Microstructure and Mechanical Properties of the Dactylopodites of the Chinese Mitten Crab (Eriocheir Sinensis)

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Article Microstructure and Mechanical Properties of the Dactylopodites of the Chinese Mitten Crab (Eriocheir sinensis)

Ying Wang 1, Xiujuan Li 1,* ID , Jianqiao Li 1 and Feng Qiu 2 ID 1 Key Laboratory of Bionic Engineering, Ministry of Education, Jilin University, Changchun 130025, China; [email protected] (Y.W.); [email protected] (J.L.) 2 Key Laboratory of Automobile Materials, Ministry of Education, Department of Materials Science and Engineering, Jilin University, Changchun 130025, China; [email protected] * Correspondence: [email protected]; Tel.:+86-431-8509-5760

Received: 1 April 2018; Accepted: 21 April 2018; Published: 26 April 2018

Abstract: The dactylopodites of the Chinese mitten crab (Eriocheir sinensis) have evolved extraordinary resistance to wear and impact loading after direct contact with rough surfaces or clashing with hard materials. In this study, the microstructure, components, and mechanical properties of the dactylopodites of the Chinese mitten crab were investigated. Images from a scanning electron microscope show that the dactylopodites’ exoskeleton was multilayered, with an epicuticle, exocuticle, and endocuticle. Cross sections and longitudinal sections of the endocuticle revealed a Bouligand structure, which contributes to the dactylopodites’ mechanical properties. The main organic constituents of the exoskeleton were chitin and protein, and the major inorganic compound was CaCO3, crystallized as calcite. Dry and wet dactylopodites were brittle and ductile, respectively, characteristics that are closely related to their mechanical structure and composition. The ﬁndings of this study can be a reference for the bionic design of strong and durable structural materials.

Keywords: dactylopodite; microstructure; composition; mechanical properties

1. Introduction After hundreds of millions of years of evolution, the functional properties of parts of organisms tend to have optimal structural and material characteristics, as well as excellent adaptability and longevity [1,2]. The strong exoskeletons of decapod crustaceans such as crabs and lobsters are an adaption to harsh environments [3] and play important roles in supporting the body, resisting impacts from external forces, providing protection from external erosion, predators, and disease, and preventing the evaporation of water [4–6]. The Chinese mitten crab (Eriocheir sinensis) is a widespread freshwater crustacean. It has an impressive ability to move on hard and deformable terrain, such as mire. Due to their unique strong shell and means of locomotion, Chinese mitten crabs are able to adapt to a variety of complex terrains, a characteristic that has attracted widespread attention from researchers [7–10]. Most studies of crustaceans’ hard shells have focused on pincers and other parts [3,8], whereas studies of pereiopods have focused mostly on gait, kinematics, and energy conversion [11,12]. However, in adaptation to complex terrains, in addition to the Chinese mitten crabs’ means of locomotion, their dactylopodites, which come into contact with the terrain during movement, also play an important role [13]. As the end parts of the pereiopods, dactylopodites are in direct contact with rough ground and clash with hard materials such as gravel. They possess excellent mechanical properties and unique biological characteristics, such as shape, structure, and composition. Studies on the dactylopodites of the Chinese mitten crab are scarce.

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Appl. Sci. 2018, 8, 674 2 of 13 unique biological characteristics, such as shape, structure, and composition. Studies on the dactylopodites of the Chinese mitten crab are scarce. ThisThis paper paper presents presents an an analysis analysis of of the the shape, shape, structure, structure, components, components, and and mechanical mechanical properties properties of theof dactylopodites the dactylopodites of Chinese of Chinese mitten crabs,mitten helping crabs, us helping to further us tounderstand further understand the excellent the adaptability excellent andadaptability athletic ability and athletic of these ability crustaceans. of these Thecrustaceans. research willThe provideresearch newwill bionicprovide design new bionic methods design for strongmethod ands for durable strong structural and durable materials, structural which materials, would be which applied would to the be development applied to the and development preparation ofand walking preparation mechanism of walkingdesign with mechanism optimal performance, design with optimization optimal performance, of environmental optimization adaptation, of andenvironmental minimum energy adaptation, consumption and minimum on soft energy road terrain. consumption on soft road terrain.

2.2. ExperimentalExperimental MethodsMethods

2.1.2.1.Preparation Preparation ofof Dactylopodite Dactylopodite Samples Samples from from Chinese Chinese Mitten Mitten Crabs Crabs ChineseChinese mittenmitten crabscrabs (Figure(Figure1 )1) were were purchased purchased from from an an aquatic aquatic products products market market in in Changchun, Changchun, China.China. SevenSeven crabscrabs werewere selected,selected, ofof whichwhich fourfour werewere malemale (two(two werewere larger,larger, twotwo werewere smaller)smaller) andand × threethree were were female. female. TableTable1 1shows shows the the crabs’ crabs’ shell shell sizes sizes (length (length ×width) width) and and their their weights. weights. Studies Studies havehave shownshown thatthat thethe secondsecond andand thirdthird pairspairs ofof paraeiopodsparaeiopods areare importantimportant forfor movement;movement; therefore,therefore, thethe secondsecond andand thirdthird paraeiopods paraeiopods werewere selectedselected asas thethe subjects subjects of of study. study. TheThe dactylopoditesdactylopodites werewere removedremoved from from the the propodite–dactylopodite propodite–dactylopodite joints joints (as (as shown shown in in Figure Figure1) 1) for for experimental experimental use. use.

FigureFigure 1. 1.Chinese Chinese mitten mitten crab. crab.

TableTable 1. 1.Parameters Parameters of of Chinese Chinese mitten mitten crab crab specimens. specimens. Dimension Specimen Dimension Weight (g) Specimen Length (mm) Width (mm) Weight (g) 1 (male) Length71.21 (mm) Width64.03 (mm) 167 2 (male)1 (male) 68.42 71.21 64.0362.38 167 174 3 (male)2 (male) 57.46 68.42 62.3852.73 174 92 3 (male) 57.46 52.73 92 4 (male) 55.74 51.61 81 4 (male) 55.74 51.61 81 5 (female)5 (female) 54.81 54.81 51.32 77 77 6 (female)6 (female) 56.12 56.12 51.18 75 75 7 (female)7 (female) 51.50 51.50 49.22 63 63 MeanMean ± SD± SD59.32 59.32 ± 7.44± 7.44 54.64 ± ± 5.96 104.14104.14± 46.18 ± 46.18

2.2.2.2.Biological Biological CharacteristicsCharacteristics ofof Chinese Chinese Mitten Mitten Crabs Crabs

2.2.1.2.2.1.Microstructure Microstructure of of the the Dactylopodites Dactylopodites ToTo obtain obtain high-quality high-quality microstructure microstructure images, images, the surface the surface of the of specimens the specimens to be observed to be observed should beshould flat, and be flat, the structureand the structure should be should clear. Whenbe clear. samples When weresamples taken, were a Cartesian taken, a Cartesian coordinate coordinate grid was establishedgrid was established with the dactylopodite with the dactylopodite as the origin as (Figure the origin2). The (Figure dactylopodites 2). The dactylopodites were divided into were fivedivided segments into five along segments the x-axis along with the a surgical x-axis with blade, a surgical and the filmblade, and and muscles the film inside and weremuscles removed. inside Thewere segments removed. were The marked segments 1, 2, were 3, 4, andmarked 5 in order1, 2, 3, of 4 decreasing, and 5 in cross-sectionalorder of decreasing diameter, cross left-sectional to dry naturally,diameter, and left then to dry sprayed naturally, gold. a Thend thenstructures sprayed of the gold. cross The sections structures were of observed the cross with sections a scanning were

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observed with a scanning electron microscope (SEM) (Zeiss EVO 18, Cambridge, England). When electronobserving microscope the longitudinal (SEM) (Zeiss section EVO 18, structure, Cambridge, the UK). segments When were observing cut the along longitudinal the axis section of the structure,dactylopod theite. segments were cut along the axis of the dactylopodite.

FigureFigure 2.2. CoordinateCoordinate gridgrid withwith ChineseChinese mittenmitten crabcrab dactylopodites.dactylopodites.

2.2.2.2.2.2. CompositionComposition ofof thethe DactylopoditesDactylopodites X-rayX-ray diffraction diffraction (XRD)(XRD) (Model(Model D/Max D/Max 2500PC 2500PC Rigaku, Rigaku, Tokyo,Tokyo, Japan)Japan) and and Fourier Fourier transformtransform infraredinfrared (FTIR) (FTIR) analyses were were conducted conducted to to identify identify the the components components of the of theChinese Chinese mitten mitten crab crabdactylopodites dactylopodites.. SamplesSamples forfor XRDXRD werewere preparedprepared byby breakingbreaking fragmentsfragments fromfrom thethe dactylopodites, dactylopodites, removingremoving thethe musclemuscle tissue,tissue, dryingdrying thethe fragmentsfragments naturally,naturally, andand crushingcrushing andand grindinggrinding themthem intointo aa powderpowder inin aa mortarmortar untiluntil thethe particleparticle sizesize waswas aboutabout 4545µ μm.m. TheThe detectiondetection apparatusapparatus waswas anan X-rayX-ray diffractometerdiffractometer withwith Cu-KCu-Kαα radiation radiation (λ(λ= = 1.5405981.540598A). A). ForFor thethe FTIRFTIR analysis,analysis, thethe drieddried dactylopoditesdactylopodites werewere crushedcrushed andand groundground intointo aa ﬁnefine powderpowder andand thenthen scatteredscattered evenlyevenly onon aa glassglass slideslide andand compactedcompacted toto formform aasample. sample. AA ShimadzuShimadzu FnR-8400SFnR-8400S (Shimadzu(Shimadzu Corporation, Corporation, Kyoto, Kyoto, Japan) Japan) was was used used to detectto detect the the components. components. The The frequency frequency range range of the of experimentthe experiment was was 400 to400 4000 to 4000 cm− cm1. −1.

2.3.2.3. Three-PointThree-Point BendingBending TestsTests TheThe secondsecond andand thirdthird pairspairs ofof dactylopoditesdactylopodites werewere selectedselected forfor three-pointthree-point bendingbending teststests andand divideddivided intointo drydry and wet samples. To To prepare prepare the the dry dry samples, samples, the the dactylopodites dactylopodites were were placed placed in inseparate separate aluminum aluminum boxes, boxes, and and the the weights weights of of all all the the dactyli dactyli and boxes were measured measured with with an an electronicelectronic balancebalance (JJ323BC,(JJ323BC, GG && G,G, Suzhou,Suzhou, China).China). TheThe samplessamples werewere thenthen putput intointo dryingdrying boxesboxes atat 8080◦ C°C and and dried dried for for 4 h. 4 Theh. The weights weights were were then then recorded, recorded, and theand samples the samples were returned were returned to the drying to the boxesdrying for boxes another for hour.another If nohour. change If no was change recorded, was recorded, drying was drying stopped; was otherwise, stopped; otherwise, drying continued drying untilcontinued the weight until didthe weight not change. did not change. ToTo prepareprepare thethe wet samples, the the segmented segmented dactylopodites dactylopodites were were soaked soaked in inwater water about about 1 h. 1 To h. Toprevent prevent moisture moisture loss loss and and to to prevent prevent the the samples samples becoming becoming soft soft after after soaking, which wouldwould affectaffect theirtheir mechanicalmechanical properties,properties, the the tests tests were were completed completed within within 4 4 h. h. ConsideringConsidering thethe actualactual workingworking conditionsconditions ofof thethe specimen,specimen, thethe testtest positionspositions werewere selectedselected andand markedmarked asas 2/52/5 from from the the top top of of the the dactylopodite dactylopodite (Figure (Figure 2).2). The The maximum maximum diameter diameter d dandand length length l atl ateach each marked marked place place on on the the specimens specimens were were measured measured with with a VernierVernier caliper.caliper. Three-pointThree-point bendingbending experimentsexperiments were were carriedcarried out out using using a a small small in in situsitu tension tension tester tester (Changchun (Changchun Mechanical Mechanical Science Science Institute,Institute, maximum maximum load load 50 50 N, N, 220 220 V, V, 50 50 Hz). Hz). At At least least ﬁve five samples samples were were selected selected for eachfor each test. test. The testThe spantest span was 15was mm, 15 andmm, the and pressure the pressure head moved head moved at 3 mm/min. at 3 mm/min. Each sample Each sample was ﬁxed was on fixed the holders, on the thenholders, a pressure then a head pressure was moved head was towards moved it driven towards by a it motor. driven When by a the motor. indenter When was the exposed indenter to was the sample,exposed the to computerthe sample, began the computer to automatically began recordto automatically the load on record the sample the load and on the the displacement sample and data. the Thedisplacement tests were data. stopped The when tests were the samples stopped reached when the the samples yield point reached or broke. the yield point or broke.

Appl. Sci. 2018, 8, 674 4 of 13 Appl. Sci. 2018, 8, x FOR PEER REVIEW 4 of 12 Appl. Sci. 2018, 8, x FOR PEER REVIEW 4 of 12 3.3. Results Results and and D Discussioniscussion 3. Results and Discussion 3.13.1.. Structural Structural P Propertyroperty 3.1. Structural Property 3.1.13.1.1.. Microstructure Microstructure of of Cross Cross Secti Sectionon of of a a D Dactylopoditeactylopodite 3.1.1. Microstructure of Cross Section of a Dactylopodite AA layered layered structure structure is is characteristic characteristic of of the the exoskeletons of of crustaceans [14]. [14]. Numerous Numerous studies studies A layered structure is characteristic of the exoskeletons of crustaceans [14]. Numerous studies havehave shown shown that that most most exoskeletons exoskeletons include include an epicuticle, an epicuticle, an exocuticle, an exocuticle, and an and endocuticle an endocuticle[9,10,15] . have shown that most exoskeletons include an epicuticle, an exocuticle, and an endocuticle [9,10,15].TheThe SEM results SEM showed results that showed a cross that section a cross of a section Chinese of mitten a Chinese crab dactylopodite mitten crab dactylopoditecontains three [9,10,15].The SEM results showed that a cross section of a Chinese mitten crab dactylopodite containslayers, as three shown layers, in Figure as shown3. The in epicuticle,Figure 3. The which epicuticle, is a thin which layer ofis a wax thin on layer the outerof wax surface on the ofout theer contains three layers, as shown in Figure 3. The epicuticle, which is a thin layer of wax on the outer surfacedactylopodite, of the dactylopodite, is easy to peel offis easy in the to dry peel state. off Thein the middle dry state. layer The is the middle exocuticle, layer and is the the exocutic inner layerle, surface of the dactylopodite, is easy to peel off in the dry state. The middle layer is the exocuticle, anisd the the endocuticle. inner layer is the endocuticle. and the inner layer is the endocuticle.

Figure 3. Multilayered structure of Chinese mitten crab dactylopodite. FigureFigure 3. 3. MultilayeredMultilayered structure structure of of Chinese Chinese mitten crab dactylopodite. Figure 4 shows the cross-sectional structures of various parts of a dactylopodite. The thickness Figure 4 shows the cross-sectional structures of various parts of a dactylopodite. The thickness ratiosFigure between4 shows the exocuticle the cross-sectional and the structures endocuticl ofe various differed parts among of across dactylopodite. sections with The thickness different ratios between the exocuticle and the endocuticle differed among cross sections with different diameters.ratios between Figure the 4a exocuticleshows a cross and section the endocuticle of the first section differed of amongthe specimen cross sectionsshown in with Figure different 2. The diameters. Figure 4a shows a cross section of the first section of the specimen shown in Figure 2. The thicknessesdiameters. of Figure the exocuticle4a shows and a cross endocuticle section of were the 382.4 ﬁrst sectionand 304.6 of theμm, specimen respectively, shown yielding in Figure a ratio2. thicknesses of the exocuticle and endocuticle were 382.4 and 304.6 μm, respectively, yielding a ratio ofThe 1.26. thicknesses Figure 4bof shows the exocuticle a cross section and endocuticle of the third weresection 382.4 of the and specimen 304.6 µm, in respectively, Figure 2, in which yielding the a of 1.26. Figure 4b shows a cross section of the third section of the specimen in Figure 2, in which the thicknessesratio of 1.26. of Figure the exocuticle4b shows and a cross endocuticle section of were the third310.4 sectionand 383.5 of theμm, specimen respectively. in Figure The 2diameter, in which of thicknesses of the exocuticle and endocuticle were 310.4 and 383.5 μm, respectively. The diameter of thethe thirdthicknesses section of of the the exocuticle sample and was endocuticle smaller than were that 310.4 of the and first 383.5 section,µm, respectively. and the thickness The diameter ratio the third section of the sample was smaller than that of the first section, and the thickness ratio betweenof the third the sectionlayers was of the 0.81. sample When was the smaller cross-sectional than that diameter of the ﬁrst was section, reduced, and the the thickness thickness ratios ratio between the layers was 0.81. When the cross-sectional diameter was reduced, the thickness ratios betweenbetween the the exocuticle layers was and 0.81. the Whenendocuticle the cross-sectional were correspondingly diameter reduced. was reduced, the thickness ratios betweenbetween the exocuticle and the endocuticle were correspondingly reduced.

Figure 4. Thickness of the exocuticle and endocuticle at different positions along a Chinese mitten Figure 4. Thickness ofof thethe exocuticle exocuticle and and endocuticle endocuticle at at different different positions positions along along a Chinese a Chinese mitten mitten crab crab dactylopodite: (a) Section at l = 5 mm, (b) Section at l = 15 mm. crabdactylopodite: dactylopodite (a) Section: (a) Section at l = at 5 l mm, = 5 mm (b), Section (b) Section at l = at 15 l = mm. 15 mm. For the same cross section, the thickness of the exocuticle and endocuticle changed with the For the same cross section, the thickness of the exocuticle and endocuticle changed with the concave–convex variation of the dactylopodite. Figures 3 and 4a show the microstructure of a cross concave–convex variation of the dactylopodite. Figures 3 and 4a show the microstructure of a cross

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For the same cross section, the thickness of the exocuticle and endocuticle changed with the concave–convexAppl. Sci. 2018, 8, x variation FOR PEER REVIEW of the dactylopodite. Figures3 and4a show the microstructure5 of 12 of a cross section at l = 5 mm. The concave and convex areas are marked A and B, respectively, section at l = 5 mm. The concave and convex areas are marked A and B, respectively, in Figure 5. The in Figure5 . The thickness of the exocuticle at position A was 140.7 µm, which was only 36.79% thickness of the exocuticle at position A was 140.7 μm, which was only 36.79% of the exocuticle of the exocuticle thickness at position B. That is, the structural proﬁle was uneven within a single thickness at position B. That is, the structural profile was uneven within a single section of sectiondactylopodite. of dactylopodite. Appl. Sci. 2018, 8, x FOR PEER REVIEW 5 of 12

section at l = 5 mm. The concave and convex areas are marked A and B, respectively, in Figure 5. The thickness of the exocuticle at position A was 140.7 μm, which was only 36.79% of the exocuticle thickness at position B. That is, the structural profile was uneven within a single section of dactylopodite.

FigureFigure 5. Concave 5. Concave (A ()A and) and convex convex ((BB) areas of of the the dactylopodite dactylopodite seen seen in cross in cross section. section.

Figure 6 shows the layered structure of the cross section of the dactylopodite. The SEM results showFigure no6 shows obvious the stratified layered structure structure in of the the exocuticle cross section layer of (Figure the dactylopodite. 6a); however, The a stratified SEM results showstructure no obvious was clear stratifiedFigure from 5. C structuretheoncave outside (A) and in to convex the the exocuticleinside (B) areas of ofthe the layer endocuticle dactylopodite (Figure 6layer,seena); in however, ascross shown section. ain stratified Figure 6b structure. was clearIt from can be the seen outside in Figure to the 6c that inside the ofendocuticle the endocuticle is overlapped layer, by as multiple shown inlayers Figure of fibers6b. that are Figure 6 shows the layered structure of the cross section of the dactylopodite. The SEM results boundedIt can be by seen amorphous in Figure calcium6c that thebodies. endocuticle The thickness is overlapped of the fiber by layer multiple was about layers 6 ofμm, fibers and the that are show no obvious stratified structure in the exocuticle layer (Figure 6a); however, a stratified boundedthickness bystructure amorphousof the was interlayer clear calciumfrom was the outsideabout bodies. 1to μm, the The inside forming thickness of the a endocuticlevery of dense, the fiber layer, twisted, layeras shown plywood was in Fi aboutgure-like 6 6bstructure. µm, and the thicknessknown of as the aIt Bouligandcan interlayer be seen instructure Figure was about 6 c[16]. that 1Eachtheµ endocuticlem, layer forming contains is overlapped a very an array dense, by of multiple independent twisted, layers plywood-likeof fiber fibers bundles. that are structureThe knownenlarged asbounded a Bouligand image by showsamorphous structure that calcium the [ fiber16 ].bodies. bundles Each The layer thickness in the contains same of the fiber anfiber array layer layer arewas of independent alignedabout 6 μm, along and fiberthe the same bundles. angle,thickness which ca ofuses the theinterlayer fiber layerwas about to intersect 1 μm, forming with the a veryadjacent dense, layers. twisted, Each plywood group-like of fiberstructure bundles The enlarged image shows that the fiber bundles in the same fiber layer are aligned along the same consistsknown of many as a Bouligand filamentous structure fibers [16]. with Each a helical layer contains twist structure,an array of asindependent shown in fiberFigure bundles. 6d. The The cross angle,section whichenlarged of causesthe fiber image the bundle shows fiber thatlayerreveals the to fibertiny intersect bundlespores between with in the the same fibers, adjacent fiber as layershown layers. are in aligned EachFigure groupalong 6e. theInterestingly, of same fiber bundles consistssome of fibersangle, many whichare filamentous interwoven causes the infiber fibers an layer orderly with to intersect manner a helical with into twist the crisscrossed adjacent structure, layers. crossing as Each shown groupframesin of (Figure Figurefiber bundles 6fd.), whichThe cross consists of many filamentous fibers with a helical twist structure, as shown in Figure 6d. The cross sectionincreases of the the fiber structure’s bundle toughness, reveals tiny stability, pores and between strength. fibers, as shown in Figure6e. Interestingly, section of the fiber bundle reveals tiny pores between fibers, as shown in Figure 6e. Interestingly, some fibers are interwoven in an orderly manner into crisscrossed crossing frames (Figure6f), some fibers are interwoven in an orderly manner into crisscrossed crossing frames (Figure 6f), which which increasesincreases the the structure’s structure’s toughness, toughness, stability, stability, and strength. and strength.

Figure 6. Cont.

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Figure 6. LayeredFigure 6. microstructureLayered microstructure of crosssections of crosssections of Chinese of Chinese mitten mitten crab crab dactylopodite: dactylopodite: (a( a))Exocuticle of dactylopodite,Exocuticle ( bof) dactylopodite endocuticle, ( ofb) dactylopodite,endocuticle of dactylopodite (c) Bouligand, (c) Bouligand structure structure of endocuticle, of endocuticle (,d ) twisted (d) twisted filaments of fiber, (e) porosities between the fibers, (f) crossing frames. filamentsFigure of 6.fiber, Layered (e) porosities microstructure between of the crosssections fibers, (f) crossingof Chinese frames. mitten crab dactylopodite: (a) ExocuticleFigure3.1.2. Microstructure 6. ofLayered dactylopodite microstructureof Longitudinal, (b) endocuticle ofSection crosssections of of dactylopodite Dactylopodite of Chinese, ( c) Bouligand mitten structure crab dactylopodite of endocuticle: (a,) (d) twisted filaments of fiber, (e) porosities between the fibers, (f) crossing frames. 3.1.2. MicrostructureExocuticleThe SEMof dactylopodite of results Longitudinal show, ( b that) endocuticle Section the longitudinal ofof dactylopodite Dactylopodite sections , of(c ) dactylopoditesBouligand structure also hadof endocuticle a layered , (structured) twisted (Figure filaments 7). Theof fiber fibrous, (e) playersorosities of thebetween endocuticle the fibers are, densely(f) crossing arranged frames (Figure. 8a), and its 3.1.2. Microstructure of Longitudinal Section of Dactylopodite The SEMsurface results is densely show covered that with the elliptic longitudinal pore canals. sectionsThe pore canals of dactylopodites are formed by thealso crossing had of a layered structure3.1.2.The (FigurefiberMicrostructure SEMbundles7 ). results The (A inof fibrous Figure showLongitudinal 8 thatb layers), with the Section a of longitudinalhelical the of endocuticleband Dactylopodite in sections each pore are of canal. densely dactylopodites The spiral arranged ribbon also (Figure(B had in Figure a 8 layereda), and its 8b) is a pore canal tubule; these small tubes can transport nutrients across the exocuticle to new surfacestructure isThe densely (FigureSEM resultscovered 7). The show fibrous with that elliptic layers the longitudinalof pore the endocuticle canals. sections The are pore ofdensely dactylopodites canals arranged are formed also(Figure had by8 a a), the layeredand crossing its exoskeletons [17]. Meanwhile, soft and continuous tissues help increase the toughness of the of fibersurfacestructure bundles is (Figuredensely (A in7). covered FigureThe fibrous with8b), withellipticlayers a ofpore helical the canals. endocuticle band The in pore eachare denselycanals pore are canal.arranged formed The (Figure by spiral the crossing8a ribbon), and itsof (B in exoskeleton. This porous structure can be observed in the fiber layers of many crustaceans, including fiber bundles (A in Figure 8b), with a helical band in each pore canal. The spiral ribbon (B in Figure Figuresurface8b)the is is aedible densely pore crab canal covered (Cancer tubule; paguruswith these elliptic) [18], small thepore green tubescanals. crab can The(Carcinus transport pore maenascanals nutrients) are[19], formed the acrossAmerican by the the lobstercrossing exocuticle of to new8 exoskeletonsfiberb) is bundles (Homarus a pore canal(Aamericanus [17 in]. Figure tubule; Meanwhile,) [20], 8 theseb and), with Scapharca small soft a helical tubesand subcrenata continuousband can transport[21].in each tissuespore nutrients canal. help The across increase spiral the ribbon exocuticle the toughness (B in to Figure new of the exoskeleton.exoskeletons8b) is a pore This canal[17]. porous Meanwhile, tubule; structure these soft can small andbe tubes observed continuous can transport in the tissues fiber n utrients help layers increase acrossof many the the crustaceans, toughnessexocuticle to of including new the the edibleexoskeleton.exoskeletons crab (This Cancer[17]. porous Meanwhile, pagurus structure)[ 18 soft can], theand be observed green continuous crab in the (tissuesCarcinus fiber layers help maenas increaseof many)[19 crustaceans, the], the toughness American includin of the lobsterg the edible crab (Cancer pagurus) [18], the green crab (Carcinus maenas) [19], the American lobster (Homarusexoskeleton. americanus This)[ porous20], and structureScapharca can be subcrenata observed [in21 the]. fiber layers of many crustaceans, including (theHomarus edible americanus crab (Cancer) [20], pagurus and Scapharca) [18], the subcrenata green crab [21]. ( Carcinus maenas) [19], the American lobster (Homarus americanus) [20], and Scapharca subcrenata [21].

Figure 7. Layered structure in longitudinal section of dactylopodite.

Figure 7. Layered structure in longitudinal section of dactylopodite. Figure 7. Layered structure in longitudinal section of dactylopodite. Figure 7. Layered structure in longitudinal section of dactylopodite.

Figure 8. Cont.

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Figure 8. Microstructure of longitudinal section of dactylopodite: (a) Layered structure in exocuticle, Figure 8. Microstructure of longitudinal section of dactylopodite: (a) Layered structure in exocuticle, Figure(b ,8.c) Microstructurepore canal and pore of longitudinal canal tubule. section of dactylopodite: (a) Layered structure in exocuticle, (b,c) pore canal and pore canal tubule. (b,c) pore canal and pore canal tubule. The tip of the dactylopodite is the part that makes the most frequent contact with the ground, Therubbing tip of and the colliding dactylopodite with sand is and the gravel.part that It possesses makes makes the high most most strength frequent and abrasion contact resistance.with the ground, As shown in Figure 9a, unlike the structure of the other parts of the dactylopodite, the exocuticle rubbing and and colliding colliding with with sand sand and and gravel. gravel. It possesses It possesses high high strength strength and and abrasion abrasion resistance. resistance. As As shownaccounts in Figure for most9a, of unlike the exoskeleton the structure at the of tip,the withother a parts thickness of the about dactylopodite, three times that the exocuticle of the shownendocuticle. in Figure The 9a ,exocuticle unlike the at the structure tip is highly of the condensed, other parts with of pore the channels dactylopodite, crossing thethrough exocuticle it accounts for most of the exoskeleton at the tip, with a thickness about three times that of the accounts(Figure for 9 b most).The endocuticle of the exoskeleton structure isat similar the tip, to that with in the a thicknessother parts aboutof the dactylopodites, three times that with ofa the endocuticle.Bouligand The The structure, exocuticle exocuticle as shown at at the the in tip Figure tip is is highly highly9c. This condensed, condensed,kind of structure with with porehas pore high channels channels strength crossing but crossing maintains through through it (itFigure (Figuresufficient 9b9b).The).The toughness endocuticle endocuticle to prevent structure structure the dactylopodites is is similar similar to to thatthat from in in being the the other fractured parts by of shock the dactylopodites, [16]. withwith aa Bouligand structure, as shown in Figure 99c.c. ThisThis kindkind ofof structurestructure hashas highhigh strengthstrength butbut maintainsmaintains sufficientsufﬁcient toughness to prevent the dactylopodites fromfrom beingbeing fracturedfractured byby shockshock [[16].16].

Figure 9. Longitudinal section of the tip of Chinese mitten crab dactylopodite: (a) Layered structure at tip of dactylopodite, (b) highly condensed structure of exocuticle, (c) Bouligand structure in endocuticle.

3.2. Material Properties Figure 9. Longitudinal section of the tip of Chinese mitten crab dactylopodite: (a) Layered structure FigureXRD 9. Longitudinal and FTIR were section used of the to tip analyze of Chinese the material mitten crab composition dactylopodite: of the (a) Layered exoskeleton structure of the at at tip of dactylopodite, (b) highly condensed structure of exocuticle, (c) Bouligand structure in tipdactylopodites. of dactylopodite, The (resultsb) highly of condensedXRD analysis structure show a of narrow exocuticle, band (c )peak Bouligand at 2θ = structure 29.5° with in endocuticle.a full width endocuticle. at the half maximum (FWHM) of 0.27 and peak intensity of 180, indicating a crystalline structure. In 3.2. Materialaddition, Properties smaller diffraction peaks were seen at 2θ = 36.1°, 36.6°, 39.6°, 39.7°, 43.3°, and 48.8°, as 3.2. Material Properties XRD and FTIR were used to analyze the material composition of the exoskeleton of the XRD and FTIR were used to analyze the material composition of the exoskeleton of the dactylopodites. The results of XRD analysis show a narrow band peak at 2θ = 29.5◦ with a full dactylopodites. The results of XRD analysis show a narrow band peak at 2θ = 29.5° with a full width width at the half maximum (FWHM) of 0.27 and peak intensity of 180, indicating a crystalline structure. at the half maximum (FWHM) of 0.27 and peak intensity of 180, indicating a crystalline structure. In addition, smaller diffraction peaks were seen at 2θ = 36.1°, 36.6°, 39.6°, 39.7°, 43.3°, and 48.8°, as

Appl. Sci. 2018, 8, 674 8 of 13

Appl. Sci. 2018, 8, x FOR PEER REVIEW 8 of 12 In addition, smaller diffraction peaks were seen at 2θ = 36.1◦, 36.6◦, 39.6◦, 39.7◦, 43.3◦, and 48.8◦, shown in Figure 10. Comparative analysis [22] revealed that calcite is the predominant form of as shown in Figure 10. Comparative analysis [22] revealed that calcite is the predominant form of CaCO3 in the exoskeleton. CaCO3 in the exoskeleton.

FigureFigure 10. 10. ResultsResults of of XRD XRD analysis. analysis.

StudiesStudies have have shown shown that that chitin chitin is is a a common common substance substance in in the the exoskeleton exoskeleton of of crustaceans crustaceans [23,24]. [23,24]. KayaKaya et et al. al. [2 [255]] used used FTIR FTIR analysis analysis of of the the exoskeleton exoskeleton of of the the blue blue crab crab ( (Callinectes sapidus sapidus)) and and found found −−1 −−11 OO–H–H stretching at wavewave numbernumber 34323432 cmcm ,, CH33 andand CH 2 stretching at 29192919 cmcm ,, C=O C=O secondary secondary amideamide stretching stretching at at 1657 1657 cm cm−−1, 1N, N–H–H bending bending and and C– C–NN stretching stretching at at1550 1550 cm cm−1, −C1,–O C–O–C–C stretching stretching at 1064at 1064 cm cm−1, − and1, and C–H C–H ring ring stretching stretching at at 890 890 cm cm−1.− 1The. The FTIR FTIR results results for for the the grooved grooved tiger tiger prawn prawn ((PenaeusPenaeus semisulcatus) showedshowed that that the the same same functional functional groups groups appeared appeared at wave at wave numbers numbers 3438, 3438, 2918, 2918,1656, 1562,1656, 1072,1562, and1072, 899 and cm 899−1, respectivelycm−1, respectively [26]. In [2 the6]. In exoskeleton the exoskeleton components components of Gammarus of Gammarus argaeus , argaeusabsorption, absorption peaks were peaks shown were at 3433, shown 2913, at 3433, 1655, 1554, 2913, 1064,1655, and 1554, 894 1064, cm− 1and[27], 894 showing cm−1 [2 characteristics7], showing characteristicsof the same functional of the same groups. functional groups. FigureFigure 1111 shows the FTIR results of the Chinese mitten crab dactylopodite. The The graph graph shows shows absorptionabsorption peaks peaks at 3440, 1635, 1408, 1075, 870, and 560 cm −11. .Compared Compared with with the the results results above, above, it it can can bebe concluded concluded that the absorption peak ofof 34403440 cmcm−−1 isis O O–H–H stretching, 1635 cmcm−−11 iiss C=O C=O secondary secondary amideamide stretching, stretching, 1408 1408 cm −1 1isis N N–H–H bend bend and and C C–N–N stretching, stretching, 1075 1075 cm−1 1isis C C–O–O stretching stretching in in the the −−11 −−11 phasephase ring, and 870 cm isis closer closer to to CH CH ring ring stretching. stretching. At 2930 cmcm , ,weak weak CH CH33 stretchingstretching and and CH CH2 stretchingstretching are are seen. seen. TheThe above above analysis analysis suggests suggests that the that organic the organic components components of the exoskeleton of the exoskeletonof the dactylopodites of the dactylopoditesof the Chinese of mitten the Chinese crab are mitten mainly crab chitin are andmainly protein chitin ﬁber and and protein that fiber the inorganic and that componentthe inorganic is componentmainly CaCO is mainly3 in the CaCO form of3 in crystallized the form of calcite. crystallized calcite.

3.33.3.. Mechanical Mechanical P Propertiesroperties FigureFigure 12 illustratesillustrates thethe stressstress condition condition of of hitting hitting the the ground ground and and the the supporting supporting state state when when the theChinese Chinese mitten mitten crab crab is moving, is moving, ignoring ignoring the the force force of frictionof friction and and considering considering only only the the effect effect of theof thesupport support reaction. reaction. Figure Figure 12a 12 showsa shows the statethe state when when the tipthe oftip dactylopodite of dactylopodite (T) comes (T) comes into contactinto contact with withthe ground, the ground, and Figureand Figure 12b shows12b shows the statethe state of the of footstepthe footstep during during the supportingthe supporting phase. phase. When When the thedactylopodites dactylopodites are are in contactin contact with with the the ground ground and and supporting, supporting, a bending a bending moment moment will will form form at theat theend end of the of dactylopodite the dactylopodite O’, due O’, todue the to impulsive the impulsive force and force the and support the support reaction reactionFN. If the FN bending. If the bending strength of the dactylopodite is insufficient, a bending fracture will occur. To observe the mechanical properties of dactylopodites, three-point bending tests were conducted on dry and wet specimens.

Appl. Sci. 2018, 8, 674 9 of 13 strength of the dactylopodite is insufﬁcient, a bending fracture will occur. To observe the mechanical propertiesAppl. Sci. 2018 of, 8 dactylopodites,, x FOR PEER REVIEW three-point bending tests were conducted on dry and wet specimens.9 of 12 Appl. Sci. 2018, 8, x FOR PEER REVIEW 9 of 12

FigureFigure11 11.. FTIRFTIR resultsresults forfor thethe dactylopoditedactylopodite exoskeleton.exoskeleton. Figure11. FTIR results for the dactylopodite exoskeleton.

Figure 12. Forces on a dactylopodite during locomotion. (a) hitting the ground; (b) the supporting Figurestate. 12. Forces on a dactylopodite during locomotion. (a) hitting the ground; (b) the supporting Figure 12. Forces on a dactylopodite during locomotion. (a) hitting the ground; (b) the supporting state. state. The specimens for the three-point bending test were the second and third pairs of dactylopodites, whichThe were specimens divided for into the dry three-pointthree and-point wet bending samples. test Table were 2 the shows second the and parameters third pairs of ofthe dactylopodites,dactylopodites samples. The, whichtests were werewere carried divideddivided out into into by dry andry andin and situ wet wettensile samples. samples. tester. Table TableEach2 shows sample2 shows the was parametersthe fixed parameters on ofthe the holders, of samples. the samples. and The the tests spanThe weretestswas were carried15 mm. carried outThen by out load an by in an on situ i itntensile situ with tensile pressure tester. tester. Each head Each sample driven sample was by ﬁxed was a motor fixed on the aton holders, a the constant holders, and speed theand span the until span was the 15wassamples mm. 15 mm. Then were Then load broken. onload it with on it pressure with pressure head driven head driven by a motor by a atmotor a constant at a constant speed until speed the until samples the weresamples broken. were broken. Table 2. Parameters of dactylopodite samples used for three-point bending tests. Table 2. Parameters of dactylopodite samples used for three-point bending tests. Table 2. Parameters of dactylopodite samples used forParameters three-point bending tests. Samples Moisture Content Max. Outer DiameterParametersParameters Inner D iameter Length SamplesSamples MoistureMoistureϕ Content (%) Content MaxMax.. OuterOuterD (mm) Diameter Diameter InnerInnerD D (mm) Diameteriameter Lengthl Length(mm) Dry φϕ(%) (%)0 2.71DD(mm) (mm) ± 0.28 1.39DD (mm) ±(mm) 0.046 32.62l (mm)l (mm) ± 1.95 DryDryWet 41.06 00 ± 4.64 2.712.712.50± ±±0.28 0.28 0.15 1.391.31 1.39 ±±± 0.046 0.0050.046 32.6230.8832.62 ±± ± 1.951.161.95 WetWet 41.0641.06± ±4.64 4.64 2.502.50± ±0.15 0.15 1.31 1.31 ±± 0.0050.005 30.8830.88 ± ±1.161.16 The bending strength of a dactylopodite, which can be calculated by Equation (1), is affected by a numberThe bending of factors, strength including of a dactylopodite,the structure, composition,which can be and calculated size. The by cross Equation section (1) ,of is theaffected Chinese by amitten number crab of dactylopoditefactors, including is irregularly the structure, oblong composition, with curved and grooves size. The on cross its circumference section of the and Chinese has a mittenvariable crab cross dactylopodite section arc structure.is irregularly Its composition oblong with is curved nonhomogeneous. grooves on its The circumference section modulus and hasof the a variablebending cross section section is difficult arc structure. to calculate Its composition accurately. Thereforeis nonhomogeneous. the cross section The section of dactylopodite modulus of wasthe bendingapproximated section asis difficult annular to in calculate calculation. accurately. Thus M Therefore is proportional the cross to section load F ofand dactylopodite span L, and was the approximated as annular in calculation. Thus M is proportional to load F and span L, and the

Appl. Sci. 2018, 8, 674 10 of 13

The bending strength of a dactylopodite, which can be calculated by Equation (1), is affected by a number of factors, including the structure, composition, and size. The cross section of the Chinese mitten crab dactylopodite is irregularly oblong with curved grooves on its circumference and has a variableAppl. Sci. 2018 cross, 8, section x FOR PEER arc structure.REVIEW Its composition is nonhomogeneous. The section modulus of10 theof 12 bending section is difﬁcult to calculate accurately. Therefore the cross section of dactylopodite was approximatedmodule of bending as annular section in calculation. is anti-correlated Thus M is with proportional the third to load powerF and of span diameterL, and of the section module D ofapproximately. bending section The is bending anti-correlated stress can with be thecalculated third power accordingly of diameter: of section D approximately. The bending stress can be calculated accordingly:M FL / 4  b   3 4 , (1) MW D (FL1/4 ) / 32 = = σb 3 4 , (1) where M is the bending moment, W is theW moduleπD of(1 bending− α )/32 section, F is the load applied, L is span, 4 whereα indicatesM is the d/D bending, π(1 − α moment,)/32 ≈ 0.092W .is the module of bending section, F is the load applied, L is span, α indicatesFigured /13D shows, π(1 − aα set4)/32 of ≈curves0.092. for bending stress–displacement. The bending stress of the dry specimensFigure increases13 shows linearly a set of with curves the fordisplacement bending stress–displacement. of indenter, reaches a The maximum bending value stress of of56.781 the dryMPa specimens, then drops increases rapidly linearly and decreases with the at displacementabout 0.553 mm of indenter,. The bending reaches load a maximumof the wet specimen value of 56.781increases MPa, with then displacement drops rapidly and and reaches decreases a maximum at about of 0.553 85.525 mm. MPa The at 0.780 bending mm load, then of begins the wet to specimendecrease increasesslowly and with drops displacement dramatically and at reaches 1.357 amm maximum. The results of 85.525 show MPa that at the 0.780 dry mm, dactylopodites then begins toexhibited decrease typical slowly characteristics and drops dramatically of brittle atmaterial, 1.357 mm. and The the resultschange show of the that bending the dry stress dactylopodites curve was exhibitedlinear and typical tended characteristics to break within of brittle a small material, displacement and the change. The ofwet the specimens bending stress were curve distorted was linearwhen andloaded tended during to break the test, within so the a small maximum displacement. bending Thestress wet occurred specimens after were a longer distorted displacement when loaded. The duringslow decrease the test, soof thethe maximum bending stress bending in stressthe later occurred period after of the a longer test also displacement. showed that The as slow the decreaseindenter ofdisplacement the bending increased, stress in the even later though period the of tensile the test stress also showedon the back that of as the the dactylopodite indenter displacement increased and the specimen deformed to a large degree, it did not break, indicating the strength of the wet increased, even though the tensile stress on the back of the dactylopodite increased and the specimen deformeddactylopodite. to a large degree, it did not break, indicating the strength of the wet dactylopodite.

FigureFigure 13. 13.Curves Curves of of bending bending stress–displacement. stress–displacement.

Figure 14 shows the statistics of the maximum value of bending stress and the corresponding Figure 14 shows the statistics of the maximum value of bending stress and the corresponding displacement of indenter moved. Comparing the average maximum of dry and wet samples, the displacement of indenter moved. Comparing the average maximum of dry and wet samples, maximum bending stress they were able to withstand were about 63.339 ± 9.483 MPa and 81.817 ± the maximum bending stress they were able to withstand were about 63.339 ± 9.483 MPa and 15.884 MPa, respectively, which indicated an excellent mechanical property of Chinese mitten crab 81.817 ± 15.884 MPa, respectively, which indicated an excellent mechanical property of Chinese dactylopodite. The bending stress of the wet samples was 29.17% higher than that of the dry mitten crab dactylopodite. The bending stress of the wet samples was 29.17% higher than that of samples. The displacement of indenter reflects the deformation of samples, which indicates they the dry samples. The displacement of indenter reﬂects the deformation of samples, which indicates have better toughness with greater deformation under the higher load. In the figure, the average they have better toughness with greater deformation under the higher load. In the ﬁgure, the average displacements for dry and wet samples are approximately 0.594 ± 0.044 mm and 0.698 ± 0.078 mm, displacements for dry and wet samples are approximately 0.594 ± 0.044 mm and 0.698 ± 0.078 respectively, at the time of stress reaching the maximum. It is well known that biomechanical mm, respectively, at the time of stress reaching the maximum. It is well known that biomechanical properties depend greatly on hydration [28]. The results of the three-point bending experiment properties depend greatly on hydration [28]. The results of the three-point bending experiment show show that moisture content influenced the bending strength of the dactylopodites and that the failure stress as greater for the wet dactylopodites than for the dry ones, which is consistent with the results conducted by Hepburn et al. on wet and dry specimens of mud crab (Scylla serratu) [29].

Appl. Sci. 2018, 8, 674 11 of 13 that moisture content inﬂuenced the bending strength of the dactylopodites and that the failure stress as greater for the wet dactylopodites than for the dry ones, which is consistent with the results

Appl.conducted Sci. 2018 by, 8, x Hepburn FOR PEER etREVIEW al. on wet and dry specimens of mud crab (Scylla serratu)[29]. 11 of 12

Figure 14. Comparison of stress and displacement for dry and wet samples samples..

44.. Conclusions In thisthis work,work, thethe structure structure and and mechanical mechanical properties properties of theof the dactylopodites dactylopodites of Chinese of Chinese mitten mitten crab werecrab were investigated. investigated. The conclusionsThe conclusions are as are follows. as follows.

1. TheThe exoskeleton exoskeleton of of the the dactylopodites dactylopodites of Chinese of Chinese mitten mitten crab is crab multilayered, is multilayered, with an epicuticle, with an epicuticle,exocuticle exocuticle and endocuticle. and endocuticle. The thickness The thickness ratio of each ratio layer of each is related layer is to related the section to the diameter section diameterand the concave and the andconcave convex and aspects convex of aspects the proﬁle. of the As profile. the cross-sectional As the cross diameter-sectional decreases, diameter decreases,the thicknesses the thicknesses of the exocuticle of the andexocuticle endocuticle and endocuticle reduce and reduce increase, and respectively. increase, respectively. For a given Forsection, a given the section, thickness the of thickness the concave of the area concave is less thanarea thatis less of than the convex that of area.the convex area.

2. CrossCross sections sections and and longitudinal longitudinal sections sections of the endocuticleof the endocuticle of the exoskeleton of the of exoskeleton the dactylopodites of the dactylopoditesreveal a Bouligand reveal structure, a Bouligand which structure, benefits their which mechanical benefits properties.their mechanical Each layer properties. of the fibrous Each layerlayer of consists the fibrous of several layer consists regularly of arrangedseveral regularly fiber bundles. arranged Many fiber porebundles. canals Many are pore distributed canals areregularly distributed in the regular fiber layer,ly in the in which fiber layer, pore canalin which tubules pore transport canal tubules nutrients transport while nutrients increasing while the increasingtoughness the of the toughness inner epidermis. of the inner epidermis. 3. The main organic constituents of the exoskeleton of the dactylopodites are chitin and protein, 3. The main organic constituents of the exoskeleton of the dactylopodites are chitin and protein, and the major inorganic compound is CaCO3 crystallized as calcite. and the major inorganic compound is CaCO3 crystallized as calcite. 4. Due to their structure and composition, the dry dactylopodites had characteristics of brittle 4. Due to their structure and composition, the dry dactylopodites had characteristics of brittle materials, whereas the wet dactylopodites had characteristics of ductile materials. materials, whereas the wet dactylopodites had characteristics of ductile materials. Author Contributions: Xiujuan Li and Jianqiao Li conceived and designed the initial idea; Xiujuan Li analyzed andAuthor interpreted Contributions: experimentsXiujuan data; Li andYing Jianqiao Wang wrote Li conceived the article and manuscript; designed the Feng initial Qiu idea; revised Xiujuan the Limanuscript analyzed forand publication. interpreted experiments data; Ying Wang wrote the article manuscript; Feng Qiu revised the manuscript for publication. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. U1601203) U1601203)and Postdoctoral and Postdoctoral Science Foundation Science Foundation of China (No. of China 801161050414). (No. 801161050414). ConflictsConflicts of Interest: The authors declare no no conflict conflict of of interest. interest.

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