Morphology and Material Composition of the Mouthparts of Stromatium Unicolor Olivier 1795 (Coleoptera: Cerambycidae) for Bionic Application

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Article Morphology and Material Composition of the Mouthparts of Stromatium unicolor Olivier 1795 (Coleoptera: Cerambycidae) for Bionic Application

Roberto D. Martínez * , Luis-Alfonso Basterra , Luis Acuña and José-Antonio Balmori

Timber Structures and Wood Technology Research Group, UVa, 47014 Valladolid, Spain; [email protected] (L.-A.B.); [email protected] (L.A.); [email protected] (J.-A.B.) * Correspondence: [email protected]

Received: 19 April 2020; Accepted: 25 June 2020; Published: 27 June 2020

Abstract: Research Highlights: The novelty of this study is the deep analysis of the morphologic, geometric and mechanical performance of longhorn beetle larvae mouthparts. Furthermore, a metal nano identification of jaw reinforced parts was made. Background and Objectives: Analysis of insect mechanical properties has shown an important application in the develop of bionic technologies such as new materials, industrial machines and structural concepts. This study aims to determine the mechanical and geometric properties of longhorn beetle (Stromatium unicolor Olivier 1795) larvae mouthparts to improve the development of innovative cutting tools. In addition, this study obtains a nano identification of metals in the cuticle of the mouthparts, which will enable the development of new nontoxic and sustainable preservation agents against xylophagous insects based on nanoparticles. Materials and Methods: five third-larval-stage samples of Stromatium unicolor were used to study its mandible morphologic, geometric and mechanical properties. To this end, mouthparts were analyzed by several microscopic techniques using a scanning electron microscope, a stereomicroscope and an optical microscope. Composition analysis was performed using with two Analytical-Inca X-ray detectors, dispersive energy spectroscopy and dispersive wavelength spectroscopy. Results: The main geometric parameters of the insect jaw are the edge angle (β = 77.3◦), maximum path depth of the insect (120 µm), length (800 µm) and mouthpart movement, which were identified and measured. The chemical analysis results of the jaw tissues shows the presence of zinc and manganese. Conclusions: The geometry and angles of the mouthparts can be applied in the fabrication of bionic self-sharpening cutting tools. Molecular compounds that form the reinforcing elements in the jaws can be used to develop wood preservatives based on nanometals and metal absorption and metabolism inhibitors.

Keywords: bionics; mouthparts; microstructure; nanoindentation properties; working eﬃciency

1. Introduction Bionic science studies nature and natural organisms to understand their geometry and physical, chemical, structural and material properties underlying biologic principles. These bionic concepts are found to transfer biologic solutions to engineering, medicine, smart technology and general science. This knowledge has been well developed as a scientific approach to design new technology and optimize existing ones. Thus, currently, it is possible to find hundreds of examples of bionic influence on our daily lives that involve the transformation of the principles of nature into technology [1–4]. In the last decades, bionic science witnessed a huge increase. The combination of new imaging analysis techniques such as synchrotron-generated X-rays, microtomography, laser scanning microscopy, energy disperse spectroscopy and scanning electron microscopy has enabled further studies of the morphology

Forests 2020, 11, 715; doi:10.3390/f11070715 www.mdpi.com/journal/forests Forests 2020, 11, 715 2 of 14 and mechanical properties of live animals, plants and insects. These techniques have resulted in new knowledge about the chemical composition and nanoindentation properties of specific biologic structures. All of these advances have strongly developed the comprehension of bionic materials and structures [5,6]. Insect structure and function investigations have shown promise for use in mechanical, informational and intelligential new bionic devices [7]. Almost 99% of insects can fly; their aerodynamic behavior and flight skills have been repeatedly applied in mechanical engineering to promote new design concepts for aerodynamic or flight vehicles [8,9]. Other applications of insect bionics have been to develop new composite materials, more rigid and strong materials, materials that save weight [10,11] or to inspire new structure designs [12]. Therefore, the biomechanical and morphology analysis of insect mouthparts can directly contribute to cutting-edge engineering developments. Insects—especially their larvae—can easily cut and eat vegetables and wood of different hardness. This ease of cutting depends on their well-formed, strong and sharply shaped mouthparts, which have evolved to chew different natural materials [13–15]. In all cases, the mouthparts—whose main function is to chew—must be stiffer, harder, stronger and more resistant than the food to prevent breakage, wear or deformation. Similarly, it must have a good cutting tool [16,17]. However, insect mouthparts are not always as simple as most metal-cutting tools. Links have been established between tooth geometry and angle of attack, distance, obliqueness, etc., and the effects of these parameters and the forces required to cut natural materials. Research on the basic structure of insect mouthparts, type of jaw, geometry, hardness and elemental content can help solve problems such as the coating of cutting tools or more efficient cutting designs [18]. Thus, several investigations have analyzed the geometric characteristics and shapes of different insect mouthparts to enhance the performance and design of bionic cutting tools. For example, the insect species Locusta migratoria manilensis [19] and Gryllotalpa orientalis [20] were studied to improve the design of the cutting mechanics of agricultural machines. Cyrtotrachelus sp. inspired the design of bionic blades in the food industry [21]. Another focus of research is the analysis of cuticle nano properties. In 1982, Hillerton et al. studied the Locusta migratory insect to find the hardness of different parts of the mouthparts. This study found that some area of the incisor teeth was much harder than other parts of the mandibular cuticle, including two shear surfaces, which suggests that hard surfaces form cutting edges that bite and due to the way that the jaws move, they are also self-sharpening [22]. Several investigations have analyzed the microstructure and elements that insects incorporate into their cuticles as reinforcement to increase their resistance. Numerous studies have identified the presence, type and location of metals in the cuticles of certain groups of insects [23–25]. These elements are normally zinc, manganese, iron, calcium and chlorine, and they are frequently found in high concentrations in the cuticle of various insect structures. Among other structures, jaws frequently contain significant amounts of these metals [26–28]. The concentrations of metals at the cutting edges can improve the mechanical properties of insect jaws [29]. Currently, this nano-identification is applied in the design of bionic blades [30,31]. However, the most promising application of this nano-identification is the development of nontoxic and sustainable preservation agents to apply durable wood treatments with nanoparticles. Some investigations have studied the application of metal nanoparticles by pressure treatment as an effective antimicrobial and antifungal wood preservation method [32–34]. In addition, this treatment can be effective against different xylophagous insects; nanoparticles of specific metals can chemically interact with the metals in the cuticle to weaken the mouthparts and prevent feeding [35–37]. This study aims to determine the specific material, morphologic and mechanical properties of larval-stage longhorn beetle (Stromatium unicolor Olivier 1795) insect mouthparts. Knowledge of the geometric parameters such as the edge angle, relief angle, detachment angle or type of cut that the jaw makes when eating will improve the technology of current cutting tools. In addition, the nano-identification of metal present in the cuticle of mouthparts may enable us to develop Forests 2020, 11, 715 3 of 14 new sustainable preservation agents against xylophagous insects based on metallic nanoparticles,

Forestsmetal 2020 absorption, 11, x FOR and PEER metabolism REVIEW inhibitors. 3 of 13

2. Materials and Methods 2. Materials and Methods Stromatium unicolor ThThisis study was performed on on five five larvae (third stage stage)) of Stromatium unicolor (Olivier,(Olivier, 1795) 1795),, which were collected in the town of Nombela (Toledo, Spain),Spain), Figure1 1.. ThisThis speciesspecies isis aa longhornlonghorn Quercus ilex beetle whose larvae feed on holm oak ( Quercus ilex L.). Each larva was used to study a specific specific type of parameter, and it was necessary to perform different different histological preparations that destroy destroyeded the sample. The trace of the cutcut inin thethe holmholm oakoak wood wood by by the the larvae larvae was was studied studied by by observing observing the the walls walls of ofthe the pierced pierced galleries. galleries.

(a) (b)

FigureFigure 1. 1. StromatiumStromatium unicolor unicolor (Olivier,(Olivier, 1795) 1795) ( a) i imagomago (F: f female;emale; M: m male);ale); ( b) larvae.larvae.

The larvae selectionselection waswas basedbased on on a a biologic biologic and and evolutionary evolutionary criterion: criterion: the the largest large larvaest larvae are are the thestrongest strongest and and best best adapted adapted to their to their environment. environment Since. Since their their function function at these at these stages stages is to is pierce to pierce the thewood wood and and feed feed on it,on the it, best-adaptedthe best-adapted larvae larvae will will have have better better wood wood cutting cutting characteristics. characteristics. The low low number number of of larvae larvae used used in the in research the research was mainly was mainly due to due three to reasons: three reasons: first, the first,difficulty the difficultyof finding of larvae finding in larvae their last in their stage last since stage they since were th extractedey were extracted from holm from oak holm firewood. oak firewood. As it does As itnot does come not from come a hatchery from a larva, hatchery their larva, availability their was availability limited. was Second, limited. taking Second, into consideration taking into considerationthe costs of testing the costs the techniques of testing the and techniques equipment and used equipment for a large used number for a of large tests number would of require tests woulda disproportionate require a dispro economicportionate contribution economic compared contribution to the compared objective to set. the Moreover, objective finally,set. Moreover, in other finally,investigations in other to determine investigations the geometric to parametersdetermine of the insects geometric [8,10,19 ,21 parameters,30,31,38,39 ], of these insects were [8,10,19,21,30,31,carried out with38, one39 to], fivethese larvae. were carried out with one to five larvae. What has has been been done done is is a asimplification simplification since, since, if a if larva a larva has has reached reached its last its lastlarval larval stages, stages, the characteristicsthe characteristics of the of jaw the are jaw close are-t close-to-,o-, or above or- above-average.average. This is due This to is the due act to that the if actthe thatjaw is if not the efficientjaw is not in ecutting,fficient the in cutting,insect could the insect not have could reached not have that reachedstage because that stage of the because inability of to the feed. inability to feed.We are aware of the limitations in this type of study using a single sample, due to the dimensionalWe are variabilityaware of the depending limitations on the in this larval type stage, of studybut further using biometric a single studies sample, with due a to large the numberdimensional of individuals variability at depending different g onrowth the larval stages stage, will advance but further the morphometricsbiometric studies of with the head a large of xylophagousnumber of individuals insects. at different growth stages will advance the morphometrics of the head of xylophagous insects. 2.1. Obtaining The Profile of The Jaw and Perpendicular Cut of The Musculature. Paraffin Inclusion 2.1. Obtaining The Profile of The Jaw and Perpendicular Cut of The Musculature. Paraffin Inclusion First, a larva was selected and sectioned perpendicularly a few millimeters below the prothorax to obtainingFirst, a larvathe head was, as selected shown and in F sectionedigure 2b,d perpendicularly. a few millimeters below the prothorax to obtainingTo obtain the a precise head, as and shown clean in-cut Figure section,2b,d. the larvae were prepared using the paraffin embedding techniqueTo obtain. First, a precisethey were and dehydrated clean-cut section, in an ethanol the larvae immersion were prepared; then, the using sample the paras wereffin stained embedding with atechnique. solution of First, ethanol they and were methylene dehydrated blue. in anBefore ethanol introducing immersion; the then,larva thee into samples a paraffin were bath, stained ethanol with wasa solution extracted of ethanol from the and tissues methylene with a blue. xylene Before treatment. introducing Xylene the was larvae used into as aan para intermediateffin bath, ethanol liquid betweenwas extracted ethanol from and the paraffin tissues since with it ais xylene miscible treatment. in both. Once Xylene the was paraffin used solidified, as an intermediate it was removed liquid from the mold and dissected by using a scalpel to expose the head of the larvae. Then, cuts were made with a microtome until the desired plane was exposed, as shown in Figure 2.

Forests 2020, 11, 715 4 of 14 between ethanol and paraffin since it is miscible in both. Once the paraffin solidified, it was removed from the mold and dissected by using a scalpel to expose the head of the larvae.

ForestsThen, 2020, 11 cuts, x FOR were PEER made REVIEW with a microtome until the desired plane was exposed, as shown in Figure4 of 132.

(a) (b)

FigureFigure 2. LarvaLarvaee cutting cutting analysis analysis.. ( (aa)) Cut Cut perpendicular perpendicular to to the the musculature. musculature. Plane Plane where where the the cut cut was was mademade;; ( b)) r resultingesulting cut cut;; ( c) m mandibleandible profile. proﬁle. Plane where the cut was made made;; (d) resultingresulting cut.

2.2.2.2. Surface Surface and and Elemental Elemental Composition Composition Analysis Analysis CompositionComposition analysis analysis was was performed performed using using an an FEI FEI Quanta Quanta 200 200 ( (ThermoThermo Fisher Fisher Scientific Scientific,, Alcobendas, Spain) Spain) scanning scanning electron electron microscope microscope that that operates operates with three with vacuum three vacuum modes (high modes vacuum, (high vacuum,low vacuum low and vacuum ambient and mode), ambient secondary mode), and secondary backscattered and backscattered electron detectors electron for all detectors vacuum modesfor all vacuumand the modes integrated and analysisthe integrated system analysis Oxford system Instruments Oxford Analytical-Inca Instruments Analytical with two-Inca X-ray with detectors, two X- raywhich detectors can be, which simultaneously can be simultaneously and alternatively and alternatively used: dispersive used: energydispersive spectroscopy energy spectroscopy (EDS) and (dispersiveEDS) and d wavelengthispersive wavelength spectroscopy spectroscopy (DWS). Before (DWS testing,). Before the testing, samples the samples were washed were washed with alcohol with alcoholat different at different graduations graduations (70◦, 90(70◦,°, 96 90◦°,and 96° and 100 ◦100).° The). The cephalic cephalic capsule capsule and and prothoraxprothorax assembly assembly andand an extruded mandible were observed. The The surface surface characteristics characteristics and and distributions distributions of of different different chemicalchemical compositions compositions were were studied. studied. After After different different areas areas were were found, found, specific specific points points were were selected selected,, where sem semiquantitativeiquantitative chemical microanalyses were performed.

2.3.2.3. Measurement Measurement Methodology Methodology AA stereomicroscope and and an an optical microscope microscope with with an an image image digiti digitizingzing system system were were used to perform the geometric characteri characterizationzation of the mandibles. The The images were subsequentl subsequentlyy analy analyzedzed usingusing the the Image Image-Pro-Pro Plus Plus 4.0 4.0 software. software. With With this this software, software, after after calibration calibration with with a a specific speciﬁc standard, standard, measurementsmeasurements of of length, angles, etc etc.. were were obtained. The The software software manufacturer manufacturer estimates estimates that that the the errorerror made in the measurement using this meth methodologyodology is 3% of the measure.

3. Results and Discussion

3.1. Elemental Composition of The Mandibles The contrast difference in the strip next to the cutting edge in Figure 3 indicates that the mandible cut-area is reinforced with inclusions of elements of higher atomic weight than the remainder of the head and body. Studies of the cutting edges show a change in the composition of the jaw tissues [26].

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3. Results and Discussion

3.1. Elemental Composition of The Mandibles The contrast diﬀerence in the strip next to the cutting edge in Figure3 indicates that the mandible cut-area is reinforced with inclusions of elements of higher atomic weight than the remainder of the head and body. Studies of the cutting edges show a change in the composition of the jaw tissues [26]. Forests 2020, 11, x FOR PEER REVIEW 5 of 13

(a) (b)

FigureFigure 3.3. ImageImage ofof thethe mandiblemandible obtained by ( a) secondary electron electron detection detection and and ( (bb)) backscattered backscattered electron detection.electron detection. The contrast The reveals contrast diﬀ revealserences differences in the chemical in the composition. chemical composition.

A semiquantitative chemical microanalysis using X-rayX-ray wavelength dispersion spectrometry showshoweded that the tissues that experience the most significant signiﬁcant stress during the the bite are reinforced by elements such such as as zinc, zinc, manganese manganese and and chlorine chlorine (Figure (Figure4). The 4). analysisThe analysis of the rakeof the face rake showed face show meagreed meagreamounts amoun of zinctsand of zinc manganese and manganese and the and absence the absence of chlorine. of chlorine. If the metabolism of these metallic elements by the insect can be limited or prevented using chelating substances substances [38] [38,], the the tissues tissues involved involved in cutting in cutting the wood the wood are not are reinforced not reinforced by them by, which them, whichaffects atheﬀects cutting the cutting and feeding and feeding capacity capacity of the of the larva larva and and protect protectss the the wood wood from from attacks attacks by xylophagous insects.

(a)

(b) (c)

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(a)

(b) (c)

(d) (e)

Figure 4.4. SemiquantitativeSemiquantitative chemical chemical microanalysis microanalysis po pointsints through X-ray wavelength dispersion spectrometry. ( (aa)) Location Location of of the the analysis analysis po pointsints on on the the cut surfaces; ( (b)) plots of the chemicalchemical microanalysis results of of the the flank ﬂank of of the the jaw jaw from from the the (c ()c cutting) cutting edge, edge, (d) ( drake) rake face face of the of the jaw jaw and and (e) (remainse) remains of wood of wood in the in the jaw. jaw.

3.2. Study ofof TheThe Mandible Mandible Movement: Movement: Joints, Joints, Musculature Musculature and Footprintand Footprint of The of Bite The Bite Each tooth tooth or jawor jaw has twohas strongly two strongly chitinized chitinized hemispheres, hemispheres, which is similar which to the head is similar of a femur to bone. These hemispheres are housed in separate cavities that are also strongly chitinized in the head the head of a femur bone. These hemispheres are housed in separate cavities that are (Figure5). Thus, each tooth rotates through 2 points or joints that form an axis, and each jaw has aalso degree strongly of freedom chitinized of movement in the in the head cephalic (Figu capsulere 5). (rotation). Thus, each The cephalic tooth rotates capsule hasthrough 3 degrees 2 ofpoints freedom or sincejoints it that performs form vertical, an axis, horizontal and ea andch jaw extension has a movements. degree of Itfreedom appears similarof movement to a robot within the polar cephalic coordinate capsule movements. (rotation). The cephalic capsule has 3 degrees of freedom since it performs vertical, horizontal and extension movements. It appears similar to a robot with polar coordinate movements.

Forests 2020, 11, x FOR PEER REVIEW 6 of 13

(d) (e)

Figure 4. Semiquantitative chemical microanalysis points through X-ray wavelength dispersion spectrometry. (a) Location of the analysis points on the cut surfaces; (b) plots of the chemical microanalysis results of the flank of the jaw from the (c) cutting edge, (d) rake face of the jaw and (e) remains of wood in the jaw.

3.2. Study of The Mandible Movement: Joints, Musculature and Footprint of The Bite Each tooth or jaw has two strongly chitinized hemispheres, which is similar to the head of a femur bone. These hemispheres are housed in separate cavities that are also strongly chitinized in the head (Figure 5). Thus, each tooth rotates through 2 points or joints that form an axis, and each jaw has a degree of freedom of movement in the cephalic capsule (rotation). The cephalic capsule has 3 Forestsdegrees2020 of, 11 freedom, 715 since it performs vertical, horizontal and extension movements. It appears similar7 of 14 to a robot with polar coordinate movements.

(a) (b)

Figure 5. 5. MouthpartsMouthparts of of the larva: ( (aa)) Optical Optical microphotography of of the mandible;mandible; ( b) electronelectron micrograph of the cephalic capsule. The The m mandiblesandibles are highlighted in green green;; the joints and axis of rotation are highlighted inin red.red.

Inside the the larva larva head head are are multiple multi muscles.ple muscles Most. ofMost them of are them not crucial are not in crucial the cutting in movementsthe cutting andmovements are therefore and are not therefore the object not of the this object study. of Wethis only study. focus We ononly the focus main on actuator the main muscles. actuator muscles. Three groups of muscles muscles are are mainly mainly involved involved inin the the cutting cutting movement: movement: The adductors are responsible for the closing movement of the jaw and therefore are the most responsible for the cutting action.action. T Thehe abductors are accountable for the mandibular opening opening,, and the extension muscles of the cephalic capsulecapsule causecause itsits movementmovement outwardoutward and and into into the the prothorax prothorax and and are are responsible responsible for for bringing bringin itg closerit closer to the to the wood wood and andsupporting supporting its perpendicular its perpendicular reaction reaction force. As force. shown As inshown Figure in6, F theigure adductor 6, the Forestsmusclesadductor 2020 have , muscles11, xa FOR much PEERhave higher REVIEW a much volume higher than volume the other than muscles the other since muscles they provide since thethey main provide cutting the power.7 mainof 13 cutting power.

(a) (b)

Figure 6. OpticalOptical micrographs: micrographs: (a (a)) longitudinal longitudinal and and ( (bb)) cross cross-section-section of of the the cephalic capsule and prothorax. The The a adductordductor muscles muscles (AD) (AD) are are highlighted highlighted in in yellow, yellow, the abductor muscles (AB) are highlighted in in light light blue blue,, and and the the exit exit and and entrance entrance muscles muscles of of the the cephalic cephalic capsu capsulele are are highlighted highlighted in orange.in orange.

TThehe difference diﬀerence in muscle power is observed in the size of the tendons that transmit the the effort eﬀort to the jaw. Figure Figure 77 showsshows thethe abductorabductor tendontendon highlightedhighlighted inin greengreen onon thethe leftleft andand thethe adductoradductor tendontendon on the right right,, which is seve severalral times larger than the tendon on the left.left.

Figure 7. Electron micrograph of the right mandible. The tendons are highlighted in green and the joints and axis of rotation are highlighted in red.

To pierce the wood, the larvae use two main opening and closing movements. First, by contracting the muscle bundles in the prothorax, the cephalic capsule leaves the prothorax (similar to the head of a turtle emerging from its shell) to bring the jaw closer to the wood. While performing this movement, the larva opens the jaw using the abductor muscles. Once the open mouth makes contact with the wood, it exerts pressure and closes the jaws with the adductor muscles to cut.

3.4. Tool Footprint (Mandible) As shown in Figure 8, the jaw cut is not clean. It produces tears in the wood fibers and breaks at different depths due to bending and shear stresses. The estimated moisture content of the holm oak wood in which the larva was feeding was approximately 16%.

Forests 2020, 11, x FOR PEER REVIEW 7 of 13

(a) (b)

Figure 6. Optical micrographs: (a) longitudinal and (b) cross-section of the cephalic capsule and prothorax. The adductor muscles (AD) are highlighted in yellow, the abductor muscles (AB) are highlighted in light blue, and the exit and entrance muscles of the cephalic capsule are highlighted in orange.

The difference in muscle power is observed in the size of the tendons that transmit the effort to Foreststhe jaw.2020 Figure, 11, 715 7 shows the abductor tendon highlighted in green on the left and the adductor tendon8 of 14 on the right, which is several times larger than the tendon on the left.

Figure 7. Electron micrograph of the right mandible. T Thehe t tendonsendons are highlighted in green and the joints and axis ofof rotationrotation are highlightedhighlighted inin red.red.

ToTo pierce pierce the the wood, wood, the thelarvae larva useetwo use main two openingmain opening and closing and movements. closing movements First, by contracting. First, by thecontracting muscle bundlesthe muscle in the bundles prothorax, in the the prothorax, cephalic capsulethe cephalic leaves capsule the prothorax leaves the (similar prothorax to the (similar head of ato turtle the head emerging of a turtle from emerging its shell) tofrom bring its theshell) jaw to closer bring to the the jaw wood. closer While to the performing wood. While this performing movement, thethis larvamovement, opens thethe jawlarva using open thes the abductor jaw using muscles. the abductor Once the muscles. open mouth Once the makes open contact mouth with makes the wood,contact it with exerts the pressure wood, it and exerts closes pressure the jaws and with closes the the adductor jaws with muscles the adductor to cut. muscles to cut.

3.3.3.4. Tool Footprint (Mandible)(Mandible) As shown in FigureFigure8 8,, thethe jawjaw cutcut isis notnot clean.clean. ItIt producesproduces tearstears inin thethe woodwood ﬁbersfibers andand breaksbreaks atat didifferentﬀerent depths due to bending and shear stresses.stresses. The estimated moisture content of the holm oak wood in which the larva was feeding was approximately 16%. Forestswood 2020in which, 11, x FOR the PEER larva REVIEW was feeding was approximately 16%. 8 of 13

(a) (b)

Figure 8. Electron micrographs of the cut footprint made by the insect in a cross section of holm oak wood. Torn cells of polygonalpolygonal sections are observed. ( a) Whole footprint;footprint; (b) detaildetail of the latewood zone in the annual ring.

FigureFigure9 9aa shows shows that that the the footprint footprint begins begins to smoothlyto smoothly deepen, deepen, takes tak a semicirculares a semicircular path, path and ends, and inends an in almost an almost vertical vertical wall, wall, which which leaves leav aes type a type of burr. of burr. Since Since this this footprint footprint is notis not symmetrical, symmetrical, it isit is conceivable that the cut is made by one jaw at a time. To more quickly visualize the characteristics, Figure 9b shows a larva bite simulation.

(a) (b)

Figure 9. Optical microphotography of the cut footprint (a) with shadow play (bite length F = 800 μm) and (b) a photographic montage of the larva cutting.

From the study of the joints, muscles, tendons and footprint of the mandibles, it can be deduced that the larva primary cutting mechanism consists of using one of the jaws as an anchor or stop. Simultaneously, the other performs chip removal, which describes a semicircular movement until the jaws touch each other. This mechanism can be observed in the imprint of the mandibular tool left on the wood. We say that it is its primary cutting mechanism because since it is a living being, it can change its modus operandi depending on its needs, the shape of the wood, the hardness of the wood, etc.

3.5. Cut Type Each jaw rotates through 2 points or joints that form an axis. The cutting edge and articular axis form a plane, and the axes of the two teeth are parallel, as shown in Figure 10. Therefore, the cutting

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(a) (b)

Figure 8. Electron micrographs of the cut footprint made by the insect in a cross section of holm oak wood. Torn cells of polygonal sections are observed. (a) Whole footprint; (b) detail of the latewood zone in the annual ring. Forests 2020, 11, 715 9 of 14 Figure 9a shows that the footprint begins to smoothly deepen, takes a semicircular path, and ends in an almost vertical wall, which leaves a type of burr. Since this footprint is not symmetrical, it conceivableis conceivable that that the the cut cut is is made made by by one one jaw jaw at at a a time. time. ToTo moremore quicklyquickly visualizevisualize the characteristics characteristics,, FigureFigure9 9bb showsshows aa larvalarva bitebite simulation.simulation.

(a) (b)

Figure 9. OpticalOptical microphotography microphotography of the cut footprint (a) with shadow play (bite(bite lengthlength F = 800800 μm)µm) and (b) a photographic montage of the larvalarva cutting.cutting.

From the study of the joints, muscles, tendons and footprint of the mandibles, it can be deduced that the larva primary cutting mechanism mechanism consists consists of of using using one one of of the the jaws jaws as as an an anchor anchor or or stop. stop. Simultaneously,Simultaneously, the the other other performs performs chip chip removal, removal, which which describ describeses a semicircular a semicircular movement movement until until the thejaws jaws touch touch each each other other.. This Thismechanism mechanism can be can observed be observed in the inimprint the imprint of the mandibular of the mandibular tool left tool on leftthe wood. on the We wood. say Wethat say it is that its itprimary is its primary cutting cutting mechanism mechanism because because since it since is a living it is a livingbeing, being,it can itchange can change its modus its modus operandi operandi depending depending on its needs, on its needs,the shape the of shape the wood, of the wood,the hardness the hardness of the wood of the, wood,etc. etc.

3.4.3.5. Cut Type

ForestsEach 2020, 11jaw , x FORrotates PEER through REVIEW 2 points or joints that form an axis. The cutting edge and articular 9 ofaxis 13 form a plane, and the axes of the two teeth are parallel, as shown in FigureFigure 10.. Therefore, the cutting edge of the mandible is perpendicular to the direction of the cut. Thus, the cutting movement of the edge of the mandible is perpendicular to the direction of the cut. Thus, the cutting movement of the insect is orthogonal.

Figure 10. MandibularMandibular joints joints and axes of rotation locations.

3.6. Geometric Characterization of The Mandibles

3.6.1. Geometric Parameters The wedge angle of the mandible near its plane of symmetry is β = 77.3°. The angle between the rake face and the segment that joins the cutting edge with the muscular insertion of the mandible is δ = 9.6°. The position and dimensions of the jaws were characterized from segments A, B and C and at angles a and b, as shown in Figure 11.

(a) (b)

Figure 11. Distances and angles: (a) Jaw wedge angle β = 77.3° and the angle between segment A and the rake face c = 9.6°; (b) A = 1249 µm is the distance between cutting edge and insertion of the adductor, B = 1496 µm is the distance between the cutting edge and the articulation axis radius of rotation of the jaw, C = 2539 µm is the joint distance, a = 44.8° is the angle between segments A and B and b = 30.9° is the declination angle between segments B and C.

The bite length (F = 800 μm) was obtained by measuring the length of the cutting footprint in the wood (Figure 9a).

3.6.2. Radius and Arches The radii that describe the flank face, cutting edge and radius of the edge were identified and measured, as shown in Figure 12.

Forests 2020, 11, x FOR PEER REVIEW 9 of 13 edge of the mandible is perpendicular to the direction of the cut. Thus, the cutting movement of the insect is orthogonal.

Forests 2020, 11, 715 10 of 14 Figure 10. Mandibular joints and axes of rotation locations.

3.5.3.6. Geometric Characteri Characterizationzation of The Mandibles 3.5.1. Geometric Parameters 3.6.1. Geometric Parameters The wedge angle of the mandible near its plane of symmetry is β = 77.3 . The angle between the The wedge angle of the mandible near its plane of symmetry is β = 77.3°.◦ The angle between the rake face and the segment that joins the cutting edge with the muscular insertion of the mandible is rake face and the segment that joins the cutting edge with the muscular insertion of the mandible is δ = 9.6 . The position and dimensions of the jaws were characterized from segments A, B and C and at δ = 9.6°.◦ The position and dimensions of the jaws were characterized from segments A, B and C and angles a and b, as shown in Figure 11. at angles a and b, as shown in Figure 11.

(a) (b)

Figure 11. DistancesDistances and and angles: angles: (a ()a Jaw) Jaw wedge wedge angle angle β =β 77.3= 77.3° and◦ and the theangle angle between between segment segment A and A theand rake therake face face c = c 9.6°= 9.6; (b◦;() Ab)A = 1249= 1249 µmµ mis is the the distance distance between between cutting cutting edge edge and and insertion insertion of of the adductoradductor,, B = 1496 µmµm is the distance between the cutting edge and the articulation axis radius of

rotation of the jaw,jaw, CC == 2539 µµmm is the joint distance,distance, aa == 44.8°◦ isis the the angle between segments A and B and b = 30.930.9°◦ isis the the declination declination angle angle between between segments segments B B and and C. C.

The bite lengthlength (F(F= = 800800µ μm)m) was was obtained obtained by by measuring measuring the the length length of theof the cutting cutting footprint footprint in the in woodthe wood (Figure (Figure9a). 9a).

3.5.2.3.6.2. Radius and Arches The radii that describedescribe the flank flank face, cutting edge and radius o off the edge were identifiedidentified and measured, as shown in FigureFigure 12.. The arc of the cutting edge is offset from the axis of symmetry of the jaw (Figure 12c). The maximum depth of the cut of the insect (D = 120 µm) was deduced from the distance between the line tangent to the center of the arc of the cutting edge and the line formed by its ends as shown in Figure 12d. Forests 2020, 11, x FOR PEER REVIEW 10 of 13

The arc of the cutting edge is offset from the axis of symmetry of the jaw (Figure 12c). The maximum depth of the cut of the insect (D = 120 μm) was deduced from the distance between the lineForests tangent2020, 11 ,to 715 the center of the arc of the cutting edge and the line formed by its ends as shown11 of in 14 Figure 12d.

(a) (b)

Figure 12. The r radiusadius and arches of the mandibles: ( (aa)) Radius Radius of of curvature curvature of of the the flank ﬂank r1 r1 = 566566 μmµm;; (b) r radiusadius of the arc of the cutting edge r2 = 574 μmµm;; ( c) e edgedge radius r3 = 33 μmµm;; ( (dd)) a anglengle of the the tangent tangent

toto the the cutting arc with the longitudinal axis of the jaw d = 106.5106.5°◦;;( (dd)) m maximumaximum cut cut depth depth of the the insect insect bite D = 120120 μm.µm.

3.6.3.3.5.3. Cutting Cutting Edge Edge The cutting edge of this species is smooth without protuberances or defined deﬁned shapes shapes,, as shown inin F Figureigure 13. In In other other species, species, the the cutting cutting edge edge has has periodic periodic forms forms;; for for example, Schmidt, H. and ParaParameswaran,meswaran, N. N. comment commenteded that that the the cutting cutting edge edge of of the the jaw jaw of of HylotrupesHylotrupes bajulus bajulus L.L. describe describedd a zigzaga zigzag of of approximately approximately 120° 120 ◦[39[39].]. This This particular particular insect insect feeds feeds on on coniferous coniferous wood wood with with densities densities on 3 the order of 450 450–600–600 kg /m3..

Forests 2020, 11, 715 12 of 14 Forests 2020, 11, x FOR PEER REVIEW 11 of 13

(a) (b)

Figure 13. ((aa)) Smooth Smooth cut cut edge edge detail detail;; ( (bb)) d detailetail of the the rake rake face without texture. The The cracks are due to drying of the specimen specimen..

4. Conclusions Conclusions The material, morphology, geometry and mechanical properties of the mouthparts of longhorn beetle ( Stromatium unicolor unicolor)) in in its its larva larva stage stage were were analy analyzedzed in in this this study study,, which will increase the knowledge of the cutting mechanics of this insect insect.. The primary cutting mechanism mechanism of of the the larva larva consists consists of of using using one one of of the the jaws as an anchor or stop. Simultaneously,Simultaneously, the the other other jaw jaw performs performs chip removal, chip removal, which describeswhich describ a semicirculares a semicircular movement movementuntil they comeuntil together.they come The together. analysis The of analysis the footprint of the in footprint the wood in concludesthe wood concludes that the cut that produced the cut producedby the larva’s by the jaw larva is not’s jaw clean is not and clean has and a rough has a surface rough surface finish, sincefinish, it since produces it produces tears in tears the in fibers the fibanders breaks and breaks at diff aterent different depths depths due to due bending to bending and shear and shear stresses. stresses. The tissuesThe tissues of the of insect the insect jaw thatjaw thatare directlyare directly involved involved in cutting in cutting are subjected are subjected to higher to higher stresses, stresses including, including wear wear stress, stress than, thethan other the othertissues. tissues Therefore,. Therefore, they are they reinforced are reinforced with higher with atomic higher weight atomic elements weight suchelements as zinc such and as manganese. zinc and manganese.Meanwhile, theMeanwhile inner tissues, the that inner are tissues not directly that are involved not directly in shear involved stresses in are shear not reinforced. stresses are not reinforced.Fromthe analysis of the musculature and joints of the larva, of the three principal muscle bundles that interveneFrom the inanalysis the cutting of the movement, musculature the adductors and joints are of responsible the larva, for of the the closing three principal movement muscle of the bundlesjaw and therefore that intervene are the in main the responsible cutting movement, muscle bundle the adductors for the action are of responsible the cut. To pierce for the the closing wood, movementthe larva uses of the two jaw main and movements. therefore are the main responsible muscle bundle for the action of the cut. To pierceThe jointthe wood, analysis the andlarva geometric uses two main characterization movements.of the mandibles showed that the cutting movementThe joint of the analysis insect isand orthogonal geometric and characteri that thez cuttingation of edge the of mandibles the larva’s showed jaw issmooth. that the cutting movementThe most of the significant insect is mandiblesorthogonal geometric and that the parameters cutting edge were of also the found.larva’s jaw is smooth. TThehe most results significant of this study mandibles serve as geometric a reference parameters in the investigation were also offound. this specific species. Possible linesThe of research results of to this follow study in theserve future as a reference include studies in the investigation of the non-stick of this surface specific characteristics species. Possible of the linesjaws, of molecular research compounds to follow in that the formfuture the include reinforcing studies elements of the non in the-stick chitin surface matrix, characteristics inhibitors of of metal the jaws,absorption molecular and metabolism compounds as that wood form preservatives the reinforcing and joint elements research inof the di ff chitinerent species matrix, ofinhibitors xylophages of metalthat attack absorption different and species metabolism of wood. as wood preservatives and joint research of different species of xylophages that attack different species of wood. Author Contributions: Conceptualization, R.D.M.; data curation, R.D.M.; formal analysis, R.D.M.; funding Authoracquisition, Contributions: R.D.M., J.-A.B. Conceptuali and L.A.; Investigation,zation, R.D.M R.D.M.;.; data curation, methodology, R.D.M. R.D.M.;; formal Project analysis, administration, R.D.M.; funding R.D.M.; resources, R.D.M.; supervision, R.D.M.; writing—original draft, R.D.M., J.-A.B., L.A. and L.-A.B.; writing—review acquisition, R.D.M., J.-A.B. and L.A.; Investigation, R.D.M.; methodology, R.D.M.; Project administration, & editing, R.D.M., J.-A.B., L.A. and L.-A.B. All authors have read and agreed to the published version of R.D.M.the manuscript.; resources, R.D.M.; supervision, R.D.M.; writing—original draft, R.D.M., J.-A.B., L.A. and L.-A.B.; writing—review & editing, R.D.M., J.-A.B., L.A. and L.-A.B. All authors have read and agreed to the published Funding: This research received no external funding. version of the manuscript. Acknowledgments: We thank Unidad Docente de Patología forestal y Conservación de madera de ETSI de Montes Funding:de La Universidad This research Polit receivedécnica de no Madrid, external Spain; funding. Unidad Docente de Industrias de los Productos Forestales de Acknowledgments: We thank Unidad Docente de Patología forestal y Conservación de madera de ETSI de Montes de La Universidad Politécnica de Madrid, Spain; Unidad Docente de Industrias de los Productos

Forests 2020, 11, 715 13 of 14

EUIT Forestal de la Universidad Politécnica de Madrid, Spain; Unidad Docente de Zoología y Enfermedades y Plagas de la EUIT Forestal de la Universidad Politécnica de Madrid, Spain; Eduard Vives for his advice and Sara Garrido Espinosa for the photo retouching and digital montage. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

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