Morphology of the pterothoracic musculature in Paraneoptera and its phylogenetic implication (Insecta: Neoptera).

Although the monophyly of Paraneoptera (=hemipteroid orders or Acercaria, composed of Psocodea, Thysanoptera and Hemiptera) has been widely accepted morphologically, the results from molecular phylogenetic and phylogenomic analyses contradict this hypothesis. In particular, phylogenomic analyses provide strong bootstrap support for the sister group relationship between Psocodea and Holometabola, that is, paraphyly of Paraneoptera. Here, we examined the pterothoracic musculature of Paraneoptera, as well as a wide range of other neopterous insect orders, and analysed its phylogenetic implication. By using the synchrotron microcomputed tomography (µCT) and parsimony-based ancestral state reconstruction, several apomorphic conditions suggesting the monophyly of Paraneoptera, such as the absence of the II/IIItpm7, IIscm3, IIIspm2 and IIIscm3 muscles, were identified. In contrast, no characters supporting Psocodea + Holometabola were recovered from the thoracic muscles. These results provide additional support for the monophyly of Paraneoptera, together with the previously detected morphological apomorphies of the head, wing base, and abdomen.

Comparative Morphology of the Wing Base Structure Illuminates Higher-Level Phylogeny of Holometabola.

Phylogenetic relationships among Holometabola have been the subject of controversy. The value of the wing base structure in phylogenetic analysis has been demonstrated but remains largely underexplored and scarce in studies of Holometabola. We studied the phylogenetic relationships among Holometabola (excluding Siphonaptera), focusing exclusively on wing base structure. Cladistic assessments were conducted using 53 morphological data points derived from the bases of both the forewing and hindwing. The results of wing base data revealed a sister relationship between Hymenoptera and remaining orders. The sister-group relationships between Strepsiptera and Coleoptera, Mecoptera and Diptera, Trichoptera and Lepidoptera, and Neuropterida and Coleopterida were corroborated. In Neuropterida, our results recovered the sister relationship between Megaloptera and Neuroptera, as well as the monophyly of Megaloptera.

The morphology of the free-living females of Strepsiptera (Insecta).

The morphology of the adult free-living females of Mengenilla moldrzyki and Eoxenos laboulbenei (Strepsiptera, Mengenillidae) was documented with µCT-based 3D reconstructions and histological serial sections. External and internal features of both species are characterized by far-reaching specialization and structural simplification. The well-developed mandibles are moved by large muscles. Other mouthparts and their corresponding musculature are simplified or absent. The brain is partly shifted into the prothorax. It is followed by a single postcerebral ganglionic complex also containing the subesophageal ganglion and an unpaired abdominal nerve. Postcephalic sclerites are absent, except for the plate-like pronotum and small pleural sclerites. Wings and associated muscles are missing. The lumina of the large midgut and the anterior hindgut are disconnected. Seven bulb-shaped Malpighian tubules in M. moldrzyki is the highest number yet described for Strepsiptera. The 10-segmented abdomen lacks appendages. An unpaired birth organ opens ventrally on abdominal segment VII. The entire body cavity is filled with numerous freely floating eggs, 1386 in the specimen of M. moldrzyki and 721 in E. laboulbenei. Genital ducts, defined gonads, and genital glands are missing. The morphology of female Mengenillidae is discussed with respect to sexual dimorphism and structural features of the postembryonic stages. Phylogenetic implications are outlined.

Ultrastructure of the dorsal ocellus of Bittacus planus larvae (Mecoptera: Bittacidae) with evolutionary significance.

The Bittacidae are unique in holometabolous insects in that their larvae bear a dorsal ocellus on the frons. The fine structure of the dorsal ocellus, however, has not been investigated to date. Here, the ultrastructure of the larval dorsal ocellus was studied in the hangingfly Bittacus planus Cheng, 1949 using light, scanning, and transmission electron microscopy. The dorsal ocellus of the larvae comprises a cornea, corneagenous cells, and retinula cells. The cornea is a laminated structure. A layer of corneagenous cells is located below the cornea. Numerous retinula cells are arranged tightly beneath the corneagenous cells. The retinula cells modify their adjacent membranes into numerous linear microvilli, which form an analogue of the rhabdom among adjacent retinula cells. The results show that the dorsal ocellus of larval Bittacidae is a highly vestigial organ and appears to be degenerating during the postembryonic development. The presence of the vestigial dorsal ocellus is likely to represent an ancestral plesiomorphy of holometabolous insects, providing new evidence for exploring the evolutionary origin of holometabolous larvae.

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.

Ultrastructural 3D reconstruction of the smallest known insect photoreceptors: The stemmata of a first instar larva of Strepsiptera (Hexapoda).

Stemmata of strepsipteran insects represent the smallest arthropod eyes known, having photoreceptors which form fused rhabdoms measuring an average size of 1.69 × 1.21 × 1.04 µm and each occupying a volume of only 0.97-1.16 µm3. The morphology of the stemmata of the extremely miniaturized first instar larva of Stylops ovinae (Strepsiptera, Stylopidae) was investigated using serial-sectioning transmission electron microscopy (ssTEM). Our 3D reconstruction revealed that, despite different proportions, all three stemmata maintain the same organization: a biconvex corneal lens, four corneagenous cells and five photoreceptor (retinula) cells. No pigment-containing cell-types were found to adjoin the corneagenous cells. Whereas the retinula cells are adapted to the limited space by having laterally bulged median regions, containing mitochondria and the smallest nuclei yet reported for arthropods (1.37 µm3), special adaptations are found in the corneagenous cells which have cell volumes down to 1 µm3. The corneagenous cells lack nuclei and pigment granules and bear only a few mitochondria (up to three) or none at all. Morphological adaptations due to miniaturization are discussed in the context of photoreceptor function and the visual needs of the larva.

The lobula plate is exclusive to insects.

Just one superorder of insects is known to possess a neuronal network that mediates extremely rapid reactions in flight in response to changes in optic flow. Research on the identity and functional organization of this network has over the course of almost half a century focused exclusively on the order Diptera, a member of the approximately 300-million-year-old clade Holometabola defined by its mode of development. However, it has been broadly claimed that the pivotal neuropil containing the network, the lobula plate, originated in the Cambrian before the divergence of Hexapoda and Crustacea from a mandibulate ancestor. This essay defines the traits that designate the lobula plate and argues against a homologue in Crustacea. It proposes that the origin of the lobula plate is relatively recent and may relate to the origin of flight.

Discovery of wing imaginal discs in the penultimate instar of the lacewing Mallada desjardinsi (Insecta: Neuroptera: Chrysopidae) with histological notes on postembryonic imaginal disc development.

Holometabolous insects are alternatively named "Endopterygota" because, in the larvae of many taxa, the wing primordia in the lateral regions of the meso- and metathoracic segments form more or less invaginated structures called wing imaginal discs. Holometabolous insects exhibit differential developmental timing of the wing during ontogeny. The condition in which wing growth is deferred until the end of larval life has been considered ancestral, whereas early disc formation has been recognized as the derived condition. Even though wing disc development in holometabolous insects has been studied extensively in select groups, many questions remain about the development of the wing imaginal disc in the orders Raphidioptera, Megaloptera, Neuroptera, and Mecoptera. To clarify whether the wing imaginal disc of Neuroptera is typical of the derived condition, we examined the ontogeny of the wing imaginal discs in the lacewing Mallada desjardinsi histologically. Using both light microscopy and transmission electron microscopy, we were able to recognize wing imaginal discs in the penultimate larval instar (prefinal larval instar) of this species. To date, neuropteran insects have been characterized as having late-forming wing imaginal discs. However, our findings show that the developmental pattern of the wing imaginal discs within the Neuroptera represents a more derived pattern of development in the Holometabola than was assumed previously.

The highly rearranged karyotype of the hangingfly Bittacus sinicus (Mecoptera, Bittacidae): the lowest chromosome number in the order.

Cytogenetic features of the hangingfly Bittacus sinicus Issiki, 1931 were investigated for the first time using C-banding and DAPI (4',6-diamidino-2-phenylindole) staining. The karyotype analyses show that the male B. sinicus possesses the lowest chromosome number (2n = 15) ever observed in Mecoptera, and an almost symmetric karyotype with MCA (Mean Centromeric Asymmetry) of 12.55 and CVCL (Coefficient of Variation of Chromosome Length) of 19.78. The chromosomes are either metacentric or submetacentric with their sizes decreasing gradually. Both the C-banding and DAPI+ patterns detect intermediate heterochromatin on the pachytene bivalents of B. sinicus, definitely different from the heterochromatic segment at one bivalent terminal of other bittacids studied previously. The male meiosis of B. sinicus is chiasmate with two chiasmata in metacentric bivalents and one in the submetacentric bivalent. The sex determination mechanism is X0(♂), which is likely plesiomorphic in Bittacidae. Two alternative scenarios of karyotype origin and evolution in Bittacus Latreille, 1805 are discussed.

The endoparasitic larval stages of Eoxenos laboulbenei: An atypical holometabolan development (Strepsiptera, Mengenillidae).

Endoparasitic larval stages of Eoxenos laboulbenei were documented with different techniques, with a main focus on the male tertiary larva. Two discrete endoparasitic stages occur, the secondary and the tertiary larva. The presence of large compound eyes and externally visible wing buds in the tertiary larva is a unique feature within Holometabola. The brain with large optic lobes is followed by a single postcephalic ganglionic complex. The cephalic musculature is greatly reduced but pharyngeal dilators and muscles associated with the mouth field are present. Postcephalic sclerites are absent except for the pronotum. The segmented legs bear filiform pretarsal claws. The indirect flight muscles fill up a large part of the metathorax. The 10-segmented abdomen lacks appendages. Pleural folds are present on the thorax and abdomen. The digestive tract is characterized by a very short oesophagus. The large midgut and the narrow hindgut are disconnected. Six short Malpighian tubules are present. Large testes fill out almost the entire abdomen. In contrast to the tertiary larva, the muscles of the secondary larva are not fully differentiated. Cephalic appendages are present as bud-shaped anlagen. The legs lack a pretarsal claw. The developmental transformations are outlined and discussed, also with respect to phylogenetic implications.

An unusual 100-million-year old holometabolan larva with a piercing mouth cone.

Holometabola is a hyperdiverse group characterised by a strong morphological differentiation between early post-embryonic stages (= larvae) and adults. Adult forms of Holometabola, such as wasps, bees, beetles, butterflies, mosquitoes or flies, are strongly differentiated concerning their mouth parts. The larvae most often seem to retain rather plesiomorphic-appearing cutting-grinding mouth parts. Here we report a new unusual larva preserved in Burmese amber. Its mouth parts appear beak-like, forming a distinct piercing mouth cone. Such a morphology is extremely rare among larval forms, restricted to those of some beetles and lacewings. The mouth parts of the new fossil are forward oriented (prognathous). Additionally, the larva has distinct subdivisions of tergites and sternites into several sclerites. Also, the abdomen segments bear prominent protrusions. We discuss this unusual combination of characters in comparison to the many different types of holometabolan larvae. The here reported larva is a new addition to the 'unusual zoo' of the Cretaceous fauna including numerous, very unusual appearing forms that have gone extinct at the Cretaceous-Palaeogene boundary.

A general theory of genital homologies for the Hexapoda (Pancrustacea) derived from skeletomuscular correspondences, with emphasis on the Endopterygota.

No consensus exists for the homology and terminology of the male genitalia of the Hexapoda despite over a century of debate. Based on dissections and the literature, genital skeletomusculature was compared across the Hexapoda and contrasted with the Remipedia, the closest pancrustacean outgroup. The pattern of origin and insertion for extrinsic and intrinsic genitalic musculature was found to be consistent among the Ectognatha, Protura, and the Remipedia, allowing for the inference of homologies given recent phylogenomic studies. The penis of the Hexapoda is inferred to be derived from medially-fused primary gonopods (gonopore-bearing limbs), while the genitalia of the Ectognatha are inferred to include both the tenth-segmental penis and the ninth-segmental secondary gonopods, similar to the genitalia of female insects which comprise gonopods of the eighth and ninth segments. A new nomenclatural system for hexapodan genitalic musculature is presented and applied, and a general list of anatomical concepts is provided. Novel and refined homologies are proposed for all hexapodan orders, and a series of groundplans are postulated. Emphasis is placed on the Endopterygota, for which fine-grained transition series are hypothesized given observed skeletomuscular correspondences.

The tracheal system in post-embryonic development of holometabolous insects: a case study using the mealworm beetle.

The tracheal (respiratory) system is regarded as one of the key elements which enabled insects to conquer terrestrial habitats and, as a result, achieve extreme species diversity. Despite this fact, anatomical data concerning this biological system is relatively scarce, especially in an ontogenetic context. The purpose of this study is to provide novel and reliable information on the post-embryonic development of the tracheal system of holometabolous insects using micro-computed tomography methods. Data concerning the structure of the respiratory system acquired from different developmental stages (larvae, pupae and adults) of a single insect species (Tenebrio molitor) are co-analysed in detail. Anatomy of the tracheal system is presented. Sample sizes used (29 individuals) enabled statistical analysis of the results obtained. The following aspects have been investigated (among others): the spiracle arrangement, the number of tracheal ramifications originating from particular spiracles, the diameter of longitudinal trunks, tracheal system volumes, tracheae diameter distribution and fractal dimension analysis. Based on the data acquired, the modularity of the tracheal system is postulated. Using anatomical and functional factors, the following respiratory module types have been distinguished: cephalo-prothoracic, metathoracic and abdominal. These modules can be unambiguously identified in all of the studied developmental stages. A cephalo-prothoracic module aerates organs located in the head capsule, prothorax and additionally prolegs. It is characterised by relatively thick longitudinal trunks and originates in the first thoracic spiracle pair. Thoracic modules support the flight muscles, wings, elytra, meso- and metalegs. The unique feature of this module is the presence of additional longitudinal connections between the neighbouring spiracles. These modules are concentrated around the second prothoracic and the first abdominal spiracle pairs. An abdominal module is characterised by relatively thin ventral longitudinal trunks. Its main role is to support systems located in the abdomen; however, its long visceral tracheae aerate organs situated medially from the flight muscles. Analysis of changes of the tracheal system volume enabled the calculation of growth scaling among body tissues and the volume of the tracheal system. The data presented show that the development of the body volume and tracheal system is not linear in holometabola due to the occurrence of the pupal stage causing a decrease in body volume in the imago and at the same time influencing high growth rates of the tracheal system during metamorphosis, exceeding that ones observed for hemimetabola.

Regulatory Pathways Controlling Female Insect Reproduction.

The synthesis of vitellogenin and its uptake by maturing oocytes during egg maturation are essential for successful female reproduction. These events are regulated by the juvenile hormones and ecdysteroids and by the nutritional signaling pathway regulated by neuropeptides. Juvenile hormones act as gonadotropins, regulating vitellogenesis in most insects, but ecdysteroids control this process in Diptera and some Hymenoptera and Lepidoptera. The complex crosstalk between the juvenile hormones, ecdysteroids, and nutritional signaling pathways differs distinctly depending on the reproductive strategies adopted by various insects. Molecular studies within the past decade have revealed much about the relationships among, and the role of, these pathways with respect to regulation of insect reproduction. Here, we review the role of juvenile hormones, ecdysteroids, and nutritional signaling, along with that of microRNAs, in regulating female insect reproduction at the molecular level.

Capturing the Phylogeny of Holometabola with Mitochondrial Genome Data and Bayesian Site-Heterogeneous Mixture Models.

After decades of debate, a mostly satisfactory resolution of relationships among the 11 recognized holometabolan orders of insects has been reached based on nuclear genes, resolving one of the most substantial branches of the tree-of-life, but the relationships are still not well established with mitochondrial genome data. The main reasons have been the absence of sufficient data in several orders and lack of appropriate phylogenetic methods that avoid the systematic errors from compositional and mutational biases in insect mitochondrial genomes. In this study, we assembled the richest taxon sampling of Holometabola to date (199 species in 11 orders), and analyzed both nucleotide and amino acid data sets using several methods. We find the standard Bayesian inference and maximum-likelihood analyses were strongly affected by systematic biases, but the site-heterogeneous mixture model implemented in PhyloBayes avoided the false grouping of unrelated taxa exhibiting similar base composition and accelerated evolutionary rate. The inclusion of rRNA genes and removal of fast-evolving sites with the observed variability sorting method for identifying sites deviating from the mean rates improved the phylogenetic inferences under a site-heterogeneous model, correctly recovering most deep branches of the Holometabola phylogeny. We suggest that the use of mitochondrial genome data for resolving deep phylogenetic relationships requires an assessment of the potential impact of substitutional saturation and compositional biases through data deletion strategies and by using site-heterogeneous mixture models. Our study suggests a practical approach for how to use densely sampled mitochondrial genome data in phylogenetic analyses.

Body-enlarging effect of royal jelly in a non-holometabolous insect species, Gryllus bimaculatus.

Honeybee royal jelly is reported to have body-enlarging effects in holometabolous insects such as the honeybee, fly and silkmoth, but its effect in non-holometabolous insect species has not yet been examined. The present study confirmed the body-enlarging effect in silkmoths fed an artificial diet instead of mulberry leaves used in the previous literature. Administration of honeybee royal jelly to silkmoth from early larval stage increased the size of female pupae and adult moths, but not larvae (at the late larval stage) or male pupae. We further examined the body-enlarging effect of royal jelly in a non-holometabolous species, the two-spotted cricket Gryllus bimaculatus, which belongs to the evolutionarily primitive group Polyneoptera. Administration of royal jelly to G. bimaculatus from its early nymph stage enlarged both males and females at the mid-nymph and adult stages. In the cricket, the body parts were uniformly enlarged in both males and females; whereas the enlarged female silkmoths had swollen abdomens. Administration of royal jelly increased the number, but not the size, of eggs loaded in the abdomen of silkmoth females. In addition, fat body cells were enlarged by royal jelly in the silkmoth, but not in the cricket. These findings suggest that the body-enlarging effect of royal jelly is common in non-holometabolous species, G. bimaculatus, but it acts in a different manner than in holometabolous species.

Cloning, phylogeny, and expression analysis of the Broad-Complex gene in the longicorn beetle Psacothea hilaris.

Seven isoforms of Broad-Complex (PhBR-C), in which the sequence of the zinc finger domain differed (referred to as Z1, Z2, Z3, Z2/Z3, Z4, Z5/Z6, and Z6, respectively), were cloned from the yellow-spotted longicorn beetle Psacothea hilaris. The Z1-Z4 sequences were highly conserved among insect species. The Z5/Z6 isoform was aberrant in that it contained a premature stop codon. Z6 had previously only been detected in a hemimetabola, the German cockroach Blattella germanica. The presence of Z6 in P. hilaris, and not in other holometabolous model insects such as Drosophila melanogaster or Tribolium castaneum, suggests that Z6 was lost multiple times in holometabolous insects during the course of evolution. PhBR-C expression levels in the brain, salivary gland, and epidermis of larvae grown under different feeding regimens were subsequently investigated. PhBR-C expression levels increased in every tissue examined after the gut purge, and high expression levels were observed in prepupae. A low level of PhBR-C expression was continuously observed in the brain. An increase was noted in PhBR-C expression levels in the epidermis when 4th instar larvae were starved after 4 days of feeding, which induced precocious pupation. No significant changes were observed in expression levels in any tissues of larvae starved immediately after ecdysis into 4th instar, which did not grow and eventually died.

Permian ancestors of Hymenoptera and Raphidioptera.

The origin of Hymenoptera remains controversial. Currently accepted hypotheses consider Hymenoptera as the first side branch of Holometabola or sister-group to Mecopteroidea. In contrast, fossils confirm the idea of Martynov that Hymenoptera are related to Megaloptera and Raphidioptera. Hymenoptera have descended along with Raphidioptera from the earliest Megaloptera, the Permian Parasialidae. A related new family, minute Nanosialidae from the Permian of Russia is supposedly ancestral to Raphidioptera. The fusion of the third ovipositor valvulae is shown to be not a synapomorphy of Neuropteroidea. Parasialids and nanosialids bridge the gap between megalopterans and snakeflies; all can be classified into a single order, Panmegaloptera nom. n., including a new suborder Siarapha for Nanosialidae. The earliest megalopterans and their descendants, Raphidioptera and Hymenoptera, have passed through a "miniaturization bottleneck", likely a common macroevolutionary mechanism.

The first mitochondrial genome for caddisfly (insecta: Trichoptera) with phylogenetic implications.

The Trichoptera (caddisflies) is a holometabolous insect order with 14,300 described species forming the second most species-rich monophyletic group of animals in freshwater. Hitherto, there is no mitochondrial genome reported of this order. Herein, we describe the complete mitochondrial (mt) genome of a caddisfly species, Eubasilissa regina (McLachlan, 1871). A phylogenomic analysis was carried out based on the mt genomic sequences of 13 mt protein coding genes (PCGs) and two rRNA genes of 24 species belonging to eight holometabolous orders. Both maximum likelihood and Bayesian inference analyses highly support the sister relationship between Trichoptera and Lepidoptera.

Snakefly diversity in Early Cretaceous amber from Spain (Neuropterida, Raphidioptera).

The Albian amber from Spain presently harbors the greatest number and diversity of amber adult fossil snakeflies (Raphidioptera). Within Baissopteridae, Baissoptera? cretaceoelectrasp. n., from the Peñacerrada I outcrop (Moraza, Burgos), is the first amber inclusion belonging to the family and described from western Eurasia, thus substantially expanding the paleogeographical range of the family formerly known from the Cretaceous of Brazil and eastern Asia. Within the family Mesoraphidiidae, Necroraphidia arcuatagen. et sp. n. and Amarantoraphidia ventolinagen. et sp. n. are described from the El Soplao outcrop (Rábago, Cantabria), whereas Styporaphidia? hispanicasp. n. and Alavaraphidia imperterritagen. et sp. n. are describedfrom Peñacerrada I. In addition, three morphospecies are recognized from fragmentary remains. The following combinations are restored: Yanoraphidia gaoi Ren, 1995, stat. rest., Mesoraphidia durlstonensis Jepson, Coram and Jarzembowski, 2009, stat. rest., and Mesoraphidia heteroneura Ren, 1997, stat. rest. The singularity of this rich paleodiversity could be due to the paleogeographic isolation of the Iberian territory and also the prevalence of wildfires during the Cretaceous.