Isolation and characterization of microsatellites in giant freshwater prawn Macrobrachium rosenbergii

Microsatellite loci were characterized in a freshwater prawn from enriched genomic library using six biotinylated probes: (AG)₁₀, (TG)₁₀, (CAA)₁₀, (CAG)₁₀, (GAT)₁₀ and (TAC)₁₀. Primers for DNA amplification were designed and synthesized for 20 loci. Ten loci were polymorphic with the number of alleles ranging from five to 17 alleles per locus and the observed heterozygosity ranging from 0.27 to 0.83 per locus. Developed microsatellite primers should prove useful for selective breeding programs and population genetic studies of freshwater prawn.     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Isolation and characterization of microsatellite loci in giant freshwater prawn, Macrobrachium rosenbergii

This paper reports on isolation and characterization of 8 microsatellite loci from a partial genomic library of Macrobrachium rosenbergii using DIG labeled dinucleotides, (GT)₁₅ and (CT)₁₅ as probes. Primers were designed and PCR conditions optimized for repeat arrays. Polymorphism was studied using 24 individuals collected from the wild. All the loci were polymorphic with number of alleles ranging from 3 to 5 and observed heterozygosity 0.50–0.85. All the loci except one were in agreement with Hardy–Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers should prove useful for characterization of natural population as well as brood stock management of this species.     

Microsatellite loci in the eastern form of the giant freshwater prawn (Macrobrachium rosenbergii)

Six microsatellite loci were identified and characterized in the eastern form of the widespread and commercially important giant freshwater prawn (Macrobrachium rosenbergii). The loci were detected by randomly screening for dinucleotide and trinucleotide repeat units within a partial genomic library developed for the species. In a sample of 29 prawns, number of alleles and heterozygosity per locus ranged from 12 to 18 and from 0.66 to 0.90, respectively. These markers provide powerful tools for the conservation and management of wild stocks, the improvement of cultured stocks of M. rosenbergii, and for investigating evolutionary processes underlying genetic divergence among populations.     

Development of microsatellite markers for blunt snout bream Megalobrama amblycephala using 5'-anchored PCR

A rapid method for isolating microsatellite loci in blunt snout bream, based on the 5'-anchored polymerase chain reaction technique, revealed 522 microsatellite loci (consisting of 442 dinucleotide, 4 trinucleotide and 76 tetranucleotide repeats). Of the 25 loci characterized, 10 turned out to be highly polymorphic. The number of alleles per locus ranged from 3 to 17 while the expected heterozygosity ranged from 0.4899 to 0.9355 in population of selected strain F(7 ) and from 0.5786 to 0.9556 in wild population from Lake Liangzi. These markers are useful as tools for the detection of genetic variation levels in selected strains and wild populations of blunt snout bream for germplasm conservation.     

Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (～ 3 kb long) revealed the presence of tandem and inverted repeats. The ～ 3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ～ 3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.     

Hyperglycemic responses to cold shock in the freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the x-organ sinus gland complex, is primarily involved in carbohydrate metabolism; biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Marked elevations of hemolymph glucose titers, which peaked within 2 h, were observed in both intact and bilaterally eyestalk-ablated prawns, Macrobrachium rosenbergii, when they were transferred directly from their optimal temperature of 28 °C to lower temperatures close to their lethal limit. Hyperglycemia can therefore be considered a characteristic response in this species under cold shock. Involvement of biogenic amines in the hyperglycemic response was also demonstrated. Hyperglycemic effects of epinephrine, dopamine and serotonin were mediated through CHH at the eyestalk level, but the response under cold shock was not exclusively mediated through CHH. It is suggested that factor(s) other than CHH are involved in the hyperglycemic response, possibly norepinephrine or/and octopamine. Accepted: 24 October 1998     

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

Crustacean insulin‐like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N‐linked glycan, was synthesized by the combination of solid‐phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N‐linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin‐like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four‐disulfide insulin‐like peptides.     

Identification and characterization of a hyperglycemic hormone from freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone stored in the sinus gland of eyestalks, primarily regulates carbohydrate metabolism and also plays significant roles in reproduction, molting and other physiological processes. In the freshwater giant prawn, Macrobrachium rosenbergii, an injection of X-organ sinus gland (XOSG) extract evoked a hyperglycemic response, peaked in 1 h. The hyperglycemic effect of the eyestalk extract was maximal at the dose of 0.5 eyestalk equivalent. CHH fractionated by RP-HPLC, in M. rosenbergii was identified by its hyperglycemic activity and partial amino acid sequence, and the molecular weight of 8534 was determined by matrix-assisted laser desorption ionization mass spectrometry--time of flight analysis (MALDI-TOF). The amino acid sequence of the first 25 residues of CHH showed 72% homology with the first 25 residues of CHH A and CHH B of the American lobster Homarus americanus.     

Isolation and characterization of microsatellite loci from three cryptic species of Ceratosolen emarginatus

From an enriched (AG)_n/(AC)_n library, we developed nine polymorphic microsatellite loci for three cryptic species of the fig‐pollinating wasp Ceratosolen emarginatus. We genotyped one population for each of the three cryptic species across all the nine loci. In total, 204 alleles were detected from the three cryptic species of C. emarginatus. The observed heterozygosity was 0.755 ± 0.034, 0.653 ± 0.030 and 0.603 ± 0.073 in C. emarginatus populations A, B and C, respectively; the expected heterozygosity was 0.850 ± 0.031, 0.724 ± 0.035 and 0.702 ± 0.104, respectively. No linkage disequilibrium was found between any two loci of all three cryptic species. The newly isolated microsatellite markers will be very useful for estimating the genetic variation within and among the cryptic species and for revealing the mechanisms of speciation and inbreeding coexistence hypothesis of the cryptic species.     

罗氏沼虾体内两种病毒颗粒的分离、纯化与核酸特性

从患肌肉白浊症状的罗氏沼虾幼苗体内分离纯化得到两种大小不同的病毒颗粒.这两种病毒颗粒均为对称的20面体结构,表面光滑,无囊膜,对氯仿不敏感.一种是直径为26nm～27nm的颗粒,在氯化铯中的密度为132g/cm3,病毒基因组含两段单链的RNA,分别为30kb和12kb,具有诺达病毒科成员的特征.一种是直径为14nm～16nm的颗粒,在氯化铯中的密度为133g/cm3,含有一段大小为09kb的单链RNA,拟为卫星病毒样颗粒或辅助病毒.     

Lead toxicity at different life stages of the giant prawn (Macrobrachium rosenbergii,de Man): considerations of osmoregulatory capacity and histological changes in adult gills

This study evaluated the acute toxicity of lead in different life stages of the freshwater prawn Macrobrachium rosenbergii, determined the effect of sublethal Pb concentrations on osmoregulatory capacity (OC), measured the Pb level in gills, and investigated the effect of Pb on the structure of the gills of adult prawns. The 24-, 48-, and 96-h LC₅₀ values for Pb to M. rosenbergii increased progressively with increasing life stage, from post-larvae (PL), juvenile to adult. The 24- and 48-h LC₅₀ values for post-larvae ranged from 0.01- to 0.09-mg Pb/L. The 24-, 48-, and 96-h LC₅₀ values for Pb were lower at 12 ppt than those at 0 ppt for either the juveniles or the adults. At 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.47-, 0.58-, and 2.03-mg Pb/L, respectively. Meanwhile, at 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.63-, 4.44-, and 7.98-mg Pb/L, respectively. In adults, the OC values of controls and prawns exposed to 2- and 4-mg Pb/L at 0 ppt were not significantly different. The OC of prawns exposed to and 2-mg Pb/L at 12 ppt increased by 72 and 109% from the OC of the control prawns. At media 12 ppt, the OC value of prawns exposed to 1-mg Pb/L was significantly different from that of prawns exposed to 2-mg Pb/L. The concentrations of Pb in gill tissues increased significantly in Pb exposed prawns both at 0 and 12 ppt. The level of Pb in gills of prawns exposed to 2-mg Pb/L at 12 ppt was not significantly different from those exposed at 0 ppt. The severe toxic actions of Pb were noted in gills of prawns exposed in media 12 ppt. Hyperplasia and necrosis were observed in gill lamellae, resulting in abnormal gill tips after Pb exposure at media 12 ppt. Since the effect of Pb is more pronounced in higher salinity (12 ppt) than in freshwater (0 ppt) it is clear that aquaculture of M. rosenbergii should be conducted in freshwater ponds.     

Isolation and characterization of microsatellite loci in the finless porpoise (Neophocaena phocaenoides)

Eight dinucleotide microsatellite loci were isolated from the finless porpoise (Neophocaena phocaenoides). Analysis of 30 individuals showed the number of alleles ranged from four to 21 with observed heterozygosity ranging from 0.300 to 0.833, and expected heterozygosity ranging from 0.437 to 0.932. Cross‐species amplification was tested in four other cetacean species. These microsatellite markers would be valuable tools for population genetic studies of finless porpoises and other cetacean species.     

Cells producing insulin-like androgenic gland hormone of the giant freshwater prawn, Macrobrachium rosenbergii, proliferate following bilateral eyestalk-ablation

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 μm in diameter), stain strongly with hematoxylin-eosin (H&;E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 μm in diameter), stain lightly with H&;E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction.     

An in vitro study of metal ion-induced lipid peroxidation in giant fresh water prawn Macrobrachium rosenbergii (de MAN)

Though metal ions are essential components of many cellular functions, their overexposure to organisms lead to oxidative stress through the formation of reactive oxygen species (ROS). Lipid peroxidation (LPX) is the oxidative deterioration of membrane lipids and considered as an index of oxidative stress. In the present study in vitro effect of various metals (FeCl₃, FeSO₄, CuSO₄, CdCl₂, and ZnSO₄) on the lipid peroxidation of gills and hepatopancreas of Giant Freshwater prawn, Macrobrachium rosenbergii, was compared with respect to dose and duration. The results clearly indicate that among all the metals investigated, FeCl₃ and CdCl₂ are more potent in inducing LPX, and FeCl₃ is more toxic than FeSO₄ in inducing LPX in the hepatopancreas. ZnSO₄ exhibits a moderate toxicity while CuSO₄ is least toxic and also inhibits LPX at higher concentration. Thus results of the present investigation suggest that all the metal ions investigated in the present study are capable of inducing oxidative stress in gills and hepatopancreas of M. rosenbergii     

Isolation and characterization of 18 new polymorphic microsatellite loci for the swordfish, Xiphias gladius

Eighteen microsatellite loci (13 di- and 5 tri-repeats) were isolated from swordfish and characterized in two populations from the Atlantic Ocean and Mediterranean Sea. The number of alleles per locus ranged from two to 29 and the observed heterozygosity from 0.302 to 0.953. All but one locus conformed to Hardy-Weinberg expectations and there was no evidence for linkage disequilibrium between loci.     

Effects of All-trans retinoic acid on germ cell development of embryos and larvae of the giant freshwater prawn, Macrobrachium rosenbergii

Embryos of the giant freshwater prawn, Macrobrachium rosenbergii, were treated with 1, 10 or 50 μg ml⁻¹ all-trans retinoic acid (AtRA) for 2 days. Survival and hatching rates were not affected. However, an increase in the number of primordial germ cells (PGCs), progenitors of gametes, and a slightly more advanced stage of the gonads were found in those treated with 10 or 50 μg ml⁻¹ AtRA. Newly hatched larvae were treated with 0.1, 0.5 or 1 μg ml⁻¹ AtRA for 2 days. Survival rates were lower in those treated with 0.5 or 1 μg ml⁻¹ AtRA; nevertheless, the gonads were slightly more developed. The results indicated that AtRA, an active metabolite of vitamin A, affected germ cell and gonad development of embryos and the larvae of giant freshwater prawn.     

基于锚定PCR技术对10种重要农业害虫微卫星DNA位点的筛选及其特征分析

微卫星DNA广泛存在于真核和原核生物的基因组中,具有多态性丰富、易于检测等特点,在遗传图谱的构建、动植物遗传学育种等方面被广泛应用。利用5′锚定PCR技术对桃小食心虫、桃蛀螟、玉米螟、二点委夜蛾、花蓟马、黄胸蓟马、棕榈蓟马、斑翅果蝇、稻水象甲、扶桑绵粉蚧10种重要农业害虫进行微卫星DNA筛选,并分析各个物种的微卫星DNA的特点。不同物种的阳性克隆率、微卫星比率、克隆效率和冗余率存在不同程度的差异;在核苷酸碱基数目上,主要为二核苷酸重复序列,占重复位点总数的93．2％～100％,三、四核苷酸重复位点较少。在二核苷酸重复位点中, CA/TG重复位点最为丰富,占重复位点总数的89．2％～100％。这与使用的锚定引物密切相关;10种害虫的微卫星DNA平均重复次数为6．7～8．9次,其中玉米螟具有最高的微卫星DNA重复次数（34次）;在序列类型上,完全型序列占上述害虫序列总数的91．0％～100％。5′锚定PCR技术能够快速挖掘害虫的微卫星DNA位点,本研究结果为这些害虫微卫星位点的进一步利用奠定了基础。