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.

Regio- and stereoselective (3 + 2)-cycloaddition reactions of nitrones with cyclic allenes.

An effective approach to access functionalized 2H-cyclonona(deca)[d]isoxazoles and 15-oxo-3,10-methanobenzo[b][1]azacyclododecines has been developed by the reaction of N-aryl-C,C-bis(methoxycarbonyl)nitrones with cyclonona(deca)-1,2-dienes in a one-pot fashion. The reaction of N-aryl-C-(phenylcarbamoyl)nitrones with these allenes proceeds strictly regioselectively giving the mixtures of diastereomeric isoxazolidines containing a double bond at the C4-position of the isoxazolidine ring. The quantum chemical calculations show that the regioselectivity of these reactions is in good agreement with the reactivity indices of the considered compounds.

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.

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.

Morphology and scaling of compound eyes in the smallest beetles (Coleoptera: Ptiliidae).

The coleopteran family Ptiliidae (featherwing beetles) includes some of the smallest insects known with most of the representatives of this family measuring less than 1 mm in body length. A small body size largely determines the morphology, physiology, and biology of an organism and affects the organization of complex sense organs. Information on the organization of the compound eyes of Ptiliidae is scarce. Using scanning electron microscopy we analyzed the eyes of representatives of all subfamilies and tribes and provide a detailed description of the eye ultrastructure of four species (Nephanes titan, Porophila mystacea, Nanosella sp. and Acrotrichis grandicollis) using transmission electron microscopy. The results are compared with available data on larger species of related groups of Staphyliniformia and scale quantitative analyses are performed. The eyes of Ptiliidae consist of 15-50 ommatidia 6-13 µm in diameter and all conform to the apposition acone type of eye with fused rhabdoms of banded organization. Each ommatidium has the typical cellular arrangement present also in the eyes of larger staphyliniform beetles, but strongly curved lenses, short cones, reduced pigment cells, a high density of pigment granules and certain modifications of the rhabdom seem typical of ptiliid eyes. Allometric analyses show that as body size decreases, the number of facets drops more steeply than their average size does.

Cognitive abilities with a tiny brain: Neuronal structures and associative learning in the minute Nephanes titan (Coleoptera: Ptiliidae).

Revealing the effect of brain size on the cognitive abilities of animals is a major challenge in the study of brain evolution. Analysis of the effects of miniaturization on brain function in the smallest insects is especially important, as they are comparable in body size to some unicellular organisms and next to nothing is known about their cognitive abilities. We analyse for the first time the structure of the brain of the adult featherwing beetle Nephanes titan, one of the smallest insects, and results of the first ethological experiments on the capacity of learning in this species. N. titan is capable of associative learning, in spite of the structural modification in its nervous system and the greatly reduced number of neurons compared to the nervous systems of larger insects. Microinsects can become useful model organisms for neurobiology. On the one hand, the structural simplicity and extremely small size of their central nervous system make it possible to study it very efficiently. On the other hand, their learning capacity and retained principal cognitive abilities make them suitable objects for behavioural experiments.

Between extreme simplification and ideal optimization: antennal sensilla morphology of miniaturized Megaphragma wasps (Hymenoptera: Trichogrammatidae).

One of the major trends in the evolution of parasitoid wasps is miniaturization, which has produced the smallest known insects. Megaphragma spp. (Hymenoptera: Trichogrammatidae) are smaller than some unicellular organisms, with an adult body length of the smallest only 170 µm. Their parasitoid lifestyle depends on retention of a high level of sensory reception comparable to that in parasitoid wasps that may have antennae hundreds of times larger. Antennal sensilla of males and females of Megaphragma amalphitanum and M. caribea and females of the parthenogenetic M. mymaripenne are described, including sensillum size, external morphology, and distribution. Eight different morphological types of sensilla were discovered, two of them appearing exclusively on female antennae. Two of the types, sensilla styloconica and aporous placoid sensilla, have not been described previously. Regression analyses were performed to detect and evaluate possible miniaturization trends by comparing available data for species of larger parasitoid wasps. The number of antennal sensilla was found to decrease with the body size; M. amalphitanum males have only 39 sensilla per antenna. The number of antennal sensilla types and sizes of the sensilla, however, show little to no correlation with the body size. Our findings on the effects of miniaturization on the antennal sensilla of Megaphragma provide material for discussion on the limits to the reduction of insect antenna.

The scaling and allometry of organ size associated with miniaturization in insects: A case study for Coleoptera and Hymenoptera.

The study of the influence of body size on structure in animals, as well as scaling of organs, is one of the key areas of functional and evolutionary morphology of organisms. Most studies in this area treated mammals or birds; comparatively few studies are available on other groups of animals. Insects, because of the huge range of their body sizes and because of their colossal diversity, should be included in the discussion of the problem of scaling and allometry in animals, but to date they remain insufficiently studied. In this study, а total of 28 complete (for all organs) and 24 partial 3D computer reconstructions of body and organs have been made for 23 insect species of 11 families and five orders. The relative volume of organs was analyzed based on these models. Most insect organs display a huge potential for scaling and for retaining their organization and constant relative volume. By contrast, the relative volume of the reproductive and nervous systems increases by a considerable factor as body size decreases. These systems can geometrically restrain miniaturization in insects and determine the limits to the smallest possible body size.