GFS = genulexor sclerite, conj = conjunctiva between the site of origin of the tibial flexor tendon and the distoventral margin of the femur, HL = Heitler's lump, SLP = semilunar process, distal to the left). Fig. 2. Open in new tabDownload slide The femoro-tibial joint of the hind leg of the locust, Omocestus haemorrhoidalis (Charpentier, 1825) (Orthoptera: Acrididae); (A–C) closing of the joint, (D) open joint, (E) closed joint (conjunctiva is located between Heitler's lump and GFS), (F) open joint (CLSM), (G) same at greater magnification (GFS = genulexor sclerite, conj = conjunctiva between the site of origin of the tibial flexor tendon and the distoventral margin of the femur, HL = Heitler's lump, SLP = semilunar process, distal to the left). Although it was suspected, it has never been shown that the tibial flexor sclerite of other jumping insects would participate in similar locks and catapult mechanisms (Barth 1954, Furth and Suzuki 1990a, Betz et al. 2007). Instead, it is now speculated that the tibial flexor sclerite in these taxa might be involved in strengthening the tendon, altering the working angle of the flexor system or simply protect the ventral portion of the femoro-tibial joint (Nadein and Betz 2016, 2018). These hypotheses are also supported by the fact that the sclerite is not only found in jumping but also in grasping insect legs, which would not utilize a catapult mechanism (Furth and Suzuki 1990a,b). Although the tibial flexor sclerite across jumping and grasping insects seems structurally equivalent, its relative position to the site of origin of the tibial flexor tendon tendon is variable (Furth and Suzuki 1990a). The tibial flexor tendon is an invagination of the single layer epithelium at the femoro-tibial conjunctiva and is connected to the ventral tibial wall with a resilin rich ligament, the genuflexor sclerite (Snodgrass 1956). In Alticini leaf beetles and in Orthoptera, the tibial flexor sclerite is the atrophied genuflexor sclerite (Fig. 3C–F; Snodgrass 1956, Furth and Suzuki 1990a, Nadein and Betz 2016), whereas in jumping curculionids, the tibial flexor sclerite is the atrophied basal region of the tibial flexor tendon (Fig. 3G and H; Nadein and Betz 2018). Fig. 3. Open in new tabDownload slide Line drawings showing the three major types of locking mechanisms in the ventral portion of the femoro-tibial joint. Left: extended position; Right: flexed position. (A, B) TFS over Heitler's lump without conjunctiva in between (grasping legs); (C, D) GFS locked at tip of femur, no conjunctiva in between (Alticini); (E, F) GFS over Heitler's lump with conjunctiva in-between (Orthoptera); (G, H) TFS locked at tip of femur, conjunctiva in between; GFS = genuflexor sclerite, TFS = tibial flexor sclerite, distal to the left. Fig. 3. Open in new tabDownload slide Line drawings showing the three major types of locking mechanisms in the ventral portion of the femoro-tibial joint. Left: extended position; Right: flexed position. (A, B) TFS over Heitler's lump without conjunctiva in between (grasping legs); (C, D) GFS locked at tip of femur, no conjunctiva in between (Alticini); (E, F) GFS over Heitler's lump with conjunctiva in-between (Orthoptera); (G, H) TFS locked at tip of femur, conjunctiva in between; GFS = genuflexor sclerite, TFS = tibial flexor sclerite, distal to the left. While examining ethanol preserved beetle, fly, and hymenopteran specimens with enlarged fore or hind femora, we discovered that if the specimen died with fully flexed legs, we were not able to open (unflex) the femoro-tibial joint easily, whereas in specimens that died without fully flexed legs, the joints could usually be easily moved. We also observed in specimens with transparent femoral cuticle that the tibial flexor sclerite and the ventral tibial wall is locked together in some specimens preventing the straightening of the femoro-tibial joint. In this study, we examined the femoro-tibial joint of 13 grasping and jumping insect taxa combining simple dissection and cutting edge 3D visualization techniques, to evaluate lock presence in the modified femoro-tibial joints and to better understand structural equivalencies of the anatomical structures that might be involved in these locks. This study demonstrates that simple observations using classical dissection techniques still play an important and unavoidable role in insect morphology even in the age of noninvasive 3D reconstruction techniques. Materials and Methods We have examined grasping legs with enlarged femora in taxa where modifications of the tibial flexor tendon have never been reported (diopsid flies, shore flies, and torymid wasps) and reexamined jumping (Alticini, Chrysomelidae and Rhamphini, Curculionidae) and a grasping (Bruchinae, Chrysomelidae) beetles in which the tendon sclerotizations have been reported (Furth and Suzuki 1990a,b) but their involvement in the ventral femoral lock has been dubious (Alticini, Rhamphini) or were never proposed (Bruchinae, Chrysomelidae). We have recorded our dissections with bright field microscopy and visualized dissection results with Confocal Laser Scanning Microscopy. For 3D reconstruction, we applied synchrotron-based micro–computer tomography (SR micro-CT) and to explore the fine structure of the tibial flexor sclerite, we applied scanning and transmission electron microscopy. Specimens for the present study (Table 1) were stored in 75% ethanol and were transferred to anhydrous glycerol on a concave coverslip for dissection and CLSM and are deposited in the UNH Collection of Insects and Arachnids (UNHC). Anatomical terms in are mapped to anatomical concepts in the Hymenoptera Anatomy Ontology (Supplementary URI Table). Table 1. Specimens examined Order Taxon(number of specimens), Family Function Specimen data Sclerite involved in lock Study technique Unlocking/ locking Diptera Teleopsis dalmanni (Wiedemann, 1830) (15), Diopsidae grasping, kicking UCL lab culture (MALAYSIA: KL) TFS fore, middle and hind legs, dissection, video, CLSM, SR-µCT; https://doi.org/10.6084/m9.figshare.9820595.v1 +/+ Diptera Sphyracephala brevicornis (Say, 1817) (6), Diopsidae grasping, kicking (?) USA: New Hampshire Durham 43.135, −70.933 TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820487.v1 +/+ Diptera Ochthera sp. mantis-group (6♀), Ephydridae grasping USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820484.v1 +/+ Hymenoptera Podagrion sp. 1. (6), Torymidae grasping, kicking GERMANY TFS hind legs SR-µCT of hind legs +/+ Hymenoptera Podagrion sp. 2. (3), Torymidae grasping, kicking USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS hind and middle legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820475.v2 +/+ Orthoptera Gryllus campestris Linnaeus, 1758 (3), Gryllidae jumping HUNGARY: Hortobágy GFS hind legs, dissection, SR-µCT −/− Orthoptera Omocestus (Omocestus) haemorrhoidalis (Charpentier, 1825) (5), Acrididae jumping, kicking HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820598.v1 +/− Coleoptera Disonycha xanthomelas (Dalman, 1823) (5),Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820613.v2 +/− Coleoptera Chaetocnema minuta F. E. Melsheimer, 1847 (6), Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, CLSM +/− Coleoptera Longitarsus sp. (3), Chrysomelidae jumping HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection +/− Coleoptera Caryobruchus gleditsiae (Linnaeus, 1763) (2), Bruchinae, Chrysomelidae grasping USA: FL:Coll. Co. Wiggins Pass Rec. Area 10 mi N Naples.XII-3, 1-1992, R.M. Reeves, rotten wood on beach TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820466.v1 +/+ Coleoptera Orchestes mixtus Blatchley & Leng, 1916 (2), Curculionidae jumping USA, VT. Lamoille Co. Wolcott, Lamoille Riv. 5-26-2009. T. Murray TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820616.v1 +/? Hymenoptera Schlettererius cinctipes (Cresson, 1880) (4), Stephanidae kicking, grasping (?) USA: CA: S. BRDO. Co. Jenks LK. Rd., 2105m, 34,9′48″N: 116,51′43″W; ex. Abies log coll. 28.I.06. Emerge iv.06, F. Reuter TFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820562.v1 +/+ Order Taxon(number of specimens), Family Function Specimen data Sclerite involved in lock Study technique Unlocking/ locking Diptera Teleopsis dalmanni (Wiedemann, 1830) (15), Diopsidae grasping, kicking UCL lab culture (MALAYSIA: KL) TFS fore, middle and hind legs, dissection, video, CLSM, SR-µCT; https://doi.org/10.6084/m9.figshare.9820595.v1 +/+ Diptera Sphyracephala brevicornis (Say, 1817) (6), Diopsidae grasping, kicking (?) USA: New Hampshire Durham 43.135, −70.933 TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820487.v1 +/+ Diptera Ochthera sp. mantis-group (6♀), Ephydridae grasping USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820484.v1 +/+ Hymenoptera Podagrion sp. 1. (6), Torymidae grasping, kicking GERMANY TFS hind legs SR-µCT of hind legs +/+ Hymenoptera Podagrion sp. 2. (3), Torymidae grasping, kicking USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS hind and middle legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820475.v2 +/+ Orthoptera Gryllus campestris Linnaeus, 1758 (3), Gryllidae jumping HUNGARY: Hortobágy GFS hind legs, dissection, SR-µCT −/− Orthoptera Omocestus (Omocestus) haemorrhoidalis (Charpentier, 1825) (5), Acrididae jumping, kicking HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820598.v1 +/− Coleoptera Disonycha xanthomelas (Dalman, 1823) (5),Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820613.v2 +/− Coleoptera Chaetocnema minuta F. E. Melsheimer, 1847 (6), Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, CLSM +/− Coleoptera Longitarsus sp. (3), Chrysomelidae jumping HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection +/− Coleoptera Caryobruchus gleditsiae (Linnaeus, 1763) (2), Bruchinae, Chrysomelidae grasping USA: FL:Coll. Co. Wiggins Pass Rec. Area 10 mi N Naples.XII-3, 1-1992, R.M. Reeves, rotten wood on beach TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820466.v1 +/+ Coleoptera Orchestes mixtus Blatchley & Leng, 1916 (2), Curculionidae jumping USA, VT. Lamoille Co. Wolcott, Lamoille Riv. 5-26-2009. T. Murray TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820616.v1 +/? Hymenoptera Schlettererius cinctipes (Cresson, 1880) (4), Stephanidae kicking, grasping (?) USA: CA: S. BRDO. Co. Jenks LK. Rd., 2105m, 34,9′48″N: 116,51′43″W; ex. Abies log coll. 28.I.06. Emerge iv.06, F. Reuter TFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820562.v1 +/+ Open in new tab Table 1. Specimens examined Order Taxon(number of specimens), Family Function Specimen data Sclerite involved in lock Study technique Unlocking/ locking Diptera Teleopsis dalmanni (Wiedemann, 1830) (15), Diopsidae grasping, kicking UCL lab culture (MALAYSIA: KL) TFS fore, middle and hind legs, dissection, video, CLSM, SR-µCT; https://doi.org/10.6084/m9.figshare.9820595.v1 +/+ Diptera Sphyracephala brevicornis (Say, 1817) (6), Diopsidae grasping, kicking (?) USA: New Hampshire Durham 43.135, −70.933 TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820487.v1 +/+ Diptera Ochthera sp. mantis-group (6♀), Ephydridae grasping USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820484.v1 +/+ Hymenoptera Podagrion sp. 1. (6), Torymidae grasping, kicking GERMANY TFS hind legs SR-µCT of hind legs +/+ Hymenoptera Podagrion sp. 2. (3), Torymidae grasping, kicking USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS hind and middle legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820475.v2 +/+ Orthoptera Gryllus campestris Linnaeus, 1758 (3), Gryllidae jumping HUNGARY: Hortobágy GFS hind legs, dissection, SR-µCT −/− Orthoptera Omocestus (Omocestus) haemorrhoidalis (Charpentier, 1825) (5), Acrididae jumping, kicking HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820598.v1 +/− Coleoptera Disonycha xanthomelas (Dalman, 1823) (5),Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820613.v2 +/− Coleoptera Chaetocnema minuta F. E. Melsheimer, 1847 (6), Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, CLSM +/− Coleoptera Longitarsus sp. (3), Chrysomelidae jumping HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection +/− Coleoptera Caryobruchus gleditsiae (Linnaeus, 1763) (2), Bruchinae, Chrysomelidae grasping USA: FL:Coll. Co. Wiggins Pass Rec. Area 10 mi N Naples.XII-3, 1-1992, R.M. Reeves, rotten wood on beach TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820466.v1 +/+ Coleoptera Orchestes mixtus Blatchley & Leng, 1916 (2), Curculionidae jumping USA, VT. Lamoille Co. Wolcott, Lamoille Riv. 5-26-2009. T. Murray TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820616.v1 +/? Hymenoptera Schlettererius cinctipes (Cresson, 1880) (4), Stephanidae kicking, grasping (?) USA: CA: S. BRDO. Co. Jenks LK. Rd., 2105m, 34,9′48″N: 116,51′43″W; ex. Abies log coll. 28.I.06. Emerge iv.06, F. Reuter TFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820562.v1 +/+ Order Taxon(number of specimens), Family Function Specimen data Sclerite involved in lock Study technique Unlocking/ locking Diptera Teleopsis dalmanni (Wiedemann, 1830) (15), Diopsidae grasping, kicking UCL lab culture (MALAYSIA: KL) TFS fore, middle and hind legs, dissection, video, CLSM, SR-µCT; https://doi.org/10.6084/m9.figshare.9820595.v1 +/+ Diptera Sphyracephala brevicornis (Say, 1817) (6), Diopsidae grasping, kicking (?) USA: New Hampshire Durham 43.135, −70.933 TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820487.v1 +/+ Diptera Ochthera sp. mantis-group (6♀), Ephydridae grasping USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS fore and middle legs, dissection, video, CLSM; https://doi.org/10.6084/m9.figshare.9820484.v1 +/+ Hymenoptera Podagrion sp. 1. (6), Torymidae grasping, kicking GERMANY TFS hind legs SR-µCT of hind legs +/+ Hymenoptera Podagrion sp. 2. (3), Torymidae grasping, kicking USA: Texas Bracketville 29312, −100637 III.20–22.2010 YPT TFS hind and middle legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820475.v2 +/+ Orthoptera Gryllus campestris Linnaeus, 1758 (3), Gryllidae jumping HUNGARY: Hortobágy GFS hind legs, dissection, SR-µCT −/− Orthoptera Omocestus (Omocestus) haemorrhoidalis (Charpentier, 1825) (5), Acrididae jumping, kicking HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820598.v1 +/− Coleoptera Disonycha xanthomelas (Dalman, 1823) (5),Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820613.v2 +/− Coleoptera Chaetocnema minuta F. E. Melsheimer, 1847 (6), Chrysomelidae jumping USA: NH, Dover, Bellamy Rd. 43.172, −70.809 v.17-v.19.2019, YPT I. Miko GFS hind legs, dissection, CLSM +/− Coleoptera Longitarsus sp. (3), Chrysomelidae jumping HUNGARY: Bács-Kiskun Bugacpusztaháza 46.696945°, 19.601822° Aug.10.2014 alkaline meadow sweeping Deans and Mikó GFS hind legs, dissection +/− Coleoptera Caryobruchus gleditsiae (Linnaeus, 1763) (2), Bruchinae, Chrysomelidae grasping USA: FL:Coll. Co. Wiggins Pass Rec. Area 10 mi N Naples.XII-3, 1-1992, R.M. Reeves, rotten wood on beach TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820466.v1 +/+ Coleoptera Orchestes mixtus Blatchley & Leng, 1916 (2), Curculionidae jumping USA, VT. Lamoille Co. Wolcott, Lamoille Riv. 5-26-2009. T. Murray TFS hind legs (dry specimens), dissection, video; https://doi.org/10.6084/m9.figshare.9820616.v1 +/? Hymenoptera Schlettererius cinctipes (Cresson, 1880) (4), Stephanidae kicking, grasping (?) USA: CA: S. BRDO. Co. Jenks LK. Rd., 2105m, 34,9′48″N: 116,51′43″W; ex. Abies log coll. 28.I.06. Emerge iv.06, F. Reuter TFS hind legs, dissection, video; https://doi.org/10.6084/m9.figshare.9820562.v1 +/+ Open in new tab Terminology for cuticular elements follows Klass and Matushkina (2012) and Ronquist and Nordlander (1989). We used the term sclerite for less flexible areas of the exoskeleton that are connected to each other by more flexible conjunctivae (=arthrodial membrane, =membrane). We identified these elements by manipulating the exoskeleton using insect pins and forceps. Terminology of anatomical structures in the femoro-tibial joint follows Furth and Suzuki (1990a), Snodgrass (1956) and Betz et al. (2007). We have classified sclerites on the ventral region of the femoro-tibial joint based on their relative position to the site of origin of the tendon of the tibial flexor apodeme, which corresponds to an invagination on the distal femoral margin. We used the term tibial flexor sclerite (TFS, Furth and Suzuki 1990a) for slcerotized elements on the tibial flexor tendon and the term genuflexor sclerite (GFS, Snodgrass 1956, =Lever's triangular plate, =tibial flexor sclerite sensu Furth and Suzuki 1990a,b; Betz et al. 2007; Nadein and Betz 2016) for slcerotized elements between the site of origin of the tibial flexor tendon and the proximal tibial margin. The Heitler's lump for the flattened invagination on the ventral femoral wall proximal to the site of origin of the femoro-tibial conjunctiva (Fig. 3A, B, E, and F), the femoral abutment for the resilin rich distal projection at the distal margin of the ventral femoral wall (Fig. 3C and D; =femoral abutment of Lever's triangular plate, Betz et al. 2007) and the internal protrusion for the rim at the distal margin of the ventral femoral wall (Fig. 3G and H). Both the femoral abutment and the internal protrusion are distal to the site of origin of the femoro-tibial conjunctiva. We have introduced the new term genuflexor apodeme for the invagination on the distal tibial end of the genuflexor sclerite that is sclerotized and is adjacent to the external wall of the tibia (GFS: Fig. 4A and D, and Fig. 6A) and the ventral lock of the femoro-tibial joint that refers to a lock between the ventral femoral wall and a sclerite that originates from the femoro-tibial conjunctiva (genuflexor sclerite or tibial flexor sclerite). The terminology for muscles follows Snodgrass (1956). We use the term lock to refer to two sclerite surfaces that are involved in a locking mechanism. Fig. 4. Open in new tabDownload slide Synchrotron based micro-CT micrographs of the femoro-tibial joints of the stalk-eyed fly, Teleopsis dalmanni (Wiedemann, 1830). A–D, male fore leg; E, female fore leg; F, G, male middle and hind legs (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, fe-tifld = distal tibial flexor muscle (potential trigger or release muscle), fe-tiflp = proximal tibial flexor muscle, fem = femur, tib = tibia, A–D, distal to the left; E–F, distal to the top. Fig. 4. Open in new tabDownload slide Synchrotron based micro-CT micrographs of the femoro-tibial joints of the stalk-eyed fly, Teleopsis dalmanni (Wiedemann, 1830). A–D, male fore leg; E, female fore leg; F, G, male middle and hind legs (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, fe-tifld = distal tibial flexor muscle (potential trigger or release muscle), fe-tiflp = proximal tibial flexor muscle, fem = femur, tib = tibia, A–D, distal to the left; E–F, distal to the top. We dissected ethanol-preserved and dried (card mounted) specimens. One part of the ethanol-preserved specimens were transferred to anhydrous glycerol and longitudinally bisected with Personna razor blades (Edgewell Operations, Allendale). Another part of ethanol-stored specimens and all dried specimens were bleached and rehydrated in 35% H2O2 (Sigma Aldrich, Burlington, MA) for 24 h and then transferred to anhydrous glycerol (Mikó et al. 2016). Specimens were dissected in glycerol with Dumont 5# forceps (Fine Science Tools, Foster City, CA), insect pins (#2), Vannas Spring Scissors with 2mm cutting edge (Fine Science Tools, Foster City, CA) and Personna razor blades on concavity slides in anhydrous glycerol using an Olympus SZX16 stereomicroscope equipped with a 2X objective providing a 230× magnification (Olympus Corporation of the Americas, Center Valley, PA) and a Huvitz HSZ-ZB700 stereo-microscope (Huvitz BD, Gyeonggi-do, Republic of Korea). We observed the movement/interaction between the proximal tibial flexor tendon and the ventral femoral wall while moving (straightening and bending) the femoro-tibial joint through the bleached cuticle of H2O2-treated specimens or viewing the internal side of bisected specimens. Then we detached (severed) muscle sites of origin and repeated the observations while moving the joint. If we found a lock mechanism between the tibial flexor tendon and the ventral femoral wall, we tried to unlock/relock the catch by straightening the joint or by using an insect pin as a lever to dislodge the locking sclerites. Videos were taken on an Olympus SZX16 stereo-microscope and a Huvitz HAZ-ZB700 stereo-microscope with a Canon EOS 70D and a Canon Rebel DSLR camera (Canon USA Inc. Melville, NY), respectively. Stacks of bright field images were taken manually on an Olympus CX41 microscope (Olympus Corporation of the Americas, Center Valley, PA) with a Canon EOS 70D DSLR camera attached and the images were combined using the Align and Stack All (DMap) algorithm of ZereneStacker (Version 1.04 Build T201404082055; Zerene Systems LLC, Richland, WA). Sample preparation for confocal laser scanning microscopy (CLSM) followed Mikó and Deans (2013). Specimens were imaged between two #1.5 coverslips with an Olympus FV10i confocal laser-scanning microscope (CLSM, Olympus Corporation of the Americas, Center Valley, PA) at the Microscopy and Cytometry Facility at the Huck Institute of Life Sciences at the Pennsylvania State University and with a Nikon A1R-HD CLSM at the University of New Hampshire Instrumentation Center. With the Olympus FV10i, we used three excitation wavelengths, 405, 473, and 559 nm, and detected the autofluorescence using two channels with emission ranges of 490–590 and 570–670 nm (Fig. 2). On the Nikon A1R-HD, we either used a preset (confocal) with three excitation wavelengths, 408.9, 487.4, and 559.9 nm, and three emission ranges of 435–470, 500–540, and 570–645 nm (Fig. 1) or used one excitation wavelength 487 nm laser with emission ranges defined using the A1-DUS spectral detector, 500–560 and 570–630 nm (Figs. 3E and F, and 4–6). The resulting image sets were assigned pseudo-colors that reflected the fluorescence spectra. Volume-rendered micrographs and media files were created using FIJI (Schindelin et al. 2012) and Nikon NIS-Elements AR v. 5.02.01. Synchrotron X-ray tomography (SR-µCT) was performed at the UFO imaging station of the Karlsruhe Institute of Technology (KIT) light source. The specimens were either critical point dried (Gryllus campestris & T. dalmanni) or scanned in 70% ethanol (Podagrion sp.). For each scan, 2,500 (G. campestris & Podagrion sp.) or 3,000 (T. dalmanni) equiangularly spaced radiographic projections were acquired in a range of 180°. A parallel polychromatic X-ray beam was spectrally filtered by 0.2 mm Al to obtain a peak at about 15 keV. The detector consisted of a thin, plan-parallel lutetium aluminum garnet single crystal scintillator doped with cerium (LuAG:Ce), optically coupled via a Nikon Nikkor 85/1.4 photo-lens to a pco.dimax camera with a pixel matrix of 2008x2008 pixels (dos Santos Rolo et al. 2014). The magnification was set to 10X (Gryllus sp. & Podagrion sp.) and 20X (T. dalmanni), resulting in effective pixels sizes of 1.22 and 0.61 µm. Tomographic reconstruction was performed with the GPU-accelerated filtered back projection algorithm implemented in the software framework UFO (Vogelgesang et al. 2012). 3D reconstruction of tomographic data were performed using Amira (version 5.4.3, FEI) for volume segmentation and rendering. For transmission electron microscopy (TEM), legs were removed from adult flies and fixed in 2% paraformaldehyde (PFA), 1.5% glutaraldehyde in 0.1M phosphate buffered solution (PBS) for 1.5 h at room temperature. After three 10-min washes in 0.1M PBS, the fixed tissue was transferred to 1% osmium tetroxide (OsO4) for 45 min, followed by a 10-min buffer (PBS) wash and two 10-min washes in double distilled H2O (ddH2O) and then to 2% uranyl acetate (UO2(CH3COO)2·2H2O) for 15 min, followed by three 10-min washes in ddH2O. The legs were then dehydrated through an ethanol (EtOH) series (5 min at 25% EtOH, 5 min at 75% EtOH, 5 min at 90% EtOH, 5 min at 100% EtOH). This was followed by four 10-min washes in 100% EtOH and three in propylene oxide (C3H6O). The legs were then embedded in the epoxy resin, Agar 100 (Agar Scientific, UK) in a stepwise manner, being transferred to 2 parts propylene oxide: 1 part Agar 100 resin for 1.5 h and then 1 part propylene oxide: 2 parts Agar 100 resin for 1.5 h. The samples were left in 100% Agar 100 for 8–16 h at room temperature before the Agar 100 was replaced and the samples placed in resin in molding blocks at 60°C, to harden for 48 h. Results Fully flexed femoro-tibial joints with enlarged femora in the studied taxa were locked and difficult or impossible to open even if the site of origin of the tibial flexor muscle has been destroyed. Based on the involved sclerotic elements and their interaction with the ventral femoral wall and the femoro-tibial conjunctiva, we identified four major lock types at the ventral portion of the femoro-tibial joint. type 1—TFS Lump-engaging (Figs. 3A and B, and 4–8; Supp Videos 1, 2, 6 and appropriate figshare links from Table 1 (Diptera, Hymenoptera and Caryobrichus gleditschiae) [online only]). In taxa with grasping legs, the lock is between the tibial flexor sclerite and the Heitler's lump. In the locked position, the anteroventral portion of the convex ventral surface of the sclerite is in physical contact with the Heitler's lump. We were able to release the lock after extending the leg multiple times in Schlettererius. In the diopsids, Ochthera, Caryobruchus, and Podagrion, we were not able to open the joint without forcing the tibial flexor sclerite over the Heitler's lump with an insect pin. While moving the sclerite over the lump, we observed that it stuck multiple times at different positions of the lump (as if they were two sides of a velcro tape). By pulling the tibial flexor muscle, we were able to move the sclerite over the Heitler's lump and thereby secure it in a reengaged locked position in all taxa. We were able to unlock and lock the joint multiple times. The proximal portion of the femoro-tibial conjunctiva between the site of origin of the tibial flexor tendon and the distoventral margin of the femur is not located in between the tibial flexor sclerite and the Heitler's lump when the joint is in a locked position. In the hymenopteran and dipteran specimens, the genuflexor apodeme is well developed, and the external tibial wall is angled at the point of its attachment with the apodeme (Figs. 4A and D, and 6A). The tibial flexor sclerite has a melanized center that is covered ventrally by a transparent (glass-like) ventral layer that is in physical contact with the dorsal surface of the Heitler's lump in all grasping taxa. In T. dalmanni, the melanized center of the tibial flexor sclerite is electron dense (darker on TEM images) while the transparent ventral layer is electron lucent (core, covp, covd: Fig. 7A–D), the ventral surface of the TFS is heavily sculptured (Fig. 6B–F), the Heitler's lump is T-shaped in cross section (HL: Fig. 3A, D, and E, 4A and C, 5G, 6A, and 7E and F) and lacks enlarged epithelial cells on its internal (dorsal) surface. The genuflexor sclerite is resilin-rich while the tibial flexor sclerite and the Heitler's lump are not containing resilin based on the presence/absence of blue autofluorescence in response to UV light (407 nm, Fig. 4E–H) in the diospids, Podagrion and Ochthera. In diopsids, 10–15 fibers of the tibial flexor muscle (inserting on the internal surface of the genuflexor sclerite) are oriented vertically and arise to reach the femoral wall distally to the tibial flexor sclerite when the femoro-tibial joint is fully bent (flexed) while these fibers are oriented proximodistally similarly to more proximal fibers in not fully bent legs (fe-tifld: Fig. 4A and D; Supp Video 6 [online only]). Fig. 5. Open in new tabDownload slide Bright field images and CLSM micrographs of the femoro-tibial joint of grasping insect legs. A–D, T. dalmanni, female, fore leg; E–F, Ochthera sp., female, fore leg; G, Podagrion sp., female, hind leg; H, T. dalmanni, male, fore leg (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, ten = tendon of the femoro-tibial muscle, con = femoro-tibial conjunctiva; cov = glassy ventral layer of the tibial flexor sclerite; fe-tifl = tibial flexor muscle, pit = pit corresponding to the invagination of the femoro-tibial flexor tendon, tib=tibia). Fig. 5. Open in new tabDownload slide Bright field images and CLSM micrographs of the femoro-tibial joint of grasping insect legs. A–D, T. dalmanni, female, fore leg; E–F, Ochthera sp., female, fore leg; G, Podagrion sp., female, hind leg; H, T. dalmanni, male, fore leg (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, ten = tendon of the femoro-tibial muscle, con = femoro-tibial conjunctiva; cov = glassy ventral layer of the tibial flexor sclerite; fe-tifl = tibial flexor muscle, pit = pit corresponding to the invagination of the femoro-tibial flexor tendon, tib=tibia). Fig. 6. Open in new tabDownload slide Synchrotron-based micro-CT micrographs showing the femoro-tibial joint of grasping legs. A–F, T. dalmanni, male, fore leg; G, H, Podagrion sp., female, hind leg (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, con = femoro-tibial conjunctiva, cnd = condyles [pivot points], tib = tibia, fem = femur, A–F, distal to the right, H, distal to the left). Fig. 6. Open in new tabDownload slide Synchrotron-based micro-CT micrographs showing the femoro-tibial joint of grasping legs. A–F, T. dalmanni, male, fore leg; G, H, Podagrion sp., female, hind leg (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, con = femoro-tibial conjunctiva, cnd = condyles [pivot points], tib = tibia, fem = femur, A–F, distal to the right, H, distal to the left). Fig. 7. Open in new tabDownload slide SEM micrographs showing anatomical structures at the femoro-tibal joint of the fore leg of male T. dalmanni. A, internal (dorsal) view, B, ventral view, C, external (ventral view), D, ventral view, E and F, lateral view (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, pit = pit corresponding to the invagination of the femoro-tibial flexor tendon, fe-tifld = distal tibial flexor muscle [potential trigger or release muscle], fe-tiflp=proximal tibial flexor muscle, distal to the left). Fig. 7. Open in new tabDownload slide SEM micrographs showing anatomical structures at the femoro-tibal joint of the fore leg of male T. dalmanni. A, internal (dorsal) view, B, ventral view, C, external (ventral view), D, ventral view, E and F, lateral view (GFS = genuflexor sclerite, TFS = tibial flexor sclerite, HL = Heitler's lump, pit = pit corresponding to the invagination of the femoro-tibial flexor tendon, fe-tifld = distal tibial flexor muscle [potential trigger or release muscle], fe-tiflp=proximal tibial flexor muscle, distal to the left). type 2—GFS Latch-engaging (Figs. 3C and D, and 9; Supp Video 5 and appropriate figshare links from Table 1 (Coleoptera: Alticinae) [online only]). In Alticini, the lock is between the genuflexor sclerite and the ventral femoral wall distal to the site of origin of the posterior portion of the femoro-tibial conjunctiva. Only the distal end of the genuflexor sclerite is in physical contact with the femoral abutment. The femoral abutment contains a distal sclerite and bends ventroapically when the genuflexor sclerite is unlocked (s: Fig. 9A–F). We were able to unlock the joint by extending the tibia multiple times. By pulling the tibial flexor muscle, we were not able to relock the joint. The femoro-tibial conjunctiva is not in between the interlocking sclerite surfaces and the ventral surface of the genuflexor sclerite is not connected to the ventral femoral wall. Fig. 8. Open in new tabDownload slide TEM and CLSM micrographs showing the femoro-tibial joint in T. dalmanni (TFS = tibial flexor sclerite, core = electron dense core of TFS, cov = electron lucent external surface [coating] on the ventral portion of the TFS, col = electron dense external region [coating] on the lateral portion of the TFS, tib = tibia, fe-tifld = distal tibial flexor muscle [potential trigger or release muscle], fe-tiflp = proximal tibial flexor muscle, distal to the left). Fig. 8. Open in new tabDownload slide TEM and CLSM micrographs showing the femoro-tibial joint in T. dalmanni (TFS = tibial flexor sclerite, core = electron dense core of TFS, cov = electron lucent external surface [coating] on the ventral portion of the TFS, col = electron dense external region [coating] on the lateral portion of the TFS, tib = tibia, fe-tifld = distal tibial flexor muscle [potential trigger or release muscle], fe-tiflp = proximal tibial flexor muscle, distal to the left). Fig. 9. Open in new tabDownload slide Femoro-tibial joint of the hind legs in Alticini (Chrysomelidae) and Rhamphini (Curculionidae). A, B, Longitarsus sp., A, genuflexor sclerite in a locked position, B, genuflexor sclerite in an unlocked position, C, D Disonycha xanthomela, C, genuflexor sclerite in a locked position, D, genuflexor sclerite in an unlocked position, E, F, Orchestes mixtus, E, tibial flexor sclerite in an unlocked position, F, tibial fexor sclerite in a locked position (GFS = genuflexor scelite, TFS = tibial flexor sclerite, s = distal sclerotic element of the femoral abutment [=femoral abutment of Lever's trinagular plate], vfw = ventral femoral wall, distal to the left). Fig. 9. Open in new tabDownload slide Femoro-tibial joint of the hind legs in Alticini (Chrysomelidae) and Rhamphini (Curculionidae). A, B, Longitarsus sp., A, genuflexor sclerite in a locked position, B, genuflexor sclerite in an unlocked position, C, D Disonycha xanthomela, C, genuflexor sclerite in a locked position, D, genuflexor sclerite in an unlocked position, E, F, Orchestes mixtus, E, tibial flexor sclerite in an unlocked position, F, tibial fexor sclerite in a locked position (GFS = genuflexor scelite, TFS = tibial flexor sclerite, s = distal sclerotic element of the femoral abutment [=femoral abutment of Lever's trinagular plate], vfw = ventral femoral wall, distal to the left). type 3—GFS Lump-engaging. In the locust (Figs. 2, 3E and F, and 10; Supp Video 4 and appropriate figshare links from Table 1 (Orthoptera) [online only]), similar to flea beetles, the lock is between the external surface of the genuflexor sclerite and a process on the internal surface of the Heitler's lump. The external surface of the genuflexor sclerite is concave and limited proximally by a ridge. We were able to release the lock after extending the tibia multiple times. We were able to relock the joint by pulling the tibial flexor sclerite. The proximal portion of the femoro-tibial conjunctiva (proximal in relation to the site of origin of the tibial flexor tendon) is in between the genuflexor sclerite and the Heitler's lump when the joint is in a locked position (Figs. 2D and E, and 3E and F). The internal surface of the Heitler's lump is covered with enlarged epithelial cells (HL: Fig. 2D). The elements of the locking mechanism (Fig. 10A–F) are present in Gryllus, we did not find specimens with fully flexed and locked femoro-tibial joint and similarly to the locust, and we were not able to lock the joint by pulling the tibial flexor muscle. Fig. 10. Open in new tabDownload slide Synchrotron-based micro-CT micrographs showing the hind leg femoro-tibial joint of Gryllus campestris (slp = semilunar process, fem = femur, tib = tibia, HL = Heitler's lump, GFS = genuflexor sclerite, con = femoro-tibial conjunctiva, fe-tifl = tibial flexor muscle, fe-tiex = tibial extensor muscle, lines marked with E, F show the sites of sections on E and F, distal to the left). Fig. 10. Open in new tabDownload slide Synchrotron-based micro-CT micrographs showing the hind leg femoro-tibial joint of Gryllus campestris (slp = semilunar process, fem = femur, tib = tibia, HL = Heitler's lump, GFS = genuflexor sclerite, con = femoro-tibial conjunctiva, fe-tifl = tibial flexor muscle, fe-tiex = tibial extensor muscle, lines marked with E, F show the sites of sections on E and F, distal to the left). type 4—TFS Latch-engaging. In Rhamphini curculionids (Figs. 3G and H, 9E and F; Supp Video 3 and appropriate figshare links from Table 1 (Coleoptera: Curculionidae) [online only]), the lock is between the tibial flexor sclerite sclerite and the ventral femoral wall distal to the site of origin of the posterior portion of the femoro-tibial conjunctiva. We were not able to open the joint without forcing the tibial flexor sclerite over the internal protrusion with an insect pin. By pulling the tibial flexor muscle, we were not able to relock the joint. The femoro-tibial conjunctiva connects the proximal end of the internal protrusion with the distal end of the tibial flexor sclerite and lays in between the interlocking sclerite surfaces (Fig. 9F). The ventral surface of the tibial flexor sclerite sclerite is not connected to the ventral femoral wall by a broad ligament. Discussion Besides locusts, the presence of ventral locks in the femoro-tibial joints have never been undoubtedly evinced in insects (Furth and Suzuki 1990a, Burrows and Wolf 2002, Betz et al. 2007, Hustert and Baldus 2010). Using simple manipulations in glycerol-stored specimens, we were able to show that locking mechanisms are present in the atrophied legs of the examined jumping and grasping insects except in Gryllus. These locks are either 1) between the internal surface of the ventral femoral wall and the internal surface of the tibial flexor sclerite (Fig. 3A and B), or 2) between the internal surface of the ventral femoral wall and the external surface of the genuflexor sclerite (Fig. 3C and D), or 3) between the external surfaces of the ventral femoral wall and the external surface of the genuflexor sclerite (Fig. 3E and F), or between the external surfaces of the ventral femoral wall and the internal surface of the tibial flexor sclerite (Fig. 3G and H). These types also differ in the position and the size of the locking surfaces, the presence or absence of the tibio-femoral membrane in between the locking surfaces, and numerous other modifications. The first type occurs in grasping insects (Diptera, Hymenoptera, Bruchinae) and the second to fourth types in jumping insects (Orthoptera, Alticini Chrysomelidae, and Rhamphini Curculionidae). These observations clearly demonstrate that, albeit the presence of a ventral lock in the tibio-femoral joint of enlarged legs is universal in insects, different lineages achieve this mechanical function using different solutions. Furth and Suzuki (1990b) have observed that the tendon of the tibial flexor muscle is enlarged in some bruchine and oedemerid taxa with grasping (holding) hind legs. They did not discover the ventral femoral lock and concluded that the atrophied tendon might be related to the increased stress caused by the extended contraction of the enlarged tibial flexor muscle (Furth and Suzuki 1990b). According to our study, the enlarged portion of the tibial flexor muscle (the tibial flexor sclerite) of bruchines is involved in the ventral femoro-tibial locking mechanism and helps to keep the femoro-tibial joint in a flexed position for an extended period of time. We found similar locks in grasping Hymenoptera and Diptera taxa where holding for an extended period of time might play a crucial role in their biology. In their paper, de la Motte and Burkhardt (1983) describe diopsid males, where the larger opponent (Diopsis subnotata) catches the smaller one (Megalabops rubicunda) by the eye stalk through the use of “tibia-femur pincers” as a grasping mechanism that is capable of locking an object. They also observed numerous Cyrtodiopsis (in literature sometimes referred to as Teleopsis) individuals with missing eye-stalks and leg segments and they suspect aggressive encounters as reasons, i.e., they are capable of breaking off each other's eye stalks. Based on our observations, this behavior occurs rarely and the few individuals seen with broken eye stalks soon die; stalk-eyed flies rather reach out to try to grasp the supporting legs of conspecific males as reported by Wickler and Seibt (1972) during fights. It is also reported that they grab and flip each other off the surface—in particular off free hanging root hairs where they accumulate in the evenings, and they also jab each other with their extended legs (Panhuis and Wilkinson 1999). Diopsid females are often competing for nesting sites or food resources and although not as expressed as in males, they also exhibit aggressive behavior with the involvement of striking with fore legs (Burkhardt and de la Motte 1983, Al-khairulla et al. 2003, Bath et al. 2015). Females of multiple distantly related chalcidoid taxa use their hind legs to secure their body position (Cowan 1979, Grissell and Goodpasture 1981) while depositing eggs in the host. Perhaps the most intriguing of them is the example of Lasiochalciia igiliensis (Chalcididae) as in this species the female holds the mandible of antlion larvae apart while depositing her eggs through the less sclerotized regions between the head and pronotum (Steffan 1961). Other species use their legs for securing their body on their host during dispersal. Phoresis has been reported in torymid Podagrion species, where the females grasp the wing of their mantid hosts (Bordage 1913, Xambeu 1881). Although grasping has never been described in Podagrion males, they often kick each other as part of their aggression behavior similarly to chalcidid females (Cowan 1979, Grissell and Goodpasture 1981). Among flies, Ochthera species are well characterized by their enlarged fore femur and sickle-shaped tibia representing typical raptorial legs (Clausen 1977). They are predators of smaller aquatic insect larvae and have been reported as important natural enemies of black flies and mosquitoes (Travis 1947, Minakawa et al. 2007). Ochthera flies use their “prehensive” fore legs to secure their prey items while they are probing and consuming them (Deonier 1972), but the enlarged fore femur is also used as a waving device during their courtship and aggressive interactions (Eberhard 1992). A promising candidate for future study is the apocritan family Stephanidae. Although grasping behavior has never been reported for these wasps (Hausl-Hofstätter and Bojar 2016), the presence of robust teeth on the ventral surface of their hind femora indicates that they might be used for grasping. Both males and females of stephanids have been reported to kick with their middle and hind legs during intraspecific fights (Hausl-Hofstätter and Bojar 2016). Genuflexor Sclerite, Tibial Flexor Sclerite, Heitler's Lump, and Femoral Abutment The key components of the ventral femoro-tibial locks are atrophied sclerites at the tibial flexor tendon (tibial flexor sclerite) or the femoro-tibial conjunctiva distal to the tendon (genuflexor sclerite). In flea beetles and in orthopterans, the atrophied sclerite that participates in the lock is the genuflexor sclerite, and which is located distal to the site of insertion of the tibial flexor tendon. Although the genuflexor sclerite can be found in almost all insects, it is more or less sclerotized and in numerous cases it is not involved in any locking mechanism (e.g., Apis mellifera; Snodgrass 1956). The genufexor sclerite is continuous to the tibial flexor tendon and connects the tibial flexor sclerite to the tibial base, and has an important mechanical function (the tendon that arises from the femoro-tibial conjunctival would perhaps destroy the conjunctiva without the presence of the genuflexor sclerite). Furth and Suzuki (1990a) proposed that the tibial flexor sclerite (in their paper they used this term for both the genuflexor sclerite and the tibial flexor sclerite) might protect the ventral side of the femoro-tibial joint. The protective function might be possible, but this function is not restricted to taxa with atrophied genuflexor sclerite. The Heitler's lump on the ventral femoral wall is a cuticular invagination in grasping insects and in orthopterans. The lump is proximal to the site of origin of the femoro-tibial conjunctiva and is more or less flattened (pressed against the femoral wall). The femoral abutment and the internal protrusion are distal to the femoro-tibial conjunctiva. The Heitler's lump has largely been ignored in grasping insects, as the focus has been on the gross morphological description of tibial flexor sclerites in earlier works (Furth and Suzuki 1990a) and has only been mentioned on a single illustration for grasping heteropterans (Ranatra sp., Gorb 1995, fig. 11d). We found that the femoral abutment in flea beetles are more complex than has been described, as it is movable and has a sclerotic component. In the dissection experiment of the bisected femoro-tibial joint (Supp Video 5 [online only]), it is clearly visible that when we unlocked the genuflexor sclerite, the pivot changed its shape which might explain why we were not able to relock this joint: proper backfolding of the pivot most likely requires an orchestrated movement of the tibial flexor muscle, and perhaps even the extensor muscles and the tibial extensor apodeme. Friction Enhancing Modifications on the Lock Surfaces Friction between the interacting sclerite surfaces must play an important role in keeping the femoro-tibial joint locked. Consequently, understanding the mechanical properties of the included sclerite surfaces should be the requisite of any studies that aim to understand the biomechanics of the systems that involves these locks. Surprisingly, earlier studies mostly failed to provide a detailed description of the fine structure of the interacting sclerite surfaces, including the perhaps most well-studied Heitler's lump, that of the locust. A pad of soft tissue has been reported from the ventral surface of the genuflexor sclerite in bush crickets (Burrows and Morris 2003) that is suspected by the authors to enhance the impact of the Heitler's lump on the lever of the tibial flexor muscle. We found that the genuflexor sclerite in Gryllus have a thick ventral pad (GFS: Fig 10) similar to bush crickets. We did not find a similar pad in the locust; however, the surface of the Heitler's lump is covered with a thick layer of columnar epithelial cells with unknown mechanical properties (Fig. 2G). We did not find cricket specimens with fully locked femoro-tibial joints, neither were able to relock the joint, supporting the hypothesis that these insects, unlike locusts, do not possess the ventral lock in the femoro-tibial joint. A locust can only kick and jump if the femoro-tibial joint is fully flexed and the lock is activated, whereas crickets are able to kick and jump even with partially flexed femoro-tibial joints (Burrows and Morris 2003). Comparative biomechanical study will be of value to better understand the evolutionary differences that led to the distinct jumping behaviors in crickets and locusts. In grasping taxa, the ventral ⅓ of the tibial flexor sclerite is transparent, lacks resilin, and has cardinally different electron microscopy properties than the melanized core of the sclerite. The surface of the sclerite is heavily sculptured. Since unlike in orthopterans, the proximal region of the femoro-tibial conjunctiva is not stuck in between the tibial flexor sclerite and the Heitler's lump, better understanding of surface friction in these taxa is especially important. Unlike Alticini and Orthoptera, in many grasping taxa, the joint cannot be unflexed only by pulling the tibia away from the femur, but we have to actively move over the tibial flexor sclerite through the Heitler's lump as an obstacle. This joint can be relocked multiple times while we were forcing the sclerite over the lump indicating that larger surface area of both the lump and the slcerite has an increased surface frictional property. Presence or Absence of Trigger Muscles Burrows (1969) mentioned that releasing a lock not necessarily require the presence of newly evolved trigger or release muscles to carry the additional load because slight modifications of the already present antagonistic muscles can act as triggers in mantis shrimps. Similarly, Heitler (1974) concluded that, although putative release accessory muscles can be found in locusts, these muscles are most likely not involved in the release of the lock. Rather, he proposed that contraction of the antagonistic extensor might be enough to release the lock. It has been proposed (based on their innervation pattern) that these muscles do not lift the TFS out from the locked position, but might have a stabilizing function in jumping systems (Nishino 2004). The differently oriented 10–15 muscle fibers that inserts on the genuflexor sclerite in diopsid flies (fe-tifld: Figs. 3D, and 7E and F) and clearly for a separate, fan-shaped morphological unit in fully flexed femoro-tibial joint might represent trigger portions of the tibial flexor muscle. This band can not be separated and thus observed in not-fully-flexed legs, indicating that similar muscles might have been simply overlooked in other taxa examined, and we should perhaps put more emphasis to properly describe these patterns in future works. How to Confirm the Presence of a Lock In earlier studies, the presence/absence of locking mechanisms in the femoro-tibial joint was inferred indirectly based on slow motion video recordings, and spatial relationships between anatomical structures on static images. Heitler (1974) suspected the presence of a lock in locust legs based on his experiment in which he pulled the flexor muscles until femoro-tibial joint was fully flexed and then measured the force required to reopen the joint. H2O2 bleaching is perhaps the most crucial part of our dissection-based approach as it allowed us to see how different sclerties interact while we move the joint. We assumed that a locking mechanism is present in the femoro-tibial joint only if we have seen two interacting sclerites preventing the joint to open in fully flexed legs after the flexor muscle origin was detached from the femur. The ability to relock the joint by pulling the flexor muscle is only an additional piece of evidence for the presence of the lock, and we have to acknowledge that by pulling the flexor muscles, we cannot model properly the natural contraction of the muscles. The flexor muscle fibers are grouped in multiple, distinct bundles, whose neural control, strength, and speed remain to be described. Importance of Simple Observations in the 21st-Century Morphology The advent of micro-CT-based, high-resolution 3D reconstruction tremendously accelerated the collection of morphological data and made insect morphology accessible for a broader range of students (Betz et al. 2007, Deans et al. 2012). However, micro-CT based methods, at least today, did not substitute perfectly traditional dissection-based techniques and histology. Albeit there is some convincing development in in vivo X-ray imaging techniques (dos Santos Rolo et al. 2014, Xu et al. 2016), dissections are still the most available and perhaps the most accurate methods to visualize the motion of anatomical systems both in live and dead specimens. Besides observing motion of elements, to define functional units of the skeleton also requires classical methods as the tissue-specific contrast of X-ray based methods is usually not sufficient enough to separate more and less flexible cuticular elements (sclerites and conjunctivae) from each other. Nadein and Betz (2016, 2018) have used highly sophisticated, noninvasive imaging techniques to analyze the femoro-tibial complex. However, using basic dissections techniques, we hereby revealed two key elements of this system that they were not able to capture properly: the presence of a ventral lock in the femoro-tibail joint and the lack of broad ligament (bl, broad ligament: figs. 8E and F, and 10 in Nadein and Betz 2018) between the internal surface of the ventral femoral wall and the genuflexor sclerite. Nadein and Betz (2018), most likely, considered the ventral, transparent layer on the tibial flexor sclerite on their CLSM micrograph as the connection between the sclerite and the internal surface of the ventral femoral wall. This would be interesting to further explore as we did not find resilin in this structure in the newly examined grasping taxa. Conclusion and Future Directions The ventral lock of the femoro-tibial joint reveals a remarkable parallel implementation of the physical mechanism to create a grasping and a jumping function. The building blocks of this system, genuflexor sclerite, ventral femoral wall and tibial flexor tendon are obviously present in most insects. Descriptive analysis based on static images can suggest the presence of a locking mechanism and our dissection-based experimental technique, but by studying moving parts under the microscope, we can reveal the workings of a locking apparatus and describe its functioning in detail. Due to the simplicity of this manual approach, it offers a chance to the wide scientific community to test various species representing diverse clades of insects. Until micro-CT techniques can be applied to live animals in sufficient resolution, the best solution is to utilize the wide variety of existing anatomical techniques (CLSM, SEM, TEM, etc.) in combination with the traditional dissections-under-the-microscope technique that allows us to manipulate, e.g., joints in a manner that provides information on their live role while in motion. Such future studies may range from jumping leaf beetles to leafhoppers and grasping (predatory) water scorpions, heteropterans, and robber flies. Acknowledgments Mark Turmaine's help (UCL Biosciences Electron Microscopy Facility) with SEM and TEM imaging is greatly appreciated. We thank Mark Townley (University of New Hampshire) and Missy Hazen (Pennsylvania State University) for their assistance in CLSM. We acknowledge the KIT light source for provision of instruments at their beamlines and we would like to thank the Institute for Beam Physics and Technology (IBPT) for the operation of the storage ring, the Karlsruhe Research Accelerator (KARA). Marie-Curie Fellowship (GENORN221592) supported AP and MF, Research at KIT was supported by the German Federal Ministry of Education and Research by grants 05K12CK2 (UFO2) and 05K13VK5 (ASTOR). AP acknowledges support from NERC (NE/G00563X/1, NE/R010579/1) and EPSRC grants (EP/F500351/1, EP/I017909/1). 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TI - Jumping and Grasping: Universal Locking Mechanisms in Insect Legs
JO - Insect Systematics and Diversity
DO - 10.1093/isd/ixz018
DA - 2019-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/jumping-and-grasping-universal-locking-mechanisms-in-insect-legs-499d4UxFXI
VL - 3
IS - 6
DP - DeepDyve
ER -