Mechanism of PINK1 activation by autophosphorylation and insights into assembly on the TOM complex.

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

Temporal control of RNAi reveals both robust and labile feedback loops in the segmentation clock of the red flour beetle.

Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.

Mapping the functional form of the trade-off between infection resistance and reproductive fitness under dysregulated immune signaling.

Immune responses benefit organismal fitness by clearing parasites but also exact costs associated with immunopathology and energetic investment. Hosts manage these costs by tightly regulating the induction of immune signaling to curtail excessive responses and restore homeostasis. Despite the theoretical importance of turning off the immune response to mitigate these costs, experimentally connecting variation in the negative regulation of immune responses to organismal fitness remains a frontier in evolutionary immunology. In this study, we used a dose-response approach to manipulate the RNAi-mediated knockdown efficiency of cactus (IκBα), a central regulator of Toll pathway signal transduction in flour beetles (Tribolium castaneum). By titrating cactus activity across four distinct levels, we derived the shape of the relationship between immune response investment and traits associated with host fitness, including infection susceptibility, lifespan, fecundity, body mass, and gut homeostasis. Cactus knock-down increased the overall magnitude of inducible immune responses and delayed their resolution in a dsRNA dose-dependent manner, promoting survival and resistance following bacterial infection. However, these benefits were counterbalanced by dsRNA dose-dependent costs to lifespan, fecundity, body mass, and gut integrity. Our results allowed us to move beyond the qualitative identification of a trade-off between immune investment and fitness to actually derive its functional form. This approach paves the way to quantitatively compare the evolution and impact of distinct regulatory elements on life-history trade-offs and fitness, filling a crucial gap in our conceptual and theoretical models of immune signaling network evolution and the maintenance of natural variation in immune systems.

NHA1 is a cation/proton antiporter essential for the water-conserving functions of the rectal complex in Tribolium castaneum.

More than half of all extant metazoan species on earth are insects. The evolutionary success of insects is linked with their ability to osmoregulate, suggesting that they have evolved unique physiological mechanisms to maintain water balance. In beetles (Coleoptera)-the largest group of insects-a specialized rectal ("cryptonephridial") complex has evolved that recovers water from the rectum destined for excretion and recycles it back to the body. However, the molecular mechanisms underpinning the remarkable water-conserving functions of this system are unknown. Here, we introduce a transcriptomic resource, BeetleAtlas.org, for the exceptionally desiccation-tolerant red flour beetle Tribolium castaneum, and demonstrate its utility by identifying a cation/H+ antiporter (NHA1) that is enriched and functionally significant in the Tribolium rectal complex. NHA1 localizes exclusively to a specialized cell type, the leptophragmata, in the distal region of the Malpighian tubules associated with the rectal complex. Computational modeling and electrophysiological characterization in Xenopus oocytes show that NHA1 acts as an electroneutral K+/H+ antiporter. Furthermore, genetic silencing of Nha1 dramatically increases excretory water loss and reduces organismal survival during desiccation stress, implying that NHA1 activity is essential for maintaining systemic water balance. Finally, we show that Tiptop, a conserved transcription factor, regulates NHA1 expression in leptophragmata and controls leptophragmata maturation, illuminating the developmental mechanism that establishes the functions of this cell. Together, our work provides insights into the molecular architecture underpinning the function of one of the most powerful water-conserving mechanisms in nature, the beetle rectal complex.

TGFß/activin-dependent activation of Torso controls the timing of the metamorphic transition in the red flour beetle Tribolium castaneum.

Understanding the mechanisms governing body size attainment during animal development is of paramount importance in biology. In insects, a crucial phase in determining body size occurs at the larva-pupa transition, marking the end of the larval growth period. Central to this process is the attainment of the threshold size (TS), a critical developmental checkpoint that must be reached before the larva can undergo metamorphosis. However, the intricate molecular mechanisms by which the TS orchestrates this transition remain poor understood. In this study, we investigate the role of the interaction between the Torso and TGFß/activin signaling pathways in regulating metamorphic timing in the red flour beetle, Tribolium castaneum. Our results show that Torso signaling is required specifically during the last larval instar and that its activation is mediated not only by the prothoracicotropic hormone (Tc-Ptth) but also by Trunk (Tc-Trk), another ligand of the Tc-Torso receptor. Interestingly, we show that while Tc-Torso activation by Tc-Ptth determines the onset of metamorphosis, Tc-Trk promotes growth during the last larval stage. In addition, we found that the expression of Tc-torso correlates with the attainment of the TS and the decay of juvenile hormone (JH) levels, at the onset of the last larval instar. Notably, our data reveal that activation of TGFß/activin signaling pathway at the TS is responsible for repressing the JH synthesis and inducing Tc-torso expression, initiating metamorphosis. Altogether, these findings shed light on the pivotal involvement of the Ptth/Trunk/Torso and TGFß/activin signaling pathways as critical regulatory components orchestrating the TS-driven metamorphic initiation, offering valuable insights into the mechanisms underlying body size determination in insects.

The BTB transcription factor, Abrupt, acts cooperatively with Chronologically inappropriate morphogenesis (Chinmo) to repress metamorphosis and promotes leg regeneration.

Many insects undergo the process of metamorphosis when larval precursor cells begin to differentiate to create the adult body. The larval precursor cells retain stem cell-like properties and contribute to the regenerative ability of larval appendages. Here we demonstrate that two Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (BTB) domain transcription factors, Chronologically inappropriate morphogenesis (Chinmo) and Abrupt (Ab), act cooperatively to repress metamorphosis in the flour beetle, Tribolium castaneum. Knockdown of chinmo led to precocious development of pupal legs and antennae. We show that although topical application of juvenile hormone (JH) prevents the decrease in chinmo expression in the final instar, chinmo and JH act in distinct pathways. Another gene encoding the BTB domain transcription factor, Ab, was also necessary for the suppression of broad (br) expression in T. castaneum in a chinmo RNAi background, and simultaneous knockdown of ab and chinmo led to the precocious onset of metamorphosis. Furthermore, knockdown of ab led to the loss of regenerative potential of larval legs independently of br. In contrast, chinmo knockdown larvae exhibited pupal leg regeneration when a larval leg was ablated. Taken together, our results show that both ab and chinmo are necessary for the maintenance of the larval tissue identity and, apart from its role in repressing br, ab acts as a crucial regulator of larval leg regeneration. Our findings indicate that BTB domain proteins interact in a complex manner to regulate larval and pupal tissue homeostasis.

The Hox gene Antennapedia is essential for wing development in insects.

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. We propose, therefore, that insect wing development occurs in an Antp-dependent manner. This article has an associated 'The people behind the papers' interview.

Chitin deacetylases are necessary for insect femur muscle attachment and mobility.

Muscle attachment sites (MASs, apodemes) in insects and other arthropods involve specialized epithelial cells, called tendon cells or tenocytes, that adhere to apical extracellular matrices containing chitin. Here, we have uncovered a function for chitin deacetylases (CDAs) in arthropod locomotion and muscle attachment using a double-stranded RNA-mediated gene-silencing approach targeted toward specific CDA isoforms in the red flour beetle, Tribolium castaneum (Tc). Depletion of TcCDA1 or the alternatively spliced TcCDA2 isoform, TcCDA2a, resulted in internal tendon cuticle breakage at the femur-tibia joint, muscle detachment from both internal and external tendon cells, and defective locomotion. TcCDA deficiency did not affect early muscle development and myofiber growth toward the cuticular MASs but instead resulted in aborted microtubule development, loss of hemiadherens junctions, and abnormal morphology of tendon cells, all features consistent with a loss of tension within and between cells. Moreover, simultaneous depletion of TcCDA1 or TcCDA2a and the zona pellucida domain protein, TcDumpy, prevented the internal tendon cuticle break, further supporting a role for force-dependent interactions between muscle and tendon cells. We propose that in T. castaneum, the absence of N-acetylglucosamine deacetylation within chitin leads to a loss of microtubule organization and reduced membrane contacts at MASs in the femur, which adversely affect musculoskeletal connectivity, force transmission, and physical mobility.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

Shaking hands is a homeodomain transcription factor that controls axon outgrowth of central complex neurons in the insect model Tribolium.

Gene regulatory mechanisms that specify subtype identity of central complex (CX) neurons are the subject of intense investigation. The CX is a compartment within the brain common to all insect species and functions as a 'command center' that directs motor actions. It is made up of several thousand neurons, with more than 60 morphologically distinct identities. Accordingly, transcriptional programs must effect the specification of at least as many neuronal subtypes. We demonstrate a role for the transcription factor Shaking hands (Skh) in the specification of embryonic CX neurons in Tribolium. The developmental dynamics of skh expression are characteristic of terminal selectors of subtype identity. In the embryonic brain, skh expression is restricted to a subset of neurons, many of which survive to adulthood and contribute to the mature CX. skh expression is maintained throughout the lifetime in at least some CX neurons. skh knockdown results in axon outgrowth defects, thus preventing the formation of an embryonic CX primordium. The previously unstudied Drosophila skh shows a similar embryonic expression pattern, suggesting that subtype specification of CX neurons may be conserved.

The neuroblast timer gene nubbin exhibits functional redundancy with gap genes to regulate segment identity in Tribolium.

The neuroblast timer genes hunchback, Krüppel, nubbin and castor are expressed in temporal sequence in neural stem cells, and in corresponding spatial sequence along the Drosophila blastoderm. As canonical gap genes, hunchback and Krüppel play a crucial role in insect segmentation, but the roles of nubbin and castor in this process remain ambiguous. We have investigated the expression and functions of nubbin and castor during segmentation in the beetle Tribolium. We show that Tc-hunchback, Tc-Krüppel, Tc-nubbin and Tc-castor are expressed sequentially in the segment addition zone, and that Tc-nubbin regulates segment identity redundantly with two previously described gap/gap-like genes, Tc-giant and Tc-knirps. Simultaneous knockdown of Tc-nubbin, Tc-giant and Tc-knirps results in the formation of ectopic legs on abdominal segments. This homeotic transformation is caused by loss of abdominal Hox gene expression, likely due to expanded Tc-Krüppel expression. Our findings support the theory that the neuroblast timer series was co-opted for use in insect segment patterning, and contribute to our growing understanding of the evolution and function of the gap gene network outside of Drosophila.

Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis.

In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.

Life history correlations and trade-offs resulting from selection for dispersal in Tribolium castaneum.

Dispersal is an important facet of the life history of many organisms and is, therefore, subject to selective pressure but does not evolve in isolation. Across nature, there are examples of dispersal syndromes and life history strategies in which suites of traits coevolve and covary with dispersal in combinations that serve to maximize fitness in a given ecological context. The red rust flour beetle, Tribolium castaneum, is a model organism and globally significant post-harvest pest that relies on dispersal to reach new patches of ephemeral habitat. Dispersal behaviour in Tribolium has a strong genetic basis. However, a robust understanding of the relationship between dispersal and other life-history components, which could elucidate evolutionary processes and allow pest managers to control their spread and reduce the impact of infestation, is currently lacking. Here, we use highly replicated lines of T. castaneum previously artificially selected for divergent small-scale dispersal propensity to robustly test several important life history components: reproductive strategy, development time, and longevity. As predicted, we find that a suite of important changes as a result of our selection on dispersal: high dispersal propensity is associated with a lower number of longer mating attempts by males, lower investment in early life reproduction by females, slower development of later-laid offspring, and longer female life span. These findings indicate that correlated intraspecific variation in dispersal and related traits may represent alternative life history strategies in T. castaneum. We therefore suggest that pest management efforts to mitigate the species' agro-economic impact should consider the eco-evolutionary dynamics within multiple life histories. The benefits of doing so could be felt both through improved targeting of efforts to reduce spread and also in forecasting how the selection pressures applied through pest management are likely to affect pest evolution.

Immigration delays but does not prevent adaptation following environmental change: experimental evidence.

In today's rapidly changing world, it is critical to examine how animal populations will respond to severe environmental change. Following events such as pollution or deforestation that cause populations to decline, extinction will occur unless populations can adapt in response to natural selection, a process called evolutionary rescue. Theory predicts that immigration can delay extinction and provide novel genetic material that can prevent inbreeding depression and facilitate adaptation. However, when potential source populations have not experienced the new environment before (i.e., are naive), immigration can counteract selection and constrain adaptation. This study evaluated the effects of immigration of naive individuals on evolutionary rescue using the red flour beetle, Tribolium castaneum, as a model system. Small populations were exposed to a challenging environment, and 3 immigration rates (0, 1, or 5 migrants per generation) were implemented with migrants from a benign environment. Following an initial decline in population size across all treatments, populations receiving no immigration gained a higher growth rate one generation earlier than those with immigration, illustrating the constraining effects of immigration on adaptation. After 7 generations, a reciprocal transplant experiment found evidence for adaptation regardless of immigration rate. Thus, while the immigration of naive individuals briefly delayed adaptation, it did not increase extinction risk or prevent adaptation following environmental change.

A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

Prediction and analysis of cis-regulatory elements in Dorsal and Ventral patterning genes of Tribolium castaneum and its comparison with Drosophila melanogaster.

Insect embryonic development and morphology are characterized by their anterior-posterior and dorsal-ventral (DV) patterning. In Drosophila embryos, DV patterning is mediated by a dorsal protein gradient which activates twist and snail proteins, the important regulators of DV patterning. To activate or repress gene expression, some regulatory proteins bind in clusters to their target gene at sites known as cis-regulatory elements or enhancers. To understand how variations in gene expression in different lineages might lead to different phenotypes, it is necessary to understand enhancers and their evolution. Drosophila melanogaster has been widely studied to understand the interactions between transcription factors and the transcription factor binding sites. Tribolium castaneum is an upcoming model animal which is catching the interest of biologists and the research on the enhancer mechanisms in the insect's axes patterning is still in infancy. Therefore, the current study was designed to compare the enhancers of DV patterning in the two insect species. The sequences of ten proteins involved in DV patterning of D. melanogaster were obtained from Flybase. The protein sequences of T. castaneum orthologous to those obtained from D. melanogaster were acquired from NCBI BLAST, and these were then converted to DNA sequences which were modified by adding 20 kb sequences both upstream and downstream to the gene. These modified sequences were used for further analysis. Bioinformatics tools (Cluster-Buster and MCAST) were used to search for clusters of binding sites (enhancers) in the modified DV genes. The results obtained showed that the transcription factors in Drosophila melanogaster and Tribolium castaneum are nearly identical; however, the number of binding sites varies between the two species, indicating transcription factor binding site evolution, as predicted by two different computational tools. It was observed that dorsal, twist, snail, zelda, and Supressor of Hairless are the transcription factors responsible for the regulation of DV patterning in the two insect species.

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT.

The maturation of RAS GTPases and approximately 200 other cellular CAAX proteins involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CAAX motif, proteolytic cleavage of the AAX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate. This final step is catalysed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryote-specific integral membrane enzyme that resides in the endoplasmic reticulum. ICMT is the only cellular enzyme that is known to methylate prenylcysteine substrates; methylation is important for the biological functions of these substrates, such as the membrane localization and subsequent activity of RAS, prelamin A and RAB. Inhibition of ICMT has potential for combating progeria and cancer. Here we present an X-ray structure of ICMT, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor, at 2.3 Å resolution. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for the cytosolic methyl donor, S-adenosyl-l-methionine, and for prenylcysteine substrates, which are associated with the endoplasmic reticulum membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localizations together in a membrane environment-a relatively uncharacterized but defining feature of many integral membrane enzymes.

Boron compounds are effective on Tribolium castaneum (Coleoptera: Tenebrionidae): Reduced lipogenesis and induced body weight loss.

In the current study, we investigated the insecticidal efficacy of two borates, disodium octaborate tetrahydrate (Etidot-67) and calcium metaborate (CMB) via surface application or diet delivery on the red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae). The application method did not change the boron-related mortality, but CMB was more effective than Etidot-67. At the highest dose, it took around 13 days to reach the highest mortality (≥98.1%) for CMB, while it was 19 days for Etidot-67 (≥95.8%). Both boron compounds led to a significant reduction in triglyceride levels in parallel to the downregulation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), the two primary genes involved in de novo lipogenesis, while they also induced body weight loss. In conclusion, the current study indicated the insecticidal potential of boron compounds but CMB is more promising and more effective in controlling T. castaneum, while lipogenesis is inhibited and weight loss is induced by boron compounds.

The zona pellucida protein piopio regulates the metamorphosis and reproduction in Tribolium castaneum.

The zona pellucida domain protein piopio (Pio) was only reported to mediate the adhesion of the apical epithelial surface and the overlying apical extracellular matrix in Drosophila melanogaster, but the developmental roles of Pio were poorly understood in insects. To address this issue, we comprehensively analyzed the function of Pio in Tribolium castaneum. Phylogenetic analysis indicated that pio exhibited one-to-one orthologous relationship among insects. T. castaneum pio had a 1236-bp ORF and contained eight exons. During development pio was abundantly expressed from larva to adult and lowly expressed at the late stage of embryo and adult, while it had more transcripts in the head, epidermis, and gut but fewer in the fat body of late-stage larvae. Knockdown of pio inhibited the pupation, eclosion, and reproduction of T. castaneum. The expression of vitellogenin 1 (Vg1), Vg2, and Vg receptor (VgR) largely decreased in pio-silenced female adults. Silencing pio increased the 20-hydroxyecdysone titer by upregulating phm and spo expression but decreased the juvenile hormone (JH) titer through downregulating JHAMT3 and promoting JHE, JHEH-r4, and JHDK transcription. These results suggested that Pio might regulate the metamorphosis and reproduction via modulating the ecdysone and JH metabolism in T. castaneum. This study found the novel roles of pio in insect metamorphosis and reproduction, and provided the new insights for analyzing other zona pellucida proteins functions in insects.

A unique Malpighian tubule architecture in Tribolium castaneum informs the evolutionary origins of systemic osmoregulation in beetles.

Maintaining internal salt and water balance in response to fluctuating external conditions is essential for animal survival. This is particularly true for insects as their high surface-to-volume ratio makes them highly susceptible to osmotic stress. However, the cellular and hormonal mechanisms that mediate the systemic control of osmotic homeostasis in beetles (Coleoptera), the largest group of insects, remain largely unidentified. Here, we demonstrate that eight neurons in the brain of the red flour beetle Tribolium castaneum respond to internal changes in osmolality by releasing diuretic hormone (DH) 37 and DH47-homologs of vertebrate corticotropin-releasing factor (CRF) hormones-to control systemic water balance. Knockdown of the gene encoding the two hormones (Urinate, Urn8) reduces Malpighian tubule secretion and restricts organismal fluid loss, whereas injection of DH37 or DH47 reverses these phenotypes. We further identify a CRF-like receptor, Urinate receptor (Urn8R), which is exclusively expressed in a functionally unique secondary cell in the beetle tubules, as underlying this response. Activation of Urn8R increases K+ secretion, creating a lumen-positive transepithelial potential that drives fluid secretion. Together, these data show that beetle Malpighian tubules operate by a fundamentally different mechanism than those of other insects. Finally, we adopt a fluorescent labeling strategy to identify the evolutionary origin of this unusual tubule architecture, revealing that it evolved in the last common ancestor of the higher beetle families. Our work thus uncovers an important homeostatic program that is key to maintaining osmotic control in beetles, which evolved parallel to the radiation of the "advanced" beetle lineages.