An experimental test of eutardigrade preparation methods for light microscopy

Abstract

Given the constantly growing number of described tardigrade species and the scarcity of taxonomically meaningful morphological characters that these microinvertebrates exhibit, in many cases morphometric traits are the primary or even the only means of classic species differentiation, especially between closely related taxa. However, the validity of comparisons is contingent on the assumption that the mounting does not significantly affect the dimensions of morphometric traits, meaning that specimens mounted on different microscope slides may be compared with confidence. Despite the importance of the accuracy of morphometric measurements in tardigrade taxonomy and a long-standing supposition that the pressure exerted by the coverslip on a tardigrade specimen mounted on a microscope slide may deform some of the taxonomically important morphometric traits, the effect of mounting on specimen quality has not yet been addressed conclusively. In this paper we experimentally tested nine mounting methods using a lab-bred clonal strain of Milnesium cf. alpigenum Ehrenberg, 1853. Among all known tardigrades, species of the genus Milnesium Doyère, 1840 exhibit the widest buccal tubes, thus they provide the most sensitive gauge of deformation caused by coverslip pressure. Moreover, using equal-age clonal animals reared under uniform lab conditions allowed us to minimize background noise in the data. Our study showed that among the nine mounting methods tested, thermal inactivation of animals at 60 °C for 30 min followed by briefly pressing the coverslip with an entomological pin produced specimens fixed optimally for morphometry and imaging (i.e. maximally stretched and levelled but not deformed).