Seasonal climatic variability shapes immune responses and infection risks in the common bluetail damselfly

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Herberstein, Md Kawsar Khan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7912881/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Oecologia → Version 1 posted 4 You are reading this latest preprint version Abstract Understanding how a changing climate influences host-parasite interactions is important to predict disease-driven extinction risks. Insect immune responses are sensitive to seasonal climatic factors such as temperature, humidity, and rainfall. The influence of seasonal climatic fluctuations on insect immune responses and parasite prevalence remains poorly understood. To address this gap, we studied seasonal variation in immune response and endoparasite (protozoan gregarine) prevalence (proportion of infection) in Ischnura heterosticta damselflies. Damselflies may have greater food and nutrition available in warmer seasons; therefore, we predicted higher melanisation and lower gregarine prevalence in warmer months. In accordance with our prediction, we found stronger melanisation and lower gregarine prevalence in summer. We further found that melanisation increased with air temperature and decreased with rainfall and humidity. On the other hand, gregarine prevalence decreased with air temperature and increased with humidity and rainfall in females but not in males. Our study provided evidence that natural seasonal variation in climatic factors impacted host-parasite interactions and infection prevalence across seasons. While short-term warming during favourable seasons may enhance host immune response, long-term or extreme climate change might disrupt host-parasite relationship by altering resource availability, humidity patterns, or insect thermal limits, thereby contributing to seasonal declines in host populations. Insect immunity encapsulation host-pathogen interaction commensalism seasonality anthropogenic climate change Figures Figure 1 Figure 2 Figure 3 Introduction Seasonal fluctuation in climatic conditions may contribute to the decline of insects worldwide by increasing disease prevalence and spreading diseases (see Harvey et al., 2020 ). Insects' immune responses are influenced by climatic factors such as temperature, humidity, and rainfall (Martin and Hillyer, 2024 ). Climate change-driven increase in temperature can impact insects' immune responses and infections across the spatiotemporal scale (Paul et al., 2024 ; Reece et al., 2017 ). Seasonal air temperature can alter prevalence, intensity of parasitism, and fitness costs imposed by parasites. Understanding how climate change would impact parasitism and the cost of parasitism across seasons is crucial for determining the local decline of the population across seasons. Studying variation of parasitism across seasons and how climatic local factors impact this variation provides an excellent platform to estimate how changes in seasonal climatic factors might impact population decline across seasons. Climatic factors such as temperature, rainfall, and humidity impact various aspects of insect life history traits, including immune response (Cohen et al., 2020 ; da Silva et al., 2021 ; LoScerbo et al., 2020 ; Mlynarek et al., 2015 ). Higher temperatures can increase metabolic costs and energy required to perform basal physiological functions, thereby reducing resources available for immune responses, consequently increasing parasitism or diseases in warmer months (Khan & Rolff, 2025 ). For example, damselfly larvae ( Coenagrion puella ) exposed to heatwaves showed reduced energy reserves and lowered immunity (Tüzün and Stoks, 2021 ). On the other hand, higher temperatures can increase the availability of food and nutrition available which can increase resources for immune response, thereby reducing parasitism or diseases in warmer months. For example, stronger immune responses were detected under higher temperatures in mosquitoes (see Murdock et al., 2012 ) and sepsid flies (Gourgoulianni et al., 2023 ). Similarly, in Lestes forcipatus and Ischnura elegans damselflies, stronger immune responses are observed in warmer seasons (Robb and Forbes, 2005 ; Raczyński et al., 2022 ). Impact of temperature, therefore, could increase or prevalence and intensity of parasitism, the direction and extent depend on the host-parasite systems and the local climatic conditions. Insect immune responses include cellular and humoral components, with melanisation playing a major role in humoral defence against pathogens. The prophenoloxidase (proPO) cascade drives melanin production, encapsulating pathogens and removing parasites from the insect body (see Khan and Rolff, 2025 ; Ilvonen et al., 2018 ; Siva–Jothy, 2000 ). Environmental factors affect individual and population immune responses (measured as PO activity or degree of melanisation) thereby modify infection rates in insects (Carter et al., 2021 ; Ismail et al., 2024 ; Scharsack and Franke, 2022 ). Little is, however, the impact of climate fluctuations on insect immune response and seasonal parasite prevalence, albeit of high importance for determining disease risk and seasonal decline of insects under a changing climate. Here, we aim to understand the pattern of immune response and parasite prevalence across seasons and determine the underlying climatic drivers. We hypothesised that immune response and parasitism would vary with temperature across seasons. Specifically, 1) higher temperatures in warmer months may increase immune response and reduce parasitism if thermal conditions promote physiological activity and development; alternatively, 2) parasitism may increase in warmer months if high temperatures reduce host immune investment or favour parasite development. We tested these hypotheses using the Australian common bluetail damselfly ( Ischnura heterosticta ) and the endoparasite gregarine (Apicomplexa: Protozoa) as a host-parasite study system. We determined melanisation as an index of immune response and measured gregarine prevalence across seasons. Methods and materials Host-parasite study system Damselflies are semi-aquatic insects and hosts to endoparasite gregarines (Zawal and Dyatlova, 2008 ), which are transmitted by drinking water contaminated with Gregarine oocysts, or through ingesting contaminated prey such as flies (Hecker et al., 2002 ). In the damselfly gut, gregarine oocysts develop into sporozoites, which attach to the damselfly posterior gut epithelium, then transform into mature trophozoites, ultimately developing into reproductive gametocysts (Baker III, 2023 ). Gregarines may damage the damselfly gut lining and reduce damselfly fitness, such as impacting their survival and lower egg production in females (Cordoba-Aguilar and Munguía-Steyer, 2013 ; Kaunisto et al., 2017 ). We studied seasonal variation in immune response and gregarine parasite prevalence in Ischnura heterosticta damselflies. Ischnura heterosticta is a medium size (body size: 33.7 ± 0.08 mm) damselfly belonging to the Coenagrionidae family (Haque et al., 2025 ). In the field, male I. heterosticta are distinguished by a black and blue head and thorax, and a black abdomen with blue bands (Fig. 1 a). I. heterosticta females initially resemble males in colour and turn grey as they mature (Fig. 1 a). This species is widely distributed throughout Australia and found in lentic and lotic habitats which are naturally parasitised by Arrenurus water mites and protozoan gregarines (Paul et al., 2022 ; 2024 ). Study site We collected damselflies from a natural population located on the Wallumattagal campus of Macquarie University, NSW, Australia. The study site is a small artificial lake with an area of approximately 895 m 2 and a perimeter of 212 m. The lake is permanent with stagnant water flow. We surveyed the study site every month from March 2024 to February 2025, covering the entire flight season of this species. No permits were required for damselflies collection from the site, as this species is not a protected species and the field site is not part of a national park. Determining immune response We captured damselflies from the field with insect-catching nets (dimensions: 1260 mm handle, 456 mm diameter hoop, 81 cm long net bag) while walking along the edge of the water body and adjacent grasslands. For each sampling day, we covered the same study area and spent approximately 30 minutes collecting 35–40 damselflies (14 sampling days with 10 control and 30–35 experimental damselflies each day for a total n = 470). We transported the damselflies to the laboratory within five minutes of capture and placed them in a plant growth chamber (Plant growth facility, Macquarie University, NSW, Australia) for acclimation for two hours. We set the temperature at 25°C and relative humidity at 80% and the light: dark cycle was set for 16:8 h. We quantify the immune response in damselflies using the encapsulation response assay (Nagel et al., 2011 ). We used a needle holder to insert a sterile 3 mm (filament insertion depth was 2 mm) long nylon monofilament (diameter 0.20 mm; treated with fine sandpaper) into the body of the experimental damselflies (Koskimäki et al., 2004 ; Nagel et al., 2011 ; Rantala et al., 2000 ). We insert the nylon filament into the thorax below the lateral stripe (Fig. 1 b). We maintained the consistency of the length of inserted filaments throughout the experimental procedures. Control animals were not manipulated. We kept all damselflies individually in a plastic drinking cup (100 ml) covered with a cotton mesh and a wood dowel for perching. We placed the cups in the growth chamber for 24 h (studies showed melanisation reaction occurred within 24 hours after implanting nylon inserts; Galko and Krasnow, 2004 ; Tang, 2009 ), after which we recorded the survival status of control and experimental animals (dead, or alive). We removed the nylon from the experimental animals with the needle holder, and the piece of nylon was placed in ethanol (70%) in an Eppendorf tube (0.5ml). Damselflies were euthanised in -30°C and randomly selected experimental individuals (n total = 140) from each sampling were dissected to determine the presence of gregarines. We photographed the nylon filaments at 3.2x with an OLYMPUS SZX16 stereo microscope under standard lighting using OLYMPUS cellSens imaging software. We took the filaments' images from three different angles and used ImageJ software to calculate the amount of melanin present on the insert. We measured the melanin in the form of a greyscale value (average darkness) from the filament part, which was inserted into the thorax of damselflies. We also measured the greyscale value of the part of the insert that remained outside of the damselfly thorax to check whether all the images have similar values. In ImageJ, we converted the RGB image to 8-bit greyscale and considered 0 as pure black and 255 as pure white (the lower greyscale value indicated a higher amount of melanin present on the insert). We took the average of greyscale values from three images and subtracted it from 255 (called reverse greyscale value) to make it easier to interpret (lower greyscale value, lower melanisation) (Ferguson and Sinclair, 2017 ). We collected monthly maximum and minimum temperature data for 2024–2025 from the Bureau of Meteorology (BOM: http://www.bom.gov.au/climate/data/index.shtml ) and calculated monthly average temperature (°C), rainfall (mm), and humidity (%) for each month that we surveyed. The average autumn, spring, and summer temperatures of this study area during our collection period were 19.83 ± 1.67°C, 19.28 ± 2.61°C, and 24.06 ± 0.48°C, respectively. Average autumn, spring, and summer rainfall levels were 132.84 ± 62.96 mm, 44.54 ± 9.79 mm, 66.84 ± 37.82 mm, respectively. Average autumn, spring, and summer relative humidity were 60.70 ± 14.15%, 53.49 ± 9.16% and 60.1 ± 5.81%, respectively. Statistical analyses We applied the DurgaDiff function of the Durga R package to determine mean differences of greyscale value (as a measure of melanisation) and gregarine prevalence between sexes and across seasons (Khan and McLean, 2024 ). This R package calculated 95% confidence intervals of the mean difference by bootstrapping 1000 times. We applied a generalized linear model (GLM) to identify the effect of climatic factors (monthly average temperature, rainfall, and humidity) on melanisation and gregarine prevalence. We fitted the GLM models with melanisation/gregarine prevalence as the response variables, and climatic factors as fixed effects. We analysed all data in R version 4.0.3 (R Core Team, 2020 ) using packages “lme4” (Bates et al., 2014 ), “performance” (Lüdecke et al., 2021 ), and “Durga” (Khan and McLean, 2024 ). We used the Durga package for estimating and plotting effect sizes (Khan and McLean, 2024 ). All values are estimated ± standard error. For model description, please see the supplementary information. Results Overall, the nylon treatment did not differentially affect the mortality of experimental damselflies compared to control damselflies, and 94.89% of animals died after 24 hours in the growth chamber. Therefore, the observed melanisation is likely to reflect baseline immune capacity rather than short-term stress responses induced by captivity. Melanisation and gregarine prevalence between sexes across seasons Melanisation is higher in female than males (mean difference in greyscale value: 3.96, 95% CI [0.45, 8.19], Fig. 2 a). Similarly, gregarine prevalence was higher in females than males (mean difference: 0.54, 95% CI [0.4, 0.66], Fig. 2 d). Melanisation was higher in summer (average greyscale value: 98.80 ± 12.43) and spring (93.68 ± 16.66) and lower in autumn (75.15 ± 19.20) for both sexes (Fig. 2 b, Fig. 2 c; Table 1 ; also see supplementary for GLM results). Gregarine prevalence was relatively higher during spring (66.66%) and autumn (54.76% than in summer (45.09%) (Fig. 2 d, Fig. 2 e; Table 1 ; also see supplementary for GLM results). Table 1 Mean differences showing the variation in melanisation of female and male I. heterosticta damselflies across seasons. Sex Response variable Group difference Mean difference 95% CI Female Melanisation Summer – Autumn 26.53 [20.09, 32.22] Summer - Spring 1.34 [-4.5, 6.41] Male Summer – Autumn 20.72 [13.61, 27.25] Summer - Spring 9.60 [4.48, 14.43] Female Gregarine prevalence Spring - Autumn 0.05 [-0.06, 0.18] Spring - Summer 0.09 [0, 0.2] Male Spring - Autumn 0.13 [0.02, 0.26] Spring - Summer 0.27 [0.04, 0.46] Correlation of melanisation and gregarine prevalence with climatic factors Melanisation is positively correlated with temperature (GLM, estimate = 1.63 ± 0.50, z = 3.25, p = 0.001; Fig. 3 a), but negatively correlated with rainfall (GLM, estimate = − 0.07 ± 0.02, z = -2.67, p = 0.008; Fig. 3 b) and humidity (GLM, estimate = − 0.58 ± 0.13, z = -4.35, p < 0.0001; Fig. 3 c) in females. Melanisation is negatively correlated with humidity in males (GLM, estimate = − 0.31 ± 0.13, z = -2.28, p = 0.02; Fig. 3 c), but not with temperature and rainfall (Table 2 ; Fig. 3 (a-b)). Table 2 Correlation of melanisation and gregarine prevalence with monthly average temperature, rainfall, and relative humidity across seasons in female and male I. heterosticta damselflies. Sex Response variable Fixed effect Estimate Std. Error z value P value Female Melanisation Monthly average temperature 1.63 0.50 3.25 0.001 Rainfall -0.07 0.02 -2.67 0.008 Relative humidity -0.58 0.13 -4.35 < 0.0001 Male Monthly average temperature 0.68 0.52 1.31 0.19 Rainfall -0.02 0.02 -0.92 0.35 Relative humidity -0.31 0.13 -2.28 0.02 Female Gregarine prevalence Monthly average temperature -0.22 0.12 -1.81 0.07 Rainfall 0.001 0.006 0.20 0.83 Relative humidity 0.01 0.02 0.39 0.69 Male Monthly average temperature -0.09 0.08 -1.06 0.28 Rainfall -0.003 0.004 -0.76 0.44 Relative humidity -0.05 0.02 -2.37 0.01 Gregarine prevalence showed negative correlation with monthly average temperatures in females (GLM, estimate = − 0.22 ± 0.12, z = -1.81, p = 0.07; Fig. 3 d), but not in males (Table 2 ; Fig. 3 d). In females, gregarine prevalence was positively with rainfall (Table 2 , Fig. 3 e) and humidity (Table 2 , Fig. 3 f), but negatively correlated with rainfall (Table 2 , Fig. 3 e) and humidity (GLM, estimate = − 0.05 ± 0.02, z = -2.37, p = 0.01; Fig. 3 f) in males. Discussion We found that females and males had higher melanisation which was correlated with lower gregarine prevalence in summer compared to spring and autumn. Melanisation increased with temperature but decreased with rainfall and humidity, whereas gregarine prevalence was higher in females throughout seasons and weakly negatively correlated with temperature in females. We further found that melanisation is higher in seasons when gregarine prevalence is lower. Seasonal variation of melanisation and correlation with climatic factors Our study showed that melanisation in I. heterosticta damselflies was higher in summer compared to spring and autumn. Consistent with our findings, immune responses in cricket ( Allonemobius socius ) were also higher during warmer months (Fedorka et al., 2013 ). Insects are likely able to mount a stronger immune response in summer (see Adamo and Lovett, 2011 ), due to greater resource availability, which enables a relatively higher investment in immunity (Hangartner et al., 2013 ; Kiss et al., 2020 ; Rivera-Rea et al., 2022 ). For instance, food sources for damselflies, such as Diptera, Hymenoptera, and Coleoptera, are more abundant during the summer (Lim et al., 2021 ). Increased food availability enhances nutrition, which in turn boosts immune responses in damselflies and other insects (Leung, B et al., 2001; Kiss et al., 2020 ). Direct experiments are needed to unlink the effect of temperature from season to understand the influence of temperature and food availability on host fitness under warming conditions. We found that climatic factors, such as temperature, rainfall, and humidity, influenced melanisation, with temperature being positively, and rainfall and humidity negatively correlated with melanisation. Our study supports previous findings where higher temperatures enhanced melanisation in tropical species or those adapted to warmer climates, as seen in Sepsis thoracica , Galleria mellonella , and Sarcophaga africa (Gourgoulianni et al., 2023 ; Mastore et al., 2019 ). Summer temperatures have a positive association with phenol-oxidase (PO) enzyme activity- a key enzyme of melanin production, as shown in the Caribbean termite Nasutitermes acajutlae (Fuller et al., 2011 ) and mealworm larvae Tenebrio molitor (Catalán et al., 2012 ). Similarly, the negative correlation between melanisation and rainfall or humidity was also recorded in the Parnassius clodius butterfly (Zaman et al., 2019 ). Drier conditions increase the expression of proPO and consequently increase PO activity in burying beetles (Urbański et al., 2021 ), which may explain the observed seasonal variation in melanisation in our study. Seasonality of gregarine prevalence and correlation with climatic factors We found higher gregarine prevalence in spring compared to autumn and summer. Our study corroborates previous findings of higher parasitism in cooler months, i.e., water mite infection in damselflies (Paul et al., 2024 ; Robb and Forbes, 2005 ) and endoparasite infections in mosquitoes during cooler periods (Farner et al., 2025 , preprint; Trzebny et al., 2024 ). Spring’s lower temperature, rainfall, and humidity may drive this seasonal shift in gregarine prevalence in damselflies by influencing gregarine life history traits. Spring conditions increase development, density, and infectivity of the free-living stages of gregarines (Paul et al., 2024 ; Trzebny et al., 2024 ), a pattern also observed in other endoparasites such as Lambornella clarki in mosquitoes (Ismail et al., 2024 ) and gregarine Blabericola migrator infection in cockroaches (Kolman et al., 2015 ). While lower rainfall and humidity were linked to higher parasitism in damselflies in our study, the correlation was weak, as seen in microsporidian occurrence in mosquitoes (Trzebny et al., 2024 ). Wetter conditions may still contribute to increased infection prevalence by increasing parasite abundance (Shearer and Ezenwa, 2020 ; Trzebny et al., 2024 ), oocyst viability, transmission, or greater host exposure to parasites. Additionally, climatic impact on the host's immune responses can also influence the prevalence of gregarine infection. Does melanisation reduce gregarine prevalence? Our results revealed an inverse relationship between melanisation and infection across seasons (Fig. 2 , Fig. 3 ; also see supplementary information: Correlation of melanisation, month temperature, and their interaction with gregarine prevalence). Crickets (Fedorka et al., 2013 ) and dung flies (Gourgoulianni et al., 2023 ) showed stronger immune responses in warmer seasons, which correlated with lower infection risks. Warmer conditions enhanced melanin-producing enzyme activity, aiding parasite clearance. Conversely, damselflies exhibited weaker immune responses in spring, increasing their susceptibility to gregarines, resulting in higher gregarine prevalence. These findings support that seasonal shifts in immune responses shaped infection risks in I. heterosticta damselflies, higher immune response correlates to lower parasite infection, and vice versa. Conclusion Overall, we showed seasonal dynamics in melanisation and gregarine prevalence, with higher melanisation and lower gregarine prevalence in the warmer season. These findings highlight that seasonal climatic fluctuations shape host-parasite interactions, providing valuable insights into patterns of disease dynamics and insect fitness. Declarations Statement of diversity and inclusion We believe, support, and practice equity, diversity, and inclusion in science and everywhere (Rößler et al., 2020). We come from different countries, nationalities, residency, ethnicity, and cultural backgrounds (Bangladesh, Austria, and Australia), and the neurodivergent community. We represent different career stages (Graduate student, Early career researcher, and Professor). One or more of the authors self-identifies as a member of the LGBTQI+ community and represents a religious minority in science. Acknowledgements We acknowledge the Wallumattagal clan of the Dharug nation, the traditional custodians of the lands where Macquarie University is located, and the damselflies were collected. We thank Muhammad Masood and Wenfeng Ren for guiding us during work in the Plant Growth Facility (PGF), Macquarie University. We gratefully acknowledge the support and space provided by Microscopy Unit Manager Sue Lindsay in taking microscopic images of the nylon filament inserts. The authors thank their families, friends, and well-wishers for their empathy and support when most needed. Funding MKK was supported by a Humboldt Postdctoral Fellowship Conflicts of interest The authors declare no competing interests. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material All data for analysis are deposited in Figshare and can be accessed with the private link: https://figshare.com/s/acd36adb9201843828a2. Code availability All codes used to analyse the data of this study are deposited in Figshare and can be accessed via following link: https://figshare.com/s/acd36adb9201843828a2. Authors' contributions SP- conceived, designed, analysed, executed the experiment, and wrote original manuscript. MTH- analysed the data, executed the experiments, edited the manuscript. MEH- conceived, designed original ideas, edited the manuscript. MKK- conceived, designed original ideas, analysed the data, edited the manuscript. References Adamo SA, Lovett MM (2011) Some like it hot: the effects of climate change on reproduction, immune function, and disease resistance in the cricket Gryllus texensis . J Exp Biol 214(12):1997–2004. https://doi.org/10.1242/jeb.056531 Baker III (2023) J.D., Patterns of Gregarine Parasitism in Damselflies (Master’s Thesis). 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Evol Appl 14(1):24–35. https://doi.org/10.1111/eva.13041 Urbański A, Walkowiak-Nowicka K, Nowicki G, Chowański S, Rosiński G (2021) Effect of short-term desiccation, recovery time, and capa–pvk neuropeptide on the immune system of the burying beetle Nicrophorus vespilloides . Front Physiol 12:671463. 10.3389/fphys.2021.671463 Yadav N, Upadhyay RK (2023) Global effect of climate change on seasonal cycles, vector population, and rising challenges of communicable diseases: a review. J Atmospheric Sci Res 6. https://doi.org/10.30564/jasr.v6i1.5165 Zaman K, Hubert MK, Schoville SD (2019) Testing the role of ecological selection on colour pattern variation in the butterfly Parnassius clodius . Mol Ecol 28:5086–5102. https://doi.org/10.1111/mec.15279 Zawal A, Dyatlova ES (2008) Parasitizing on damselflies (Odonata: Coenagrionidae) by water mite (Acari: Hydrachnidia) larvae from Odessa province (Southwestern Ukraine). Nat Montenegrina 7:453–462 Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Oecologia → Version 1 posted Reviewers agreed at journal 03 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor assigned by journal 22 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7912881","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":539101726,"identity":"c638eaff-5c68-4138-8e51-4b8e682c649f","order_by":0,"name":"Shatabdi Paul","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-2884-2600","institution":"Macquarie University","correspondingAuthor":true,"prefix":"","firstName":"Shatabdi","middleName":"","lastName":"Paul","suffix":""},{"id":539101727,"identity":"0aae8380-d3f1-4663-ae4c-1a03b4cda8cb","order_by":1,"name":"Md Tangigul Haque","email":"","orcid":"","institution":"Macquarie University","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Tangigul","lastName":"Haque","suffix":""},{"id":539101728,"identity":"cbbd0fd9-59e4-4ca8-aed3-2a5971e7884a","order_by":2,"name":"Marie E. 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07:11:51","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136780,"visible":true,"origin":"","legend":"","description":"","filename":"OECOD25005630structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/ff2cbeaaa1b11ce89e5e3353.xml"},{"id":95866433,"identity":"58a02cf8-b033-48f6-a0dc-830b0066e83c","added_by":"auto","created_at":"2025-11-13 19:21:35","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147198,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/0a9ceabc6fc3ca8b45e6e6da.html"},{"id":96242184,"identity":"13726fb8-3b45-4833-be22-0cd80f21d3e1","added_by":"auto","created_at":"2025-11-19 07:12:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":603533,"visible":true,"origin":"","legend":"\u003cp\u003ePhotograph (a), showing a mating pair of \u003cem\u003eIschnura heterosticta\u003c/em\u003e damselfly (male colour blue and immature female mimic male colour); Photograph (b), showing a male damselfly with a nylon filament inserted; (c) 8 bit microscopic image of an insert after ~24 hours of experiment removed from a female damselfly; (d) an image of endoparasite gregarine (associative form of young gamonts and mature gamonts) (Clopton and Hays, 2006). Image credit © S. Paul.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/e694c6c30ed96c77e62d58ed.png"},{"id":96241727,"identity":"da8ef0b9-f451-42d3-a1e3-02aec3d6c3e0","added_by":"auto","created_at":"2025-11-19 07:11:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":299122,"visible":true,"origin":"","legend":"\u003cp\u003eMelanisation response (greyscale value) and gregarine prevalence in \u003cem\u003eI. heterosticta\u003c/em\u003e damselflies across seasons. Melanisation response and variation in gregarine prevalence (a), (d) between sexes; across seasons (b), (e) in females; and (c), (e) in males. In the upper panel of plots (a-c), the black circle represents the mean, and the vertical bar represents confidence intervals (CI) of both sexes across seasons. In (a), coloured dots represent melanisation, and each coloured circle in (b) and (c) represents a sampling event across seasons for females and males, respectively. In the lower panel, the triangle represents the mean difference, vertical line represents the 95% CI of the mean difference from 1000 bootstraps. Boxplots (d-f), showing the difference in gregarine prevalence between sexes and across seasons, where bold lines indicate the median, and bottom and top borders depict the 25th and 75th percentiles. The error bars extend downward from the first quartile to the minimum and upward from the third quartile to the maximum data points. We used a subset of data (n=10 from each sampling event) to determine the percentage of damselflies infected.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/60c8106aa69038c28410e6dc.png"},{"id":96241927,"identity":"ebee31f9-cf20-4f28-9a1f-328dfc76a213","added_by":"auto","created_at":"2025-11-19 07:11:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":526700,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of melanisation response (greyscale value) and gregarine prevalence in \u003cem\u003eI. heterosticta\u003c/em\u003e damselflies with climatic factors (monthly average temperature, rainfall, and humidity). Plots (a-c) and (d-f) show the correlation of melanisation and gregarine prevalence with climatic factors across three seasons in females and in males, respectively. Each circle represents a sampling event. The fitted lines represent the overall trend of the data points. We used a subset of data (n=10 from each sampling event) for calculating the effect of climatic factors on gregarine prevalence.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/256dfe0ac5f58afa39a0f2e4.png"},{"id":105755970,"identity":"faeab5f7-be3b-45e7-8d68-0cdf92856bfc","added_by":"auto","created_at":"2026-03-30 16:33:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2370916,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/03e0b27e-7678-499e-b335-9f788b577231.pdf"},{"id":95866434,"identity":"568e2ea9-c5e3-451e-8c4a-4de905b9c519","added_by":"auto","created_at":"2025-11-13 19:21:35","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5831608,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7912881/v1/da0b05e972e66d13f6644cfa.docx"}],"financialInterests":"","formattedTitle":"Seasonal climatic variability shapes immune responses and infection risks in the common bluetail damselfly","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSeasonal fluctuation in climatic conditions may contribute to the decline of insects worldwide by increasing disease prevalence and spreading diseases (see Harvey et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Insects' immune responses are influenced by climatic factors such as temperature, humidity, and rainfall (Martin and Hillyer, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Climate change-driven increase in temperature can impact insects' immune responses and infections across the spatiotemporal scale (Paul et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Reece et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Seasonal air temperature can alter prevalence, intensity of parasitism, and fitness costs imposed by parasites. Understanding how climate change would impact parasitism and the cost of parasitism across seasons is crucial for determining the local decline of the population across seasons. Studying variation of parasitism across seasons and how climatic local factors impact this variation provides an excellent platform to estimate how changes in seasonal climatic factors might impact population decline across seasons.\u003c/p\u003e\u003cp\u003eClimatic factors such as temperature, rainfall, and humidity impact various aspects of insect life history traits, including immune response (Cohen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; da Silva et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; LoScerbo et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mlynarek et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Higher temperatures can increase metabolic costs and energy required to perform basal physiological functions, thereby reducing resources available for immune responses, consequently increasing parasitism or diseases in warmer months (Khan \u0026amp; Rolff, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For example, damselfly larvae (\u003cem\u003eCoenagrion puella\u003c/em\u003e) exposed to heatwaves showed reduced energy reserves and lowered immunity (T\u0026uuml;z\u0026uuml;n and Stoks, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, higher temperatures can increase the availability of food and nutrition available which can increase resources for immune response, thereby reducing parasitism or diseases in warmer months. For example, stronger immune responses were detected under higher temperatures in mosquitoes (see Murdock et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and sepsid flies (Gourgoulianni et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, in \u003cem\u003eLestes forcipatus\u003c/em\u003e and \u003cem\u003eIschnura elegans\u003c/em\u003e damselflies, stronger immune responses are observed in warmer seasons (Robb and Forbes, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Raczyński et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Impact of temperature, therefore, could increase or prevalence and intensity of parasitism, the direction and extent depend on the host-parasite systems and the local climatic conditions.\u003c/p\u003e\u003cp\u003eInsect immune responses include cellular and humoral components, with melanisation playing a major role in humoral defence against pathogens. The prophenoloxidase (proPO) cascade drives melanin production, encapsulating pathogens and removing parasites from the insect body (see Khan and Rolff, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ilvonen et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siva\u0026ndash;Jothy, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Environmental factors affect individual and population immune responses (measured as PO activity or degree of melanisation) thereby modify infection rates in insects (Carter et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ismail et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Scharsack and Franke, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Little is, however, the impact of climate fluctuations on insect immune response and seasonal parasite prevalence, albeit of high importance for determining disease risk and seasonal decline of insects under a changing climate.\u003c/p\u003e\u003cp\u003eHere, we aim to understand the pattern of immune response and parasite prevalence across seasons and determine the underlying climatic drivers. We hypothesised that immune response and parasitism would vary with temperature across seasons. Specifically, 1) higher temperatures in warmer months may increase immune response and reduce parasitism if thermal conditions promote physiological activity and development; alternatively, 2) parasitism may increase in warmer months if high temperatures reduce host immune investment or favour parasite development. We tested these hypotheses using the Australian common bluetail damselfly (\u003cem\u003eIschnura heterosticta\u003c/em\u003e) and the endoparasite gregarine (Apicomplexa: Protozoa) as a host-parasite study system. We determined melanisation as an index of immune response and measured gregarine prevalence across seasons.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHost-parasite study system\u003c/h2\u003e\u003cp\u003eDamselflies are semi-aquatic insects and hosts to endoparasite gregarines (Zawal and Dyatlova, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), which are transmitted by drinking water contaminated with Gregarine oocysts, or through ingesting contaminated prey such as flies (Hecker et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In the damselfly gut, gregarine oocysts develop into sporozoites, which attach to the damselfly posterior gut epithelium, then transform into mature trophozoites, ultimately developing into reproductive gametocysts (Baker III, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Gregarines may damage the damselfly gut lining and reduce damselfly fitness, such as impacting their survival and lower egg production in females (Cordoba-Aguilar and Mungu\u0026iacute;a-Steyer, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kaunisto et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe studied seasonal variation in immune response and gregarine parasite prevalence in \u003cem\u003eIschnura heterosticta\u003c/em\u003e damselflies. \u003cem\u003eIschnura heterosticta\u003c/em\u003e is a medium size (body size: 33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mm) damselfly belonging to the Coenagrionidae family (Haque et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the field, male \u003cem\u003eI. heterosticta\u003c/em\u003e are distinguished by a black and blue head and thorax, and a black abdomen with blue bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). \u003cem\u003eI. heterosticta\u003c/em\u003e females initially resemble males in colour and turn grey as they mature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This species is widely distributed throughout Australia and found in lentic and lotic habitats which are naturally parasitised by \u003cem\u003eArrenurus\u003c/em\u003e water mites and protozoan gregarines (Paul et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStudy site\u003c/h3\u003e\n\u003cp\u003eWe collected damselflies from a natural population located on the Wallumattagal campus of Macquarie University, NSW, Australia. The study site is a small artificial lake with an area of approximately 895 m\u003csup\u003e2\u003c/sup\u003e and a perimeter of 212 m. The lake is permanent with stagnant water flow. We surveyed the study site every month from March 2024 to February 2025, covering the entire flight season of this species. No permits were required for damselflies collection from the site, as this species is not a protected species and the field site is not part of a national park.\u003c/p\u003e\n\u003ch3\u003eDetermining immune response\u003c/h3\u003e\n\u003cp\u003eWe captured damselflies from the field with insect-catching nets (dimensions: 1260 mm handle, 456 mm diameter hoop, 81 cm long net bag) while walking along the edge of the water body and adjacent grasslands. For each sampling day, we covered the same study area and spent approximately 30 minutes collecting 35\u0026ndash;40 damselflies (14 sampling days with 10 control and 30\u0026ndash;35 experimental damselflies each day for a total n\u0026thinsp;=\u0026thinsp;470). We transported the damselflies to the laboratory within five minutes of capture and placed them in a plant growth chamber (Plant growth facility, Macquarie University, NSW, Australia) for acclimation for two hours. We set the temperature at 25\u0026deg;C and relative humidity at 80% and the light: dark cycle was set for 16:8 h.\u003c/p\u003e\u003cp\u003eWe quantify the immune response in damselflies using the encapsulation response assay (Nagel et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). We used a needle holder to insert a sterile 3 mm (filament insertion depth was 2 mm) long nylon monofilament (diameter 0.20 mm; treated with fine sandpaper) into the body of the experimental damselflies (Koskim\u0026auml;ki et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nagel et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rantala et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). We insert the nylon filament into the thorax below the lateral stripe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We maintained the consistency of the length of inserted filaments throughout the experimental procedures. Control animals were not manipulated. We kept all damselflies individually in a plastic drinking cup (100 ml) covered with a cotton mesh and a wood dowel for perching. We placed the cups in the growth chamber for 24 h (studies showed melanisation reaction occurred within 24 hours after implanting nylon inserts; Galko and Krasnow, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Tang, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), after which we recorded the survival status of control and experimental animals (dead, or alive). We removed the nylon from the experimental animals with the needle holder, and the piece of nylon was placed in ethanol (70%) in an Eppendorf tube (0.5ml). Damselflies were euthanised in -30\u0026deg;C and randomly selected experimental individuals (n \u003csub\u003etotal\u003c/sub\u003e = 140) from each sampling were dissected to determine the presence of gregarines.\u003c/p\u003e\u003cp\u003eWe photographed the nylon filaments at 3.2x with an OLYMPUS SZX16 stereo microscope under standard lighting using OLYMPUS cellSens imaging software. We took the filaments' images from three different angles and used ImageJ software to calculate the amount of melanin present on the insert. We measured the melanin in the form of a greyscale value (average darkness) from the filament part, which was inserted into the thorax of damselflies. We also measured the greyscale value of the part of the insert that remained outside of the damselfly thorax to check whether all the images have similar values. In ImageJ, we converted the RGB image to 8-bit greyscale and considered 0 as pure black and 255 as pure white (the lower greyscale value indicated a higher amount of melanin present on the insert). We took the average of greyscale values from three images and subtracted it from 255 (called reverse greyscale value) to make it easier to interpret (lower greyscale value, lower melanisation) (Ferguson and Sinclair, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We collected monthly maximum and minimum temperature data for 2024\u0026ndash;2025 from the Bureau of Meteorology (BOM: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bom.gov.au/climate/data/index.shtml\u003c/span\u003e\u003cspan address=\"http://www.bom.gov.au/climate/data/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and calculated monthly average temperature (\u0026deg;C), rainfall (mm), and humidity (%) for each month that we surveyed. The average autumn, spring, and summer temperatures of this study area during our collection period were 19.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u0026deg;C, 19.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u0026deg;C, and 24.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u0026deg;C, respectively. Average autumn, spring, and summer rainfall levels were 132.84\u0026thinsp;\u0026plusmn;\u0026thinsp;62.96 mm, 44.54\u0026thinsp;\u0026plusmn;\u0026thinsp;9.79 mm, 66.84\u0026thinsp;\u0026plusmn;\u0026thinsp;37.82 mm, respectively. Average autumn, spring, and summer relative humidity were 60.70\u0026thinsp;\u0026plusmn;\u0026thinsp;14.15%, 53.49\u0026thinsp;\u0026plusmn;\u0026thinsp;9.16% and 60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.81%, respectively.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eWe applied the DurgaDiff function of the Durga R package to determine mean differences of greyscale value (as a measure of melanisation) and gregarine prevalence between sexes and across seasons (Khan and McLean, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This R package calculated 95% confidence intervals of the mean difference by bootstrapping 1000 times. We applied a generalized linear model (GLM) to identify the effect of climatic factors (monthly average temperature, rainfall, and humidity) on melanisation and gregarine prevalence. We fitted the GLM models with melanisation/gregarine prevalence as the response variables, and climatic factors as fixed effects. We analysed all data in R version 4.0.3 (R Core Team, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) using packages \u0026ldquo;lme4\u0026rdquo; (Bates et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), \u0026ldquo;performance\u0026rdquo; (L\u0026uuml;decke et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u0026ldquo;Durga\u0026rdquo; (Khan and McLean, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We used the Durga package for estimating and plotting effect sizes (Khan and McLean, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). All values are estimated\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. For model description, please see the supplementary information.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOverall, the nylon treatment did not differentially affect the mortality of experimental damselflies compared to control damselflies, and 94.89% of animals died after 24 hours in the growth chamber. Therefore, the observed melanisation is likely to reflect baseline immune capacity rather than short-term stress responses induced by captivity.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMelanisation and gregarine prevalence between sexes across seasons\u003c/h2\u003e\u003cp\u003eMelanisation is higher in female than males (mean difference in greyscale value: 3.96, 95% CI [0.45, 8.19], Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Similarly, gregarine prevalence was higher in females than males (mean difference: 0.54, 95% CI [0.4, 0.66], Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Melanisation was higher in summer (average greyscale value: 98.80\u0026thinsp;\u0026plusmn;\u0026thinsp;12.43) and spring (93.68\u0026thinsp;\u0026plusmn;\u0026thinsp;16.66) and lower in autumn (75.15\u0026thinsp;\u0026plusmn;\u0026thinsp;19.20) for both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; also see supplementary for GLM results). Gregarine prevalence was relatively higher during spring (66.66%) and autumn (54.76% than in summer (45.09%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; also see supplementary for GLM results).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean differences showing the variation in melanisation of female and male \u003cem\u003eI. heterosticta\u003c/em\u003e damselflies across seasons.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResponse variable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGroup difference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMean difference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95% CI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eMelanisation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSummer \u0026ndash; Autumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[20.09, 32.22]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSummer - Spring\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[-4.5, 6.41]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSummer \u0026ndash; Autumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[13.61, 27.25]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSummer - Spring\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[4.48, 14.43]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eGregarine prevalence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpring - Autumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[-0.06, 0.18]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpring - Summer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[0, 0.2]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpring - Autumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[0.02, 0.26]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpring - Summer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[0.04, 0.46]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCorrelation of melanisation and gregarine prevalence with climatic factors\u003c/h3\u003e\n\u003cp\u003eMelanisation is positively correlated with temperature (GLM, estimate\u0026thinsp;=\u0026thinsp;1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50, z\u0026thinsp;=\u0026thinsp;3.25, p\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), but negatively correlated with rainfall (GLM, estimate = \u0026minus;\u0026thinsp;0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, z = -2.67, p\u0026thinsp;=\u0026thinsp;0.008; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and humidity (GLM, estimate = \u0026minus;\u0026thinsp;0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, z = -4.35, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) in females. Melanisation is negatively correlated with humidity in males (GLM, estimate = \u0026minus;\u0026thinsp;0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, z = -2.28, p\u0026thinsp;=\u0026thinsp;0.02; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), but not with temperature and rainfall (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a-b)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelation of melanisation and gregarine prevalence with monthly average temperature, rainfall, and relative humidity across seasons in female and male \u003cem\u003eI. heterosticta\u003c/em\u003e damselflies.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResponse variable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFixed effect\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEstimate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStd. Error\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ez value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e\u003cb\u003eMelanisation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eMonthly average temperature\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eRainfall\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-2.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eRelative humidity\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-4.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMonthly average temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRainfall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eRelative humidity\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-2.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e\u003cb\u003eGregarine prevalence\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eMonthly average temperature\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRainfall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRelative humidity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMonthly average temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-1.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRainfall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eRelative humidity\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-2.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eGregarine prevalence showed negative correlation with monthly average temperatures in females (GLM, estimate = \u0026minus;\u0026thinsp;0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, z = -1.81, p\u0026thinsp;=\u0026thinsp;0.07; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), but not in males (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In females, gregarine prevalence was positively with rainfall (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) and humidity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), but negatively correlated with rainfall (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) and humidity (GLM, estimate = \u0026minus;\u0026thinsp;0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, z = -2.37, p\u0026thinsp;=\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) in males.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found that females and males had higher melanisation which was correlated with lower gregarine prevalence in summer compared to spring and autumn. Melanisation increased with temperature but decreased with rainfall and humidity, whereas gregarine prevalence was higher in females throughout seasons and weakly negatively correlated with temperature in females. We further found that melanisation is higher in seasons when gregarine prevalence is lower.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSeasonal variation of melanisation and correlation with climatic factors\u003c/h2\u003e\u003cp\u003eOur study showed that melanisation in \u003cem\u003eI. heterosticta\u003c/em\u003e damselflies was higher in summer compared to spring and autumn. Consistent with our findings, immune responses in cricket (\u003cem\u003eAllonemobius socius\u003c/em\u003e) were also higher during warmer months (Fedorka et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Insects are likely able to mount a stronger immune response in summer (see Adamo and Lovett, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), due to greater resource availability, which enables a relatively higher investment in immunity (Hangartner et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kiss et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rivera-Rea et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, food sources for damselflies, such as Diptera, Hymenoptera, and Coleoptera, are more abundant during the summer (Lim et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Increased food availability enhances nutrition, which in turn boosts immune responses in damselflies and other insects (Leung, B et al., 2001; Kiss et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Direct experiments are needed to unlink the effect of temperature from season to understand the influence of temperature and food availability on host fitness under warming conditions.\u003c/p\u003e\u003cp\u003eWe found that climatic factors, such as temperature, rainfall, and humidity, influenced melanisation, with temperature being positively, and rainfall and humidity negatively correlated with melanisation. Our study supports previous findings where higher temperatures enhanced melanisation in tropical species or those adapted to warmer climates, as seen in \u003cem\u003eSepsis thoracica\u003c/em\u003e, \u003cem\u003eGalleria mellonella\u003c/em\u003e, and \u003cem\u003eSarcophaga africa\u003c/em\u003e (Gourgoulianni et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mastore et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Summer temperatures have a positive association with phenol-oxidase (PO) enzyme activity- a key enzyme of melanin production, as shown in the Caribbean termite \u003cem\u003eNasutitermes acajutlae\u003c/em\u003e (Fuller et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and mealworm larvae \u003cem\u003eTenebrio molitor\u003c/em\u003e (Catal\u0026aacute;n et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Similarly, the negative correlation between melanisation and rainfall or humidity was also recorded in the \u003cem\u003eParnassius clodius\u003c/em\u003e butterfly (Zaman et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Drier conditions increase the expression of proPO and consequently increase PO activity in burying beetles (Urbański et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which may explain the observed seasonal variation in melanisation in our study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSeasonality of gregarine prevalence and correlation with climatic factors\u003c/h2\u003e\u003cp\u003eWe found higher gregarine prevalence in spring compared to autumn and summer. Our study corroborates previous findings of higher parasitism in cooler months, i.e., water mite infection in damselflies (Paul et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Robb and Forbes, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and endoparasite infections in mosquitoes during cooler periods (Farner et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, preprint; Trzebny et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Spring\u0026rsquo;s lower temperature, rainfall, and humidity may drive this seasonal shift in gregarine prevalence in damselflies by influencing gregarine life history traits. Spring conditions increase development, density, and infectivity of the free-living stages of gregarines (Paul et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Trzebny et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), a pattern also observed in other endoparasites such as \u003cem\u003eLambornella clarki\u003c/em\u003e in mosquitoes (Ismail et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and gregarine \u003cem\u003eBlabericola migrator\u003c/em\u003e infection in cockroaches (Kolman et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). While lower rainfall and humidity were linked to higher parasitism in damselflies in our study, the correlation was weak, as seen in microsporidian occurrence in mosquitoes (Trzebny et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Wetter conditions may still contribute to increased infection prevalence by increasing parasite abundance (Shearer and Ezenwa, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Trzebny et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), oocyst viability, transmission, or greater host exposure to parasites. Additionally, climatic impact on the host's immune responses can also influence the prevalence of gregarine infection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDoes melanisation reduce gregarine prevalence?\u003c/h2\u003e\u003cp\u003eOur results revealed an inverse relationship between melanisation and infection across seasons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; also see supplementary information: Correlation of melanisation, month temperature, and their interaction with gregarine prevalence). Crickets (Fedorka et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and dung flies (Gourgoulianni et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed stronger immune responses in warmer seasons, which correlated with lower infection risks. Warmer conditions enhanced melanin-producing enzyme activity, aiding parasite clearance. Conversely, damselflies exhibited weaker immune responses in spring, increasing their susceptibility to gregarines, resulting in higher gregarine prevalence. These findings support that seasonal shifts in immune responses shaped infection risks in \u003cem\u003eI. heterosticta\u003c/em\u003e damselflies, higher immune response correlates to lower parasite infection, and vice versa.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, we showed seasonal dynamics in melanisation and gregarine prevalence, with higher melanisation and lower gregarine prevalence in the warmer season. These findings highlight that seasonal climatic fluctuations shape host-parasite interactions, providing valuable insights into patterns of disease dynamics and insect fitness.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatement of diversity and inclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe believe, support, and practice equity, diversity, and inclusion in science and everywhere (R\u0026ouml;\u0026szlig;ler et al., 2020). We come from different countries, nationalities, residency, ethnicity, and cultural backgrounds (Bangladesh, Austria, and Australia), and the neurodivergent community. We represent different career stages (Graduate student, Early career researcher, and Professor). One or more of the authors self-identifies as a member of the LGBTQI+ community and represents a religious minority in science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the Wallumattagal clan of the Dharug nation, the traditional custodians of the lands where Macquarie University is located, and the damselflies were collected. We thank Muhammad Masood and Wenfeng Ren for guiding us during work in the Plant Growth Facility (PGF), Macquarie University. We gratefully acknowledge the support and space provided by Microscopy Unit Manager Sue Lindsay in taking microscopic images of the nylon filament inserts. The authors thank their families, friends, and well-wishers for their empathy and support when most needed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMKK was supported by a Humboldt Postdctoral Fellowship\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data for analysis are deposited in Figshare and can be accessed with the private link: https://figshare.com/s/acd36adb9201843828a2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll codes used to analyse the data of this study are deposited in Figshare and can be accessed via following link: https://figshare.com/s/acd36adb9201843828a2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP- conceived, designed, analysed, executed the experiment, and wrote original manuscript. MTH- analysed the data, executed the experiments, edited the manuscript. MEH- conceived, designed original ideas, edited the manuscript. MKK- conceived, designed original ideas, analysed the data, edited the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdamo SA, Lovett MM (2011) Some like it hot: the effects of climate change on reproduction, immune function, and disease resistance in the cricket \u003cem\u003eGryllus texensis\u003c/em\u003e. J Exp Biol 214(12):1997\u0026ndash;2004. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.056531\u003c/span\u003e\u003cspan address=\"10.1242/jeb.056531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaker III (2023) J.D., Patterns of Gregarine Parasitism in Damselflies (Master\u0026rsquo;s Thesis). 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Nat Montenegrina 7:453\u0026ndash;462\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oecologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oeco","sideBox":"Learn more about [Oecologia](https://www.springer.com/journal/442)","snPcode":"442","submissionUrl":"https://submission.nature.com/new-submission/442/3","title":"Oecologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Insect immunity, encapsulation, host-pathogen interaction, commensalism, seasonality, anthropogenic climate change","lastPublishedDoi":"10.21203/rs.3.rs-7912881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7912881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding how a changing climate influences host-parasite interactions is important to predict disease-driven extinction risks. Insect immune responses are sensitive to seasonal climatic factors such as temperature, humidity, and rainfall. The influence of seasonal climatic fluctuations on insect immune responses and parasite prevalence remains poorly understood. To address this gap, we studied seasonal variation in immune response and endoparasite (protozoan gregarine) prevalence (proportion of infection) in \u003cem\u003eIschnura heterosticta\u003c/em\u003e damselflies. Damselflies may have greater food and nutrition available in warmer seasons; therefore, we predicted higher melanisation and lower gregarine prevalence in warmer months. In accordance with our prediction, we found stronger melanisation and lower gregarine prevalence in summer. We further found that melanisation increased with air temperature and decreased with rainfall and humidity. On the other hand, gregarine prevalence decreased with air temperature and increased with humidity and rainfall in females but not in males. Our study provided evidence that natural seasonal variation in climatic factors impacted host-parasite interactions and infection prevalence across seasons. While short-term warming during favourable seasons may enhance host immune response, long-term or extreme climate change might disrupt host-parasite relationship by altering resource availability, humidity patterns, or insect thermal limits, thereby contributing to seasonal declines in host populations.\u003c/p\u003e","manuscriptTitle":"Seasonal climatic variability shapes immune responses and infection risks in the common bluetail damselfly","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 19:21:31","doi":"10.21203/rs.3.rs-7912881/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-03T13:05:15+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-03T10:20:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-22T14:26:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oecologia","date":"2025-10-21T05:23:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oecologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oeco","sideBox":"Learn more about [Oecologia](https://www.springer.com/journal/442)","snPcode":"442","submissionUrl":"https://submission.nature.com/new-submission/442/3","title":"Oecologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"575c6ed7-f8b8-4480-821d-9dea57da1426","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:30:19+00:00","versionOfRecord":{"articleIdentity":"rs-7912881","link":"https://doi.org/10.1007/s00442-026-05882-w","journal":{"identity":"oecologia","isVorOnly":false,"title":"Oecologia"},"publishedOn":"2026-03-24 16:10:24","publishedOnDateReadable":"March 24th, 2026"},"versionCreatedAt":"2025-11-13 19:21:31","video":"","vorDoi":"10.1007/s00442-026-05882-w","vorDoiUrl":"https://doi.org/10.1007/s00442-026-05882-w","workflowStages":[]},"version":"v1","identity":"rs-7912881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7912881","identity":"rs-7912881","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}