calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (acari:...

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Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida) ROY A. NORTON State University of New York College of Environmental Science and Forestry, Syracuse, NY 13210, U.S.A. AND VALERIE M. BEHAN-PELLETIER Biosystematics Research Centre, Agriculture Canada, Ottawa, Ont., Canada KIA OC6 Received September 7, 1990 NORTON, R. A., and BEHAN-PELLETIER, V. M. 1991. Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida). Can. J. Zool. 69: 1504-15 11. Hardening of adult oribatid mite cuticle by the deposition of crystallinecalcium salts is demonstratedby a combinationof X-ray diffraction and X-ray dispersion methods, in conjunction with experimental acid decalcification. Calcite (calcium carbonate) is the mineral deposited by mites representating the Ptyctima, but three species of Enarthronota (Eniochthonius minutissimus, Archoplophora rostralis, and Prototritia major) deposit whewellite, a form of calcium oxalate. The latter is deposited even in individuals living in base-poor environments such as sphagnum bogs, and probably derives from crystals originally precipitated by the fungal food of these mites. Our observations provide the first strong evidence of cuticular hardening by mineralization in the Arachnida, and the only known instance of the use of whewellite as a general intracuticular hardening agent in arthropods. A brief survey of representative oribatid mites, examined for strong birefringence under polarized light, suggested that each of the three ptychoid lineages (Ptyctima, Mesoplophoridae, Protoplophoridae) evolved cuticular mineralization independently. NORTON, R. A., et BEHAN-PELLETIER, V. M. 1991. Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida). Can. J. Zool. 69 : 1504-151 1. Le durcis~ement~de la cuticule des oribates adultes par accumulation de cristaux 'de calcium a CtC dCmontrC par une combinaison de mCthodes : diffraction aux rayons X, dispersion aux rayons X et dCcalcification B l'acide selon un procCdC expkrimental. Les Ptyctima accumulent de la calcite (carbonate de calcium), mais trois espkces d'Enarthronota (Eniochthonius minutissimus,Archoplophora rostralis et Prototritia major) accumulent de la whewellite, une forme d'oxalate de calcium. Cette forme de cristaux est accumulCe meme par les individus qui vivent dans des milieux pauvres en bases, comme les tourbikres B sphaignes, et dCrive probablement de cristaux prCcipitCs B l'origine par les champignons consommCs par ces mites. Ces observations constituent les premikes indications certaines d'un durcissement cuticulaire par minkralisation chez les arachnides et c'est la premike fois qu'on constate que la whewellite sert d'agent de durcissement intracuticulaire gCnCral chez des - arthropodes. Un bref examen de la birkfringence chez des oribates types exposCs B une lumikre polariske a permis de constater que les trois lignCes ptychoides (Ptyctima, Mesoplophoridae, Protoplophoridae) ont acquis leur mCcanisme de minkralisation cuticulaire de faqon indkpendante. [Traduit par la rCdaction] Introduction Mites of the suborder Oribatida are small (100 k m - 1 mm) ubiquitous arachnids, most of which are mycophagous or saprophagous inhabitants of organic soil layers. They are well known for having a hardened cuticle in the adult instar, though there are numerous exceptions. Hardening is an effective defense against many small potential predators such as mesostigmatic mites, and is especially effective when coupled with adaptations that protect vulnerable articulations. The latter range from simple tecta (hard cuticular projections covering a single articulation) to the complex behavioral-morphological adaptations associated with ptychoidy (see below) or moveable pteromorphs (Schmid 1988). Hardening seems to be of lesser significance in defense against some predaceous mites with powerful chelicerae, such as the bdellid Cyta latirostris (Alberti 1973), or ectoparasitic larvae of the erythraeid genus Leptus, the needle-like chelicerae of which can penetrate sclerites (Norton et al. 1988). Some predaceous insects are strong enough either to break through hardened cuticle or to force cutting mouthparts into protected articulating membranes. These include beetles of the families Ptiliidae and Scydmaenidae (Riha 1951 ; Schmid 1988) and some small ants of the myrmicine genus Pheidole (E. 0. Wilson, personal communication, 1989). the process of protein sclerotization, which produces the melanin by-products that give adult oribatid mites their typical brown to black color, as well as the common name "beetle mites." An alternative possibility, that mineralization is the principal hardening process in some oribatid mite species or lineages, has not been directly addressed in the literature. This is surprising, since the relatively high calcium content of adult cuticle in some species has been suspected or known for several decades (Klima 1956; Wallwork 1973). During general studies of cuticular birefringence in oribatid mites, we discovered striking optical activity in the hardened cuticle of certain light-colored species that was suggestive of the presence of heavy deposits of crystalline calcium salts. We investigate this possibility later, after briefly reviewing other locations and sources of cuticular birefringence. Specifically, our objectives were to (i) document the high calcium content of strongly birefringent cuticles and test the stability of these properties in an acid bath; (ii) identify the specific calcium salt(s) involved; and (iii) examine general taxonomic and ecological patterns in the distribution of mineralization in oribatid mites. Cuticular birefringence and its causes Optical activity under polarized light has been a well-known feature of some cuticular structures in the mite order Acari- In oribatid mites, hardening is usually assumed to result from formes (to which the Oribatida belongs) since the pioneering Rinted in Canada I lmprimt au Canada Can. J. Zool. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/23/14 For personal use only.

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Page 1: Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida)

Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida)

ROY A. NORTON State University of New York College of Environmental Science and Forestry, Syracuse, NY 13210, U.S.A.

AND

VALERIE M. BEHAN-PELLETIER Biosystematics Research Centre, Agriculture Canada, Ottawa, Ont., Canada KIA OC6

Received September 7, 1990

NORTON, R. A., and BEHAN-PELLETIER, V. M. 1991. Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida). Can. J. Zool. 69: 1504-15 1 1.

Hardening of adult oribatid mite cuticle by the deposition of crystalline calcium salts is demonstrated by a combination of X-ray diffraction and X-ray dispersion methods, in conjunction with experimental acid decalcification. Calcite (calcium carbonate) is the mineral deposited by mites representating the Ptyctima, but three species of Enarthronota (Eniochthonius minutissimus, Archoplophora rostralis, and Prototritia major) deposit whewellite, a form of calcium oxalate. The latter is deposited even in individuals living in base-poor environments such as sphagnum bogs, and probably derives from crystals originally precipitated by the fungal food of these mites. Our observations provide the first strong evidence of cuticular hardening by mineralization in the Arachnida, and the only known instance of the use of whewellite as a general intracuticular hardening agent in arthropods. A brief survey of representative oribatid mites, examined for strong birefringence under polarized light, suggested that each of the three ptychoid lineages (Ptyctima, Mesoplophoridae, Protoplophoridae) evolved cuticular mineralization independently.

NORTON, R. A., et BEHAN-PELLETIER, V. M. 1991. Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida). Can. J. Zool. 69 : 1504-151 1.

Le durcis~ement~de la cuticule des oribates adultes par accumulation de cristaux 'de calcium a CtC dCmontrC par une combinaison de mCthodes : diffraction aux rayons X, dispersion aux rayons X et dCcalcification B l'acide selon un procCdC expkrimental. Les Ptyctima accumulent de la calcite (carbonate de calcium), mais trois espkces d'Enarthronota (Eniochthonius minutissimus, Archoplophora rostralis et Prototritia major) accumulent de la whewellite, une forme d'oxalate de calcium. Cette forme de cristaux est accumulCe meme par les individus qui vivent dans des milieux pauvres en bases, comme les tourbikres B sphaignes, et dCrive probablement de cristaux prCcipitCs B l'origine par les champignons consommCs par ces mites. Ces observations constituent les premikes indications certaines d'un durcissement cuticulaire par minkralisation chez les arachnides et c'est la premike fois qu'on constate que la whewellite sert d'agent de durcissement intracuticulaire gCnCral chez des

- arthropodes. Un bref examen de la birkfringence chez des oribates types exposCs B une lumikre polariske a permis de constater que les trois lignCes ptychoides (Ptyctima, Mesoplophoridae, Protoplophoridae) ont acquis leur mCcanisme de minkralisation cuticulaire de faqon indkpendante.

[Traduit par la rCdaction]

Introduction Mites of the suborder Oribatida are small (100 km - 1 mm)

ubiquitous arachnids, most of which are mycophagous or saprophagous inhabitants of organic soil layers. They are well known for having a hardened cuticle in the adult instar, though there are numerous exceptions. Hardening is an effective defense against many small potential predators such as mesostigmatic mites, and is especially effective when coupled with adaptations that protect vulnerable articulations. The latter range from simple tecta (hard cuticular projections covering a single articulation) to the complex behavioral-morphological adaptations associated with ptychoidy (see below) or moveable pteromorphs (Schmid 1988).

Hardening seems to be of lesser significance in defense against some predaceous mites with powerful chelicerae, such as the bdellid Cyta latirostris (Alberti 1973), or ectoparasitic larvae of the erythraeid genus Leptus, the needle-like chelicerae of which can penetrate sclerites (Norton et al. 1988). Some predaceous insects are strong enough either to break through hardened cuticle or to force cutting mouthparts into protected articulating membranes. These include beetles of the families Ptiliidae and Scydmaenidae (Riha 195 1 ; Schmid 1988) and some small ants of the myrmicine genus Pheidole (E. 0. Wilson, personal communication, 1989).

the process of protein sclerotization, which produces the melanin by-products that give adult oribatid mites their typical brown to black color, as well as the common name "beetle mites." An alternative possibility, that mineralization is the principal hardening process in some oribatid mite species or lineages, has not been directly addressed in the literature. This is surprising, since the relatively high calcium content of adult cuticle in some species has been suspected or known for several decades (Klima 1956; Wallwork 1973).

During general studies of cuticular birefringence in oribatid mites, we discovered striking optical activity in the hardened cuticle of certain light-colored species that was suggestive of the presence of heavy deposits of crystalline calcium salts. We investigate this possibility later, after briefly reviewing other locations and sources of cuticular birefringence. Specifically, our objectives were to (i) document the high calcium content of strongly birefringent cuticles and test the stability of these properties in an acid bath; (ii) identify the specific calcium salt(s) involved; and (iii) examine general taxonomic and ecological patterns in the distribution of mineralization in oribatid mites.

Cuticular birefringence and its causes Optical activity under polarized light has been a well-known

feature of some cuticular structures in the mite order Acari- In oribatid mites, hardening is usually assumed to result from formes (to which the Oribatida belongs) since the pioneering

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Page 2: Calcium carbonate and calcium oxalate as cuticular hardening agents in oribatid mites (Acari: Oribatida)

NORTON AND 1

work of Grandjean (1935). Two synonyms of Acariformes (Actinochitinosi, Actinotrichida) refer to this property (Grandjean 1936; van der Hammen 196 l), as does the name of a more inclusive taxon of arachnids, Actinochaeta, in the classification of Zachvatkin (1952). In nearly all acariform mites, mec hanoreceptive setae and their homologues (e.g ., eupathidia, famuli, rutella) possess a birefringent central column or cylinder (if hollow) that extends through all or part of the structure's length.' Other structures with a birefringent core include the paired claws, empodium, and distal parts of the cheliceral digits, all of which Grandjean (1947) considered to be of setal origin. The optically active material in these structures, termed actino- pilin (previously actinochitin), is deposited in the lumen after formation of the setal cuticle (Grandjean 1969). Some of its optical properties have been characterized by Grandjean (1947, 1969; see also van der Hammen 1961), but its chemistry and significance remain unknown.

A second cuticular component that may be birefringent is the cerotegument. This non-chitinous secretion (see Alberti et al. 1981), deposited above the epicuticle proper, is developed to varying degrees in most oribatid taxa, but usually shows no optical activity. In the Malaconothridae and Phenopelopoidea, however, it is highly birefringent (Grandjean 195 16; Norton and Behan-Pelletier 1986), and there are other oribatid mite lineages in which the property exists unreported. For example, the network of cerotegument on the noJogaster and prodorsal sclerite (aspis) of Rhysotritia curticephala (Jacot) is strongly birefrin- gent, as is the less abundant but similar cerotegument of Rhyso- tritia duplicata (Grandjean). Grandjean ( 1953, p. 158) described this net-like formation in R. duplicata, but did not mention optical activity. He speculated that the reticulate cerotegument pattern outlined the underlying epidermal ("hypodermal' ') cells, a result of secretion being greatest at the cell borders, but ultrastructural evidence is not a~ai lable .~

The general body cuticle can also exhibit birefringence, though this has not yet been noted in the literature. Such birefringence is not as striking as that of actinopilin or optically active ceroteguments, but it can be considerable; mites may glow strongly enough to make optical enhancement techniques that rely on polarization (such as Nomarski interference contrast) ineffective. Optical activity seems to be relatively uniform at low magnification (Figs. 1 and 3), but at higher magnification (400X and above) the pattern is irregular, usually like a jigsaw puzzle that completely covers the hardened parts of the cuticle (Figs. 2 and 4). Individual patches are bordered with a narrow isotropic black line, and a patch that glows brightly with the specimen in one position will be dark after a 45" rotation, then return to the same brightness after another 45" turn. Altering the focal plane along with rotation causes patches to change shape slightly, indicating a deep three-dimensional structure. Optical sectioning shows this to occur in the procuticle, though we attempted no corroborating ultrastructural studies. Such birefringence is typical of mineral crystallites that serve a structural, hardening role in arthropod cuticle (Richards 195 1; Neville 1975).

'Solenidia, the other major type of setiform sense organ of acariform mites, are optically inactive; these appear to be multiporous chemoreceptors.

2 ~ a r k e l and Meyer (1959) criticized Grandjean's interpretation, suggesting that the pattern was too irregular to be accounted for in this way, but they were misinterpreting the object of Grandjean's discus- sion. Grandjean's statements clearly dealt with the cerotegument, whereas the former authors were referring to a much finer "shag- reened" intracuticular pattern that is discussed later.

Methods The stability of optical activity in oribatid mite cuticles was tested by

bathing alcohol-preserved adult specimens (predetermined to exhibit high birefringence) in a decalcifying bath of 5% nitric acid held in a warming oven at 40°C. Tested taxa included the enarthronotes Enioch- thonius minutissimus (Berlese), Mesoplophora sp., Archoplophora rostralis (Willmann), and Prototritia major (Jacot), along with two representative Ptyctima, Atropacarus striculus (Koch) and Rhysotritia ardua (Koch). Mites were removed from the bath and examined at 1- or 2-h intervals under polarized light (see below) to investigate loss of birefringence.

The relative calcium content of adult mites with birefringent (E. minutissimus, At. striculus) and optically inactive (Liacarus sp., Oppia nitens Koch) cuticles was examined by X-ray dispersion. Absolute quantification was not attempted. Whole preserved specimens were removed from 70% ethanol, and critical-point dried; some E. minutissimus were subjected to nitric acid decalcification prior to drying. Specimens were mounted on scanning electron microscope stubs covered by a carbon disc, using two-sided tape. A small piece of copper foil was attached to each stub to standardize the equipment. The stub, with specimens and foil, was examined in a Model IS1 DS 130 scanning electron microscope at 20 kV. This system was operated in conjunction with an energy-dispersive Traycor Northern 5500 X-ray spectrometer, using a semiquantitative sequence method. Calcium concentration in an individual mite may vary by a factor of about five (Todd et al. 1974) depending on the part of the body examined, so we consistently made our spot analyses mid-dorsally in the anterior region of the notogaster.

Mineral identification was by X-ray powder diffraction. Whole specimens of E. minutissimus, Ar. rostralis, P. major, R. ardua, and At. striculus, along with exoskeleton fragments of Steganacarus magnus (Nicolet) and the brachypyline Liacarus sp., were removed from 70% ethanol, allowed to air-dry, and then crushed to a fine powder between two frosted-glass slides. This powder was then placed on an amorphous silica fibre powder mount and subjected to X-ray diffraction, utilizing a 57.3-rnm Debye-Scherrer powder camera with Ni-filtered Cu radiation, and a 1- to 3-h exposure.

Specimens used in the taxonomic survey of cuticular birefringence were part of the Canadian National Collection (Ottawa) or in the personal collection of R.A.N. All were examined in polarized light on Nikon Optiphot microscopes, and a Richert Polyvar microscope was used for photomicrography. Based on other results, the presence of strong birefringence was assumed to indicate mineralization. Gross effects of preservation on birefringence were examined by comparing specimens of E. minutissimus killed immediately prior to the study with some preserved in 70% ethanol for 5 years. No diminution of optical activity was detected.

Results Stability and form of cuticular birefringence

The birefringence of actinopilin in setae, claws, and cheliceral digits was unaffected by nitric acid treatment, as was that of optically active ceroteguments. Failure of the acid to penetrate is not a viable explanation for retention of birefringence, since optical activity did not diminish in cut or broken structures. Grandjean (1 947, 1969) has discussed the resistance of actino- pilin (and its birefringence) to chemical solutions, even those powerful enough to dissolve the cuticle proper. In separate experiments, after 2 days in 20% nitritic acid the cerotegument and actinopilin of Trimalaconothrus glaber (Michael) and Eupelops silvestris (Jacot) retained their original optical activity. In contrast, cuticular birefringence was always strongly dimin- ished or nearly lost after only several hours of acid treatment, suggesting that it derived from acid-soluble mineral salts. These same cuticles became soft and flexible after treatment; the loss of their original brittle consistency suggests that the leached mineral salts were responsible for hardening.

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1506 CAN. J. ZOOL. VOL. 69, 1991

X-ray dispersion yielded striking calcium peaks for each tested specimen that had an optically active cuticle (E. minutis- simus, At. striculus). Scans of Oppia nitens detected calcium, but at low concentration: approximately 1-5% of that in the species exhibiting birefrigence. In contrast, the cuticle of Liacarus sp. showed a distinct calcium peak, about 1040% of that of the species exhibiting birefringence, even though it is optically inactive. In decalcified specimens of E. minutissimus that had lost their birefringence, no calcium above background levels could be detected.

Thus, mineralization by some crystalline calcium salt is the source of optical activity and probably the source of hardness in these birefringent cuticles. We expected X-ray diffraction analysis to detect calcite (CaCO,), since this mineral is found in other arthropod groups (see below). This proved true for members of the Ptyctima, a mostly xylophagous group common- ly called "box mites" (At. striculus, S. magnus, and R. ardua). However, the three members of the Enarthronota (E. minutis- simus, Ar. rostralis, and P. major) had no detectable calcite; rather, their calcium was crystallized as whewellite (CaC2*H20), a monohydrous form of calcium oxalate. The considerable amount of calcium in the cuticle of Liacarus sp. is not detectable by means of X-ray diffraction, and thus is assumed to be deposited as an amorphous (non-mineral) compound. The absence of birefringence is consistent with this observation.

Taxonomic survey b

Under polarized light, we examined preserved specimens of each family represented in our collections for cuticular birefrin- gence. In the following summary, the numbers given after the generic or familial names indicate the number of species represented in our studies. See Marshall et al. (1987) for references relating to synonymies and other taxonomic matters. Descriptions of birefringence are subjective; we did not attempt to quantify optical activity in this brief survey. Our purpose was instead to identify lineages in which this property was strongly expressed, suggestive of mineralization. In each case, birefrin- gence noted as "weak" was restricted to contours rather than face-on surfaces, and is probably not due to mineral deposition.

The Palaeosomata is generally considered the earliest deriva- tive major oribatid taxon; the cuticle is isotropic or very weakly birefringent in the Palaeacaridae (Palaeacarus, 1); Ctenacaridae (Ctenacarus, 1 ; Beklemishevia, 1 ; Archeonothridae (Zachvat- kinella, 1); Acaronychidae (Acaronychus, 1); and Aphelacaridae (Aphelacarus, 1).

For the enarthronote taxon Protoplophoroidea the procuticle was strongly birefringent only in Prototritia. That of other Protoplophoridae examined (undetermined members of Proto- plophora and Cryptoplophora) is isotropic. In the Sphaeroch- thoniidae, the sister-family of the Protoplophoridae (Norton et al. 1983), very weak birefringence is present. Other members of the Protoplophoroidea examined (Haplochthonius, Paralycus, Cosmochthonius) have isotropic procuticles. Phyllozetes spp. possess a curious mineralization of the epicuticle which is discussed elsewhere (Norton and Behan-Pelletier 199 1).

Strong birefringence is much more common in the cuticle of mites of the Hypochthonioidea (sensu Norton 1984) than in that of the Protoplophoroidea. The sister-families Eniochthoniidae (Eniochthonius, 3) and Mesoplophoridae (Mesoplophora, 7; Archoplophora, 1 ; Apoplophora, 1 ; and Dudichoplophora, 1) include mites with some of the most strongly birefringent cuticles observed in this study (Figs. 1-4). Members of the family Hypochthoniidae are only weakly birefringent (Hypoch- thonius, 2; Eohypochthonius, 2).

Among Ptyctima examined, cuticular birefringence is wide- spread, though it is often not quite as striking as in Prototritia, Eniochthonius, or the Mesoplophoridae. Within the Phthira- caridae the degree of birefringence varies widely among (and sometimes within) genera. It is strong in Hoplophorella (4), Atropacarus (3), and Steganacarus (I), but virtually absent from Phthiracarus (4). Among three Hoplophthiracarus species examined, H. costa (McFarlane & Sheals) and H. histricinus (Berlese) have thick, birefringent cuticles, but the cuticle of H. paludis Jacot is thinner and isotropic.

Birefringence also seems to be common in the Euphthira- caroidea. Euphthiracarus (4) and Rhysotritia (3) have moderate to strong cuticular birefringence, whereas in Microtritia (2) birefringence is weak or absent. All Oribotritiidae examined showed a modest degree of birefringence (Protoribotritia, 1; Mesotritia, 2; Maerkelotritia, 2; Oribotritia, 2; Perutritia, l), and the cuticle of Synichotritiidae (Synichotritia, 2) is strongly birefringent.

Birefringence has been noted in the cuticle of a variety of brachypyline oribatid mites, but we have not conducted a comprehensive survey. It seems to be especially common in the genus Carabodes, moderate optical activity being regularly exhibited by several species sampled: gibbiceps, forsslundi, areolatus, clavatus, jloridus, and falcatus. In mites of two other species, C. granulatus and C. femoralis, the cuticle was nearly isotropic. In the related family Otocepheidae, specimens of Fissicepheus exhibited rather strong birefringence, but not those of other family members (Dolicheremaeus, Otocepheus, Papillocepheus, . Plenotocepheus).

Although our study dealt primarily with adults, we examined immature specimens of several taxa having strongly mineralized adult cuticle. In Archoplophora rostralis and Rhysotritia sp., only the adults have mineralized cuticle. In contrast, all instars of Eniochthonius minutissimus have heavy deposits which are indistinguishable from those of adults.

Discussion "Corroded, ' ' "shagreened, ' ' and opalescent cuticles

There are several published observations on oribatid mite cuticle that can be explained by the presence of mineral deposits. For example, Grandjean (1933) described the "corroded" nature of the cuticle of Mesoplophora pulchra Sellnick under bright- field illumination, and the absence of such a structure in a recently eclosed adult. The "corroded" pattern in this and other Mesoplophora species is identical with the birefringence pattern seen under polarized light (cf. Fig. 4) and is presumably caused

FIGS. 1-4. Adult Hypochthonioidea under plane-polarized light, showing the strong birefringence that is considered evidence of procuticular mineralization. Individual crystallites are small, irregular, and occupy much of the depth of the procuticle. Fig. 1. Eniochthonius minutissimus (Berlese) (Eniochthoniidae), lateral aspect, slightly broken. Fig. 2. Cuticle of same specimen, enlarged. Fig. 3. Archoplophora rostralis (Willmann) (Mesoplophoridae), lateral aspect, slightly broken. Fig. 4. Cuticle of same specimen, enlarged. Both specimens are alcohol preserved and untreated; the plane of polarization is not indicated.

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NORTON AND BEHAN-PELLETIER 1507

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1508 CAN. J. ZOOL. VOL. 69, 1991

by varying refractivity of adjacent crystallites. The delay in the appearance of the pattern is expected, since minerals are deposited as the adult cuticle matures.

Similarly, Make1 and Meyer (1959) noted a so-called "shagreened" pattern in the cuticle of alcohol-preserved Rhysotritia specimens (their Figs. l c and 16) under bright-field illumination. They found that the pattern could be lost during treatment (e.g., clearing in lactic acid), and Grandjean (1953, calling it a "corroded" pattern) suggested that it develops as the adult matures. Each observation probably relates to mineraliza- tion. Both dissolution of the pattern under acid treatment and gradual development of mineralization patterns during matura- tion of the adult cuticle are expected. We doubt that the loss of pattern during preparatory treatment is due to a "wetting" phenomenon as Miirkel and Meyer (1959) suggested.

Grandjean (1954, p. 312 and his Fig. 2E) noted that under diffuse light at high magnification (400X or higher) the outer layer of procuticle of the prodorsum and major notogastral plates of Prototritia glomerata (Grandjean) is penetrated by regular conical intrusions with a different refractive index and stronger dye affinity. He suggested that the pattern resulted from the presence of two different kinds of chitin, but we believe that mineralization is involved, the conical intrusions perhaps representing unmineralized cuticle.

Mineralization may be responsible for not only the cuticular birefringence and rigidity of mites of the phthiracarid genus Atropacarus, but also their often-noted opalescence in reflected light. After treatment with nitric acid, the opalescence was lost, along with optical activity and hardness. The cuticle of certain members of Carabodes (a genus with cuticular birefringence in many species) exhibits metallic iridescence (Grandjean 195 la; personal observations), but a possible relationship with mineral- ization has not been examined.

Calcium oxalate as a biogenic mineral The monohydrate calcium salt of oxalic acid, whewellite, is

commonly associated wth fungi and certain higher plant tissues (Arnott 1973; Lowenstam and Weiner 1983). It is a pathogenic biomineral of vertebrates, being one of the precipitates that form calculi, as kidney stones, for example. But among animals, it is known as a normally formed biomineral only in the Arthropoda, particulary the Insecta. It forms in the egg shells of some phasmids and in the oothecae (egg cases) of certain cockroaches and mantids (Richards 195 1; Neville 1975). Ours seems to be the first record of whewellite being used as a hardening agent in arthropod cuticle. According to Lowenstam and Weiner (1983), the dihydrate form of calcium oxalate, weddellite, is more widely distributed in animals (mollusks, echinoderms, chordates) but is not known from arthropods. Though the specific mineral was not identified, Kovoor (1978) believed that some form of calcium oxalate might compose the crystals encrusting the mesodermal endosternite of harvestmen (Phalangida).

Phylogenetic aspects of calcium mineralization in oribatid mite cuticle

The deposition of crystalline calcium salts to harden cuticle is a mechanism with an uneven distribution among extant Arthro- poda (Richards 195 1 ; Neville 1975). The few previous records of calcium oxalate were noted above. In contrast, deposition of crystalline calcium carbonate is the predominant hardening process in the Crustacea, and is common in some higher taxa of Diplopoda. It is rare among insects, where the compound occurs as intra- or extra-cuticular deposits in the larvae of a few fly taxa. With the exception of the oribatid mites, cuticular harden-

ing by mineralization is unknown in the Arachnids, a group whose members are mostly fluid-feeding predators with probably little access to large quantities of calcium. Oribatid mites represent the most significant deviation from this arachnid life- style; their food consists mostly of fungi and decaying structural material of higher plants, and is ingested in particulate form (Schuster 1956; Norton 1985 and references therein). Oribatid mites thus have access to the considerable quantities of calcium found in these resources, and have been studied from the standpoint of their role in ecosystem cycling of this element (see below).

We are not the first to mention the deposition of calcium carbonate in oribatid mite cuticle, but we are the first to publish direct evidence that it occurs in the form of calcite. Wallwork (1 973) reported a high ash content in the cuticle of Steganacarus magnus adults investigated by means of bomb calorimetry; he suggested that this was due to the presence of calcium carbonate deposits, but offered no proof, nor did he suggest a link with cuticular hardening. In an unpublished thesis, Sanders (1982) inferred that calcium carbonate is the compound responsible for cuticular hardening in Euphthiracarus sp. Specifically, he found that (i) gentle decalcification procedures used during histological studies changed the cuticle from brittle to leathery; (ii) treatment with strong acid (1 N HCl) produced effervescence (assumed to be due to carbon dioxide); (iii) strong silver-calcium exchange (indicating the presence of either calcium carbonate or calcium phosphate) occurred when cuticle was treated with silver nitrate in the Von Kossa histochemical technique.

As a hardening mechanism, protein sclerotization with concomitant melanization is certainly the rule in oribatid mite adults, strong mineralization being less common. In crustaceans and millipedes there seems to be an inverse relationship between the amounts of protein and calcite in cuticle (Neville 1975). Extrapolating to oribatid mites, we would expect those with strongly birefringent cuticles (i.e., with heavy mineral deposits) to be relatively light in color, and soft when decalcified; this is generally true for the early derivative taxa. The cuticle of adult members of Pr~totritia, Eniochthoniidae, and most Mesoplo- phoridae is heavily mineralized and brittle, and they are rather pale brown or yellowish mites. Also, adults of the most highly birefringent Phthiracaridae (e.g, Atropacarus, Steganacarus, Hoplophorella) and Euphthiracaroidea (e.g., Synichotritia) exhibit little coloration. Even mineralized cuticles with reddish- brown pigmentation may not possess scleroprotein, as demon- strated for Euphthiracarus by Sanders (1982). In more derived taxa, however, there are some species in which both sclerotiza- tion and mineralization seem to be contributing to cuticular hardness. Several Carabodes species, for example, are strongly melanized, yet exhibit cuticular birefringence.

Cuticular mineralization appears to be a derived state in oribatid mites. As a simple test of this hypothesis we examined specimens of at least one species from each family of "Endeo- stigmata' ' (various potential outgroups) de~cribed~and found no evidence of cuticular birefringence. Within the Oribatida, however, the distribution of birefringence and the two forms of calcium salts involved show patterns suggestive of multiple origins. The ptychoid groups, which when disturbed cover the retracted legs and podosomal structures by an operculum-like prodorsum, are a good example.

Each of the three independently derived ptychoid lineages, Protoplophoridae, Mesoplophoridae, and Ptyctima, contains species with marked mineral content. As might be expected, deposition of crystallites is restricted in some species to regions

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of the body where it can serve a protective purpose. For example, Grandjean (1954) noted that not all cuticular plates of the protoplophorid mite Prototritia glomerata had the "corroded," chambered structure that we found to correlate with birefrin- gence and mineral deposition. Rather, this type of structure was restricted to the prodorsum and notogastral shields, those exposed when the mite contracts into its globular defensive posture. These same plates are the only birefringent regions of the body cuticle of its congener P. major, and are presumably the only regions hardened by the deposition of whewellite. In contrast, the coxisternal, subcapitular, and leg sclerites are isotropic and presumably unmineralized. A similar morphologi- cal distribution of birefringence exists in those Ptyctima that deposit calcite.

However, distribution is different in the Mesoplophoridae, in which the sclerotized leg, coxisternal, and subcapitular cuticles are almost as strongly birefringent (as heavily mineralized) as those of the prodorsum and notogaster. The Eniochthoniidae (the sister-family of the Mesoplophoridae), which has a similar distribution of birefringence, is not ptychoid, and all exposed sclerites would seem to be logical sites for mineralization.

In a phylogenetic context, the mineralization of Prototritia spp. and various Ptyctima seems to have been independently derived (i.e., after the evolution of ptychoidy), and morpho- logically it only develops where it is functionally advantageous. In contrast, mineralization is an ancestral state in the group Mesoplophoridae + Eniochthoniidae, and its physical distribu- tion reflects that of the group's non-ptychoid common ancestor (i.e., it evolved before ptychoidy).

Perhaps the most interesting general evolutionary questions relating to mineralization are the following: what advantage does it impart over sclerotization, and why is it especially common in ptychoid mites? Sclerotization (i.e., protein tanning) has a presumed advantage in higher insects because it adds less mass to the cuticle than does mineralization, an important consider- ation in flying animals. Heavily sclerotized cuticle is also harder than mineralized cuticle (Neville 1975). In contrast, mineraliza- tion may be less energy expensive than sclerotization, though it is probably under enzymatic control to some degree (Neville 1975) and has some energy costs.

Though the first oribatid mites and their ancestors were probably soft-bodied, the most strongly mineralized procuticles are found in taxa considered to be rather early derivative, often loosely considered the "lower oribatid mites.'' Perhaps mineral- ization represents an evolutionary divergence that provided an adequate solution for these few groups. Oribatid mites are rather slow-moving relative to their potential predators, and this is especially true of the ptychoid families, in which heavy mineral- ization is most common. Hardening would seem to be advanta- geous in such mites, and the lower mass associated with scleroti- zation may be less significant than in lineages of more active species.

Ecological considerations The sequestering of calcium in oribatid mite cuticle has been

mostly reported in the context of nutrient dynamics (e.g., Todd et al. 1974; Gist and Crossley 1975a; Carter and Cragg 1976, 1977; Crossley 1977; Lowrey 1980; Frater 1980; Wallwork 1983). Using X-ray dispersion methods, substantial amounts of calcium (suggested to be about 10 000 - 70 000 ppm) have been measured in adults of common forest soil species (Crossley 1977 and references therein). The highest concentrations were reported for Liacarus specimens, but our data suggest that they

have at most half the calcium concentration of species exhibiting birefringence, none of which were previously examined. Standardization problems exist with these methods, however, Using atomic absorption spectroscopy, Frater (1 980) measured calcium concentration in Phthiracarus aflnis (a species of Ptyctima that does not exhibit birefringence) as 1490 ppm, about 20 times lower than the estimates of Crossley (1977) for unidentified Phthiracaroidea. It is not clear, however, whether her data relate to the whole mite or only its cuticle.

Not all oribatid mites sequester much calcium, as shown by the low value of 1 13 ppm for Platynothrus peltifer reported by Frater (1980). Still, the fragmentary data available suggest that as a group, oribatid mites have high calcium requirements relative to other soil microarthropods. Gist and Crossley (1975b) suggested that these mites process a significant portion of the calcium pool in some ecosystems, though Seastedt (1 984) suggested that their standing crop biomass is usually too low to allow them much direct influence on calcium cycling.

Another ecological question relates to the habitat distribution of oribatid mites with heavily mineralized cuticle. Are they absent from base-poor habitats such as acidic sphagnum bogs? Using the genus Hoplophthiracarus as an example, of the three species examined in the present study, only H. paludis does not have birefringent cuticle and this species is an inhabitant of bogs in eastern North America. Birefringence in other members of the Phthiracaridae (Steganacarus magnus, Atropacarus striculus) was shown above to result from the presence of crystalline calcium carbonate, which is expected to be relatively unavailable in such environments. The pattern is not clear-cut, however; Eniochthonius minutissimus and Archoplophora rostralis are often found in acidic sphagnum bogs, as well as in forest habitats (Schuster 1960; Marshall et al. 1987), yet their cuticles are strongly mineralized. How could this be possible? The answer might lie in the type of calcium salt deposited, a form of calcium oxalate.

Oxalic acid has been known for over a century to be formed and secreted by fungi as a by-product of incomplete carbo- hydrate metabolism (Foster 1949), and it has been considered of importance in non-enzymatic wood decay by brown-rot fungi (Schmidt et al. 1981). It is an ecologically important compound which reacts with calcium in solution in the environment to form crystalline precipitates externally on the hyphae, in either monohydrate (whewellite) or dihydrate (weddellite) form, as discussed by Graustein et al. (1977). These authors found such crystals on all hyphae examined from the litter region of five diverse temperate forest types, as well as on hyphae growing interstitially in the A horizon. These crystals are recalcitrant, having only slight solubility in water; thus, they represent a means for retaining calcium in the organic horizons of soils.

In contrast, rapid release of calcium from such crystals is mediated by the digestive action of soil mycophages (Cromack et al. 1977; Crossley 1977). Based on our unpublished observa- tions of the gut contents of preserved specimens, Eniochthonius, Archoplophora, and Prototritia are mycophagous. Using the terminology of Lowenstam and Weiner (1983), we therefore suggest that they harden their cuticle via a normal "matrix- mediated" mineralization process, but use calcium oxalate appropriated from free crystals that were themselves "biologi- cally-induced" by the secretory action of fungi.

Perhaps crystals induced by the action of fungi represent a principal calcium source for both whewellite and calcite deposi- tion in oribatid mites. The possibility that minerals are with- drawn from the cuticle of E. minutissimus prior to molting needs

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to be investigated, but the large quantities of calcium required for either type of mineralization may have to be obtained during the short period of time between molting and cuticular hardening. In this case a highly concentrated calcium source would seem to be necessary. Calcium oxalate crystals are produced by wood-decay fungi (e.g., Dumas et al. 1989) and may therefore be available to xylophagous species such as members of the Ptyctima. There may be times during the year, however, when even adult Ptyctima, including Steganacarus magnus, are predominantly mycophagous (Anderson 1975; Behan-Pelletier and Hill 1983) and presumably active outside decaying wood. If fungi are indeed an important calcium source for the Ptyctima, the oxalate would need to be either excreted or metabolized, rather than being used in mineralization as in the Enarthronota.

Are calcium oxalate crystals available in the acidic environ- ment of a sphagnum bog? We found no direct answers in the literature, though Cochrane ( 1958, p. 144) mentioned the general view that "oxalic and other acids are formed maximally when available carbon cannot be used for growth," a situation that perhaps obtains in nutrient-poor bogs. However, Foster (1949) noted that oxalate production by fungi is diminished in acidic media; this is due to the enzymatic degradation of oxalate at low pH (Cochrane 1958). Also, Graustein et al. (1977) suggested that small calcium oxalate crystals might not last long in base-poor environments, even if the compound has low solubility. We know little about the distribution of mites such as E. minutis- simus or Ar. rostralis in 6ogs, but perhaps there are microsites where abiotic and biotic conditions are favorable for crystal growth.

Conclusions The sequestering of calcium in oribatid mite cuticle is

probably widespread both taxonomically and ecologically. In niost instances the precipitate is unknown, but perhaps takes an unmineralized form such as amorphous hydrous carbonate, the common form of amorphous calcium precipitate in arthropods (Lowenstam and Weiner 1983). Crystalline deposits heavy enough to induce strong birefringence are found mostly in mites of three distinct lineages: the Ptyctima and the enarthronote superfamilies Hypochthonioidea and Protoplophoroidea. We have few data on the distribution of particular forms of calcium salts, but the apparent pattern is for mineralization in the Ptyctima, which is generally considered a xylophagous taxon, to result from the deposition of calcite (calcium carbonate). In contrast, mineralized Enarthronota, a predominantly mycophag- ous taxon, deposit whewellite (calcium oxalate), and this occurs even in base-poor sphagnum bogs.

Acknowledgements Drs. E. E. Lindquist, Biosystematics Research Centre, Ottawa,

D. E. Walter, U.S. Department of Agriculture Horticultural Research Laboratory, Orlando, G. 0 . Evans, West Sussex, England, and M. J. Mitchell, State University of New York College of Environmental Science and Forestry (SUNY-CESF), offered constructive criticism of the manuscript, and an anony- mous reviewer was particularly influential. Ms. B. Bissett, Biosystematics Research Centre, Ottawa, provided technical assistance. Mr. E. Bond, Electron Microscopy Centre, Agricul- ture Canada, Ottawa, and Mr. A. Roberts, Geological Survey of Canada, Ottawa, conducted the X-ray dispersion and X-ray diffraction analyses, respectively, and provided advice. Dr. J. Dalingwater, University of Manchester, and Drs. J. Worrall and D. Griffin, SUNY-CESF, provided helpful comments and

literature references, and Dr. E. 0 . Wilson, Harvard University, Cambridge, kindly allowed us to cite unpublished information on ant predators of oribatid mites. We are grateful to all.

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