Efferocytosis and extrusion of leukocytes determine the progression of early mycobacterial pathogenesis.

Macrophages and neutrophils are the first responders to invading pathogens and contribute strongly to the host defense against intracellular pathogens. The collective interplay and dynamic interactions between these leukocytes are to a large extent not understood. In the present study, we have investigated their role using a combination of confocal laser-scanning and electron microscopy in a zebrafish model for tuberculosis, a local Mycobacterium marinum infection in the tissue of the larval tail fin. Our results show that neutrophils are efficient in phagocytosis of mycobacteria and that they contribute largely to their dissemination. Macrophages appear to play a major role in efferocytosis, phagocytosis of dead cells that contain bacterial content. Phagocytic cells with large bacterial aggregates are formed that can be extruded out of the tissue after cell death. Alternatively, these excessively infected cells can undergo necrosis leading to immediate recruitment of surrounding leukocytes and subsequent phagocytosis of released bacteria. Our data show that these necrotic burst events result in progression of the infection, whereas extrusion abates the infection.

Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum.

In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.

Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors.

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:ß-glucuronidase reporter expression was down-regulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley (Hordeum vulgare) could act as a repressor on an intron sequence in the KNOX gene Hooded/Barley Knotted3 (Bkn3) and that AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis (Arabidopsis thaliana) could repress KNOTTED-LIKE FROM ARABIDOPSIS THALIANA2 (KNAT2) promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with down-regulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNA interference silencing of OsGRF3, OsGRF4, and OsGRF5 resulted in dwarfism, delayed growth and inflorescence formation, and up-regulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants.

The biodistribution of polystyrene nanoparticles administered intravenously in the chicken embryo.

Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18-19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.

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.

Nanoplastics causes extensive congenital malformations during embryonic development by passively targeting neural crest cells.

Nanomaterials are widespread in the human environment as pollutants, and are being actively developed for use in human medicine. We have investigated how the size and dose of polystyrene nanoparticles affects malformations in chicken embryos, and have characterized the mechanisms by which they interfere with normal development. We find that nanoplastics can cross the embryonic gut wall. When injected into the vitelline vein, nanoplastics become distributed in the circulation to multiple organs. We find that the exposure of embryos to polystyrene nanoparticles produces malformations that are far more serious and extensive than has been previously reported. These malformations include major congenital heart defects that impair cardiac function. We show that the mechanism of toxicity is the selective binding of polystyrene nanoplastics nanoparticles to neural crest cells, leading to the death and impaired migration of those cells. Consistent with our new model, most of the malformations seen in this study are in organs that depend for their normal development on neural crest cells. These results are a matter of concern given the large and growing burden of nanoplastics in the environment. Our findings suggest that nanoplastics may pose a health risk to the developing embryo.

Interplay between calcium and sarcomeres directs cardiomyocyte maturation during regeneration.

Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.

Single-walled carbon nanotubes as scaffolds to concentrate DNA for the study of DNA-protein interactions.

Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration.

Specificity of the innate immune responses to different classes of non-tuberculous mycobacteria.

Mycobacterium avium is the most common nontuberculous mycobacterium (NTM) species causing infectious disease. Here, we characterized a M. avium infection model in zebrafish larvae, and compared it to M. marinum infection, a model of tuberculosis. M. avium bacteria are efficiently phagocytosed and frequently induce granuloma-like structures in zebrafish larvae. Although macrophages can respond to both mycobacterial infections, their migration speed is faster in infections caused by M. marinum. Tlr2 is conservatively involved in most aspects of the defense against both mycobacterial infections. However, Tlr2 has a function in the migration speed of macrophages and neutrophils to infection sites with M. marinum that is not observed with M. avium. Using RNAseq analysis, we found a distinct transcriptome response in cytokine-cytokine receptor interaction for M. avium and M. marinum infection. In addition, we found differences in gene expression in metabolic pathways, phagosome formation, matrix remodeling, and apoptosis in response to these mycobacterial infections. In conclusion, we characterized a new M. avium infection model in zebrafish that can be further used in studying pathological mechanisms for NTM-caused diseases.

Gold nanoparticles decorated with ovalbumin-derived epitopes: effect of shape and size on T-cell immune responses.

Gold nanoparticles (GNPs) can be manufactured in various shapes, and their size is programmable, which permits the study of the effects imposed by these parameters on biological processes. However, there is currently no clear evidence that a certain shape or size is beneficial. To address this issue, we have utilised GNPs and gold nanorods (GNRs) functionalised with model epitopes derived from chicken ovalbumin (OVA257-264 and OVA323-339). By using two distinct epitopes, it was possible to draw conclusions regarding the impact of nanoparticle shape and size on different aspects of the immune response. Our findings indicate that the peptide amphiphile-coated GNPs and GNRs are a safe and versatile epitope-presenting system. Smaller GNPs (∼15 nm in diameter) induce significantly less intense T-cell responses. Furthermore, effective antigen presentation via MHC-I was observed for larger spherical particles (∼40 nm in diameter), and to a lesser extent for rod-like particles (40 by 15 nm). At the same time, antigen presentation via MHC-II strongly correlated with the cellular uptake, with smaller GNPs being the least efficient. We believe these findings will have implications for vaccine development, and lead to a better understanding of cellular uptake and antigen egress from lysosomes into the cytosol.

Peptide modified mesoporous silica nanocontainers.

Here we report a new peptide modified mesoporous silica nanocontainer (PMSN) as a novel controlled release system. The peptides are part of a stimuli responsive nanovalve and ensure an efficient cellular uptake.

The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis.

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms.

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation.

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.

Macrophages target Salmonella by Lc3-associated phagocytosis in a systemic infection model.

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type.

Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish.

By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes. To identify the myc/YFP-expressing cell type, we re-examined specificity of described myeloid markers by multiplex fluorescent in situ hybridization, showing that lcp1 can be considered as a general leukocyte marker, csf1r as a macrophage-specific marker, and mpx and lyz as neutrophil-specific markers. Subsequent colocalization analysis defined the YFP-positive cells as a subset of the neutrophil population. Using real-time confocal imaging we demonstrate that these cells migrate to sites of inflammation and are involved in innate immune responses towards infections, including Mycobacterium marinum-induced granuloma formation.

Characterization of mucosal biofilms on human adenoid tissues.

OBJECTIVES: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids. STUDY DESIGN: Prospective study. METHODS: A total of 39 specimens of adenoidectomy were removed from children with chronic and/or recurrent otitis media. The specimens were prepared for light microscopy using Gram staining, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Double staining was performed with CLSM to visualize both the bacteria and the glycocalyx matrix. Nine adenoids on which bacterial biofilms were visualized with CLSM were used for identification of bacterial species by 16S-DNA polymerase chain reaction (PCR) amplification and homology analysis. RESULTS: Of the 39 adenoids investigated, 22 (54%) showed evidence of mucosal biofilms. Gram staining, SEM and CLSM showed the presence of bacterial cells, organized in bacterial microcolonies. CLSM with double staining demonstrated mucosal biofilms by showing the presence of both bacteria and the glycocalyx. The use of 16S-DNA polymerase chain reaction (PCR) amplification and subsequent sequence analyses identified the presence of Corynebacterium argentoratense, Streptococcus salivarius, Micrococcus luteus, and Staphylococcus aureus. CONCLUSIONS: This study demonstrates that adenoid tissue in children with chronic or/and recurrent otitis media contains mucosal biofilms in 54% of the cases. The existence of living bacteria has been demonstrated. Further studies are required to describe the panel of bacteria that can be harbored in the biofilms present in adenoids and the mechanisms involved in the physiopathology of otitis prone children.

Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.

OBJECTIVES: To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis. DESIGN: Prospective study. SETTING: Tertiary university-based referral center. PATIENTS: Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis. INTERVENTIONS: Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen. RESULTS: Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils. CONCLUSION: Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.

Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles.

Protein delivery into the cytosol of cells is a challenging topic in the field of nanomedicine, because cellular uptake and endosomal escape are typically inefficient, hampering clinical applications. In this contribution cuboidal mesoporous silica nanoparticles (MSNs) containing disk-shaped cavities with a large pore diameter (10 nm) are studied as a protein delivery vehicle using cytochrome-c (cytC) as a model membrane-impermeable protein. To ensure colloidal stability, the MSNs are coated with a fusogenic lipid bilayer (LB) and cellular uptake is induced by a complementary pair of coiled-coil (CC) lipopeptides. Coiled-coil induced membrane fusion leads to the efficient cytosolic delivery of cytC and triggers apoptosis of cells. Delivery of these LB coated MSNs in the presence of various endocytosis inhibitors strongly suggests that membrane fusion is the dominant mechanism of cellular uptake. This method is potentially a universal way for the efficient delivery of any type of inorganic nanoparticle or protein into cells mediated by CC induced membrane fusion.

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.