Journal of Insect Physiology

Adult females of the two-spotted spider mite, Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in T. urticae is accompanied by reproductive arrest and the orange body coloration that arises from the accumulation of astaxanthin esters. How these two traits are coordinated at the molecular level remains poorly understood. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using liquid chromatography-tandem mass spectrometry, followed by RNA interference (RNAi) of candidate genes. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of the genes encoding TuPDS and TuLCPS markedly reduced orange pigmentation as well as {beta}-carotene and astaxanthin contents, demonstrating that these enzymes are required for diapause-associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. In addition to the suppression of orange pigmentation, RNAi of the TuPLAT10 gene restored reproduction even under diapause-inducing conditions and selectively reduced TuPDS and TuLCPS protein levels, despite the absence of sequence similarity to their genes. We propose that TuPLAT10 acts as a lipid-allocation switch that, in response to photoperiodic information, partitions fatty acids between astaxanthin esterification and yolk lipid supply, thereby coupling reproductive arrest and carotenoid pigmentation during diapause in T. urticae.

Adaptation to local environments enables species to thrive in diverse and challenging habitats. Steep elevational gradients provide a compelling natural adaptation laboratory, because abiotic conditions change progressively over short geographical differences. Given that elevation can strongly reshape physiology and behavior of insects, neuromodulatory systems offer a promising lens through which to examine elevation-specific adaptation. We challenged the hypothesis that adaptation to elevation involves octopaminergic signaling in honey bees (Apis mellifera), an important pollinator species occupying different elevations along East African mountains. We collected foragers from two distinct elevations at Mount Kenya (1,150 m and 1,900 m above sea level) and analyzed elevation-dependent changes in octopaminergic signaling. Tissue-specific analysis revealed a striking upregulation of all three octopamine {beta} receptor genes in the thoracic flight muscles and elevated octopamine brain concentrations at high elevation. Expression differences in the brain and fat body were rather modest. We subjected CRISPR/Cas9-mediated octopamine {beta}2 receptor knockouts to cold stress to study the function of octopaminergic signaling in thermoregulation. Loss of AmOAR{beta}2 reduced both the slope and amplitude of heating phases, indicating altered thermogenic dynamics. Together, these results identify the octopaminergic system as a central neuromodulatory regulator of thermogenic performance across elevations in honey bees. More broadly, our study highlights how modulation of conserved aminergic signaling pathways can shape physiological resilience to environmental gradients, pointing to a general mechanism by which insects adapt to changing thermal landscapes. Highlights- Bees from high and low elevation differ in expression of octopamine {beta} receptor genes and octopamine brain concentrations - CRISPR/Cas9-mediated octopamine receptor knockout alters thermogenic behavior - Octopaminergic signaling emerges as a key neuromodulator in thermal adaptation to elevation in honey bees Significance statementAnimals living along mountain gradients must cope with rapidly changing temperatures, yet the mechanisms enabling this adaptation remain poorly understood. We show that honey bees from higher elevations have increased brain octopamine levels and enhanced expression of octopamine receptors in heat-producing flight muscles. Using gene editing, we demonstrate that disrupting one key receptor alters how bees generate heat under cold stress. These findings identify octopamine signaling as a central regulator of thermogenesis and reveal a mechanism by which insects adjust to colder environments. More broadly, our results highlight how conserved neuromodulatory systems can fine-tune physiological performance, offering insight into how insects may respond to changing climates and expanding environmental extremes.

Arboreal insects have developed various strategies to navigate their discontinuous habitats. Many insects, including leafhoppers, katydids, and praying mantises, exhibit the ability to actively leap across their leafy platforms and land on a distant substrate. This behavior is especially important for non-winged insects, including nymphal forms of winged insects, which cannot fly between these substrates. To make a targeted jump, an animal must first orient towards the target, estimate the target distance and angular location, and jump with the appropriate take-off speeds and angles to land on their intended substrate. In three-dimensional space, jumping from one point to another requires estimating distance, as well as azimuthal and elevational angles. Jumping insects such as mantises typically reorient their bodies on the substrate to align with the azimuthal direction of the target. This behavior effectively reduces the task to a two-dimensional problem, in which they must estimate only the distance to the target and its elevational angle. Many insects, including praying mantises, perform rhythmic lateral head movements called peering before performing a targeted jump. Although previous studies suggest that mechanisms such as motion parallax while peering are used for distance estimation, the full repertoire of behaviors that enable mantises to jump to arbitrarily located substrates remains unclear. We hypothesized that mantises have distinct behaviors for distance and elevation angle estimation, which enable them to independently modulate their take-off speeds and angles before jumping. To test this hypothesis, we developed behavioral assays in which mantises were placed on a launch platform and jumped to a target platform positioned at variable distances and angles. Using this apparatus, we filmed the jumps of Giant Asian mantis nymphs (Hierodula spp.) with high-speed videography and tracked body parts to quantify take-off speed and angle. Because mantis jumps are ballistic, their trajectories can be modeled as projectile motion. Our results indicate that mantises estimate target distance and elevation angle using two separate behavioral strategies: distance is assessed through peering maneuvers that generate motion parallax, whereas elevation angle is determined through visual fixation of the target accompanied by specific postural adjustments. By combining these behaviors, mantises modulate the magnitude and direction of propulsive force to achieve successful jumps.

In many tropical areas, seasonal rainfall leads to distinct dry and wet seasons. Many butterflies developing under wet season conditions develop into adults with large ventral eyespots on the wing margins, whereas those developing under dry season conditions have smaller or no eyespots. In greener, wet season habitats, larger eyespots can divert predator attacks toward the wing margins, while reduced eyespot size improves camouflage in the dry leaf litter-dominated habitat during the dry season. However, the dry season is also characterised by higher desiccation stress than the wet season. We hypothesised that larvae developing under dry season conditions develop into adults with higher desiccation tolerance than those reared under wet season conditions. We tested this by rearing larvae of the butterfly Mycalesis mineus under simulated dry and wet season conditions and assaying the desiccation tolerance of the resulting adults. Butterflies reared in dry conditions survived longer under desiccation stress, lost lesser water during pupal-adult metamorphosis, and were heavier than those reared in wet conditions. We also tested the correlation between eyespot size and desiccation tolerance. A negative correlation between the traits would be expected if similar developmental pathways regulate them. Consistent with this expectation, individuals with smaller eyespots had higher desiccation tolerance. Our results demonstrate plasticity in desiccation tolerance, and suggest that predator avoidance and desiccation tolerance traits may share similar developmental pathways.

O_LITerrestrial insects are vulnerable to desiccation due to their small body size. Because insects lose most water through cuticular evaporation, cuticular traits strongly influence desiccation tolerance. Individuals with greater cuticular melanisation, i.e., darker ones, are hypothesised to tolerate desiccation better than less melanised ones. C_LIO_LIIn many butterflies, pupal colour is plastic - individuals pupating on leaves tend to be greener, while those that pupate away from leaves (off-leaf), such as on tree bark or defoliated twigs, tend to be browner. Brown pupae are hypothesised to have more cuticular melanin and are expected to experience higher desiccation stress than leaf-borne green pupae. Thus, plasticity in pupal melanisation may be an adaptation against desiccation. We tested this in the butterfly Eurema blanda. C_LIO_LIWe demonstrate that individuals pupating on on-leaf substrates are greener than those pupating on off-leaf substrates, and that desiccation stress is higher in the off-leaf substrates, a microenvironment typical of brown pupae, than in typical green pupae. Using Raman spectroscopy, we show that brown, but not green, pupal cuticles contain melanin. C_LIO_LIFollowing this, we obtained greener and browner pupae by manipulating substrate colour. When subjected to desiccation stress, browner pupae survived better than greener ones. There was no correlation between pupal colour and survival in the absence of desiccation stress. Thus, melanisation appears to confer a survival advantage to pupae by increasing desiccation tolerance. C_LIO_LISurvival under desiccating conditions was inversely related to water loss. Interestingly, melanisation did not correlate with water loss, suggesting that melanisation helps tolerate desiccation through physiological mechanisms not directly related to water loss reduction. C_LIO_LIOur findings reveal an additional, crucial, adaptive value of pupal colour plasticity, a trait that has been studied primarily from an anti-predatory perspective. C_LI

Many marine invertebrates are characterized by broad and highly plastic thermal limits, though the dynamic molecular mechanisms that enable extended thermal acclimation remain poorly understood. A classic example is the green crab (Carcinus maenas), which is a prolific and damaging non-indigenous species. Using a 22-day thermal exposure to cold (5{degrees}C), ambient (13{degrees}C), or warm (30{degrees}C) temperatures, we characterized plastic shifts in C. maenas performance using respirometry and time-to-right. We then used untargeted metabolomics and lipidomics analysis of heart tissues from days 4 and 22 to identify the molecular mechanisms underpinning plastic responses over time. Crabs at 30{degrees}C exhibited higher oxygen consumption rates than counterparts at 5{degrees}C. Interestingly, oxygen consumption rate increased over time at both temperatures, indicating thermal plasticity of aerobic respiration. Temperature-dependent metabolic reprogramming was employed by crabs to sustain aerobic respiration across temperature. Catabolism of branched-chain amino acids was important for energy production at elevated temperatures, while catabolism of arginine may have sustained the minimal energy needs of crabs exhibiting metabolic depression at cold temperatures. Righting response was positively correlated with temperature, and did not exhibit any changes over time. Lipidome remodeling consistent with homeoviscous adaptation could have enabled motor activity across temperature. Higher abundances of saturated and monounsaturated lipids likely provided structural integrity to cell membranes at 30{degrees}C, while lower abundances of these compounds may have enabled membrane fluidity at 5{degrees}C. Our work demonstrates the importance of ongoing molecular reprogramming in long-term acclimation, even when whole-animal physiology remains relatively stable. Summary StatementThis study demonstrates how the highly invasive green crab regulates metabolite and lipid pathways over time to maintain physiological performance across different temperatures.

Cuticular hydrocarbons (CHCs) are essential for insect waterproofing, yet how they change seasonally in social insects remains poorly understood. Due to its distinct seasonal worker phenotypes (summer and winter bees) and diverse subspecies, the western honey bee (Apis mellifera) is an ideal model to study seasonal CHC plasticity across populations with distinct local adaptations. We performed a common garden experiment to investigate the seasonal plasticity in CHC profiles across five European subspecies (A. m. carnica, A. m. iberiensis, A. m. ligustica, A. m. macedonica, A. m. ruttneri). We compared the CHC composition of workers performing tasks inside ("in-hive") or outside ("out-hive") the colony during summer and winter. Notably, out-hive workers consistently exhibited more waterproofing CHC profiles compared to in-hive workers, regardless of season or subspecies. The persistence of this stereotypical task-related differentiation in long-lived winter bees, which largely lack an age-based division of labor, indicates a robust, age-independent regulatory mechanism linked to the environment faced by the workers rather than a simple response to seasonal desiccation pressure. Moreover, we demonstrate CHC seasonal plasticity for the first time in honey bees. However, these seasonal shifts in hydrocarbon classes and chain length were not uniform; they varied across subspecies and critically depended on the task the workers performed.

Gastropods are a highly diverse and often overlooked taxonomic group of significant ecological and economic importance. Some terrestrial gastropods are critical pests of commercial agriculture and home gardens worldwide. Malacopathogenic nematodes offer an effective biological control method of managing pest slugs and snails as a natural enemy. Pellioditis (syn. Phasmarhabditis) hermaphrodita and Pellioditis (syn. Phasmarhabditis) californica are two species of biocontrol nematodes that have been commercialized, sold as Nemaslug(R) and Nemaslug(R) 2.0 respectively on three continents. Although there is interest in bringing Nemaslug(R) products to the US, they are currently not permitted due to limited knowledge on their North American distribution and effects on non-target and native species. In this study, we investigated the impact of P. hermaphrodita and P. californica on Ariolimax columbianus across two slug-host life stages, in laboratory infectivity assays. The objectives were to 1. determine whether P. hermaphrodita and P. californica nematodes impact survival of A. columbianus, and 2. evaluate whether there are differential effects on survival in juvenile and adult life stages of A. columbianus, in laboratory infectivity trials. We found that P. hermaphrodita caused significant mortality in A. columbianus with 100% mortality observed in both juvenile and adult slug hosts. The P. californica treatment had significant effects on the juvenile A. columbianus group only, with 80% mortality. By contrast, only 16% of unexposed control juveniles and 4% of control adult slugs died during the experiment. These results indicate that P. hermaphrodita and P. californica are lethal to the native, non-target Pacific banana slug (A. columbianus) under laboratory conditions, with mortality differing between juvenile and adult host life stages. Given the ecological importance of A. columbianus, these findings raise concerns for potential non-target effects of P. californica and P. hermaphrodita on terrestrial gastropod communities and emphasize the need for testing biocontrol agents against multiple life stages.

Giant pangolin (Smutsia gigantea) is one of the least studied pangolin species worldwide, with no published hematological and biochemical data available. We report the first blood parameters from a rehabilitated adult male from Campo Maan National Park (southern Cameroon). Hematological and biochemical findings are described and discussed in relation to available data from other pangolin species. These preliminary results provide the first reference framework for this species and highlight their relevance for clinical assessment, health monitoring, and conservation management.

Multimodal communication plays a central role in animal behavior, particularly when individuals must integrate information from different sensory channels to make rapid decisions. In aquatic environments, chemical and visual cues differ markedly in their spatial and temporal properties, such that chemical signals may be constrained by limited spatial resolution and temporal instability, potentially requiring visual information to reliably guide social decisions. In decapod crustaceans, both cue types are known to mediate reproduction, yet their relative contribution to mate-location behavior remains unclear. Here, we tested how visual and chemical cues from males influence mate-location behavior in females of the prawn Macrobrachium rosenbergii. Females were placed in a central arena and exposed to four stimulus configurations combining visual cues (a life-size photograph of a male or a control background) and chemical cues (water from an aquarium with or without a male). Attraction was quantified as the time spent in each half of the arena. Females showed no directional preference when exposed to chemical cues alone or when visual and chemical cues were spatially incongruent. In contrast, females spent significantly more time near male-associated stimuli only when visual and chemical cues were spatially congruent. These results indicate that mate-location behavior in this species depends on multimodal integration with a strong contextual dependence on visual information, which appears to gate the effectiveness of chemical cues. Spatially congruent multimodal signals are therefore necessary to guide orientation during mate search, suggesting that disruption of visual or chemical information in aquaculture systems may impair mating efficiency.

Background and AimsPlants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. MethodsLarvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC- qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key ResultsLarvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. ConclusionsInsect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Issue SectionOriginal article

Within species, individuals of the same age can differ in size. Previously, parental genetics, nutrition, space, and social interactions have been suggested to explain different growth rates. However, direct effects of larger individuals on the physiology and growth of smaller individuals are poorly understood. In this study, we investigated how larger individuals of the marine worm Platynereis dumerilii can impact the growth of smaller conspecifics. Comparing growth distributions in communally and individually reared worms, we show that larger worms suppress the growth of smaller ones. Furthermore, we were able to demonstrate that this suppression is chemically mediated. The chemical cue does not originate from faeces but is water soluble, stable for several days and smaller than 3 kDa. Our findings highlight the importance of non-reproduction related chemical signalling, showing evidence that dominant individuals can chemically suppress the growth of their conspecifics. This study provides new insights into how hierarchy can be established and maintained in a population and is particularly relevant for the growing community studying this model species.

There is disagreement on whether secondary endosymbionts are found in the major cereal pest aphid, Rhopalosiphum padi. Some papers report a diversity of secondary bacterial endosymbionts while others have failed to find evidence of these bacteria in this species. Here we revisit this issue by summarizing the relevant literature and through additional sampling of the species in Australia, China and Denmark using a combination of molecular approaches. We find a general absence of secondary endosymbionts beyond the obligate endosymbiont Hamiltonella defensa in R. padi. While the inconsistency in survey results may reflect rapid changes in endosymbiont turnover in populations and/or the impact of ecological factors such as host plant type on endosymbiont diversity, we are concerned that technical issues may be at least partly responsible for inconsistencies in the literature. This leads us to emphasize the importance of multiple sources of evidence required to establish and characterize endosymbiont infections, including PCR and qPCR assays, DNA Sanger sequencing and 16SrRNA gene metabarcoding. We note that several major aphid pests show a low incidence of secondary endosymbionts which raises issues about the importance of these endosymbionts in aphids that constitute pests, even though endosymbionts can in some cases increase host fitness and therefore pest impact.

Plant-gall wasp systems provide unique models for studying multitrophic interactions and unique developmental trajectories, yet standardized laboratory protocols for maintaining wild rose hosts (Rosa spp.) and sustaining gall inducers (Diplolepis spp.) are lacking. We developed and tested a method for growing and maintaining translocated individuals of Rosa canina, R. rubiginosa, R. spinosissima, R. gallica, R. tomentosa, and R. pendulina under laboratory conditions over three consecutive years (2023-2026). The goal was to have a constant supply of plant host material for reliably producing galls of D. rosae and D. mayri for experimental use. The protocol integrates soil and substrate composition, photoperiod and humidity regimes, pruning, dormancy management, and controlled exposure to gall-inducing wasps. More than 75% of rose individuals survived the full 3-year period, with consistent annual gall induction across some of the species. This work represents the first reproducible laboratory method for long-term maintenance of wild rose hosts and controlled gall induction by Diplolepis species, while also providing a transferable framework for maintaining perennial woody hosts and experimentally manipulating specialized plant-insect interactions under laboratory conditions, thereby providing a platform for ecological, physiological, and evolutionary studies on these interactions.

Sex ratio is a key demographic parameter shaping population dynamics and evolutionary trajectories. In biocontrol agents, demographic bottlenecks during species introduction to a new habitat and subsequent mass rearing can elevate inbreeding, potentially biasing sex ratios through sex-specific mortality associated with inbreeding depression. Moreover, reproductive endosymbionts such as Wolbachia are known to manipulate host reproduction and further skew sex ratios. However, the relative contributions of these processes to sex-ratio variation remain poorly resolved. In this study, we evaluated the effects of cross-generational full-sibling inbreeding and Wolbachia infection on sex ratio and key life-history traits in the biocontrol beetle Zygogramma bicolorata using controlled laboratory crosses across three generations. Inbreeding did not significantly alter offspring sex ratio, which remained close to parity across generations, while pupal mortality increased in later generations, consistent with delayed expression of inbreeding depression. Adult body weight remained largely unaffected by inbreeding. Wolbachia infection was detected in a subset of females and was associated with a modest but significant increase in female-biased offspring production, although the effect was variable across lineages. Strain typing identified a single supergroup A Wolbachia, consistent with previous descriptions of the wBic strain from this species. These findings indicate that sex-ratio variation in introduced populations of Z. bicolorata is not driven by inbreeding alone but instead emerges from the interaction between demographic processes and symbiont-mediated effects, providing crucial insights for optimizing biocontrol programs where sex-ratio stability is essential for population establishment and persistence. SignificanceSex ratio is a key determinant of population growth and stability - the essential parameters determining success of biocontrol programs. Yet, the mechanisms shaping sex-ratio variation remain poorly resolved. Using controlled crosses in Zygogramma bicolorata, we show that short-term inbreeding does not directly alter sex allocation, despite inducing delayed fitness costs through increased pupal mortality. In contrast, Wolbachia infection contributes to female-biased offspring production, although with variable outcome across lineages. These findings demonstrate that sex-ratio variation in Z. bicolorata arises from the interaction of demographic processes and symbiont effects, rather than a single mechanism, with important implications for predicting the establishment, persistence, and efficacy of mass-reared biocontrol populations.

RDL (Resistance to dieldrin) is a GABA-gated chloride channel that was first described as target of the insecticide dieldrin. Despite dieldrin being discontinued for decades because of its environmental per-sistence and health concerns, Rdl resistance mutations (A296S, A296G) continue at high frequencies in natural populations of the malaria mosquito Anopheles gambiae complex across Africa, suggesting a selective advantage. We have recently shown that RDL acts as a critical modulator of mosquito auditory sensitivity. Because acoustic recognition is essential for mate acquisition in An. gambiae, we hypothesized that these mutations confer a pleiotropic effect on mating success in the field, mediated through altered acoustic sensitivity, with potential consequences for sexual selection. We first provide laboratory evidence that resistance mutations enhance auditory behaviours of An. gambiae and show that the effect of environmental noise on mating success depends on the male Rdl genotype. We then conduct field collections in the city of Bangui (Central African Republic) and surrounding rural areas, revealing the presence of Rdl resistant alleles and their association with the urban environment, and within the city, with the noisiest locations. We also show decreased mating success of susceptible females with increasing noise levels, suggesting detrimental effects. Together, our findings support that Rdl resistance mutations enhance auditory function and mating success in acoustically challenging environments. We propose that this auditory advantage may contribute, together with other selective pressures such as cross-selection by other insecticides, to the persistence of these alleles in nature and may facilitate urban colonization by malaria vectors. Our study reveals, for the first time, an unintended evolutionary consequence of insecticide use, where a resistance mutation has been co-opted to enhance sensory performance and ecological adaptation, with significant implications for vector management strategies.

The presence of multiple discrete color patterns within a species has captivated evolutionary biologists for more than a century, especially when such polymorphism is confined to one sex. The brown anole Anolis sagrei exhibits a female-limited polymorphism in dorsal patterning, which is controlled by allelic variation at the autosomal gene CCDC170. Here, we present and test a threshold model that can explain why the polymorphism is female-limited. We hypothesize that allelic variation at the CCDC170 locus affects only female color pattern because this gene is co-expressed with its neighboring gene ESR1, highly expressed in female, but not male, embryos. By manipulating embryonic estradiol levels, we show that genetic males can be induced to express the polymorphism according to allelic variation at the CCDC170 locus, which is naturally masked by low expression levels of this gene. Inversely, treating genetic females with fadrozole, which depletes estradiol, leads to monomorphic patterns irrespective of genotype, as for natural males. Using RT-qPCR, we demonstrate that these effects are accompanied by a direct influence of estradiol and fadrozole on gene expression levels of CCDC170 and ESR1, thereby validating the threshold model. Our results suggest that the CCDC170-ESR1-locus is part of a mechanistic link between the morph-determining and the sex differentiation systems and provide a causal explanation for the developmental origin of a sex-limited color polymorphism.

Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites. IMPORTANCETrypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.

Tickborne diseases are a significant burden in many parts of the world. In the upper Midwestern United States, Lyme disease is the most common tickborne disease. It is carried by Ixodes scapularis. This vector can also transmit the pathogens causing anaplasmosis, babesiosis, ehrlichiosis, and several more tickborne diseases in this region. There is also concern for other tick species, such as Amblyomma americanum, that are expanding their ranges northward. We launched a citizen science passive tick surveillance program in 2024 to investigate tick species ranges in the upper Midwest, as well as the pathogens carried by I. scapularis. We received over 12,000 ticks in the first two years of this program, primarily from Wisconsin. While we received submissions of adult A. americanum outside of their endemic range, we did not see evidence of establishment in our study area. We measured pathogen prevalence in adult female I. scapularis (n=707) and observed 51% positivity for Borrelia burgdorferi, 9% for Babesia microti, 9% for Anaplasma phagocytophilum, and 3% for Ehrlichia muris eauclairensis. Multiple pathogens were identified in 14% of tested specimens, and significant associations were observed between B. burgdorferi and B. microti, and B. burgdorferi and E. muris eauclairensis. Pathogen prevalences varied across time and geography. Our results can begin to inform risk assessment for tickborne diseases in our region. A non-technical version of this document with interactive maps is available here: https://storymaps.arcgis.com/stories/8008c9d710b5400599f3c6cf88b2c546 Our online data dashboard is available here: redcap.link/TICS

Development and use of assisted reproductive technologies (ARTs) in non-domestic species provides novel tools for species conservation. As a first step towards in vitro embryo production, we developed an OPU technique for two antelope species, scimitar horned oryx (Oryx dammah) and roan antelope (Hippotragus equinus) utilizing a custom-made needle guide and existing OPU equipment utilized by livestock and human practitioners. Females were anesthetized and placed in sternal recumbency for transvaginal OPUs. Prior to OPUs (36 - 45 hours), SHO and roan were either hormonally stimulated with follicle stimulating hormone (FSH, 140 or 250IU) as a single injection or not. A total of 32 and 26 OPUs were completed in SHO (n=10) and roan (n=7), respectively, representing one to four OPUs per animal at monthly intervals. A total of 141 oocytes were recovered from 215 follicles in SHO and 31 oocytes from 58 follicles in roan. FSH dose (250IU) increased (P<0.05) the number of follicles aspirated and the number of oocytes recovered in SHO. No effects of FSH were observed in roan (P>0.05). Good quality oocytes were recovered from all females and procedures were conducted in four consecutive months with no evidence of scar tissue buildup or reduced capacity to recover quality oocytes. These ARTs can be used to develop in vitro embryo production tools for population management and the preservation of female genetics; bolstering genetic diversity and guarding against extinction.