Organization of the central nervous system and innervation of cephalic sensory structures in the water bear Echiniscus testudo (Tardigrada: Heterotardigrada) revisited

Abstract

The tardigrade brain has been the topic of several neuroanatomical studies, as it is key to understanding the evolution of the central nervous systems in Panarthropoda (Tardigrada + Onychophora + Arthropoda). The gross morphology of the brain seems to be well conserved across tardigrades despite often disparate morphologies of their heads and cephalic sensory structures. As such, the general shape of the brain and its major connections to the rest of the central nervous system have been mapped out already by early tardigradologists. Despite subsequent investigations primarily based on transmission electron microscopy or immunohistochemistry, characterization of the different regions of the tardigrade brain has progressed relatively slowly and open questions remain. In an attempt to improve our understanding of different brain regions, we reinvestigated the central nervous system of the heterotardigrade Echiniscus testudo using anti‐synapsin and anti‐acetylated α‐tubulin immunohistochemistry in order to visualize the number and position of tracts, commissures, and neuropils. Our data revealed five major synapsin‐immunoreactive domains along the body: a large unitary, horseshoe‐shaped neuropil in the head and four neuropils in the trunk ganglia, supporting the hypothesis that the dorsal brain is serially homologous with the ventral trunk ganglia. At the same time, the pattern of anti‐synapsin and anti‐tubulin immunoreactivity differs between the ganglia, adding to the existing evidence that each of the four trunk ganglia is unique in its morphology. Anti‐tubulin labeling further revealed two commissures within the central brain neuropil, one of which is forked, and additional sets of extracerebral cephalic commissures associated with the stomodeal nervous system and the ventral cell cluster. Furthermore, our results showing the innervation of each of the cephalic sensilla in E. testudo support the homology of subsets of these structures with the sensory fields of eutardigrades.

Serial Homology and Segment Identity in the Arthropod Head

“…The presence of a single brain neuromere in both tardigrades ( Smith et al. 2017 ; Gross et al. 2021 ) and in lower-stem arthropods ( Cong et al.…”

“…This single neuromere has been attributed to the protocerebrum, as in the brain of tardigrades ( Smith et al. 2017 ; Gross et al. 2021 ).…”

in vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada

“…Fluorescent proteins fused with target proteins possibly enable us to reveal not only the subcellular localization but also the protein dynamics during dehydration and rehydration without sample loss. For example, GCaMP will reveal neural activity and networks, building on the tardigrade anatomy that was revealed through IHC and electron microscopy imaging [29][30][31][32][33][34][35][36]50,51 .…”

“…Therefore, the actual subcellular localization in tardigrade cells and the dynamics during anhydrobiosis, as well as the tissue specificity of these expressions in tardigrades, remain elusive. IHC has been used widely in tardigrades to reveal cell morphology, such as muscle and neural systems, and the localization of tardigrade protein in the whole body [29][30][31][32][33][34][35][36] , but it nevertheless cannot be used to observe the dynamics of proteins and cells in a living tardigrade.…”

“…Therefore, the actual subcellular localization in tardigrade cells and the dynamics during anhydrobiosis, as well as the tissue specificity of these expressions in tardigrades, remain elusive. IHC has been used widely in tardigrades to reveal cell morphology, such as muscle and neural systems, and the localization of tardigrade protein in the whole body [29][30][31][32][33][34][35][36] , but it nevertheless cannot be used to observe the dynamics of proteins and cells in a living tardigrade.In the present study, we have developed an in vivo expression system, designated the TardiVec system, using a promoter that originated from an anhydrobiotic tardigrade, Ramazzottius varieornatus; it allows the introduction of exogenous genes such as GFP for live imaging in tardigrades. Tardigrade vectors show distinct expression patterns depending on the promoter sequence, and the GFP signals were retained for more than 10 days in tardigrades.…”