The hierarchical organisation of cuticle in the basis-coxa joint of terrestrial isopods: variations in structure and composition adapt the cuticle for movement, support and stability

This research on crustacean cuticle focuses on a correlation between architecture and function of a joint connecting a pereiopod (walking leg) to the body in isopods. In general, research on biological materials aims to provide an answer as to how life has evolved different functional materials and made efficient use of them. In the Arthropoda, with a view to understanding the ultrastructure and composition of the taxon’s diverse cuticular elements, a wide range of skeletal elements for example those belonging to insects and crustaceans have been subjected to investigation. While it can be similarly structured and composed, the cuticle serves diverse functions for the animal by slightly modifying its structures and components resulting in a great diversification of cuticles adapted to functions ranging, for example, from mechanical protection and movement to vision, respiration as well as defence. In crustaceans, the incorporation of mineral into the cuticle distinguishes them from members of other arthropod groups; generally, crustacean cuticle is, as a result, harder and stiffer than cuticles of other arthropods and, by having mineral, there is one more degree of freedom for cuticular variation in crustaceans. Groups in the Crustacea that are a focus of biological materials research include Decapoda, Amphipoda and Isopoda. In isopods, a variety of skeletal elements has been studied, including cuticles of the tergite, mandible, eye as well as joint. Joints offer researchers an opportunity to look into a cuticle where both a fixed and a moveable element can be observed. The joint in this study is of a ball-and-socket type, with the end of the basis (the most proximal segment of the pereiopod in isopods) acting as a ball moving within the confinement of the sternal plate, which acts as a socket of the joint. In this study, the end of the basis is termed condyle and the cuticle surrounding the socket in the sternal plate simply called socket. In between the condyle and socket lies a thin cuticle, called arthrodial membrane, which ensures flexible movement of the former against the latter. Isopod species that are a subject of this work include Helleria brevicornis Ebner, 1868, Tylos europaeus Arcangeli, 1938, and Porcellio scaber Latreille, 1804. The animals live in different habitats. H. brevicornis are found in forests near rivers on islands of the Tyrrhenian See, while T. europaeus roam the upper zones of sandy shores of the European Mediterranean and Atlantic coasts and P. scaber are adapted to drier climates and inhabit mesic habitats. Phylogenically, the former two are closely related belonging to the family Tylidae, while P. scaber belong to the Crinochaeta. In defence, P. scaber cling tightly to the substrate or run away from predators, while the two tylids role their bodies up exposing only the hard, dorsal part to the outside while keeping the soft, ventral part unexposed. The three different species were chosen for the joint study to compare similarities and differences that may arise from phylogenic distance, habitats, and/or defence tactics. Like in many other cuticles, the joint cuticle is composed of an epicuticle, exocuticle and endocuticle. The epicuticle contains lipids, waxes, proteins and alcohols. It forms scales and hairs, protects the animals from osmotic stress and, in terrestrial species, prevents desiccation. Layers that constitute the bulk of the cuticle are the exo- and endocuticle. They are helicoidally arranged in which chitin-protein fibrils/fibres are oriented parallel to one another in one plane but rotate at an angle from one plane to the next resulting in a so-called twisted plywood structure or Bouligand structure. Variations in structure as well as in chemical composition occur between the hard cuticle of the condyle and socket and the soft cuticle of the arthrodial membrane, with edges of the hard cuticle acting as transitional points. The region containing hard cuticle is called the central region in reference to its position at the centre of the condyle/socket cuticle. The central region contains a thin epicuticle, heavily mineralised and organically sparse distal exocuticle, and proximal exocuticle and endocuticle in which chitin-protein fibrils are dense and each individual fibril is surrounded by mineral. In terms of thickness of the major layers, there are some variations between species. While, in all species, the endocuticle is the major layer accounting for more than half of the total thickness of the whole cuticle, in H. brevicornis and T. europaeus, the distal exocuticle is much thicker than the proximal exocuticle but in P. scaber, thickness relation of the two exocuticular layers is the reverse. Mineral in fractured surfaces of the distal exocuticle has different morphologies. In the two tylid species, polygonal structures are observed alternating with smooth faces, whereas the fracture face in P. scaber is smooth or plate-like. Unlike in the proximal exocuticle and endocuticle, the mechanism governing mineral formation in the distal exocuticle seems to be independent of the organic phase. The crystal morphology is a result of mineral grains growing in a competitive manner leading to the fracture face of the distal exocuticle resembling that of inorganic minerals. The joints are mineralised by calcium carbonate, and to a much smaller extent, calcium phosphate. The calcium carbonate occurs in two different phases, crystalline calcite and amorphous calcium carbonate (ACC), with the former mineralising the outer periphery of the cuticle and the latter the inner part. Since these mineral phases have distinct mechanical properties, this leads to the outer part, i.e. the distal exocuticle, being harder and stiffer than the inner part, i.e. proximal exocuticle and endocuticle. At edges of the joint, the region near the arthrodial membrane, the distal exocuticle disappears and the epicuticle becomes many-fold thicker. This together with the higher concentration of amorphous calcium phosphate results in the edge having higher compliance than the central region, which is crucial to its being a transitional cuticle to the soft arthrodial membrane. In regions of the joint where the condyle and socket come into frequent contact, their edges are large to support the weight of the opposing structure. To prevent delamination of the epicuticle as a result of the frequent contact, the epicuticle at the edge sends extension downwards firmly anchoring its material to the subjacent exocuticle. This modification is common in H. brevicornis and T. europaeus but very rare in P. scaber and when such modification is observed, the epicuticular extensions are much shorter than those of the tylids. In P. scaber, an unusual fibril orientation is observed in the epicuticle at the edge. Instead of having the usual Bouligand structure, the fibrils do not twist between planes running parallel along the rim of the condyle forming a “rope-like” structure, which helps sustain higher loads that arise from the species’ higher agility. In the arthrodial membrane, the cuticle is much thinner than the condyle and socket cuticle, with thickness of the exo- and endocuticle greatly reduced, while that of the epicuticle remains comparably thick to that observed at the edge. The reduction in thickness of the exo- and endocuticle coupled with their much less indistinguishable structure likely results in the arthrodial membrane being much softer and more flexible than the condyle and socket cuticle. Important functions of the epicuticle are retained because, despite a requirement to be soft and flexible, the arthrodial membrane still needs to be impermeable to water, for which the epicuticle serves a primary role. It appears that differences observed in the joint are rather species-specific, with the joint cuticles of the two tylids comparable to each other, while differing from that of P. scaber, reflecting the close phylogenetic relationship of the former two against P. scaber as well as the different defence strategies, that is, rolling into spheres in H. brevicornis and T. europaeus and clinging to the substrate or running away in P. scaber. What has been found in this study helps us understand more the structure and composition of joint cuticle that is specialised for stable connection and guidance in relative movements of exoskeletal elements. Its function in movement, coordinated by the condyle, the socket and the arthrodial membrane connecting the two together, is reflected in the joint architecture. By seeing the changes occurring in the cuticle as we go from the hard central region to the softer edge and eventually to the even softer and more flexible arthrodial membrane, we have a better understanding of the structure/composition-function relationship of the different layers that constitute a crustacean cuticle

New insights from Thailand into the maternal genetic history of Mainland Southeast Asia

Tai-Kadai (TK) is one of the major language families in Mainland Southeast Asia (MSEA), with a concentration in the area of Thailand and Laos. Our previous study of 1234 mtDNA genome sequences supported a demic diffusion scenario in the spread of TK languages from southern China to Laos as well as northern and northeastern Thailand. Here we add an additional 560 mtDNA genomes from 22 groups, with a focus on the TK-speaking central Thai people and the Sino-Tibetan speaking Karen. We find extensive diversity, including 62 haplogroups not reported previously from this region. Demic diffusion is still a preferable scenario for central Thais, emphasizing the expansion of TK people through MSEA, although there is also some support for gene flow between central Thai and native Austroasiatic speaking Mon and Khmer. We also tested competing models concerning the genetic relationships of groups from the major MSEA languages, and found support for an ancestral relationship of TK and Austronesian-speaking groups