ACTA

ZOOLOGICA

SINICA

ACTA ZOOLOGICA SINICA

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Vol. 47  No.2        Apr. 2001

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ABSTRACT

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The southern boundary of the palaearctic realmin china and adjacent countries

Robert S. Hoffmann

(Division of Mammals, Department of Systematic Biology, National Museum of Natural History

Smithsonian Institution, Washington D. C. , USA ) 

Abstract  A method is described for determining objectively the boundaries of biogeographic regions of various magnitudes through site sampling. Species presence or absence at selected sites in the vicinity of proposed boundaries is combined with assignment of those species to alternative regions based on the concepts of distributional range, and phylogenetic/ecological affinity. Iterative sampling permits definition of both linear boundaries and broader transition zones between biogeographic regions. [Acta Zoologica Sinica  47(2):121-131,2001, In English]

Key words  Palaearctic, Indomalaya, Biogeography, Phylogeny, Range, Affinity

E-mail: Hoffmann.Robert@nmnh.si.edu

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Analysis on immunoglobulin induced by inoculation of Aeromonas  hydrophila  in grass carp ( Ctenopharyngoden idellus Cuvier ET Valencinnes)

LI Ya-Nan

(Department of Biotechnology, College of Life Sciences, Zhejiang University, Hangzhou 310012, China ) 

Abstract  Grass carp (Ctenopharyngoden idellus ) is an economically important fresh water fish in China. Although a significant amount of effort has been devoted to study the development of vaccines against infectious diseases in this fish, little was known about the biological features of its immunoglobulin. We induced the immune response in Grass carp with the vaccine consisting of inactivated bacterial Aeromonas hydrohila. The increased level of immunoglobulin in response to the vaccination challenge was observed. The immunological and biochemical features of the immunoglobulin protein were characterized.

We used one-year fish C. idellus of 100 to 150 grams of body weight. Each fish in the experimental group received A. hydrophila vaccine (strain TPS-30 one billion inactivated bacteria per ml) at a dose of 0.5 ml. Vaccine solution was injected into the fish muscle under the back fins. Controlled group of fish received no treatment, but were raised for the same period of time. Sever and 14 days after the first injection, blood was collected from the tail arteries and sera was separated. The activity and specificity of immunoglobulin in fish sera were tested with standard agglutination assays conventionally. Immunoglobulin proteins were isolated from fish sera either by the fractionation of saturated ammonium sulfate or by Sephadex G-200 gel column chromatography and by high pH discontinuous polyacrylamide gel electrophoresis.

Antigen-specific agglutination testing revealed that the highest antibody titer was present in the fraction precipitated with 35 % saturated ammonium sulfate, or in the first elution peak of chromatography from the first collected tube. The results by electrophoresis indicated that the components having antigen-specific agglutination were mainly located on the B band of the electrophoretic gel. After being treated with 2-mercaptoethanol, the agglutination acvtivity for components dropped greatly.

By comparison with non-immunized control group, electrophoresis under non-reducing conditions identified an extra B-band with Mr = 309 kD in the immunized samples. When this B-band was isolated and subjected to electrophoresis under denaturing conditions, two separate polypeptides were detected with Mr of 52 kD and 26 kD, respectively, which should be identical of the molecular weights of fish IgG heavy and light chains. Therefore, we conclude that the A. hydrophila induced B-bind likely represents a fish IgM class polymerized with four IgG molecules.  [Acta Zoologica Sinica  47(2):132-138, 2001]

Key words   Grass carp ( Ctenopharyngoden idellus ), Aeromonas hydrophila (AH), Immune serum, Immunoglobulin, Agglutination

E-mail: yananli@21cn.com

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Phylogeny of ten species of chinese gekkonid lizards (Gekkonidae:Lacertilia) inferred from

12 s rDNA DNA sequences

HAN De-Min  ZHOU Kai-Ya  WANG Yi-Ouan

( Institute of Genetic Resources, Nanjing Normal University, Nanjing 210097, China ) 

Abstract  The studies on phylogenetic relationships of gekkonid lizards have been largely based on morphological characters before, including the structure of digits and internal anatomy. In China, the phylogeny among some gekkonid lizards based on karyotype, nucleolar organizer and LDH (lactic dehydrogenase) isozyme was reported. However, nucleotide sequence data have not yet been employed to study the phylogeny of gekkonid lizards. In the present study, phylogenetic relationship among 10 Chinese gekkonid lizard species was inferred based on sequences of 12S rRNA gene fragment.

1. Material and method

Total DNA was extracted from about 50 mg of the muscle tissue from 10 gekkonid lizards ( Gekko japonicus,  G. swinhonis, G. hokouensis,  G. gecko,  Cyrtopodion elongatus ,  C. russowi ,  Teratoscincus roborowskii, Hemidactylus bowringii, H. frenatus and Gehyra rnutilata ) collected in China. The samples were either frozen ( - 20¡æ ), or ethanol or formalin preserved ( Table 1 ). Two primers L1091 and H1478 (Kocher et al., 1989) were used for amplification and sequencing. Sequencing were performed manually using silver-staining technique or automatically in an ABI Prism 310 Genetic Analyzer.

All sequences were aligned with the program of Clustal w (Thompson et al., 1994). The DNA sequences were analyzed with the MEGA program (1.02 edition). For distance estimation, we deleted sites with gaps in each pairwise comparison (Table 2). The phylogenetic trees of NJ, UPGMA and MP were reconstructed with the same program based upon Juke-Cantor distance, with bootstrap test (1 000 replications) (Fig. 1).

Result and discussion

The sequences have been deposited in GenBank (accession numbers: AF236819 and Aligned sequences of 421 available base pairs (including 201 variant sites) were obtained AF323510¡«323518) The percentage of sequence divergence (sequence difference / (sequence size) among the five genera was 0.228¡«0.282, within the genera 0.005¡«0.263. Tricotomous clusters were obtained by those methods in all three phylogenetic trees: The first cluster contained the sister group of G. mutilata and G. gecko; The second cluster contained the monophyletic lineage of other three species of Gekko; The third cluster contained the remaining five species.

All three phylogenetic trees reconstructed indicate that G. mutilata is closely related to the genus Gekko, especially G. gecko. This result is in agreement with the traditional systematic classification.

The feet of the Cyrtopodion (=Cyrtodactylus ) geckos are of creep moving type, which adapts to the desert habitat. Which have with dilated toes and hyperextended digits, the feet are adapted to arboreal life. Though the two genera differ not only in morphology but also in habitat use, they cluster in all three phylogenetic trees. This outcome supports the result of Russell (1976), who considered the muscle, bone and tendon systems of the feet and digits developed along with the scansors as well as setae and pointed out that the antepenultimate phalanxes of both Cyrtodactylus and Hemidactylus were very small and erect and the extremely reduced nature of this particular phalanx was unique to the cyrtodactyl-hemidactyl linege.

The results of cladistic study of the Gekkonoidea (Kluge, 1987) indicated that Teratoscincus was the sister-lineage to the remaining taxa of Gekkonidae, and a new subfamily, Teratoscincinae, should be established. Our result did not support Kluge's hypothesis. The genetic distances between T. roborowskii and other gekkonid lizards based upon the percentage divergence of the DNA sequences was similar to those among the other geckos. T. roborowskii was a sister group to the clade constituted by the two species of Cyrtopodion in UPGMA tree, or clustered with Cyrtopodion and Hernidactylus in a monophyletic group in MP tree.

There were studies on karytype, nucleolar organizer and LDH isozyme of G.japonicus, G. swinhonis and G. hokouensis (Chen et al., 1986; Guo et al., 1995; Shen et al., 1996). However, the phylogenetic relationships of these Gekko speices have not yet been resolved. Our molecular data indicated that G. hokouensis and G. swinhonis clustered as the sister group of G.japonicus. The molecular data also supported the validity of G. hokouensis.

Although G.gecko was included in the clade constituted by the other three species of Gekko in MP tree, the sequence divergences of 12S rRNA gene fragment between G. gecko and the other Gekko species (P = 0. 259¡«0.263) were higher than those among different genera of gekkonid lizards. Additionally, it clustered with G. mutilata in the NJ and UPGMA trees. Therefore, the phylogenetic position of G. gecko remains unresolved. [Acta Zoologica Sinica  47(2):139-144,2001]

Key words  Gekkonidae, 12S rRNA gene, Molecular phylogeny

E-mail: kyzhounj@jlonline.com

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Sequencing SRY  gene of  Muntiacus  muntiak  vaginalis  and

a phylogenetic comparison with some of Artiodactyla

GUO Jin-Hu^*  SHAN Xiang-Nian  CHANG Qing  YU Duo-Wei

(Department of Biology, Nanjing Normal University, Nanjing 210097, China)

WU Jing-Yang

(Department of Biology, Nanjing Railway Medical College, Nanjing 210009, China)   

Abstract  With human SRY gene primers of the conserved region, the SRY gene fragment of M. m. vaginalis homologous to human SRY was amplified by PCR. In male M. m. vaginalis, a gene fragment of 221 bp homologous to human SRY was identified by DNA dot hybridization. By ligating pGEM^® T Vector with the PCR products of SRY gene of M. m. vaginalis and transforming the bacteria DH5a and clony hybridization with human SRY probe, the positive clone with M. m. vaginalis SRY gene fragment was identified gene, the SRY fragment of this clone was sequenced. The sequences identities of SRY gene fragments of all animals of Artiodactyla available from GenBank were compared with the M. m. vaginalis SRY gene fragment. The phylogenetic tree was constructed and analyzed by UPGMA method and it was studied at the level of taxonomics and evolution. [Acta Zoologica Sinica  47(2):145-149, 2001]

Key words  M. m. vaginalis, SRY gene, Sex determination, Artiodactyla, DNA sequence, Phylogeny

E-mail: golden-tiger@990.net

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Effects of photoperiod on growth and development of pupsin Brandt's voles ( Microtus brandti)

LIU Wei^*  FANG Ji-Ming

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

Abstract  Most of small mammals, including Brandt's in temperate zone, voles( Microtus brandti ), reproduce and rear offspring during seasons with mild environmental conditions and abundant food. It is assumed that animals bearing offspring during the breeding season increase their reproductive fitness and that reproduction at other periods results in fewer surviving progeny and possible energy crises for the parents. It, so that, is important that the environmental cues employed by animals to forecast the optimal breeding season are termed the proximate factors or cues of seasonal breeding. Photoperiod is the most common environmental factor used by north temperate mammals for timing reproduction. Brandt's voles' puberty and somatic growth are delayed by as many as 20 weeks in offspring late compared with early in the natural breeding season. The present study was the first measurement of some somatic and reproductive traits by comparing Brandt's voles pups born and housed in long (LD:14L: 10D) versus short (SD:10L: 14D) day photoperiod from birth to 28 days age or in 60 days age (The parents of LD and SD pups respectively housed exceeded 4 weeks in long and short day photoperiod after pairing). The goal was to determine whether photoperiod affect rate of growth and development of Brandt's voles offspring.

The results showed that photoperiod had no significant effect on the litter size at birth (t = 1.21, df= 18,P>0.05) ,the litter size at weaning(t = 1.43,df= 18,P>0.05) and the mean survival rate of pups per litter at weaning( t = 1.38, df = 18, P > 0.05). Compared with SD pups, however, it is during eye-opening period (postnatal day 10¡«14) or after eye-opening (exceeded postnatal day 14) that LD offspring matured more rapidly with respect to body weight (W), body length (L) and relative fatness (Kwl = W/L ) with the development of pups. Additionally, gonad-somatic index [GSI' = sin^-1 (w_g/w)^1/2 ¡Á100% , W_g: gonad weight, W: body weight], including paired testes index (GSI_t '), paired epididymal index (GSI_e ') and seminal vesicle index (GSI_v'), were greater for LD 60-day-age male pups than those for SD group(t-test, GSI_t', t = 3.309, df=14,P<0.01;GSI_e',t =3.622,df= 14,P<0.01;GSI_v', t =3.379, df= 14, P<0.01). In contrast, the pelage depth and under hair length of 60-day-age SD offspring were thicker or longer than those of LD ones( t-test, t = - 5.185, df = 14, P < 0.01; t = - 2. 415, df = 14, P < 0.05, respectively). The present study indicated that several traits of vole pups were influenced by postnatel photoperiod or dam's photoperiod history. The results suggest that photoperiod or day-length induce the seasonal changes of growth and development of Brandt's voles pups, and likely facilitates adaptation of voles to seasonally varying environmental condition. [Acta Zoologica Sinica  47(2):150-157, 2001]

Key words  Brandt's voles (Microtus brandti), Photoperiod, Body weight, Body length, Relative fatness, Gonad development, Pelage

*E-mail: 1457@263.net

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Population structur of the Przewalski's gazelle around the Qinghai lake, China

JIANG Zhi-Gang^*  LI Di-Qiang  WANG Zu-Wang

(Institute of Zoology, The Chinese Academy of Sciences, Beijing 100080, China)

ZHU Sheng-Wu  WEI Wang-Hong

(Northwest Plateau Institute of Biology, The Chinese Academy of Sciences, Xining 810001, China) 

Abstract  The Przewalski's gazelle (Procapra przewalskii ) is endemic to China. The historical distribution of the Przewalski's gazelle covered an area in central and northwest China. It was reported that there were only some 350 Przewalski' s gazelles lived around the Qinghai lake in mid-1980's. The critical status of the gazelle has aroused attention worldwide. The Species Surviving Commission of World Conservation Union ranked the status of the species as CR (critically endangered) in the IUCN 1996 Red List of Endangered Species. However, the ecology and status of the gazelle have never been documented thoroughly. To rescue this unique gazelle, we need to understand its population ecology and to find out the proximate causes accounting for the population declines in the species. We formed a research team in 1994 to monitor the behavior and population dynamics of the Przewalski's gazelle around Qinghai lake. New evidence indicated that Bird island population is now at the brink of extinction, while the other two populations are declining. In 1997, the Hudong-Ketu population consisted of 29£¥ males, 50 % females and 21% juveniles; the Yuanzhe population consisted of 29 % males, 43 % of females and 27 % of juveniles. Bird island population is threatened by habitat loss, desertification, wolf predation and poaching. Decline of Hudong-Ketu population in 1997 is due primarily to wolf predation; compared with the Yuanzhe population, in which wolf predation was absent, the ratio of post reproductive season surviving juveniles to adult females was 20 % lower. When the wolves were hunted down in 1998, the gazelle population increased. Yuanzhe population is facing lose of the habitat and poaching. Human activity and high juvenile mortality are major threats to survival of the Przewalski's gazelle around Qinghai lake. Przewalski's gazelle is a flagship species in the eastern part of the Qinghai-Tibetan plateau, and also an indicating species of the ecotone between typical steppe and desert. Przewalski's gazelle lives in a habitat with special biodiversity. We propose the following conservation measures: (1) to establish a special reserve for the Przewalski's gazelle; (2) To establish a habitat corridor that links the Hudong-Ketu and Yuanzhe populations; (3) To study the wolf-gazelle relationship and to control the density of wolf in the area; (4) To actively search for other plausible living Przewalski's gazelle population in other regions and to look for ex situ conservation sites. [Acta Zoologica Sinica  47(2):158-162, 2001]

Key words  Przewalski's gazelle(Procapra przewalskii ), Endangered animals, Population dynamics

*E-mail: jiangzg@panda.ioz.ac.cn

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Immune response of soft-shelled turtle (Trionyx sinensis ) to bacterin T3

YANG Xian-Le^*

(Key Lab. of Ecology and Physiology in Aquacutrure, Ministry of Agriculture, Shanghai Fisheries University,

Shanghai 200090, China)

ZHOU Jian-Guang  AI Xiao-Fei  KE Fu-EN

(Yangtze River Fisheries Institute, Jiangzhou 434000, Hubei, China) 

Abstract  The immune response in soft-shelled turtle (Trionyx sinensis) immunized with bacterin T3, was determined with indirect agglutination titer (IAT) and percent relative protection (PRP). In the vaccinated turtle, the strong immune response to bacterin T3 was produced. Six groups of 1-year-old turtles and 19 groups of 2-years-old turtles were respectively vaccinated by bacterin T3. At the 30^th day, the PRP and IAT of the 1-year-old turtles were to 61.8 ¡À 11.2 (6) % and 403.3 ¡À 165.4 (6), yet the corresponding values of 2-years-old turtles were 82.6 ¡À 15.2(19) % and 1 978.1 ¡À 716.4(19) respectively. According to the test, the PRP and IAT of 2-years-old turtles were higher than those of 1-year-old turtles. The study demonstrated that the effective phase of immune response to bacterin T3 was 3 days. At the 20^th day, the IAT value is up to 1768.0 ¡À 447.6(4). Which maintained for about 10 days and decreased to the initial level on the 60^th day. The results proved that the immunologic memory of the turtles was strong. The secondary enhanced immunization began at the 30^th day of the primary immunization. The IAT increased quickly and was 5 760.0 ¡À 2 790.0(4) at the 16^th day of the secondary immunization, which reached the second peak value that was nine times as high as the primary peak value. The IAT decreased to 4 266.7 ¡À 1 478.0(3) at the 30^th day, 2 986.5 ¡À 1 955.3(3) at the 60^th day, and 1280.0 ¡À0(2) at the 70^th day. The result still has no significant difference compared to the peak value of the secondary immunization (0.2< P < 0.3), yet having significantly higher than that of immunization (0.01 < P < 0.02). From these facts, we can concluded that the decreasing speed of antibody of the secondary immunization was clearly slower than increasing speed. The result suggests that the character of immune response of Soft-shelled turtle lies between fish and bird, but it is more like the latter. [Acta Zoologica Sinica  47(2):163-169, 2001]

Key words  Soft-shelled turtle ( Trionysc sinensis ), Bacterin T3, Immune response

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

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Promotive effects of GABA on acid secretion from isolated mouse stomach in vitro

XU Xiang-Gui^*  YANG Zai-Fu  HUANG Shen-He  YI Xiao-Hong

( Department of Biology, Suzhou Railway Teachers' College, Suzhou 215009, Jiangsu, China ) 

Abstract  To explore the effects and the mechanism of¦Ã-aminobutyric acid (GABA) on gastric acid secretion (GAS) of isolated mouse stomach with perfused lumen, 12 cm H₂0 column intragastric pressure-provided. Whole stomach preparations from mice were incubated in buffer at 37¡æ in vitro, and perfusate was measured for pH with a pHS-3 type pH meter. The results shown that GABA (1 ¡«10¡Á10^-7 mol/L) and baelofen (Bac) ( 0.6¡«9.6¡Á10^-7mol/L) significantly promoted GAS in a concentration-dependent manner, whereas cimetidine (Cim) (2¡«20¡Á10^-7mol/L) potently inhibited GAS in the same manner. Picrotoxin (Pic) (3 x 10-7mol/L) did not affect basal gastric acid secretion (BGAS) and the promotive effect of GABA on GAS. Phaclofen (Phac) (0.6¡Á10^-7mol/L) completely inhibited the promotive effect of GABA on GAS. Cim did not completely eliminate the promotive effects of GABA and Bae on GAS in isolated mouse stomach. Above results suggest that in mouse, GABA may promote GAS in isolated stomach, possibly by affecting GABAB receptors on both cholinergic neurons and non-neuronal cells, such as parietal cells and some endocrine cells. GABA may stimulate parietal cells to secrete acid directly or indirectly. [Acta Zoologica Sinica  47(2):170-175, 2001]

Key words  Isolated mouse stomach, ¦Ã-aminobutyric acid, Gastric acid secretion, GABAB receptor

E-mail: tdsy@suda.edu.cn

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In vivo and in vitro immunological localization of DHEA in rat submaxillary gland

YAO Bing¢Ù  HUANG Wei-QuanO  ZHANG Chong-Li¢Ú  WAN Jiang-Hua¢Ù

(¢Ù Department of Histology and Embryology, The Fourth Military Medical University, Xi'an 710032, China )

(¢ÚState key Laboratory of Reproductive Biology, Institute of Zoology,

The Chinese Academy of Sciences, Beijing 100080, China )    

Abstract  The submaxillary is situated below the floor of the mouth just beneath the body of the mandible. The secretory portion of the gland is composed of tubulo-alveolar acini of the mucous and serous types, their secretion contains many enzyme which hydrolyzes the polysaccharide starch into the disaccharides maltose and isomaltose. Previous studies have described that rat submaxillary could not only secret digestive fluid but also synthesize many biological activated substance, such as nerve growth factor (NGF), epithelial growth factor (EGF), retina nodal cell nerve induced factor, 5-HT and GnRH etc. DHEA is the precursor of sexual hormone, this substance and its combination can be situated in the rat brain whose suprarenal or testis is ablated. Recent studies have found that DHEA could also exist in adrenal gland, uterus and testis. However, whether DHEA could exist in submaxillary remains unknown. This study was undertaken to demonstrate the localization of DHEA in submaxillary gland and the epithelial cells from submaxillary gland cultured in serum free medium. The paraffin and culture sections were washed by PBS (pH 7.1, 0.01 mol/L)for five minutes three times, and incubated in methanol-H202 for 20 min to remove endogenous peroxidase and then washed by PBS(pH7.1, 0.01 mol/L) 5 minutes three times. They were then stained according to the immunohistochemical ABC method. Tissue sections were incubated at 4¡æ for 24 hr in the primary antibodies of rabbit anti-DHEA antibody and rabbit anti-keratin antibody(l:100 dilution),respectively. The secondary antibody, biotin-labeled horse anti-rabbit IgG(1:200 dilution) was incubated at room temperature for one hour and ABC complex(l:100 dilution) incubated at room temperature for 30 min. The negative control tissue sections were incubated with normal rabbit serum and phosphate buffer salt as primary antibodies. The results showed that the epithelial cells cultured in serum free medium exhibited keratin positive reaction, the positive substance was distributed in cytoplasm and the nuclei were negative. The glandular epithelial cells of serous acinus and all gland ducts produced DHEA positive immunoreaction. Similarly, the epithelial cells of submaxillary gland cultured in serum free medium showed DHEA immunoreactivity. The positive immunoreactive substance was distributed in the cytoplasm with negative nuclei. In control test, the epithelial cells of serous acinus and of excretory duct in rat submaxillary gland showed negative immunoreaction. The results indicated that the cells cultured in serum free medium were submaxillary gland epithelial cells, and these cells also showed DHEA positive reaction, which suggeste that the DHEA may be synthesized in the serous acinus of submaxillary gland of rat and it may play an important role in the regulation of digestive function. [Acta Zoologica Sinica  47(2):176-178, 2001]

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In situ hybridization detection of cholecystokinin mrna in cerebral

cortex of audiogenic seizure-prone rats

NI Hong^¢Ù*  WANG Shou-Biao^¢Ú  XU Luo^¢Ú  TANG Ming^¢Ú

(¢Ù Department of Neurology, Children' Hospital, College of Medicine,

Zhejiang University, Hangzhou 310003, China )

(¢ÚDepartment of Physiology, College of Medicine, Oingdao University, Oingdao 266021, Shandong , China ) 

Abstract  Using in situ hybridization method, we examined CCK mRNA positive signal in temporal cerebral cortex of audiogenic seizure-prone rats(P77 PMC)before and after a single or multiple seizures. The results were as follows: (1) CCKmRNA levels were elevated significantly in temporal cortex after a single or multiple seizures; (2) CCK mRNA level in multiple consecutive seizure rats was lower than that in a single seizure rats. The increased CCK mRNA positive signal indicates that CCK mRNA in cerebral cortex may play a role in seizure-associated processes. The decreased CCK mRNA levels after multiple consecutive seizures may suggest differential regulation of CCK transcription by single and multiple seizures. [Acta Zoologica Sinica  47(2):179-181, 2001]

Key words  Cholecystokinin, mRNA, In situ hybridization, Seizure, P₇₇PMC, Cerebral cortex

 *E-mail: nyr@yeah.netmailto:nyr@yeah.net

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Cytological changes of sperm and egg nuclei during  fertilization in  Penaeus chinensis  

KANG Xian-Jiang^*  WANG Suo-An

(Department of Biology, Hebei University, Baoding 071002, China)

DU Nan-Shan  LAI Wei

(Department of Biology, East China Normal University , Shanghai 200062, Chnia )    

Abstract  When a female prawn spawns, stored spermatozoa were released from the thelycum and mixed with the eggs by the beating of the pleopods. The spermatozoa binded the egg with their spike top by random. At the time when the jelly coat was formed, and before the first polar body was emitted, the sperm penetrated into the egg by crossing the plasma membrane. After the sperm penetrated into the egg, its nucleus was transformed into a big male pronucleus.

The eggs of the prawn at spawning were in meiotic metaphase I stage, and the meiotic apparatus was positioned parallel to the egg surface. After the egg was activated, the meiotic apparatus rotated and positioned perpendicularly to the egg surface. The chromosomes drawn by the spindle microtubule separated. The chromatin near the plasma membrane was surrounded and formed the first polar body. After fertilization membrane was lifted, the secondary egg emitted the second polar body into the perivitelline space.

Following the extrusion of the second polar body, the haploid female chromatin formed a female pronucleus. The male pronucleus formation was earlier than the female's. The male and female pronuclei moved to the center of the egg, and matched into a association nucleus. The chromosomes of the first mitotic apparatus separated 50 min after fertilization and 2-cell embryo was formed by plasma membrane invagination. [Acta Zoologica Sinica  47(2):182-189, 2001]

Key words  Penaeus chinensis, Fertilization, Sperm nucleus, Egg nucleus, Cytology

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

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Changes of the morphological and chemical substances of the egg of

Pirata piraticus during embryonic development

PENG Yu^¢Ù*  HU Cui^¢Ú  ZHAO Jing-Zhao^¢Ù  CHEN Jian^¢Ù

(¢Ù School of Life Science, Hubei University, Wuhan 430062, China)

(¢Ú Department of Plant Protection, Zhejiang University, Hangzhou 310029, China)    

Abstract  We studied the morphological characteristics and the changes of biochemical substances in the embryos during embryonic development in Pirata piraticus. At 28¡æ, embryonic development took 144 hr, with 42 hr at early embryo stage, 33 hr at metameric phase, 27 hr at inversion phase and 42 hr at pre-larva phase.

The early embryo stage ranges from the Beginning of the first cleavage to blastodisc stage. During this period, the nucleus division occurred in the center of the zygocyte firstly and then these divided nuclei migrated into the edge where they were surrounded by cytoplasm thus formed into the blasterderm. About 23 hours later, the blastula contracted. In the meantime, most blastocytes aggregated in the upper of the embryos and came to hlastodisc. Afterwards, these cells differentiated into three kinds of shapes and located in different position. About 30 hours after oviposition, the cumulus was formed.

During the metameric phase, the blastodisc prolonged and formed a lycotropal germinal band, which gradually differentiated into head lobe, caudal lobe and the middle five somites and made further development of the primodia of chelicera, taster and walking legs.

About 72 hours after oviposition, in the middle of the germinal band, a median groove appeared and gradually widened, which suggested that the embryo developed into the inversion stage. In this stage, embryonic lateral plate moved towards the dorsal-lateral surface and concrescent in the dorsal line. The walking legs differentiated seven segments. The filators were developed from the rudiments of appendages in the fourth and fifth somites. At the end of this stage, the embryos had the typical morphological characters of spiders.

The heart and stomach were developed in larva phase and the larva can move slowly. Three pairs of filators came to be formed in 11- 12 days following oviposition. The first instar larva molted one time thus enter the second instar stage. The spider in larva phase before the second instar stage couldn't catch foods, so they supported themselves By yolk.

With the development of the spider, wet weight of egg, and water, fat contents of embryos started to decrease at about 24 hours post oviposition. The contents of protein began to decline at 48 hours after oviposition. However, the content of carbohydrate had little change. [Acta Zoologica Sinica  47(2):190-195, 2001]

Key words  Pirata piraticus, Embryonic development, Egg

E-mail: pengyu@smtp.njau.edu.cn

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EXPRESSION AND DEVELOPMENT OF RAT PREPROTHYROTROPIN-RELEASING HORMONE  AND ITS RECEPTORS IN THE TESTI OF RAT

LI Zhen^¢Ù*  LIU Xin-Ping^¢Ú  ZHANG Yuan-Qiang^¢Ù  XU Ruo-Jun^¢Û

(¢Ù Department of Histology and Embryology, The Fourth Military Medical University, Xi'an 710032, China )

(¢ÚDepartment of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi'an 710032, China )

(¢Û Department of Zoology, University of Hong Kong, Hong Kong)    

Abstract  Recently, testis has been known as a source of numerous hypothalamic neuropeptides, including thyrotrophin-releasing hormone (TRH). We have found that TRH receptor (TRH-R) mRNA was identified in leydig cells of mouse testis using in situ hybridization. In order to investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R) in rat testis, and to study their function in reproduction and development, we designed oligonucleotide primers from the sequences of rat hypothalamus prepro (pp)TRH and pituitary TRH-R cDNA for reverse transcription-polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed and expression carried out in E. coli DH5a cells. Based on the developmental pattern of sexual maturation of the rat, ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats of 8, 15, 20, 35, 60 and 90 days of age by real time quantitative RT-PCR. SDS-PAGE showed that the supernatant of PGEX-ppTRH demonstrated a new obvious protein band of 60 kD and that of PGEX-TRH-R demonstrated 40 kD, which were of the same as the expected size of fusional proteins respectively. The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). PpTRH and TRH mRNA signals were detected on day 15 and increased progressively on day 20, 35, 60 and 90. These Results suggest that rat testis specifically express TRH and TRH-R, and the transcription of ppTRH and TRH-R gene in the rat testis is development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on physiological significance of TRH and TRH-R expression in rat testis. [Acta Zoologica Sinica  47(2):132-138, 2001]

Key words  Rat, Testis, Preprothyrotrophin-releasing hormone, Thyrotrophin-releasing hormone, RT-PCR

E-mail: zhen.li@263.net

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Effect of gonadotropinson  in  vitro  maturation of oocyte and cumulus

expansion in kunming white mouse

CHEN Yong  XIA Guo-Liang  SU You-Qiang  WANG Hai-Bin  HE Chen

( College of Biological Sciences, China Agricultural University, Beijing 100094, China )     

Abstract  The present experiments were conducted to evaluate the effect of gonadotropins on Kunming white mouse oocyte maturation and the relationship between cumulus cell expansion and oocyte maturation in vitro. The cumulus cell-enclosed oocytes (CEO) were collected from gonadotropins-stimulated Kunming white mouse ovaries and were cultured for 24 hr in 4 mmol/L hypoxthantine (HX)-supplemented medium (HX-medium) and treated with different agents in the following designs: (1) CEO were treated with FSH (10¡«500 IU/L) to find the optimal dosage of FSH on the oocyte maturation; (2) CEO were treated with 100 IU/L FSH for the different time (0, 10, 30, 60 and 120 min) and then transferred to a new HX-medium for the further culture to figure out the time effect of FSH on oocyte maturation and cumulus cell expansion; (3) CEO were treated with different dose of hCG (0¡«400 IU/L) or hCG plus FSH to evaluate the effect of hCG on oocyte maturation and cumulus cell expansion. The results revealed that FSH stimulated oocyte resumption of meiosis and cumulus expansion of cumulus cell-enclosed oocyte(CEO)in the presence of 4 mol/[, hypoxathine(HX), with maximal effect occurring at a dosage of 100 IU/L. The oocyte meiotic resumption could be induced by 30 min FSH presentation. By 10 hr after FSH treatment, cumulus cells had already apparently expanded, whereas the significant increase of GVBD did not appear until 16¡«20 hr in culture. Nearly all the oocytes with cumulus cell expansion underwent the meiotic resumption. But those CEO whose cumulus cell did not expanded didn't undergo GVBD. Low doses of hCG failed to affect the oocyte maturation and cumulus expansion of CEO, however, high doses of hCG could partially inhibit the oocyte maturation stimulated by FSH. Our results indicate that, in the presence of HX, FSH stimulated significantly cumulus expansion and oocyte maturation of CEO. hCG had no stimulation effect on the oocyte meiotic resumption. The cumulus cell expansion occurred 6 to 8 hr before the oocyte resumption of meiosis. We conclude that oocyte meiotic resumption stimulated by FSH in the presence of HX in vitro is initiated by cumulus expansion, whereas cumulus mass complete expansion is not needed for the maturation of oocyte. [Acta Zoologica Sinica  47(2):203-208, 2001]

Key words  Kunming white mouse, Oocyte resumption of meiosis, Cumulus expansion, Gonadotropins

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Age-specific  changes of proliferation and apoptosis in the

seminiferous epithelium of laboratory mice

ZHANG Jian^¢Ù  GAO Fu-Lu^¢Ú  ZHI Hui-Ying^¢Û  DUAN Xiang-Lin^¢Ù£ª

(¢Ù Life Science of College, Hebei normal University, Shijiazhuang 050016, China )

¢ÚHistory and Embryology Department, Chengde Medical University, Chengde 067000, Hebei,

(¢ÛBiochemistry Department, Hebei Medical University, Shijiazhuang 050017, China)     

Abstract  The ultrastructure, cell apoptosis and proliferation of the spermatogenic cells in mice during a series of development stages from 15th day of fetus to 10th month after birth were observed with a variety of techniques, such as immunocytochemistry (ICC), TUNEL method for detection of apoptotic cells in situ, electron microscopy and analysis of morphometry. The results demonstrated that: (1)The PCNA positive spermatogonium were found at 15th day of fetus, then decreased from 18th day of fetus to the 5th day postnatal. At postnatal 7th day,the PCNA positive spermatogonium were found again, at the same time , the spermatogonium were partly found positive till 6th month after birth; (2)At 3rd day after birth, the pyknotic chromatin in nucleus and progressive degeneration in plasmic structures were observed. The number of apoptotic cells reached its peaks on postnatal 21st day. Conclusion: (1)Surface densities of PCNA-positive cells reached their peaks on 14th day after birth, but the number of apoptotic cells reached its peaks on 21st day postnatal, so the apoptotic peak was late the proliferating peak for a week, and related to the particular period in cycle of seminiferous epithelium; (2)The spermatogium were found move from center to the periphery of the seminiferous tubule and arrange disorder to order; (3)The spermatogonium remained replicating activity of DNA during the developing stage. [Acta Zoologica Sinica  47(2):132-138, 2001]

Key words  Mouse, Spermatogenic cell, Proliferation, Apoptosis, Age-specific change

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Microstructure and microchemistry in otolith of Ariake ice fish (Salanx ariakensis)

XIE Yu-Hao  TANG Zuo-Peng  XIE Han  LI Bo

(Research Institute for FRESHWATER fisheries of liaoning PROVINCE, Liaoyang 111000, Liaoning, China)

ZHANG Shi-Dong  YU Fu

(Fisheries Management Station of Zhenan District, Dandong 118001, Liaoning, China)    

Abstract  Adult Ariake icefish (Salanx ariakensis ) in Yalujiang river estuary were collected in September 1998. After biological measurements of the samples, the sagittae were removed from the fish. One of the sagittae from each individual was sectioned to examine its microstructure under microscope, the other one was polished and coated with carbon for measurement of the concentration ratio of strontium and calcium in otoliths by using a wavelength dispersive X-ray electron microprobe (EMP-820Q). In the analysis of Sr and Ca, CaSO4 and SrSO4 were used as standards.

The long radius Y(¦Ìm) of the otolith was linearly related to the fish length X (SL, mm), and the equation total from 31 individuals (SL 116¡«157 mm) was Y = 2.7216X + 9.2292, r = 0.9178, P<0.01. The mean diameters of core and primordum in otolith were 22.55 ¡À 2.88¦Ìm and 6.95 5 1.30¦Ìm, respectively. The number of daily growth increments in otolith of 13 adults ranged from 305 to 354. The transition zone (mark ring)occured appromately at 45¡«70 days increments. The width of daily growth increments was from 0.94¦Ìm ¡«1.14¦Ìm, the largest width at 190¡«220 days increments and the smallest width appeared after 280 increments. The first peak of Sr/Ca concentration ratio appeared at the 5^th¡«7^th measurement point that coincided with the time of migration of the juvenile fish to the sea for overwintering. While the second peak appeared from the measurement points 28 to 32 which coincided well with the time of the fish anadromous migration for spawning. The changes of physiological and ecological condition during ontogenic development of the fish lead to the Sr/Ca ratio alteration in the otolith, but the drastic change of salinity in habitability water that was the dominant factor causing the Sr/Ca ratio significantly increase. [Acta Zoologica Sinica  47(2):215-220, 2001]

Key words  Ariake icefish ( Salanx ariakensis), Otolith, Microstructure, Microchemeistry

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Observation on microstructure and ultrostructure of the venomous gland

in stingray  (Dasyatis laevigalus Chu)

         LIU Xiao-Ping^*  ZHANG Ke-ling  YU Ye-Jun  FANG Li-Hua WANG Hai-Qing  SUN Yan-Zeng

(Department of Histology and Embryology, Qingdao University Medical College, Qingdao 266021, Shandong , China )    

Abstract  In order to study the mechanism of Stingrays Dasyatis laevigalus Chu sting, the structure of the venomous gland of the Stingray was observed under light microscope and electromicroscope. The results showed that the venom tissue was the stratified epithelium in the ventral lateral grooves. The epithelium consisted of about 4 layers of cells from the base to the surface. The cells in stratum basale were basophilic cuboidal cells which rested on the basement membrane. The cells above the stratum basale were polygonal cells with central nucleus and cytoplasm filled with rough endoplasmic reticulum, ribosome, golgi apparatus and tonofibril. There were a few eosinophilic cells in HE staining scattered among the polygonal cells. Eosinophilic cells above the polygonal cells to the surface of the epithelium were about 10 layers and were also polygonal profile and were filled with many membrane-bound, electron-lucent secretory granules (venom granules)in cytoplasma. Some free ribosomes, rough endoplasmic reticulium, golgi complexes and tonofilament were observed in the eosinophilic cells. Near the superficial cells the venom granules were fused. The superficial cell were flat which cytoplasm were filled with fused venom granules and degenerating mitochondria. The cell membrane next to free surface thickened significantly like keratose. Melanocytes, langerhans cells and merkel cells were also observed in the venom epithelium. The results suggest that the substances in the membrane-coated, electron-lucent granules in eosinophilic cells may be the key factor in the envenomation by Stingrays. [Acta Zoologica Sinica  47(2):221-225, 2001]

Key words  Stingray (Dasyatis laevigalus Chu), Venomous gland, Microstructure and ultrastructure

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A new species of the genus Paa from southeast xizang, china(Amphibia: ranidae)

FEI Liang^*  YE Chang-Yuan

(Chengdu Institute of Biology, the Chinese Academy of Sciences, Chengdu 610041, China)

Abstract  Spiny frogs from Hengduan Mountains and Himalayas of China, i.e. types of Paa chayuensis from Chayu and Medog, southeast Xizang (Tibet) are studied. The results indicate that Paa chayuensis from Chayu is a valid species which differs from Paa arnoldi Dubois; Paa chayuensis from Medog differs from that one in Chayu and any of other described species of genus Paa. Therefore Paa chayuensis from Medog is considered as a new species, Paa medogensis sp.nov.

Paa medogensis Fei and Ye, sp.nov.

Holotype: CIB8370106, adult male (SVL 79.0 mm), collected at Medog, Xizang (ca: 29¡ã22'N, 95¡ã35'E), in China, alt. 1100 m, on July 19, 1983, by LI Sheng-Quan.

Paratypes: Nine adult males (CIB73II0013, 0018, 8370105, 8370134, 8370139¡«141, 8370147148), 4 adult females (CIB73II0010¡«12, 8370100), four subadults and some tadpoles collected at Maniweng, Medog, alt. 1 000¡«1 100 m, on July 16, 1973 and on July 19, 1983. One adult female (CIB7430080) from Hanmi, Medog, alt. 2 100 m, on August 23, 1974; three adult females CIB8370024¡«25, 8370149 collected at 62th km. from Medog to Bomi, on July 19, 1983.

Diagnosis: This new species Paa medogensis is closely similar to Paa chayuensis, but it is distinguished from the latter by the following characters: ¢Ùlarger body size, snout-vent length (SVL) of adult males 62¡«79 mm (mean 71 mm), SVL of adult females 71¡«114 mm (mean 86 mm); ¢Úanterior 2/3 part of body dorsum smoother although with a few elongated warts arranged in longitudinal rows, posterior 1/3 part rough, with many small spine warts; ¢Ûthroat smooth, without granules; ¢Üthe space between two vomerine teeth patches nearly equal to the length of a vomerine teeth patch; ¢Ýtympanum hidden; ¢Þthe color of dorsum of living adults dark olive, or with four greenish longitudinal bands; ¢ßmale chest with two transverse spine groups, each group with about 20 spines. The latter with smaller body size, SVL of adult males 52¡«65 mm (mean 58.5 mm), that of adult females 59¡«80 mm (mean 69 mm); the upper sides of head and body densely covered with round spine warts; throat with round small warts; two vomerine teeth patches mostly touched each other; tympanum small and clear; body dorsum green olive, parts with wart dark olive; male chest with many spines, and appearing two oval patches, each patch with 33¡«56 spines.

The type specimens are kept in Chengdu Institute of Biology, the Chinese Academy of Sciences. [Acta Zoologica Sinica  47(2):226-230, 2001]

Key words  Amphibia, Ranidae, Paa medogensis sp. nov.

*E-mail: xiefeng6@mail.sc.cninfo.net

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Female reproductive output and neonate characteristics in a viviparous

water snake  ( Sinonatrix annularis )

JI Xiang^¢Ù*  XU  Xue-Feng^¢Ù¢Ú  DU Wei-Guo^¢Ù

(¢Ù Department of Biology, Hangzhou Normal College, Hangzhou 310036, China )

(¢Ú Department of Biochemistry, Chuzhou Normal College, Chuzhou , Anhui 239012, China)

[Acta Zoologica Sinica  47(2):231-234, 2001]

Key words  Colubridae, Sinonatrix annularis, Reproductive output, Litter size, Neonate

*E-mail:xji@mail.hz.zj.cn 

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In vitro cultivation of the entomogenous nematode Ovomermis Sinensis

WANG Guo-Xiu  CHEN Qu-Hou

(College of Life Sciences, Central China Normal University, Wuhan 430079, China)

CHEN Guo

(Biology Control Laboratory, Chinese Academy of Agricultural Sciences, Beijing  100081, China) 

Abstract The insect-parasitic nematode, Ovomermis sinensis Chen et al., 1991 has the potential for the biological control of a range of insect pests due to many insect sepecies are susceptible to this nematode. For example Leucania separata , Heliothis armigera , Prodenia litura , Spodoptera exigua , Plutella xylostella , Pieris rapae, Agrotis ypsilon, Euxoa segetum etc. The insect host invariably dies when the juvenile nematode completes its parasitic development and exits from the host's hemocoel. There is an important significance for studying the mass cultivation of O. sinensis for field trials. This paper describes the techniques of in vitro cultivation of O. sinensis .

Gravid females of O. sinensis collected from sand were washed three times with an antibiotic solution of 200 U penicillin/ml and 200 ¦Ìg/ml streptomycin/ml per ml solution. Surface washed gravid female were placed in 25 ml culture tubes containing 1¡«10 ml deionized water, 100 U penicillin/ml, and 100 ¦Ìg streptomycin/ml for oviposition. Eggs were collected and their surface were sterilized by immersing them for 5¡«10 min in 0.13 % ¡«0.5 % sodium hypochlorite solution and rinsing four times with sterile distilled water. Surface sterilized eggs were incubated at 26¡À1¡æ in the culture media. The preparasites which emerged from those eggs were utilized for culture work. The four media were tested and they were TC-199, TC-199 + MK, TC-100 or Grace supplemented with 20% heat inactivated fetal bovine serum respectively. The pH of the media were adjusted to pH6.8¡«7.0. The culture medium was changed or replenished every 3¡«6 days by withdrawing the supernatant and replacing it with a portion of fresh medium. In vitro growth of O. sinensis parasites was monitored in TC-199 supplemented with 20 % heat inactivated fetal bovine serum under differing condition of crowding. Crowded cultures (150¡«200 parasites per tube) were set up in early days of in vitro (length of nematodes( 10 mm), whereas crowed culture (50¡«70 parasites per tube) and uncrowded cultures (2¡«7 parasites per tube) were tested in the late days of in vitro cultivation (length of nematodes>10 mm). Cultures were examined daily with an inverted microscope for nematode growth and activity.

The results showed that the best growth obtained among the test media was TC-199 supplemented with 20% heat inactivated fetal bovine serum. In this medium, most nematodes can grow for up to three months, reaching a development stage similar to 8¡«9-day-old parasites grown in vivo in the armyworm (Leucania separata walker). Maximum size of the juveniles was 55.1 mm in length and 204.13 ¦Ìm in width at the widest point and the results to date have yielded a 20-fold increase in body volume from the preparasitic to the parasitic stage. Two molts were observed in vitro, with the first occurred within the egg, and the second occurred after 6¡«8 days in culture. The stylet was lost. Trophosome developed prominently, and some of the nematodes developed a caudal appendage. However, no gonadial development was observed.

Population density were investigated. In vitro length of nematodes (<10 mm) that had developed in early days of cultivation under crowed condition was significantly greater than that of uncrowed nematodes. the late stage of cultivation growth of uncrowded nematodes was greater than that of nematodes of crowded matodes.

The experiment proved that terrestrial mermithid nematode, 0. sinensis can live and oviposit in water. The idea that terrestrial mermithid nematode cannot survive in water because lack of 02 is not supported.

Key word  Ovomermis sinensis, Mermithid, In vitro cultivation

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