Macrobiotus Richtersi Research Articles

The Tardigrada of Southern Africa, with the Description ofMinibiotus harrylewisi, a New Species from KwaZulu-Natal, South Africa (Eutardigrada: Macrobiotidae)

ABSTRACT Nine species of tardigrades (Phylum Tardigrada) were found in moss and lichen samples in KwaZulu-Natal, South Africa, with two species being also present in Lesotho. Macrobiotus richtersi, Minibiotus intermedius, Hypsibius convergens, Ramazzottius theroni, Milnesium tardigradum, and Echiniscus duboisi were previously known to be part of the southern African fauna, Macrobiotus iharosi has not been reported before from this region. One species, Macrobiotus cf. echinogenitus, could be identified only to species complex because eggs were not found. One South African species, Minibiotus harrylewisi sp. n., is new to science and is described and illustrated. It differs from other Minibiotus species in its adult cuticle and its egg ornamentation. It most closely resembles M. furcatus, from which it differs in having smooth lunules on leg IV, smaller eyes, smaller and more rounded posterior cuticular pores, and eggs with a smooth shell and much longer, non-bifurcate processes. These new records bring to ...

Tardigrade Resistance to Space Effects: First Results of Experiments on the LIFE-TARSE Mission on FOTON-M3 (September 2007)

The Tardigrade Resistance to Space Effects (TARSE) project, part of the mission LIFE on FOTON-M3, analyzed the effects of the space environment on desiccated and active tardigrades. Four experiments were conducted in which the eutardigrade Macrobiotus richtersi was used as a model species. Desiccated (in leaf litter or on paper) and hydrated tardigrades (fed or starved) were flown on FOTON-M3 for 12 days in September 2007, which, for the first time, allowed for a comparison of the effects of the space environment on desiccated and on active animals. In this paper, we report the experimental design of the TARSE project and data on tardigrade survival. In addition, data on survival, genomic DNA integrity, Hsp70 and Hsp90 expressions, antioxidant enzyme contents and activities, and life history traits were compared between hydrated starved tardigrades flown in space and those maintained on Earth as a control. Microgravity and radiation had no effect on survival or DNA integrity of active tardigrades. Hsp expressions between the animals in space and the control animals on Earth were similar. Spaceflight induced an increase of glutathione content and its related enzymatic activities. Catalase and superoxide dismutase decreased with spaceflight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades molted, and females laid eggs. Several eggs hatched, and the newborns exhibited normal morphology and behavior.

Macrobiotus (Eutardigrada, Macrobiotidae) from the Great Smoky Mountains National Park, Tennessee/North Carolina, USA (North America): two new species and six new records

As part of the All Taxa Biodiversity Inventory (http://www.dlia.org), we are conducting a large-scale multihabitat inventory of tardigrades in the Great Smoky Mountains National Park (GSMNP) in Tennessee and North Carolina, USA. Here we report our findings for the genus Macrobiotus (Eutardigrada, Macrobiotidae). Two new species, Macrobiotus martini sp. nov. and Macrobiotus halei sp. nov., are described from moss, lichen, soil and leaf litter samples. Macrobiotus martini sp. nov. differs from all other species of the Macrobiotus hufelandi group by having a very simple buccal armature without bands of teeth, very large elliptical cuticular pores, unique characteristics of the egg, and other morphometric characters. Macrobiotus halei sp. nov. differs from other species in the Macrobiotus richtersi group by having very small cuticular tubercles, well-developed macroplacoids, unique characteristics of the egg, and other morphometric characters. Seven additional Macrobiotus species have been identified in the GSMNP: M. harmsworthi Murray, 1907, M. hufelandi Schultze, 1834, M. islandicus Richters, 1904, M. montanus Murray, 1910, M. pallarii Maucci, 1954, M. recens Cuénot, 1932, and M. tonollii Ramazzotti, 1956. All of these except M. harmsworthi are new records for the national park. In addition, M. pallarii is a new record for North Carolina, M. islandicus is a new record for North Carolina and Tennessee, M. montanus is a new record for the eastern USA, and M. recens is a new record for the USA.

Foraging theory and partial consumption in a tardigrade–nematode system

We investigated foraging behavior of a carnivorous eutardigrade, Macrobiotus richtersi, living in soil or fresh water sediments, where the distribution of nematode prey is heterogeneous and where, consequently, the value of information about prey availability should be high. We directly observed diet choice in various mixtures of 2 size classes of nematodes Acrobeloides nanus, A1 (small prey) and A2 (large prey), differing in profitability (biomass divided by handling time) but not in digestive quality. At various prey densities of A2, we measured how much of each prey item was consumed as a function of search time. Additionally, we derived cumulative biomass uptake rates per single A2 prey from 154 complete feeding acts, and we used the marginal value theorem (MVT) to predict optimal residence time and prey exploitation as a function of environmental quality and search time. Macrobiotus richtersi did not preferentially select the more profitable size class A2 but increased its biomass uptake rate by modifying the amount consumed per prey item (partial consumption): when encounter rates were high and there were more high-quality prey, the tardigrade abandoned food more quickly and consumed less biomass per captured prey. We conclude that 1) hungry tardigrades under food-rich or food-limited conditions maximize biomass gain according to MVT and 2) tardigrades differentiate at least 2 prey situations, high supply (at high encounter rates) and low supply (at moderate and low prey densities). 3) Partial consumption, performed under food-rich conditions or by a satiated predator, leads to a reduction of prey numbers up to 3 times as much as under food-poor conditions.

Distribution of Tardigrades in Florida

Abstract The distribution of terrestrial tardigrades in the Gulf Coast states of the United States is poorly known. Only one species has been reported in Florida. In this study, bryophyte samples (47 identified species) from trees and shrubs were collected from all 67 Florida counties. Twenty tardigrade species were found: Echiniscus cavagnaroi, E. kofordi, E. virginicus, Pseudechiniscus suillus, Ramazzottius baumanni, Diphascon (Diphascon) pingue, Macrobiotus echinogenitus, two species of Macrobiotus cf. harmsworthi, Macrobiotus cf. hibiscus, Macrobiotus hufelandi, two species of Macrobiotus cf. hufelandi, Macrobiotus islandicus, Macrobiotus occidentalis, Macrobiotus richtersi, Macrobiotus tonollii, Minibiotus fallax, Minibiotus intermedius, and Milnesium tardigradum. The distribution of tardigrade species was not correlated with Florida's ecological regions. Tardigrade species richness declined from north to south in peninsular Florida.

Role of tardigrades in the suppressive service of a soil food web

In a previous study, soil suppressiveness to populations of the plant-parasitic nematode Meloidogyne incognita was correlated with abundance of omnivore and predatory nematodes. In the current study, performance of the regulatory function switched from nematodes to tardigrades when predatory nematodes disappeared due to predation pressure and/or unfavorable environmental conditions. Two tardigrade species, Macrobiotus richtersi and M. harmsworthi effectively suppressed nematode populations. While there were significant changes in the soil food web over time, the regulatory function was maintained. Under experimental conditions, M. richtersi consumed 61 nematodes per day, indicating that tardigrades may significantly reduce nematode biomass. The impact of tardigrades on soil food web dynamics and their relationships with nematodes are discussed.

Energy allocation in two species of Eutardigrada

To improve our knowledge on life histories in tardigrades and the energy allocated for their reproduction and growth, we have studied two species (Macrobiotus richtersi and Hypsibius convergens) differing in evolutionary histories, diet and ways of oviposition. For both species we considered a bisexual population dwelling in the same substrate. In both species we investigated energy allocations in males with a testis rich in spermatozoa and females, each with an ovary containing oocytes in advanced vitellogenesis. The age of the specimens was estimated on the basis of buccal tube length and body size. Body and gonad areas were calculated using an image analysis program. In both species females reach a larger size than males. Macrobiotus richtersi has both a significantly longer buccal tube and wider body area than H. convergens. Statistical analyses show that the buccal tube has a positive correlation with body area and gonad area. For an estimate of the relative energy allocated for reproduction in one reproductive event (relative reproductive effort = RRE), we have used the ratio between gonad area and body area. In males of both species, the absolute amount of energy and the RRE is statistically lower than that of females. Males and females of H. convergens have a RRE higher than those of M. richtersi. In M. richtersi, the gonad increases proportionally more when animals are large (old), whereas in H. convergens this direct relationship is not detectable. In M. richtersi the energy allocated for a reproductive event increases during the life of the females. In males, the increase of the gonad size is progressive during the animal life. In each reproductive event, females of H. convergens allocate a lower amount of energy in absolute value when compared to M. richtersi. Nevertheless, when considering the RRE, their investment is higher than that of M. richtersi.

Ultrastructure of the digestive system of Ramazzottius tribulosus and Macrobiotus richtersi (Eutardigrada) in relationship with diet

The ultrastructure of the digestive system of tardigrades was already described in some species, but it has never been studied in relationship to diet. We performed ultrastructural analyses of the midgut and hindgut of phytophagous Ramazzottius tribulosus and zoophagous Macrobiotus richtersi. In addition, the foregut of R. tribulosus was analyzed. New ultrastructural details have been observed. Among them are: (a) distinct transverse pillar-like structures, lacking in electron-dense and compact cuticle of the buccal tube; (b) a hole or groups of holes sometimes present in the buccal tube; (c) a large cavity within each of the salivary glands where secreted mucus accumulates; and (d) already found in zoophagous Isohypsibius prosostomus, one valve, formed by folds of the pharynx and located at the transition from pharynx to esophagus. In both analyzed species the increase of midgut surface is identified by two orders of folds of the gut wall and by microvilli. In R. tribulosus there are many first-order folds and few second-order folds, whereas in M. richtersi the opposite pattern is found. A peritrophic membrane and microvilli with a well developed glycocalyx are found only in the midgut lumen of R. tribulosus. The density of microvilli and the ratio between the real surface with microvilli and the hypothetical surface without microvilli is lower in zoophagous M. richtersi and I. prosostomus than in phytophagous R. tribulosus. All of these data represent an indirect indication of differences in digestive physiology between phytophagous and zoophagous tardigrade species. The shape of the hindgut is similar in both species and the lumen of the hindgut looks like a heartshaped cavity with some narrow cell evaginations.

A long-term study of population dynamics of tardigrades in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst

Variation of abundance, dominance and diversity of tardigrades were studied over a period of 54 months in a carpet of the moss Rhytidiadelphus squarrosus that covered a lawn in the Black Forest (Germany). Altogether 19,909 individuals belonging to 24 species were extracted from the moss. Macrobiotus hufelandi (56%), Macrobiotus richtersi (18%) and Diphascon pingue (12%) were the most abundant and dominant species. Dominances of the other species varied between 0.01 to 2.15%. Generally, species diversity (Shannon-Index and evenness) was highest during the winter. A temporal variation in numbers with a decline in winter and an increase in spring until fall was seen in M. hufelandi, D. pingue and less clear in M. richtersi. Three species (Diphascon rugosum, Hypsibius dujardini, Hypsibius cfr. convergens) showed a cyclic variation with clear peaks in wintertime. The moist season favoured species that were considered as hygrophilous in the literature, whereas the relatively dry sunny seasons promoted the relative increase of euryhygric species. The composition of the tardigrade community was strikingly robust over the years. Fourteen species were always present during the entire period of investigation. However, changes in the species composition over the years, and cyclical temporal presence of certain species stress the significance of long-term studies to estimate species richness in a given habitat. Application of the Spearman rank correlation test to the data set of the whole study period showed different correlations between the mean number of tardigrades as well as the mean number of individuals of selected species (dominance >1) and rainfall, humidity and temperature. Correlations confirmed some of the ecological dependences of tardigrade species known from literature. Conflicting classifications suggest dependence from other variables or perhaps presence of ecotypes.

Predicting and testing functional responses: An example from a tardigrade–nematode system

Abstract Numerous studies have empirically measured consumer functional responses or theoretically developed response models, but whether these models can quantitatively predict observed data has hardly been tested. We perform such a test for the terrestrial predator–prey system Macrobiotus richtersi (Tardigrada)– Acrobeloides nanus (Nematoda). For two different size classes of A. nanus , we report a functional response as measured in the laboratory and quantitatively compare it to predictions of three models with different degrees of complexity: (1) the disc equation which does not include satiation effects; (2) the steady-state satiation (SSS) equation which assumes a constant level of predator satiation; and (3) the satiation model which accounts for prey depletion and increasing predator satiation over the course of the experiments. We parameterized these models with data that were measured independently of the functional response experiments. In both prey-size classes, the predictions of the satiation model matched the observations best, and the match came close to that of logistic regressions fitted to the observations. Thus, the parameterized satiation model seems to include the most important determinants of the functional response in our focal system. For understanding functional responses, we need more studies that compare data to independently derived model predictions.

Tardigrade species composition in young soils and some aspects on life history of Macrobiotus richtersi J. Murray, 1911

Summary In an afforested coal-mining site at Berzdorf, Germany, the soil tardigrade community was investigated. Tardigrade densities ranged from 300 to 33,600 individuals m −2 . Tardigrade distribution was very aggregated with standard deviations of up to 220% of mean densities. Fourteen species belonging to five eutardigrade genera were identified. Soils were dominated by the carnivorous tardigrade Macrobiotus richtersi that contributed 60% of overall densities. A comparison between nine soils of different forest ages (4–46 years) and tree species (poplar, alder, pine, mixed deciduous and mixed coniferous) showed no relationship of tardigrade species distribution and abundance to tree species, thickness of organic or litter layer, humus form, pH or soil moisture. Further, no correlation between nematode biomass and Macrobiotus richtersi abundances were detected. The age of afforested soils, however, affected tardigrade densities: younger soils (7–17 years) yielded higher tardigrade numbers compared to 46-year-old sites. Life history investigations on a strain of parthenogenetic Macrobiotus richtersi demonstrated a significant influence of temperature on egg development, survival rate, body growth and generation time. Hatching, first to fourth molts and maturing, on the other hand, were dependent upon body size alone. The simple, new culture method for carnivorous tardigrades described has successfully been utilized for parthenogenetic Macrobiotus richtersi for 6 years.

Phenotypic Variations in the Life History of Two Clones of Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

A comparative study of life history traits of two clones (CDMr01 and CDMr02) of a triploid thelytokous apomictic population of the eutardigrade Macrobiotus richtersi has been carried out. Both clones were reared under the same lab conditions: temperature of 14 °C, photoperiod of 12 h/12 h (L/D), and nematodes ad libitum as food. Statistical analysis of the life history traits considered has indicated interclonal variability. The two clones were significantly different in the number of eggs per clutch (fertility), number of eggs laid per female per life span (fecundity), hatching percentage of eggs and hatching time. Similarities between clones have been observed with regard to life span, total number of ovipositions per life span, and age at first oviposition. In addition, significant differences in hatching time, with eggs hatched over a long period, were found within each clone. Interclonal variability in life history traits indicated the effects of genetic factors, whereas intraclonal variability reflected the effects of environmental factors. The evolutionary and adaptive significance of the life history phenotypic variations is discussed.

Three new species of eutardigrades from the Seychelles

Three new species of tardigrades are described from the Seychelles: Macrobiotus pseu‐docoronatus, Macrobiotus danielisae, and Hypsibius seychellensis. Macrobiotus pseudocoronatus sp. n. belongs to the Macrobiotus harmsworthi group but it can be distinguished by the presence of very small cuticular tubercles. It also differs from some other species in the group by having eye spots, or by characters of the claws, or by details of the bucco‐pharyngeal apparatus, or by some characters of the eggs. Macrobiotus danielisae sp. n. belongs to the Macrobiotus richtersi group. It differs from the other species in the group by the characteristics of the cuticular sculpture and, from some of them, or by lacking eye spots, or by characters of the claws, or by details of the bucco‐pharyngeal apparatus, or by some characters of the eggs. Hypsibius seychellensis sp. n. differs from Hypsibius dujardini (Doyère, 1840) by having a slightly narrower buccal tube of more uniform width (in H. dujardini the posterior portion of the tube is wider than the anterior portion), and shorter claws. It differs from H. iskandarovi Tumanov, 1997 by lacking lunules and cuticular bars on the first three pairs of legs. It differs from Hypsibius allisoni Horning, Schuster & Grigarick, 1978 by having a septulum instead of an almost invisible microplacoid, and more slender claws with a longer basal portion.

Predator–prey interaction in soil food web: functional response, size-dependent foraging efficiency, and the influence of soil texture

In a series of laboratory experiments, we presented carnivorous Macrobiotus richtersi (Tardigrada, Macrobiotidae) with nematode prey to assess their importance as predator. We investigated consumption rate for (a) different prey densities (10–400 prey individuals), (b) different prey biomasses (22–80 ng), (c) different prey species (Pelodera teres, Rhabditidae, versus Acrobeloides nanus, Cephalobidae) and (d) different environments (2-D agar surface versus 3-D sand fractions of three different textures). M. richtersi consumed up to 4.6 μg nematode prey in 4 h, that is, 43% of the tardigrade’s body mass. Predation rate was positively correlated with prey density. The optimal prey in the present investigation was the biggest prey because it yielded the highest biomass uptake per time. In addition, the size of M. richtersi played an important role in consumption rate. Bacterivorous nematodes reacted differently to attack. Even in a water film on stiff agar where nematode agility was limited, a vigorous undulation reaction of P. teres led to a measurable reduction in consumption rate. A. nanus, in contrast, showed little response to attack. Microcosm experiments with sands of different particle size demonstrated that M. richtersi is able to chase and consume small bacterivorous nematodes in a 3-D soil matrix. However, consumption rate in sand microcosms was significantly reduced compared with pure agar. The sand matrix improved nematode agility and possibly provided small pores as refuge for the nematodes. The lowest consumption rate was observed in fine sand. Effects of predatory tardigrades on nematode numbers in the field are discussed.

Retention of embryonated eggs in parthenogenetic Macrobiotus richtersi J. Murray, 1911 (Eutardigrada)

According to the current opinion, bisexual tardigrades lay their eggs before pronuclear fusion and partenogenetic tardigrades before cleavage and differentiation. Meiosis (or a corresponding ameiotic oocyte division in many parthenogenetic strains) stops at the first metaphase and starts again after oviposition. Tardigrades, therefore, are considered as being exclusively oviparous (for review see Marcus 1929; Bertolani and Rebecchi 1999; see also Suzuki 2003). When checking a series of permanent preparations enclosed in Faure’s medium made by one of us (K.H.) Dr. H. Dastych, Hamburg, called our attention to a female of Macrobiotus richtersi that obviously carried two embryonated eggs and, thus, differed remarkably from other gravid females of the same population. The specimens of the series came from a long-term culture on agar and were fed with nematodes (see Hohberg, in prep.). During April 1999–December 2003 no males were detected. Juvenile females isolated from others continuously produced young without males when they reached maturity. Therefore, we assume that the population is thelitokous parthenogenetic. In the wild and in culture gravid females reach the remarkable length of up to 860 mm (smallest gravid females measured 450 mm) and carry up to 27 mature oocytes in their ovaries. More than once we observed

First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae).

Chromosome behaviour during male meioses has been studied in two bisexual amphimictic populations of two tardigrade species, namely Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae). Both bisexual populations exhibit a diploid chromosome number 2n=12 and no sex chromosomes were identified. DAPI staining and C-banding data indicate that all chromosomes of the bisexual population of R. coronifer are acrocentric. In both species, at male meiotic prophase, all six bivalent homologous chromosomes are aligned side by side along their length and show no evidence of chiasmata. However, in the oocytes of both species a chiasma is generally present in each bivalent at diplotene stage. Lack of recombination is previously unknown in tardigrades, but is a well known phenomenon in many other metazoans where it is always restricted to the heterogametic sex. In tardigrades there is no evidence of heterochromosomes, but it does not mean that in tardigrades, the heterogametic sex does not exist. The adaptive and evolutionary significance of achiasmatic meiosis is discussed.

Banding techniques on tardigrade chromosomes: the karyotype of Macrobiotus richtersi (Eutardigrada, Macrobiotidae).

This work represents the first attempt to define tardigrade chromosomes using banding techniques. Macrobiotus richtersi, a eutardigrade morphospecies with amphimictic diploid and thelytokous triploid cytotypes, was used as a model. Prime consideration was given to oocyte chromosomes because they are larger than those of spermatocytes and of mitotic chromosomes. With Giemsa staining, the chromatids of the 6 bivalents of the diploid cytotypes and those of the 17-18 univalents of the triploid cytotypes were very similar to each other and appeared rod- or flame-shaped. In the amphimictic strain, a chiasma was generally present in each bivalent at diplotene, whereas there were no chiasmata in the oocyte prophase chromosomes of the triploid strain. Both in diploid and triploid cytotypes, C-banding and fluorescence showed a heterochromatic centromeric band on the telomere of each chromosome oriented towards the spindle pole, indicating that all of them were acrocentric. Silver staining showed the presence of a NOR in only a pair of chromosomes, close to the centromeric C-banded site. NOR was particularly evident in the oocyte prophases. Other silver positive regions, corresponding to the kinetochore, were located on all other chromosomes on the telomeres towards the spindle pole.

Rearing Tardigrades: Results and Problems

We report our first results of attempts to rear four species of eutardigrades inhabiting different substrates, feeding on different kinds of food and characterized by different sexual conditions and modes of reproduction. Attempts were carried out to follow individual terrestrial carnivorous (Macrobiotus richtersi, M. joannae) and limnic herbivorous (Diphascon cf. scoticum; Isohypsibius monoicus) species. Carnivorous leaf litter-dwelling species were reared in small dishes containing agar as substrate and bacteriophagous nematodes as food. Five generations were obtained with the triploid thelytokous strain of M. richtersi, whereas three generations were obtained with the hermaphrodite species M. joannae. Diphascon cf. scoticum and I. monoicus were reared in small dishes containing algae as food and substrate. Several generations were obtained for both species. Males were never found in D. cf. scoticum and I. monoicus was hermaphroditic. Specimens isolated from hatchings were maintained and reproduced in both species, demonstrating parthenogenesis in the first one and self-fertilization in the latter. Consideration of the problems and on the future applications of tardigrade rearing are discussed.

Seasonal and Altitudinal Variation in the Distribution and Abundance of Tardigrada on Dugger Mountain, Alabama

A seasonal survey of the distribution of terrestrial tardigrades on Dugger Mountain, Alabama, was conducted during the time period from April 1997 through April 1998. Cryptogams from five trees ( Quercus alba), three on north-facing slopes and two on south-facing slopes, were sampled seasonally at three stations (645 m, 410 m, 183 m) along an unnamed tributary of the South Fork of Terrapin Creek. Trees were chosen based on their location outside the riparian zone. Tardigrades were extracted from the samples, mounted individually in Hoyer's medium, and identified to species with phase microscopy. Seasonal and altitudinal variations in the distribution of the populations on the north- and south-facing slopes were determined. Present on Dugger Mountain were tardigrades belonging to 12 species ( Macrobiotus cf. areolatus/tonollii, Macrobiotus cf. echinogenitus, Macrobiotus islandicus, Macrobiotus richtersi, Minibiotus intermedius, Milnesium tardigradum, Diphascon pingue, Hypsibius pallidus, Echiniscus cf. arctomys, Echiniscus virginicus, Pseudechiniscus ramazzottii, and Pseudechiniscus suillus). Due to the small numbers of individuals of each species, the total numbers of tardigrades of all species were pooled. There were no significant differences in the mean number of species or the mean number of all tardigrades per sample at each station (altitude). However, seasonal differences in both abundance and number of species were detected in pooled samples due to the high numbers collected in spring 1997.

Remarks on some tardigrades of the African fauna with the description of three new species ofMacrobiotusSchultze 1834

Ten species of tardigrades are recorded. Three of them, Macrobiotus radiatus, Macrobiotus vanescens, and Macrobiotus iharosi are new for science; two species, Macrobiotus sapiens Binda & Pilato 1984 and Isohypsibius kristenseni Pilato et al. 1989, are new for the African fauna. Macrobiotus radiatus n. sp. differs from the other species of the harmsioorthi group in the characters of the eggs and in other characters regarding various structures (buccal armature, or placoids length, or the insertion point of the stylet supports, or the claws length). Macrobiotus vanescens n. sp. is similar to Macrobiotus richtersi Murray 1911 and to Macrobiotus peteri Pilato et al. 1989 but differs from them in some characters regarding the bucco-pharyngeal apparatus, the claws and the eggs. Macrobiotus iharosi n. sp. differs from Macrobiotus echinogenitus Richters 1904 in the characters of the eggs, and from Macrobiotus sapiens Binda & Pilato 1984 in some characters of the bucco-pharyngeal apparatus and of the claws.