Journal of Insect Physiology

The convergent lady beetle Hippodamia convergens is widespread in the Americas and considered an important beneficial insect due to its use in pest control. Early work on this species characterized the beetles as freeze-avoidant (freeze-intolerant), suggesting they survive low winter temperatures by physiologically preventing ice formation to temperatures as low as -15{degrees}C. Here, we show that H. convergens can be freeze-tolerant if ice formation occurs at relatively high temperatures. There was 100% survival following inoculative freezing at -0.5{degrees}C and exposure to -3{degrees}C for 20 hours, as well as freezing that spontaneously occurred in fed beetles exposed to -4{degrees}C for 4 hours. Males exposed to 0{degrees}C or -4{degrees}C for 4 hours had similar mating behaviours (latency to first mating, copulation duration) as control beetles exposed to 4{degrees}C, although sample sizes were too small to determine whether freezing itself had an effect on these behaviours. Several putative cryoprotectants were detected in fat body tissue of H. convergens: glycerol, proline, trehalose, and myo-inositol - in order of abundance. Exposure to -3{degrees}C for 20 hours, whether frozen or unfrozen, did not statistically affect cryoprotectant accumulation, although there was a trend towards increased glycerol following freezing. This study is the first to describe inoculative freeze tolerance in a lady beetle and establishes a baseline for future studies that examine the mechanisms underlying this freeze tolerance and the effects of freezing on reproductive behaviour. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/744689v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c0382org.highwire.dtl.DTLVardef@12a5932org.highwire.dtl.DTLVardef@145f4adorg.highwire.dtl.DTLVardef@1c2cb22_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHippodamia convergens can tolerate freezing at high subzero temperatures C_LIO_LIMating behaviour of males is normal after chilling or freezing C_LIO_LICryoprotectant accumulation may support overwintering survival C_LI

The physiology of ectotherms can change substantially during acclimation to changing environmental temperature. The role of transcription in acclimation responses has been well-established, but it remains unclear whether transcriptional regulation generally reflects abrupt changes after surpassing temperature thresholds, or whether transcript abundance is a relatively monotonic, continuous function of acclimation temperature. In this study we exposed adult male Drosophila subobscura flies to four different 96-hour acclimation treatments at temperatures that were not acutely stressful but ranged from relatively cold (10{degrees}C) to relatively warm (27{degrees}C) with respect to standard rearing conditions. Transcriptome sequencing of whole-body homogenates (mRNAseq) revealed a massive, transcriptome-wide response across acclimation temperatures, with a marked overrepresentation of genes that were continuously and monotonically up- and down-regulated in response to increasing acclimation temperature. Though some genes showed more complex relationships consistent with putative threshold responses, a high percentage of the differentially expressed transcriptome (42%) showed continuous and strictly monotonic relationships. Functional enrichment suggested continuous up-regulation of spermatogenesis-related transcripts with increasing temperature and continuous up-regulation of oxidative phosphorylation-related transcripts with decreasing temperature, illustrating contrasting patterns consistent with previous studies of thermal sensitivity of male reproduction and metabolic compensation in the cold. Thus, continuous thermal sensitivity of transcription is a hallmark of acclimation in D. subobscura that likely underlies the continuous thermal sensitivity of downstream physiological processes. We also provide evidence for shared transcriptomic responses across short-term acclimation (this study) vs. published results for long-term, developmental acclimation.

The global expansion of Black Soldier Fly (BSF), Hermetia illucens (L.), production systems for organic waste management has increased the need to better understand its reproductive biology in order to optimize mass-rearing programs. In this study, we macroscopically characterized chronological morphological changes in the internal genitalia of adult H. illucens reared on a sustainable alternative larval diet based on coffee pulp and corn bran. Morphological assessments and dissections were conducted on female and male reproductive tracts at 3, 6, and 9 days after adult emergence. Overall, the general organization of both reproductive systems was consistent with previous descriptions. A notable observation was the presence of a tripartite fertilization chamber in females, composed of three distinct compartments apparently associated with the three spermathecae. The functional significance of this anatomical organization remains to be determined. Females showed progressively advanced ovarian development and reached the clearest morphological indicators of reproductive maturity at 9 days, while males showed the greatest testicular distention and opacity at the same age. Compared with maturation times reported in previous studies using conventional larval diets, these observations suggest a later pattern of reproductive maturation under the coffee pulp-based diet. The observed differences may be associated with the nutritional composition and carbohydrate-to-protein balance of the larval diet. These results provide chronological and iconographic information that may contribute to the optimization of laboratory rearing protocols and the use of coffee by-products in H. illucens bioconversion systems.

Planning husbandry tasks and experiments with Drosophila melanogaster requires converting a target date into development times that depend on the rearing temperature. This calculation needs to be done for each cross, genotype, and temperature, and the risk of error grows quickly. Available laboratory management tools let users register stocks, crosses, and track them, but they do not create schedules based on a clear, adjustable thermal model. To fill that gap, we developed DrosoTracker, a self-contained web application that works offline and predicts Drosophila development with a thermal summation model recalibrated through regression on data from Powsner (1935) (T0 = 11.78 {degrees}C, DD = 116.38 {degrees}C{middle dot}days, R{superscript 2} = 0.997). The model offers an optional two-level calibration driven by user observations. A wild-type strain first adjusts the model to the laboratorys own conditions. Then each genotype is calibrated against that reference using a random-effects shrinkage estimator that accounts for measurement error and between-batch variability. The model creates schedules for husbandry tasks, evaluates adult cohort survival with the Kaplan-Meier estimator and the log-rank test, and calculates sample size for lifespan studies using Schoenfelds formula. The quantitative components were checked against independent references, including Rs survival package and manual calculations. Ongoing work is focused on validating the calibrated model using cohorts specifically bred for this purpose. DrosoTracker runs entirely in the browser, stores data locally, and is available in English and Spanish.

BACKGROUND: Varroa destructor is the major ectoparasite of honey bees and a vector of viral pathogens. Because pathogen transmission and exposure to contact-active acaricides depend on mite host contacts, understanding the factors governing host residence time is important for both disease epidemiology and pest management. We quantified host residence time under varying bee densities and host-type compositions. RESULTS: Mean residence time was 9.48 h across 312 host-residence events. Mites remained on individual hosts for only 2.36 h on Day 1 but approximately 11-14 h from Day 2 onward. A generalized linear mixed model showed a strong positive effect of day on residence time ({beta} = 0.341, SE = 0.044, P < 0.001), corresponding to an approximately 41% increase in residence time per day. Excluding Day 1 eliminated this effect (P = 0.16), indicating that the temporal pattern was driven primarily by the initial exposure period. Reconstructing Day 1 observations to an 8-hour schedule confirmed that this pattern was not an artifact of observation frequency. Neither host type nor bee density affected residence time, and mite occupancy of nurse bees matched host availability. CONCLUSION: Host residence time was governed primarily by initial exposure rather than host identity or moderate crowding. The results identify a previously undescribed exploratory phase immediately after mites enter a novel adult-bee population. Because shorter residence times imply more frequent host switching, these findings improve our understanding of pathogen transmission dynamics and may help explain variation in the performance of contact-based Varroa control strategies.

Circadian clocks enable mosquitoes to anticipate recurring environmental variations and coordinate behaviors critical for survival and disease transmission, such as locomotion, reproduction, host-seeking, and blood-feeding, with times of day when performance is maximal. In Aedes aegypti, locomotor activity follows a robust diurnal rhythm shaped by endogenous circadian clocks and environmental cues, among which light has been shown to be the primary source of temporal information. While early studies established the role of light in regulating locomotor activity, behavior, oviposition and pupation, it remains unclear which features of a light cycle drive changes in circadian rhythms. This question is increasingly relevant as Ae. aegypti is frequently exposed to artificial and dynamic lighting conditions in urban environments. Here, we investigated how transient changes in light schedules influence circadian rhythms in locomotor activity by systematically manipulating the timing, duration, and direction of light exposure. Using a high-throughput assay, we tested over 1900 individuals, including wild-type and timeless knockout mutants, and showed that a single day of al tered lighting is sufficient to induce robust phase shifts, with no evidence of masking effects. A 6-hour light pulse was sufficient to re-entrain mosquitoes regardless of the timing of the pulse, and phase shifts were primarily driven by the offset time of the light pulse, indicating that light-offset acts as a major zeitgeber. Together, these findings challenge conventional assumptions about the timescale of circadian synchronization and highlight the remarkable plasticity of mosquito behavior in response to anthropogenic light. Eventually, these effects could explain the rapid adaptation of the species to urban environments and have potential consequences for disease transmission dynamics.

IntroductionChanges in vector control strategies alter the selection pressures exerted on natural populations of Anopheles gambiae s.l. and may influence the dynamics of insecticide resistance mechanisms. However, the consequences of discontinuing indoor residual spraying (IRS) campaigns on the evolution of Ace-1-mediated resistance remain poorly documented under natural conditions. This study aimed to assess the spatiotemporal evolution of resistance to pirimiphos-methyl following the cessation of IRS and to investigate evidence consistent with the existence of a biological cost associated with Ace-1-mediated resistance. MethodsNatural populations of An. gambiae s.l. were collected between 2017 and 2023 in three districts in Burkina Faso that had undergone IRS campaigns (Kampti, Solenzo, and Kongoussi). Susceptibility tests with pirimiphos-methyl (0.25%) were conducted out following WHO protocols, and a subsample of exposed mosquitoes was genotyped to detect the Ace-1 G119S mutation. Spatiotemporal trends in mortality, allele frequencies and genotypes were analyzed according to the pre-IRS, IRS, and post-IRS periods. The association between the Ace-1 genotype and survival following exposure to pirimiphos-methyl was assessed using logistic regression, while the concordance between phenotypic and molecular indicators of resistance was examined using Spearmans correlation. ResultsThe susceptibility of An. gambiae s.l. populations to pirimiphos-methyl was gradually restored after the discontinuation of IRS at all sites. At the same time, the frequencies of the Ace-1 119S resistance allele declined sharply, particularly in Kampti and Solenzo, while they remained low in Kongoussi throughout the study period. Mosquitoes carrying resistant genotypes had a significantly higher probability of survival after exposure to pirimiphos-methyl than susceptible homozygotes, with resistant homozygotes (RR) exhibiting the greatest survival advantage (OR = 27.62; 95% CI: 6.91-110.45; p < 0.001). A significant negative correlation was observed between the frequency of the Ace-1 119S allele and phenotypic mortality ({rho} = -0.48; p = 0.033), indicating a concordance between the two indicators of resistance. The progressive decline in allele frequencies, the decreasing prevalence of resistant genotypes, and the concomitant restoration of susceptibility are field observations consistent with the existence of biological costs associated with Ace-1-mediated resistance. ConclusionThis study provides field evidence consistent with the existence of biological costs associated with Ace-1-mediated resistance in natural populations of An. gambiae s.l. These results underscore the value of an adaptive resistance management strategy based on alternating selection pressures and could guide future vector control strategies, particularly if indoor residual spraying campaigns or other interventions relying on organophosphates were reintroduced.

In vertebrates, top-down visual attention is a cognitive process where internal goals modulate the tuning of peripheral sensory systems. This leads to increased perceived contrast to both goal-relevant objects and areas of the visual field that are attended. Such a system would also be beneficial to bees, enabling them to detect and recognise the most profitable flowers in their environment. We tested whether bumblebees possess a top-down attentional system resembling that seen in vertebrates. We trained two groups of bees to collect rewards under high contrast targets. To potentially induce a difference in attention while searching for the targets, one group received a higher concentration of sucrose rewards compared to the other. During tests, the targets were presented with a series of lower contrasts to measure the contrast sensitivity curves of the bees induced by the different learnt reward levels. We predicted a stronger effect of any attention-like process on contrast sensitivity in the high reward group. We also repeated this experiment with the neonicotinoid pesticide imidacloprid dissolved in the sucrose rewards to test whether this affects bee attention. Across all test contrasts, higher rewards significantly increased bee accuracy when locating targets, lowered contrast thresholds and reduced the latency to make first choices. Imidacloprid reduced bee accuracy but did not influence first choice latency. These results suggest that learnt floral rewards can influence bee behavioural contrast sensitivity in a manner resembling vertebrate top-down attention and that imidacloprid may modulate this through effects on their nervous system.

Riparian environments are characterised by a high diversity of arthropod species, linking the aquatic with the terrestrial ecosystem. One of the dominant arthropod predators in this ecotone, carabid beetles are of particular interest as they may also act as facultative scavengers, feeding on carrion deposited along the shoreline. To test whether carabids feed on carrion, we examined the feeding preferences of the abundant riparian carabid Bembidion elongatum in a laboratory feeding trial, offering freshly killed and 24 hours post mortem Drosophila melanogaster. We subsequently assessed the detection probability of ribosomal prey RNA and DNA in predator gut contents using Drosophila-specific RT-PCR and PCR assays, and quantified nucleotide abundance by quantitative real-time PCR at 0, 3, 6, and 12 hours post-feeding. In the feeding experiment, B. elongatum showed a significant preference for fresh over carrion prey. Following consumption, the quantities of prey DNA and RNA in the predators gut declined over a 12-hour post-feeding period. However, no differences were detected in prey DNA or RNA quantities between individuals fed fresh prey and those fed carrion. Only immediately after consumption was the DNA:RNA ratio significantly lower in individuals fed fresh prey compared to those fed carrion while this difference was not observed at later time points. Overall, our results indicate that ingested ribosomal prey RNA in predators is present in high quantities, and that the DNA:RNA ratio is not a suitable indicator for distinguishing between consumption of carrion and fresh prey.

Gene model for the ortholog of Insulin-like peptide 3 (Ilp3) in the D. pseudoobscura Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. 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.

Cultivar susceptibility strongly influences the epidemiology of vector-borne plant diseases, and understanding cultivar-specific variation can inform management strategies. This is particularly relevant for flavescence doree, an incurable grapevine disease associated with a phytoplasma and transmitted by the leafhopper Scaphoideus titanus. In this study, we investigated the susceptibility of the main Swiss varieties, by combining controlled insect-mediated inoculation experiments with complementary field analyses conducted at progressively finer spatial scales. Together, these approaches allowed us to compare both infection probability and phytoplasma relative titre under standardised transmission conditions with disease incidence and relative titre under natural epidemiological conditions. For most cultivars, laboratory results were broadly consistent with field observations. However, a marked discrepancy emerged in the relative infection pattern between the two main grapevine cultivars grown in Switzerland: Chasselas and Pinot Noir. Under controlled conditions, they did not differ significantly in either their probability of infection or the phytoplasma relative titre, indicating no detectable difference in susceptibility to phytoplasma infection. In contrast, Pinot Noir consistently showed higher disease incidence than Chasselas under natural conditions. This pattern was observed across all spatial scales examined, from regional surveys to neighbouring vineyard plots, and was mirrored by higher phytoplasma relative titres. Importantly, under controlled conditions, S. titanus mortality during the one-week inoculation period was significantly higher on Chasselas than on Pinot Noir, indicating that Chasselas may provide a less favourable host for S. titanus. Together, these findings support the hypothesis that differences in field disease incidence between these cultivars may arise from differences in vector performance rather than intrinsic susceptibility to phytoplasma infection. This highlights the importance of considering plant-vector interactions, alongside susceptibility to infection, when assessing cultivar-specific vulnerability to vector-borne plant diseases.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. 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.

Elucidating the biological and molecular mechanisms that govern male fertility and mating behavior in mosquitoes is critical for optimizing genetic and sterile insect technique-based vector control strategies. Here, we examined age-related changes in male reproductive capacity in Anopheles culicifacies, using female egg output as an indirect indicator of male fertility. Our results demonstrated that male reproductive age follows a non-linear pattern of fertility. Morphometric analysis from emergence to day 13 post-eclosion revealed a strong correlation between seminal vesicle capacity and female fecundity, suggesting that age-dependent gonadal development directly influences reproductive potential. At the molecular level, we identified AcLITAF6 as a key regulator of male reproductive homeostasis. RNAi-mediated knockdown of AcLITAF6 impaired apoptosis-associated and phagocytic clearance, reduced sperm viability, and decreased female productive outcomes. Conclusively, we reveal a previously unrecognized role of LITAF in sperm quality control and male reproductive fitness, highlighting AcLITAF6 as a potential target for mosquito population suppression strategies.

Many insect populations are experiencing thermal stress as a result of global change, making it imperative to investigate how their relationship with other organisms will be impacted by heat disturbances. Microbial symbionts, such as bacteria, have the potential to enhance or inhibit an insect's thermal tolerance. Solitary bee larvae host bacteria within their food stores ("pollen provisions"), which have been shown to benefit survival and development; however, it is unclear how heatwaves brought about by climate change will impact their relationships with these bacterial partners. In this study, we subjected blue orchard bee (Osmia lignaria) eggs and larvae to a 4-day heatwave (35 {degrees}C daytime:22 {degrees}C nighttime) or kept them at control temperatures (25 {degrees}C daytime:15 {degrees}C nighttime), then returned all bees to control temperatures for a 5-day recovery period. We assessed bacterial communities within pollen provisions and larval development stage pre-heatwave (Day 0), immediately post-heatwave (Day 4), and following the recovery period (Day 9). Bacterial community composition, diversity, and abundance were resilient to heat stress, but larval bees developed faster when subjected to a heatwave. This finding refutes the hypothesis that bacteria within pollen provisions modulate blue orchard bee responses to heat, suggesting instead that developmental effects could be more largely shaped by bee physiology or interactions with microorganisms other than bacteria.

Orchid bees (Euglossini, Apidae) are Neotropical insects whose species-level identification can depend on minute morphological characters, some difficult to see or analyze. In such cases, DNA barcoding may facilitate identification by comparing standardized DNA sequences with reference libraries. The mitochondrial cytochrome c oxidase I (COI) marker widely used in animals does not, however, provide uniform species-level resolution across bee lineages. We developed an adaptive-length nuclear ribosomal DNA (nrDNA) barcoding framework based on overlapping Nanopore-sequenced markers spanning approximately 500 to 5500 bp for 114 Euglossini species. By matching barcode length to specimen quality, material with varied preservation histories was processed within a single analysis. Leave-k-out validation with IDTAXA achieved more than 96% identification success for two longer barcodes, while performance was lower for the shortest. Combining barcode lengths within one reference library maintained high identification success, and confidence filtering reduced overclassification when species were absent from the reference library. For orchid bees, this framework permits affordable high-throughput identification and supports targeted taxonomic verification and revision. Combining adaptive barcode lengths in one analytical framework offers a general design principle for long-read reference-library construction. Its performance must now be tested in other groups.

Tree shrews (Tupaia belangeri) are increasingly used in comparative neuroscience, yet their respiratory physiology remains poorly characterized. We quantified spontaneous breathing and respiratory rhythm variability in awake adult tree shrews (n = 10; 5 males, 5 females) using whole-body plethysmography. Respiratory frequency decreased by approximately 16% with acclimatization to the recording chamber, while respiratory timing, body-mass-normalized respiratory amplitude, inspiratory flow, and minute ventilation remained relatively stable. After acclimatization, mean respiratory parameters were similar between sexes, but short-term breath-to-breath variability (SD1) was greater in males than females, whereas SD2 was comparable. These findings establish baseline respiratory characteristics in awake tree shrews and identify sex-dependent differences in short-term respiratory rhythm stability.

Allee effects are positive relationships between components of individual fitness and the number or density of individuals in a population. Negative density effects are well documented in Drosophila melanogaster across life stages, while Allee effects are rare and largely confined to the larval stages. However, despite the costs of high density, adult flies are found to exhibit attraction to same-sex conspecifics, suggesting some fitness value to the presence of conspecifics. We measured fitness related traits of singly mated-females housed at same-sex densities of 1, 2, or 10 and found clear reductions in lifetime reproductive output at low densities. Additionally, these females concentrated reproductive effort within early adulthood, albeit without improving estimates of fitness. When housed at variable densities, females altered their reproductive output in response to immediate densities, but were unable to improve it unless remating was possible, indicating an interaction between mating and density. Overall, our findings suggest that reproductive plasticity in response to the presence of conspecifics mediates positive effects of density on fitness in female Drosophila melanogaster. Understanding the physiological and ecological bases of such plasticity may help explain the evolution of social tendency in the fly.

The influences of the gut microbiome on animal physiology are well-documented, yet the developmental timing of microbial colonization and its long-term consequences remain poorly understood. In this study, we investigated how the timing of bacterial colonization during development affects transcriptional programming and phenotypic outcomes in adult Drosophila melanogaster reared on a common, rich diet. Using RNA-seq analysis on whole flies colonized either as newly hatched larvae or as newly eclosed adults, we observed minor but distinct transcriptional responses dependent on when flies were colonized. Both embryonic and adult colonization were associated with ~ 25 to ~ 200 differentially expressed genes compared to axenic controls, with the majority upregulated and enriched for immune-response genes, suggesting that colonization establishes a broader immune competence. However, only 10 genes showed persistent differential expression that was not normalized by introducing bacteria to adult axenic flies, including mitochondrial genes, the adipokinetic hormone (Adh), and a putative secreted neuropeptide. Overall, these findings suggest that Drosophila development on a rich diet is largely robust to the timing of bacterial colonization but that certain metabolic effects may occur.

Animals reproduce across a spectrum from oviparity to viviparity. Internal gestation and live birth have inherent advantages despite their costs. However, comparative analyses of key drivers, such as resilience to dehydration, lack taxonomic breadth. In this study, we assessed whether live birth improves the ability to proliferate in environments with limited access to water. To do so, we used the viviparous cockroach, Diploptera punctata, as a model of viviparity to assess dehydration-induced damage during extended periods of water deprivation. During pregnancy, maternal survival did not decline during dry periods compared with non-pregnant individuals, suggesting that pregnancy has minimal impact on resistance to dehydration stress. When mothers are deprived of water for 2 weeks, they show an increase in osmolality after one week, while their embryos show no change in osmolality until after two weeks, at which point abortions occur. Periods of dehydration increased the duration of pregnancy, likely due to a slower production of in utero milk, but there was no change in the size of progeny. After birth, viviparous D. punctata newborns showed increased survival under dry conditions compared to two other ovoviviparous species, which are much smaller and dehydrate more quickly. The combined impact of increased dehydration resistance during pregnancy and during the first independent phase indicates a benefit of prolonged viviparity in Diploptera punctata. This study provides evidence that viviparity in animal systems could enable progeny survival during drought.

Honey bees can swim on the water surface toward dark regions, a behavior known as scototaxis that may facilitate escape from water. Although this behavior has been reported in both honey bees and solitary bees, variation among honey bee species remains poorly understood. We compared scototaxis during swimming in four honey bee species representing two nesting types: open-nesting (Apis florea and A. dorsata) and cavity-nesting (A. cerana and A. mellifera). Individual bees were released into a water-filled arena containing a dark sector, and their landing angles were recorded. All species exhibited significant orientation toward the dark sector. However, open-nesting species showed significantly stronger orientation than cavity-nesting species. No significant differences were detected between replicate colonies within species or between species within the same nesting type, whereas differences between nesting types were highly significant. Hierarchical clustering based on orientation strength placed Osmia, a solitary cavity-nesting bee from our previous study, in the same behavioral cluster as the two open-nesting Apis species rather than the cavity-nesting honey bees. We also measured swimming duration, distance, and velocity, but found no consistent differences between nesting types. These results demonstrate substantial interspecific variation in swimming scototaxis. The behavioral clustering is consistent with the hypothesis that strong scototaxis represents an ancestral trait that has been reduced in the derived A. cerana/A. mellifera lineage.