giganteus and Typopeltis dalyi (Arachnida: Thelyphonida)...et al. 1993; Schmidt et al. 2000; Haupt &...

Elemental characterization of the exoskeleton in the whipscorpions Mastigoproctusgiganteus and Typopeltis dalyi (Arachnida: Thelyphonida)

James Gallant and Rick Hochberga

Department of Biological Sciences, University of Massachusetts Lowell, Lowell, Massachusetts 01854, USA

Abstract. The arthropod cuticle is a structurally diverse secretion that is largely composedof lipids, proteins, and a-chitin that function together in protection, prey capture, and as askeletal framework for efficient and diverse means of locomotion. Aquatic, aerial, and ter-restrial arthropods are well known to enrich their cuticles with trace elements that are pro-posed to strengthen or otherwise enhance specific regions of the exoskeleton. In this study,we provide evidence that whipscorpions (vinegaroons) enrich their exoskeleton with up to14 trace elements. We use energy-dispersive x-ray spectroscopy (EDS) to provide the firstcomparisons of trace element composition in two species of Thelyphonida: Mastigoproctusgiganteus (female) and Typopeltis dalyi (female and male). We analyzed the chemical com-position of eight regions that have sensory, locomotory, taxonomic, protective, and/orpredatory significance: the flagellum, prosoma, tarsal claws of the walking legs, tarsi of theantenniform legs, chelicerae, and the terminal three segments of the pedipalps. Our resultsreveal the presence of 14 trace elements across both species (12 elements in T. dalyi, 10 ele-ments in M. giganteus), and only four elements are present at significant levels (≥1%weight): Si, Cl, Ca, and Zn. The flagellum, antenniform leg tarsi, and prosoma keel lackthese elements in both species, while the chelicerae of all species are enriched with Ca, Zn,and Cl, and the tarsal claws are enriched with Zn. Significantly, we note the presence of Siin the prosomal carapace (but not the keel) of males and females of Typopeltis only, whichappears to be the first evidence of this transition metal in the arthropod exoskeleton. Wediscuss the significance of these chemical enrichments in whipscorpions and providehypotheses about their functional significance.

Additional key words: EDS, exoskeleton, arthropod, metallization, biomineralization

Whipscorpions are a small order of Arachnida(Thelyphonida) with slightly more than 100described species that inhabit tropical, subtropical,and arid environments across the globe. Whipscorpi-ons are part of the larger clade Pedipalpi that con-sists of Amblypygi (whip spiders), Thelyphonida(whipscorpions), and Schizomida (short-tailed whip-scorpions) (Harvey 2002). Species in the clade havea highly conserved morphology, including a pair ofstrong, raptorial pedipalps and a pair of antenni-form first legs, which no longer function in locomo-tion but instead have evolved a sensory function.Within the Pedipalpi, the orders Thelyphonida andSchizomida together form the clade Uropygi, largelycharacterized by a segmented pygidium and a pairof defensive glands (also known as acid glands, anal

glands, and pygidial glands) (Shultz 1990; McGoni-gle 2013). Species of Thelyphonida are largelydefined by the presence of two pygidial ommatoidsand muscularized, mobile, chelate fingers on theirpedipalps (Shultz 1990). The pygidial glands are themost distinctive feature of uropygids, and give thely-phonids one of their common names—vinegaroons.These glands secrete an acid cocktail of mostlyacetic acid (vinegar) and caprylic acid (octanoicacid) (Eisner et al. 1961; Itokawa et al. 1981; Hauptet al. 1993; Schmidt et al. 2000; Haupt & M€uller2004) that can be directed at potential predators inthe form of a spray.

In general, whipscorpions live in warm and humidenvironments, and so are best known from countriesin Australasia that harbor rich tropical forests (Har-vey 2002). The high humidity of these regions isthought to decrease the desiccation stress of manyarthropods and increase the quality (moisture

aAuthor for correspondence.E-mail: [email protected]

Invertebrate Biology x(x): 1–15.© 2017, The American Microscopical Society, Inc.DOI: 10.1111/ivb.12187

http://orcid.org/0000-0002-5567-5393

http://orcid.org/0000-0002-5567-5393

http://orcid.org/0000-0002-5567-5393

content) of the sediment they use as burrows (Punzo2006; Punzo & Ludwig 2006; Hembree 2013). Spe-cies of Typopeltis are common in the tropics, but todate, only T. crucifer POCOCK 1894 has been the sub-ject of any detailed investigations (Yogi & Haupt1977; Itokawa et al. 1981; Haupt 1996a; Schmidtet al. 2000; Kuwada et al. 2001). A distantly relatedspecies, M. giganteus (LUCAS 1835), has received sig-nificantly more attention. This species is uniqueamong whipscorpions in that it lives in both humidand arid environments and is native to North Amer-ica. Specimens of M. giganteus are regularly avail-able via the pet trade, and so the species has beenthe subject of several studies on chemical secretions(Eisner et al. 1961; Schmidt et al. 2000), ecology(Punzo 2006; Punzo & Ludwig 2006; Carrel & Britt2009; Hembree 2013), functional morphology(Shultz 1991, 1992; Klußmann-Fricke & Wirkner2016), physiology (Horne 1969; Ahearn 1970; Craw-ford & Cloudsley-Thompson 1971; Haupt et al.1980), reproductive biology and life history (Wey-goldt 1970, 1971; Wolff et al. 2015), and sensorybiology (Patten 1919; Haupt 1996a).

To date, there are few details on the structure andcomposition of the whipscorpion cuticle despite itsimportance in sensory biology (Barth & Stagl 1976),maintaining water balance in xeric environments(Ahearn 1970), and as a skeletal framework forunique forms of locomotion and burrowing (Shultz1991, 1992, 1993; Hembree 2013). Based on histo-logical observations of the exoskeleton in Typopeltis(unidentified sp.), whipscorpions have a multilayeredcuticle consisting of a waxy epicuticle up to 2 lmthick atop a much thicker internal cuticle up to73 lm thick (Krishnakumaran 1961, 1962). Theinternal layer is subdivided into exocuticle, mesocu-ticle, and outer and inner endocuticles, based onstaining properties and structural characteristicssuch as the presence of lamellae, vertical striae, andfiber bundles. To date, there are no ultrastructuralstudies of the whipscorpion cuticle, and only a sin-gle study has examined the elemental makeup of thewhipscorpion exoskeleton (Schofield 2001).

In 2001, Schofield examined the chemical compo-sition of the exoskeleton in 16 species of arachnidsincluding one species of whipscorpion (unidentified).In addition to the elements nitrogen (N), carbon(C), and oxygen (O) that make up the major cuticlecomponents of both proteins and alpha chitin, Scho-field also noted the presence of the transition metalzinc (Zn) that was incorporated into the chelicerae,pedipalps, and tarsal claws. The presence of transi-tion metals in these appendages follows a commonpattern of elemental enrichment noted for other

arachnids, which also tend to add some alkalineearth metals. For example, Schofield et al. (1989)discovered Zn and manganese (Mn) in the cheliceraeof the spider Araneus diadematus CLERCK 1758, andPoliti et al. (2012) discovered Zn, Mn, iron (Fe),copper (Cu), calcium (Ca), and chlorine (Cl) in thefangs of the spider Cupiennius salei KEYSERLING

1877. Scorpions also have Zn and Fe in their che-licerae (Schofield et al. 1989) as does Halobisiumoccidentale BEIER 1931, a marine pseudoscorpion(Gallant et al. 2016). These observations highlightthe common phenomenon of chemical enrichment,and more specifically, metal enrichment, in the cuti-cle of arthropods, and often in regions that areimportant in locomotion, defense, and predation(Hillerton & Vincent 1982; Schofield et al. 1989,2003; Fawke et al. 1997; Schofield 2001; Lichteneg-ger et al. 2003; Romano et al. 2007; Politi et al.2012; Erko et al. 2013; Amini et al. 2014; Gallantet al. 2016). Such studies of elemental enrichmentrely on a variety of methods (e.g., energy-dispersivex-ray spectroscopy [EDS, EDX], wavelength disper-sive x-ray spectroscopy, and particle-induced x-rayemission), but not all methods have the same sensi-tivities, so strict comparisons (e.g., weight percent-ages, details of enrichment) are often not possible.Still, they can provide important insights into thechemical diversity of the arthropod exoskeletonfrom ecological and functional perspectives, and somay have utility in our greater understanding ofarthropod cuticle evolution.

In this study, we apply EDS to characterize theelemental composition of the cuticle in two speciesof whipscorpions: female adults of M. giganteus,and male and female adults of T. dalyi POCOCK

1900. While our species selections were based solelyon specimen availability, we nonetheless tested twohypotheses regarding the types of enrichment andtheir locations in whipscorpions, independent of tax-onomic relationships: (1) transition metals are themajor trace elements used in enrichment; and (2) thehighest abundance of trace elements are found inregions that require increased hardness such asappendages that function in prey capture (e.g.,chelicerae and pedipalps).

Methods

We obtained specimens of M. giganteus (n=4)from American suppliers and dried specimens of T.dalyi (n=8) from insectworld.com. Importantly,we note that insectworld.com listings include the siteof collection (Thailand), and they also includedincorrect taxonomic identifications (as species of

Invertebrate Biologyvol. x, no. x, xxx 2017

2 Gallant & Hochberg

Thelyphonus). While we cannot verify the origins ofthe collections, we could verify the taxonomiesbeginning with Rowland & Cooke (1973) to cor-rectly identify the specimens as members of Typo-peltis. We then used the type descriptions byPocock (1900) and Kraepelin (1900), and redescrip-tions by Haupt (1996b,c) to verify our species iden-tifications, along with assistance from colleagues(see Acknowledgments). Photographs of key taxo-nomic characters are provided in Fig. 1. Specimensof M. giganteus were euthanized in a glasscontainer containing a cotton ball soaked in chloro-form. Specimens were then fixed in 2.5% glu-taraldehyde in 0.1 mol L�1 phosphate buffer salinefor 72 h and then dissected (soft tissues were usedfor another study). Appendages and cuticular struc-tures were removed from the specimens, dehydratedin a desiccator for 24 h, mounted on an aluminumstub with carbon tape, and placed back in a desic-cator for another 72 h. Dried specimens (Typopel-tis) were dissected and mounted on an aluminumstub with carbon tape and placed in a desiccatorfor 24 h. All mounted specimens were coated with8 nm of gold or gold-palladium alloy with a Den-ton Vacuum Desk IV and examined with a JEOLJSM-7410F field emission scanning electron micro-scope (FE-SEM) in the Materials CharacterizationLaboratory (MCL) at the University of Mas-sachusetts Lowell. The FE-SEM includes theEDAX Genesis XM2 Imaging System (Mahwah,New Jersey, USA) with a 10 mm2 Si(Li) energy-dis-persive spectroscopy (EDS) detector with SUTWwindow for the detection of all chemical elementsdown to beryllium (Be). Calibration of the EDSsystem was performed by MCL staff. The quantifi-cation method used for elemental analysis was stan-dardless quantification with the built-in ZAF(atomic number, absorption, and fluorescence)matrix correction factors, with an automatedBremsstrahlung background subtraction andnormalization to 100%.

Images and spectra were collected at 20 kV with aprobe current of 15 lA and emission current of10 lA. At the 20-kV-beam voltage, the x-rayabsorption effects of the ultrathin Au coating layerwere not considered in the quantitative analysis ofthe EDS data beyond the matrix (ZAF correctionfactor) contribution of Au. A probe current of15 lA is required for EDS analysis to generate suffi-cient x-ray excitation of the sample by the electronbeam coming from a cold cathode field emissiongun. No visible sample damage was observed withinthe duration of the analysis. Importantly, we didnot polish our specimens to give them flat surfaces,

which is normally performed for quantitative EDSand leads to improved accuracy of elemental data(Mukhopadhyay 2003). Our reasoning for not pol-ishing was twofold: the surfaces were irregular inshape and size, thus making polishing both difficultand time-consuming; we also wanted to collect ele-mental data from the most exterior surface (epicuti-cle) inwards, which would have been damaged bythe polishing techniques we had available. All ele-mental data were added to Excel software to calcu-late the mean % weights of each element�1standard deviation (SD). While quantitative accu-racy down to 0.01% can be achieved with EDS, werounded our percentage weights to the nearest 1%(see Supporting information, Appendices S1–S3)because our surfaces were not polished and thus theaccuracy could be misleading. The EDS scans arealso time-consuming and costly, so we chose to scanonly a few regions on all specimens that play a rolein protection, locomotion, predation, or havetaxonomic relevance.

To keep analyses consistent between specimensand species, 19 scans were taken from each speci-men at positionally similar (but not necessarily iden-tical) locations (see Figs. 1, 2). The followingregions were scanned: prosoma (one scan of the dor-sal keel and one scan of the dorsal carapace); flagel-lum (one scan at a random location); walking legs2–4 (one scan each of the distal and middle regionsof the tarsal claws, and one scan on the tertiaryspike); antenniform leg tarsi, leg 1 (random scan ofdistal segment); and chelicerae (one scan at distal,middle, and proximal regions). On the pedipalps,scans were taken of the patellar and tibial apophy-ses, as well as the entire tarsus. On the tarsus andtibia, scans were taken at distal (tip), medial (body),and proximal areas. On the patellar apophyses,scans were taken distally and on the teeth of all spe-cies. On specimens of T. dalyi, scans were taken onthe modification to the apophysis (spike), while themiddle (body) region of the apophysis was scannedon specimens of M. giganteus.

Results

We focused on the identity and locations of thetrace elements detected by EDS. The elements C, N,and O were present in all specimens and assumed tobe part of the protein–chitin matrix characteristic ofthe arthropod exoskeleton, and so were excludedfrom the descriptions below. The background car-bon tape was scanned for all specimens to determineif any trace element signals were detected, but onlyC and O were present. And while all preserved


Whipscorpion exoskeleton 3

specimens were cleaned of debris, we also scannedthe soil substrate that housed our living specimensof M. giganteus. The substrate had various elementspresent, including Ca, Fe, aluminum (Al), silicon(Si), and potassium (K). Below, we provide separatedescriptions of all species, including separatedescriptions of males and females of T. dalyi, tohighlight the differences in weight percentages of thetrace elements. As noted in the methods, weight per-centages are presented as mean�1 SD. For the pur-poses of this study, we consider an element presentwhen its weight percentage was 1% or higher, keep-ing in mind that there was variability when includ-ing the SD (e.g., an element of 2.45 � 0.49% wasrounded to 2%).

Typopeltis dalyi, male (Figs. 1A–D, 3, 4)

Four specimens were analyzed with major regionsphotographed (Figs. 1A–D, 4). Twelve trace ele-ments were consistently present across four speci-mens: Al, Si, Cl, K, Ca, Zn, fluorine (Fl), sodium(Na), magnesium (Mg), phosphorus (P), sulfur (S),and nickel (Ni). Two elements, Mg and S, while pre-sent at noise levels, were never present above 1%weight and thus were excluded from our descrip-tions below and in Fig. 3. Five other elements (Al,Fl, K, Ni, and P) were only present at select loca-tions, but never averaged >1% in any region of thecuticle. Four elements >1% weight were present: Si,Cl, Ca, and Zn.Flagellum. No trace elements (>1%) were present

on the body of the flagellum or the setae (Fig. 3A).The setae were sparse along the flagellum, and thecuticle itself had a scale-like appearance, with eitherhexagonal or pentagonal scale-like structures thatwere imbricated (Figs. 1A, 4A).Antenniform tarsus. No trace elements were pre-

sent above 1% weight (Fig. 3B). The tarsi were cov-ered in setae (Fig. 4B).Prosoma. The carapace of the prosoma contained

Si (5�2%) (Fig. 3C), but the keel did not containany trace elements. The keel extended longitudinallybetween the eyes and was mostly smooth with some

dimpling (Figs. 1B, 4C). The remainder of the pro-soma carapace, however, was roughly textured andhad sparse setae.Walking leg tarsus claw. Two elements, Cl and

Zn, were present. Zinc was present across the entireclaw including the distal (5�1%) and middle regions(3%), as well as the spike (6�1%) (Fig. 3D). Chlo-rine was present on the distal region of the claw(1%), absent from the middle region, and presenton the spike (1%). The cuticle of the claws wassmooth; bristles were present proximal to the tarsalclaw (Fig. 4D).Chelicera. Three elements were present: Cl, Ca,

and Zn (Fig. 3E). The proximal region had no traceelements, while the middle region only had Ca(1%). The distal region had the highest level ofbiomineralization with Cl (3%) and Zn (11%) pre-sent (Fig. 3E). The chelicerae had a smooth surfacebut were surrounded by setae (Fig. 4E).Pedipalp tarsus. Only Zn was present. It was pre-

sent in the distal (3%) and middle (1%) regions(Fig. 3F). There were no trace elements proximally.The lateral surfaces of the tarsi were mostly smoothwith some dimpling, while the medial section wasirregular and had a ridge extending down the center.Both medial and lateral cuticles were sparselycovered in setae (Fig. 4F).Pedipalp tibia. Silicon and Zn were present. Zinc

was present in the distal region (8�4%), while Siwas present proximally (2�1%) (Fig. 3G). The mid-dle region had no trace elements (Fig. 4G). Thecuticle of the tibia was similar to the tarsus, but wasmore irregular on the medial region, with a series oftooth-like ridges extending along the proximo-distalaxis. Setae were present as bristles and pore hairs(Fig. 4G).Pedipalp patella. Silicon and Zn were present.

The distal region had Zn (5�1%), while the teethhad both Si (2�1%) and Zn (3�2%) (Fig. 3H). Thespike region had no elements present (Fig. 3H). Thepatella structure was indicative of the genus Typo-peltis, as there was a large modification of theapophysis (Fig. 1C). The teeth extended along theouter edge, with the spike opposite them (Fig. 4H).

Fig. 1. Typopeltis dalyi from Thailand. Dried adult specimens that show taxonomic, sex-specific, and functional char-acters, some of which were analyzed with EDS. A. Male specimen, dorsal view. B. Male specimen, close-up of middor-sal keel between median eyes. C. Male specimen, dorsal view of the distal region of the pedipalps showing themodified patellar apophysis (mpa) diagnostic of males of the species. D. Male specimen, ventral view of anterioropisthosoma showing genital region. E. Female specimen, dorsal view. F. Female specimen, ventral view of anterioropisthosoma showing genital region. 1, 1st antenniform appendage (modified 1st walking appendage); 2–4, walking legs;aop (gp), anterior opercular plate (genital plate); fl, flagellum; le, lateral eyes; ke, middorsal keel; me, median eyes;mpa, modified patellar apophysis; op, opisthosoma; pa, patellar apophysis; pop, posterior opercular plate; pp,pedipalps; pr, prosoma; pt, patella; ta, tarsus; ti, tibia.



There were no setae present in the distal region.However, the ventral side of the patella included asmall number of setae in two lines extending alongthe proximo-distal axis (Fig. 4H).

Typopeltis dalyi, female (Figs. 1E,F, 3, 5)

Four specimens were analyzed with some regionsphotographed (Figs. 1E,F, 5). Twelve elements wereconsistently present across four specimens: Na, Mg,Al, Si, P, S, Cl, K, Ca, Mn, Fe, and Zn. Threeelements, P, S, and Mn, while present at noiselevels, were never present above 1% weight and thusexcluded from the descriptions below and in Fig. 3.Five elements (Na, Mg, Al, K, and Fe) wereonly present at a few locations, but never averaged>1% in any region. Four elements (Si, Cl, Ca,and Zn) were present at various locations andaveraged >1%.Flagellum. There were no trace elements present

(Fig. 3A). Setae were sparse and primarily sensoryin structure; more specifically, they were small, hadthin pore hairs, and had few long bristles (Fig. 1E).Antenniform tarsus. No trace elements were pre-

sent (Fig. 3B). The tarsi were covered in setae,although they appeared to be solely bristles, with noother forms visible (Fig. 5A).Prosoma. The prosoma contained only Si. The

carapace had Si (2�1%), while the keel had no traceelements (Fig. 3C). The keel was a fairly smoothstructure that extended between the eyes and hadminimal dimpling (Figs. 1E, 5B). There were twosetae located between the posterior end of the keeland the central eyes. The remainder of the prosomacarapace, however, was roughly textured, and hadsmaller setae that were sparsely distributed(Fig. 5B).Walking leg tarsal claw. Two elements, Cl and Zn,

were present. Zinc was present across the entire clawbut varied by weight: distal region (7�1%), middleregion (4�1%), and spike (10�3%) (Fig. 3D). Addi-tionally, Cl was present on the spike (1%) and distal(1%) regions of the claw, but absent from the middle(Fig. 3D). The cuticle on the claws was smooth, withbristles present only prior to the tarsal claw itself(Fig. 5C).

Chelicera. Three elements were present: Cl, Ca,and Zn. The proximal region had no trace elements,while the middle region had only Ca (1%) (Fig. 3E).The distal region had the highest level of biominer-alization, with both Cl (3�1%) and Zn (11�2%)(Fig. 3E). The chelicerae were smooth, but the sur-rounding cuticle was covered in filtering setae(Fig. 5D).Pedipalp tarsus. Two elements were present only in

the distal region of the tarsi: Cl (1%) and Zn (7�1%)(Fig. 3F). No trace elements were detected in the mid-dle or proximal regions (Fig. 3F). The exterior of thetarsi was smooth with sparse setae, while the interiorsection was irregular and had a ridge extending downthe center. There were rows of bristles and pore hairson either side of the ridge (Fig. 5E).Pedipalp tibia. The tibiae had two elements pre-

sent: Cl and Zn. While both were present in the dis-tal region (Cl, 2�1%; Zn, 5�1%), there were notrace elements in the middle or proximal regions(Fig. 3G). Both edges of the tibia had a row of teethextending down their length, with setae on eitherside (Fig. 5F). The more distal row of teeth lined upperfectly with the row of teeth in the tarsus, likelyproviding extra grip between the two segments. Theexterior cuticle had setae sparsely located on it, andappeared to dimple in multiple (Fig. 5F).Pedipalp patella. The patellae contained only Zn.

All regions contained Zn including the teeth(4�1%), distal end (6�1%), and spike (7�1%)(Fig. 3H). The teeth extended along the outer edgeand the spike opposite them (Fig. 5G). There wereno setae present in these areas. However, the ventralside of the patella included a variety of setae extend-ing along its teeth. In addition, setae were denselygrouped near the distal end of the spike, but lessabundant at the proximal end (Fig. 5G).

Mastigoproctus giganteus, female (Figs. 2, 3, 6)

Four specimens were examined with majorregions photographed (Figs. 2, 6). Ten elementswere present across all four animals: Na, Mg, Al,Si, P, S, Cl, K, Ca, and Zn. Four elements, Na, Mg,Al, and K, never exceeded >1% and so are excludedfrom the descriptions below and in Fig. 3. Three

Fig. 2. Mastigoproctus giganteus from the USA. Live adult specimen showing taxonomic and functional areas analyzedwith EDS. A. Live female specimen. B. Dorsal view of prosoma, anterior end. C. Pedipalp, with focus on patella. D.

Pedipalp, with focus on tibia and tarsus. 1, 1st antenniform appendage (modified 1st walking appendage); 2–4, walkinglegs; fl, flagellum; me, median eyes; op, opisthosoma; pa, patellar apophysis; pp, pedipalps; pr, prosoma; pt, patella; se,setae; ta, tarsus; te, tarsal teeth; ti, tibia; tr, trochanter.



Fig. 3. Elemental percentages (in atomic weight percentage) of eight individual whipscorpion regions (A–H) by speciesand/or sex. Histogram bars represent the major chemical elements: Si, black; Cl, white; Ca, gray; Zn, striped. MG,Mastigoproctus giganteus; TDF, female of Typopeltis dalyi; TDM, male of T. dalyi.



elements, Si, P, and S, were only present at a fewlocations but never averaged >1% throughout anyregion in the specimens. Three trace elements wereconsistently present >1%: Ca, Cl, and Zn.Flagellum. No trace elements were present

(Fig. 3A). The setae were sparse and were primarilysensory in structure. Most setae formed small, thinpore hairs and fewer formed long bristles (Fig. 6A).

Antenniform tarsus. No trace elements were pre-sent (Fig. 3B). Tarsi were covered in setae, althoughthey appeared to be solely bristles, with no otherforms (Fig. 6B).Prosoma. No trace elements were present

(Fig. 3C). The keel was a fairly smooth structurethat extended between the eyes and had minimaldimpling. The remainder of the prosoma carapace,however, had a waved texture, with setae sparselylocated throughout (Fig. 6C).Walking leg tarsal claw. Only Zn was present at

significant levels. Zinc was present across the entireclaw: distally (3�1%), in the middle (2�1%), aswell as on the spike (2%) (Fig. 3D). The cuticle onthe claws was smooth, with bristles present onlyprior to the tarsal claw itself (Fig. 6D).Chelicera. Three elements, Cl, Ca, and Zn were

present. The proximal region had no trace elements,while the middle region had Cl (1%) and Ca (1%)(Fig. 3E). The distal region had the highest level ofbiomineralization with Cl (3�1%) and Zn (10�3%)(Fig. 3E). The chelicerae were mostly smooth withvarious filtering setae toward the mouth (Fig. 6E).Pedipalp tarsus. Two elements were present, Cl

and Zn. Both were present in the distal regions (Cl,2�1%; Zn, 7�1%), but there were no trace ele-ments in the middle or proximal regions (Fig. 3F).The tarsus had a ridge of teeth extending down thedorsal and ventral sides, with the ventral teeth beingmore prominent. The lateral surface of the tarsuswas mostly smooth, with setae regularly dispersedthroughout. The medial section had lines of setaeextending down the middle (Fig. 6F).Pedipalp tibia. The tibiae had two trace elements,

Cl and Zn. Both were present in the distal regions(Cl, 1%; Zn, 7�2%), but there were no trace ele-ments in the middle or proximal regions (Fig. 3G).The structure of the tibia was similar to the tarsus,but shorter, with teeth extending down dorsal andventral sides (Fig. 6G). The ventral teeth were moreprominent, but the dorsal teeth were larger in thetibiae than the tarsi. The medial surface wassmoother, with a group of setae each extending onthe dorsal and ventral sides, but the central regionwas mostly devoid of setae.Pedipalp patella. Chlorine and Zn were present.

The distal region had both elements present (Cl,2%; Zn, 7�1%), as did the teeth (Cl, 1%; Zn,6�1%) (Fig. 3H). The middle region had no traceelements (Fig. 3H). The patellae were irregular andrough unlike the tarsi and tibiae (Fig. 6H). Theteeth were again on the ventral side, but fewer innumber than on the tarsi or tibiae. There were vari-ous setae randomly distributed on the medial side of

Fig 4. Electron micrographs of EDS-scanned regions ofmale T. dalyi. A. Scale-like surface of flagellum. B. Anten-niform tarsus. C. Prosoma carapace. D. Leg tarsal claw.E. Chelicerae. F. Pedipalp tarsus. G. Pedipalp tibia. H.

Pedipalp patella. di, distal region; ey, eye; ke, keel; mi,middle region; pr, proximal region; sp, spike; te, teeth.



the patellae and more regularly spaced on the lateralsurface.

Discussion

The arthropod cuticle is a multifunctionalexoskeleton that is traditionally described as multi-laminate, with a thin epicuticle over a thicker

procuticle that may be subdivided into exocuticle,mesocuticle, and endocuticle based on structuraland histochemical criteria (Neville 1975). In theirsimplest forms, these laminae consist of a matrix ofproteins, lipids, and/or chitin (Neville 1975). Stabi-lization of the protein component often comes fromtanning or enrichment (biomineralization) with vari-ous chemical elements, particularly the alkaline

Fig. 5. Electron micrographs of EDS-scanned regions offemale Typopeltis dalyi. A. Antenniform tarsus. B.

Prosoma carapace. C. Leg tarsal claw. D. Chelicerae. E.Pedipalp tarsus. F. Pedipalp tibia. G. Pedipalp patella. di,distal region; ey, eye; ke, keel; mi, middle region; pr,proximal region; sp, spike; te, teeth.

Fig. 6. Electron micrographs of EDS-scanned regions offemale Mastigoproctus giganteus. A. Flagellum. B.

Antenniform tarsus. C. Prosoma carapace. D. Leg tarsalclaw. E. Chelicerae. F. Pedipalp tarsus. G. Pedipalp tibia.H. Pedipalp patella. di, distal region; ey, eye; ke, keel; mi,middle region; pr, proximal region; sp, spike; te, teeth.



earth metals such as Ca and the transition metalssuch as Mn and Zn (Neville 1975; Vincent 2002).Marine arthropods are well known to strengthentheir cuticle by depositing Ca in the form of calciumcarbonate and calcium phosphate (e.g., Bentov et al.2016). Non-marine (freshwater, terrestrial) arthro-pods such as some arachnids, insects, and myri-apods may also strengthen their cuticle with Ca(e.g., Vohland et al. 2003), sometimes in the form ofcalcium oxalate (Norton & Behan-Pelletier 1991).Most arthropods are assumed to obtain Ca throughtheir diets (e.g.,Vohland et al. 2003), and in low-cal-cium environments, may be able to form Ca reserves(Greenaway 1985). More common to terrestrialarthropods, though, appears to be the incorporationof various transition metals into their cuticles, gen-erally in the form of Zn and Mn (Schofield & Lev-ere 1989; Schofield et al. 1989; Schofield 2001;Cutler & McCutchen 2006). The arachnids, as asubset of the terrestrial fauna, are known to miner-alize their exoskeletons with Zn, Fe, and Mn, whichare assumed to function as hardening agents andmay therefore contribute to the strength of appen-dages used in predation such as the chelicerae andpedipalps (reviewed in Schofield 2001; Gallant et al.2016). Despite these observations, the chemical com-positions of arachnid exoskeletons are poorlyknown, and to date, there are only two briefdescriptions of elemental enrichment in species ofUropygida (Schofield 2001; Cutler & McCutchen2006). Here, we examined two species of vinega-roons (Uropygida: Thelyphonida) in detail to deter-mine whether, how, and where they enrich theircuticles.

Elemental enrichment

Our analyses detected up to 12 trace elements inthe cuticle of male and female Typopeltis dalyi, and10 trace elements in M. giganteus. These elementsare assumed to be in the epi- and/or exocuticularregions, as a constraint of EDS is the depth atwhich the electrons can penetrate (~4 lm), and thewhipscorpion cuticle has been measured at 73 lm(e.g., adult Thelyphonus sp., 2 lm epicuticle, 18 lmexocuticle, >50 lm mesocuticle+endocuticle; Krish-nakumaran 1962). The most abundant (by %weight) trace elements for both T. dayli and M.giganteus were Ca, Cl, and Zn; both sexes of Typo-peltis also had Si, which was found at non-signifi-cant levels in M. giganteus. Our analyses revealedthat the majority of regions we scanned (13 of 18/19) have some level of elemental enrichment, andthese were generally confined to the distal regions of

various appendages. Only six regions lacked evi-dence of significant enrichment (>1%): the flagellum,prosoma keel, antenniform legs, proximal region ofthe chelicerae, proximal region of the pedipalp tarsi,and the middle region of the tibiae. Zinc is consis-tently present in two regions of both species: themiddle region of the walking legs’ tarsal claws andthe distal region of the chelicerae. Five additionallocations have the same chemical enrichment inboth sexes of T. dayli: the middle region of the che-licerae (Ca), the distal and spike regions of thewalking legs’ tarsal claws (Cl, Zn), the distal regionof the pedipalps’ patellae (Zn), and the prosomacarapace (Si). While other trace elements were alsopresent in most of these regions, their levels werealways below 1%, and in many cases could not bedistinguished from “noise” levels, and so are notconsidered in detail here.

Zinc is the most abundant trace element in allthree whipscorpions, present primarily in the tarsalclaws as well as the distal regions of the pedipalpsand chelicerae. Zinc was also noted in the tarsalclaws, chelicerae, and pedipalps of an unidentifiedvinegaroon (Schofield 2001), and in the palpal clawof a schizomid (also known as short-tailed whipscor-pion, part of the Uropygida: Cutler & McCutchen2006). In our studies, we note that Cl is almostexclusively present in scans where Zn is present. Pre-vious research on other arthropods has also showna correlation between these two elements, althoughCl levels found in the present study are much lowerthan previous findings (Schofield 2001; Schofieldet al. 2003; Politi et al. 2012). A plot (not shown) ofZn versus Cl in atomic percentage (R2=0.679)reveals a 2.5:1 ratio of Zn:Cl. From a total of 57scans across both species, 13 scans reveal both Znand Cl, 13 scans reveal Zn without Cl (Fig. 3D,F–H), and one scan reveals Cl without Zn (individualscans not shown). These data support the hypothesisthat the presence of Zn does not indicate the pres-ence of Cl, but Cl does indicate the presence of Zn.These correlations may have importance for futurestudies of elemental enrichment in arachnids oncetests of hardness, elasticity, and other mechanicalproperties begin to functionally characterize theimportance of different elements in the exoskeletons(e.g., Raabe et al. 2005; Barbakadze et al. 2006;Sachs et al. 2006).

Prior to this study, Si was not known from anycontemporary arthropod’s exoskeleton (currentlyonly known as a result of diagenesis, e.g., Anomalo-caris; see Paterson et al. 2011), but we have evidencethat it is present in the prosoma carapace of bothsexes of T. dalyi. We note that Si was also detected



in M. giganteus but at non-significant levels. Inmales of T. dalyi, Si is also present in the tibia andpatella of the pedipalps. Importantly, we do notknow the specific origins of the dried specimens usedin these analyses (other than collected in Thailand),nor if any postmortem treatments were performed,so we cannot be certain if the Si is natural or artifi-cial. However, the EDS analyses revealed that theoverall chemical composition of T. dalyi is similarto that of M. giganteus (laboratory raised), and sowe predict that Si is naturally present in theexoskeleton of Typopeltis. And while the function ofthe Si remains unknown, this element has beenshown to increase elasticity in biological materialsthat contain a significant Si component, such as pineseedlings (Emadian & Newton 1989). Elasticity isdue, in part, to the orientation of the Si crystals(Hopcroft et al. 2010), which is of course unknownin our specimens. If the Si does in fact add elasticityto the exoskeleton, then its function in the prosomamay be related to ecdysis. Whipscorpions exit theirexuvium by backing out of the region between theprosoma and opisthosoma, and therefore the addedSi component may increase flexion to ease the ecdy-sial process in some species. Additionally, we notethe presence of Si in the tibia and patella of thepedipalps in males of T. dalyi only; we are uncertainhow Si and its potential in adding elasticity to thesearticles might alter their function.

The only other significant (by % weight) trace ele-ment in the vinegaroon exoskeleton is calcium. Thisalkaline earth metal is important in the enrichmentof the exoskeleton of marine and terrestrial pancrus-taceans as calcite, amorphous calcium carbonate,and calcium phosphate (e.g., Ziegler 1994; Beckeret al. 2005; Romano et al. 2007; Ziegler et al. 2007;Erko et al. 2013; Amini et al. 2014). However, theabundance and form of Ca in arachnids are not wellcharacterized and only a few studies document thepresence of Ca in species of Araneae (Schofield2001), Ricinulei (Cutler & McCutchen 2006), andScorpiones (Schofield et al. 2003; Politi et al. 2012).Here, we show that both species of whipscorpionhave Ca present at one specific location: the middleregion of the chelicerae. The fact that Ca is presentin identical regions of the chelicerae in both speciessuggests it plays a significant role in their function,but what this role may be is unknown. Otherarthropods such as some semi-terrestrial pancrus-taceans (isopods) and fully terrestrial insects andmillipedes also incorporate Ca into their cuticles,and their levels of enrichment are often muchhigher. For example, the isopods Armadilidium vul-gare LATREILLE 1804 and Porcellio scaber LATREILLE

1804 have Ca contents >10% (Becker et al. 2005),while two rainforest millipedes have cuticles with21–25% Ca by weight (Vohland et al. 2003). Stillmost terrestrial arthropods do not appear to calcifytheir exoskeletons to the same extent as marine pan-crustaceans (e.g., Hillerton & Vincent 1982; Fawkeet al. 1997; Schofield et al. 2003), but whether this isdue to a lack of Ca in their prey or some otherfunction of Ca availability or physiological process-ing by the animals remains unknown.

Functional ramifications of enrichment for appendages

The common theme with elemental enrichmentamong whipscorpions, and in fact among all arach-nids, is that structures that function in locomotion,predation, and defense are almost always supportedby elements such as Zn that are known or assumedto strengthen the exoskeleton (Politi et al. 2012;Erko et al. 2013; Amini et al. 2014; Gallant et al.2016). In addition, there appears to be a gradient ofenrichment in these appendages or structures wherethe more distal regions receive increased support rel-ative to proximal regions. For example, the distalregion of the chelicera; the tarsus, tibia, and patellaof the pedipalp; and the tarsus of the leg are allreinforced, while proximal and middle regionsreceive little or no support. Appendages that serve asensory role (antenniform legs and flagellum) do notreceive any significant elemental support.

Whipscorpions have three pairs of walking legs,with tarsi that closely resemble those of relatedarachnids, especially schizomids, amblypygids, andspiders (Dunlop 2000). They have a three-forkedtarsus, with two major claws and a tertiary spike,which allows them to grasp surfaces extraordinarilywell. Therefore, the spike and distal regions of theclaws are the primary points of contact that whip-scorpions use for locomotion, and these regions arereinforced with specific elements in the same manneramong species (e.g., Schofield 2001; this study).While the middle region of the tarsal claws does notcome into contact with the substratum as much asthe distal region, it is still reinforced in a similarmanner. As a whipscorpion can hold its entireweight on one claw for a short period of time (un-publ. data), the entire claw needs to be strongenough to support the weight of the animal.

Whipscorpion pedipalps are the primary appen-dages for prey capture. These appendages are con-structed of five articles: the coxa, trochanter, femur,patella, tibia, and tarsus. We examined the distalthree articles, as they are often the first to makecontact with potential prey; however, we



acknowledge that all articles play a role in grasping,and the trochanters may be significant in immobiliz-ing prey. Significantly, the distal region of thesethree articles is highly enriched, while the middleregions receive low levels of support (Zn in the tarsiof male T. dalyi) or no incorporation at all. Thepatellar teeth were also found to have high enrich-ment. The teeth and distal regions of the articles allmake contact with potential prey and/or predators,and therefore need to inflict damage (puncture orcrush their prey) when doing so. The teeth increasethe surface area of the articles, thereby increasingfriction between predator and prey, and decreasingthe ability of prey to escape. The presence of Zn inthese regions therefore makes functional sense, as itcan be used to strengthen the cuticle in regionswhere high-stress interactions might take place.

Maceration of prey is performed by the che-licerae, which are formed of a fixed article that isdensely covered in setae, and a mobile terminal digit(Dunlop 1994). The mobile digit is the only regionvisible through the setae, and for this reason it wasthe area we examined with EDS. The digit is heavilyenriched with both Cl and Zn (>10%) in all species.The middle region of the digit, as previously stated,is the only region with Ca enrichment. The distalregion of the digit is the primary point of contactduring mastication and feeding; this region mustpenetrate the integument of their prey (often otherarthropods) prior to the release of digestiveenzymes. Therefore, a heavily biomineralized digit isto be expected in a predator such as a whipscorpion.Similar elemental enrichments of the terminal articleare present in other arachnid chelicerae includingspiders (Schofield et al. 1989; Schofield 2001; Politiet al. 2012; Erko et al. 2013), scorpions (Schofield2001), and pseudoscorpions (Gallant et al. 2016).

Conclusions

Examining the elemental enrichment of two whip-scorpions provides insight into how ecology and evo-lution affect the mineralization of the thelyphonidcuticle. Both species showed enrichments characteris-tic of other arachnids such as the incorporation of amixture of transition and alkaline earth metals in thechelicerae, pedipalps, and tarsal claws of the legs.However, the depth of this analysis and its importantnew findings (e.g., the presence of Si) reveals that thechemical profile of the thelyphonid exoskeleton is stillincompletely known, and highlights the need for addi-tional studies that examine comparable (i.e., homolo-gous) regions in other arachnids. In general, theminimal differences in enrichment among the

whipscorpions studied here suggests that mineraliza-tion is a conservative physiological phenomenon thattranscends broad environmental differences (e.g.,tropical vs. xeric). Appendages and structures thatare important in locomotion, prey capture, anddefense are consistently enriched with hardening traceelements across all taxa.

Acknowledgments. The authors are grateful to the twoanonymous reviewers who provided both technical adviceon the methods and recommendations to improve theoverall quality of the manuscript. The authors also thankDr. Earl Ada (UML) for his assistance with training onthe SEM-EDS, and we owe thanks to Dr. Lorenzo Pren-dini and Scientific Assistant Jeremy Huff (AmericanMuseum of Natural History) for confirming the identityof our dried specimens. The authors have no conflict ofinterest to declare.

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Supporting information

Additional Supporting information may be found in theonline version of this article:

Appendix S1. Elemental data (in % weight) for differentanatomical regions in male of Typopeltis dalyi.Appendix S2. Elemental data (in % weight) for differentanatomical regions in female of Typopeltis dalyi.Appendix S3. Elemental data (in % weight) for differentanatomical regions in female of Mastigoproctus giganteus.



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