Miniaturization is an evolutionary trend observed in many animals. Some arachnid groups, such as spiders and mites, demonstrate a strong tendency toward miniaturization. Some of the most miniaturized spiders belong to the family Anapidae. In this study, using light and confocal microscopy and 3D modelling, we provide the first detailed description of the anatomy of a spider of the genus Rayforstia, which is only 900 µm long. In comparison with larger spiders, Rayforstia has no branching of the midgut in the prosoma and an increased relative brain volume. In contrast to many miniature insects and mites, the spider shows no reduction of whole organ systems, no allometry of the digestive and reproductive systems, and also no reduction of the set of muscles. Thus, miniature spider shows a more conserved anatomy than insects of a similar size. These findings expand our knowledge of miniaturization in terrestrial arthropods.

Methods of three-dimensional electron microscopy have been actively developed recently and open up great opportunities for morphological work. This approach is especially useful for studying microinsects, since it is possible to obtain complete series of high-resolution sections of a whole insect. Studies on the genus Megaphragma are especially important, since the unique phenomenon of lysis of most of the neuron nuclei was discovered in species of this genus. In this study we reveal the anatomical structure of the head of Megaphragma viggianii at all levels from organs to subcellular structures. Despite the miniature size of the body, most of the organ systems of M. viggianii retain the structural plan and complexity of organization at all levels. The set of muscles and the well-developed stomatogastric nervous system of this species correspond to those of larger insects, and there is also a well-developed tracheal system in the head of this species. Reconstructions of the head of M. viggianii at the cellular and subcellular levels were obtained, and of volumetric data were analyzed. A total of 689 nucleated cells of the head were reconstructed. The ultrastructure of M. viggianii is surprisingly complex, and the evolutionary benefits of such complexity are probably among the factors limiting the further miniaturization of parasitoid wasps.

Anucleate animal cells are a peculiar evolutionary phenomenon and a useful model for studying cellular mechanisms. Anucleate neurons were recently found in one genus of miniature parasitic wasps of the family Trichogrammatidae, but it remained unclear how widespread this phenomenon is among other insects or even among different tissues of the same insect species. We studied the anatomy of miniature representatives of another parasitic wasp family (Hymenoptera: Mymaridae) using array tomography and found two more species with nearly anucleate brains at the adult stage. Thus, the lysis of the cell bodies and nuclei of neurons appears to be a more widespread means of saving space during extreme miniaturization, which independently evolved at least twice during miniaturization in different groups of insects. These results are important for understanding the evolution of the brain during miniaturization and open new areas of studying the functioning of anucleate neurons.