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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.

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 is a widespread trend in animal evolution. In insects, the effects of miniaturization have been described at all the levels of body organization, from major rearrangements of organ systems to fundamental changes at the cellular level. The results of studies on the anatomy of tiny beetles, wasps, booklice, thrips, and strepsipterans have contributed significantly to understanding the process of miniaturization in animals. However, not all the orders of microinsects have been sufficiently well investigated so far. In this paper, the internal structure of the adult male of one of the smallest nematocerans, the midge Corynoneura scutellata Winnertz, 1846 (Diptera: Chironomidae), is described in detail for the first time using 3D computer reconstructions based on serial histological sections. Despite the tiny body size, most organ systems retain the ground plan typical of the larger representatives of related groups. The tracheal system of C. scutellata has not lost its high level of organization, being represented by large tracheal trunks and a branching system of tracheae. The ganglia of the central nervous system are concentrated, while the head muscles and those associated with the intestine are reduced. The changes in the relative volumes of the organ systems in tiny dipterans are analyzed in comparison with microinsects from other orders. The previously revealed general patterns of organ allometry are valid for tiny dipterans as well, but the reproductive system of C. scutellata has a considerably smaller relative volume, as compared with that of microinsects from other orders.

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.

Extreme miniaturization implies a high degree of optimization, rendering the retention of non-functional organs almost impossible. Two unique non-porous placoid sensilla on the antennae of females of Megaphragma were described in the literature. Placoid sensilla in Hymenoptera have an olfactory function and always bear pores; the apparent absence of pores therefore raises the questions whether such sensilla are functional in Megaphragma and whether their surface sculpture had been sufficiently well examined. We examined in detail the external microsculpture and internal ultrastructure of the placoid sensilla using Focused Ion Beam Scanning Electron Microscopy and Scanning Electron Microscopy with various types of sputtering and show that these sensilla actually have a porous cuticle and are innervated by 11 or 12 neurons with branched cilia, which is typical of olfactory sensilla. Comparison of various methods of electron microscopy allows us to conclude that for an accurate determination of the morphofunctional types of sensilla, especially in miniature insects, it is necessary to study both the internal ultrastructure of the sensilla and their external morphology using carefully selected scanning electron microscopy methods.

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.

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.

Flight speed generally correlates positively with animal body size1. Surprisingly, miniature featherwing beetles can fly at speeds and accelerations of insects three times as large2. We show here that this performance results from a previously unknown type of wing motion. Our experiment combines three-dimensional reconstructions of morphology and kinematics in one of the smallest insects, Paratuposa placentis (body length 395 μm). The flapping bristled wing follows a pronounced figure-eight loop that consists of subperpendicular up and down strokes followed by claps at stroke reversals, above and below the body. Computational analyses suggest a functional decomposition of the flapping cycle in two power half strokes producing 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 wing beat cycle, making elastic energy storage obsolete. This novel flight style evolved during miniaturization may compensate for costs associated with air viscosity and helps explain how extremely small insects preserved superb aerial performance during miniaturization. Incorporating this flight style in artificial flappers is a challenge for designers of micro aerial vehicles.

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.

Flight speed is positively correlated with body size in animals

The wings of Ptiliidae, the coleopteran family containing the smallest free-living insects, are analyzed in detail for the first time. A reconstruction of the evolutionary sequence of changes associated with miniaturization is proposed. The wings of several species are described using light microscopy and scanning electron microscopy. The morphology and scaling are analyzed in comparison with larger representatives of related groups. The wings of all studied ptiliids show some degree of ptiloptery (feather-like shape, typical for extremely small insects). In larger ptiliids the wing contains at least five veins, has a wide blade, and bears a marginal fringe of 200-300 setae; in the smallest species it has three veins or fewer, a narrow blade, and about 40 setae along the margin. The setae are brush-like; peculiar outgrowths, denser towards the apex, increase the effective diameter of the setae. Morphometric analysis shows that the geometry of the wings and their elements strongly differs from those of other staphyliniform beetles, suggesting that the aerodynamics of the feather-like wings may also differ distinctly from the usual pattern.

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.

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.

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.

This article describes the morphology and ultrastructure of the brain of the greenhouse thrips, Heliothrips haemorrhoidalis (Thripidae), studied in a series of histological sections with the use of 3D reconstructions and TEM. The structural peculiarities of the brain organization and its features associated with body miniaturization as well as its quantitative characteristics (the number of cells and the volume of individual centers) are described for the first time in both the adult and the first-instar nymph of H. haemorrhoidalis. It is suggested that the limits of the nervous system miniaturization may be different in holo- and hemimetabolous insects.

The third communication in this series describes the structure of the brain in one of the smallest psocopterans, Liposcelis bostrychophila (Liposcelididae), studied in a series of histological sections with the use of 3D reconstructions and TEM. Like all microinsects, L. bostrychophila shows both oligomerization and concentration of ganglia, an increased relative volume of the brain, and a reduced number and size of neurons. In addition, reduction of one of the optic lobes is noted. The structural features of the neuropil brain centers in the early and late first-instar nymphs are described.

Aerodynamic force generation capacity of the wing of a miniature beetleParatuposa placentisis evaluated using a combined experimental and numerical approach. The wing has a peculiar shape reminiscent of a bird feather, often found in the smallest insects. Aerodynamic force coefficients are determined from a dynamically scaled force measurement experiment with rotating bristled and membrane wing models in a glycerin tank. Subsequently, they are used as numerical validation data for computational fluid dynamics simulations using an adaptive Navier–Stokes solver. The latter provides access to important flow properties such as leakiness and permeability. It is found that, in the considered biologically relevant regimes, the bristled wing functions as a less than$$50\%$$50%leaky paddle, and it produces between 66 and$$96\%$$96%of the aerodynamic drag force of an equivalent membrane wing. The discrepancy increases with increasing Reynolds number. It is shown that about half of the aerodynamic normal force exerted on a bristled wing is due to viscous shear stress. The paddling effectiveness factor is proposed as a measure of aerodynamic efficiency.Graphic abstract

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 springtailMesaphorura 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 ofM.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 ofM. sylvaticahave 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.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals to 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, the gene content of M. amalphitanum compared to other chalcid wasps is remarkably conserved. Among the very rare cases of apparent gene loss is centrosomin, which encodes an important centrosome component; the absence of this protein might be related to the large number of anucleate neurons in M. amalphitanum. Intriguingly, we also observed significant changes in M. amalphitanum transposable element dynamics over time, whereby an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Thus, 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.

Miniaturization is an evolutionary process that is widely represented in both invertebrates and vertebrates. Miniaturization frequently affects not only the size of the organism and its constituent cells, but also changes the genome structure and functioning. The structure of the main heat shock genes (hsp70 and hsp83) was studied in one of the smallest insects, the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) parasitic wasp, which is comparable in size with unicellular organisms. An analysis of the sequenced genome has detected six genes that relate to the hsp70 family, some of which are apparently induced upon heat shock. Both induced and constitutively expressed hsp70 genes contain a large number of introns, which is not typical for the genes of this family. Moreover, none of the found genes form clusters, and they are all very heterogeneous (individual copies are only 75-85% identical), which indicates the absence of gene conversion, which provides the identity of genes of this family in Drosophila and other organisms. Two hsp83 genes, one of which contains an intron, have also been found in the M. amalphitanum genome.

Anatomy of the adult and the first-instar nymph of Liposcelis bostrychophila (Psocoptera, Liposcelididae) is described in detail for the first time based on series of sections and 3D computer reconstruction. Despite its tiny size, no strong structural simplification is observed in Liposcelis. The main features of the anatomy of this species associated with miniaturization are the merging of individual skeletal elements and simplification of the endoskeleton in the nymph, lack of the midgut muscles, simplification of the tracheal and circulatory systems, oligomerization and concentration of ganglia, asymmetry of the central nervous system in the nymph, and the absence of several muscles. Changes in the relative volumes of organs during the postembryonic development of Liposcelis are analyzed and compared with other minute insects.

Anatomy of adults of Trichogramma evanescens (Hymenoptera: Trichogrammatidae) is described in detail for the first time based on series of sections and 3D computer reconstruction. The complex structure of the exoskeleton and musculature (except for reductions of individual muscles) is preserved in T. evanescens despite its tiny size. Considerable simplification is observed in the structure of the intestine, tracheal and circulatory systems, and in the reduced number of Malpighian tubules. Thus, the smallest Hymenoptera (Mymaridae and Trichogrammatidae) demonstrate both the effects of miniaturization universal for insects (reduction of the head endoskeleton, circulatory and respiratory systems) and specific ones.

The mouthparts and musculature of the head of Sericoderus lateralis are described. It is shown that the mouthparts of minute beetles preserve their complex structure and are not significantly affected by miniaturization. Such stability allows these features to be used in macrosystematics of groups with variable body size.

Anatomy of the adult fairyfly Anaphes flavipes (Hymenoptera, Mymaridae) is described in detail for the first time, based on series of sections and 3D computer reconstruction. Despite its tiny size, A. flavipes does not demonstrate strong morphological simplification, except for the merging of certain skeletal elements, a slight simplification of endoskeleton, lack of midgut muscles, a reduced number of Malpighian tubules, simplification of the tracheal and circulatory systems, asymmetry of the central nervous system, and the absence of several muscles.

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.

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 ofScydosella musawasensisHall, 1999 (Coleoptera: Ptiliidae), the smallest free-living insect, using scanning electron microscopy. The antenna ofS. musawasensisbears 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 ofS. 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 ofS. musawasensisand 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.

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.

One of the major trends in the evolution of parasitoid wasps is miniaturization, which has produced the smallest known insects.Megaphragmaspp. (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 ofMegaphragma amalphitanumandM. caribeaand females of the parthenogeneticM. mymaripenneare 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. amalphitanummales 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 ofMegaphragmaprovide material for discussion on the limits to the reduction of insect antenna.

The smallest known beetle Scydosella musawasensis Hall is recorded for the second time. Precise measurements of its body size are given, and it is shown that the smallest examined representative of this species has a length of 325 µm.

Miniaturization, or decrease in body size, is a widespread trend in animal evolution and one of the principal directions of evolution in insects. It is known that body size, especially extremely small, is a key characteristic that largely determines the morphology, physiology, and biology of animals. Miniaturization leads to considerable reorganizations of structure in insects, affecting almost all organs and tissues. In the smallest insects, comparable in size to unicellular organisms, modifications arise not only at the level of organs but also at the cellular level. Miniaturization is accompanied by allometric changes in many organ systems. The consequences of miniaturization displayed by different insect taxa include both common and unique changes.

Miniaturization of organisms is one of the most interesting evolutionary phenomena. This is a very common process and is widely represented in multicellular organisms: invertebrates (nemertean, nematodes, brachiopods, mollusks, arachnids, and insects), and vertebrates (fishes, amphibians, birds, and even mammals). Miniaturization leads to a significant reduction in the body size and simplification of some systems and organs without loss of species viability. Typically, this complex evolutionary modification is associated with occupation of the new ecological niches by the species. Reduction can affect not only the overall size of the organism but also the size of its cells as well as promote changes in the genome and the structure of specific genes. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) is considered one of the smallest insects described to date. Therefore, it represents an attractive object for the studies of genetic aspects of body size reduction during evolution. Whole genome sequencing of M. amalphitanum and subsequent genome annotation and transcriptome assembly allow us to define the changes in the genetic material during organism miniaturization in relation to the larger Hymenoptera species. Our preliminary analysis revealed that genome size of M. amalphitanum is 346 Mb and transcriptome size is 27.5 Mb.

Here is the first description of the visual organ of Acrotrichis grandicollis, a member of Ptiliidae family that includes the smallest free-living insects. The apposition eyes of A. grandicollis have the acone-type crystalline cone; a fused rhabdom is formed by eight retinula cells and surrounded by primary pigment cells. Secondary pigment cells are located only in the distal part of the ommatidium under the lens. The eye consists of about 50 facets about 12 μm in diameter. The A. grandicollis ommatidium ultrastructure has been compared with that of large Coleoptera. The results obtained enabled us to emphasize the compound eye specific features associated with the small body size.

Miniaturization leads to considerable reorganization of structures in insects, affecting almost all organs and tissues. In the smallest insects, comparable in size to unicellular organisms, modifications arise not only at the level of organs, but also at the cellular level. Miniaturization is accompanied by allometric changes in many organ systems. The consequences of miniaturization displayed by different insect taxa include both common and unique changes. Because the smallest insects are among the smallest metazoans and have the most complex organization among organisms of the same size, their peculiar structural features and the factors that limit their miniaturization are of considerable theoretical interest to general biology.

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.

In this first of three articles we show the construction of the articular part of the elytron, the root. The root bears a conspicuous field of campaniform sensilla. This field was studied using light and scanning electron microscopes. The diversity of shape of the field among beetles, types of orientation of elongated sensilla within the field, individual variability of their number among conspecifics are demonstrated. Elongated sensilla point to the junction of the elytron with the second axillary plate. Presumably, they monitor twist movement in this junction, which is possible if the elytron is open. The goal of the whole project is to reveal the effect of both structure and function of the hind wings and elytra on the morphology of this mechanosensory field. Our data on allometric relationships between the animal size and quantitative characteristics of the field in normally flying beetles provide an important background for further functional analysis of this sensory organ. We selected 14 series of several species belonging to the same taxon but differing in size from big to small. It is revealed that the area of the sensory field is directly proportional to the elytral area, whereas the number of sensilla is proportional to the square root of the elytral area. Despite the great range in the elytral area (1500 times) in series of selected species the area of an external pit or cap of a single sensillum varies only 25-fold. The density of sensilla per unit area of the sensory field increases with decrease of the elytral area. (C) 2014 Elsevier Ltd. All rights reserved.

The head morphology of larvae of two undescribed species of the corylophid genus Holopsis were examined. Both are associated with the same basidiomycete host Ganoderma cf applanatum. Whereas the round and convex adults are very similar, one of the disc-shaped larvae is characterized by an elongate weevil-like snout, which is a unique feature in larval beetles. The posterior head region, the mouthparts and the general configuration of the musculature are similar in the larvae of both species. However, in the rostrate Holopsis sp. 1 most muscles are either widened along the longitudinal axis or elongated. Moreover, an additional bundle of M. frontobuccalis posterior is present, which strengthens the pharyngeal pumping apparatus. Both species share an unusual connection between the prepharynx and pharynx. This is a potential autapomorphy of the genus. The larval cephalic morphology of Holopsis sp. 2 and the corylophine genus Sericoderus is quite similar. However, they differ in some muscular features and in the configuration of the foregut. Holopsis species are associated with Basidiomycetes. Whether this is an ancestral condition in Corylophidae remains ambiguous due to conflicting phylogenetic hypotheses and the largely unknown biology of the Australian subfamily Periptyctinae. Several features of Holopsis are likely plesiomorphic and possibly related with the association with basidiomycetes. However, the larval rostrum of sp. 1 is doubtlessly derived, and could have a performance advantage over other species feeding on the spores of Ganoderma cf applanatum including Holopsis sp. 2.