Material composition of the endophytic ovipositor in the damselfly Calopteryx splendens (Odonata, Calopterygidae)

Material composition of the endophytic ovipositor in the damselfly Calopteryx splendens (Odonata, Calopterygidae) Natalia Matushkina a,1 , Stanislav N. Gorb b,2 , Wencke Krings b,c,d,e,*,3 a Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Hlushkova Avenue 2, 03127 Kyiv, Ukraine b Department of Functional Morphology and Biomechanics, Zoological Institute, Kiel University, Am Botanischen Garten 1–9, 24118 Kiel, Germany c Department of Cariology, Endodontology and Periodontology, University of Leipzig, Liebigstraße 12, 04103 Leipzig, Germany d Department of Electron Microscopy, Institute of Cell and Systems Biology of Animals, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany e Department of Mammalogy and Paleoanthropology, Leibniz Institute for the Analysis of Biodiversity Change, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany ARTICLE INFO Keywords: Nanoindentation Cuticle Elemental composition CLSM Biomechanics Ovipositor ABSTRACT Natural selection has favoured the incorporation of ions, including transition metals, in materials of various biological structures susceptible to mechanical fracture to enhance their failure and wear resistance. With regards to insects, only a few taxa have been investigated. The objective of this study was to analyse the biomechanical properties of the ovipositor in the damselfly Calopteryx splendens (Harris, 1780) (Odonata, Zygoptera, Calopterygidae) through nanoindentation and to ascertain the elemental composition gradient within the cuticle using energy-dispersive X-ray spectroscopy. This research represents the first report indicating that the damselfly ovipositor exhibits a gradient in the mechanical properties of the cuticle, with Young’s modulus ranging from approximately 3.0 to 7.0 GPa and hardness from 0.1 to 0.3 GPa. These properties highly correlate with the contents of copper and magnesium, both of which increase in the distal direction. The results also suggests that the mechanical properties of the cuticle are significantly influenced by the degree of sclerotization revealed by confocal laser scanning microscopy. These findings propose that the material properties of the ovipositor cuticle in C. splendens may have adapted to enhance piercing capability and to reduce the risk of structural failure during insertion of eggs in plant substrates. 1. Introduction The insect cuticle is a biological material of a wide range of structural diversity, complex chemical compositions, variable biomechanical properties, and multiple functions (e.g., Vincent, 2002; Vincent and Wegst, 2004; Stamm et al., 2021). In several cuticular structures prone to mechanical fracture, such as ovipositors, natural selection has favoured the incorporation of ions, including zinc, manganese, and iron, to enhance the hardness and wear resistance of the cuticle. Scanning electron microscopy combined with energy dispersive X-ray analysis has been used to examine the differences in metal composition observed in the ovipositor of cicadas (Lehnert et al., 2019) and certain Hymenoptera (Vincent and King, 1995; Quicke et al., 1998; Quicke et al., 2004; Polidori et al., 2013; Kundanati and Gundiah, 2014; Baumann et al., 2018), as well as in the secondary ovipositor of flies (Polidori and Wurdack, 2019). The distribution of metals in the ovipositor is generally similar, with the highest concentrations observed in the apical region, where the cutting serrations are located (Vincent and King, 1995; Quicke et al., 1998; Polidori et al., 2013; Kundanati and Gundiah, 2014; Baumann et al., 2018), but the gradients in mechanical properties remain largely unclear. Odonata are among the most ancient winged insects (Kohli et al. 2021; Osozawa and Nel, 2024). Representatives of the group of so-called ’endophytic’ Odonata (Zygoptera, Anisozygoptera and Anisoptera: Aeshnidae) oviposit in a plesiomorphic manner by inserting eggs into slits in living and dried plant tissues using their ovipositors (Matushkina * Corresponding author at: Department of Functional Morphology and Biomechanics, Zoological Institute, Kiel University, Am Botanischen Garten 1–9, 24118 Kiel, Germany. E-mail address: [email protected] (W. Krings). 1 https://orcid.org/0000-0002-5426-8016. 2 https://orcid.org/0000-0001-9712-7953. 3 https://orcid.org/0000-0003-2158-9806. Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys https://doi.org/10.1016/j.jinsphys.2025.104813 Received 8 April 2025; Accepted 17 April 2025 Journal of Insect Physiology 163 (2025) 104813 Available online 17 April 2025 0022-1910/© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). and Stetsun, 2025). These endophytic ovipositors are characterised by sharply pointed valves (=gonapophyses) with serrations on their exposed surfaces, which serve to facilitate the penetration process (Klass, 2008; Matushkina, 2008; Matushkina and Lambret, 2011). However, the material properties of ovipositors in Odonata have not been investigated so far. The main objective of this study was to describe the biomechanical properties of the ’endophytic’ ovipositor in a damselfly Calopteryx splendens (Harris, 1780) (Odonata, Zygoptera, Calopterygidae) through nanoindentation and to ascertain the elemental composition gradient within the cuticle using energy-dispersive X-ray spectroscopy. We discuss potential correlations between biomechanical characteristics and sclerotization patterns of the ovipositor cuticle revealed by confocal laser scanning microscopy, and then compare our findings with cuticle characteristics retrieved from other insect groups to discuss the structural, biomechanical, functional and evolutionary implications of insect cuticle adaptation to specific mechanical loads. 2. Material and methods 2.1. Specimens and documentation One adult female of Calopteryx splendens (collected by Dmytro Lukashov on 01/06/2005 in the village of Nedanchichi, Chernihiv Oblast, Ukraine) was used for scanning electron microscopy (SEM). Four additional adult females (one collected by Lyudmila Garmanchuk on 10/ 08/2024 in the village of Obolonne, Chernihiv Oblast, Ukraine; three by Friedrich Sick on 01/06/1965 at Schmalstede (Eider), Germany) were used for confocal laser scanning microscopy. Four adult females (one collected by Stanislav N. Gorb in 07/1983 in Sudak, Crimea, Ukraine; three by Friedrich Sick on 01/06/1965 at Schmalstede (Eider), Germany) were used for nanoindentation and energy dispersive X-ray spectroscopy. All insects were air dried and stored in paper envelopes prior investigation. 2.2. Scanning electron microscopy For documentation with scanning electron microscopy (SEM), the terminal abdominal segments of one female were dissected in the median plane in order to isolate the ovipositor valves. The cuticle components were macerated in 10 % KOH, rinsed in distilled water, dehydrated in a graded ethanol series and in acetone, critical point dried (OM CPD 7501), mounted on aluminium stubs with double sided adhesive carbon tape, and coated with gold–palladium (OM-SC7640). Scanning electron micrographs of the ovipositor valves were taken using a Zeiss EVO 50 SEM (SNSD, Dresden). The background of ovipositor valves on resulting images was corrected using brightness/contrast in Adobe Photoshop 26.0.0. 2.3. Confocal laser scanning microscopy To visualize the cuticle autofluorescence with confocal laser scanning microscopy (CLSM), four specimens were prepared. First, the ovipositor was carefully removed from the specimens and cleaned by a short ultrasonic bath in 70 % EtOH. Each valve was positioned on glass slides and encircled with a plastic reinforcement ring. The rings were filled with glycerine (≥99.5 %, water-free; Carl Roth GmbH and Co. KG, Karlsruhe, Germany) and sealed with a glass cover slip. The valves were imaged using a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany), following the protocol outlined by Michels and Gorb (2012). Four solid-state lasers with wavelengths of 405 nm, 488 nm, 555 nm, and 639 nm were employed. Emission filters, either bandpass or longpass, were used to transmit light at wavelengths of 420–480 nm, ≥490 nm, ≥560 nm, and ≥640 nm. For multi-colour imaging, autofluorescence from the 405 nm laser was assigned blue, the 488 nm laser was assigned green, and both the 555 nm and 639 nm lasers were assigned to 50 % red (as per Michels and Gorb, 2012). The images were then merged using maximum intensity projection with the Zeiss Efficient Navigation (Zen) software (Carl Zeiss MicroImaging GmbH, Jena, Germany). Valves from three individuals were scanned connected to each other, one side in medial and one in lateral view. Valves from the other specimen were separated and scanned individually in medial and lateral view. Here, the same settings were used, which enables comparison of the autofluorescence signals between images. 2.4. Nanoindentation To assess the mechanical properties of the exocuticle under native conditions, nanoindentation tests were conducted on the valves from the specimens used for CLSM. The upper and lower valves were carefully separated, dried at room temperature, and then attached to nanoindentation sample holder with wax, which hardened under room temperature. One set was attached by its lateral side, so that the medial side was on display, and one set was attached by its medial side, so that the lateral side was on display. For control, we also studied two styli of two lateral valves (gonocoxite 9) with nanoindentation and elemental analysis, each at 30 localities. The stylus was treated with the same protocol as the valves. The cuticle structures were rewetted prior to testing, as water is known to significantly influence mechanical properties, generally lowering both the Young’s modulus (E; elastic modulus) and hardness (H) values (Klocke and Schmitz, 2011). To rehydrate the samples, they were placed in a sealed plastic box containing wet paper towels for two hours, creating a humid environment. Nanoindentation was performed using an SA2 nanoindenter (MTS Nano Instruments, Oak Ridge, Tennessee, USA) equipped with a Berkovich indenter tip and a dynamic contact module (DCM) head. The H and E values were determined from force-distance curves using the continuous stiffness mode, with the same settings (e.g., a Poisson’s ratio of 0.3) as in previous studies on chitinous radular teeth (Krings et al., 2019; Gorb and Krings, 2021) or on insect cuticle (Krings and Gorb, 2023; Krings et al., 2024; Klunk et al., 2024; Birkenfeld et al., 2024; Matsumura et al., 2024; Roze et al., 2024). All tests were carried out under normal room conditions, with each indent and corresponding force-distance curve manually controlled. E and H values were measured at penetration depths of 500–900 nm to ensure the exocuticle was tested. Approximately 100 values were collected at various indentation depths for each site, and these were averaged to produce one H and one E mean value per indent. A total of 28–30 locations were tested per valve and side (along the length of each valve, see Fig. 2 for the specific locations). 2.5. Energy dispersive X-ray spectroscopy We used energy dispersive X-ray spectroscopy (EDX, EDS) to analyse the sites previously tested by nanoindentation. After indentation, the samples were carefully detached from the wax and cleaned by a short ultrasonic bath in 70 % EtOH, mounted on SEM sample holders with double sided adhesive carbon tape and coated with a 5 nm platinum layer, following established procedures (Krings and Gorb, 2023; Krings et al., 2024; Klunk et al., 2024; Birkenfeld et al., 2024; Matsumura et al., 2024; Roze et al., 2024). Elemental composition was then analysed using a Zeiss LEO 1525 SEM equipped with an Octane Silicon Drift Detector (SDD) (TEAM microanalysis system; EDAX Inc., New Jersey, USA). Due to the indent from nanoindentation, we could not test the exact same locality, but we tested the region next to the indent. Each analysis was performed under consistent settings (20 kV acceleration voltage, 15 mm working distance, 60 µm lens opening, 30 s measuring time per point). Copper calibration was done before each analysis, meaning the results are semi-quantitative. Small regions (4x4 µm) were analysed to obtain detailed elemental data. N. Matushkina et al. Journal of Insect Physiology 163 (2025) 104813 2 In the cuticle, we determined and measured the content (atomic % and weight %) to two decimal places of aluminium (Al), carbon (C), calcium (Ca), sodium (Cl), copper (Cu), fluorine (F), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), nitrogen (N), sodium (Na), oxygen (O), phosphorus (P), platinum (Pt), sulphur (S), silicon (Si), and zinc (Zn). Elements such as H, C, N, and O (found in chitin and proteins), the coating element Pt, and the polishing powder component Al were not discussed in the results. Due to the overlap of phosphorus (P) and platinum (Pt) peaks in the spectrum, the software was unable to distinguish between the two, so P content was evaluated in combination with Pt (PPt). Although sputter coating introduced some artefacts, we followed this protocol to confirm the success of the measurements. If an extremely high or no Pt content was detected, the measurement was excluded. To estimate the proportion of P, we measured 15 areas of pure epoxy, yielding a platinum content of 0.12 ±0.02 atomic %. Additionally, tests on the epoxy confirmed the absence of other relevant elements (e.g., Ca, Si, Mg, Mn, Zn), ruling out artefacts from sample preparation. 2.6. Statistical analysis Data on mechanical properties and elemental composition was plotted in graphs with JMP Pro, Version 14 (SAS Institute Inc., Cary, USA). Relationships between parameters were visualized and correlation coefficients calculated. 3. Results 3.1. Autofluorescence The ovipositors consisting of the lower valves (=gonapophyses 8) and upper valves (=gonapophyses 9) possessed cutting ridges and teeth in an apical half on the lateral (=external) surfaces (Fig. 1A, B). We recorded strong autofluorescence in the apical regions, shown as a red colour, when excited with both the 555 nm and 639 nm lasers (Fig. 1C, D). More blueish-yellow-greenish colours of the basal regions of the ovipositors, where there are no cutting devices, indicated an autofluorescence under 405 nm and 488 nm laser wavelengths (Fig. 1C, E, F, G). The lateral sides of the lower valves showed generally a more uniform and stronger red colouration than the medial sides of the structure (Fig. 1 E, F). In lateral view, the bases of the lower valves exhibited a strong blue signal, as answer to the 405 nm laser, fading ventrally and posteriorly first into green, as answer to the laser of 488 nm wavelength, and then posteriorly into the strong red signal (Fig. 1E). In medial view, the lower valves showed generally a strong green signal with an exception of their red coloured dorsal edges along most of its length, where the groove-like parts of the olistheter articulations, the aulax, Fig. 1. Scanning electron microscopy (A, B) and confocal laser scanning microscopy (C-H) images of the ovipositor of Calopteryx splendens (Odonata, Calopterygidae). A, E. Lateral view on the isolated lower valves. B, G. Dorso-lateral and lateral views on the isolated upper valves, respectively. C. Lateral view on the interconnected lower and upper valves. D. Medial view on the interconnected lower and upper valves. F. Medial view on the isolated lower valve. H. Ventro-medial view on the isolated upper valve. E-H. Images were taken with the same settings (i.e., laser intensities, etc.) and autofluorescence signals are thus comparable. Abbreviations: A, anterior direction; al, aulax (groove-like ventral part of the olistheter articulation); D, dorsal direction; fhs, field of sensilla on expanded base of upper valve; lv, lower valve (gonapophysis 8); P, posterior direction; uv, upper valve (gonapophysis 9); V, ventral direction. Arrowheads indicate the compact area of the green autofluorescing cuticle on the apical region of the lower valve (see text). Scale bars: 200 µm. N. Matushkina et al. Journal of Insect Physiology 163 (2025) 104813 3 were located (Fig. 1F, al). The ventral edges of the lower valves showed extended cuticle regions that autofluoresced predominantly in green color (Fig. 1F). In the upper valves, both the ventro-lateral and dorsomedial edges showed a strong red signal, whereas the bases of the valves exhibited a strong blue one, associated with the presence of a field of sensilla on the external surface (Fig. 1C, G, fhs). The medial surfaces of the upper valves showed a blue to greenish-blue signal (Fig. 1G, H). The apices of all valves exhibited the strongest red colorations from both the lateral and medial sides. A small and clearly defined cuticle area, which autofluoresced green, was observed on the outer surface of each lower valve subapically (Fig. 1C, E, arrowhead). 3.2. Elemental composition The detected elements included C (carbon), Ca (calcium), Cl (chlorine), Cu (copper), F (fluorine), Fe (iron), K (potassium), Mg (magnesium), Mn (manganese), N (nitrogen), O (oxygen), PPt (phosphorus and platinum), S (sulphur), Si (silicon), and Zn (zinc). Their proportions were measured accordingly. Among these, the following elements were abundant in each ovipositor, listed from the highest to lowest proportions: F (means: 1.06–1.11 atomic %), Mg (0.65–0.88), Cu (0.41–0.54), PPt (0.26–0.27), Cl (0.03–0.04), Zn (0.03), Fe (0.03), Mn (0.02), Si (0.02), Ca (0.01), K (0.01), and S (0.00–0.011) (refer to Supplementary Table 1 for the values for each locality and each ovipositor). In the control structures, the stylus, we found F with 1.03 ±0.25 atomic %, followed by PPt (0.21 ±0.14), K (0.24 ±0.15), S (0.13 ± 0.10), Mg (0.11 ±0.07), Cu (0.06 ±0.03), Fe (0.04 ±0.03), Cl (0.04 ± 0.03), Ca (0.04 ±0.02), Zn (0.03 ±0.02), Mn (0.02 ±0.02), and finally Si (0.02 ±0.01). 3.3. Mechanical properties Young’s modulus (E; elastic modulus) quantifies the stiffness of a solid material, describing the relationship between tensile stress and axial strain and indicating the material’s ability to transmit force, which Fig. 2. A-B. Schematic illustrations of the localities for nanoindentation and elemental analysis in medial and ventral view. C. Results from nanoindentation (Young’s modulus E, in GPa) for each specimen A–D. D. Results from elemental analysis for cupper (Cu) and magnesium (Mg) content for each specimen A–D. The tested localities are designated in the schematic illustrations (A–B). For values, see Supplementary Table 1. Abbreviations: A, anterior direction; D, dorsal direction; P, posterior direction; V, ventral direction. N. Matushkina et al. Journal of Insect Physiology 163 (2025) 104813 4 affects its puncturing behavior and resistance to failure (for a review on puncture mechanics, see Anderson, 2018). Hardness (H), on the other hand, measures resistance to local plastic deformation caused by the normal force. In the tested ovipositors, E ranged from 3.01 to 8.06 GPa and H from 0.10 to 0.29 GPa (refer to Fig. 2 and to Supplementary Table 1 for the values for each locality). In the lower valve, H ranged from 0.10 to 0.27 GPa, while E ranged from 2.85 to 7.27 GPa. In the upper valve, H ranged from 0.10 to 0.29 GPa, while E ranged from 2.72 to 8.06 GPa. E of the control structure, the stylus, was found to be 3.55 ±0.12 GPa and H to be 0.14 ±0.17 GPa. In general, the tips of the upper and lower valves were harder and stiffer than the remaining areas of the valves except for the medial upper valve basis, which showed similar E and H values as the tip (Fig. 2, Supplementary Table 1). The lateral cuticle of the upper valve was softer and more flexible in its middle part if compared to the basis and tip. The medial cuticle of the upper valve (tested at the rail-like rachis of the olistheter) was stiffer and harder than the lateral cuticle. In the lower valve, this pattern was the opposite; the lateral cuticle was harder and stiffer than the medial cuticle. 3.4. Correlation between parameters Most parameters showed low, moderate or negative correlations (see Supplementary Table 2 for correlation coefficients) or relationships (see Fig. 2 and Supplementary Fig. 1). There was a very high positive correlation between H and E (r =0.97 for all specimens together; r = 0.96–0.99 for each individual specimen). Both mechanical properties also correlated very highly with the concentration of Mg (with H: r = 0.73, with E: 0.73 for all specimens together; with H: r =0.66–0.83, with E: 0.66–0.84 for each individual specimen) and Cu (with H: r =0.90, with E: 0.89 for all specimens together; with H: r =0.86–0.96, with E: 0.86–0.96 for each individual specimen). In the control structure, the stylus, E and H correlated highly positively with 0.96. Both mechanical properties did not correlate with any element. 4. Discussion Odonata are exclusively carnivorous insects, nevertheless they provide one of the earliest examples of plant-insect interactions, demonstrating the persistence of the long-standing non-trophic relationship between insects and plants (Romero-Lebr´ on et al., 2022). A notable characteristic of this is the behavioural similarity exhibited by fossil Odonata and their modern relatives with regard to egg-laying habits when they laid their eggs in plants, i.e. endophytically (Romero-Lebr´ on et al., 2023). The process of endophytic oviposition in Odonata involves the cutting of plant tissues and insertion of eggs into prepared slits. This behaviour has been recorded in representatives of all three suborders of modern Odonata, which have been documented to oviposit in live and rotten plants (Matushkina and Gorb, 2002a). Adult Odonata are closely associated with waters, because of their aquatic larvae. This means that the broad spectrum of fully terrestrial, semiaquatic, and aquatic plant species is available for ovipositing females. Some species have been observed to have preferences in selecting plant materials for oviposition (Martens, 1992, 1993, 1994, 2001; Wildermuth, 1993; Grunert, 1995; Lambret et al., 2015a, 2015b). How structure and material properties of the ovipositor are combined in a specific morpho-functional adaptation for oviposition in plant substrates of different biomechanical properties has remained poorly understood in Odonata, despite extensive research on this topic (e.g. Matushkina and Gorb, 2002b, 2007; Lambret et al., 2015a, 2015b; Matushkina et al., 2016). Plant tissue penetration experiments demonstrated an existence of significant positive correlation between mechanical properties of the ovipositor and preferred oviposition substrates (Matushkina and Gorb, 2007a,b). In these experiments, the bending stiffness of the upper valve of the ovipositor was estimated in force measurements carried out with a load cell force transducer. Among seven studied damselfly species, the target species of this research, Calopteryx splendens, was observed to have the lowest estimates of the ovipositor bending stiffness, which was found to correlate with using relatively soft aquatic plant substrata for egg laying, like Nuphar lutea leaves. However, material composition and mechanical properties of the ovipositor cuticle of Odonata were not estimated before present study. The arthropod cuticle is a composite material composed primarily of chitin and associated proteins, incorporating various components, which together determine the material properties which results in both stiff and hard or soft and flexible regions. Its mechanical properties vary significantly, ranging from KPa to GPa, influenced by factors such as the specific region tested and the water content, as water plays a crucial role in modulating these properties (Vincent and Wegst, 2004; Klocke and Schmitz, 2011; Stamm et al., 2021; Krings et al. 2021a, 2021b). Estimates of the mechanical properties of the ovipositor cuticle by the nanoindentation have been available for only a few insect taxa. Our ovipositor sample with a Young’s modulus (E) of ~ 3–7 GPa and a hardness (H) of ~ 0.1–0.3 GPa was especially hard and stiff in comparison with ovipositors of both the polinatory and parasitic fig wasps with an E of ~ 0.5–1.7 GPa (Kundanati and Gundiah, 2014). The measured properties of the damselfly ovipositor are similar to those of the mature dry locust ovipositor with an E of ~ 5 GPa, who dig the soil to lay eggs in (Das et al., 2022), and the honeybee stinger with an E of ~ 7–10 GPa and a H of ~ 0.3–0.5 GPa, who use the stinger as a defence device (Miao et al., 2020). However, in comparison with mandibles of a predatory dragonfly larva, with an E of ~ 9 GPa and a H of ~ 0.9 GPa (Kundanati et al., 2021), the ovipositor sample we tested was softer and more flexible. Thus, the ovipositor cuticle of Calopteryx splendens appeared to be stiff and hard enough to cut the plant tissues and to deposit eggs in the prepared plant tissue slits. The mechanical properties of the cuticle, as reviewed by Politi et al. (2019), are influenced by factors such as the degree of sclerotization from quinone binding (Hopkins and Kramer, 1992; Andersen, 2010), the chitin microstructure, mineral content, and the presence of proteins, transition metals, or halogens (e.g., Hillerton and Vincent, 1982; Schofield et al., 2002). Mechanical property gradients within the cuticle can arise from these varied sources (see Liu et al., 2017, for a review on these gradients and their origins). The degree of sclerotization and its impact on mechanical properties have been studied across various arthropod cuticle structures. Using autofluorescence signals obtained through CLSM following Michels and Gorb’s (2012) protocol, researchers commonly assess cuticle regions with specific material compositions: sclerotized, stiff cuticle is associated with signals obtained from 555 nm and 639 nm laser excitation, which appear as red in CLSM images. Weakly-sclerotized chitin shows signals from 488 nm excitation, depicted in green, indicating regions that are flexible and relatively tough. Resilin-rich regions, containing elastic and flexible proteins like resilin (Andersen and Weis-Fogh, 1964; Michels et al., 2016) seems to emit strong autofluorescence with 405 nm excitation, seen as blue in CLSM images. With CLSM method, the presence of resilin cannot be entirely proved, but autofluorescence in the UV/blue region of wavelengths provides important information about the presence of non-crosslinked cuticle presumably supplemented by resilin. This method is widely used for detecting soft and flexible regions of the insect cuticle (see below). Regions with a mixture of resilin and weakly-sclerotized chitin display overlapping color, often appearing brown, yellow, or pink. This autofluorescence-based approach (Michels and Gorb, 2012) has been applied to a variety of chitinous structures, including insect endocuticle (Wang et al., 2019), wings (Ma et al., 2022), foot attachment structures (Peisker et al., 2013), thoracic structures (Casey et al., 2022), antennae (Saltin et al., 2019), genitalia (Stetsun et al., 2019; Stetsun and Matushkina 2020; Matsumura et al., 2021b), and mouthparts N. Matushkina et al. Journal of Insect Physiology 163 (2025) 104813 5 (Weihmann and Wipfler, 2019; Matsumura et al., 2021a; Lehnert et al., 2021; Sun et al., 2021; Wei et al., 2022). Also, dragonfly structures have been examined with this protocol (Büsse and Gorb, 2018; Preuss et al., 2024). This relationship between autofluorescence signals and material properties has previously been validated in ladybird beetle leg attachment devices (Peisker et al., 2013), chitinous radular teeth (Krings et al., 2022), parameres of male genitalia (Matsumura et al., 2024), and insect mouthparts (Krings and Gorb, 2023; Krings et al., 2024; Roze et al., 2024). Consistent with these findings, this study also confirms that blue to green regions are softer and more flexible (e.g., the basis of the lower valve) compared to red-coloured regions, which are stiffer and harder (e. g., the tips of the valves), which indicates that the mechanical properties are highly influenced by the degree of sclerotization. We detected a compact area of the green autofluorescing cuticle, which is surrounded by the red autofluorescing cuticle, on the outer surface of the lower valve near the apex (Fig. 1C,E, arrow). In parasitoid wasps, the area of softer cuticle flanked by areas of stiffer cuticle on the lower valves has been considered as part of the mechanism of controlled steering of the ovipositor during its movement within the substrate (Cerkvenik et al., 2019). However, such an interpretation seemed unlikely for Calopteryx splendens due to differences in movement pattern of ovipositor valves between Odonata and Hymenoptera, and therefore this finding deserved further investigation. Unlike other arthropod groups, such as crustaceans, the insect cuticle contains relatively low levels of inorganic material. However, calcium and magnesium may exist in crystalline form and contribute to biomineralization, as biomineralization has been recently determined in ant and beetle exoskeletons (Leschen and Cutler, 1994; Li et al., 2020). Insect cuticle is often enriched with transition metals (copper, iron, manganese, zinc) along with colocalized halogens (chlorine) and alkaline earth metals (calcium, magnesium), especially in structures susceptible to structural failure or wear, like mouthparts (Hillerton and Vincent, 1982; Hillerton et al., 1984; Quicke et al., 1998; Fawke et al., 1997; Schofield et al., 2002, 2021; Morgan et al., 2003; Cribb et al., 2008a, 2008b; Jorge et al., 2017; Polidori et al., 2020; Laiolo et al., 2021; Lehnert et al., 2022; Reiter et al., 2023; Krings and Gorb, 2023; Krings et al., 2024; Birkenfeld et al., 2024; Klunk et al., 2024; Roze et al., 2024; Lehnert, 2024; Polidori et al., 2024; Betz et al., 2024; Sevarika and Romani, 2024; Wegst et al., 2024), claws (Zhang et al., 2019), parameres of male genitalia (Matsumura et al., 2024), or ovipositors (Vincent and King, 1995; Quicke et al., 1998, 2004; Polidori et al., 2013; Kundanati and Gundiah, 2014; Baumann et al., 2018; Lehnert et al., 2019; Polidori and Wurdack, 2019). These metals could potentially contribute to cuticle durability as they could act as cross-links, which are known to reinforce structures (Schofield, 2001, 2005; Lichtenegger et al., 2003; Birkedal et al., 2006; Degtyar et al., 2014; Jorge et al., 2017; Schofield et al., 2021). In many studies, notably zinc, manganese, calcium, and magnesium, have been found to enhance hardness and thus wear resistance, but also stiffness and thus the reduction of structural failure (Hillerton et al., 1982; Hillerton and Vincent, 1982; Edwards et al., 1993; Schofield et al., 2002, 2021; Cribb et al., 2008a; Andersen, 2010; Vega et al., 2017; Zhang et al., 2019; Kundanati et al., 2020; Johnston et al., 2022; Krings and Gorb, 2023; Krings et al., 2024, Matsumura et al., 2024; Klunk et al., 2024; Birkenfeld et al., 2024; Roze et al., 2024). In the ovipositor of Calopteryx splendens, significant correlations were found between copper and magnesium content and the mechanical properties, with correlation values ranging from 0.83 to 0.96 (refer to Fig. 2 for relationships between copper and magnesium and mechanical properties; refer to Supplementary Table 2 for correlation coefficients). In our control structure, the stylus, having E values of 3.47 ±0.13 GPa and H values of 0.07 ±0.11 GPa, no elements correlated with the mechanical properties, indicating that copper and magnesium are important for increasing mechanical property values. The presence of higher magnesium in the tip is similar to observations made in the ovipositors of certain Cicadoidea and Diptera (Lehnert et al., 2019; Polidori and Wurdack, 2019), as well as the presence of higher amounts of copper in their tips seems similar to the ovipositors of gall-inducing wasps (Polidori et al., 2013). Copper was also detected in stingers of Polistes wasps (Vespidae) and a honeybee Apis mellifera (Apidae) stings in concentration <0.3 wt% (Baumann et al., 2018). In wood drilling wasps Sirex noctilio Fabricius, 1773 (Siricidae) and Megarhyssa nortoni (Cresson, 1864) (Ichneumonidae), transition metals (manganese and zinc, respectively) were found in those regions of their ovipositors that would be subjected to the highest levels of abrasion (Vincent and King, 1995). In several aculeate Hymenoptera, minor concentrations of iron, zinc, manganese, titanium and copper, ranging from 0.02 to 1.5 wt%, were predominantly detected in the distal region of the stinger, especially along the longitudinal edges. Additionally, a marginally positive relationship between the presence of barbs and the concentration of zinc was observed (Baumann et al., 2018). A correlation between magnesium and an increase in mechanical property values has been previously documented for mantis mandibles (Roze et al., 2024). Here, our correlations suggest that copper and magnesium could contribute to strengthening the cuticle in the ovipositor of Calopteryx splendens, possibly through cross-linking mechanisms (e.g. Degtyar et al., 2014; Liu et al., 2017, Politi et al., 2019). However, it remains unclear, whether these elements serve specifically as cross-linkers or are present in a crystalline form within the cuticle, as EDX analysis does not provide information about their bonding state. This study is the first to report that the damselfly ovipositor exhibits a gradient in the stiffness and hardness of the cuticle and in content of some transition metals, both increasing in the distal direction. This may imply an existence of adaptation of the cuticle material properties to the enhancement of piercing abilities of the ovipositor and at the same time reduction of structural failure which may occur during the cutting of oviposition substrates. As the present study has been conducted on one species of damselfly, the results may not apply to all Odonata. Therefore, it will be important to examine, whether similar effects exist in other species, particularly those that lay eggs in different types of substrates, like stiffer (such as lignified tree branches) vs. softer (such as moss clusters) plant materials, vegetation vs. soil, as well as to investigate whether taxonomically mediated differences exist between Odonata lineages. Thus, our findings should be regarded as requiring further investigation with a larger sample size of odonate taxa. Funding statement. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 7 Declaration of generative AI in scientific writing. No Ai was used. 5. Ethics Not applicable. CRediT authorship contribution statement Natalia Matushkina: Conceptualization, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft. Stanislav N. Gorb: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Software, Writing – review & editing. Wencke Krings: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. Declaration of competing interest The authors declare the following financial interests/personal N. Matushkina et al. Journal of Insect Physiology 163 (2025) 104813 6 relationships which may be considered as potential competing interests: Wencke Krings reports article publishing charges was provided by University of Hamburg. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We are grateful for the help of Elke Woelken, Institute of Cell and Systems Biology of Animals, Universit¨ at Hamburg, Germany, for her support with the SEM, and Greta Huttegger, Leibniz Institute for the Analysis of Biodiversity Change, Hamburg, Germany, for the analysis of the elemental composition. We would like to thank Dmytro Lukashov and Lyudmila Garmanchuk, Taras Shevchenko, National University of Kyiv, Ukraine, and Thies Büscher, Kiel University, Germany, for providing dragonfly specimens used in this study. We are thankful for the constructive comments of the anonymous reviewers and the editor. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jinsphys.2025.104813. 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