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Project titleEffects of miniaturization on neuroanatomy and brain efficiency in microinsects

AffiliationFederal State Budgetary Educational Institution of Higher Education Lomonosov Moscow State University,

KeywordsInsects, miniaturization, brain, enucleate neurons, Megaphragma, Hymenoptera, connectomics

Annotation
Miniaturization is not only a common trend in the evolution of animals, but also one of the main trends in the development of biomimetics. The shift from the study of individual neurons to the reconstruction and analysis of large neural networks at the cellular level is one of the most important areas of modern neuroscience. The large size of the brain and the high number of neurons in most model species make this work extremely difficult. Microinsects, by contrast, share a combination of minute size, a small number of neurons, and preserved brain functionality, which makes them convenient models for connectomics and other advanced areas of neuroscience (Makarova et al., 2021). Despite their tiny size and the number of neurons by orders of magnitude smaller than in other animals, microinsects have been shown to be capable of associative learning and long-term memory (Hoedjes et al., 2012; Hoedjes and Smid, 2014; Woude et al., 2018; Polilov et al. ., 2019). A key place among microinsects is occupied by Megaphragma (Hymenoptera: Trichogrammatidae), one of the smallest egg parasitoid wasps, for three species of which anucleate neurons and the unique phenomenon of lysis of the bodies and nuclei of up to 95% of all brain cells at the late stages of pupal development were described (Polilov, 2017). As part of the implementation of our previous project, we also discovered the presence of an almost anucleate nervous system in representatives of another family, Mymaridae. Thus, the phenomenon of lysis of the bodies and nuclei of brain cells turned out to be widespread among the smallest insects. A detailed study of the structure and ultrastructural organization of the brain of these insects is of fundamental importance for understanding the organization, functioning, and evolution of the brain of microinsects. In the course of this project, we plan to perform a comprehensive study of the structure of the brain of the smallest insects, paying special attention to the structure of the associative centers of the Megaphragma brain, as centers for integrating information incoming from the sense organs. With unique three-dimensional electron microscopy data on the entire head of Megaphragma, we will proceed to reconstruct the connectome of the mushroom bodies and the central complex, which will make it possible to model the cellular composition and projections of the sense organs to the key centers of the brain. A separate objective of the project will be a comprehensive study of the plasticity of the brain of microinsects using the example of Trichogramma telengai (Trcihogrammatidae), a miniature egg parasitoid wasp. Experiments will be carried out to study the efficiency of learning and different forms of memory in artificially bred and natural lines of Trichogramma with different body sizes, using a setup developed by us for ethological experiments on microinsects. A large-scale analysis of the effects of body size on the structure of the brain of parasitoid wasps will be carried out and an assessment will be made of changes associated with the effects of brain size on learning efficiency. The results of studying the brain of miniature insects will be of fundamental importance in understanding the structure of an animal brain and optimization of its neural pathways during miniaturization; it may also be useful for applied neurobiological problems in the development of biomorphic neural networks and processors.

Expected results
The result of the project will include the first comprehensive study of the effects of body size on the structure of the brain and its associative centers in miniature insects and the effects of brain size on the efficiency of learning and of various forms of memory. The methods and approaches we use fully advise the world level of neurobiological research and even exceed it, thanks to the unique species that we study. The availability of original data allows us to reveal the structure of miniature neural pathways and peculiar features of the functioning of the most optimized brain, which is a promising area for modern connectomics. The results of our previous project have shown that the unique phenomenon of anucleate neurons occurs not only in representatives of the genus Megaphragma of the family Trichogrammatidae (Megaphragma amalphitanum, M. mymaripenne and M. caribea), but has also been found by us in two representatives of the family Mymaridae. This project will include the first comparative analysis of the organization of the brain and neuropilar centers in the smallest hymenopterans that display the phenomenon of anucleate neurons. The brain structure will be studied in two genera of the family Mymaridae, Camptoptera and Eofoersteria. Particular attention will be paid to the volumetric analysis of the brain and its functional regions. A comparative analysis of the structure and modular organization of the associative centers of miniature hymenopterans with anucleate neurons will be carried out using data on those microinsects in which the phenomenon of the lysis of nuclei in neurons has not been revealed. Expanding the range of objects and preparing a comprehensive study of insects with anucleate neurons will provide new data on the laws of brain scaling in different groups of insects and a better understanding of the principles and causes of the lysis of nuclei in neurons at the final stages of ontogeny. The study of the structure and principles of functioning of miniature neural networks is one of the most important areas of connectomics. Mapping of the nuclei of neurons belonging to the key associative centers of the brain of Megaphragma (mushroom bodies and central complex) will be carried out as part of the project for all xyz measurements, based on the unique full series of sections of the head of Megaphragma made on a dual beam focused ion beam scanning electron microscope (FIB-SEM) with a resolution of 8 nm per pixel. The ultrastructural organization of nucleus-containing cells of the associative centers of the brain will be studied in detail. An ultrastructural analysis and reconstruction of some of the neurites of anucleate cells of the associative centers of the brain will be carried out. Quantitative analysis of the nuclei of neurons of the associative centers of the brain will allow us to estimate the percentage of nucleus-containing cells. Based on the data obtained during the annotation of synaptic connections, an analysis of neuronal connections in nucleus-containing cell clusters will be performed. On the basis of three-dimensional reconstructions, a comparative morphological analysis of subcellular elements of nucleus-containing and anucleate neurons and a quantitative and volumetric assessment of key organelles will be performed. The obtained morphological data on the structure of anucleate neurons contained in the associative centers of the brain will be of great fundamental importance for understanding the nature and functioning of anucleate nervous systems, and can become the basis for the formation of new areas of research in neuromodeling. A separate objective of the project will be to study the plasticity of the brain in microinsects and the effects of the organization of the brain of miniature insects on their cognitive abilities. In the course of the project, complex comparative ethological experiments will performed on the miniature wasp Trichogramma telengai (Hymenoptera: Trichogrammatidae) in a multi-purpose thermal arena developed by us to study the ability to learn and retain memory. Several laboratory lines will be used in the experiments, including individuals with the maximum range of body sizes, some of them representing specially selected lines with the smallest and largest bodies. Bidirectional artificial selection will be performed to study the effects of body size on brain structure and learning efficiency. After the experiments, the structure of the brain of studied specimens will be analyzed using confocal microscopy, matrix tomography, and three-dimensional modeling. A comparative analysis of the volumetric parameters of the brain of microinsects will be carried out, with an emphasis on the features of scaling of key neuropilar centers. An analysis of the number of cells in the brain will be performed. Such an integrated approach will allow us to analyze the influence of body size on the structure of the brain and its functional areas, reveal the qualitative and quantitative features of their organization, and their impact on the learning rate and memory duration. The data obtained will expand our understanding of the effects of miniaturization on the cognitive abilities of animals and will allow us to determine the structural factors that limit the minimum size of a functional brain. The study of the structural organization of the brain of the smallest insects with different body sizes will provide new data on the principles of scaling of nerve centers, determine the nature of the relationship between brain size and the degree of development of associative centers and centers of sensory modalities, and supplement the factors limiting the miniaturization of the brain in insects. Comparative analysis of evolutionary and static brain allometry in microinsects will create an important context for connectomics, which in the future may open up new opportunities for modern areas of biotechnology and bioinformatics, such as modeling neural networks and studying their scaling.

Annotation of the results obtained in 2022
The study of the effect of the miniaturization on the neuroanatomy and efficiency of the brain of microinsects in the first year of the project included three areas: the study of the structure of the anucleate brain in a new family of microhymenopterans; study of the cellular structure of the key associative centers of the brain (mushroom bodies); and studying the effect of the body size on the ability to form different types of memory in minute wasps. As part of the implementation of the first direction of the project, the structure of the brain of the miniature representatives of the Mymaridae family was studied and it was found that their central nervous system is practically devoid of a cell cortex and contains only about 700 nuclei, of which more than 90% belong to the brain, which is comparable to the number of nuclei in the CNS and brain in species of the genus Megaphragma. The size of the preserved nuclei is very small, averaging only 1.4 µm. Due to the absence of a cell cortex, the relative brain volumes of the studied fairy wasps are comparable to those of larger related groups, and, like species of the genus Megaphragma, represent an exception to Haller's rule. The relative brain neuropil volume in species of the genus Camptoptera is 96%, which is also an exception to the rule of the neuropilar brain constant described for adults. Thus, the lysis of neuronal nuclei appears to be a more common miniaturization phenomenon, independently developed at least twice in different groups of insects. At the same time, microinsects retain complex forms of behavior and are capable of learning, which indirectly indicates the functionality of anucleate neurons. In the course of work in the second direction of the project, the structure of the mushroom bodies of the miniature wasp Megaphragma viggianii was studied. Kenyon cells of one of the mushroom bodies units were reconstructed and analyzed. It was shown that out of 56 Kenyon cells, only two have nuclei, the rest are anucleate. In anucleate neurons, the absence of the Golgi apparatus was noted. Electron-dense structures were also found, apparently being RNA-protein complexes that serve as a reserve source of RNA in anucleate neurons. While most of the neurons in the adults of Megaphragma lack nuclei, the remaining nucleated neurons are not preserved by chance, but due to their position and function in the connectome. Thus, the study of anucleate neural circuits is of great interest as an answer to the question of the functional significance of the nuclei of neurons in the adult brain. The third strand of the project involved studying the effect of body size on associative learning and memory abilities in T. telengai using a microinsect thermal arena. Traces of riders' memory persisted up to 6 hours after associative learning, which may indicate the presence of both short-term and consolidated forms of memory. Preliminary comparisons of cognitive abilities between small and large T. telengai show better memory retention in small individuals. Further research into the behavior of the microinsects and the anatomy of their nervous system will decipher the fundamental neural mechanisms that allow their nervous system to shrink without loss of cognitive abilities. The complex approach we have developed, which includes a detailed study of the structure of anucleate nervous systems and the cellular organization of key associative centers of the brain associated with cognitive abilities and memory in insects, along with ethological experiments to study the consequences of genetic variability in the relative size of the brain on its morphology, will allow us to analyze brain plasticity in microinsects, to determine the nature of the influence of body size on the structure of associative centers, to highlight the qualitative and quantitative features of the organization, as well as their influence on the learning rate and memory duration. The results of the project will provide answers to fundamental questions related to the impact of miniaturization on the vital activity of organisms, and will provide a theoretical basis for the practical development of neuromorphic systems.

Publications

1. Fedorova M.A., Farisenkov S.E., Timokhov A.V., Polilov A.A. Ассоциативное обучение и память наездников Trichogramma telengai (Hymenoptera, Trichogrammatidae) ЗООЛОГИЧЕСКИЙ ЖУРНАЛ, Т 102, № 3, стр. 284-290 (year - 2023) https://doi.org/10.31857/S0044513423030054

2. Polilov A.A., Hakimi K.D., Makarova A.A. Extremely small wasps independently lost the nuclei in the brain neurons of at least two lineages Scientific Reports, 13:4320 (year - 2023) https://doi.org/10.1038/s41598-023-31529-4

3. Desyatirkina I.A. Ультраструктурная организация безъядерных и ядросодержащих нейронов Megaphragma viggianii (Hymenoptera: Trichogrammatidae) Сборник тезисов докладов XVI съезда Русского энтомологического общества, Товарищество научных изданий KMK, 59 (year - 2022) https://doi.org/10.5281/zenodo.6976546

4. Fedorova M.A., Polilov A.A. Зависимость эффективности обучения и сохранения памяти от размеров тела у Trichogramma telengai (Hymenoptera: Trichogrammatidae) Сборник тезисов докладов XVI съезда Русского энтомологического общества, Товарищество научных изданий KMK, 93 (year - 2022) https://doi.org/10.5281/zenodo.6976546

5. Hakimi K.D. Особенности строения центральной нервной системы миниатюрных наездников из родов Camptoptera и Eofoersteria (Hymenoptera: Mymaridae) Материалы XXХ Международной научной конференции студентов, аспирантов и молодых ученых "Ломоносов 2023", 1 (year - 2023)

6. Makarova A.A. Влияние миниатюризации на метаморфоз ЦНС Megaphragma vigianii (Trichogrammatidae) Сборник тезисов докладов XVI съезда Русского энтомологического общества, Товарищество научных изданий KMK, 59 (year - 2022) https://doi.org/10.5281/zenodo.6976546