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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.3 June. 2002

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ABSTRACT

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Bird migration: the present view of evolution, control, and further development as global warming progresses

Peter Berthold

(Max Planck Research Centre for Ornithology, Vogelwarte Radolfzell, Schloss, Moeggingen, D-78315 Radolfzell, Germany)

Abstract Studies during recent decades have shown that bird migration in general is to a great extent under direct genetic control. There is evidence indicating the existence of an innate migratory drive as well as genetic control of (i) the onset, duration and end of the migration period, (ii) the amount of migratory activity, a genetically prescribed parameter that determines the distance over which the bird flies, (iii) the migration directions and (iv) physiological parameters, in particular fat deposition during the migratory period, but also those determining which individuals will migrate and which will not in forms that are obligatorily partial migrants. A two-way selection experiment has shown that within only a few generations partial migrants can be converted by selection into pure migrants or nonmigrants. Selection for a new migration direction, leading to new winter quarters, can occur equally rapidly in free-living birds. At least in the species most closely studied so far (the Blackcap, sylvia atricapilla), the attributes "migrant" or "nonmigrant" are inherited in conjunction with the characteristic amount of migratory activity (as a time program), and the former are evidently controlled by a threshold mechanism. A new bird-migration theory postulates that even in forms that at present are exclusively migratory, selection for lower levels of migratory activity can cause a threshold to be crossed, below which nonmigrants appear. Accordingly, conversion of a population from migratory to nonmigratory can occur by selection with a transition through partial migration. This intermediate stage is prevalent among the recent bird species; it appeared early in the evolution of organisms and in the case of birds was probably acquired from ancestral, pre-avian forms. Model calculations indicate that with strictly directed selection, migratory birds can be converted to sedentary in about 40 years and conversely. This explains the changes in migratory behaviour currently observable in so many bird species in the course of global warming; in particular, migratoriness is decreasing in various respects at higher latitudes. The new theory also enables us to predict the ways in which avifaunas are likely to be restructured if warming continues; these scenarios are briefly outlined. [Acta Zoologica Sinica 48(2):291-301, 2002, in English]

Key words Birds, Migration, Evolution, Genetics, Global warming

E-mail: berthold@vowa.ornithol.mpg.de

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Anti-predation does not fully explain grouping in the Chinese water deer (Hydropotes inermis)

SUN Li-Xing

(Department of Biological Sciences, Central Washington University, Ellensburg, WA 98926-7537, USA)

Abstract The grouping behavior of the Chinese water deer (Hydropotes inermis) was studied at Poyang Lake National Nature Reserve, Jiangxi, China. I made three predictions to test the hypothesis that grouping is an anti-predatory strategy: (1) group size is negatively related to vegetation cover, (2) animals in groups have more time to feed than solitary animals, and (3) animals spend less time in vigilance when they are in groups than when they are alone. Results showed that group size was largest in the mating season and smallest in the parturition season. Deer tended to form larger groups when they were in short grass areas than in tall grass areas. Hence, the first prediction is supported. However, solitary deer and deer in groups did not differ in time allocated to feeding or vigilance. Therefore, predictions 2 and 3 are not supported. Time budget analysis revealed that females with young allocated less time to feeding, but more time to walking during the non-mating season. During the mating season, females grouped with males spent less time feeding than solitary females, and solitary males spent more time looking for females than did males grouped with females during the mating season. In light of the complications found in the grouping behavior of the water deer, the anti-predatory hypothesis seems to be insufficient as a reason for the deer to group. Other socio-ecological factors are also important in determining the grouping behavior of this species. [Acta Zoologica Sinica 48(2):302-308, 2002, in English]

Key words water deer (Hydropotes inermis), grouping behavior, anti-predation, group size, time-budget

E-mail: lixing@cwu.edu

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Foraging strategy of przewalski's gazelle (Procapra przewalskii)ŞŹ

LIU Bing-Wan JIANG Zhi-Gang

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

Abstract We studied the foraging strategy of the Przewalski's gazelle in Gonghe and Haiyan counties around the Qinghai Lake region from August 1998 to August 2000. We observed the foraging activities of about 200 Przewalski's gazelle individuals in the fields with the focal\|animal sampling method, and determined the plant biomass above ground in habitats of the Przewalski's gazelle with a systematic quadrate sampling method and used the microhistological analysis of fecal samples to assess the diet composition of the Przewalski's gazelle. We found that foraging behavior is one of the dominant behaviors of the PrzewalskiŹđs gazelle, which made up 40.02% -65.58% of the daily activity time of the gazelles. Przewalski's gazelle prefered to active at dawn and dusk, however, more than 82.9% of their active time at dawn and dusk were foraging. The Przewalski's gazelle liked to graze in the grassland of low human disturbance and abundant foods. Five plant families, Graminae, Leguminosae, Cypaeraceae, Comsitae, Rosaece, made up more than 60 percent of the diets of Przewalski gazelle whole year around, but there were significant differences among the seasonal food compositions of Przewalski's gazelle (P<0.01). When the food abundance decreased to below 155.3 g/m², those plant species that were never or seldom eaten by the Przewalski's gazelle during the plant g period green appeared in the foods of the Przewalski's gazelle. The proportion of those substitute foods in the diet of Przewalski's gazelle increased while the aboveground biomass decreased. The Przewalski gazelle generally grazed in the ecotones between grasslands and sandbanks. It preferred to forage in the place with foods of high quantity, of less human disturbance and good over conditions. The Przewalski gazelle also liked to graze in habitats within less than 1 km from the fenced pasture but they seldom grazed in the fenced pastures. Based on the above results, the following conclusions were obtained: human activity and food abundance affected the foraging strategy of the Przewalski's gazelle significantly. [Acta Zoologica Sinica 48(2):309-316, 2002]

Key words Przewalski's gazelle(Procapra przewalskii), Foraging strategy, Qinghai Lake, Human activity, Alpine ecosystem

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

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Hoarding behavior of eurasian nutcracker (Nucifraga caryocatact) and its role in seed dispersal of Korean pine (Pinus koraiensis)

LU Chang-Hu

(College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China)

Abstract We studied the seed hoarding behavior of Eurasian nutcrackers from 1994-1997 at Liangshui Nature Reserve, which located at Xiaoxinganling Mountains. It was founded for conserving the virgin Korean pine hard wood forest. Eurasian nutcrackers mainly feed on the Korean pine seed from autumn to next early spring. When cones become ripe, nutcrackers begin to harvest seeds and store a lot for winter and next spring. Nutcrackers are good at collecting seeds from the cone and swallowing. Occasionally, nutcrackers stop to crack some seeds and eat the kernel, seed coat become two half parts. After eating some seeds in the tree, nutcrackers carry 30-50 seeds with their sublingual pouch to some places 2-5 km far generally, and cach the seeds 2.5-3.5 cm deep under ground in common. Nutcrackers jab caches with bill on the ground, 2-5 times for one cache. 2-4 seeds are buried under each cache generally. Cache sites mainly are found at 8 types of habitats: roadside (forest edge), open area around protecting station, Larix plantation, Abies plantation, Pinus koraiensis plantation, Picea plantation, Populus-Betula natural regeneration and natural Korean pine-hard wood. One nutcracker can store 16 000 seeds in one harvest season (about 40 days in research site). Nutcrackers do not recover all the caches and reforage all seeds. Untouched seeds may germinate in the second spring in about 18 months since being cached. 1-year and 2-year seedlings, which usually appear with 2-4 seedlings in one cluster, are found in all the 8 types of habitats around research sites, but saples (higher than 3 m) are only found in Larix plantation, Populus-Betula natural regeneration and natural Korean pine-hard wood. Such situation is decided by growing characterstics of Korean pine. Nutcracker is an effective disperser of the Korean pine seeds. [Acta Zoologica Sinica 48(2):317-321, 2002]

Key words Eurasian nutcracker(Nucifraga caryocatact), Korean pine(Pinus koraiensis), Hoarding behavior, Seed dispersal

E-mail:lchanghu@yahoo.com

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Ant fauna in the northwestern regions of China

CHANG You-De^* HE Da-Han

(Agricultural College, Ningxia University,Yinchuan 750105, China)

Abstract The ant species in different vegetation sites and main typical mountains such as desert forest Helan Mountains, humid forest Liupan Mountains and high-cold shrub Qilian Mountains in Gansu, Qinghai Provinces, Ningxia Hui and Inner Mongolia Autonomous Regions were collected from 1997 to 2000. The total investigated area was more than 900 000 km² and total 1 000 species numbers were collected. 79 species belonging to 16 genera and three subfamilies were recorded. 15 genera, typically Formica, Myrmica, Lasius, Proformica, Cataglyphis, Messor and Polygergus distribute in Palaearctic Realm; nine genera, typically Solenopsis, Tapinoma, Liotopum, Camponotus mainly distribute in Oriental Realm. At genus level, Inner Mongolia, Xinjiang region components were 15 genera of which Tetramorium, Proformica and Cataglyphis were typical; North China region components were 15 genera only except Cardiocondyla. Each of Qinghai-Xizang region and Northeast China region components was four genera of which Formica and Lasius are typical. Southwest and South China regions components play no significant role. At species level, components of North China and Inner Mongolia-Xinjiang regions were dominant and were 51 and 49 species respectively. Components of Middle, Northeast China and Qinghai-Xizang regions were subdominant and were 17, 15, 15 species respectively. The similarities of ant communities in 9 different types of grassland, that is typical grassland(loess plateau area), desert grassland, steppe grassland, typical desert, humid forest(Liupan Mountains), high-cold shrub, desert swamp, desert forest(Helan Mountains)and desert oasis, were analyzed by principle component analysis (PCA). Results showed that desert grassland, steppe desert, typical desert and desert oasis ant communities were significantly close to each other to form an integrated group, while desert forest(HelanMountains) ant community which was however far away from the above integrated group and other four ant communities were isolated from other each. Under the guidelines from the results above, the ant fauna of investigated area was divided into six ant regions which were loess plateau ant region, desert ant region subdivided into desert grassland ant subregion, steppe desert ant subregion, typical desert ant subregion and desert oasis ant subregion, desert swamp ant region, highŞ˛cold shrub ant region, Liupan Mountains ant region and Helan Mountains ant region. In addition, 79 ant species were clustered into 10 ecological species groups by PCA. [Acta Zoologica Sinica 48(2):322-332, 2002]

Key words Ant fauna, Ecological distribution, Ant community similarity, Ecological species group, Northwest China

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

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Genetic structure of edge population in przewalski's rock partridge (Alectoris magna)

HOU Peng WEI Ming ZHANG Li-Xun LIU Nai-Fa^*

(School of Life Science, Lanzhou University, Lanzhou 30000, China)

Abstract Alectoris magna is endemic to China, which distributes in arid region and semi-arid zone from western Qinghai, central Gansu to western Ningxia Huizu Autonomous Region. Because of the environmental changes, some populations of A.magnaŞŤhave expanded their range to regions of recent deforestation by humans in southern Gansu, and have colonized to form new edge populations. In this paper the hypervariable domain ˘ń of the mtDNA control egion (D-oop) from A. magna populations in edge and central region was studied. In 39 A. magna samples and five A. chukar outgroups, the entire mtDNA D-loop was PCR-mplified. The 456-457 nucleotides of the DNA D-loop domain ˘ń have been sequenced by dideoxy chain-termination method. The 16 variable sites(3.5% of the entire sequence) defined 15 haplotypes. The edge dispersal population has three haplotypes, and shared M6 with other two populations. Its haplotype frequency was 0.108 in the edge dispersal population. M4 and M5 were only found in the edge dispersal population, their frequencies were 0.081 and 0.027 respectively. The population 1 and population 2 of the central population each had 7 haplotypes, sharing M6. The proportion of haplotype and genetic diversity were 37.5% and 0.594 respectively in the edge dispersal population. In the population 1 and population 2 the proportions of haplotype were 46.7% and 50.0% respectively and the genetic diversity separately 0.729 and 0.78 6. This result suggested that the edge dispersal population had lower proportions of haplotype and genetic diversity than the two central geographical populations (population 1 and 2). [Acta Zoologica Sinica 48(2):333-338, 2002]

Key words Przewalski's rock partridge (Alectoris magna),Edge dispersal population, Genetic structure, Mitochondrial DNA(mtDNA), Genetic diversity, Central geographical population

*E-mail:Liu-nf @sina.com

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Interspecific phylogenesis and intraspecific genetic differences of genus Megalobrama: bluntnose black bream (m.amblycephala), Guangdong black bream (M.hoffmanni) and black bream (M.terminalis)

LI Si-Fa^* ZHU Ze-Wen ZOU Shu-Ming ZHAO Jin-Liang CAI Wan-Qi

(Key Laboratory of Ecology and Physiology in Aquaculture of Ministry of Agriculture,

Shanghai Fisheries University,Shanghai 200090, China)

Abstract We studied interspecific phylogenetic relationships and intraspecific genetic variations of three species in genus Megalobrama, M.amblycephala, M.terminalis and M.hoffmanni by morphologic discrimination analysis, enzyme analysis and RAPD analysis. The results showed that: (1) there were no significant differences on meristic characters of the three breams, but measurable parameters ratio and truss framwork of the three species presented a close relationships between M.amblycephala and M. terminalis, and obvious differences between them and M.hoffmanni; (2) the band s-Mdh-D of enzyme MDH existed in M.amblycephala and M. terminalis, but not in M.hoffmanni. The RAPD primer S11 could amplify species-specific bands in each of three species. These enzyme band and PCR amplified bands all could be used as interspecific molecular markers;(3) the same conclusion could be made on interspecific relationships of the three species by morphologic discrimination analysis, enzyme analysis and RAPD analysis, namely, M.hoffmanni had significant differences with M.amblycephala and M.terminalis, and there were small differences between M.amblycephala and M.terminalis;(4) allozyme and RAPD analysis revealed that the genetic diversity within species of M.terminalis was significantly higher than that of M.amblycephala and M.hoffmanni.ŞŤ[Acta Zoologica Sinica 48(2):339-445, 2002]

Key words Bluntnose black bream (Megalobrama amblycephala), Guangdong black bream (M.hoffmanni), Black bream (M.terminalis), Morphology, Enzyme analysis, RAPD, Phylogenesis

*E-mail:lisifak@online.sh.cn

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Gabaergic inhibition can sharpen frequencytuning of auditory cortical neurons in big brown bat

(Eptesicus fuscus)

CHEN Qi-Cai^˘Ů* P. H-S JEN^˘ÚWU Fei-Jian^˘Ů

(˘ŮDepartment of Biology, Central China Normal University, Wuhan 430079, China)

(˘ÚDivision of Biological Sciences, University of MissouriŞ˛Columbia, MO 65211, USA)

Abstract Nine adult big brown bats were used in this experiment.The GABAergic inhibition sharpening the frequency tuning of primary auditory cortical (AC) neurons was studied with the multi-barrel electrode for iontophoresis and two-tone stimulation method.We observed that excitatory frequency tuning curves appeared three types: the single-peak opening, multi-peak opening, and single-peak closed. The inhibitory inputs to AC neurons could be activated by inhibitory tone and they had their best frequencies.The inhibitory frequency tuning curve or inhibitory frequency-intensity response area at the low limb or/and high limb of an excitatory frequency tuning curve could be tested and obtained according to effective degree of GABAergic inhibition on the excitatory response induced by the tone of 10 dB above minimum threshold and by systematic change of inhibitory tone intensity.The result showed that GABAergic inhibition could decrease the firing rate of excitatory response to 10 dB stimulation above minimum threshold and increase the inhibitory effectiveness with the elevation of inhibitory tone intensity.GABA ergic inhibition disinhibited during iontophoresis of bicuculline could make the excitatory frequency tuning curve to become the wider, the multi-peak tuning curve to become the single-peak, and single-peak closed tuning curve to become the single-peak opening tuning curve.Therefore, the results described above demonstrated that GABAergic inhibition participate in the formation of inhibitory input to AC neuron, it could help to sharpen the frequency tuning of the central auditory neurons, and this is the reason that frequency tuning curves become the Ą°pencil-shapedĄą and Ą°spindle-shapedĄą, and that central frequency tuning is more sensitive than peripheral. [Acta Zoologica Sinica 48(2):346-352, 2002]

Key words Big brown bat (Eptesicus fuscus), GABAergic inhibition, Sharpening frequency tuning, AC neuron

*E-mail:chenqc@ccnu.edu.cn

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Angiotensin ˘ň-induced vascular endothelial growth factor regulates pericardial lymphatic stomata and mesothelial sinusoid in European edible frog (Rana esculenta)

LI Ji-Cheng^˘Ů* SHEN-Yi^˘ŮBruno TOTA^˘ÚGiovanni ZUMMO^˘Ű

(˘Ů Department of Lymphology, Institute of Cell Biology & Tissue Engineering , Zhejiang University, Hangzhou310031,China)

(˘Ú Institute of Cell Biology, University of Calabria, Cosenza, Italy)

(˘Ű Institute of Anatomy, Palermo University School of Medicine, Palermo, Italy)

Abstract Angiotensin ˘ň (Ang˘ň) plays a role in many vascular diseases. In the present study, the effect of Ang˘ň on vascular endothelial growth factor (VEGF) regulating the pericardial lymphatic stomata and the pericardial mesothelium was investigated in the European edible frog (Rana esculenta). Ang˘ň+VEGF and VEGF were injected into the peritoneal cavity in 10 frogs respectively. The results were obtained by using scanning electron microscopy and computerized imaging analysis. Scatteredly distributed pericardial lymphatic stomata were found on the parietal pericardium with a few mesothelial sinusoid in the frogs. In the VEGF group, which consisted of 5 frogs, the average diameter of the stomata, the average distribution density of the stomata and the sinusoid areas were 1.50 ŚĚm, 8.25/0.1 mm² and 2 443.95 ŚĚm²/0.1 mm² respectively, which were much higher than those in control group (P<0.01). Ang˘ň induced a significant increase of the effects of VEGF on the stomata diameter (2.81 ŚĚm), the stomata distribution density (28.82/0.1 mm²) and the sinusoid areas (3 866.14 ŚĚm²/0.1 mm²), which were much higher than those in the VEGF group (P<0.05). VEGF strongly regulated the pericardial lymphatic stomata by increasing their numbers, diameters, and higher distribution density and also increased the sinusoid area with the result of the higher permeability of the pericardium. Ang˘ň enhanced VEGF-induced the stomata and the sinusoid, which would speed up material transfer of the pericardipal cavity and pericardial leaking from the myocardium so as to play an import ant role in preventing myocardial interstitial edema. These findings suggest that Ang˘ň may potentiate VEGF-induced regulatory activity of the pericardial lymphactic stomata and sinusoid through a increase in the gene expression of VEGF. [Acta Zoologica Sinica 48(2):353-357, 2002]

Key words Frog(Rana esculenta), Pericardial lymphatic stomata, Angiotenisn ˘ň, Vescular endothelial growth factor

*E-mail: lijc@mail.hz.zj.cn

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Lipotropy effect of betaine in swine

XU Zi-Rong FENG Jie

(Key Laboratory for Molecular Animal Nutrition of Ministry of Education,Zhejiang University, Hangzhou310029,China)

Abstract Fat liver is a common disease and always harmful to animal health. As an effective methyl donor, betaine can affect fat and protein metabolism. Although lots of work focused on the fat deposition influenced by betaine in animal's body has been published,little information is available about the effect of betaine on lipid metabolism and lipotropy in animal liver. Swine is easy to accumulate fat in organ and tissue especially in the finishing stage(60.90 kg). According to this, 150 pigs(DurocĄÁLandraceĄÁYorkshire) which weighed about 63 kg each were chosen and allocated to five treatments with three replicates in a randomize d block design to determine the dose-response effects of betaine(0, 1 000, 1 250, 1 500 and 1 750 mg/kg) on lipid metabolism and lipotropy in liver at high dietary energy when pigs reach 93 kg. The protein, lipid, free carnitine, long-chain acylcarnitine, lecithin level in liver and free fatty acid,cholesterol content in serum were determined after feeding experiment. The results showed that betaine increased protein content in swine liver especially in 1 250 and 1 750 mg/kg groups in which it was significantly increased by 4.47% and 4.27%(PŁź0.05)respectively as compared with the control group. There was no significant effect on lipid content in swine liver among the groups but it showed an evident downtrend with the enhanced betaine dosage. The addition of betaine increased free carnitine and long-chain acylcarnitine in liver,which were increased most significantly in the 1 750 mg/kg group by 30.18%(PŁź0.01) and 44.44%(PŁź0.01) respectively compared with the control group. Lecithin in liver was also enhanced by betaine with the most remarkable mcrease by 25.29% (P<002) at the dose of 1 250 mg/kg betaine. The results also showed that free fatty acid was increased by 21.27%(PŁź0.01) and cholesterol was decreased by 9.72%(PŁź0.05) in serum in the group of 1 750 mg/kg betaine, but there was no considerable variation in triglyceride level in all the trail groups. These results suggested that betaine regulate lipid metabolism and had lipotropic effect. Betaine influenced the content of methylated products,free carnitine and long-chain acylcarnitine, the important carriers of long-chain fatty acid,and modulated the ratio between the two Substances. It also increased the lecithin level in liver and therefore improve the lipid metabolism in liver of swine. [Acta Zoologica Sinica 48(2):358-362, 2002]

Key words Swine, Lipotropy, Betaine, Lecithin, Carnitine

Role of P16 protein and germ cell apoptosis in rhesus monkey azoospermia or oligozoospermia induced by heat stress or testosterone undecanoate

ZHOU Xin-Chang^˘Ů HU Zhao-Yuan^˘ŮLI Yin-Chuan^˘Ů ZOU Ru-Jin^˘ÚLIU Yi-Xun^˘Ů*

(˘Ů State Key Laboratory of Reproductive Biology,Institute of Zoology,Chinese Academy of Sciences,Beijing 100080,China)

(˘Ú Kunming Institute of Zoology,Chinese Academy of Sciences, Kunming 650223,China)

Abstract We investigated the functional relationship between the role of P16 protein and germ cell apoptosis in heat stress or TU-induced azoospermia or oligo-zoospermia. The TUNEL result showed that the apoptotic signals after treatment increased and the strongest apoptotic signals appeared in the germ cells of the cryptorchid testes on day 5 or in the TU-treated monkey testes on day 30.The immunohistochemical result showed that both heat stress and TU induced expression of P16 protein mainly in spermatogonia,and also in other type of germ cells and sertoli cell at the later stage of germ cell apoptosis,namely on day 10 after operation or on day 60 after TU injection respectively, suggesting that P16 protein may inhibit the spermatogonia mitosis at the later stage of the germ cell apoptosis. The above results indicated that both heat stress and TU treatment were capable of inducing germ cell apoptosis at the early stage on one hand,and inhibiting spermatogonia mitosis at the later stage in the testis on the other hand,which might result in azoospermia or oligozoospermia. [Acta Zoologica Sinica 48(2):363-367, 2002]

Key words Rhesus monkey(Macaca mulatta),Azoospermia/Oligozoospermia,Mitosis,P16,Heat stress,TU

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

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Effects of the donor blastomere cell cycle stages on the development of rabbit

nuclear transplantation embryos

WANG Yong-Sheng^˘ŮZENG Shen-Ming^˘Ů ZHU Shi-En^˘ŮTIAN Jian-Hui^˘ŮYU Wen-Li^˘Ů

ZHANG Zhong-Cheng^˘ŮCHEN Yong-Fu^˘Ů SHANG Jia-Ji^˘Ú WEI Jian-Sheng^˘Ú

(˘Ů College of Animal Science and Technology,China Agricultural University,Beijing 100094,China)

(˘Ú Hongkong Wiss Biotechnology Center, China)

Abstract We studied the effects of donor cell cycle stages on the development of rabbit nuclear transfer embryos.The donor nuclei were from early rabbit embryos that were treated with chemicals to synchronize blastomeres at different cell cycle stages.16-cell rabbit embryos were recovered from the oviducts of New Zealand white rabbit does administrated with FSH and hCG for superovulation. The embryos were cultured to 32-cell stage in TCM199 with Earle's salt supplemented with 10% FBS,1.25 mmol/L Na pyruvate,0.1 mmol/L EDTA,0.75 ŚĚg/L penicillin and 0.50 ŚĚg/L streptomycin(RM199).The embryos were treated with 0.5% pronase to separate blastomeres. The blastomeres without chemical treatment were used as non-synchronized donor cells.The cells at G1 stage were isolated from the 16-cell embryos that were cultured first in RM199 containing 1.00 ŚĚg/ml nocodazole for 12 h,and then in RM199 containing 0.20 ŚĚg/ml aphidicolin for 8 h. The blastomeres at S stage were is olated from the 16-cell embryos that were cultured first in RM199 containing 1.00 ŚĚg/ml nocodazole for 12 h,and then in RM199 for 6 h.The blastomeres at G2 phase were isolated from the 16-cell embryos that were cultured first in RM199 containing 1.00 ŚĚg/ml nocodazole for 12 h,then in RM199 for 8 h,and finally in 1.00 ŚĚg/ml cycloheximide for 8 h. The M˘ň oocytes were recovered from superovulated New Zealand white rabbit does at 14.15 h after hCG injection and mechanically enucleated as recipient cytoplasm.

The G1,S,G2 stage or non-synchronized blastomeres were transferred into the enucleated oocytes,respectively. The oocyte-blastomere couplets were electrically fused and activated using three DC pulses of 2.0 kV/cm at 5 min intervals after AC(10V,1MHz)for 5 -10 s in 0.29 mol/L mannitol solution containing 0.1 mmol/L CaCl₂,0.1 mmol/L MgSO₄ and 10 mmol/L histidine(pH=7.0). The reconstituted embryos were transferred into RM199 and cultured in humidified air with 5% CO₂ at 38Ąć to observe the in vitro developmental potentials of nuclear transplantation embryos.The fusion rates were 95.4%,91.2%,90.9% and 91.1% in G1,S,G2 and non synchronized group,respectively.There was no statistical difference among different groups(P>0.05). The result implied that cell fusion did not have relationship to the cell cycle stage of the two cells. However,the cell cycle stages of donor nuclei significantly affected the development of nuclear transplantation embryos. The activation rate in G1 group(87.3%)was significantly higher than that in S(79.6%),G2(63.6%)or non-synchronized group (78.1%). The rates of cleavage,morula,blastocyst,hatched blastocyst and blastocyst cell number of cloning embryos from G1 group(84.9%,71.5%,58.0%,35.1% and 126ĄŔ4.8)were significantly higher than in S(74.4%,30.3%,19.3%,6.8% and 118.8ĄŔ3.5),G2(60.0%,16.9%,16.3%,4.7% and 110.6ĄŔ5.8)or non-synchronized group(73.2%,49.4%,32.2%,12.6% and 120.5ĄŔ4.4)(P<0.05). The embryos from G2 nucleihad poor developmental potential in vitro. The rates of cleavage,morula and blastocyst cell number were significantly lower than other groups(P<0.05). The in vivo development of cloning embryos from different cell cycle nuclei was observed in this experiment. A total of 144 nuclear transplantation embryos from G1 stage were transferred into 12 recipient does.Six of them became pregnant and gave birth 19 live pups.The pupping rate was 13.2%. It was significantly higher than S(5.8%,7/120),G2 group(0,0/126)or non-synchronized donor nuclei(5.3%,8/150) (P<0.05). The cloning embryos from G2 nuclei could not develop to normal offspring although a few cloning embryos developed to blastocysts. Nevertheless,there was no statistical difference in developmental potential of cloning embryos between S and non-synchronized group,indicating that the cell cycle stage of early rabbit embryonic cells is in S stage.

The results in this experiment show that the cycle stages of donor cells significantly affect the developmental potential of rabbit cloning embryos. The G1 nuclei can successfully support the development of cloning embryos in vitro and in vivo. Although the embryos from S nuclei can develop to live offspring the pupping rate significantly decreases.The embryos from G2 nuclei have poor developmental rate in vitro and in vivo. [Acta Zoologica Sinica 48(2):368-374, 2002] ŞŤ

Key words Rabbit, Cell cycle,Nuclear transplantation,Development,Embryo

* E-mail:zengsm@cau.edu.cn

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Cloning and sequence analysis of silkworm(bombyx mori) mitochondrial ND2, CO˘ńand tRNA genes

LIAO Shun-Yao LIU Yun-Qiang LU Cheng^*ŞŞZHOU Ze-Yang XIANG Zhong-Huai

(The Key Sericultural Laboratory of Agricultural Ministry, Southwest Agricultural University,

Chongqing 400716,China)

Abstract Mitochondrial genome of silkworm (Bombyx mori) is a small, self-replicating and diverse genome, as that of other animals. This paper wanted to clone, sequence and analyze silkworm's mitochondrial genome DNA (BmtDNA). We cloned and sequenced a 3 468 bp fragment derived from EcoR˘ň and Hind˘ó double digestion of BmtDNA. Using the basic local alignment search tool (BLAST) on line, the fragment sequence was searched for homologous genes in GenBank, EMBL and DDBJ nucleotides database. And its structure was analyzed by Editseq program from DNAstar software. The sequence contains three protein-coding genes (i.e.ND2, CO˘ń and 5Ąäend of CO˘ň gene), 6 tRNA genes and 1 not fully confirmed tRNA gene. The homology of ND2 gene sequence between Bombyx mori and Drosophila melanogaster is about 69.7%, while the homology of CO˘ń and 5Ąäend of CO˘ň gene sequences between B.mori and D. melanogaster is about 83.8% and 80% respectively. This result indicates that the sequences of cytochrome oxidase(CO) genes are more conservative than those of nicotinamide adenine dinucleotide dehydrogenase(ND) genes. The primary and secondary structures of the 6 tRNA genes are different from those corresponding tRNA gene stuctures of D. melanogaster except that the secondary structures of the tRNA^Gln genes are similar between the two species. [Acta Zoologica Sinica 48(2):375-383, 2002]

Key words Silkworm(Bombyx mori), Mitochondrial genome, 3.5 kb fragment, Genome organization, Gene structure

* E-mail:lucheng@swau.cq.cn

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Annuli characters of Selincuo schizothoracine fish (Gymnocypris selincuoensis)

in Selincuo Lake,Qinghai-Tibetan Plateau

CHEN Yi-Feng^*HE De-Kui DUAN Zhong-Hua

(Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072)

Abstract Covering an area of 1 640 km², the Selincuo Lake lies in the Qinghai-Tibetan Plateau (31Ąă34Ąä-31Ąă57ĄäN, 88Ąă33Ąä-89Ąă21ĄäE) and is the second largest lake in Tibet. With an altitude of 4 530 m above sea level, the lake has an ice-free period from early April to middle October. The Selincuo schizothoracine fish ŞŤ(Gymnocypris selincuoensis) is the only fish in the lake and its body is mostly scaleless, remaining only several degenerated scales along the lateral line and on part of pectoral girdle, but the anal scales (scales lies on either side of the anal fin) are enlarged and well developed. The present study describes characters of annulus from a suite of calcified structures percular bones, vertebrae anal scales, dorsal fin spines and otoliths (from 258 individuals). Both opercular bones and vertebrae could not be used to determine age, because only their margin had several opaque and translucent zones. In most scales recorded as uninterpretable some annili could not to be recognized because of partial or complete resorption and abrasion which caused by reproductive activities in adult individuals. In other case no clear pattern of alternating zones of widely- and closely-spaced circuli could be seen, mostly du to strong irregularities in the arrangement of the circuli. The result of scales' records could be misleading, because the position of the first annulus cuold not be determined consistently, as well as the present of irregular circuli. The sectioned fin spines and otoliths showed the clear pattern of opaque- and translucent-zones which could validated as annual for age determination. In all interpretable sectioned fin spines and otoliths the first annulus was usually identifiable, unlike that in scales. There presented regular pseudoannuli or checks in the arrangement of sectioned fin spines and otoliths of mature individuals, often 4-6 sub-zones in each opaque zone. In sectioned otoliths of juveniles, there were daily growth increments and checks or discontinuities which arranged in lunar cycle manner. There were 20-25 daily rings between adjacent checks and 4-6 checks in every opaque zone. Under light microscopy, daily growth increments and checks decrease or cease in translucent zones which deposited during periods of slow growth in winter. In contrast, the result using sectioned otoliths was more accurate than using anal scales and sectional dorsal fin spines for age determination. [Acta Zoologica Sinica 48(2):384-392, 2002]

Key words Selincuo schizothoracine (Gymnocypris selincuoensis), Annuli characters, Otolith, Dorsal fin spine, Anal scale, Tibet

*E-mail:chenyf@ihb.ac.cn

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Microstructure and ultrastructure of the peripheral blood cells of European eel (Anguilla anguilla)

ZHOU Yu^* GUO Wen-Chang YANG Zhen-Guo ZOU Xiao-Huan

ZHANG Kai WEN Xing-Hao WANG Tie-Dong

(Faculty of Animal Sciences and Veterinary Medicine,The Quartermaster University of PLA,Changchun 130062,China)

Abstract We studied the peripheral blood cells of one\|year\|old European eel (Anguilla anguilla) using light microscopy and transmission electron microscopy. For morphology,peroxidase-Giemsa was used. The morphological study revealed that the peripheral blood has the following cell types: erythrocyte, neutrophil, lymphocyte and thrombocyte. Erythrocytes are 11.82ĄŔ0.75 ŚĚm in length and 7.84ĄŔ0.83 ŚĚm in width,oval shape and contain oval nuclei and dark gray cytoplasm. They were almost completely devoid of organelle,except for a few vesicles and small mitochondria.Neutrophils were 11.73ĄŔ1.45 ŚĚm in length and 10.73ĄŔ1.14 ŚĚm in width,round or oval,containing round,bilobed or trilobed nuclei and whitish cytoplasm.There were three special granules in neutrophils. Special granules A were round or oval shape,possess thread-like,cotton-like or crystalline structrures, special granules B were long oval or rod-like,dark stained and possess cotton-like structures,which paralleled to the long axia. Special granules C were round or oval,possessed light gray structures. The ultrastructural study revealed that the neutrophils include three types of cells according to their special granules(A,B and C special granules): type ˘ń(included A,B and C special granules)Ą˘type ˘ň(included A and C special granules)and type ˘ó(included C special granules).A greater amount of mitochondria,small tubules which resemble smooth endoplasmic reticulum,and numerous oval on elongated granules which contain a striated or crystalline structure,were observed in neutrophils although neutrophils are similar to monocytes in size and shape.Monocytes were of 10.78ĄŔ1.55ŚĚm in length and 9.74ĄŔ1.75 ŚĚm in width,containing round or oval nuclei. A greater amount of vesicles and a few mitochondria were observed. Thrombocytes of 1.97ĄŔ0.71 ŚĚm in length and 4.02ĄŔ0.74 ŚĚm in width,were round,oval,spindle or tadpole-like shape. Deep indentations of the plasmalemma,which gave appearance of vacuoles, were often seen.Lymphocytes were clearly different from thrombocytes by the presence of large number of free ribosomes.Neither eosinophils nor basophils were found directly in peripheral blood.The nuclear division of erythrocyte and monocyte were observed in peripheral blood indicating that erythroblasts and young monocytes could enter the peripheral blood.The size of thrombocyte was the smallest while that of the neutrophil was the largest and the number of neutrophils was the least while that of lymphocytes was the most in the blood cells. [Acta Zoologica Sinica 48(2):393-401, 2002]

Key words European eel(Anguilla anguilla),Blood cell, Microstructure,Ultrastructure

E-mail: zhouyu69@sina.com

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Histochemistry and ultrastructure of the kidney in disk abalone (Haliotis discus hannai)

CUI Long-Bo^* ZHOU Xue-Ying LIU Chuan-Lin LU Yao-Hua

(Department of Biochemistry, Yantai University, Yantai 264005, Shandong, China)

Abstract The structure and function of kidney in the disk abalone Haliotis discus hannai were studied with histological and histochemical methods and transmission electron microscopy. The abalone had a pair of kidneys, the left was larger than the right. The kidney consisted of a number of tubules and collecting ducts. Many tubules, which differ in size and round or flat in shape, converged into a collecting duct. The tubule epithelium was cuboid or low columnar. Round nucleuses were located near apex of the epithelial cells. In the tubule epithelial cells, obvious brush border consisted of a number of microvilli and quite a few cilia were observed in the free margin. The microvilli were a typical structure of absorptive cells. The tubule epithelial cells, therefore, might reabsorb or absorb selectively water or certain ions from their filtrate. The cilia might be conducive to draining the excretion by their swing. The apical cytoplasm of the tubule epithelial cell contained many small refractile spherules above the nucleus. The spherules were blue by method of Turnbull's blue, which showed that they eld iron. Under transmission electron microscope, the spherules were enclosed by the membrane, 0.8ĄŞ1.8 ŚĚm in diameter, and contained scattered substance whose electron density was low. The spherules holding iron might have something to deal with detoxification. Rich blood spaces were present among the tubules. There was only a layer of basement membrane between the epithelial cells and the blood spaces. The components in the blood might get into the epithelial cells only by passing through the basement membrane. The basal plasma membrane of the tubule epithelial cell formed numerous infoldings that was a typical structure by which the epithelial cells could transport actively and showed alkaline phosphatase activity by histochemical method that was related to trans-membrane transport of materials. It showed that the tubule epithelial cells might transport certain substances or metabolite within blood into cells, then excrete them into the tubule lumen. There were many mitochondria in the tubule epithelial cell which were located mainly in the apex and the basal region and around the nucleus. The epithelial cells did not contain glycogen and showed acid phosphatase activity, but show non-specific esterase activity in the apex by histochemical method. The non-specific esterase in the cells might conduce to detoxification. Ą°Body of membranous capsuleĄą is the typical structure of the cells. The larger Ą°body of membranous capsuleĄą was located between the nucleus and the infoldings of the basal plasma membrane. It was unregular in shape and acidophilic by H.E. staining. The body was deep blue by mercury-bromophenol blue method, which showed that it contained abundant protein. Under transmission electron microscope, it consisted of a great number of canaliculi and flat capsules enclosed by two layers of unit membrane. The canaliculi or the flat capsules were 30ĄŤ80 nm in diameter or height. The shape and arrangement of the canaliculi or the flat capsules in some localities were very regular. The canaliculi were round and the flat capsules appeared in various shape. The flat capsules were arranged mainly wave-like, some were crossways or in right angle. The special structureĄŞĄ°body of membranous capsuleĄąĄŞhad not been observed in other cells, and its nature was not clear. According to its location above the infoldings of the basal plasma membrane of the cell, we reason that the Ą°body of membranous capsuleĄą may be related to the processing of metabolic product in the cell just transported from blood. Except cilia increasing in number and Ą°body of membranous capsuleĄądecreasing in size, the histochemistry and ultrastructure of the epithelial cells of the collecting ducts were similar to those of the epithelial cells of the tubules. A number of cilia but no Ą°body of membranous capsuleĄą were observed in the epithelial cells of the kidney sac. A few of mucous cells were present in the epithelium of the collecting duct and the kidney sac. They secreted mucus that lubricates the wall of the duct and the sac, thus contributing to drain the excretion. There were some germ-cells and other substance in the sac lumen. [Acta Zoologica Sinica 48(2):402-407, 2002]

Key words Abalone (Haliotis discus hannai), Kidney, Histochemistry, Ultrastructure

*E-mail: lbcui@163.com

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Morphogenetic role of the micronucleus during conjugation in the hypotrichous ciliate

(Pseudourostyla cristata)

JIN Li-PeiŞŹLIU Xiao-Yi JIN Hua-Zhong

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

Abstract Sexual reproduction of the hypotrichous ciliate Pseudourostyla cristata takes place only between complementary mating types. To assess the participation of the micronucleus in cortical development during sexual reproduction, several amicronucleate cell lines were generated by amputation of cells from four stocks which belonged in four different but complementary mating types between any two of them. These amicronucleate cell lines were 2am1 and 2am2 from stock 2, 4am1 from stock 4, 28am1 from stock 28 and 46am1 from stock 46 respectively. The conjugation of amicronucleates between two mating types was induced and researched. In the meantime, the conjugation between micronucleates, which included stock 2, 4, 28, 46 and micronucleate cell lines 2m1 from stock 2, 4m1 from stock 4 and 28m1 from stock 28 derived also by amputation of cells, was used as control. To follow the course of development of the cortex and the nuclei was by protargol staining.

During normal conjugation, micronucleate cells reorganized their cortical structure for four rounds, each time resorbing the pre-existing ciliature and producing a new set. In contrast, during conjugation among amicronucleates, the cells developed from pairing to formation of spherical paracysts in a manner similar to that situation among micronucleates but discontinued their development before initiation of the second cortical reorganization. They could complete the first cortical reorganization like micronucleate cells did. At the end of the reorganization, the exconjugants again did not possess undulating membrane (UM); only half of the cells possessed the oral cirrus; the cytopharynx was underdeveloped; there were only 6 rows of dorsal bristles, as usual. However, there were some evident differences compared with micronucleates. First, it was clear that the conjugating pairs formed fewer than among micronucleates. Second, differentiation and positioning of the ciliature primordia in amicronucleates became abnormal, such as: some bars of the primordium of marginal cirri rows were not divided into the single cirri; the frontal cirri differed from each other in size and arranged disorderly and unsystematically; there was only one row of the ventral cirri in some section; the primordia of adoral zone membranelles (AZMP) in length at the same development stage of the first reorganization were shorter; the number of AZM membranelles and frontal-ventral-transverse cirri (FVT) after the reorganization was more reduced than in the normal; and finally, the exconjugants, without losing all or almost all ciliature after the first reorganization, transformed into the paracysts and perished inevitably before initiation of the second reorganization. On the other hand, it was clear that there were no new nucleogenesis in the amicronucleate conjugants and rearrangement of the genomic DNA sequences of macronuclear development in the exconjugants.

The results suggest that although the micronucleus is not absolutely required for the first reorganization as in asexual cycle, yet it still maintains some somatic functions in the process. On the other hand, these results show that initiation of the second cortical reorganization depends on signals from the macronuclear anlage after chromosomal polytenization and genomic rearrangement. The developmental signals required for initiating the first and the second rounds of reorganization are therefore different. The above considerations suggest that the character of the first reorganization is asexual and the second one is the `true' cortical reorganization unique to sexual reproduction. [Acta Zoologica Sinica 48(2):408-414, 2002]

Key words Hypotrichous ciliate(Pseudourostyla cristata), Micronucleus, Sexual reproduction, Morphogenesis

E-mail: lpjin@21cn.com

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A headspace sampling method and its application in analysis of volatiles of anal glands of the

American mink (Mustela vision)

ZHANG Jian-Xu ZHANG Zhi-Bin^* WANG Rui CHEN Yi CAO Xiao-Ping

WANG Fu-Sheng WANG Zu-Wang

(State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture,

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

Abstract We designed a headspace sampling apparatus to trap volatile compounds from mammalian scent glands. By using this apparatus together with solvent desorption and gas chromatography-mass spectrometry, we analyzed the anal gland secretions from American mink (Mustela vision). From GC-MS data, five compounds were tentatively identified. Their molecular weights and formula were : (1)102(C₅H₁₀S), (2)104(C₅H₁₂S), (3) 102(C₅H₁₀S),(4)136(C₅H₁₂S₂)and (5)134(C₅H₁₀S₂). (1), (3) and (5) were 2,2-dimethylthietane, 2-ethylthietane and 3,3-dimethyl-1,2-dithiacyclopentane, respectively, which have been identified in previous studies. Moreover, 2,2-dimethylthietane was predominant and there was no sex-specific compounds. These results consistent with the previous reports indicated that our head space sampling method was reliable. There were two new compounds (2) and (4) that have not been detected in other Mustela species. This suggested that the apparatus was more sensitive and the method is very useful and unique.

Key words American mink (Mustelia vision), Headspace sampling method, Anal sac secretion, Volatile compounds

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

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Influence of temperature on egg incubation of the wild Chinese alligator (Alligator sinensis)

HE Li-Jun^˘ŮWANG Xiao-Ming^˘Ů*DING You-Zhong^˘ŮSHAO Min^˘ÚWANG Guo-Hong^˘Ú

XIE Wan-Shu^˘ÚJohn THORBJARNARSON^˘Ű

(˘ŮDepartment of Biology,East China Normal University, Shanghai 200062,China)

(˘ÚAnhui Research Center for Chinese Alligator Reporduction,Xuanzhou 242000,Anhui,China)

(˘ŰWildlife Conservation Society, New York 10460, USA)

Abstract Influence of temperature on egg incubation of the wild Chinese alligator was studied during the summer seasons of 1999 and 2000(July to October and August to September, respectively)at three observation sites, Zhuangtou of Xuanzhou County,Shuangkeng and Zhongqiao of Jingxian county, Anhui Province. One nest at each of the above sites was monitored, bearing 19,17 and 19 eggs,respectively. According to the width of the band on the eggs, we estimated the dates that the eggs were laid. We recorded both the nest temperature and atmosphere temperature with an automated temperature data-logger (HOBO-Temp) during the whole observation period. We found that the average nest temperature maintained around 25-26Ąć during incubation throughout day and night,within over two thirds of time in each day the nest temperature being higher than the atmosphere temperature. Analysis of our data revealed that alligator nests have buffering effect on the inside temperature. Higher nest temperature (27-30Ąć)increased the hatching rate. It was also noticed that the hatching rate varied considerably from site to site(between 0-100%). We conclude that climate is an important factor influencing egg hatching in the wild, and prolonged rainy season which caused large temperature fluctuation can decrease the hatching rate of the alligator eggs.

Key words Chinese alligator(Alligator sinensis),Eggs, Incubation, HOBO-Temp

*E-mail:wxming@public3.sta.net.cn

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Cloning and sequence analysis of SRY-HMG box from raccoon

YANG Xiao-Juan^˘Ů YANG Yu-Hua^˘ŮZHANG Yi-Zheng^˘Ů* CHEN Hong-Wei^˘Ú FEI Li-Song^˘Ú SONG Yun-Fang^˘Ú HE Guang-Xin^˘Ú

(˘Ů Laboratory for Molecular Biology,Sichuan University,Chengdu 610064,China)

(˘ÚChengdu Zoo,Chengdu 610081,China)

Abstract We amplified the 197 bp HMGŞ˛box sequence by using Polymerase Chain Reaction (PCR) with primers according to the known SRY sequence from male raccoon genomic DNA, and then cloned and sequenced. The nucleic acid and amino acid sequence comparison between raccoon and other mammals revealed high conservation of the SRY-HMG box in mammals (about 80%), which implied that the DNA-binding activity was crucial to mediate the action of SRY in sex determination. The variation of raccoon HMG-box sequence mainly occurred on purine by replacement and missense mutation, which implicated that only the HMG-box protein with advanced structure had efficient activity. The homology and difference between mammals' HMG-box was accorded to the evolution systematic tree.

Key words Raccoon (Procyon lotor), SRY HMG-box, PCR sequence analysis

*E-mail: nic3602@scu.edu.cn

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RAPD ANALYSIS OF TEN STRAINS OF MICE

LI Shu-Rong^˘Ů CHEN Yi-Sheng^˘ŮWEI Hong^˘Ú*

(˘Ů Institute of Pathology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China)

(˘Ú Center for Laboratory Animals, Third Military Medical University, Chongqing 400038, China)

Abstract Genetic variation and relationship in ten common strains of mice were studied with RAPD. We attempted to identify DNA polymorphisms that distinguish the ten strains of mice using 80 random primers,21 of the 80 primers generated polymorphic profile among the mice. Each of the strains could easily be distinguished from the others using these polymorphic primers. The results indicated that (1)among the ten strains, the higher genetic distances were detected between DBA/2 and T739,615,C57BL/6J, the genetic distances were 0.162, 0.166,and 0.168 respectively; (2) KM had a closer relationship with TA2,BALB/c,BALB/c-nu, the geneticdistances were 0.060, 0.066 and 0.065 respectively; (3)BALB/c and BALB/c-nu had the closest relationship. These results coincided with the history of origins and breeds suitable for mouse genetic monitoring and strains identification.

Key words Mouse, RAPD, Genetic monitoring, Phylogenetic relationship

*E-mail:weihong@mail.tmmu.com.cn

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