Genomic and morphological evidence converge to resolve the enigma of Strepsiptera

The phylogeny of insects, one of the most spectacular radiations of life on earth, has received considerable attention. However, the evolutionary roots of one intriguing group of insects, the twisted-wing parasites (Strepsiptera), remain unclear despite centuries of study and debate. Strepsiptera exhibit exceptional larval developmental features, consistent with a predicted step from direct (hemimetabolous) larval development to complete metamorphosis that could have set the stage for the spectacular radiation of metamorphic (holometabolous) insects. Here we report the sequencing of a Strepsiptera genome and show that the analysis of sequence-based genomic data (comprising more than 18 million nucleotides from nearly 4,500 genes obtained from a total of 13 insect genomes), along with genomic metacharacters, clarifies the phylogenetic origin of Strepsiptera and sheds light on the evolution of holometabolous insect development. Our results provide overwhelming support for Strepsiptera as the closest living relatives of beetles (Coleoptera). They demonstrate that the larval developmental features of Strepsiptera, reminiscent of those of hemimetabolous insects, are the result of convergence. Our analyses solve the long-standing enigma of the evolutionary roots of Strepsiptera and reveal that the holometabolous mode of insect development is more malleable than previously thought.     

Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.     

Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development

Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.     

Degeneration and cell regeneration in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae) during post-embryonic development

Cell death, proliferation, and differentiation in some developmental stages of insects have been studied in the midgut of ametabolous, which undergo only continuous growth, and holometabolous, which undergo complete metamorphosis. However, in hemimetabolous insects, evolutionarily intermediate between ametabolous and holometabolous, midgut reorganization during the post-embryonic development has been poorly studied. The present study evaluates the post-embryonic development of the midgut of a hemimetabolous insect, Podisus nigrispinus, to test the hypothesis that these insects have programmed cell death and proliferation followed by differentiation of regenerative cells during midgut growth from nymphs to adult. The morphometrical data showed a 6-fold increase in midgut length from the first instar nymph to the adult, which did not result from an increase in the size of the midgut cells, suggesting that the growth of the midgut occurs by an increase in cell number. Cell death was rarely found in the midgut, whereas proliferation of regenerative cells occurred quite frequently. The growth of the midgut of P. nigrispinus appears to result from the proliferation of regenerative cells present in the epithelium; unlike ametabolous and holometabolous insects, the midgut of P. nigrispinus does not undergo extensive remodeling, as shown by the low frequency of digestive cell death.     

Embryonic integument and "molts" in Manduca sexta (Insecta,Lepidoptera)

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed.     

Functions of the ecdysone receptor isoform-A in the hemimetabolous insect Blattella germanica revealed by systemic RNAi in vivo

The molecular basis of ecdysteroid function during development has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in hemimetabolous. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we show that the ecdysone receptor isoform-A (BgEcR-A) mRNA is present throughout the penultimate and last nymphal instars in all tissues analyzed (prothoracic gland, epidermis and fat body). To study the functions of BgEcR-A, we reduced its expression using systemic RNAi in vivo, and we obtained knockdown specimens. Examination of these specimens indicated that BgEcR-A during the last nymphal instar is required for nymphal survival, and that reduced expression is associated with molting defects, lower circulating ecdysteroid levels and defects in cell proliferation in the follicular epithelium. Some BgEcR-A knockdown nymphs survive to the adult stage. The features of these specimens indicate that BgEcR-A is required for adult-specific developmental processes, such as wing development, prothoracic gland degeneration and normal choriogenesis.     

On the pronymphal stage in the migratory locust (Locusta migratoria, Orthoptera, Acrididae)

The structure of the integument, somatic and visceral muscles, midgut, and Malpighian tubules were investigated at the late stages of the embryonic and early postembryonic development of the migratory locust, Locusta migratoria, to assess the organization of its pronymphal stage. In its morphogenetic features, the vermiform locust larva sometimes called the pronymph corresponds to the first nymphal instar covered with the second embryonic cuticle which has not been shed. Since the first-instar locust nymphs before and after the shedding of this embryonic cuticle differ significantly in many morphological characters, two consecutive phases of this nymphal instar can be distinguished: the first phase existing from the moment of development of the third embryonic cuticle to the shedding of the second one; the second phase existing from the shedding of the second embryonic cuticle to the formation of the cuticle of the second nymphal instar. Since the pronymphal stage should precede the nymph stage, it may be concluded that the pronymph of the locust is fully embryonized and covered with the second embryonic cuticle, which is also typical of other insects with hemimetabolous development (Konopová and Zrzavý, 2005). Therefore, it would be erroneous to refer to the vermiform first-instar nymph as the pronymph, because the two stages are separated by molting and formation of a new cuticle.     

Ultrastructure of the larval eye of the scorpionfly Panorpa dubia (mecoptera: Panorpidae) with implications for the evolutionary origin of holometabolous larvae

The evolutionary origin of holometabolous larvae is a long‐standing and controversial issue. The Mecoptera are unique in Holometabola for their larvae possessing a pair of compound eyes instead of stemmata. The ultrastructure of the larval eyes of the scorpionfly Panorpa dubia Chou and Wang, 1981 was investigated using transmission electron microscopy. Each ommatidium possesses a cornea, a tetrapartite eucone crystalline cone, eight retinula cells, two primary pigment cells, and an undetermined number of secondary pigment cells. The rhabdomeres of the eight retinula cells form a centrally‐fused, tiered rhabdom of four distal and four proximal retinula cells. The rhabdomeres of the four distal retinula cells extend distally into a funnel shape around the basal surface of the crystalline cone. Based on the similarity of the larval eyes of Panorpidae to the eyes of the hemimetabolous insects and the difference from the stemmata of the holometabolous larvae, the evolutionary origin of the holometabolous larvae is briefly discussed. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Scalariform junctions in the malpighian tubules of the insects Rhodnius prolixus (Hemiptera: Reduviidae) and Aedes taeniorhynchus (Diptera: Culicidae)

The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.     

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.     

Behavioural changes are related to moult regulation in the cockroach, Periplaneta americana

Abstract. Larval holometabolous insects show changes in behaviour (e.g. start of wandering and spinning) in specific periods of the moulting cycle in relation to definite ratios of juvenile hormone and moulting hormone (ecdysone). In hemimetabolous insects no such changes in behaviour are known. It should be investigated whether the cockroach Periplaneta americana shows changes in locomotor activity and in food and water consumption in relation to periods of ecdysone production during the last larval stage. Within a mean duration of the last larval stage of 30 days there were two periods of reduced locomotor activity: on day 9 and between days 13 and 17. From days 12–13 food consumption decreased by c . 40% up to the day 18. Water consumption decreased between days 9 and 18 by about 55%. Peaks of ecdysone production appeared after these changes of behaviour in each case. Therefore in larval Periplaneta ecdysone seems not to trigger these behavioural changes.     

PROTEIN PROFILES OF CHINESE WHITE WAX SCALE,Ericerus pela,AT THE MALE PUPAL STAGE BY HIGH‐THROUGHPUT PROTEOMICS

The Chinese white wax scale insect (Ericerus pela) is sexually dimorphic with holometabolous males and hemimetabolous females. Holometabolous insects were assumed to originate from hemimetabolous ancestors. Therefore, the male pupal stage is a major innovation compared with hemimetabolous female insects. Here, the protein profiles of the male pupae were obtained by high‐throughput proteomics and analyzed using bioinformatics methods. A total of 1,437 peptides were identified and assigned to 677 protein groups. Most of the proteins had molecular weights below 40 kDa and isoelectric points from 4 to 7. Gene Ontology terms were assigned to 331 proteins, including metabolic process, developmental process, and cellular process. Kyoto Encyclopedia of Genes and Genomes annotations identified 142 pathways and most proteins were assigned to metabolism events. Pathways involved in cell growth and death, signal transduction, folding, and sorting and degradation were also identified. Six proteins that had undergone positive selection were classified into four groups, protein biosynthesis, protein degeneration, signal transduction, and detoxification. Many of the high‐abundance proteins were enzymes involved in carbohydrate, lipid, and amino acid metabolism; signal transduction; degradation; and immunization, which indicated that metabolism, disruption, and development occurred intensely at the pupal stage. These processes are closely related to the physiological status of pupae. The results also suggested that these related proteins may be fundamental factors in the formation of pupae. This study describes pupal characterization at the molecular level and provides a basis for further physiological studies.     

Age‐specific mortality analysis of the dry forest kissing bug,Rhodnius neglectus

Age‐specific mortality patterns can be very different across insects with different life histories. Some holometabolous insects (like mosquitoes, fruit flies) show a pattern where mortality rate decelerates at older ages, whereas other holometabolous insects (bruchid beetles) and hemimetabolous insects (cotton stainers, milkweed bugs, and kissing bugs) show an age‐specific mortality pattern that increases through all ages. Kissing bugs are strictly hematophagous and are vectors of Trypanosoma cruzi Chagas, the etiologic agent of Chagas disease. Here, we tested whether cohort data from the dry forest kissing bug, Rhodnius neglectus Lent (Hemiptera: Reduviidae), supports an increase of mortality rate that decelerates with age. We analyzed the age‐specific mortality pattern of a cohort of 250 individuals of R. neglectus. We used a suite of seven models with different degrees of complexity, to model age‐dependent forms of change in mortality rate increase in R. neglectus in the laboratory. We used the Akaike model selection criterion to choose between models that consider absence or presence of mortality deceleration. Five of the seven models (logistic, Gavrilovs, Gompertz, DeMoivre, and exponential) showed a statistically significant fit to the mortality rate. Weak late‐age mortality deceleration in R. neglectus was supported by the best fit (logistic model), and this result is consistent with predictions of the disposable soma theory of senescence.     

Corpse Management in Social Insects

Undertaking behavior is an essential adaptation to social life that is critical for colony hygiene in enclosed nests. Social insects dispose of dead individuals in various fashions to prevent further contact between corpses and living members in a colony. Focusing on three groups of eusocial insects (bees, ants, and termites) in two phylogenetically distant orders (Hymenoptera and Isoptera), we review mechanisms of death recognition, convergent and divergent behavioral responses toward dead individuals, and undertaking task allocation from the perspective of division of labor. Distinctly different solutions (e.g., corpse removal, burial and cannibalism) have evolved, independently, in the holometabolous hymenopterans and hemimetabolous isopterans toward the same problem of corpse management. In addition, issues which can lead to a better understanding of the roles that undertaking behavior has played in the evolution of eusociality are discussed.     

昆虫单眼的结构和功能

大多数昆虫的视觉器官除了复眼外还有一些简单的小眼,称为单眼。昆虫成虫和半变态类若虫的单眼称为背单眼,位于头顶两复眼之间。背单眼在数目和结构上都有较大变化,但基本结构包括角膜晶体、一层角膜生成细胞(覆盖在角膜晶体上)、视网膜(由大约1000个感光细胞构成,视类群而不同)。背单眼对弱光比较敏感,但在图像感知方面的作用并不显著;它是一种“激发器官”,可以增加复眼的感知能力。全变态昆虫的幼虫既没有复眼也没有背单眼,但在其头部两侧有些类似复眼小眼的侧单眼。侧单眼的结构也与小眼相似,包括角膜,晶体和由一些视网膜细胞组成的视杆。侧单眼是完全变态类昆虫幼虫仅有的感光器官,与复眼一样,它们可以感知颜色、形状、距离等等。