1 Introduction
Larval insect infestations, particularly those caused by the house longhorn beetle, Hylotrupes bajulus L., present a significant concern not only in entomology but also in wood protection and biological degradation, underscoring the urgency of developing alternative strategies for safeguarding wooden structures against H. bajulus and similar pests. Native to Europe and now found globally, H. bajulus larvae are known to inhabit coniferous woods like Pinus sp., Abies sp., and Picea sp., yet instances in non-coniferous species such as Populus sp., Quercus sp., Acacia sp., and Salix sp. have been recorded (Vives 2000). Despite this adaptability, their predominant infestation in low-moisture, coniferous environments highlight their significant impact, particularly on European softwood structural timber, as extensively documented by Hickin (1964). This pest’s capacity to cause extensive damage to such woods has made it a focal point in numerous entomological studies (Zarco 1935; Ruiz 1942; Duffy 1953, 1957; Demelt 1966).
In the world of arthropods, particularly insects, chitin serves as a key structural component of the exoskeleton, providing protection and mechanical strength (Andersen 2010). Chitin, a polysaccharide consisting of linear chains of N-acetylglucosamine, has been well studied for its unique properties and potential applications in various fields such as biotechnology, agriculture, forestry, and medicine (Ehrlich 2019; Chen et al. 2012; Degtyar et al. 2014; Li et al. 2015; Tadayon et al. 2020; Khare et al. 2021; Schofield et al. 2021). Chitin is characterized by its versatility, enabling organisms to adapt their exoskeletons for various functions. In certain insects, such as beetles and ants, the mandibles (biting structures) demonstrate an enhanced mechanical performance due to the presence of metal ions bound to the chitin structure (Andersen 2010). Metal-reinforced chitin, or biomineralized chitin, demonstrates increased hardness, stiffness, and wear resistance in comparison to its non-mineralized counterpart, enabling these insects to efficiently cut, crush, and chew various materials (Broomell et al. 2006; Lichtenegger et al. 2010; Amini and Miserez 2013; Kundanati et al. 2020; Tadayon et al. 2020).
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The specific metal ions commonly found in metal-reinforced chitin of insect mandibles are primarily zinc (Zn) and manganese (Mn) (Lichtenegger et al. 2010; Zhang et al. 2011; Kundanati et al. 2020). These metal ions bind to the chitin structure, leading to an enhancement of the material properties, such as increased hardness, stiffness, and wear resistance. Zinc and manganese metal ions are the most well-studied in the context of metal-reinforced chitin, but some species of insects have also been found to incorporate other metal ions, such as iron (Fe), copper (Cu), or magnesium (Mg), in their chitin structures or exoskeletons for various purposes (Schofield et al. 2003, 2021; Broomell et al. 2006, 2008; Holten-Andersen et al. 2009; Andersen 2010; Lichtenegger et al. 2010; Zhang et al. 2011, 2012; Amini and Miserez 2013; Xu 2013; Degtyar et al. 2014; Kundanati and Gundiah 2014; Kundanati et al. 2019, 2020; Martínez et al. 2020). The presence and concentration of these metal ions in insect chitin structures depend on the specific species, their ecological niche, and the intended function of the reinforced cuticle. Furthermore, the distribution of metal ions may vary regionally within an insect’s exoskeleton, being more concentrated in areas that have higher requirements for mechanical strength or wear resistance, such as mandibles or other heavily-used body parts (Schofield et al. 2003; Broomell et al. 2006; Lichtenegger et al. 2010; Zhang et al. 2011, 2012; Amini and Miserez 2013; Kundanati and Gundiah 2014; Liu et al. 2014; Kundanati et al. 2019, 2020).
Among the various adaptations found in xylophagous insects, such as wood-boring beetles, termites, and certain species of ants like carpenter ants, metal-reinforced chitin in their mandibles plays a crucial role in enabling them to withstand the mechanical demands of their feeding behavior. These adaptations are essential for the survival and efficiency of these insects in their natural habitats (Hillerton and Vincent 1982; Hillerton et al. 1982; Wang et al. 2017).
The properties of metal-reinforced chitin have led to research efforts dedicated to developing biomimetic materials for various industrial applications, including the creation of more durable cutting tools and protective coatings that can better withstand mechanical wear and stress (Tong et al. 2009; Liu et al. 2014; Du and Hao 2018; Schleicher et al. 2019; Zhao et al. 2020).
Schofield et al. (2003) found that zinc and other elements are incorporated into the cuticular tools of arthropods after cuticle formation, through a network of nanometer-scale canals. Zinc mostly accumulates after eclosion, while manganese, calcium and chlorine accumulate before or during pre-ecdysial tanning. If the insect’s metabolic processing of these metallic elements could be restrained or blocked, the tissues responsible for wood cutting would not be strengthened (Broomell et al. 2006). This would prevent the larva’s wood-cutting and feeding ability, consequently safeguarding the wood from attacks by xylophagous insects. To understand the potential of this approach, it’s essential to delve deeper into the mechanisms by which metals are isolated and bound. The significance of these metallic cations extends beyond xylophagous insects. Jellison et al. (1997) studied the role that cations play in the colonization and biodegradation of wood by fungi. Wood-inhabiting fungi require cations internally for key metabolic pathways controlling growth, development, cell function, and reproduction. Limiting the fungi’s access to these cations could prevent them from colonizing and degrading the wood cell matrix.
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Metal sequestration is a process that involves capturing and isolating metal ions. One method of metal sequestration involves the use of chelating agents, which are molecules capable of binding to a metal ion via multiple coordination sites (Sanz-Luque et al. 2020). Chelating agents can effectively sequester metal ions, such as zinc and manganese, by forming stable complexes with them, preventing them from reacting with other compounds and causing deleterious effects (Pavlíková et al. 2002; Zhang et al. 2019; Regan-Smith et al. 2022). This ability to bind with metal ions serves various purposes, including preventing unwanted reactions and interactions. Two common chelating agents for zinc and manganese sequestration are EDTA (ethylenediaminetetraacetic acid) and DTPA (diethylenetriaminepentaacetic acid) (Yue et al. 2018). They form stable complexes with these metals (Zn and Mn), rendering them inactive and reducing their bioavailability. The resulting complexes can be separated and removed from various systems, including water, soil, or industrial processes (Broemmelsiek et al. 2021).
The DTPA (CAS number 67-43-6) is an aminopolycarboxylic acid that demonstrates a strong affinity for cationic metals (Sillanpää 1997). The conjugate base of DTPA potentially acts as an octodentate ligand, signifying its capacity to form up to eight coordination bonds with a metal ion (Sillanpää 1997). As a chelating agent, DTPA will surround a metal ion by forming coordination bonds and, after complexing with a metal, it still retains the ability to bind to other reactants (Sillanpää 1997). The DPTA forms stable complexes with various bivalent and trivalent metal ions such as Fe3+, Zn2+, and Mn2+. Due to this property, it is utilized in medicine for the treatment of heavy metal poisoning, in agriculture to remove Zn, and in the pulp and paper industry to inactivate metal ions (Sillanpää 1997). In the field of wood preservation, some studies have used it in conjunction with other compounds to combat decay fungi (Xu et al. 2022).
This study aims to evaluate the viability of using DTPA as both a preventive and curative treatment against the house longhorn beetle and to determine the effective dosage.
2 Materials and methods
Two trials were conducted; the first to study the mortality of newly hatched Hylotrupes bajulus L. larvae in wood treated with different doses of DTPA (Purity ≥ 99.0%, QINGDAO Eastchem INC CO., LTD), and the second to verify the mechanism of larval death. All larvae were supplied by the larval breeding facility of the Biological Testing Laboratory at the Tecnalia Research and Technological Development Centre, Gipuzkoa, Spain.
2.1 Mortality trial
To assess the mortality of newly hatched larvae, four batches of 20 pieces each of Scots pine (Pinus sylvestris L.) sapwood measuring 76 × 26 × 4 mm were prepared. These were impregnated in an autoclave with growth enhancer (aqueous solution of 10 g/l peptone and 10 g/l yeast) (2020) and three different doses of DTPA as follows:
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Batch 0 (control), impregnated with an aqueous solution of growth enhancer.
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Batch 1, impregnated with an aqueous solution of growth enhancer and 0.15 mMolar of DTPA (0.06 g/l).
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Batch 2, impregnated with an aqueous solution of growth enhancer and 1.5 mMolar of DTPA (0.60 g/l).
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Batch 3, impregnated with an aqueous solution of growth enhancer and 15 mMolar of DTPA (6.00 g/l).
The impregnation of test specimens was performed in a systematic ascending order of concentration, commencing with an aqueous solution of growth enhancer exhibiting zero concentration of DTPA to serve as a control. This graduated approach was integral to facilitating the thorough saturation of each specimen with the corresponding test solutions.
Before impregnation, each specimen was arranged within the treatment vessel. Care was taken to orient the specimens in a configuration that maximised surface exposure, typically achieved through a crosswise stacking technique. The arrangement was then ballasted using weights specifically designed to avoid the specimens’ propensity to float upon subsequent immersion in the impregnating liquid.
Each treatment vessel containing the specimens was placed within a corresponding vacuum chamber. Upon securing the vessels, a vacuum pump was engaged to reduce the internal pressure to 0.7 kPa, a level maintained over a period of 15 min. Following the designated vacuum interval, the vacuum pump was isolated by closing the corresponding stopcock. Concurrently, an alternate stopcock was opened, permitting the preservative solution to fill the treatment vessel, thus ensuring the specimens remained fully submerged for the duration of the impregnation process.
The re-equilibration of the vacuum chamber to atmospheric pressure was achieved by introducing air post-impregnation. The treatment vessels, still containing the fully immersed specimens, were then carefully extracted from the vacuum chambers. Each vessel was subsequently covered and set aside for a period of 2 h, with additional solution added as necessary to maintain complete coverage of the specimens.
After the impregnation, specimens were individually retrieved from the solution, and any excess liquid was delicately removed by lightly dabbing with filter paper, the specimens were stabilized at 20 °C and 65% relative humidity (RH) for one month. Subsequently, a hole with a diameter of 1 mm and a depth of 4 mm was drilled near the grain’s end for larval implantation. Before this procedure, the specimens were conditioned to a constant weight at 28 °C and 85% RH to minimize larval mortality during implantation.
A single one-day-old Hylotrupes bajulus L. larva was implanted in each specimen in the previously made hole, oriented towards the centre of the piece. The specimens were placed in an incubator at 28 °C and 85% RH with the hole facing upwards, and a glass slide was placed between each specimen to prevent them from touching. After 5 days, larva survival was checked by observing the presence of debris blocking the implant hole using a stereo microscope.
The 20 pieces of Scots pine with larvae were left in the incubator for 8 months. Periodic inspections were carried out every 15 days to check the attack’s progress. Once the breeding period had passed, the mortality of the larvae was checked by observing the deterioration of the specimens. If larvae had been able to grow, the wood piece would have been partially or totally destroyed.
2.2 Trial to verify the mechanism of action of DTPA
This trial aimed to have the larvae, which has a functional reinforced mandible, consume wood treated with DTPA during the interecdysial period. The hypothesis is that the larva will be unable to ecdysial due to the lack of bioavailability of necessary nutrients.
For this purpose, 20 specimens of Scots pine sapwood measuring 40 × 60 × 15 mm were impregnated with the solution used in batch 3. Similar to the previous case, after impregnation, they were allowed to stabilize at 20 °C and 65% RH for one month. Subsequently, a hole 7 mm in diameter and 25 mm deep was made for larval implantation, tailored to match the developmental stage of the larvae, ensuring appropriate space for their growth and movement. Before implantation, the wood was stabilized at 28 °C and 85% RH. The larvae were then implanted, and the hole was covered with a piece of cotton to prevent their exit. For this trial, 10 larvae weighing between 101 mg and 150 mg were used (EN 1390:2020).
After 2 months, the status of the larva inside the wood was checked. If it was still alive, it was implanted in another specimen; if it was dead, the body and the rest of the ecdysial were sought. The condition of the larva was then examined, paying particular attention to whether death had occurred at the time of moulting. The shed mandibular pieces and those present at the time of death were also analysed.
The assessment of composition was executed using an FEI Quanta 200 scanning electron microscope (Thermo Fisher Scientific, Alcobendas, Spain), which operates under three modes of vacuum (high, low, and ambient), equipped with detectors for secondary and backscattered electrons functional across all vacuum settings. The integrated analysis framework from Oxford Instruments Analytical-Inca, with dual X-ray detectors, facilitates both simultaneous and alternate use of energy dispersive spectroscopy (EDS) and wavelength dispersive spectroscopy (WDS).
Prior to the investigative phase, the larvae underwent a meticulous cleaning regimen, using a series of alcohol solutions at concentrations of 70°, 90°, 96°, and 100°, to ensure complete dehydration and removal of surface contaminants. This step was crucial for preserving the structural integrity of the specimens. Following this cleaning, the cephalic capsule and prothorax, along with the extruded mandible, were carefully prepared for examination. The specimens were then air-dried in a controlled environment to avoid any structural damage. Once fully dried, they were mounted on scanning electron microscopy (SEM) stubs using conductive adhesive, which secured them for detailed SEM analysis. This preparation was essential for investigating the surface characteristics and chemical composition distribution on the mandible, allowing for precise identification of regions for semi-quantitative chemical microanalysis.
3 Results and discussion
3.1 Mortality trial
Table 1 shows the results of the larval survival trials separated by batch. Only 3 larvae did not survive the implantation, 2 corresponding to batch 2 and 1 to batch 3, demonstrating a very high survival rate to implantation. After 8 months of incubation, 17 live larvae were found in batch 0 or “control”, 15 in batch 1, 6 in batch 2, and none in batch 3. Using the number of live larvae in batch 0 as the survival value for comparison: batch 1 had a mortality rate of 11.8%, batch 2 of 64.7%, and batch 3 of 100%.
Table 1
Number of larvae implanted and their survival by batch
Batch | DTPA dose | Implantation | Survival | Mortality relative to Batch 0 | |||
|---|---|---|---|---|---|---|---|
Initial | Confirmed | Absolute (larvae) | Relative to Confirmed | Relative to Batch 0 | |||
0 | 0.00 g/l | 20 | 20 | 17 | 85.0% | 100.0% | 0.0% |
1 | 0.06 g/l | 20 | 20 | 15 | 75.0% | 88.2% | 11.8% |
2 | 0.60 g/l | 20 | 18 | 6 | 33.3% | 35.3% | 64.7% |
3 | 6.00 g/l | 20 | 19 | 0 | 0.0% | 0.0% | 100.0% |
The lethal dose 50 (LD50) is the amount of a substance expected to cause the death of 50% of a test animal group when administered orally or dermally, and it is utilised to evaluate the toxicity of a substance. In the case of the treatment administered, the LD50 would be reached with a dose of 0.47 g/l (1.175 mMolar of DTPA) (Fig. 1).
Fig. 1
Mortality relative to batch 0 as a function of the dose
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3.2 Trial to verify the mechanism of action of DTPA
As a result of this section, we will present images of a single larva, which is the best preserved, since under the temperature and relative humidity conditions of the incubator, the remains of dead larvae degrade quickly due to the action of fungi. The result is similar, but their bodies are covered with the mycelium of the fungus, which causes a bad visualisation. Figure 2 shows a dead larva in the moulting process, with remnants of the moult attached to the body, including a noticeable mandible. The DTPA-treated larva lost its mandibles in a failed attempt to shed its skin.
Fig. 2
Larva with ecdysial interrupted by the action of DTPA
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A closer look at the right larva’s cephalic capsule under the electron microscope shows that it still has the remnants of the ecdysis, and inside it, the new mandibles are not well developed, and unlike those of the healthy larva (Fig. 3), they have no reinforced areas. The semi-quantitative chemical microanalysis using X-ray wavelength dispersion spectrometry showed that the mandibles developed during the period between ecdysis when they fed on treated wood, we can be sure that these tissues have not been reinforced as they do not show the presence of Zn, unlike the mandibles (Fig. 4). An analysis was also conducted on the mandible that the larva had prior to ecdysial, and in this case, there is the presence of Zn in these tissues.
Fig. 3
Image illustrating the localization of reinforcements in chitin, obtained using backscattered electron detection. The contrast reveals variations in chemical composition, with areas highlighted in white indicating elements of higher atomic weight. A comparison of the mandible condition is also provided, featuring a healthy larva (left) and a larva that has consumed wood treated with DTPA (right)
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Fig. 4
Semiquantitative chemical microanalysis was conducted using X-ray wavelength dispersive spectrometry. Plots representing the results of the chemical microanalysis
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DTPA blocks the bioavailability of Zn, which prevents the larva from pre-strengthening its mandibles and thus from completing the ecdysis. Being unable to feed, it dies. As demonstrated in the two trials, treating wood with DTPA protects against Hylotrupes bajulus L. larvae, both in their early stages and in more advanced developmental stages.
The observed impact of DTPA on the molting process, particularly its interference with mandible reinforcement, underscores the complexity of its action on larval development. The interruption of normal molting, as evidenced by the failed ecdysis and mandible malformation in DTPA-treated larvae, indicates a profound disruption in the essential biological processes. The deficiency in metal ions, crucial for cuticle formation and successful ecdysis, posits DTPA not merely as a toxic agent but as a disruptor of key developmental milestones in insect life cycles.
A comprehensive study is needed to understand the influence of the non-bioavailability of these cationic metals on moulting. The inability to complete moulting may be due to the essential role these cationic metals play during the process. They are integral components in various reactions that occur during moulting, either as part of hormones or as simple catalysts facilitating the formation of the new cuticle and the shedding of the old one (Keteles and Fleeger 2001; Andersen et al. 1996; Zhang et al. 2014).
Therefore, based on the results of this study, DTPA has potential applications as a treatment for preventing and mitigating wood-boring insect infestations during the larval stage. The treatment is effective against larvae at any stage.
In terms of the toxicity of the DPTA used in mammals, when compared to pyrethroids, one of the most used insecticides in wood treatments (Teng et al. 2018), it can be observed that the acute oral toxicity of DTPA in mice/rats (LD50) is 587 mg/kg, indicating low toxicity to mammals (Srivastava et al. 1986). Conversely, pyrethroid compounds such as cypermethrin, β-cyfluthrin, and tefluthrin exhibit LD50 values ranging from 1.50 to 691.83 mg/kg, 1.27 to 35.48 mg/kg, and 15.00 to 22.00 mg/kg, respectively, denoting high toxicity to mammalian subjects. Furthermore, compounds like permethrin, deltamethrin, bifenthrin, esfenvalerate, fenpropathrin, and λ-cyhalothrin, with LD50 values spanning from 30.00 to 1500.00 mg/kg, are categorized as moderately toxic, whereas resmethrin and S-bioallethrin, with LD50 values ranging from 300.00 to 2000.00 mg/kg, are identified as having lower toxicity profiles (Ensley 2018; Zhu et al. 2020).
DTPA’s relatively high LD50 suggests its reduced toxicity risk in comparison to the majority of pyrethroids. Additionally, DTPA is characterized by minimal oral absorption (Arts et al. 2018) and lacks evidence indicating chronic toxicity implications (Fukuda and Iida 1983; Fukuda et al. 1984). Potential risks may be most pronounced during pregnancy, where DTPA could potentially restrict fetal access to essential cationic metals; however, conclusive research in this area is not currently available (Arts et al. 2018). Collectively, these attributes indicate that DTPA utilization in mammalian contexts presents a reduced hazard relative to conventional pyrethroid-based treatments.
However, it is important to consider that DTPA may not provide complete protection against all wood-decaying organisms, as some fungi have developed mechanisms to use complexed metals for their metabolism (Baldrian 2003). In such cases, supplementing DTPA with additional preservation methods may be necessary to achieve optimal protection.
DTPA has a toxicity profile that is lower than traditional wood preservatives, suggesting the potential for reduced environmental impact. It can be used in combination with other wood preservation techniques, such as impregnation with water repellents, fungicides, or insecticides. The addition of DTPA to these treatments may enhance their wood-preservative properties, as indicated by the trials.
In summary, DTPA has demonstrated efficacy in complexing metal ions with a toxicity profile that may be favourable for use as a wood preservative. However, further research and development are required to fully understand its potential and limitations in preserving wood against decay.
4 Conclusion
The application of DTPA in Hylotrupes bajulus L. larvae results in high mortality rates, establishing its potential as an effective control measure. The qualitative examination revealed impaired mandible development in larvae, significantly hindering their feeding ability post-moulting. These findings indicate that DTPA represents a promising approach for preventing and managing wood-boring insect infestations during their larval stage.
Acknowledgements
The authors would like to thank Sara Garrido Espinosa from the Ministry for Ecological Transition and the Demographic Challenge of Spain for her invaluable help.
Declaration
Competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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