Journal of Insect Physiology

Many insects enter diapause, a programmed state of developmental arrest that enables survival under adverse environmental conditions. In Drosophila melanogaster Meigen, 1830, diapause is characterized by reproductive arrest and reduced intestinal growth, accompanied by suppressed intestinal stem cell (ISC) activity. Juvenile Hormone (JH) promotes ISC proliferation under favorable conditions, but its capacity to modulate stem cell dynamics during cold-induced diapause remains unclear. Here, we investigated whether JH signaling can reactivate midgut remodeling in adult females maintained at 11. At this temperature, flies exhibited pronounced gut atrophy and elevated Phospho-histone H3 (PH3+) cell abundance, consistent with temperature-dependent G2/M phase arrest JH treatment significantly increased the proportion of Delta-positive progenitor cells in the anterior (R2) and posterior (R5) midgut regions at both 11 and 25, demonstrating that JH acts as a conserved mitogen for the ISC pool irrespective of thermal environment. A trend toward reduced PH3+ accumulation in the posterior midgut following JH treatment (p = 0.061) suggests possible facilitation of mitotic exit, though this effect did not reach statistical significance. Despite cellular-level changes, JH treatment did not restore overall gut size, indicating that the 72-84 hour exposure window was insufficient for subsequent tissue hypertrophy. Additionally, we identified a recurrent cold-induced pathology of gut distension, provisionally termed Lumen Obstruction Syndrome (LOS), which was independent of JH signaling. These findings reveal an uncoupling of JH-driven stem cell expansion from gross organ growth under diapause conditions, highlighting the selective sensitivity of the ISC compartment to endocrine signaling during environmental stress.

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

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.

In many insect species, mating is coordinated through multimodal signaling, yet less obvious channels are often overlooked. In the burying beetle Nicrophorus vespilloides, chemical communication is well-documented, but the role of substrate-borne vibrational signals (stridulations) during courtship remains unknown. We investigated whether stridulation is essential for mating success through two sets of experiments. First, we found a positive correlation between the frequency of stridulations and both the number and duration of copulation events. Second, we employed a silencing experiment to test the necessity of these signals by silencing males, females, or both partners. We found no significant differences between silenced and control groups regarding the frequency or duration of physical contact and mounting events, suggesting that stridulation is not required for mate recognition or the initiation of courtship. However, the proportion of successful copulations relative to mounting events was significantly lower when females were silenced. These results suggest that while N. vespilloides relies on a redundant multimodal system that likely utilizes chemical cues to initiate mating, vibrational signals, particularly from the female, may play a critical role in facilitating successful copulation. This study provides the first evidence for the role of stridulation in the mating behavior of N. vespilloides and highlights the potential for female-mediated vibrational signaling in burying beetle courtship.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

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.

Survival analysis is a workhorse assay in Drosophila research to evaluate somatic fitness. It is indispensable in the study of ageing and insightful in immunity, metabolism, radiobiology, toxicology, ecology, and others. While conceptually simple, lifespan measurement is labour-intensive because it requires the continuous manual maintenance of large experimental cohorts. Here, we describe Drosben, an approach that combines a 3D-printed device to transfer flies from several vials simultaneously, a paper system for quick data recording and accompanying software that automatically digitalises life tables for analysis. We show that using Drosben reduces the time investment to perform lifespan assays by ~85%, with improved speed regardless of experience handling Drosophila vials. Using Drosben, we address the effects on longevity of chronic feeding of indole-acetic acid (IAA), naphthalene-acetic acid (NAA) and trimethoprim (TMP) -- compounds used to control heterologous targeted protein degradation systems. We find that IAA and NAA have noticeable deleterious effects while TMP has a small protective effect specifically in females. We further show that strong static magnetic fields do not affect Drosophila lifespan. Our work suggests that Drosben can cheaply accelerate research where lifespan is used as a life history trait.

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.

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.

BackgroundNeurulation is a fundamental process in the formation of the central nervous system (CNS). The process begins with the folding and fusion of the neural plate to form the neural tube which subsequently gives rise to the development of the brain and spinal cord. Environmental and genetic factors that disrupt neurulation can induce neural tube defects (NTDs) and consequently cause additional developmental complications, including motor impairments. Purinergic signaling is a conserved form of extracellular communication (i.e. paracrine, synaptic signaling) that plays a role in early development. This signaling is mediated by purine nucleotides and nucleosides, which activate metabotropic P2Y and ionotropic P2X purinoceptors, respectively. Distinct patterns of intracellular calcium dynamics are observed throughout vertebrate development, from fertilization through organogenesis, including neurulation. Among P2X receptors, P2X4 is an ATP-modulated, Ca2+-permeable, ligand-gated ion channel characterized by having the highest Ca2+ permeability and is known to be modulated by ivermectin (IVM). ObjectiveOur investigation focuses on assessing the effects of IVM treatment during neurulation and evaluating the impact of this drug on phenotype, motor behavior and neuromuscular junction (NMJ) structure at tadpole stage. These results were compared with those obtained following separate treatments with compounds that specifically block glycine, GABA(A) and nACh receptors, all which have been described as IVM targets. ResultsIn this study we demonstrate the transcriptional expression for both P2X and P2Y purinergic receptors during neurulation, as well as the expression of P2X4. Following IVM neurula-treatments, we observed neural tube defects (NTDs), pigmentation changes, motor paralysis and alterations in neuromuscular junction (NMJ) structure, particularly affecting axonal branching. In contrast, treatment with the blockers strychnine, bicuculline and -bungarotoxin, used to assess the involvement of GlyR, GABA(A)R and 7nAChR, respectively, failed to show similar outcomes. ConclusionsIn summary, our results highlight the critical role of purinergic signaling during early development, particularly P2X4 receptor mediated signaling during neurulation which may account for the pharmacological effects induced by the positive allosteric modulator ivermectin.

The energetic demands of courtship are central to sexual selection, but their magnitude and temporal variation remain poorly quantified in many signalling systems. We used closed-chamber respirometry and low-light video analysis to estimate courtship-associated metabolic rates in males of the bioluminescent ostracod Photeros sp. EGD. Low-activity metabolic rate varied strongly across the diel cycle: in small vessels that constrained movement, individually measured males consumed significantly more oxygen at night than during the day. We then compared oxygen consumption across vessels that differed in opportunities for movement and courtship. Metabolic rates were highest in large vessels that permitted bioluminescent courtship displays, intermediate in medium vessels that allowed swimming but not full displays, and lowest in small vessels that constrained movement. Oxygen consumption in large vessels at night was approximately 500% of small-vessel daytime rates, 280% of small-vessel nighttime rates, and 160% of medium-vessel nighttime rates. Because measurements integrated oxygen use over multi-hour intervals, these values represent time-averaged metabolic demand rather than instantaneous costs of individual light pulses or display trains. Video analyses suggested a positive association between signalling rate and oxygen consumption, although this relationship was not statistically supported in our large-vessel dataset, which had low statistical power. Together, these results show that male Photeros undergo strong diel shifts in metabolic state and that the whole-animal performance required to construct bioluminescent courtship displays may often impose substantial energetic demands.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of D. eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

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.

The plasmodium of Myxogastoria (a group of amoeboid protists) species often crawls around the forest floor to feed while searching for places to form fruiting bodies for reproduction (sporulation). Certain environmental factors that trigger sporulation have been reported; however, other unknown factors are also expected. In this study, we reported field observations of Physarum rigidum and Fuligo septica. Inspired by the field observation, we examined the effects of multiple factors on sporulation in laboratory experiments using Physarum polycephalum. We found that:(1) there was a critical body size below which sporulation did not occur under our experimental conditions and (2) the plasmodium selected its sporulation sites from the available landscape of the experimental arena: dry and low sites for the majority and dry and high sites for the minority. Further analysis revealed that they preferred the edge area at the high site. We discuss the possible ecological importance of the threshold and location preference

Gene model for the ortholog of raptor in the D. grimshawi May 2011 (Agencourt dgri_caf1/DgriCAF1) Genome Assembly (GenBank Accession: GCA_000005155.1) of Drosophila grimshawi. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

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.

Gene model for the ortholog of raptor in the May 2011 (Agencourt Dere_CAF1/DereCAF1) Genome Assembly (GenBank Accession: GCA_000005135.1) of Drosophila erecta. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Pesticides remain indispensable for global food security, yet their use must be reconciled with the preservation of biodiversity. Despite advances in developing safer pesticides, their sublethal effects and synergistic potential with co-formulants in tank-mixtures, including inert spray adjuvants, often remain poorly understood in beneficial insects like bees. In this study, we assessed the effects of acute oral exposure to field-realistic doses of SilwetTM L-77 (an organosilicone adjuvant; 0.1-2.5%), acetamiprid (a cyano-substituted neonicotinoid; 6-150 ng/bee), and sulfoxaflor (a sulfoximine; 3-27 ng/bee) on gustatory responsiveness, associative olfactory learning, and short-term memory retention in honeybees (Apis mellifera L.). While we found no significant evidence of interaction effects at the exposure concentrations tested, the highest doses of SilwetTM L-77 and sulfoxaflor alone significantly reduced gustatory responsiveness. Furthermore, all three agrochemicals alone weakly affected associative learning without impacting memory retention. Although adjuvant-insecticide mixtures did not synergistically impair bee cognition, these adjuvants can impact sensory perception. Moving beyond mortality-based assessments and including sensory-cognitive thresholds is essential for a more holistic understanding of pollinator health.

The shift to terrestrial environments in ancestrally aquatic animals is often associated with key physiological and physical changes, including shifts in respiratory physiology and in some cases, even the evolution of completely novel respiratory structures. Examining how respiration operates across a gradient of submersion states in ancestrally aquatic terrestrial animals may shed light on how complex biological traits shift under different selective regimes. In this work, we begin exploring respiration in terrestrially-adapted land crabs that still use their gills to respire while underwater. We tested the relationship between aquatic respiratory rates, body size, and sex in red devil vampire crabs (Geosesarma hagen) at two ecologically-relevant temperatures. We found small females respire more than small males at 28{degrees}C, while large females respire more than large males at 21{degrees}C. Additionally, body size is a significant factor affecting respiratory rates of both sexes at 21{degrees}C and warmer temperatures significantly increase respiration in small crabs of both sexes. Interactions between these factors also led to emerging trends that can be explained by both physiological rules, such as reproductive investment and surface-to-volume ratios and heat transfer. We also report a temperature coefficient (Q10) of 1.52 for this species, showing an expected 52% change in respiratory and metabolic rate for every 10{degrees}C increase. This work also demonstrates the importance of understanding how and to what extent biological variables like sex and body size interact with abiotic environmental factors when measuring physiological traits in ectothermic invertebrate animals.

Gene model for the ortholog of tango (tgo) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly (GenBank Accession: GCA_001277935.1) of Drosophila busckii. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.