WASPSYN: A Challenge for Domain Adaptive Synapse Detection in Microwasp Brain Connectomes.

The size of image volumes in connectomics studies now reaches terabyte and often petabyte scales with a great diversity of appearance due to different sample preparation procedures. However, manual annotation of neuronal structures (e.g., synapses) in these huge image volumes is time-consuming, leading to limited labeled training data often smaller than 0.001% of the large-scale image volumes in application. Methods that can utilize in-domain labeled data and generalize to out-of-domain unlabeled data are in urgent need. Although many domain adaptation approaches are proposed to address such issues in the natural image domain, few of them have been evaluated on connectomics data due to a lack of domain adaptation benchmarks. Therefore, to enable developments of domain adaptive synapse detection methods for large-scale connectomics applications, we annotated 14 image volumes from a biologically diverse set of Megaphragma viggianii brain regions originating from three different whole-brain datasets and organized the WASPSYN challenge at ISBI 2023. The annotations include coordinates of pre-synapses and post-synapses in the 3D space, together with their one-to-many connectivity information. This paper describes the dataset, the tasks, the proposed baseline, the evaluation method, and the results of the challenge. Limitations of the challenge and the impact on neuroscience research are also discussed. The challenge is and will continue to be available at https://codalab.lisn.upsaclay.fr/competitions/9169. Successful algorithms that emerge from our challenge may potentially revolutionize real-world connectomics research and further the cause that aims to unravel the complexity of brain structure and function.

Multiscale head anatomy of Megaphragma (Hymenoptera: Trichogrammatidae).

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.

A complete reconstruction of the early visual system of an adult insect.

For most model organisms in neuroscience, research into visual processing in the brain is difficult because of a lack of high-resolution maps that capture complex neuronal circuitry. The microinsect Megaphragma viggianii, because of its small size and non-trivial behavior, provides a unique opportunity for tractable whole-organism connectomics. We image its whole head using serial electron microscopy. We reconstruct its compound eye and analyze the optical properties of the ommatidia as well as the connectome of the first visual neuropil-the lamina. Compared with the fruit fly and the honeybee, Megaphragma visual system is highly simplified: it has 29 ommatidia per eye and 6 lamina neuron types. We report features that are both stereotypical among most ommatidia and specialized to some. By identifying the "barebones" circuits critical for flying insects, our results will facilitate constructing computational models of visual processing in insects.

Extremely small wasps independently lost the nuclei in the brain neurons of at least two lineages.

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.

Anatomy of the miniature four-legged mite Achaetocoptes quercifolii (Arachnida: Acariformes: Eriophyoidea).

Miniaturization is one of the important trends in the evolution of terrestrial arthropods. In order to study adaptations to microscopic sizes, the anatomy of the smallest insects was previously studied, but not the anatomy of the smallest mites. Some of the smallest mites are Eriophyidae. In this study we describe for the first time the anatomy of the mite Achaetocoptes quercifolii, which is about 115 µm long. For this purpose, we used light, scanning, and transmission electron microscopy and performed 3D reconstructions. The anatomy of A. quercifolii is compared with the anatomy of larger representatives of Eriophyoidea. Despite the small size of the studied species, there is no considerable simplification of its anatomy compared to larger four-legged mites. A. quercifolii has a number of miniaturization effects similar to those found in microinsects: a strong increase in the relative volume of the reproductive system, an increase in the relative volume of the brain, reduction in the number and size of cells of the nervous system. As in some larger four-legged mites, A. quercifolii undergoes midgut lysis at the stage of egg production. On the other hand, in A. quercifolii a greater number of opisthosomal muscles are preserved than in larger gall-forming four-legged mites.

The 3D ultrastructure of the chordotonal organs in the antenna of a microwasp remains complex although simplified.

Insect antennae are astonishingly versatile and have multiple sensory modalities. Audition, detection of airflow, and graviception are combined in the antennal chordotonal organs. The miniaturization of these complex multisensory organs has never been investigated. Here we present a comprehensive study of the structure and scaling of the antennal chordotonal organs of the extremely miniaturized parasitoid wasp Megaphragma viggianii based on 3D electron microscopy. Johnston's organ of M. viggianii consists of 19 amphinematic scolopidia (95 cells); the central organ consists of five scolopidia (20 cells). Plesiomorphic composition includes one accessory cell per scolopidium, but in M. viggianii this ratio is only 0.3. Scolopale rods in Johnston's organ have a unique structure. Allometric analyses demonstrate the effects of scaling on the antennal chordotonal organs in insects. Our results not only shed light on the universal principles of miniaturization of sense organs, but also provide context for future interpretation of the M. viggianii connectome.

Metamorphosis and denucleation of the brain in the miniature wasp Megaphragma viggianii (Hymenoptera: Trichogrammatidae).

Holometabolan brains undergo structural and allometric changes and complex reorganizations during metamorphosis. In minute egg parasitoids, brain formation is shifted to the late larva and young pupa, due to extreme de-embryonization. The brains of Megaphragma wasps undergo denucleation, the details of which remained unknown. We describe the morphological and volumetric changes in the brain of Megaphragma viggianii (Trichogrammatidae) during pupal development with emphasis on the lysis of nuclei and show that the absolute and relative volume of the brain decrease by a factor of 5 from prepupa to adult at the expense of the cell body rind. The first foci of lysis appear during early pupal development, but most nuclei (up to 97%) are lost between pharate adult and adult. The first signs of lysis (destruction of the nuclear envelopes) occur in pupae with red eyes. The number of lysis foci (organelle destruction and increasing number of lysosomes and degree of chromatin compaction) strongly increases in pupae with black eyes. The cell body rind volume strongly decreases during pupal development (in larger insects it increases slightly or remains unchanged). Elucidation of the lysis of nuclei in neurons and of the functioning of an anucleate brain is an important objective for neuroscience.

Revision of the World Species of Megaphragma Timberlake (Hymenoptera: Trichogrammatidae).

Megaphragma species are important models for basic organismal research, and many are potential biological control agents. We present the first extensive revision of species of the genus Megaphragma based on morphological and molecular data. Our revision includes all previously described species, 6 of which are synonymized, and 22 of which are described here as new. We also provide the first key to all species of the genus and reconstruct their phylogeny based on 28S and CO1 molecular markers. The following species are synonymized with M. longiciliatum Subba Rao: M. aligarhensis Yousuf and Shafee syn. nov.; M. amalphitanum Viggiani syn. nov.; M. decochaetum Lin syn. nov.; M. magniclava Yousuf and Shafee syn. nov.; M. shimalianum Hayat syn. nov.M. anomalifuniculi Yuan and Lou syn. nov. is synonymized with M. polychaetum Lin. The following species are described as new: M. antecessor Polaszek and Fusu sp. nov.; M. breviclavum Polaszek and Fusu sp. nov.; M. chienleei Polaszek and Fusu sp. nov.; M. cockerilli Polaszek and Fusu sp. nov.; M. digitatum Polaszek and Fusu sp. nov.; M. fanenitrakely Polaszek and Fusu sp. nov.; M. funiculatum Fusu, Polaszek, and Viggiani sp. nov.; M. giraulti Viggiani, Fusu, and Polaszek sp. nov.; M. hansoni Polaszek, Fusu, and Viggiani sp. nov.; M. kinuthiae Polaszek, Fusu, and Viggiani sp. nov.; M. liui Polaszek and Fusu sp. nov.; M. momookherjeeae Polaszek and Fusu sp. nov.; M. nowickii Polaszek, Fusu, and Viggiani sp. nov.; M. noyesi Polaszek and Fusu sp. nov.; M. pintoi Viggiani sp. nov.; M. polilovi Polaszek, Fusu, and Viggiani sp. nov.; M. rivelloi Viggiani sp. nov.; M. tamoi Polaszek, Fusu, and Viggiani sp. nov.; M. tridens Fusu, and Polaszek sp. nov.; M. uniclavum Polaszek and Fusu sp. nov.; M. vanlentereni Polaszek and Fusu sp. nov.; M. viggianii Fusu, Polaszek, and Polilov sp. nov.

Novel flight style and light wings boost flight performance of tiny beetles.

Flight speed is positively correlated with body size in animals1. However, miniature featherwing beetles can fly at speeds and accelerations of insects three times their size2. Here we show that this performance results from a reduced wing mass and a previously unknown type of wing-motion cycle. Our experiment combines three-dimensional reconstructions of morphology and kinematics in one of the smallest insects, the beetle Paratuposa placentis (body length 395 µm). The flapping bristled wings follow a pronounced figure-of-eight loop that consists of subperpendicular up and down strokes followed by claps at stroke reversals above and below the body. The elytra act as inertial brakes that prevent excessive body oscillation. Computational analyses suggest functional decomposition of the wingbeat cycle into two power half strokes, which produce a large upward force, and two down-dragging recovery half strokes. In contrast to heavier membranous wings, the motion of bristled wings of the same size requires little inertial power. Muscle mechanical power requirements thus remain positive throughout the wingbeat cycle, making elastic energy storage obsolete. These adaptations help to explain how extremely small insects have preserved good aerial performance during miniaturization, one of the factors of their evolutionary success.

Small brains for big science.

As the study of the human brain is complicated by its sheer scale, complexity, and impracticality of invasive experiments, neuroscience research has long relied on model organisms. The brains of macaque, mouse, zebrafish, fruit fly, nematode, and others have yielded many secrets that advanced our understanding of the human brain. Here, we propose that adding miniature insects to this collection would reduce the costs and accelerate brain research. The smallest insects occupy a special place among miniature animals: despite their body sizes, comparable to unicellular organisms, they retain complex brains that include thousands of neurons. Their brains possess the advantages of those in insects, such as neuronal identifiability and the connectome stereotypy, yet are smaller and hence easier to map and understand. Finally, the brains of miniature insects offer insights into the evolution of brain design.

Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects.

Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

Metamorphosis of the central nervous system of Trichogramma telengai (Hymenoptera: Trichogrammatidae).

During metamorphosis, the insect CNS undergoes both structural and allometric changes. Due to their extreme de-embryonization and parasitism, the formation of the CNS in egg parasitoids occurs at the late larval stage. Our study provides the first data on the morphological and volumetric changes of the CNS occurring during the pupal development of the parasitic wasp Trichogramma telengai Sorokina, 1987 (Trichogrammatidae). The prepupal-pupal development includes fusion and concentration of ganglia achieved by the loss of connectives. Volumetric analysis shows that during the pupal development the absolute body volume and CNS volume gradually decrease. The brain and thoracic synganglion slightly increase in volume during the pupal period and extremely decrease from late pupa to adult. The CNS neuropil volume increases from prepupa to adult. The mean cell diameter also decreases during the metamorphosis of the nervous system. The cell body rind volume decreases during pupal development; this decrease correlates with the decrease in the number of cells on the one hand and increase in the neuropilar volume on the other hand.

Sensation of the tiniest kind: the antennal sensilla of the smallest free-living insect Scydosella musawasensis (Coleoptera: Ptiliidae).

Miniaturization is a major evolutionary trend prominent in insects, which has resulted in the existence of insects comparable in size to some unicellular protists. The adaptation of the complex antennal multisensory systems to extreme miniaturization is a fascinating problem, which remains almost unexplored. We studied the antennal sensilla of Scydosella musawasensis Hall, 1999 (Coleoptera: Ptiliidae), the smallest free-living insect, using scanning electron microscopy. The antenna of S. musawasensis bears 131 sensilla; no intraspecific variation in the number or position of the sensilla has been revealed. Nine different morphological types of sensilla are described according to their external morphological features and distribution: four types of sensilla trichodea, one type of sensilla chaetica, two types of sensilla styloconica, and two types of sensilla basiconica. Morphometric analysis of the sensilla of S. musawasensis, based on measurements of the lengths and diameters of sensilla and their location and number, showed the absence of significant differences between females and males. Comparative allometric analysis of S. musawasensis and larger Coleoptera showed that the number of sensilla and the size of sensilla chaetica decrease with decreasing body size. However, the number of the types of sensilla and the length and diameter of the multiporous sensilla basiconica revealed no correlation with the body size. Comparison of the acquired data with the results of our earlier study of the antennal sensilla of some of the smallest parasitic wasps is used to put forward hypotheses on the common principles of miniaturization of the antennal sensory systems of insects.

Constant neuropilar ratio in the insect brain.

Revealing scaling rules is necessary for understanding the morphology, physiology and evolution of living systems. Studies of animal brains have revealed both general patterns, such as Haller's rule, and patterns specific for certain animal taxa. However, large-scale studies aimed at studying the ratio of the entire neuropil and the cell body rind in the insect brain have never been performed. Here we performed morphometric study of the adult brain in 37 insect species of 26 families and ten orders, ranging in volume from the smallest to the largest by a factor of more than 4,000,000, and show that all studied insects display a similar ratio of the volume of the neuropil to the cell body rind, 3:2. Allometric analysis for all insects shows that the ratio of the volume of the neuropil to the volume of the brain changes strictly isometrically. Analyses within particular taxa, size groups, and metamorphosis types also reveal no significant differences in the relative volume of the neuropil; isometry is observed in all cases. Thus, we establish a new scaling rule, according to which the relative volume of the entire neuropil in insect brain averages 60% and remains constant.

Miniaturization re-establishes symmetry in the wing folding patterns of featherwing beetles.

Most microinsects have feather-like bristled wings, a state known as ptiloptery, but featherwing beetles (family Ptiliidae) are unique among winged microinsects in their ability to fold such wings. An asymmetrical wing folding pattern, found also in the phylogenetically related rove beetles (Staphylinidae), was ancestral for Ptiliidae. Using scanning electron, confocal laser scanning, and optical microscopy, high-speed video recording, and 3D reconstruction, we analyze in detail the symmetrical wing folding pattern and the mechanism of the folding and unfolding of the wings in Acrotrichis sericans (Coleoptera: Ptiliidae) and show how some of the smaller featherwing beetles have reverted to strict symmetry in their wing folding. The wings are folded in three phases by bending along four lines (with the help of wing folding patches on the abdominal tergites) and locked under the closed elytra; they unfold passively in two phases, apparently with the help of the elasticity provided by resilin unevenly distributed in the wing and of convexities forming in the cross-sections of the unfolding wing, making it stiffer. The minimum duration of folding is 3.5 s; unfolding is much more rapid (minimum duration lowest recorded in beetles, 0.038 s). The folding ratio of A. sericans is 3.31 (without setae), which is greater than in any beetle in which it has been measured. The symmetrical wing folding pattern found in A. sericans and in all of the smallest ptiliids, in which ptiloptery is especially pronounced, is the only known example of symmetry re-established during miniaturization. This direction of evolution is remarkable because miniaturization is known to result in various asymmetries, while in this case miniaturization was accompanied by reversal to symmetry, probably associated with the evolution of ptiloptery. Our results on the pattern and mechanisms of wing folding and unfolding can be used in robotics for developing miniature biomimetic robots: the mechanisms of wing folding and unfolding in Ptiliidae present a challenge to engineers who currently work at designing ever smaller flying robots and may eventually produce miniature robots with foldable wings.

Extraordinary flight performance of the smallest beetles.

Size is a key to locomotion. In insects, miniaturization leads to fundamental changes in wing structure and kinematics, making the study of flight in the smallest species important for basic biology and physics, and, potentially, for applied disciplines. However, the flight efficiency of miniature insects has never been studied, and their speed and maneuverability have remained unknown. We report a comparative study of speeds and accelerations in the smallest free-living insects, featherwing beetles (Coleoptera: Ptiliidae), and in larger representatives of related groups of Staphylinoidea. Our results show that the average and maximum flight speeds of larger ptiliids are extraordinarily high and comparable to those of staphylinids that have bodies 3 times as long. This is one of the few known exceptions to the "Great Flight Diagram," according to which the flight speed of smaller organisms is generally lower than that of larger ones. The horizontal acceleration values recorded in Ptiliidae are almost twice as high as even in Silphidae, which are more than an order of magnitude larger. High absolute and record-breaking relative flight characteristics suggest that the unique morphology and kinematics of the ptiliid wings are effective adaptations to flight at low Reynolds numbers. These results are important for understanding the evolution of body size and flight in insects and pose a challenge to designers of miniature biomorphic aircraft.

Unusual mechanism of emission of vibratory signals in pygmy grasshoppers Tetrix tenuicornis (Sahlberg, 1891) (Orthoptera: Tetrigidae).

Acoustic communication plays an important role in the life of insects and especially in representatives of the order Orthoptera. Their vibrational signalling, unlike signalling by sound, is poorly studied. The pygmy grasshoppers Tetrix tenuicornis (Sahlberg, 1891) belonging to the ancestral family Tetrigidae (Orthoptera) can produce several types of substrate-borne vibratory signals using their mid-legs. The emission of these signals is not accompanied by visible movements of any parts of the body. The goal of our study was to elucidate the mechanism of production of these vibrations. For this, we synchronously recorded the vibratory signals and the muscle activity in various regions of the legs and thorax in freely moving males. The obtained results revealed an unusual mechanism for the emission of acoustic signals. We found that the strongest muscle activity during the emission of the vibratory signals was recorded in the mesofemur and mesotibia. According to the position of the electrode, these muscles are the flexor and extensor of the tibia, levators and depressors of the tarsus, and probably pretarsus. The motor system employed during the emission of vibratory signals was most similar to that of the jump of locusts and probably is performed as a result of co-contraction of antagonistic muscles of the tibia, tarsus, and pretarsus. The data obtained make significant additions to the presentation of a variety of insect acoustic communication systems.

A partial genome assembly of the miniature parasitoid wasp, Megaphragma amalphitanum.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect-the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.

Effects of miniaturization in the anatomy of the minute springtail Mesaphorura sylvatica (Hexapoda: Collembola: Tullbergiidae).

Smaller animals display pecular characteristics related to their small body size, and miniaturization has recently been intensely studied in insects, but not in other arthropods. Collembola, or springtails, are abundant soil microarthropods and form one of the four basal groups of hexapods. Many of them are notably smaller than 1 mm long, which makes them a good model for studying miniaturization effects in arthropods. In this study we analyze for the first time the anatomy of the minute springtail Mesaphorura sylvatica (body length 400 µm). It is described using light and scanning electron microscopy and 3D computer reconstruction. Possible effects of miniaturization are revealed based on a comparative analysis of data from this study and from studies on the anatomy of larger collembolans. Despite the extremely small size of M. sylvatica, some organ systems, e.g., muscular and digestive, remain complex. On the other hand, the nervous system displays considerable changes. The brain has two pairs of apertures with three pairs of muscles running through them, and all ganglia are shifted posteriad by one segment. The relative volumes of the skeleton, brain, and musculature are smaller than those of most microinsects, while the relative volumes of other systems are greater than or the same as in most microinsects. Comparison of the effects of miniaturization in collembolans with those of insects has shown that most of the miniaturization-related features of M. sylvatica have also been found in microinsects (shift of the brain into the prothorax, absent heart, absence of midgut musculature, etc.), but also has revealed unique features (brain with two apertures and three pairs of muscles going through them), which have not been described before.