Rapid Preparation of Living Drosophila Pupal Macrophages for Ex Vivo Imaging.

The fruit fly Drosophila is a well-established invertebrate model that enables in vivo imaging of innate immune cell (e.g., macrophage) migration and signaling at high spatiotemporal resolution within the intact, living animal. While optimized methods already exist to enable flow cytometry-based macrophage isolation from Drosophila at various stages of development, there remains a need for more rapid and gentle methods to isolate living macrophages for downstream ex vivo applications. Here, we describe techniques for rapid and direct isolation of living macrophages from mature Drosophila pupae and their downstream ex vivo preparation for live imaging and immunostaining. This strategy enables straightforward access to physiologically relevant innate immune cells, both circulating and tissue-resident populations, for subsequent imaging of signal transduction.

Telomere length is longer following diapause in two solitary bee species.

The mechanisms that underlie senescence are not well understood in insects. Telomeres are conserved repetitive sequences at chromosome ends that protect DNA during replication. In many vertebrates, telomeres shorten during cell division and in response to stress and are often used as a cellular marker of senescence. However, little is known about telomere dynamics across the lifespan in invertebrates. We measured telomere length in larvae, prepupae, pupae, and adults of two species of solitary bees, Osmia lignaria and Megachile rotundata. Contrary to our predictions, telomere length was longer in later developmental stages in both O. lignaria and M. rotundata. Longer telomeres occurred after emergence from diapause, which is a physiological state with increased tolerance to stress. In O. lignaria, telomeres were longer in adults when they emerged following diapause. In M. rotundata, telomeres were longer in the pupal stage and subsequent adult stage, which occurs after prepupal diapause. In both species, telomere length did not change during the 8 months of diapause. Telomere length did not differ by mass similarly across species or sex. We also did not see a difference in telomere length after adult O. lignaria were exposed to a nutritional stress, nor did length change during their adult lifespan. Taken together, these results suggest that telomere dynamics in solitary bees differ from what is commonly reported in vertebrates and suggest that insect diapause may influence telomere dynamics.

Histological validation of in-vivo larvicidal efficacy of marine Streptomyces sp. RD06 secondary metabolites against filariasis causing Culex quinquefasciatus and statistical media optimization for larvicidal derivatives production.

Mosquito-borne disease pandemics, such as the Zika virus and chikungunya, have escalated cognizance of how critical it is to implement proficient mosquito vector control measures. The prevention of Culicidae is becoming more difficult these days because of the expeditious imminence of synthetic pesticide resistance and the universal expansion of tremendously invasive mosquito vectors. The present study highlights the insecticidal and larvicidal efficacy of the prospective novel actinobacterium derived from the marine Streptomyces sp. RD06 secondary metabolites against Culex quinquefasciatus mosquito. The pupicidal activity of Streptomyces sp. RD06 showed LC50=199.22 ± 11.54 and LC90= 591.84 ± 55.41 against the pupa. The purified bioactive metabolites 1, 2-Benzenedicarboxylic acid, diheptyl ester from Streptomyces sp. RD06 exhibited an LC50 value of 154.13 ± 10.50 and an LC90 value of 642.84 ± 74.61 tested against Cx. quinquefasciatus larvae. The Streptomyces sp. RD06 secondary metabolites exhibited 100 % non-hatchability at 62.5 ppm, and 82 % of hatchability was observed at 250 ppm. In addition, media optimization showed that the highest biomass production was attained at a temperature of 41.44 °C, pH 9.23, nitrogen source 11.43 mg/ml, and carbon source 150 mg/ml. Compared to control larvae, the histology and confocal microscopy results showed destruction to the anal gill, lumen content, and epithelial layer residues in the treated larvae. Utilizing an eco-friendly method, these alternative inventive insecticidal derivatives from Streptomyces sp. RD06 eradicates Culex quinquefasciatus. This study highlights the promising potential of these Streptomyces sp. RD06 secondary metabolites to develop affordable and efficacious mosquito larvicides to replace synthetic insecticides in the future.

Investigation of Fungal Community Structure in the Gut of the Stag Beetle Dorcus hopei (Coleoptera; Lucanidae): Comparisons Among Developmental Stages.

Stag beetles, recognized as common saproxylic insects, are valued for their vibrant coloration and distinctive morphology. These beetles play a crucial ecological role in decomposition and nutrient cycling, serving as a vital functional component in ecosystem functioning. Although previous studies have confirmed that stag beetles are predominantly fungivores, the fluctuations in their intestinal fungal communities at different developmental stages remain poorly understood. In the current study, high-throughput sequencing was employed to investigate the dynamic changes within intestinal fungal communities at various developmental stages in the stag beetle Dorcus hopei. Results showed that microbial diversity was higher during the larval stage than during the pupal and adult stages. Furthermore, significant differences were identified in the composition of the intestinal fungal communities across the larval, pupal, and adult stages, suggesting that developmental transitions may be crucial factors contributing to variations in fungal community composition and diversity. Dominant genera included Candida, Scheffersomyces, Phaeoacremonium, and Trichosporon. Functional predictions indicated a greater diversity and relative abundance of endosymbiotic fungi in the larval gut, suggesting a potential dependency of larvae on beneficial gut fungi for nutrient acquisition. Additionally, the application of abundance-based ß-null deviation and niche width analyses revealed that the adult gut exerted a stronger selection pressure on its fungal community, favoring certain taxa. This selection process culminates in a more robust co-occurrence network of fungal communities within the adult gut, thereby enhancing their adaptability to environmental fluctuations. This study advances our understanding of the intestinal fungal community structure in stag beetles, providing a crucial theoretical foundation for the development of saproxylic beetle resources, biomass energy utilization, plastic degradation strategies, and beetle conservation efforts.

Cyp6g2 is the major P450 epoxidase responsible for juvenile hormone biosynthesis in Drosophila melanogaster.

BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.

Effects of ivermectin on development of Calliphora vicina, Robineau-Desvoidy 1830 (Diptera, Calliphoridae).

Ivermectin is one of the most widely used drugs for parasite control. Previous studies have shown a reduction in the abundance and diversity of "non-target" coprophilous organisms due to the presence of ivermectin (IVM) in bovine faecal matter (FM). Due to its breadth of behavioural habits, Calliphora vicina is a suitable dipteran species to evaluate the effects of IVM in FM. The aim of this work was to evaluate the effect of five concentrations of IVM in FM (3000, 300, 100, 30, and 3 ng/g) on the development of C. vicina. The following endpoints were evaluated: survival (between the first larval stage and emergence of new adults), larval development times to pupation and pupation times to adult, and adult emergence (% sex) and LC50. Sampling was performed from larval hatching at 60 and 120 min and at 3, 4, 5, and 12 h, and every 24 h specimens were weighed until pupae were observed. Data were analysed by ANOVA using a non-parametric Kruskal-Wallis test and as a function of elapsed development time and accumulated degree hours (ADH). Mortality at 3000 and 300 ng/g was 100% and 97%, respectively. There were statistically significant delays in adult emergence time (p = 0.0216) and in the ADH (p = 0.0431) between the control group (C) and 100 ng/g. The LC50 was determined at 5.6 ng/g. These results demonstrate the lethal and sub-lethal effects of IVM on C. vicina, while highlighting the usefulness of this species as a bioindicator for ecotoxicological studies.

Micro-CT data of complete metamorphosis process in Harmonia axyridis.

Insect metamorphosis involves significant changes in insect internal structure and is thus a critical focus of entomological research. Investigating the morphological transformation of internal structures is vital to understanding the origins of adult insect organs. Beetles are among the most species-rich groups in insects, but the development and transformation of their internal organs have yet to be systematically documented. In this study, we have acquired a comprehensive dataset that includes 27 detailed whole-body tomographic image sets of Harmonia axyridis, spanning from the prepupal to the pupal stages. Utilizing this data, we have created intricate 3D models of key internal organs, encompassing the brain, ventral nerve cord, digestive and excretion systems, as well as the body wall muscles. These data documented the transformation process of these critical organs and correlations between the origin of adult and larval organs and can be used to enhance the understanding of holometabolous adult organ genesis and offers a valuable reference model for investigating complete metamorphosis in insects.

Upscaling irradiation protocols of Aedes albopictus pupae within an SIT program in Reunion Island.

The implementation of the sterile insect technique against Aedes albopictus relies on many parameters, in particular on the success of the sterilization of males to be released into the target area in overflooding numbers to mate with wild females. Achieving consistent sterility levels requires efficient and standardized irradiation protocols. Here, we assessed the effects of exposure environment, density of pupae, irradiation dose, quantity of water and location in the canister on the induced sterility of male pupae. We found that the irradiation of 2000 pupae in 130 ml of water and with a dose of 40 Gy was the best combination of factors to reliably sterilize male pupae with the specific irradiator used in our control program, allowing the sterilization of 14000 pupae per exposure cycle. The location in the canister had no effect on induced sterility. The results reported here allowed the standardization and optimization of irradiation protocols for a Sterile Insect Technique program to control Ae. albopictus on Reunion Island, which required the production of more than 300,000 sterile males per week.

Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

miR-306-5p is involved in chitin metabolism in Aedes albopictus pupae via linc8338-miR-306-5p-XM_019678125.2 axis.

Aedes albopictus can transmit several lethal arboviruses. This mosquito has become a sever public health threat due to its rapidly changing global distribution. Chitin, which is the major component of the cuticle and peritrophic membrane (PM), is crucial for the growth and development of insect. microRNAs (miRNAs) play important roles in the posttranscriptional level regulation of gene expression, thereby influencing many biological processes in insects. In this study, an attempt was made to evaluate the role of miR-306-5p in regulating chitin metabolism in Ae. albopictus pupae. Overexpression of miR-306-5p resulted in a significantly reduced survival rate in pupae and an increased malformation rate in adults. Both in vivo and in vitro evidence confirmed the presence of the competing endogenous RNA (ceRNA) regulatory axis (linc8338-miR-306-5p-XM_019678125.2). RNAi of linc8338 and XM_019678125.2 had effects on pupae similar to those of miR-306-5p. The highest expression level of miR-306-5p was found in the midgut, and alteration in the expression of miR-306-5p, XM_019678125.2 and linc8338 induced increased transcript levels of chitin synthase 2 (AaCHS2) and decreased chitinase 10 (AaCht10); as well as increased thickness of the midgut and enlarged midgut epithelial cells. The results of this study highlight the potential of miR-306-5p as a prospective target in mosquito control and confirm that the ceRNA mechanism is involved in chitin metabolism. These findings will provide a basis for further studies to uncover the molecular mechanisms through which ncRNAs regulate chitin metabolism.

Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Unique spatially and temporary-regulated/sex-specific expression of a long ncRNA, Nb-1, suggesting its pleiotropic functions associated with honey bee lifecycle.

Honey bees are social insects, and each colony member has unique morphological and physiological traits associated with their social tasks. Previously, we identified a long non-coding RNA from honey bees, termed Nb-1, whose expression in the brain decreases associated with the age-polyethism of workers and is detected in some neurosecretory cells and octopaminergic neurons, suggesting its role in the regulation of worker labor transition. Herein, we investigated its spatially and temporary-regulated/sex-specific expression. Nb-1 was expressed as an abundant maternal RNA during oogenesis and embryogenesis in both sexes. In addition, Nb-1 was expressed preferentially in the proliferating neuroblasts of the mushroom bodies (a higher-order center of the insect brain) in the pupal brains, suggesting its role in embryogenesis and mushroom body development. On the contrary, Nb-1 was expressed in a drone-specific manner in the pupal and adult retina, suggesting its role in the drone visual development and/or sense. Subcellular localization of Nb-1 in the brain during development differed depending on the cell type. Considering that Nb-1 is conserved only in Apidae, our findings suggest that Nb-1 potentially has pleiotropic functions in the expression of multiple developmental, behavioral, and physiological traits, which are closely associated with the honey bee lifecycle.

Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

Efficiency assessment of a novel automatic mosquito pupae sex separation system in support of area-wide male-based release strategies.

This study provides a comparative analysis of two state-of-the-art automatic mosquito pupae sex sorters currently available: the ORINNO and the WOLBAKI Biotech pupae sex separation systems, which both exploit the sexual size dimorphism of pupae. In Aedes aegypti, the WOLBAKI sex sorter and the ORINNO with a sieve mesh size of 1.050 mm achieved sex separation with female contamination rates below 1%, low pupae mortality rates and high male flight capacity. However, in Ae. albopictus, there was more variability, with female contamination rates above the 1% threshold and pupae mortality reaching 27% when using the ORINNO sorter. On the other hand, the WOLBAKI sorter achieved a male pupae recovery of 47.99 ± 8.81% and 50.91 ± 11.77% in Ae. aegypti and Ae. albopictus, respectively, while the ORINNO sorter with a smaller sieve size achieved male pupae recoveries of 38.08 ± 9.69% and 40.16 ± 2.73% in Ae. aegypti and Ae. albopictus, respectively. This study provides valuable information for researchers and practitioners in the field, assisting in the selection of the most suitable system for mosquito control, management and research programs depending on their specific requirements.

Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability.

BACKGROUND: The ability of animals to regenerate damaged tissue is a complex process that involves various cellular mechanisms. As animals age, they lose their regenerative abilities, making it essential to understand the underlying mechanisms that limit regenerative ability during aging. Drosophila melanogaster wing imaginal discs are epithelial structures that can regenerate after tissue injury. While significant research has focused on investigating regenerative responses during larval stages our comprehension of the regenerative potential of pupal wings and the underlying mechanisms contributing to the decline of regenerative responses remains limited. RESULTS: Here, we explore the temporal dynamics during pupal development of the proliferative response triggered by the induction of cell death, a typical regenerative response. Our results indicate that the apoptosis-induced proliferative response can continue until 34 h after puparium formation (APF), beyond this point cell death alone is not sufficient to induce a regenerative response. Under normal circumstances, cell proliferation ceases around 24 h APF. Interestingly, the failure of reinitiating the cell cycle beyond this time point is not attributed to an incapacity to activate the JNK pathway. Instead, our results suggest that the function of the ecdysone-responsive transcription factor E93 is involved in limiting the apoptosis-induced proliferative response during pupal development. CONCLUSIONS: Our study shows that apoptosis can prolong the proliferative period of cells in the wing during pupal development as late as 34 h APF, at least 10 h longer than during normal development. After this time point, the regenerative response is diminished, a process mediated in part by the ecdysone-responsive transcription factor E93.

Scuttle fly Megaselia scalaris (Loew) (Diptera: Phoridae) endoparasitoid as a novel biocontrol agent against adult American cockroaches (Periplaneta americana).

The American cockroach, Periplaneta americana (Linnaeus, 1758) (Blattodea: Blattidae), is one of the most common pests that thrive in diverse environments and carries various pathogens, causing critical threats to public health and the ecosystem. We thus report in this study the first observation of decapitated American cockroaches as a result of infestation with scuttle fly parasitoids. Interestingly, behavioral alterations in the form of zombification-like behavior could be observed in cockroaches reared in the laboratory before being decapitated, implying that the insect targets cockroach heads. To identify this parasitoid, cockroaches' corpora were isolated in jars, and apodous larvae were observed. Larvae developed into small coarctate pupae, and adults emerged. The scuttle flies were collected and exhibited tiny black, brown, to yellowish bodies. The fly was initially identified based on its morphological properties as a member of the order Diptera, family Phoridae. To provide further insights into the morphological attributes of the phorid species, the fly was examined using a scanning electron microscope (SEM) and then identified as Megaselia scalaris accordingly. SEM analysis revealed the distinctive structure of M. scalaris concerning the head, mouth parts, and legs. Specifically, the mouth parts include the labrum, labellum, rostrum, and maxillary palps. Although further investigations are still required to understand the complicated relationships between M. scalaris and American cockroaches, our findings provide a prominent step in the control of American cockroaches using M. scalaris as an efficient biological control agent.

Gross morphology and adhesion-associated physical properties of Drosophila larval salivary gland glue secretion.

One of the major functions of the larval salivary glands (SGs) of many Drosophila species is to produce a massive secretion during puparium formation. This so-called proteinaceous glue is exocytosed into the centrally located lumen, and subsequently expectorated, serving as an adhesive to attach the puparial case to a solid substrate during metamorphosis. Although this was first described almost 70 years ago, a detailed description of the morphology and mechanical properties of the glue is largely missing. Its main known physical property is that it is released as a watery liquid that quickly hardens into a solid cement. Here, we provide a detailed morphological and topological analysis of the solidified glue. We demonstrated that it forms a distinctive enamel-like plaque that is composed of a central fingerprint surrounded by a cascade of laterally layered terraces. The solidifying glue rapidly produces crystals of KCl on these alluvial-like terraces. Since the properties of the glue affect the adhesion of the puparium to its substrate, and so can influence the success of metamorphosis, we evaluated over 80 different materials for their ability to adhere to the glue to determine which properties favor strong adhesion. We found that the alkaline Sgs-glue adheres strongly to wettable and positively charged surfaces but not to neutral or negatively charged and hydrophobic surfaces. Puparia formed on unfavored materials can be removed easily without leaving fingerprints or cascading terraces. For successful adhesion of the Sgs-glue, the material surface must display a specific type of triboelectric charge. Interestingly, the expectorated glue can move upwards against gravity on the surface of freshly formed puparia via specific, unique and novel anatomical structures present in the puparial's lateral abdominal segments that we have named bidentia.

Antimicrobial peptide cecropin B functions in pathogen resistance of Mythimna separata.

Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

Hypoxia delays steroid-induced developmental maturation in Drosophila by suppressing EGF signaling.

Animals often grow and develop in unpredictable environments where factors like food availability, temperature, and oxygen levels can fluctuate dramatically. To ensure proper sexual maturation into adulthood, juvenile animals need to adapt their growth and developmental rates to these fluctuating environmental conditions. Failure to do so can result in impaired maturation and incorrect body size. Here we describe a mechanism by which Drosophila larvae adapt their development in low oxygen (hypoxia). During normal development, larvae grow and increase in mass until they reach critical weight (CW), after which point a neuroendocrine circuit triggers the production of the steroid hormone ecdysone from the prothoracic gland (PG), which promotes maturation to the pupal stage. However, when raised in hypoxia (5% oxygen), larvae slow their growth and delay their maturation to the pupal stage. We find that, although hypoxia delays the attainment of CW, the maturation delay occurs mainly because of hypoxia acting late in development to suppress ecdysone production. This suppression operates through a distinct mechanism from nutrient deprivation, occurs independently of HIF-1 alpha and does not involve dilp8 or modulation of Ptth, the main neuropeptide that initiates ecdysone production in the PG. Instead, we find that hypoxia lowers the expression of the EGF ligand, spitz, and that the delay in maturation occurs due to reduced EGFR/ERK signaling in the PG. Our study sheds light on how animals can adjust their development rate in response to changing oxygen levels in their environment. Given that hypoxia is a feature of both normal physiology and many diseases, our findings have important implications for understanding how low oxygen levels may impact animal development in both normal and pathological situations.

The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.