Insect Biochemistry and Molecular Biology

Mosquitoes transmit diverse pathogens through repeated blood feeding, a process that subjects the midgut to significant mechanical stress and cellular damage. While hemocyte association with the midgut is known to occur following injury, the mechanisms promoting their recruitment remain poorly defined. Given the conserved roles of eicosanoid signaling in injury responses, we hypothesized that thromboxane A2 (TXA2) mediates hemocyte recruitment to the damaged midgut. Here, we demonstrate that chemically induced midgut damage significantly increases both the number of hemocytes attached to the midgut and those in circulation. Pharmacological inhibition of cyclooxygenase suppresses hemocyte recruitment whereas supplementation with a stable TXA2 analog restores the response, indicating that TXA2 signaling is required for this process. To identify candidate enzymes involved in TXA2 biosynthesis, we performed in silico docking analyses and identified two cytochrome P450 (CYP) candidates. Among these, CYP6D3 was shown to be strongly upregulated in hemocyte populations following midgut damage. RNA interference-mediated knockdown of CYP6D3 significantly reduced both hemocyte recruitment to the midgut and systemic TXB2 levels, supporting its role in the eicosanoid-mediated immune response. Together, our findings demonstrate that TXA2 signaling drives hemocyte recruitment to the damaged mosquito midgut and suggest a conserved lipid-mediated mechanism underlying insect tissue-associated immune responses that may influence vector competence.

The kynurenine pathway metabolizes tryptophan into 3-hydroxykynurenine (3-HK), a precursor for ommochrome eye pigments synthesized via the cardinal (cd) gene in mosquitoes. While cd disruption was presumed neutral, we observed fitness costs in Anopheles stephensi knock-in but not knock-out cd mutants. Here we investigated this anomaly further by assessing survival, fecundity, and midgut integrity across multiple cd mutant lines. Heterozygous knock-in lines, expressing a fluorescent marker and guide RNA for CRISPR/Cas9, exhibited reduced survival post-blood feeding, larva-to-adult survival deficits, and midgut barrier dysfunction, whereas knock-outs showed no such costs. Oral supplementation with xanthurenic acid partially rescued knock-in mortality, implicating oxidative stress linked to 3-HK metabolism. Expression analyses suggest transgene insertion effects, rather than cd disruption, underlie these fitness costs. These findings highlight the importance of evaluating insertional effects in gene drive target selection and support cd as a viable target for genetic control strategies in An. stephensi.

Sleep is a ubiquitous, yet highly variable, behavior across species. The duration and timing of sleep are influenced by ecological demands and dietary context. In the mosquito Aedes aegypti, a blood-feeding insect with specialized nutritional requirements, the relationship between feeding and sleep remains poorly understood. Here, we investigated how blood-derived dietary protein influences sleep regulation. Using postural analysis, videography, and arousal-threshold assays, we established that immobility bouts of greater than 10 minutes reliably define sleep in Ae. aegypti. Mosquitoes lacking the circadian clock gene cycle still maintained daily sleep rhythms but exhibited reduced sleep duration and heightened overall activity. Infrared activity monitoring revealed that blood-fed females showed a marked increase in sleep beginning immediately after feeding and persisting for several days, accompanied by reduced locomotor activity. Notably, this sleep elevation lasted well beyond the cessation of previously reported host-seeking phases, raising the possibility of distinct phases of opportunistic versus targeted host pursuit. To determine the dietary basis of this effect, we tested mosquitoes fed a bovine serum albumin (BSA)-based diet. BSA feeding alone was sufficient to mimic the sleep-promoting and activity-reducing effects of blood, suggesting dietary protein is a major nutritional regulator. Moreover, RNAi-mediated knockdown of the leucokinin receptor (Lkr), which has previously been associated with fluid homeostasis and feeding behavior, resulted in enhanced sleep and reduced activity, implicating mosquito LK signaling in the modulation of postprandial sleep. Together, these findings demonstrate that blood-derived proteins drive sustained increases in sleep and reductions in locomotor activity in Ae. aegypti. This work positions Ae. aegypti as a model for dissecting nutrient-specific regulation of sleep and highlights potential adaptive functions of protein-induced quiescence, such as energy conservation and predator avoidance. More broadly, it provides insight into how specialized diets shape the neural and behavioral architecture of sleep.

The yellow fever mosquito, Aedes aegypti, is a vector of Zika, Dengue, Chikungunya and yellow fever, disease-causing viruses impacting millions of people annually across the globe. Female mosquitoes transmit pathogens through serial blood-feeding, while both male and female mosquitoes feed on plant resources. Contact chemosensation (taste) guides these two feeding modes in both positive ways, feeding readily on sugar in nectar or ATP in blood, and negative ways, demonstrating aversion to chemically diverse bitter compounds in blood or sugar meals. Here, we identify a receptor, AaegGr14, that is expressed in neurons in the labellum and cibarium of the mosquito. Activation of Gr14-expressing neurons causes reduced feeding, while Gr14 and Gr14-expressing GRNs are required for aversion to bitter compounds during nectar- but not blood-feeding. This work provides a molecular and cellular on-ramp to understand bitter taste in mosquitoes and establishes that there are feeding context-dependent differences in how taste mechanisms influence blood- and nectar-feeding. Understanding the molecular and cellular basis of bitter taste in mosquitoes during nectar- and blood-feeding will help inform vector control strategies and elucidate shared principles and unique aspects of insect taste systems.

Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to pesticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94-99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including a splice-junction target site. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes, likely a consequence of reduced cleavage efficiency due to inefficient gRNA production. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings highlight that the approach used for multi-gRNA expression, target site choice and sex-specific germline environments can impact gene drive efficiency, and emphasize the need to optimize construct design within the biological context of the target species.

The electrophysiological process of auditory transduction in insects remains largely conjecture due to the unknown role of ion channels localised to the cilia, but experimental evidence supports either NompC or Nan-Iav as the auditory mechanotransduction ion channel. Here, we knocked down two key genes that code for the two candidate sound-activated ion channels using dsRNA-mediated RNA interference. We measured sound-evoked activity of the auditory nerve and intracellular electrical currents from the ciliated ending of individual auditory receptors. We found that the sound-evoked nerve activity was reduced in nompC, nan and ift88 knockdown. Using whole-cell patch clamp recordings we found that nompC and nan knockdown resulted in reduced sound-evoked transduction current. Stochastic depolarisations hypothesised to be mediated from one of the candidate mechanotransduction ion channels, either NompC or Nan-Iav, where not affected by knockdown of either channel. The discrete depolarisations are therefore mediated through another unidentified ion channel. We test the hypothesis that discrete depolarisations are graded action potentials that travel toward the soma through noise analysis of the transduction current and analysis of discrete depolarisations to voltage-steps. As a positive control we also knocked down ift88, a protein essential for transporting proteins, including ion channels, along the cilium and found both the transduction current and the discrete depolarisations decreased. Key pointsO_LIInjection of dsRNA decreased RNA of nompC and nan C_LIO_LISound-evoked nerve activity is reduced for RNAi-mediated knockdown of nompC and nan C_LIO_LINompC and Nan both contribute to the transduction current C_LIO_LIThe stochastic discrete depolarisations are not due to NompC or Nan-Iav ion channel but to a third unidentified ion channel. C_LIO_LINoise analysis of the transduction current and the discrete depolarisations suggests they are graded action potentials that travel in the direction of the soma. C_LIO_LIKnockdown of ift88 reduced both the transduction current and discrete depolarisations. C_LI Significance StatementInsects are important to understand, economically, agriculturally and medically. However, we still do not understand fundamental aspects of how insects detect their own body movements, vibrations and sound. These senses are detected by insect chordotonal organs, specialised miniaturised mechanoreceptors that convert movements into electrical signals through specialised ion channels. Previous experimental work has advocated either NompC or Nan-Iav as the mechanosensitive ion channel. Here, for the first time, we reduced the expression of both nompC and nan and measured the sound-evoked transduction current directly from neurons in a specialised auditory chordotonal organ. In contradiction to previous studies, we show that both ion channels contribute to the transduction current and find that electrical signals termed "discrete depolarisations" travel toward the soma.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.

BACKGROUNDThe soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a pest of soybean in North America that can cause significant yield loss when outbreaks are not managed. Current management tactics primarily rely on inexpensive pyrethroids, but the sustainability of this option is threatened by insecticide-resistance in A. glycines populations across the Upper-Midwest United States. Field-evolved resistance is associated with mutations in the voltage-gated sodium channel subunit h1 (vgsc-h1) gene. RESULTSFour double-stranded RNA (dsRNA) molecules, each matching the sequence of a vgsc-h1 transcript variant ("Specific dsRNAs"), were topically applied to aphids with a genotype carrying the corresponding allele. The mortality of pyrethroid resistant aphids exposed to a Specific dsRNA increased in a dose-dependent manner when applied alone or with a constant concentration of lambda-cyhalothrin, plateauing at 1000 ng ul-1. Synergism was detected between two of four combinations of the Specific dsRNAs and lambda-cyhalothrin. These results were mirrored by the topical application of a single dsRNA with the consensus sequence of all vgsc-h1 variants ("Combined dsRNA"). Mortality was consistently higher in aphids treated with either Specific dsRNA or the Combined dsRNA, alone or with lambda-cyhalothrin, compared to insecticide alone. The number of nymphs produced per female treated with the Specific or Combined dsRNA alone decreased significantly compared to untreated controls. CONCLUSIONThis study demonstrates that the topical application of dsRNAs targeting vgsc-h1 increases the susceptibility and reduces the reproductive capacity of pyrethroid resistant soybean aphids, potentially providing a novel tool for the management of insecticide-resistant aphid populations.

Redox balance is central to aerobic metabolism, yet acute heat stress can destabilize this balance by increasing metabolic rates and shifting the balance of critical electron carriers such as NADH. In early Drosophila melanogaster embryos, maintaining redox balance is particularly critical as embryos undergo a developmental redox shift and rely on oxidative phosphorylation to power nuclear divisions. Here, we assayed six isofemale D. melanogaster lines from temperate (Vermont, USA; France; Japan) and tropical (St. Kitts; Ghana; India) climates to assess metabolic responses to heat in heat-sensitive versus heat-tolerant embryos. We used untargeted LC--MS to measure 33 metabolites and the major redox couples (NADH/NAD+, NADPH/NADP+, and GSH/GSSG) at 25{degrees}C and after a 32{degrees}C heat shock. In all embryos, heat shock induced shared shifts in metabolic profiles, with increases in nucleotide monophosphates (e.g., AMP, CMP, and GMP) and amino acids (e.g., alanine, glutamic acid, serine). In contrast, redox metabolites diverged by region: heat-sensitive temperate embryos shifted toward a more oxidized state (46.6% decrease in NADH/NAD+ ratio and 4-fold increase in oxidized glutathione), while heat-tolerant tropical embryos maintained glutathione balance and increased the NADH/NAD+ ratio by 52.9%, indicating a more reduced state. These patterns are consistent with higher NADH oxidation and greater oxidative stress (inferred from oxidized glutathione) in the temperate embryos, versus better maintenance of redox balance in tropical embryos. Together, our results suggest that maintaining redox balance is a key determinant of acute heat tolerance, and healthy development overall, during early embryogenesis.

Exercise is a known remedy for metabolic disorders, including obesity and type II diabetes. In humans and some model organisms, exercise is classified into different types and intensities based on the mode of exercise and the devices used. Since Drosophila has emerged as a model for exercise research, several devices have been developed for exercise training. These devices vary in mechanisms of operation; for example, the Power Tower (PT) uses a repeated drop-and-hit mechanism, and the TreadWheel (TW) uses end-over-end rotation. This variation in mechanisms that trigger the negative geotaxis of flies could impact exercise outcomes, and it remains unclear how this variation drives the response of flies to exercise training. Stress is a known response to exercise in humans and mice. Stress in Drosophila can be induced by various conditions, including heat, cold, chemical exposure, and other environmental stressors, but there is little information on exercise-induced stress. It is also unclear whether different devices elicit distinct stress responses or whether the exercise-induced stress response is similar to other stress responses (e.g., thermal, chemical). To explore the effects of device, sex, and genotype on multiple metabolic pathways, we used an untargeted metabolomics approach to further elucidate the molecular mechanisms of exercise. As no studies have directly examined the exercise metabolome of Drosophila, this study is the first to explore the metabolome of flies post-exercise. Our results revealed that exercising on different devices elicits varying responses and reshapes the metabolome of flies. We also identified specific small compounds that may be linked to stress and inflammatory response more in the PT-exercised flies than in the TW-exercised flies. Overall, the exercise device effect interacted with sex effect and genotype, such that male and female flies have different metabolite compositions post-exercise on TW and PT.

Alternaria fungi produce various structurally diverse mycotoxins, several of which exhibit immunomodulatory properties. Among these, alternariol monomethyl ether (AME), alternariol (AOH), alterperylenol (ALTP), altertoxin I (ATX-I), and altersetin (AST) have been reported to suppress lipopolysaccharide (LPS)-induced inflammatory responses. However, the precise molecular mechanisms underlying these effects remain unclear. The present study aimed to elucidate how these selected Alternaria mycotoxins (0.1-50 M) target the NF-{kappa}B signaling pathway in THP-1 monocytes. Key components of the NF-{kappa}B cascade were analyzed by immunofluorescence microscopy, Western blotting and qRT-PCR. Nuclear translocation of NF-{kappa}B p65 and its phosphorylated form (p- NF-{kappa}B p65) was assessed by Western blot, while cytokine responses were determined at transcript (qRT-PCR) and protein (ELISA) levels. Moreover, in silico docking analyses were performed to investigate potential interactions of the toxins with IKK{beta}, and receptor-mediated crosstalk was studied using the glucocorticoid receptor (GR) antagonist RU486. Co-treatment with RU486 attenuated the immunosuppressive effects of 1 and 5 M AOH, indicating partial involvement of GR-dependent mechanisms. AME, AOH, ALTP, ATX-I, and AST increased total I{kappa}B levels while reducing its phosphorylated form. Additionally, AST and ALTP decreased the protein levels of Toll-like receptor 4 (TLR4), the I{kappa}B kinase (IKK) complex, NF-{kappa}B p65, and p- NF-{kappa}B p65. While AOH (5 M) and AST (25 M) reduced nuclear translocation of p65 and p-p65, ALTP (2 M) enhanced nuclear localization despite decreasing cytokine expression. Together, these findings suggest toxin-specific interference at multiple regulatory levels of NF-{kappa}B signaling and provide novel mechanistic insight into the immunomodulatory effects of Alternaria mycotoxins.

TULIP (TUbular LIPid binding) domain proteins (TDPs) are found in all living organisms including bacteria. They have various documented functions, some of which clearly related to their intra- or extracellular lipid transfer activities. Extracellular, BPI-related TDPs of insects (B-TDPs, also known as Takeout-related proteins), are often found in chemosensory organs, but little is known regarding their exact location or how they could contribute to olfaction or gustation. We have surveyed and updated the full set of Drosophila B-TDPs and found that roughly 50% are overexpressed in chemosensory organs. Focusing on three genes clustered on the third chromosome, we provide evidence that at least one of the encoded proteins is secreted in the lymph cavity housing the dendrites of olfactory neurons. Biochemical data give support for a putative function of B-TDPs as odorant transporters, but loss-of-function analyses also hint to a potential role as a barrier against plant-emitted terpenoids.

The interaction between volatile organic compounds (VOCs) and odorant receptors (ORs), termed VOI, constitutes the molecular basis for species to recognize chemical information in the environment. Characterization of subtle differences in VOI relationships among closely related species is crucial for understanding the rapid evolution of ORs and for identifying species-specific chemical cues relevant to pest management. However, progress in this field has been constrained by both the scarcity of experimental data and limitations in computational prediction accuracy. To address this, we developed a virtual screening-enhanced transfer learning strategy that integrates large-scale molecular docking data with sparse functional experimental data. The resulting VOI prediction model was validated through functional experiments on the pest species Bactrocera dorsalis, demonstrating its cross-species predictive capacity. Using this model, we investigated the relationship between ecological niches and olfactory sensitivity in Diptera insects from multiple perspectives, with an emphasis on patterns that may inform pest behavioral research. The model's predictive reliability was further supported by its consistency with known olfactory trends: it recapitulated the preferential responses of fruit flies and mosquitoes to esters and aromatics, respectively, and reproduced the previously reported negative correlation between olfactory and visual capacities in Drosophila. As a proof-of-concept application, we compared hematophagous and non-hematophagous mosquitoes, revealing overlapping chemical spaces but distinct patterns in their specifically recognized compounds. This study provides a reliable VOI prediction framework that reveals species-level olfactory preference patterns in Diptera, offering a computational pipeline to accelerate the discovery of behaviorally active compounds for species-specific pest monitoring and control.

Receptor tyrosine kinases (RTKs) are cell surface proteins that govern many critical cell fate decisions and their dysregulation is a major cause of diseases such as cancer. Much of what we know about how these proteins work in cells and tissues comes from model organisms such as the fruit fly Drosophila. Here, we identify and characterise a previously unstudied Drosophila receptor tyrosine kinase encoded by CG3277, which we name beanbag (beba). Ectopic beba expression activated Akt and ERK phosphorylation and produced gain-of-function phenotypes resembling those caused by other Drosophila RTKs. Using a MiMIC-derived T2A-GAL4 allele, we show that Drosophila beba is expressed in digestive, nervous and reproductive systems, in locations suggestive of potential roles in endoreplication and/or stem cell niche support. Animals transheterozygous for beba loss-of-function alleles were viable, developed at a normal rate, and showed no detectable change in enterocyte DNA content under standard conditions. However, beba loss-of-function females had fewer ovarioles, consistent with a role in the ovarian terminal filament, and males had increased testis hub cell number and hub volume, suggesting beba may regulate somatic niche architecture in the Drosophila gonad. Phylogenetic analysis places Beba within a Ret/Tor-related RTK radiation and supports the existence of a distinct Beba family in insects. Together, our data define Beba as a lineage-restricted Drosophila RTK with specialised roles in reproductive niche organisation.

Homing endonuclease genes spread by cleaving homologous chromosomes that lack the endonuclease cassette, after which repair from the endonuclease-containing chromosome converts the cut allele into a copy of the drive allele. This mechanism has provided a conceptual foundation for synthetic gene drive systems, including CRISPR-based drives, that represent promising strategies for the genetic control of insect pests. However gene drive performance depends critically on the repair pathways available in the germline of the target organism. Here, we report a set of transgenic assays originally developed as part of an attempt to establish gene targeting in the malaria mosquito Anopheles gambiae using an in vivo-generated linear targeting molecule. Although the intended FLP-mediated excision step was not achieved in the mosquito germline, analysis of the component strains revealed efficient germline activity of the rare-cutting homing endonuclease I-SceI and a striking bias towards homology-based repair of I-SceI-induced double-strand breaks. Across reporter and donor configurations, cleavage outcomes were dominated by single-strand annealing, microhomology-mediated repair, synthesis-dependent strand annealing and gene conversion-like events, with comparatively limited evidence for classical non-homologous end joining. In reciprocal crosses designed to distinguish gene conversion from gamete loss, I-SceI cleavage also produced inheritance distortion consistent with both conversion of the cleaved allele and reduced recovery of gametes carrying extensively damaged donor alleles. These findings indicate that the An. gambiae germline can strongly favour homology-dependent repair following homing endonuclease cleavage and that cleavage can also generate meiotic drive-like distortion through selective loss of damaged gametes. The results have direct relevance for the design and interpretation of homing endonuclease and CRISPR-based gene drives in malaria mosquitoes, where the balance between homology-directed repair, end joining and gamete viability will determine drive efficiency, resistance formation and transmission bias. Author summaryGene drives depend on a simple but demanding principle: a nuclease cuts one chromosome, and the cell repairs the break using the homologous chromosome as a template, copying the drive element in the process. Before CRISPR, this type of system was explored using naturally occurring homing endonucleases such as I-SceI. We attempted to develop a gene targeting system in Anopheles gambiae based on the Rong and Golic strategy, in which FLP recombinase would excise a donor molecule and I-SceI would linearise it to stimulate recombination. The full knockout technology did not work because FLP-mediated excision was not detected in the mosquito germline. However, the component tests revealed something more broadly important: I-SceI-induced breaks were repaired predominantly through homology-based pathways rather than simple end joining. We also observed inheritance distortion consistent with both gene conversion and loss of damaged gametes. These results help explain why homing-based systems can work in mosquitoes, while also highlighting why repair pathway choice and gamete viability need to be measured directly in any new drive configuration.

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

Tenuazonic acid (TeA) is one of the most frequently detected Alternaria mycotoxins in contaminated food. Despite its frequent occurrence, its immunomodulatory effects remain insufficiently characterized. Therefore, the present study investigated the impact of TeA on inflammatory signaling and cytokine regulation in monocytes and intestinal epithelial cell (IEC) models. NF-{kappa}B activity was assessed using a reporter gene assay in THP1-Lucia monocytes, while cytokine mRNA expression and protein secretion were quantified in Caco-2 and HCEC-1CT cells by qRT-PCR and ELISA, respectively. In THP-1 monocytes, TeA significantly suppressed lipopolysaccharide (LPS)-induced NF-{kappa}B activation in a concentration-dependent manner starting at 25 M, while cytotoxicity occurred only at concentrations [≥]100 M. In HCEC-1CT and differentiated Caco-2 cells, TeA increased IL-6, IL-8, and TNF- mRNA levels at non-cytotoxic concentrations ([≥]10 M). In Caco-2 cells, these transcriptional changes were accompanied by increased cytokine secretion, whereas HCEC-1CT cells showed only partial effects on the protein level after short-term exposure. Following prolonged incubation, TNF- secretion was increased and IL-6 and IL-8 secretion were slightly reduced. IL-10 remained unaffected under all conditions. Overall, TeA exerted cell type-dependent immunomodulatory effects characterized by immunoinhibitory activity in monocytes and pro-inflammatory responses in IECs, highlighting the complex immunotoxic potential of this Alternaria mycotoxin.

The tick Ixodes ricinus is the main vector of human and animal pathogens in Europe. Despite its importance in epidemiology and medicine, our understanding of physiological mechanisms controlling blood feeding, osmoregulation, or development are still limited. Here, we identify novel neuropeptide invertebrate parathyroid hormone-like peptide (iPTH) and its two receptors - PTHR1 and PTHR2 in I. ricinus. Functional aequorin-based assay confirmed specific activation of both receptors by iPTH. Using RT-qPCR we detected the PTHR1 transcript in the synganglion, while increased expression levels of PTHR2 were found in the salivary glands, hindgut and female gonads. RNA-mediated knockdown of iPTH receptors in nymphs resulted in delayed blood feeding, and a high incidence of defects in adult ecdysis. Consistent with observed phenotypes, iPTH is expressed in multiple neurons of the synganglion which project arborizing axons to the salivary glands, rectal sack and skeletal muscles. iPTH was colocalized with orcokinin-immunoreactivity (OK-IR) in all neurons that innervate these peripheral tissues. iPTH is further colocalized with tachykinin (TK) in Pd1DL1 neurons, suggesting coordinated action with other neuropeptides. Our findings indicate that iPTH signaling is required for normal feeding, development and successful ecdysis.

Fat accumulation and use is critical for sustaining life. Most insects show a typical response to feeding where fat is accumulated when sufficient sugars and other carbohydrates are consumed. Parasitoid insects are an exception, because most species do not accumulate fat when feeding on a sugar-rich diet. Studies on fat metabolism generally measure fat content early in life without considering lipid metabolism as a dynamic process that is expected to change as life progresses. In this paper, we compared fat accumulation and use throughout the lives of adult female Drosophila melanogaster and females of 5 inbred lines of the parasitoid wasp Leptopilina heterotoma. We expected that fat accumulation would take place irrespective of teneral fat content in D. melanogaster. We found that fat D. melanogaster initially used fat reserves, while lean flies economized on fat stores. Both lean and fat flies started accumulating fat after 7 days of life, indeed showing a typical response for insects. Unlike other parasitoids, L. heterotoma populations differs in fat accumulation patterns that we expected to observe also between inbred lines. In none of the inbred lines, however, did fat accumulation take place. Our results did reveal that inbred lines differed in the rate at which fat was used mainly later during life. We further confirmed that D. melanogaster pupal size was highly correlated with adult female size for both D. melanogaster and L. heterotoma. Overall, our findings for L. heterotoma provide strong evidence that genetic background has a major impact on the rate at which fat is used over a lifetime.

Cnidarians possess large number of opsins in their genomes for their various photoreceptive functions. In particular, they uniquely possess Gs-coupled opsins that induce intracellular cAMP accumulation in a light-dependent manner. These Gs-coupled opsins, cnidopsins, are powerful optogenetic tools manipulating cAMP-dependent cellular responses. In this study, we characterized a cnidopsin, named as AtCnidop3a, from the coral Acropora tenuis as a Gs-coupled and UV-sensitive bistable pigment. This cnidopsin showed a large spectral shift upon activation from absorption maxima from 395 nm to 560 nm, and the resting and activated states are interconvertible by illumination with UV (or violet) and orange light. The activated state efficiently activated Gs proteins and elevated intracellular cAMP levels in mammalian cultured cells. To engineer the opsin mutant that can be turned on and off upon long wavelength light illumination by utilizing the large spectral separation, negatively charged amino acids were introduced near the retinal Schiff base region. Among tested opsin mutants, the Y1133.28E mutant is capable of being activated by green light unlike the wild-type while retaining the property of being inactivated by orange light like the wild-type, indicating successful conversion of the opsin to a visible light sensitive bistable pigment. The visible light-induced cAMP regulation of the Y1133.28E mutant was enhanced by an additional L942.61G substitution. Our characterization and engineering of the cnidopsin revealed functional diversity of cnidarian opsins and its potential utility as optogenetic tools regulating Gs-dependent physiological responses.