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ACTA

ZOOLOGICA

SINICA

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CHINESE

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ACTA ZOOLOGICA SINICA

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Vol. 48 No.1 Jan. 2002

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ABSTRACT

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Digestive strategies of mammals

Ian D. Hume

(School of Biological Sciences A08,University of Sydney,NSW 2006, Australia)

Abstract Understanding an animal's nutritional niche is fundamental to a full appreciation of its ecology, and is important for both pest control and species conservation purposes. Carnivores have digestive systems dominated by the small intestine, which can be related to the generally high digestibility of their food. Omnivores have more complex gastrointestinal tracts, with a hindgut caecum in which some microbial fermentation takes place, and they have longer mean retention times (MRTs) of digesta. The longest MRTs are found in herbivores, in which digesta are retained and fermented by dense microbial populations in one or more regions of relative stasis. However, not all herbivores have digestive systems that maximise fibre digestibility; only ruminants, camelids and very large hindgut fermenters (rhinos, elephants) achieve this. Instead, many other herbivores (foregut fermenters such as kangaroos and small hindgut fermenters such as rabbits, voles and possums) have digestive systems that sacrifice maximal fibre digestibility for a capacity to process large amounts of forage, even when forage fibre content becomes very high. These different digestive strategies result in the wide range of nutritional niches found among mammals. [Acta Zoologica Sinica 48(1):1-19, 2002, in English]

Key words Carnivore, Herbivore, Omnivore, Caecum fermenter, Colon fermenter, Foregut fermenter, Mean retention time, Digestive strategies

E-mail: ianhume@bio.usyd.edu.au

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Effects of steppe polecat (Mustela eversmanni) odor on social behaviour and breeding of root voles (Microtus oeconomus)

WANG Zhen-Long^*

(Department of Biology, Qufu Normal University, Qufu 273165, Shandong, China)

LIU Ji-Ke

(College of Life Science, Zhejiang University, Hangzhou, 310012, China)

Abstract This study was aimed at determining how olfactory cues of mustelids affect the social behavior, foraging, reproduction and sexual hormones of root voles (Microtus oeconomus). We predicted that the presence of the odor from the main predator, the steppe polecat (Mustila eversmanni) would cause delayed breeding and changed mating behavior in root voles. We carried out the study from April to December 1997 using liveŞ˛trapped voles and steppe polecats. We carrid out the experiment in two main arenas (size: 40 cmĄÁ40 cmĄÁ40 cm) for three days first and then in breeding cages for 15 days. The test subjects were 34 female male pairs of vo les, 17 experimental and 17 control. We examined the mating and foraging behavior (the frequency and amount of food eaten) in response to the diluted urine and feces filtration from the steppe polecat. During the study, we sprayed the saw dust in the experimental arenas with the predator odor daily. We sprayed the control arenas daily with distilled water. On the 18th day when the behavioral bioassay was completed, the voles were killed and weighted, and the reproductive stage of females, the weight of female ovaries, the number of embryos, the number of sperm, and the weight of the male testes and epididymis were measured. We found that the root voles delayed breeding and significantly lowered the frequency of copulation after being exposed to the predator odor. Comparing with the control, attacking behavior decreased whereas amicable interactions increased in both males and females. Females also significantly increased the level of general activity and frequency and time spent in investigation upon the presence of the steppe polecat odor. Also, the presence of the steppe polecat odor lowered the amount of food intake and body weight in the vole. The ovary index and litter weightdecreased in females, whereas in males the sperm count in epididymis decreased significantly. [Acta Zoologica Sinica 48(1):20-26, 2002]

Key words Steppe polecat(Mustela eversmanni), Root voles(Microtus oeconomus), Social behavior, Predation risk, Delayed breeding

^*E-mail:Wangzhl2000@qfnu.edu.cn

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Odor preferences of adult male Brandt's vole (Microtus brandti)ĄŞdiscrimination of female individual scents

ZHANG Li SUN Ru-Yong FANG Ji-Ming

(Ministry of Education Key Laboratory For Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing 100875,China)

Abstract The odor preferences of adult male Brandt's voles (Microtus brandti) to spouse and adult novel females were recorded in a 30-minute 2-way odor choice test. Four kinds of odor combinations: (1)estrous spouse and estrous novel female odor; (2)estrous spouse and diestrous novel female odor;(3)diestrous spouse and estrous novel female odor;(4)diestrous spouse and diestrous novel female odors, were presented to breeding male voles. In experiment 1, 3 and 4, male voles presented more sniffing, digging, and other investigating behaviors to novel female odor than those to the spouse odor; males also spent more time in the experimental box with unfamiliar female odor than in the box with spouse female odor. In experiment 2, males preferred the estrous spouse odor over the odor of diestrous novel female, and the visit frequency to estrous spouse odor was significantly higher than the frequency of visit to diestrous novel female odor. These results indicated that male voles preferred odors of estrous females to diestrous females, and they preferred novel female scents to spouse's scents when the female odor donors were in the same estrus cycles. The results also suggested that males would prefer novel or estrous females in the mate choice. These responses are typical of a polygamous rodent species. [Acta Zoologica Sinica 48(1):27-34, 2002]

Key words Brandt's vole (Microtus brandti), Female odor, Olfactory communication, Individual discrimination, Mate choice

*E-mail: asterzhang@sina.com

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Further studies on egg incubation of an oviparous snake, Dinodon rufozonatum(Colubridae), with comments on the influence of hydric environmets

ZHANG Yong-Pu^˘Ů JI Xiang^˘Ú*ŞŞ

(˘Ů Department of Biological and Environmental Sciences, Wenzhou Normal College,Wenzhou 325003, Zhejiang, China)

(˘Ú School of Life Sciences, Hangzhou Normal College, Hangzhou 310036, China)

Abstract We incubated eggs of red banded wolf snakes (Dinodon rufozonatum) at 24 and 30Ąć on substrates with water potentials of 0 and Ł500 kPa using a 2ĄÁ2 factorial design, paying particular attention to the influence of substrate moisture and its interaction with incubation temperature on hatching success, embryonic use of energy and material and hatchling traits. All viable eggs gained mass during incubation due to absorption of water, and both thermal and hydric environments affected water exchanges between eggs and their surroundings, thereby affecting the hydric conditions inside the egg. Eggs incubated at 24Ąć gained more mass than did eggs at 30Ąć but at the same substrate water potential; eggs incubated in wetter substrates (0 kPa) gained more mass than did eggs in drier substrates (Ł500 kPa) but at the same temperature. Incubation temperature significantly affected duration of incubation, but substrate moisture and its interaction with temperature did not. In our study, both incubation temperature and substrate moisture did not affect hatching success and sex ratio of hatchlings. Deformed hatchlings were found in each treatment, the frequency being independent of treatments. Substrate moisture significantly affected size (SVL), wet mass and carcass dry mass of hatchlings, with those incubated in wetter substrates being larger in SVL, wet body mass and carcass dry mass than those in drier substrates. Variation in hatchling wet body mass among treatments stemmed mainly from variation in water contents, because there were no significant differences in total dry mass among hatchlings from different incubation conditions designed in this study. More yolk remained unutilized at hatching when eggs were incubated at 30Ąć. Hatchlings incubated at 30Ąć contained more ash than did those incubated at 24Ąć, largely because they contained more ash in the residual yolk. Hatchlings incubated at 24Ąć contained more energy than did those incubated at 30Ąć, suggesting greater energy expenditure for embryonic development at 30Ąć than at 24Ąć. Of all hatchling traits examined, only residual yolk dry mass was affected by interaction between temperature and moisture. In all treatment locomotor performance (the maximum distance of continuous locomotion) was not correlated with hatchling SVL. A two-way ANOVA indicated that there were no significant effects of incubation temperature, of substrate moisture and of their interaction on locomotor performance of hatchlings. Taken together, our results reveal that the effects of substrate moisture on incubation of D. rufozonatum egg are of minor importnce, and therefore support the conclusion that variation in hydric environments over a wide range does not have important effects on incubation of pliable-shelled reptilian eggs. [Acta Zoologica Sinica 48(1):35-49, 2002]

Key words Red-banded wolf snake (Dinodon rufozonatum), Colubridae, Egg, Incubation, Thermal and hydric environment, Hatchling trait

^*E-mail: xji@mail.hz.zj.cn

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Phylogenetic relationships of Elaphodus cephalophus and three muntiacus species revealed by mitochondrial cytochrome b nucleotide sequence

CAO Xiang-Rong^˘ŮSHU Feng-Jue^˘Ů˘ÚZHANG Xi-Ran^˘Ů BI Chun-Ming^˘ŮLI Chao-Jun^˘Ů

HU Jun^˘ŰFANG JianŞ˛Yang^˘Ű

(˘Ů College of Life Science, Nanjing Normal University,Nanjing 210097,China)

(˘Ú Institute of Genetics,Fudan University,Shanghai 200433,China)

(˘Ű Wannan FirstŞ˛Aid Centre of Wild Animal,Xiuning 245400,Anhui,China)

Abstract Phylogenetic relationships of Mutiacinae are of considerable interest because of their suitability for the study of evolutionary patterns and processes. The extant species of Mutiacinae includes Muntiacus muntjak, Muntiacus reeves, Muntiacus crinifrons, Muntiacus feae, Muntiacus rooseve torum, Muntiacus gongshanensis and Elaphodus cephalophus. Previous phylogenetic studies on morphological and palontological information for the Mutiacinae have demonstrated that the extant subfamilies of muntjak originated in Pleistocene. The earlist fossil record of Muntiacus reeves shows that this species evolved in early and mid-Pleistocence, while Muntiacus ŞŤmuntjak evolved in mid- and late-Pleistocence. Elaphodus cephalophus is a sister group to the Muntiacus which envolved in Pleistocence too. Most chromosomes of the Elaphodus cephalophus have similar characteristics with Muntiacus reeves except that 2ĄŤ3 chromosomes are homologous with Muntiacus muntjak. Evolutionary relationships among them are not well understood.Mitochondrial DNA has been used extensively for phylogenetic inference.In this study,fragments of mitochondrial DNA cytochrome b gene(each 366 base pairs in length) have been characterized from Muntiacus muntjak, Muntiacus reeves, Muntiacus crinifrons and Elaphodus cephalophus by direct sequencing of PCR products. The nuclearted divergence of the cytochrome b region was 3.55%ĄŤ4.64% among three species of Muntiacus, and 9.29%ĄŤ10.11% between Elaphodus and three species of Muntiacus. These results showed that divergence time was about 1.42ĄŤ1.84 million years among three species of ŞŤMuntiacus and Elaphodus diverged from Muntiacus about 3.7 million years ago. These results indicated that nuclearted divergency was level of inter-subfamily between Elaphodus cephalophus and Muntiacus. We obtained homologous nuclear sequence of Cervus nippon(Cervinae),ŞŤHydropotes inermis(Hydropotinae) and ŞŤAlces alces(Odocoileinae) as well as Tragelaphus scriptus(Tragulidae) for outgroup from gene bank. The phylogenetic relationship of mitochondrial DNA of seven species of Cervidae was described. Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. Results indicated that ŞŤMuntiacus muntjak, Muntiacus reeves and Muntiacus crinifrons construted a monophyletic group. The phylogenetic relationships of mitochondrial DNA suggested that Elaphodus cephalophus be more closely related with Muntiacus than other three species of subfamilies. These results are consistent with previous phylogenetic studies of mitochondrial DNA on the whole. [Acta Zoologica Sinica 48(1):44-49, 2002]

Key words Elaphodus cephalophus, Muntiacus, Mitochondrial cytochrome b nucleotide sequence, Phylogeny

*E-mail:biocell@263.net

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Neural development of vocal behavior in striated manikin (Lonchura striata swinhoei)

ZUO Ming-Xue^˘Ů* ZENG Shao-Ju^˘Ů PENG Wei-Min^˘ŮZHANG Xin-Wen^˘Ú

(˘Ů College of Life Science, Beijing Normal University, Beijing 100875, China)

(˘Ú Department of Biology, Hainan Normal College, Haikou 571158, China)

Abstract Songbird has specialized areas in the brain that are necessary for the generation of song. In striated mannikin(Lonchura striata swinhoei), the discrete nuclei, robust nucleus of the archistriatum(RA), high vocal center(HVC), lateral magnocellular nucleus of the anterior neostriatum(LMAN) and Area X, make up one part of the song system that is thought to be important for such early song acquisition. However, the relation between development of song nuclei and behavior of song learning has not been well documented. The male striated mannikin, aged from 5 days (P5) to 150 days (P150) postŞ˛hatching were studied in this experiment. We examined the development of control vocal nuclei and behavior of learned song by analyzing the spectrogram structures.The results were as follows: (1)After neural connections by day 45 were established among the HVC, RA and Area X, juvenile striated mannikin began to learn singing;(2)The striated mannikin could not get the adult song structures prior to day 80 until the volumes of HVC,RA and Area X respectively reached their adult size;(3)The volumetric growth of three song nuclei were not correlated with the process of song learning, but seem to be affected by the establishment of neural connections among the three related song nuclei. The RA, HVC, or Area X volumes increased most rapidly at different stage. Multivariate analysis of variance indicated the effect on age from P5 to P150 (P<0.05) for the RA, HVC or Area X (One-way ANOVA). However, the difference of nuclei volumes between any two groups at different stages was not always significant. The results suggested that the onset and development of song behavior of striated mannikin depend on the normal development in nuclei and the establishment of the neural connections within song control nuclei. The final volumes of the adult song nuclei may be decided mainly by inheritance and affected by the formation of normal projections within song nuclei, but not by the song behaviors. [Acta Zoologica Sinica 48(1):50-57, 2002]

Key words Striated manikin (Lonchura striata swinhoei), Song control nuclei, Neural connection, Sound spectrogram, Vocalization

*E-mail: mxzuo@bnu.edu.cn

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Changes in keratin during apoptosis of PK15 (Porcine Kidney-15) cells

CHEN Dan-Ying GAO Yun-Fei LI Li-Xia ZHAI Zhong-He

(Life Science College, Peking University, Beijing 100871, China)

Abstract Intermediate filament (IF) is a major component of cytoskeleton.Recent advances suggest that IFs not only play a cytoskeletal role in cooperation with microfilaments and microtubules, but are also involved in cell differentiation, signal transduction and gene expression.Apoptosis is a physiological form of cell death, it describes the orchestrated collapse of a cell, staging membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation and DNA degradation. How do IFs change in apoptotic process and what kind of role do they play? The answers to these questions would be of great importance to further study of their functions.

Epithelial cell PK15(Porcine Kidney-15) was induced to apoptosis by actinomycin D.The cell shapes changed from spreading to contracting and rounding during the induction process, and most of the cells dropped from the wall after induction for 96 h.DAPI staining showed the chromatin condensation and apoptotic bodies formation characterized by apoptosis in this process. At the same time Ą°DNA ladderĄą was detected by electrophoresis. Indirect immunofluorescence results showed that the fluorescence of keratin distributed unevenly in the apoptotic cells, and in some cells the keratin fluorescence contracted around the condensed chromatin. Using selective extraction combined with whole mount electron microscopy, we detected the intact keratin filament network in some area of apoptotic cells, and the filaments were obviously shortened. In consistent with the result of immunofluorescence, the keratin filaments around chromatin were condensed in comparison with filaments in other areas.Western blot analysis further showed that part of type ˘ň keratin protein degraded only partially during apoptosis, whereas type ˘ń keratin protein molecules were intact.These results suggested that in apoptotic processing the structure changes of keratin network might lead to the changes of cell shape, and might participate the formation of apoptotic bodies. [Acta Zoologica Sinica 48(1):58-63, 2002]

Key words Pig, Porcine Kidney cell,PK15,Keratin, Intermediate filament, Actinomycin D, Apoptosis

E-mail: zhaizh@plum.lsc.pku.edu.cn

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Effect of testosterone on the release and synthesis of gonadotropin hypothalamus pituitary complex in vitro in the japanese eel (Anguilla japonica)

DENG Yue-Song LIN Hao-Ran^*

(School of Life Science, Zhongshan University, Guangzhou, 510275, China)

Abstract The effects of testosterone (T) on the synthesis and release of gonadotropin (GtH) in the hypothalamus pituitary complex or pituitary alone of Japanese eel (Anguilla japonica) were investigated in vitro. The intact hypothalamus-pituitary complex (HPC), the separate hypothalamus plus pituitary (H+P) or the pituitary (P) of male eel were incubated with TC 199 medium without (control) or with different concentrations of testosterone (0.1, 1.0, or 10.0 ŚĚmol/L) for 8 h. In the control group, the GtH content of HPC group was the highest both in medium or pituitaries while the P group was the lowest. It indicated that the gonadotropin releasing hormone (GnRH) reaches pituitary directly. Incubation of pituitary alone with different concentrations of T increased the GtH content in a dose dependent manner either in medium or pituitary. We suggested that T acted directly as a positive feedback on the synthesis and release of GtH in eel pituitary. Low dosages of T (0.1 and 1.0 ŚĚmol/L) stimulated the synthesis and release of GtH in both HPC group and H+P group after 8 h incubation, indicating that there was a positive feedback effect of T on either hypothalamus or pituitary in eel; whereas a negative feedback effect of T were observed when HPC or H+P incubated with high dosage of T (10.0 ŚĚmol/L). In the highest dosage group, the inhibitory effect of GtH release was more significant than the GtH synthesis. These results demonstrated that the synthesis and release of pituitary GtH was regulated by hypothalamus via GnRH. Testosterone in male fish had feedback effects on either hypothalamus or pituitary in Japanese eel. [Acta Zoologica Sinica 48(1):64-68, 2002]

Key words Japanese eel (Anguilla japonica), Hypothalamus, Pituitary, Gonadotropin, Testosterone

*E-mail: Ls32@zsu.edu.cn

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Spermatocyte meiosis in male mule (Equus asinusĄÁEquus caballus) and hinny

(Equus caballusĄÁequus asinus)

ZHAO Zhen-Min ZHI De-Juan WANG Min-Qiang YANG Bo-Hui

(Lanzhou Institute of Animal Sciences and Veterinary Pharmaceutics, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China)

Abstract The sterility of mule (Equus asinusĄÁEquus caballus) and the phenomenon of exceptional female mules giving birth to offspring occasionally have been remaining controversal in academic cycles for a long time. The theory of chromosome diversity of parents which resulted in failure in hybrids meiosis, and denied the fact of reproduction in mules. Another theory explained the fecundity of female mules in the way that the female mule must have produced oocyte containing only a single parental set of chromosome, and the offspring from backcross would be horse (Equus caballus) or donkey (Equus asinus). But some new evidences are contradictory with the two theories mentioned above. For further studying the genetic mechanism of the reproductive isolation phenomenon of interspecific hybrid, paraffin sections of testes from 13 sexually mature male mules (Equus asinusĄÁEquus caballus) and 12 hinnies (Equus caballusĄÁEquus asinus) were prepared, and the samples of Giemsa meiosis and AgNO₃ synaptonemal complexes were observed and compared with spermatocyte meiosis of horse and donkey under light microscope and electronic microscope. We found that there

was significant individual difference in the testis development of mule. A few hybrids had better developed testis and meiosis occurred. The amount of spermatocytes in the spermatogenic tube of the testis in hybrids was significantly higher than that in the common mule. There were some spermatocytes˘ńand a few spermatocytes˘ň, but they entered the antrum of tube. There were a lot of aberrant chromosomes including fragmentation, disorder, adherence, arc, annulus, etc.ŞŤNevertheless, a few spermatocytes did complete the process of meiosis and form sperm. During the prophase of meiosis˘ń, some chromosomes at the zygoten/diploten stage existed in parallel; at the pachyten stage, chromosomes became broad, but some segments were thin and not paired; at the diploten stage, synaptonemal complexes did not divided in phase, so at the diakinesis, some chromosomes were still in the state of adherence. Chromosomes consisted of haploid, diploid and polyploid. Linear shaped materials like synaptonemal complexes of horse spermatocytes were found, but disordered and blurred, and some linear segments showed parallel double line under electronic microscope. We discussed the traditional theory of infertile mechanism of mule based on our results, and put forward a suggestion that, to recognize and understand the phenomenon of diversities of hybrids sterility, we should consider comprehensively the influences from the heterogeneous chromosomes and the interaction between heterogeneous cell nuclear and cytoplasm. [Acta Zoologica Sinica 48(1):69-74, 2002]

Key words Equus mule, Interspecific hybrid, Meiosis

E-mail: mykyc@public.lz.gs.cn

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Development of monogenean trematode (Neobenedenia girellae, Monogenea: Capsalidae)

YANG Wen-Chuan^˘Ů LI Li-Wei^˘ŮSHI Lei^˘Ů WANG Jun^˘Ú SU Yong-Quan^˘Ú

(˘Ů School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China)

(˘Ú Oceanography Department, Xiamen University, Xiamen 361005, Fujian, China)

Abstract The development of monogenean trematode (Neobenedenia girellae) (Monogenea:Capsalidae), from the maricultured fishes, was studied in Fujian province. All eggs used for this study were obtained from the parasites on the body surface of amberjack fry (Seriola dumerili) collected in floating sea cages, in Xiamen (24Ąă29ĄäN, 118Ąă03ĄäE), Fujian, China. In laboratory, the oncomiracidia hatched from eggs in four to six days at 25ĄćĄŤ28Ąć in salt water. Then, the oncomiracidia were used to infect the fish (Liza carinatus) in laboratory. After attaching to the skin of the fish host, the oncomiracidia developed to adult in 10ĄŤ14 days at the room temperature (26ĄćĄŤ30Ąć). On the sixteenth day, the adults began to release eggs. The fish host (Liza carinatus) is proved to be a new host record of N. girellae. The morphology of eggs, oncomiracidia, immature worms and adults of this monogenean was described in this paper. [Acta Zoologica Sinica 48(1):69-74, 2002]

Key words Monogenean trematode(Neobenedenia girellae), Development, Maricultured fishes

E-mail: wcyang@jingxian.xmu.edu.cn

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Structural modulation of the area between oocytes and follicular cells during

Vitellogenesis of the mud crab (Scylla serrata)

CHENG Yong-Xu^*

(Fisheries College,Shanghai Fisheries University, Shanghai 200090,China)

LI Shao-Jing WANG Gui-Zhong CHEN Xue-Lei LIN Qiong-Wu

(Department of Oceanography, Xiamen University, Xiamen 361005, Fujian,China)

Abstract Structural modulation of the area between oocyte and follicular cells during vitellogenesis (divided by primary and secondary stages) in the mud crab (Scylla serrata) was examined by electron microscopy in order to provide morphological evidence for active intro-oocytic yolk and extro-oocytic formation. Primary vitellogenic oocytes occur in 1ĄŤ9 days after the gravious crab egg being hatched at zoea stages. The primary vitellogenesis can be divided in two stages: intro-oocytic yolk formation and extra-oocytic yolk formation which associated with

pinocytotic activity. In the former stage,it was found the follicular cells showed significant streaming movement towards the surface of oocytes and gradually surrounded the oocyte membranes,there were little structural evidence of pinocytotic activity at the oolemma-follicular cells interface, demonstrated vitellogenic autonomy. The cytoplasm of oocytes were characterized by many ribosomes and intro-oocytic yolk formation inside the elements of the vesicles of the endoplamic reticulum,no other organelle were present in it.

During the latter stage, the plasma membrane of the oocytes contained many pinocytotic vesicles situated in the base of microvilli, which indicated the active extra-oocytic formation. The cytoplasm of the oocytes rich in extensive mitochondria and lipid droplets seemed to suggest the begining of the extra-oocytic vitellogenic event. In the late primary vitellogenesis, a ball and chain structure formed in the interface of oocytes and follicular cells surface because of pinocytotic role and the fuse of two cell membranes,intercellular space of oocyte appeared containing materials that resemble the pinocytotic vesicle. Ooplasmic membrane was often completely fused with that of follicular cell or even was lost and almost materials were found in the follicular cell area in the final primary vitellogenesis.

Secondary vitellogenic oocytes occurred on the 11th day and finished on the 27th day after the gravious crab egg being hatched the first larvae. During this stage, the exta-oocytic vitellogenic event took the most important role in the yolk accumulation in oocytes. At the beginning of secondary vitellogenesis, the extensive spread space of follicular cell,10ĄŤ15 ŚĚm long from basal lamina to the surface of oocyte was rapidly developed. The area of follicular was ten times larger than that of the primary vitellogenesis,and there were lots of vesicles, mitochondrias and numerous lipid droplets (usually near the surface of oocytes). There was nearly no membrane structure between the follicular cells and subjacent oocytes, so that large amount of materials of the follicular cell near the oocyte could be directly taken into the oocyte and stored as yolk bodies and lipid droplets. With the oocyte development, a membrane-like net protective screen structure of the oocyte formed,which indicated the maturity of oocyte. At the same period, it was often found that many degeneration of the oocyte characterized by the yolk lysis and might be resorbed by the conjunctive oocyte or follicular cell and consequent leakage of yolk proteins into the hemolymph.In matured oocyte surface, two distinct layers were identified,the outer layer, which appears denser than inner layer,was formed by follicular cell,probably from the numerous fibrillar materials in the cytoplasm of the follicular cell of the final stage of secondary vitellogenesis;inner layer which was finer and more lucent was formed by oocyte itself. The matured oocytes were characterized by accumulation irregular yolk bodies and lipid droplets scattered throughout the ooplasm.

This study of morphological evidences is in supports of the yolk formation in Scylla serrata oocytes, which is a combination of intra- and extra-oocytic yolk synthesis. The extra-oocytic yolk formation is by two ways. One is by massive pinocytotic activity which occurs at the oocyte surface at the primary vitellogenesis; the other is by oocyte directly taken the yolk materials in follicular cells during the secondary vitellogenesis, the extra-oocytic yolk fomation in this way accounts for most yolk accumulation in oocytes.

Key words Mud crab (Scylla serrata), Primary vitellogenesis, Secondary vitellogenesis,

*E-mail: yxcheng@shfu.edu.cn

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Morphology of sallow horseshoe crab (Tachypleus tridentatus)

LIAO Yong-Yan ^* LI Xiao-Mei

(Fisheries College of Zhanjiang Ocean University, Zhanjiang 524025, Guangdong, China)

HONG Shui-Gen

(Laboratory of Cell Biology, Xiamen University, Xiamen 361005, Fujian, China)

Abstract The North gulf and Leizhou gulf in the northern of the South China Sea is situated between 107.5ĄăĄŤ111ĄăE and 20ĄăĄŤ21.7ĄăN latitude with high temperature and a large quantity of rainfall, which belongs to oceanic climate of the torrid zone and subtropical zone. From August 1997 to August 1999, We studied the morphology of sallow horseshoe crab in the North gulf and Leizhou gulf. We found the juveniles of ŞŤTachypleus tridentatus including zooids with different sizes from 10 cm width of prosoma to 25 cm. These juveniles of Tachypleus tridentatus were called sallow horseshoe crab in the west of Guangdong province, Hainan, and the North gulf regions because of the sallow body colour. The morphology was studied in the sallow horseshoe crab by observation and comparison with other horseshoe crab. We found that the body colour of sallow horseshoe crab was yellowish brown, and its body size was small. Its middle leaves of reproductive operculum was not longer than lateral leaves. Its tail was triangular in shape, on which there was a row of little thorns. On the end of opisthosomatic carapace there were three immovable spines. There were no little thorns under two lateral ridge of its tail and on the opisthowomatic carapace, and its opisthowomatic carapace immovable spines were much longer. We also found that the back colour of body of Tachypleus tuidentatus was gradually changing from black to sallow to brown during post-embryonic development from the first instar stage to adult. When the width of prosoma of juveniles of Tachypleus tridentatus was 10ĄŤ25 cm that is in the 12ĄŤ14 instar stages of post-Ş˛embryonic development, the colour of its body was sallow. All these suggested that sallow horseshoe crab was immature Tachypleus tridentatus, not a new species or subspecies. The specimens were collected from Zhanjiang, Guangdong Province, and were preserved in the Museum of Aquatic Biology of Zhanjiang Ocean University.

Key words Sallow horseshoe crab (Tachypleus tridentatus),ŞŤJuveniles, Morphology, Taxonomy

*E-mail:lly1120@263.net

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Ultrastructure of spermatogenesis in the squat lobster (Thenus orientalis)

ZHU Dong-Fa^* LI Shao-Jing WANG Gui-Zhong

(Department of Oceanography, Xiamen University, Xiamen 361005, Fujian, China)

Abstract The spermatogenesis in the squat lobster (Thenus orientalis)ŞŤwas studied with transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The results showed that, the spermatogonium was oval in shape, in which mitochondria concentrate at one end of the cytoplasm to form a mitochondria zone. The chromatin was relatively dispersed, and some heterochromatins distributed along the nuclear envelope in the oval nucleus. The primary spermatocyte was larger than the spermatogonium, its chromatin became denser and lied at one side of the nucleus. The mitochondria zone disappeared and some mitochondria disintegrated to vesicles. The secondary spermatocyte usually contained a large vesicle between the nucleus and the cytoplasmic matrix, in which there were a large number of vesicles with the number of mitochondria decreased distinctly. The early spermatid with nucleus polarized and decondensed was smaller than the secondary spermatocyte. Many vesicles discarded their inclusions outside the early spermatid and then contributed to the development of the membrane complexes containing extensive RER (rough endoplasmic reticulum) in the cytoplasm. The mid-stage spermatid became pyramidal in overall shape, and three distinct regions were apparent:the maturing acrosome formatted by the vesicles originating from the membrane complexes at its apex, the nucleus at its basement and the cytoplasmic matrix containing extensive membrane complexes in the middle. In the late spermatid, the acrosome further developed into inner acrosomal material, outer acrosomal material and acrosomal cap. The thin, polarized and decondensed nucleus contained a large number of microtubules, and some microtubules extended into the radial arms. It was difficult to find the RER in the membrane complexes of the late spermatid with a mature acrosome. In the mature sperm,five to six radial arms packed with microtubules extend out from the plate- or bowl-like nucleus.

Key words Squat lobster (Thenus orientalis),Spermatogenesis, Ultrastructure

*E-mail:dfzhu@mail.nbptt.zj.cn

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Isolation, purification and identification of metallothionein in Chinese soft-shelled

turtle (Pelodiscus sinensis) liver

LIN Zhi-Lan^˘Ů*LI Fu-Rong^˘Ú MA Guo-Dong^˘ŮLIANG Shuang^˘Ů

(˘Ů College of Life Sciences, Peking University, Beijing 100871, China)

(˘Ú Department of Biology, Xinyang Teachers College, Xinyang 464000, Henan, China)

Abstract Metallothioneins ( MTs ) are a group of low molecular weight, metal-binding and cysteine rich proteins. MTs are widely distributed throughout living organisms and are fairly well identified in mammals, amphibians, fishes, plants, fungi, and cyanobacteria. To compare with the MT's character, the high degree of conservation of amino acid sequence and the central segments in MTs from many sources will provide significant information for investigation of MT's structure and function or the molecular evolution. Thus far, there are no reports for MT isolated from reptiles. In the present paper, we report the presence of MT from turtle liver. Each extract of Chinese soft-shelled turtle (Pelodiscus sinensis) liver after induction with subcutaneous injection of ZnSO₄, CuSO₄ or CdCl₂ was separated by gel filtration chromatography Sephadex G-50 column and subsequent DEAE Sepharose CL-6B, than Sephadex G-25 for desalination, respectively. According to the results of mass spectrometry and HPLC filtration chromatography detection, the molecular weight of the MT is shown to be about 6 300 dalton. The MT containing 61 amino acid residues has a high cysteine content (17%), aromatic amino acids (tyrosine, phenylalanine), arginine and histidine are considerably low. Zn-MT, Cu-MT or Cd-MT has an ultraviolet absorption shoulder near 220 nm, 270 nm or 250 nm, respectively. In the present work we show that the properties of turtle liver MT are similar to the mammals in molecular weight and cysteine residues of MT. However, the other properties (including the amino acid composition of MT and the capability of binding metal ions) are similar to the MT from the earthworm and yeast. The characteristic of MT in turtle lies between the MT of mammals and lower living organisms. This result presents the distinguishing characteristics of biological evolution of MT in turtle. [Acta Zoologica Sinica 48(1):107-113,2002, in English]

Key words Chinese soft-shelled turtle (Pelodiscus sinensis), Metallothionein, Isolation,Purification, Identification

*E-mail: linzhilan@263.net

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Microisolation and characterization of single chromosome in rice field eels (Monopterus albus)

JI Fu-Yun YU Qi-Xing^* GUO Yi-Qing HAI Yan ZHOU Rong-Jia LIU Li

(College of Life Science, Wuhan University, Wuhan 430072, China)

Abstract The methods of microisolation and characteristic assay of single chromosome in rice field eels ŞŤ(Monopterus albus) were established. The chromosome 3 of meiosis˘ńdiakinesis was microisolated manually with a micro-glass needle under the Olympus reverted microscope and its DNA was amplified by DOP-PCR. Several hybridizations were carried out to detect its specialities. 18 S rRNA gene of soybean labeled by Biotin-11-dUTP were hybridized with the metaphase chromosome and the DOP-PCR products dotted on the nylon membrane to map rice field eel rRNA genes. The DOP-PCR products with the positive Dot hybridization results labeled by Biotin-11-dUTP and ŚÁ-32 P-dCTP respectively were hybridized with the genomic DNA digested by different enzymes and the metaphase chromosome to further confirm its specialities. The hybridization results show that: (1) Chromosome specimen of meiosis˘ńdiakinesis is the ideal material for single chromosome microisolation; (2) The sizes of DOP-PCR products range from 200 bp to 1 000 bp, averagely 600 bp;(3) The single chromosome obtained in the study is rice field eel chromosome 3;(4) Compared with micro-manipulator and micro-laser beam, the methods developed in the study can be more widely used in ordinary laboratory for requiring no expensive instrument. In the paper, we also discuss the future application of microisolation techniques in the study of chromosome evolution, gene mapping and conserved synteny in fish. [Acta Zoologica Sinica 48(1):114-120,2002]

Key words Rice field eels (Monopterus albus), Single chromosome, Microisolation, DOP-PCR, Southern hybridization, FISH, Dot hybridization

*E-mail:davidyuqx@263.net

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Cloning and sequence analysis of a cdna fragment encoding acetylcholinesterase in cotton bollworm (Helicoverpa armigera)

REN Xiao-Xia^* HAN Zhao-Jun WANG Yin-Chang

(Key Lab of Monitoring and Management of Plant Disease and Insects, Ministry of Agriculture,

Nanjing Agricultural University, Nanjing 210095, China)

Abstract Acetylcholinesterase (AChE) is the target of organophosphate and carbamate pesticides. Organophosphate resistance is worldspread in the cotton bollwormŁŰHelicoverpa armigera (H¨šbner)ŁÝ. With the degenerate primers we amplified a 281 bp cDNA fragment of acetylcholinesterase (AChE) gene in H. armigera by reverse transcription polymerase chain reaction (RT-PCR) method using total RNA extracted from 4th larva as the template. The cDNA fragment was inserted into pGEMT vector and then cloned. The deduced amino acid sequence of AChE consisted of 94 residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shares high identity with AChE gene from other published insects and animals. The acquired sequence had 84%,79%,74%,70%,70%,72%,68%,61%,55% and 57% of amino acid residues identical to those of Leptinotarsa decemlineata(L.d.), Nephotettix cincticeps(N.c.),Anopheles stephensi(A.s.), Aedes aegypti(A.a.), Lucilia cuprina(L.c.), Drosophila melanogaster(D.m.), Musca domestica(M.d.), Meloidogyne incognita(M.i.), Torpedo californica(T.c.) and Gallus gallus(G.g.), respectively. All these results firmly established that the amplified cDNA fragment was the partial sequence of AChE gene in H. armigera. This is the first report of partial cDNA sequence of AChE in H. armigera. [Acta Zoologica Sinica 48(1):121-124,2002]

Key words Cotton bollworm (Helicoverpa armigera), RT-PCR, Acetylcholinesterase gene, cDNA cloning, Sequence analysis

* E-mail: xiaoxiaren@hotmail.com

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Changes in distribution of LCA and Cona binding glycoconjugates on

cricket (Teleogryllus emma)sperm surface

XI Geng-Si^˘Ů YANG Yue-Hong^˘Ú ZHENG Zhe-Min^˘Ů

(˘Ů Zoology Institute, Life Science College, Shaanxi Normal University, Xi'an 710062, China)

(˘Ú Basic Medical Institute, Zhejiang University, Hangzhou 310006, China)

Abstract LCA and ConA-binding glycoconjugates on cricket (Teleogryllus emma) sperm surface were detected with fluorescence microscope after FITC labelling for better understanding of the distribution of glycoconjugates during spermatogenesis and spermiogenesis.

FITC-LCA and FITC-ConA were bound on the spermatocytes, and their distribution changes in the process of spermiogenesis were observed . In the testis sperm, FITC-LCA and FITC-ConA were mainly bound on the head and neck region. That is different from the mark pattern of spermatophore sperm, in which the nucleus, neck region and front of the tail showed obvious fluorescence mark, especially the acrosome complex and neck region exhibited stronger mark.

The mark patterns of FITC-LCA and FITC-ConA were similar, though the former was distinctly clearer than the latter. But a little difference still exists in both of them. For example in the ninth stage of spermatid, FITC-LCA mark is located on the spermatid head and neck region, and FITC-ConA mark on the spermatid head, neck and front of the tail region. When fixed germ cells were treated with PBS instead of lectin solution, or fixed cells were incubated with lectin solution, which have been treated with 0.1 mol/L specific sugar inhibitor, i.e.

ŚÁ-D-mannose for FITC-LCA and FITC-ConA, andŚÁ-D-glucose for FITC-ConA, no mark was observed on the cells. Those results indicate that FITC-LCA conjugated glycoconjugates has theŚÁ-D- mannose residue, and FITC-ConA conjugated glycoconjugates has theŚÁ-D-mannose andŚÁ-D-glucose residue.

The investigations show that the changes in glycoconjugates distribution of cricket sperm is similar to those of other insects and mammals. The evidence exhibit that a common rule of the glycoconjugates distribution on the sperm surface is followed by most of animal sperm which may relate to the function of sperm physiology. [Acta Zoologica Sinica 48(1):125-130,2002]

Key words Cricket (Teleogryllus emma), Sperm, Agglutinin, Glycoconjugates

* E-mail:hsh@xjtu01.xjtu.edu.cn

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Variability of the complete mitochondrial control region of striped dolphins (Stenella coeruleualba)

YANG Guang* REN Wen-Hua NIU Ming-Hua ZHOU Kai-Ya

(College of Life Sciences, Nanjing Normal University, Nanjing 210097, China)

Abstract The mitochondial control regions from 13 striped dolphins (Stenella coeruleualba), seven from the southern Taiwan Straits (Dongshan waters) and the other six from the southern East China Sea (Pingtan waters), were amplified by using polymerase chain reaction (PCR) technique. The complete nucleotide composition of this region, ranged from 914 to 917 base pairs (bp), were determined by an ABI 310 DNA automated sequencing system to address the sequence variability. 42 variable sites, including 36 transitions, three transversions, and three indels (insertion/deletion) were found. Of the variable sites, 26 were parsimonious informative sites. Totally 11 haplotypes were identified. Of the haplotypes, six were found exclusively in Dongshan waters and the rest five only in Pingtan waters. No common haplotype was shared, and two fixed site differences were detected between haplotypes from two waters. The haplotype diversty and nucleotide diversity were high for both waters, which were 1.45%ĄŔ0.79% and 0.95 in Dongshan waters, and 1.06%ĄŔ0.51% and 0.93 in Pingtan waters respectively. A phylogenetic tree was reconstructed by using neighbor-joining (NJ) method in MEGA (Molecular Evolutionary Genetic Analysis) package. In the tree, haplotypes from the Pingtan waters constituted a monophyletic clade, whereas haplotypes from the Dongshan waters did not. Analysis of molecular variance, which was conducted by computer software AMOVA, showed an significant population differentiation and genetic structure (Śľ_st=0.35, PŁź0.01) between Dongshan waters and Pingtan waters. The haplotype diversity and nucleotide diversity of striped dolphins were higher than those found in the sympatric finless porpoise (Neophocaena phocaenoides) populations, and also higher than those of other cetacean species reported previously in other waters, including some species having high level of genetic diversity, e.g. harbor porpoises (Phocoena phocoena), humpback whales (Megaptera novaeangliae) and bottlenose dolphins (Tursiops spp.) etc. This suggested that the striped dolphins had a relatively higher genetic diversity. [Acta Zoologica Sinica 48(1):131-134,2002]

Key words Striped dolphin (Stenella coeruleualba), Mitochondrial control region, Sequence variability

*E-mail: gyang@njnu.edu.cn

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Genomic comparative analysis between the human Y chromosome-specific

heterochromatin region and fish genomes

YI Mei-Sheng YU Qi-Xing*ŞŞGUO Yi-Qing LIU Li ZHOU Rong-Jia

(College of Life Sciences, Wuhan University, Wuhan 430072,China)

Abstract The repeated DNA in the genomes of three fish species (Dario rerio, Monopterus albus and Mastacembelus aculeatus), were compared with those in human Y chromosome-specific heterochromatin by genomic comparative in situ hybridization. The results showed that the repeated DNA in the genomes of three fish species could not hybridize with those in human Y chromosome-specific heterochromatin region and the human Y chromosome had its specific repeated DNA sequence. Some problems about the evolution of the repeated DNA in human Y chromosome-specific heterochromatin region were discussed. [Acta Zoologica Sinica 48(1):135-138,2002]

Key words Human, Y chromosome, Heterochromatin, Fish genomes, Genomic comparison, Fluorescence in situ hybridization

*E-mail:yuqixing@yahoo.com

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