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Parasitoid Hymenoptera associated with necrophagous Diptera represent a potentially valuable but underexplored component of decomposition systems and forensic entomology. This study investigated parasitoid species associated with pupae of Chrysomya megacephala (Fabricius, 1794) exposed during pig cadaver decomposition in the Pampa biome of southern Brazil, and evaluated parasitoid occurrence across decomposition stages and sampling periods. Experiments were conducted in March, September, and November 2024, and January 2025 using 12 pig cadavers. Sentinel pupae were exposed daily throughout decomposition and recovered for parasitoid emergence and identification. Generalized linear mixed models were used to evaluate parasitoidism probability and species-specific occurrence across decomposition stages, while Weibull survival models were applied to analyze stage duration. A total of 5,280 pupae were exposed, of which 376 (7.12%) were parasitized, yielding 4,171 parasitoid adults. Four species were recorded: Nasonia vitripennis (Walker, 1836) (Hymenoptera: Pteromalidae), Pachycrepoideus vindemmiae (Rondani 1875) (Hymenoptera: Pteromalidae), Spalangia endius Walker, 1839 (Hymenoptera: Spalangiidae), and Tachinaephagus zealandicus Ashmead, 1904 (Hymenoptera: Encyrtidae). Parasitoidism probability was significantly lower during the fresh stage and increased from the bloated stage onward. Nasonia vitripennis showed greater contribution during the bloated stage, whereas P. vindemmiae was proportionally more frequent during the fresh stage. Decomposition progressed more rapidly during warmer months, particularly in November and January. Seasonal variation in parasitoidism was also observed, with higher rates during warmer sampling periods. These findings demonstrate that parasitoid assemblages vary across decomposition stages and environmental conditions, highlighting their ecological relevance in decomposition systems and their potential contribution to forensic entomology in subtropical environments.
In the last two decades, the field of molecular entomology has seen a shift toward next-generation sequencing techniques as a means of uncovering genetic and developmental processes. However, the standardization of methods is not well-established, and studies for insect-fungus consortia lack established protocols for advanced molecular techniques and downstream analysis compared to approaches applied in model systems involving insect-bacteria interactions. To investigate insect-microbe interactions, RNA sequencing and analysis is often used to identify genes involved in the symbiosis. But such protocols do not often consider insect-fungus systems, which vary significantly in community member abundance and/or fail to describe the details of the process from collection to data processing. This paper will introduce a comprehensive approach for RNA sequencing using two non-model insect-fungus consortia, which lack established, published protocols seen in model systems: the ambrosia beetle mutualism and cicada Massospora parasitism. The protocol includes a detailed TRIzol RNA extraction and quantification, RNA sequencing, and data processing using Nextflow pipeline software. Validation of a range of symbiotic interactions from mutualistic to parasitic is considered to justify this procedure to be utilized in a range of insect-fungus interactions with varied abundances and host interactions. Key features • Stepwise protocol for RNA extraction of samples containing insect and fungal tissue. • Novel dissection technique for beetle pupae. • Acquisition of transcriptomes of both host and symbiont with one protocol. • Direct comparisons of transcriptomes across life stages, stages of symbiosis, and/or by treatment.
Background Cutaneous leishmaniasis (CL) is an emerging and rapidly escalating public health threat in Sri Lanka, where a complex interplay of environmental, vector-related, biological, and socio-economic factors governs transmission risk. Despite the rising case burden, no validated, country-specific framework exists to systematically quantify and prioritize these risk determinants. This study aimed to develop and validate a multi-level indicator framework for assessing CL transmission risk in Sri Lanka using a combined Delphi-entropy weight approach. Method A three-level hierarchical indicator framework was initially constructed through a systematic literature review of CL risk factors. A total of 65 domain experts engaged in leishmaniasis surveillance, clinical management, entomology, and epidemiological research in Sri Lanka were invited to participate. Seventeen experts provided informed consent and completed the survey. Expert consensus was elicited using two iterative Delphi rounds. An authority coefficient (Cr) was calculated for each expert by integrating familiarity (Cs) and judgment basis (Ca) scores to weight responses proportionally to expertise. Entropy weights were derived from the degree of dispersion in expert responses to provide objective, data-driven significance scores. Comprehensive indicator weights were obtained by integrating Delphi and entropy weights. Results The final framework comprised four primary indicators, 11 secondary indicators, and 46 tertiary indicators. Among the primary indicators, biological factors received the highest normalized Delphi weight (0.312), followed by environmental factors (0.229), interventions (0.200), and social factors (0.188). The top-ranked secondary indicators were climatic features (0.194), sand flies (0.152), geographic features (0.105), and dogs (0.095). Among tertiary indicators, the highest comprehensive weights were assigned to regular monitoring of sand fly density (0.118), annual average rainfall (0.113), indoor residual spraying (IRS) (0.096), sand fly density (0.067), and screening patients through mobile clinics (0.057). Notably, three of the five highest-weighted tertiary indicators were directly related to sand fly surveillance and control, highlighting the primacy of vector management in CL transmission risk reduction. A critical data gap was identified: the absence of systematic, national-level sand fly density monitoring, which constrains real-time risk assessment capacity. Patient screening through mobile clinics also emerged as a crucial measure, particularly for rural communities with limited healthcare access. Conclusion:The Delphi-entropy weight framework provides a validated, evidence-based tool for prioritizing CL transmission risk factors in Sri Lanka. The findings highlight the urgent need to establish a systematic national sand fly surveillance programme, analogous to existing dengue vector monitoring systems, and to reinstate targeted IRS in endemic areas. This framework can guide resource allocation and evidence-based policy decisions for CL prevention and control in Sri Lanka and may serve as a model for similar resource-limited, endemic settings.
Sir Vincent Brian Wigglesworth (1899 -1994) is often cited as the leading figure most responsible for transforming entomology from a largely observational and descriptive natural science into a rigorous experimental biological discipline. His innovative investigations into the complexity of how insects work provided the foundations for modern insect physiology and endocrinology. By predicting three master hormones (prothoracicotropic hormone, ecdysone, and juvenile hormone), he managed to explain the processes of insect growth, development, and reproduction, but also established the fundamental mechanisms of neuroendocrine control across the animal kingdom. Wigglesworth's contributions were more than a collection of discoveries but constituted a unified, coherent theory of postembryonic development in insects governed by precisely timed neuroendocrine signals but extending into many other physiological processes.
Accurate minimum post-mortem interval estimation in forensic entomology relies on understanding necrophagous dipteran dispersal behavior. Post-feeding larval dispersal represents a critical phase impacting evidence recovery in forensic research. Currently, most studies suggest uniform radial dispersal patterns. Our study quantified spatial and temporal patterns of post-feeding larval dispersal in necrophagous Diptera under field conditions, examining how temperature, humidity, soil and season influence dispersal distance and directionality from a dead body. Twelve swine carcasses were deployed across wet and dry seasons in the Pedregal de San Ángel Ecological Reserve, Mexico City. Pitfall traps arranged in 9 concentric rings (1 to 7.4 m diameter) with 36 angular sectors monitored larval dispersal patterns. Among 2,545 individuals from 10 morphospecies were recorded, Lucilia sericata (Meigen) was the most dominant species (87.04%). Circular statistics rejected uniform distribution, revealing significant northwest directional preference (320.74°). Spatial distribution exhibited bimodal patterns with 50% of larvae within 130 cm and 95% within 290 cm. Dry season showed higher larvae concentration toward northwest compared to wet season. Predation by Solenopsis sp. Westwood ants during dry season forced larval aggregation in situ. Post-feeding larval dispersal exhibits consistent directional bias, complementing current uniform radial models. Our findings optimize entomological evidence collection by establishing search priorities: primary exhaustive searches within 3 m capture 95% of dispersing larvae, directional searches toward northwest up to 5 m increase recovery during wet seasons, and concentrated searches within 0.5 m maximize recovery during dry conditions when predation restricts dispersal. These quantified metrics provide operational guidelines for evidence recovery in Neotropical forensic investigations, improving search efficiency while reducing investigation time.
Although the highly prophylactic precautions and measures for keeping safe blood transfusions, transfusion-transmitted infections (TTIs) still constitute a remarkably significant risk in medicine. Few documents recorded the situation of TTIs in Dera Ismail khan (D.I. Khan) district, Pakistan so this study aimed to assess the prevalence of TTIs among blood donors in the district, to update and enhance the management of a secure blood supply. A cross-sectional observational study was conducted at the Regional Blood Centre in D.I. Khan, encompassing all eligible blood donors over three months. Screening for hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and syphilis was performed by using chemiluminescence immunoassay, while malaria parasite testing was performed by immunochromatographic technique. The results revealed that a total of 1221 people had attended the Regional Blood Center in D.I. Khan for blood donation. Only 1129 (92.46%) individuals were permitted to be screened as pre-donation scanning measures for TTIs, while the rest had medical histories that prevented them from donating. Out of 1129, eight (0.7%) were only females, and 1121 (99.29%) were males. The majority of donors, 550 (48.9%), were aged 30 years or younger. The number of voluntary donors, 87 (7.7%), was lower than the number of professional paid donors, 1042 (92.3%). The prevalence of TTIs among all donors was 60 (5.31%). The prevalence of syphilis, HBV, HCV, HIV, and malaria were 1.94%, 1.50%, 1.32%, 0.35%, and 0.17%, respectively. The infection rates among positive donors were notable for syphilis 22 (36.66%), hepatitis B 17 (28.0%), hepatitis C 15 (25.0%), HIV 4 (6.66%), and malaria 2 (3.33%). Professional paid donors showed slightly higher rates of TTIs 57 (5.47%) than that among voluntary donors 3 (3.44%). The study reflected the high prevalence of TTIs among blood donors in D. I. Khan, Pakistan. It also emphasized the role of blood screening, where the most prevalent TTIs were syphilis, HBV, HCV, HIV, and malaria. Encouraging voluntary blood donors and health education provide the community with safer blood with a higher quality.
Larval morphology of the Holarctic Boreonectes griseostriatus griseostriatus (De Geer, 1774) and of the Nearctic endemic Nectoboreus aequinoctialis (Clark, 1862) is described based on ex ovo material. General morphology and chaetotaxy of the cephalic capsule, head appendages, legs, terminal abdominal segment and urogomphi are described and illustrated for first and third instar. Phylogenetic comparison of Boreonectes Angus, 2010 and Nectoboreus Fery & Ribera, 2018 with nine other Deronectina genera was conducted in a cladistic context. Both genera are similar sharing several character states (e.g., presence of an elongate primary seta PA3, prementum non-sclerotized dorsally, presence of natatory setae on legs, elongate urogomphi, and presence of several non-sclerotized annuli on instar III urogomphomere 1) suggesting a closer relationship with Leconectes Fery & Ribera, 2018 and Mystonectes Fery & Ribera, 2018. Instar I larvae of Leconectes and Nectoboreus Fery & Ribera, 2018 share two unique character states: presence of additional setae on legs and presence of one non-sceloritized annulus on urogomphomere 1.
Madagascar's malaria burden has been increasing for several years despite continued investments in vector control. Supplemental measures to reduce vector populations and outdoor biting, such as larval source management (LSM), may be required in some settings. Madagascar piloted drone-delivered microbial larviciding using Bacillus thuringiensis israelensis (Bti) in rice fields in two districts with populations unfamiliar with drones during Feb-Jul 2022. We assessed knowledge and perceptions among purposefully selected rice farmers, rice field workers, and community members during focus group discussions (FGDs) in the two districts three to four months after the intervention. In total, 293 people participated in 39 FGDs, which were analyzed using a combined deductive-inductive approach. Despite initial concerns, both Bti-based larviciding and using drones to apply larvicides were ultimately well accepted by rice field owners and workers and community members in rice-growing areas of Madagascar. Key concerns spanned safety of Bti for people, livestock, and the environment, and harms related to drones crashing or being used for nefarious purposes. Participants perceived decreases in mosquitoes and malaria and increased rice yield related to the intervention. Receipt of project messaging appeared to differ by demographic group, with rice field workers frequently reporting having missed community meetings while working in the fields; many participants also reported that text-heavy communications were difficult to understand. Participants appreciated the involvement of personnel from health, agriculture, and environmental sectors, and they recommended expanding such intersectoral collaboration, involving trusted local officials, hiring local actors to help plan and implement the activities, and combining larvicide and fertilizer application in subsequent projects. Future LSM activities could benefit from social behavior change (SBC) efforts addressing safety and privacy concerns linked to the larvicide and the delivery method and highlight potential health and financial benefits related to decreased malaria burdens in the community, improved quality of life associated with fewer mosquito bites, the efficiency of drones in applying larvicide products, and the need to continue using ITNs. SBC will be most effective if provided early and repeatedly as needed, via channels appropriate to the community's language and literacy levels and in venues that are geographically and temporally convenient to those potentially impacted by the intervention.
A new species of the genus Micreriodes Yoshimoto is described and illustrated in the fauna of the State of Paraná, Brazil: Micreriodes lautus sp. nov. The morphological differences with Micreriodes guamensis are also analyzed. This is the first time any specimen of the genus has ever been identified at the species level outside the Pacific region.
BACKGROUND Crop pests can significantly damage crops, cause economic loss, and reduce the sustainability of agroecosystems. Control of pests is challenging because of the development of pesticide resistance, increasing environmental variation, the continual emergence of invasive pests, and growing demand for sustainable production. New ecoinformatic approaches are needed to understand the drivers of pest population dynamics and improve pest management practices. Here we analyze spatiotemporal drivers of Colorado potato beetle (CPB, Leptinotarsa decemlineata) abundance across Wisconsin potato fields using multiyear scouting data (2014-2024) linked with climate and cropping histories. We develop statistical models that account for spatial and temporal correlations while testing the effect of predictor variables, and find spatiotemporal statistical methods substantially improve fit and prediction. RESULTS After accounting for spatiotemporal random effects, we find that three predictors affected CPB abundance: cumulative growing degree days (GDD), recent potato intensity in the surrounding landscape, and winter coldest-day temperature. Cumulative GDD and potato intensity are positively associated with abundance, and warmer winter minima (higher coldest-day temperatures) are likewise associated with higher abundance, consistent with improved overwintering survival. Using the best-performing model, we generate a preliminary, statewide risk surface for Wisconsin to support regional decision-making. CONCLUSION Our results highlight the value of integrating field-level history with landscape context and explicit spatial structure when forecasting pest pressures in agroecosystems. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Anopheles stephensi, an invasive malaria vector expanding across the Horn of Africa, poses a growing public health threat. We applied a multi-faceted genomic approach combining whole genome sequencing (WGS), double-digest restriction-site associated DNA sequencing (ddRAD), and mitochondrial data to resolve population structure and adaptive evolution among An. stephensi populations from Ethiopia, Somaliland and Yemen relative to Indian lab-reared colony. WGS revealed broad-scale differentiation across the autosomal chromosomes, while ddRAD with a larger number of samples (n = 402) provided finer resolution of substructure within Ethiopia and Yemen, including sites where heightened malaria has been reported post invasion. Distinct genetic compositions in Sayhut, Yemen, indicate cryptic diversity within An. stephensi. WGS-based scans identified selective sweeps in Ethiopian populations at loci encoding homologs of esterases, serine proteases, and lipases, and in Yemeni populations at cytochrome P450 gene clusters. These results provide insight into An. stephensi diversity, particularly for locations where heightened malaria has been reported post-invasion and demonstrate that a combined WGS and ddRAD approach can offer a cost-effective option to resolve population structure and identify adaptive loci in a vector invasion scenario. Integrating these genomic approaches enhances understanding of invasion dynamics, informing surveillance and targeted vector control in urbanizing regions.
Epichloë spp. grow symbiotically within cool-season grasses and can provide crop protection against pests and drought stress. Winter cutworm (Noctua pronuba L.) is a serious insect pest of cool-season turfgrass and grass seed crops in Oregon (United States). In 2023, we conducted 2 greenhouse experiments to measure the effect of Epichloë infection in 8 cultivars of tall fescue (Schedonorus arundinaceus (Schreb.) Dumort) and perennial ryegrass (Lolium perenne L.) on N. pronuba mortality, weight gain, grass biomass, and feeding damage at 3 time points. Epichloë infection in grass plants was molecularly validated postexperiment. We did not observe an effect of Epichloë infection level or grass cultivar on N. pronuba, regardless of grass species in trial 1. In trial 2, Epichloë infection level effects remained minimal, while cultivar affected feeding damage at 3 and 12 d in both grass species. Feeding damage at 15 d did not vary among cultivars in both tall fescue and perennial ryegrass due to continuous and severe defoliation by N. pronuba. Insect mortality and weight gain were different among tall fescue cultivars. However, some cultivars were forage type, which was selected to be palatable to grazing animals, and could be preferred by the larvae. The perennial ryegrass cultivars were all turf-type and showed no variability in insect responses. The observed feeding damage variations were likely associated with cultivar-specific traits rather than Epichloë infection levels. Understanding the role of grass genotype in grass-Epichloë symbioses is critical to develop practical management for N. pronuba, and other cutworm species.
The potato cyst nematodes (PCNs), Globodera pallida and G. rostochiensis, cause substantial yield losses in potato worldwide. In the USA, Globodera ellingtonae, was described in 2008 after detection in Oregon and Idaho. Although G. ellingtonae reproduces on potato, the varieties assessed appear to be tolerant. Nonetheless, the reliable detection and discrimination of G. ellingtonae from G. pallida and G. rostochiensis is critical for regulatory decision-making, survey programs, and research, especially in regions with mixed infestations such as Idaho, Chile, Bolivia, and Argentina. Traditional morphological identification is labor-intensive, and requires specialized expertise, emphasizing the need for a rapid molecular diagnostic assay targeting G. ellingtonae so that it can be differentiated from G. pallida or G. rostochiensis. We developed a loop-mediated isothermal amplification (LAMP) assay for specific detection of G. ellingtonae based on the chorismate mutase gene, which exhibits species-specific sequence variation and alternative splicing patterns among Globodera spp. The assay showed high analytical specificity for G. ellingtonae, with no cross-reactivity observed with G. pallida or G. rostochiensis. It was completed within 30 min under isothermal conditions and demonstrated a 100-fold higher sensitivity for G. ellingtonae (100 fg) compared with conventional PCR (10 pg). This is the first report of a LAMP assay enabling rapid, sensitive, and species-specific detection of G. ellingtonae. The assay provides a simple and robust diagnostic tool suitable for integration into plant health regulatory and surveillance programs to support management of potential mixed PCN infestations.
Based on a dataset of 585 samples originating from across the Palearctic region and four genes (COI, CAD, Ca-ATPase, and 28S) for a subset of these samples, we studied the phylogenetic relationships within and between the Old World Swallowtail (Papilio machaon Linnaeus, 1758) and its closest relatives in the Palearctic region. We used Machine Learning (ML) to investigate the concordance of morphological characteristics with the molecular data. Our phylogenetic analyses showed that the Palearctic taxa everesti, archias and hippocrates formed strongly supported sister-group relationships with P. machaon. Within the Palearctic machaon a few distinct mitochondrial linages were observed, including one from the Himalayas and Central Asia, and another one that included all of the north African samples (taxa mauretanica, saharae and neosaharae) without any distinction. ML clustering was supportive of the haplotype and geographic analyses and a positive correlation was measured between average genetic phylogenetic and machine learnt specimen-image distances. Screening for Wolbachia revealed infection only in females of P. archias. We present a biogeographic scenario for the evolution of the P. machaon species group in the Palearctic region.
Trapped arthropods have been shown to benefit plants in several ways, but few studies have examined the potential costs of arthropod entanglement. Milkweeds (Asclepias spp.) have an unusual pollination system that requires relatively large packets of pollen (pollinia) to become attached to the appendages of insect pollinators and be pulled through a narrow opening in the flower. Honey bees (Apis mellifera) commonly become trapped and die with their legs still attached to milkweed flowers. In this study, we conducted a field experiment to examine how dead trapped honey bees affect floral visitation. We expected that the presence of a dead trapped bee would reduce floral visitation via two non-mutually exclusive pathways: (1) a direct deterrent effect on floral visitors and (2) an indirect deterrent effect mediated by an increased abundance of scavenging predators such as ants. The presence of a dead bee reduced floral visitation by 37% compared with controls, and this effect was stronger and more robust for honey bee visitors than non-Apis visitors. While ant densities were 51% higher on floral umbels with a dead bee and ants reduced floral visitation by 30%, our path analysis indicated that the direct deterrent pathway explained 91% of the total effect, consistent with an aversion to dead conspecifics among honey bees. Our results suggest that the lethal entanglement of honey bees is likely to incur an ecological cost for milkweed flowers, although the deterrence of honey bees could also shift the pollinator communities on milkweeds with unexpected consequences.
Genomic surveillance of Usutu virus (USUV) in blood donors is hampered by extremely low viral loads, which usually prevent reliable genome sequencing. We developed and validated a tiled amplicon-based sequencing protocol optimized for low-titer samples. Serial dilutions of four phylogenetically distinct USUV lineages showed ≥ 95% genome recovery above 100 RNA copies/µL and 65-98% recovery between 3 and 100 copies/µL. We applied the method to 27 USUV-positive blood donors from Germany (median 1.70 copies/µL), achieving lineage assignment in 74% and ≥ 70% genome coverage in 63% of samples. This approach enables routine genomic surveillance of USUV in blood donors.
Bactrocera dorsalis is an invasive pest causing severe economic losses. Its reproduction depends on sex pheromone-mediated courtship. We previously found that symbiotic Bacillus in the male rectum produce pyrazine sex pheromones (2,3,5-trimethylpyrazine/2,3,5,6-tetramethylpyrazine, TMP/TTMP) in a glucose-dependent manner, but whether host trehalose-to-glucose conversion regulates this remains unknown. Here, we show that mature males have higher rectal glucose and lower trehalose than females. Trehalase (Treh), which hydrolyzes trehalose to glucose, is enriched in the male gut and rectum, whereas trehalose-6-phosphate synthase (TPS), which catalyzes the reciprocal conversion of glucose to trehalose, is enriched in the male fat body. Inhibiting Treh pharmacologically or via RNAi reduces rectal glucose and sex pheromone levels, impairing mating success. Conversely, TPS knockdown elevates rectal glucose and sex pheromones, enhancing male competitiveness. Thus, host trehalose-glucose homeostasis controls glucose supply to symbiotic bacteria, directly regulating pheromone-mediated mating. Treh and TPS are promising targets for precision pest management.
This study formally describes a new species of Homidia Börner from Yunnan, China. Homidia quadriporasp. nov. is distinctly characterized by its purple body pigmentation, a V-shaped unpigmented area on the head, a lighter purple patch in the middle on the posterior half of the forth abdominal segment, four M-series mac-chaetae on the head, four pseudopores on the coxae of the mid leg, and a mac-chaeta m5 on the first abdominal segment. Comparative morphological and molecular analyses (using COI sequences) with phenotypically similar Homidia species corroborate its distinctiveness and support its designation as a new species. Furthermore, this research offers a crucial description of its first instar characteristics, comparing them with adult features and early stages of other Homidia species to illustrate developmental changes and differences.
Maize (Zea mays) is both an agronomically important crop and a reference model organism that has enabled the dissection of the molecular basis of plant development and environmental responses. Mass spectrometry-based proteomics provides a powerful approach to identify and quantify proteins and their post-translational modifications, facilitating the discovery of molecular mechanisms underlying complex biological processes. Unlike the study of gene expression using transcriptomics, analysis of the proteome and phosphoproteome provides direct measurement of proteins, which are responsible for driving or regulating nearly all cellular processes, thus offering a more complete picture of the cell's functional state. Over the past two decades, advancements in mass spectrometry have enabled large-scale profiling of protein abundance and phosphorylation sites in maize, improving our understanding of various biological phenomena. Here, we briefly summarize some of the major biological insights gained from maize proteome and phosphoproteome studies, and provide an overview of mass spectrometry sample preparation and acquisition/analysis workflows for the quantitative and reproducible analysis of protein abundance and phosphorylation dynamics in maize.
Climate change is shifting the distribution of insect vectors of disease, with consequences for human pathogen exposure. Predicting these range shifts requires understanding vectors' physiological limits. In North America, West Nile virus is transmitted primarily by two closely related and interbreeding Culex species-Cx. pipiens in the north and Cx. quinquefasciatus in the south-which, together with Cx. pipiens f. molestus comprise the North American Cx. pipiens Assemblage. Although the assemblage is widely distributed, little is known about how temperature shapes its species' ranges, particularly in the larval stage, where selection by temperature extremes is likely stronger than in adults. We compared larval thermal tolerance of first and fourth instars under high- and low-temperature extremes among five assemblage populations from the eastern United States. Southern Cx. quinquefasciatus populations were more tolerant of high temperatures than northern Cx. pipiens populations, whereas northern populations showed greater freezing tolerance in later stages. We also quantified larval macronutrient composition by colorimetric assay to characterize population-level differences in fitness-relevant energy stores. Populations differed in composition, but these differences did not align with thermal-tolerance patterns. Finally, we extended the first-instar high-temperature assay to two mid-latitude populations from within and near the hybrid zone in Maryland. Their thermal tolerance was intermediate to northern and southern populations', and LT50 was marginally correlated with collection-site climate after correcting for multiple comparisons. These findings provide empirical data on physiological variation within the Cx. pipiens Assemblage and inform future investigation of the genetic basis of thermal tolerance and range shifts.