Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum

The rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate:ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 10(4) g-min (fraction P1) and 10(5) g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0.4-0.6 microns in diameter and which had a mean equilibrium density of 1.22-1.24 g ml-1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 x 10(6) g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).     

Xylanases and carboxymethylcellulases of the rumen protozoa Polyplastron multivesiculatum, Eudiplodinium maggii and Entodinium sp

Endoglucanase and xylanase activities of three rumen protozoa, Polyplastron multivesiculatum, Eudiplodinium maggii, and Entodinium sp. were compared qualitatively by zymograms and quantitatively by measuring specific activities against different polysaccharides. A set of carboxymethylcellulases and xylanases was produced by the large ciliates whereas no band of activity was observed for Entodinium sp. in zymograms. Specific activity of endoglucanases from P. multivesiculatum (1.3 micromol mg prot(-1) min(-1)) was twice that of E. maggii, whereas xylanase specific activity (4.5 micromol mg prot(-1) min(-1)) was only half. Very weak activities were observed for Entodinium sp. A new xylanase gene, xyn11D, from P. multivesiculatum was reported and its gene product compared to 33 other family 11 xylanases. Phylogenetic analysis showed that xylanase sequences from rumen protozoa are closely related to those of bacteria.     

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro ) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 μg/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 μg/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2–7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 μg/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum , but not Entodinium spp. or Dip. affine , synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 μg/mg protein/min. If these reactions occur in the rumen in vivo , up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.     

The Cytology of Sheep Rumen Ciliates. II. Ultrastructure of Eudiplodinium maggii1

The anterior adoral zone of syncilia (AZS) of Eudiplodinium maggii is mounted on an extrusible peristome within a vestibulum. The peristome contains cytopharyngeal components derived from the infraciliature. These components include a crescent-shaped palisade of nematodesmata, two types of sub-membrane cytopharyngeal ribbons, and an ensheathing fibrous layer enclosing a phagoplasmic zone containing the other components. A convoluted esophagus is continuous with and extends from the posterior of the cytopharynx adjacent to the macronucleus. A posterior cytoproct has specialized cytoplasm around it and associated myoneme-like elements. The skeletal plate is composed of finely granular platelets and lies under the cortex ventral to the macronucleus. The endoplasm is separated from the ectoplasm by a fibrous boundary layer. The cortex has an external glycocalyx, a membranous layer, epiplasm, and microtubular and microfilament layers. The AZS infraciliature is of the usual cntodiniomorph type, kinetosomes linked by a sub-kinetosomal rod and with associated bifurcated kinetodesma, postciliary and transverse microtubules-the latter extending into the cytopharynx—nematodesmata, and a fibrous reticulum. A possible vestigial, somatic infraciliature consisting of short, barren kinetosomes with associated basal and cortex-directed microtubules and a periodic incomplete fiber, is found subcortically throughout the cell.     

In vitro determination of generation times for Entodinium exiguum, Ophryoscolex purkynjei and Eudiplodinium maggii

ABSTRACT. Most previously reported generation times for rumen ciliate protozoa are longer than would be required to prevent their being flushed out of the rumen. In an earlier study from this lab, using a sequential transfer procedure, generation times between 12 and 13 h were determined for both Epidinium caudatum and Entodinium caudatum . This would permit these species to be maintained in a rumen with a fluid volume turnover rate as rapid as twice a day. In this study, generation times were estimated for Entodinium exiguum (13.2 h), Eudiplodinium maggii (26.8 h), and Ophryoscolex purkynjei (29 h), by sequential transfer at both 12 and 24 h time periods. The generation time for E. exiguum is lower than reported for this and other Entodinium species as determined by logarithmic growth from a small inoculum, but similar to that obtained for Ent. caudatum using sequential transfer. Eudiplodinium maggii and O. purkynjei generation times are similar to previous estimates of 24- and 24–48 h, respectively. However, it was observed that after an adaptation period of 36 to 48 h (generally 3–4 transfers) cell concentrations decreased and generation times were markedly decreased, i.e. 12.2 h for Ent. exiguum , 15.0 h for E. maggii and 12.8 h for O. purkynjei . In a separate study, varying both the concentration of Epidinium and the quantity of substrate fed per cell had no effect on generation time.     

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen.

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 micrograms/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 micrograms/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2-7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 micrograms/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum, but not Entodinium spp. or Dip. affine, synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 micrograms/mg protein/min. If these reactions occur in the rumen in vivo, up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.     

The cryopreservation of some large ciliate entodiniomorphid protozoa taken from the rumen

M.H.P. FUNGARO, M.L.C. VIEIRA, A.A. PIZZIRANI-KLEINER AND J.L. DE AZEVEDO. 1996. Random amplified polymorphic DNA (RAPD) was used in order to analyse the relationships among 13 isolates of Metarhizium anisopliae var. anisopliae . Six of them were isolated from Deois flavopicta (Stal) (Hemiptera—Homoptera: Cercopidae) in different regions of Brazil. The other seven were isolated from soil in Paraná State in Southern Brazil. The isolates were grouped by cluster analysis using Dice similarity index. The results show that isolates of M. anisopliae var. anisopliae are extremely diverse (47% similarity) but those isolated from D. flavopicta present only a moderate degree of variation (82% similarity) when compared with the wide diversity (31% similarity) found in the group isolated from soil. These results suggest that M. anisopliae var. anisopliae has developed host specificity.     

Molecular diversity of entodiniomorphid ciliate Troglodytella abrassarti and its coevolution with chimpanzees

The entodiniomorphid ciliate Troglodytella abrassarti is a colonic mutualist of great apes. Its host specificity makes it a suitable model for studies of primate evolution. We explored molecular diversity of T. abrassarti with regard to large geographical distribution and taxonomic diversity of its most common host, the chimpanzee. We found a very low diversification of T. abrassarti in chimpanzees across Africa. Distribution of two types of T. abrassarti supports evolutionary separation of the Western chimpanzee, P. t. verus, from populations in Central and East Africa. Type I T. abrassarti is probably a derived form, which corresponds with the Central African origin of chimpanzees and a founder event leading to P. t. verus. Exclusivity of the respective types of T. abrassarti to Western and Central/Eastern chimpanzees corroborates the difference found between an introduced population of presumed Western chimpanzees on Rubondo Island and an autochthonous population in mainland Tanzania. The identity of T. abrassarti from Nigerian P. t. ellioti and Central African chimpanzees suggests their close evolutionary relationship. Although this contrasts with published mtDNA data, it corroborates current opinion on the exclusive position of P. t. verus within the chimpanzee phylogeny. The type of T. abrassarti occurring in Central and East African common chimpanzee was confirmed also in bonobos. This may point to the presence of an ancestral Type II found throughout the Lower Guinean rainforest dating back to the common Pan ancestor. Alternatively, the molecular uniformity of T. abrassarti may imply a historical overlap of the species' distribution ranges.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

The influence of ruminal concentrations of O2 and CO2 on fermentative metabolism of the rumen entodiniomorphid ciliateEudiplodinium maggii

The effects of ruminal concentrations of CO₂ and O₂ on glucose-stimulated and endogenous fermentation of the rumen ciliateEudiplodinium maggii were investigated. The principal metabolic products were butyrate, acetate, lactate, propionate, H₂, and CO₂.¹³C NMR spectroscopy revealed glycerol to be an important, but previously unidentified, fermentation product of this organism. Glucose uptake and metabolite formation rates were influenced by the headspace composition during protozoal incubations. Glucose uptake was most rapid in the presence of low O₂ in N₂ (1–3 µM O₂ dissolved in the protozoal suspension). Pathways located in the hydrogenosomes were O₂ sensitive, and low O₂ concentrations resulted in lowered acetate, H₂, and CO₂ formation. The presence of high CO₂ (65% gaseous headspace by volume) resulted in elevated acetate and butyrate formation; fumarate and propionate were similarly found to accumulate at higher concentration than previously detected in the supernatants. Results suggest that under conditions similar to those prevailing in the rumen (i.e., high CO₂),Eu. maggii produces higher levels of important ruminal volatile fatty acids, and thus its relative contribution to rumen metabolism may have been underestimated.     

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6 × 10³ and 7.8 × 10³ mL⁻¹, respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7 × 10³ and 2.4 × 10³ mL⁻¹, respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5 × 10³ mL⁻¹ for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.     

The ability of rumen ciliates, Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum, to use the murein saccharides

Murein polysaccharides may contribute to a considerable part of the dry matter of bacterial cells. Their utilization by protozoa inhabiting the rumen is, however, poorly recognized. The objective of this study was to examine the ability of three species of ciliates, i.e., Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum of digest, and ferment these saccharides. The cultivation experiments showed that the enrichment of growth medium with bacterial cell wall β-glycans increased the ciliate number (p?<?0.05). A statistically significant increase (p?<?0.01) was followed by a continuous decrease (p?<?0.01) in the percentage of individuals containing β-glycans particles after 4- and 24-h incubation of ciliates with this substrate, respectively. The enzymatic experiments confirmed the ability of the examined protozoa to digest murein. E. caudatum exhibited the highest activity (8.2 unit (U)/mg protein per min), and E. maggii, the lowest (3.0 U/mg protein per min). The production rates of volatile fatty acids by starved and fed ciliate species were 0.7 and 1.6 (E. caudatum)?pmol/ciliate cell per h, 30.5 and 42.5 (E. maggii)?pmol/ciliate cell per h, and 8.3 and 19.2 (D. affine)?pmol/ciliate cell per h (p?<?0.05).     

Effect of the rumen ciliates Entodinium caudatum, Epidinium ecaudatum and Eudiplodinium maggii, and combinations thereof, on ruminal fermentation and total tract digestion in sheep

The quantitative importance of individual ciliate species and their interaction in the rumen is still unclear. The present study was performed to test whether there are species differences in the influence on ruminal fermentation in vivo and if combinations of ciliates act additive in that respect. Six adult wethers fed a hay-concentrate diet were defaunated, then refaunated either with Entodinium caudatum (EC), Epidinium ecaudatum (EE) or Eudiplodinium maggii (EM) alone, then progressively with all possible species combinations. Feed, faeces, urine, ruminal fluid and gas were sampled for eight days always after at least 21 days of adaptation. With a linear mixed model, accounting for the 2 x 2 x 2 full factorial study design, mean marginal effect sizes, i.e., the magnitude of change in variables as caused by the presence of each ciliate species or of combinations of them, were estimated. The apparent digestibility of organic matter and neutral detergent fibre remained unaffected. The apparent N digestibility increased by 0.054 with EM (0.716 with defaunation). Ruminal ammonia increased by 1.6, 4.0 and 8.7 mmol/l in the presence of EM, EC and EE, respectively, compared to defaunation (6.9 mmol/l). In the EM + EE combination, ruminal ammonia was lower than would have been expected from an additive effect. With EE, total short-chain fatty acids increased by 23 mmol/l (100 mmol/l with defaunation), but not when EE was combined with EM. The acetate-to-propionate ratio decreased by 0.73 units in the presence of EE (4.0 with defaunation), but only when EE was the sole ciliate species in the rumen. In the presence of any ciliate species, the 16S rDNA copies of total Bacteria and major fibrolytic species decreased to 0.52- and 0.22-fold values, respectively of that found without protozoa. Total Archaea were unaffected; however, Methanobacteriales copies increased 1.44-fold with EC. The CH4-to-CO2 ratio of ruminal gas decreased by 0.036 with EM and 0.051 with EE (0.454 with defaunation). In conclusion, individual ciliates affected ruminal fermentation differently and, when different species were combined, sometimes in a non-additive manner. From the ciliates investigated, EE affected ruminal fermentation most and might play a dominant role in mixed ciliate populations.     

Effect of the rumen ciliates Entodinium caudatum,Epidinium ecaudatum and Eudiplodinium maggii,and combinations thereof,on ruminal fermentation and total tract digestion in sheep

The quantitative importance of individual ciliate species and their interaction in the rumen is still unclear. The present study was performed to test whether there are species differences in the influence on ruminal fermentation in vivo and if combinations of ciliates act additive in that respect. Six adult wethers fed a hay-concentrate diet were defaunated, then refaunated either with Entodinium caudatum (EC), Epidinium ecaudatum (EE) or Eudiplodinium maggii (EM) alone, then progressively with all possible species combinations. Feed, faeces, urine, ruminal fluid and gas were sampled for eight days always after at least 21 days of adaptation. With a linear mixed model, accounting for the 2 × 2 × 2 full factorial study design, mean marginal effect sizes, i.e., the magnitude of change in variables as caused by the presence of each ciliate species or of combinations of them, were estimated. The apparent digestibility of organic matter and neutral detergent fibre remained unaffected. The apparent N digestibility increased by 0.054 with EM (0.716 with defaunation). Ruminal ammonia increased by 1.6, 4.0 and 8.7 mmol/l in the presence of EM, EC and EE, respectively, compared to defaunation (6.9 mmol/l). In the EM + EE combination, ruminal ammonia was lower than would have been expected from an additive effect. With EE, total short-chain fatty acids increased by 23 mmol/l (100 mmol/l with defaunation), but not when EE was combined with EM. The acetate-to-propionate ratio decreased by 0.73 units in the presence of EE (4.0 with defaunation), but only when EE was the sole ciliate species in the rumen. In the presence of any ciliate species, the 16S rDNA copies of total Bacteria and major fibrolytic species decreased to 0.52- and 0.22-fold values, respectively of that found without protozoa. Total Archaea were unaffected; however, Methanobacteriales copies increased 1.44-fold with EC. The CH₄-to-CO₂ ratio of ruminal gas decreased by 0.036 with EM and 0.051 with EE (0.454 with defaunation). In conclusion, individual ciliates affected ruminal fermentation differently and, when different species were combined, sometimes in a non-additive manner. From the ciliates investigated, EE affected ruminal fermentation most and might play a dominant role in mixed ciliate populations.     

Chitinolytic enzymes of the rumen ciliate <Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>

The ability of rumen ciliates to digest chitin is clearly recognized. We investigated the chitinolytic system of the rumen ciliate Eudiplodinium maggii. The ciliates were grown in a selectively faunated sheep. They were isolated from the rumen and purified by sedimentation. A crude enzyme preparation was prepared following incubation of ciliates with antibiotics. This was done in order to reduce their contamination with intracellular bacteria. The activity of particular enzymes was examined by quantification of the products released from specific substrates. It was stated that the optimum conditions for the detected activities varied between 4.5 and 5.5 pH, and 45 and 55 °C. β-N-Acetylglucosaminidase was found as an enzyme of the highest activity (4.2 μmol/l released product per mg protein per h). The activities of endochitinase and exochitinase were almost two times lower than that of β-N-acetylglucosaminidase. Zymographic studies revealed the presence of two endochitinases, two exochitinases and two β-N-acetylglucosaminidases in the examined preparation.     

Morphogenesis of a unique pseudourostylid ciliate,Trichototaxis songi (Ciliophora,Urostylida)

Divisional morphogenesis in the freshwater spirotrichous ciliate, Trichototaxis songi Chen et al., 2007, was investigated. The main morphogenetic events are characterised as follows: (1) the parental oral apparatus is completely renewed by the independently formed oral primordium in the proter; (2) the oral primordium in the opisthe is formed on the cell surface; (3) several left cirri of the midventral pairs participate in the formation of the oral primordium in the opisthe; (4) FVT-anlage I forms the leftmost pair of the bicorona; (5) the macronuclear nodules fuse into many masses rather than a single or branched mass as described in most pseudokeronopsids; and (6) usually, two marginal anlagen develop within each left marginal row separately. However, the number of left marginal anlagen is highly variable, even differing between the proter and opisthe of the same divider. The increase in the number of left marginal anlagen is by de novo generation of small anlagen to the left of the intrakinetal left marginal anlage, whereas the decrease in number is by resorption of the old marginal row(s). We posit that Trichototaxis is an intermediate form between the Pseudourostylidae and Pseudokeronopsidae as it shares morphogenetic features with both. Additionally, as in Uroleptopsis (Uroleptopsis), FVT-anlage I forms the leftmost pair of the bicorona in Trichototaxis indicating these genera may be closely related.     

Hydrogenosomes in known species of rumen entodiniomorphid protozoa

Abstract Yeasts of the genus Kluyveromyces grew very slowly on methylamine as sole nitrogen source. Methylamine oxidase activity in cell-free extracts was very low. Under conditions known to separate methylamine oxidase from benzylamine oxidase in other yeast genera, only a single enzyme was detected in Kluyveromyces lactis . This enzyme could oxidize benzylamine, n -butylamine and (very poorly) methylamine. The enzyme lost no activity on heating at 45°C and had a high affinity and V _max for benzylamine and 1-aminoalkanes of long-chain length, with a very low affinity and V _max for methylamine. It is concluded that growth of K. lactis on methylamine involves only benzylamine oxidase, and that a methylamine oxidase of the type found in other yeasts does not occur.     

A survey of entodiniomorphid ciliates in chimpanzees and bonobos

Intestinal entodiniomorphid ciliates are commonly diagnosed in the feces of wild apes of the genera Pan and Gorilla. Although some authors previously considered entodiniomorphid ciliates as possible pathogens, a symbiotic function within the intestinal ecosystem and their participation in fiber fermentation has been proposed. Previous studies have suggested that these ciliates gradually disappear under captive conditions. We studied entodiniomorphid ciliates in 23 captive groups of chimpanzees, three groups of captive bonobos and six populations of wild chimpanzees. Fecal samples were examined using Sheather's flotation and Merthiolate‐Iodine‐Formaldehyde Concentration (MIFC) methods. We quantified the number of ciliates per gram of feces. The MIFC method was more sensitive for ciliate detection than the flotation method. Ciliates of genus Troglodytella were detected in 13 groups of captive chimpanzees, two groups of bonobos and in all wild chimpanzee populations studied. The absence of entodiniomorphids in some captive groups might be because of the extensive administration of chemotherapeutics in the past or a side‐effect of the causative or prophylactic administration of antiparasitic or antibiotic drugs. The infection intensities of ciliates in captive chimpanzees were higher than in wild ones. We suppose that the over‐supply of starch, typical in captive primate diets, might induce an increase in the number of ciliates. In vitro studies on metabolism and biochemical activities of entodiniomorphids are needed to clarify their role in ape digestion. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

悬游双眉虫(原生动物,纤毛门)无性生殖期间的形态发生

应用蛋白银染色技术研究了悬游双眉虫青岛种群无性生殖期间皮层结构和核器的演化过程,其主要特征为:后仔虫口原基独立地出现于紧靠虫体左侧第一根横棘毛的皮层下小龛,其中毛基粒不参与其它棘毛原基的形成;老的口围带发生后半部的局部重建而非整个的由前仔虫简单继承;在前仔虫中,波动膜原基来自老结构的反分化,而在后仔虫中则来自口原基;所有棘毛原基均为独立发生并与老结构没有任何关系;在前仔虫中,口棘毛(即左侧第一根额棘毛)来自于波动膜的反分化,而在后仔虫中则为独立发生;背触毛列于老的结构当中产生,并由最右一列原基演化出3根尾棘毛;两大核片段的改组带从一端向另一端移动 ,并随着两者的融合而消失.文中同时对前人有关该属发生模式的若干存疑问题做了探讨 [动物学报 54(3):517-524,2008].