Serosal Cuticle Research Articles

RNAi-mediated knockdown of papilin gene affects the egg hatching in Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Owing to the rapid adaptation of BPH to many pesticides and resistant varieties, identifying putative gene targets for developing RNA interference (RNAi)-based pest management strategies has received much attention for this pest. The glucoprotein papilin is the most abundant component in the basement membranes of many organisms, and its function is closely linked to development. In this study, we identified a papilin homologous gene in BPH (NlPpn). Quantitative Real-time PCR analysis showed that the transcript of NlPpn was highly accumulated in the egg stage. RNAi of NlPpn in newly emerged BPH females caused nonhatching phenotypes of their eggs, which may be a consequence of the maldevelopment of their embryos. Moreover, the transcriptomic analysis identified 583 differentially expressed genes between eggs from the dsGFP- and dsNlPpn-treated insects. Among them, the 'structural constituent of cuticle' cluster ranked first among the top 15 enriched GO terms. Consistently, ultrastructural analysis unveiled that dsNlPpn-treated eggs displayed a discrete and distorted serosal endocuticle lamellar structure. Furthermore, the hatchability of BPH eggs was also successfully reduced by the topical application of NlPpn-dsRNA-layered double hydroxide nanosheets onto the adults. Our findings demonstrate that NlPpn is essential to maintaining the regular structure of the serosal cuticle and the embryonic development in BPH, indicating NlPpn could be a potential target for pest control during the egg stage. © 2024 Society of Chemical Industry.

Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

LmCht5-1 and LmCht5-2 Promote the Degradation of Serosal and Pro-Nymphal Cuticles during Locust Embryonic Development.

The success of the degradation of the extraembryonic serosal cuticle and the second embryonic cuticle (pro-nymphal cuticle) is essential for the development and molting of nymph from egg in Orthoptera Locusta migratoria. Chitinase 5 is an important gene for chitin degradation in nymphs and in the egg stage. In this study, we investigated the important roles of chitinase 5-1 (LmCht5-1) and chitinase 5-2 (LmCht5-2) in the degradation of the serosal and pro-nymphal cuticles during locust embryonic development. The serosal cuticle degrades from 7-day-old embryos (E7) to E13, along with the degradation of the pro-nymphal cuticle, which begins at E12 to E14. The mRNA and protein of LmCht5-1 and LmCht5-2 are expressed during the degradation process of the serosal cuticle and the pro-nymphal cuticle. RNAi experiments at the embryonic stage show that both dsLmCht5-1 and dsLmCht5-2 contribute to the failure of development in early and late embryogenesis. Further, during the serosal cuticle molting process, ultra-structure analysis indicated that dsLmCht5-1 prevented the loss of the coarse chitin layer in the upper part in both early and late embryogenesis. Meanwhile, dsLmCht5-2 blocked the degradation of the lower fine chitin layer at the early stage and blocked the chitin degradation of loose coarse chitin in the late molting process. During the degradation of the pro-nymphal cuticle, dsLmCht5-1 suppresses chitin degradation between layers in the procuticle, while dsLmCht5-2 suppresses chitin degradation into filaments inside of the layer. In summary, our results suggest that both LmCht5-1 and LmCht5-2 contribute to the degradation of the serosal and pro-nymphal cuticles during the locust embryonic stage.

RNAi-mediated silencing of an egg-specific gene Nllet1 results in hatch failure in the brown planthopper.

The brown planthopper (BPH) is one of the most destructive agricultural pests in Asia. RNA interference (RNAi)-mediated pest management has been under development for years, and the selection of appropriate target genes is important for pest-targeted RNAi. C-type lectins (CTLs) are a class of genes that perform a variety of functions, such as the regulation of growth and development. A CTL-S protein named Nllet1, containing a single calcium ion (Ca2+ )-dependent carbohydrate-binding domain (CRD) with a conserved triplet motif QPD was identified and functionally characterized in BPH. Expression profiles at both the transcriptional and translational levels show that Nllet1 accumulates during the serosal cuticle (SC) formation period. Immunofluorescence and immunogold labeling further demonstrated that Nllet1 is located in the serosal endocuticle (en-SC). Maternal RNAi-mediated silencing of Nllet1 disrupted the SC structure, accompanied by a loss of the outward barrier and 100% embryo mortality. Injection of 10ng dsNllet1 or dsNllet1' per female adult BPH resulted in a total failure of egg hatching. Nllet1 is essential for SC formation and embryonic development in BPH, which helps us understand the important roles of CTL-Ss. Additionally, BPH eggs show high sensitivity to the depletion of Nllet1. This study indicates that Nllet1 is a promising candidate gene that can be used to develop RNAi-based control strategies at the BPH egg stage, and it can also be used as a target for developing novel ovicides. © 2022 Society of Chemical Industry.

Myracrodruon urundeuva leaf lectin damages exochorionic cells and binds to the serosal cuticle of Aedes aegypti eggs.

In recent years, lectins have been identified as alternative agents against Aedes aegypti during the aquatic phases of its life cycle. For example, chitin-binding lectin from Myracrodruon urundeuva leaf (MuLL) can function as a larvicide. In this study, we investigated whether MuLL can also act as an ovicide against this insect. Aedes aegypti eggs were incubated with MuLL for 72h to determine the concentration at which the hatching rate reduces by 50% (EC50). The effects of MuLL on the egg surface structure were evaluated using scanning electron microscopy (SEM), and the possible interaction of MuLL with the internal structures of eggs and embryos was investigated using MuLL-fluorescein isothiocyanate (FITC) conjugate. MuLL acted as an ovicidal agent with an EC50 of 0.88mg/mL. The SEM analysis revealed that eggs treated with MuLL for 24 and 48h no longer had tubercles and did not show a well-defined exochorionic network. In addition, deformation and degeneration of the surface were observed after 72h. Fluorescence microscopy showed that MuLL penetrated the eggs 48h after incubation and was detected in the upper portion of the embryo's gut. After 72h, MuLL was observed in the serosal cuticle and digestive tract. In conclusion, MuLL can function as an ovicidal agent against A. aegypti through damage to the surface and internal structures of the eggs.

Chitin synthase 1 and five cuticle protein genes are involved in serosal cuticle formation during early embryogenesis to enhance eggshells in Nilaparvata lugens.

Many holo- and hemimetabolous insects enhance their eggshells during embryogenesis by forming a serosal cuticle (SC). To date, scholarly understanding of the SC composition and SC-related gene functions has been limited, especially for hemimetabolous insects. In this study, we initially performed transmission electron microscopic (TEM) observation and chitin staining of the SC in Nilaparvata lugens, a hemimetabolous rice pest known as the brown planthopper (BPH). We confirmed that the SC was a chitin-rich lamellar structure deposited gradually during the early embryogenesis. Parental RNA interference (RNAi) against Nilaparvata lugens chitin synthase 1 (NlCHS1) in newly emerged and matured females resulted in decreases of egg hatchability by 100% and 76%, respectively. Ultrastructural analyses revealed loss of the lamellar structure of the SC in dsNlCHS1-treated eggs. According to temporal expression profiles, five cuticle protein coding genes, NlugCpr1/2/3/8/90, were specifically or highly expressed during the SC formation period, and NlugCpr1/2/3/90 were further detected in 72 h eggshells extract by ultra-performance liquid chromatography-tandem mass spectrometry/mass spectrometry. NlugCpr2/3/90 were likely three SC-specific cuticle proteins. TEM observations of the SC following parental RNAi against NlugCpr1/2/3/8/90 demonstrated that NlugCpr3/8/90 were essential for SC formation. The study provided an understanding of the SC formation process and SC-related cuticle proteins in BPHs, which offer potential targets for pest control in the egg stage as well.

The formation of a hatching line in the serosal cuticle confers multifaceted adaptive functions on the eggshell of a cicada

Insect eggshells must meet various demands of developing embryos. These demands sometimes conflict with each other; therefore, there are tradeoffs between eggshell properties, such as robustness and permeability. To meet these conflicting demands, particular eggshell structures have evolved in diverse insect species. Here, we report a rare eggshell structure found in the eggshell of a cicada, Cryptotympana facialis. This species has a prolonged egg period with embryonic diapause and a trait of humidity-inducible hatching, which would impose severe demands on the eggshell. We found that in eggs of this species, unlike many other insect eggs, a dedicated cleavage site, known as a hatching line, was formed not in the chorion but in the serosal cuticle. The hatching line was composed of a fine furrow accompanied by ridges on both sides. This furrow-ridge structure formed in the terminal phase of embryogenesis through the partial degradation of an initially thick and nearly flat cuticle layer. We showed that the permeability of the eggshell was low in the diapause stage, when the cuticle was thick, and increased with degradation of the serosal cuticle. We also demonstrated that the force required to cleave the eggshell was reduced after the formation of the hatching line. These results suggest that the establishment of the hatching line on the serosal cuticle enables flexible modification of eggshell properties during embryogenesis, and we predict that it is an adaptation to maximize the protective role of the shell during the long egg period while reducing the barrier to emerging nymphs at the time of hatching.

Increase in egg resistance to desiccation in springtails correlates with blastodermal cuticle formation: Eco-evolutionary implications for insect terrestrialization.

Land colonization was a major event in the history of life. Among animals, insects exerted a staggering terrestrialization success, due to traits usually associated with postembryonic life stages, while the egg stage has been largely overlooked in comparative studies. In many insects, after blastoderm differentiation, the extraembryonic serosal tissue wraps the embryo and synthesizes the serosal cuticle, an extracellular matrix that lies beneath the eggshell and protects the egg against water loss. In contrast, in noninsect hexapods such as springtails (Collembola) the early blastodermal cells synthesize a blastodermal cuticle. Here, we investigate the relationship between blastodermal cuticle formation and egg resistance to desiccation in the springtails Orchesella cincta and Folsomia candida, two species with different oviposition environments and developmental rates. The blastodermal cuticle becomes externally visible in O. cincta and F. candida at 22% and 29% of embryogenesis, respectively. To contextualize, we describe the stages of springtail embryogenesis, exemplified by F. candida. Our physiological assays then showed that blastodermal cuticle formation coincides with an increase in egg viability in a dry environment, significantly contributing to hatching success. However, protection differs between species: while O. cincta eggs survive at least 2 hr outside a humid environment, the survival period recorded for F. candida eggs is only 15 min, which correlates with this species' requirement for humid microhabitats. We suggest that the formation of this cuticle protects the eggs, constituting an ancestral trait among hexapods that predated and facilitated the process of terrestrialization that occurred during insect evolution.

Yellow-g and Yellow-g2 proteins are required for egg desiccation resistance and temporal pigmentation in the Asian tiger mosquito, Aedes albopictus

Eggs from Aedes mosquitoes exhibit desiccation resistance that helps them to survive and spread as human disease vectors throughout the world. Previous studies have suggested that eggshell/chorion melanization and/or serosal cuticle formation are important for desiccation resistance. In this study, using dsRNAs for target genes, we analyzed the functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the resistance to egg desiccation of the Asian tiger mosquito, Aedes albopictus, a species in which neither the timing of the melanization nor temporal development of the serosal cuticle is correlated with desiccation resistance. Injections of dsAalY-g, dsAalY-g2 or dsAalY-g/g2 (co-injection) into adult females have no effect on their fecundity. However, initial melanization is delayed by 1–2 h with the eggshells eventually becoming black similar to that observed in eggs from dsEGFP-injected control females. In addition, the shape of the eggs from dsAalY-g, -g2 and -g/g2-treated females is abnormally crescent-shaped and the outermost exochorion is more fragile and partially peeled off. dsEGFP control eggs, like those from the wild-type strain, acquire resistance to desiccation between 18 and 24 h after oviposition (HAO). In contrast, ~80% of the 24 HAO dsAalY-g and dsAalY-g2 eggs collapse when they are transferred to a low humidity environment. In addition, there is no electron-dense outer endochorion evident in either dsAalY-g or dsAalY-g2 eggs. These results support the hypothesis that AalY-g and AalY-g2 regulate the timing of eggshell darkening and are required for integrity of the exochorion as well as for rigidity, normal morphology and formation of the outer endochorion, a structure that apparently is critical for desiccation resistance of the Ae. albopictus egg.

Transcriptome of pleuropodia from locust embryos supports that these organs produce enzymes enabling the larva to hatch

BackgroundPleuropodia are limb-derived glandular organs that transiently appear on the first abdominal segment in embryos of insects from majority of “orders”. They are missing in the genetic model Drosophila and little is known about them. Experiments carried out on orthopteran insects 80 years ago indicated that the pleuropodia secrete a “hatching enzyme” that digests the serosal cuticle to enable the larva to hatch, but evidence by state-of-the-art molecular methods is missing.ResultsWe used high-throughput RNA-sequencing to identify the genes expressed in the pleuropodia of the locust Schistocerca gregaria (Orthoptera). First, using transmission electron microscopy we studied the development of the pleuropodia during 11 stages of the locust embryogenesis. We show that the glandular cells differentiate and start secreting just before the definitive dorsal closure of the embryo and the secretion granules outside the cells become more abundant prior to hatching. Next, we generated a comprehensive embryonic reference transcriptome for the locust and used it to study genome wide gene expression across ten morphologicaly defined stages of the pleuropodia. We show that when the pleuropodia have morphological markers of functional organs and produce secretion, they are primarily enriched in transcripts associated with transport functions. They express genes encoding enzymes capable of digesting cuticular protein and chitin. These include the potent cuticulo-lytic Chitinase 5, whose transcript rises just before hatching. Unexpected finding was the enrichment in transcripts for immunity-related enzymes. This indicates that the pleuropodia are equipped with epithelial immunity similarly as barrier epithelia in postembryonic stages.ConclusionsThese data provide transcriptomic support for the historic hypothesis that pleuropodia produce cuticle-degrading enzymes and function in hatching. They may also have other functions, such as facilitation of embryonic immune defense. By the genes that they express the pleuropodia are specialized embryonic organs and apparently an important though neglected part of insect physiology.

Development and ultrastructure of the thickened serosa and serosal cuticle formed beneath the embryo in the stonefly Scopura montana Maruyama, 1987 (Insecta, Plecoptera, Scopuridae)

We aimed to describe the development and ultrastructure of the thickened serosa and serosal cuticle formed beneath the embryo of Plecoptera, using Scopura montana of Scopuridae as a euholognathan representative. Using transmission electron microscopy, we found that the egg membranes were composed of a thick exochorion, a thicker endochorion consisting of two sublayers, and an extremely thin vitelline membrane. The egg membrane construction represents a groundplan feature of the euholognathan egg membranes. The serosa converges beneath the embryo to form a thickened serosa, comprising cells in a radial arrangement, in association with the formation of the amnioserosal fold. The thickened serosa then deposits the thickened serosal cuticle, consisting of four layers differing in fine structure and electron density. After achieving its secretory function, the thickened serosa then disintegrates, and the liberated serosal cells float for a short period in the peripheral region of the egg inside. Collectively, our findings should provide the basis for further characterization of the serosal structures concerned, but we were unable to corroborate previous studies assigning the thickened serosa and serosal cuticle in Plecoptera to the water absorption function.

Characterizing the embryonic development of B. hygida (Diptera: Sciaridae) following enzymatic treatment to permeabilize the serosal cuticle

Understanding the evolution of the developmental programs active during dipteran embryogenesis depends on comparative studies. As a counterpoint to the intensively investigated and highly derived cyclorrhaphan flies that include the model organism Drosophila melanogaster, we are studying the basal Diptera Bradysia hygida, a member of the Sciaridae family that is amenable to laboratory cultivation. Here we describe the B. hygida embryogenesis, which lasts 9 days at 22 °C. The use of standard fixation D. melanogaster protocols resulted in embryos refractory to DAPI staining and to overcome this, a new enzyme-based method was developed. Calcofluor-White staining of enzimatically-treated embryos revealed that this method removes chitin from the serosal cuticle surrounding the B. hygida embryo. Chitin is one of the main components of serosal cuticles and searches in a B. hygida embryonic transcriptome database revealed conservation of the chitin synthesis pathway, further supporting the occurrence of chitin biosynthesis in B. hygida embryos. Combining the enzymatic treatment protocol with the use of both DIC and fluorescence microscopy allowed the first complete description of the B. hygida embryogenesis. Our results constitute an important step towards the understanding of early development of a basal Diptera and pave the way for future evo-devo studies.

Field Distribution and Density of Culicoides sonorensis (Diptera: Ceratopogonidae) Eggs in Dairy Wastewater Habitats.

Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) is a key bluetongue virus vector in the United States. Immatures occur in mud near the edges of wastewater ponds and are understudied targets for control efforts. Eggs of C. sonorensis were collected in the morning from a dairy wastewater pond bank by taking 5-ml surface mud samples along four transects on each of six dates. Surface mud samples parallel to waterline (10-cm long, 1-cm wide, and 0.5-cm deep) were removed at 5-cm increments ranging from 15 cm below waterline up to 25 cm above waterline. Eggs were removed using MgSO4 flotation, held on moist filter paper, and scored for hatching over 3 d. Eggs hatching on days 2 and 3 were assumed to have been laid on the test night. Water levels were stable within a night according to time-lapse camera photos. Most samples from below the waterline had no eggs and were not analyzed statistically. Mean (±SE) sample moisture (25.8 ± 2.1 at 5 cm above waterline and 19.8 ± 2.6% at 25 cm above waterline) did not vary significantly by position above waterline. The highest density of viable eggs (21 eggs/5 ml), proportion of mud samples positive for viable eggs (75%), and proportion of eggs hatching (80%) were found 5 cm above waterline. Oviposition in the few hours after sunset is adaptive, allowing eggs to age, develop the serosal cuticle, and resist later desiccation. As a potential control method, reducing water levels after midnight would encourage young egg desiccation.

Darker eggs of mosquitoes resist more to dry conditions: Melanin enhances serosal cuticle contribution in egg resistance to desiccation in Aedes, Anopheles and Culex vectors.

Mosquito vectors lay their white eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, which at this moment is composed of exochorion and endochorion. Within two hours the endochorion darkens via melanization but even so eggs shrink and perish if removed from moisture. However, during mid-embryogenesis, cells of the extraembryonic serosa secrete the serosal cuticle, localized right below the endochorion, becoming the third and innermost eggshell layer. Serosal cuticle formation greatly reduces water flow and allows egg survival outside the water. The degree of egg resistance to desiccation (ERD) at late embryogenesis varies among different species: Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus eggs can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult insects, darker-body individuals show greater resistance to desiccation than lighter ones. We asked if egg melanization enhances mosquito serosal cuticle-dependent ERD. Species with higher ERD at late embryogenesis exhibit more melanized eggshells. The melanization-ERD hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type and the mutant GORO, with a dark-brown and a golden eggshell, respectively. In all cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD but egg viability outside the water is much higher in mosquitoes with darker eggshells than in those with lighter ones. The finding that pigmentation influences egg water balance is relevant to understand the evolutionary history of insect egg coloration. Since eggshell and adult cuticle pigmentation ensure insect survivorship in some cases, they should be considered regarding species fitness and novel approaches for vector or pest insects control.

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation

Mosquito eggs are laid in water but freshly laid eggs are susceptible to dehydration, if their surroundings dry out at the first hours of development. During embryogenesis of different mosquito vectors the serosal cuticle, an extracellular matrix, is produced; it wraps the whole embryo and becomes part of the eggshell. This cuticle is an essential component of the egg resistance to desiccation (ERD). However, ERD is variable among species, sustaining egg viability for different periods of time. While Aedes aegypti eggs can survive for months in a dry environment (high ERD), those of Anopheles aquasalis and Culex quinquefasciatus in the same condition last, respectively, for one day (medium ERD) or a few hours (low ERD). Resistance to desiccation is determined by the rate of water loss, dehydration tolerance and total amount of water of a given organism. The ERD variability observed among mosquitoes probably derives from diverse traits. We quantified several attributes of whole eggs, potentially correlated with the rate of water loss: length, width, area, volume, area/volume ratio and weight. In addition, some eggshell aspects were also evaluated, such as absolute and relative weight, weight/area relationship (herein called surface density) and chitin content. Presence of chitin specifically in the serosal cuticle as well as aspects of endochorion external surface were also investigated. Three features could be related to differences on ERD levels: chitin content, directly related to ERD, the increase in the egg volume during embryogenesis and the eggshell surface density, which were both inversely related to ERD. Although data suggest that the amount of chitin in the eggshell is relevant for egg impermeability, the participation of other yet unidentified eggshell attributes must be considered in order to account for the differences in the ERD levels observed among Ae. aegypti, An. aquasalis and Cx. quinquefasciatus.

Elucidation of the serosal cuticle machinery in the beetle Tribolium by RNA sequencing and functional analysis of Knickkopf1, Retroactive and Laccase2

Insects have been extraordinary successful in colonizing terrestrial habitats and this success is partly due to a protective cuticle that mainly contains chitin and proteins. The cuticle has been well studied in larvae and adults, but little attention has been paid to the cuticle of the egg. This cuticle is secreted by the serosa, an extraembryonic epithelium that surrounds the yolk and embryo in all insect eggs, but was lost in the Schizophoran flies to which Drosophila belongs. We therefore set out to investigate serosal cuticle formation and function in a beetle (Tribolium castaneum) using RNAi-mediated knockdown of three candidate genes known to structure chitin in the adult cuticle, and we aimed to identify other serosal cuticle genes using RNA sequencing. Knockdown of Knickkopf (TcKnk-1) or Retroactive (TcRtv) affects the laminar structure of the serosal cuticle, as revealed by Transmission Electron Microscopy in knockdown eggs. In the absence of this laminar structure, significantly fewer eggs survive at low humidity compared to wild-type eggs. Survival in dry conditions is also adversely affected when cross-linking among proteins and chitin is prevented by Laccase2 (TcLac-2) RNAi. Finally, we compare the transcriptomes of wild-type eggs to serosa-less eggs and find serosa-biased expression of 21 cuticle-related genes including structural components, chitin deacetylases and chitinases. Our data indicate that the serosal cuticle utilizes the same machinery for structuring the cuticle as adults. We demonstrate that the structure of the cuticle is crucial for desiccation resistance, and we put forward the serosal cuticle of Tribolium as an excellent model to study the ecological properties of the insect cuticle.

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.

When mothers anticipate: Effects of the prediapause stage on embryo development time and of maternal photoperiod on eggs of a temperate and a tropical strains of Aedes albopictus (Diptera: Culicidae)

BackgroundThe diapause of Aedes albopictus is maternally induced by photoperiod and initiates at the pharate larvae stage in eggs. This pre-diapause results in enhanced survival eggs during the winter. This study aims to disentangle the effects of photoperiod and diapause on embryonic developmental time and egg size in A. albopictus. A temperate strain capable to perform diapause and a tropical strain unable of diapause were reared at 21°C with long-(LD) and short-day (SD) lengths. Four distinct traits were studied on embryos and eggs were measured at the end of embryogenesis. ResultsThe chronologies of embryo development for both strains were influenced by maternal photoperiod, especially in the temperate strain in which the development of SD eggs took longer than LD eggs. The delay increased gradually in the temperate strain, and reached up to 38h at the end of embryogenesis. The kinetics of embryogenesis differed among the temperate and the tropical strains, each one of the 4 studied traits showing differences. For example the serosal cuticle was secreted precociously in the tropical strain. Egg width and volume are influenced by the maternal photoperiod and the strain×photoperiod interaction. For both strains, larger eggs were laid by female reared under SD when compared to LD. ConclusionsThe influence of several maternal effects was demonstrated in this study. The diapause process modifies greatly the length of embryogenesis in the temperate strain, whereas the maternal photoperiod has a direct influence on egg size and embryogenesis regardless of the strain considered. These findings provide useful data on chronology of embryonic development for integrative biology studies of egg pre-diapause stages.

Serosal cuticle formation and distinct degrees of desiccation resistance in embryos of the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus

Given their medical importance, mosquitoes have been studied as vectors of parasites since the late 1800’s. However, there are still many gaps concerning some aspects of their biology, such as embryogenesis. The embryonic desiccation resistance (EDR), already described in Aedes and Anopheles gambiae mosquitoes, is a peculiar trait. Freshly laid eggs are susceptible to water loss, a condition that can impair their viability. EDR is acquired during embryogenesis through the formation of the serosal cuticle (SC), protecting eggs from desiccation. Nevertheless, conservation of both traits (SC presence and EDR acquisition) throughout mosquito evolution is unknown. Comparative physiological studies with mosquito embryos from different genera, exhibiting distinct evolutionary histories and habits is a feasible approach. In this sense, the process of EDR acquisition of Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus at 25°C was evaluated. Completion of embryogenesis occurs in Ae. aegypti, An. aquasalis and Cx. quinquefasciatus at, respectively 77.4, 51.3 and 34.3hours after egg laying, Cx. quinquefasciatus embryonic development taking less than half the time of Ae. aegypti. In all cases, EDR is acquired in correlation with SC formation. For both Ae. aegypti and An. aquasalis, EDR and SC appear at 21% of total embryonic development, corresponding to the morphological stage of complete germ band elongation/beginning of germ band retraction. Although phylogenetically closer to Ae. aegypti than to An. aquasalis, Cx. quinquefasciatus acquires both EDR and serosal cuticle later, with 35% of total development, when the embryo already progresses to the middle of germ band retraction. EDR confers distinct egg viability in these species. While Ae. aegypti eggs demonstrated high viability when left up to 72hours in a dry environment, those of An. aquasalis and Cx. quinquefasciatus supported these conditions for only 24 and 5hours, respectively. Our data suggest that serosa development is at least partially uncoupled from embryo development and that, depending upon the mosquito species, EDR bestows distinct levels of egg viability.

The extraembryonic serosa protects the insect egg against desiccation

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane by zerknüllt1 RNAi in the beetle Tribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression of Tc-chitin-synthase1 and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates.