Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDSIKK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F. chinensis.     

Molecular characteristics and expression analysis of calreticulin in Chinese shrimp Fenneropenaeus chinensis

Calreticulin (CRT), as an endoplasmic reticulum luminal resident protein, plays important roles in Ca(2+) homeostasis and molecular chaperoning. CRT on the surface of the cell can modulate cell adhesion, phagocytosis and integrin-dependent Ca(2+) signaling. The full length cDNA of calreticulin (FcCRT) was cloned from Chinese shrimp Fenneropenaeus chinensis. It consists of 1672 bp with an open reading frame of 1221 bp, encoding 406 amino acids. This is the first reported cDNA sequence of calreticulin in Crustacea. The deduced amino acid sequence of FcCRT showed high identity with those of Bombyx mori (88%), Drosophila melanogaster (83%), Mus musculus (82%) and Homo sapiens (82%). Highest expression of FcCRT was detected in ovary by Northern blot and in situ hybridization. Different mRNA levels of FcCRT were detected at various molting stages. Expression of FcCRT was induced significantly after 3 h of heat shock treatment, reached the maximum at 4 h and dropped after that. Differential expression profiles of FcCRT were observed in hepatopancreas and haemocytes when shrimp were challenged by white spot syndrome virus (WSSV). From the above results, we inferred that FcCRT might play important roles in Ca(2+) homeostasis, chaperoning and immune function in shrimp.     

Cloning of Hsp21 gene and its expression in Chinese shrimp Fenneropenaeus chinensis in response to WSSV challenge

In the present study, a DNA sequence encoding a small heat shock protein gene (FcHsp21) in the Chinese shrimp, Fenneropenaeus chinensis, was cloned, and its expression was analyzed after white spot syndrome virus (WSSV) infection. The FcHsp21 gene contained an open reading frame (ORF) of 555 bp in length, encoding a 184 amino acid protein with a theoretical size of about 21 kDa and a predicted isoelectric point of 5.38. The mRNA of the Hsp21 had a long Poly(A) tail (748 bp) with six polyadenylation signals (AATAA) downstream from the terminator. In addition, the gene contained a relatively long intron (507 bp), which has not been described in shrimps. The intron contained a long compound type microsatellite repeat sequence. The analysis of the phylogenetics revealed that the Hsp21 was highly conserved among the genomes of animals. Our results show that the expression modes of FcHsp21 can be changed by different WSSV infection methods. The expression of FcHsp21 was inhibited by muscle-injecting WSSV, but induced by feeding WSSV.     

Infectious hypodermal and hematopoietic necrosis,a newly recognized virus disease of penaeid shrimp

Populations of the Pacific blue shrimp, Penaeus stylirostris, reared at the University of Arizona's experimental shrimp culture facility on Oahu in Hawaii from late 1980 through 1981, were severely affected by a highly acute and lethal disease of viral etiology. Also found to be susceptible to the disease were P. vannamei and P. monodon. The disease was named infectious hypodermal and hematopoietic necrosis (IHHN) disease to describe the principal lesions observed. The histopathology of acute and subacute IHHN disease in these species was dominated by the presence of conspicuous eosinophilic intranuclear-inclusion bodies of the Cowdry type A variety in ectodermally (especially the cuticular hypodermis) and mesodermally (especially the hematopoietic tissues) derived tissues that were undergoing necrosis. Electron microscopy of affected tissues demonstrated the presence of two or three types of virus-like particles with cubic morphology and diameters of 17 to 27 nm that suggest IHHN virus to be either a parvo- or picornavirus.     

Histopathology of aflatoxicosis in the marine shrimp Penaeus stylirostris and P. vannamei

The acute and subacute toxicity of aflatoxin B₁ to the marine shrimp Penaeus stylirostris and P. vannamei (Order: Decapoda, Class: Crustacea) was investigated. Experimental shrimp were exposed to a range of concentrations of the toxin directly by intramascular injection (from 2 to 160 μg aflatoxin B₁/g body weight), or by multiple per os dosing with the feed (from 53 to 300 μg aflatoxin B₁/g feed) for up to 25 days. The histopathogenesis of aflatoxicosis in the aflatoxin-exposed animals was followed and found to be time and dose dependent in the hepatopancreas, mandibular organ, and in the hematopoietic organs. Less significant and/or inconsistent lesions were also observed in other organs and tissues, but a time-dose dependency was not noted. The principal lesions of aflatoxicosis in penaeid shrimp occur in the hepatopancreas and the mandibular organ. In the former organ, subacute and acute aflatoxicosis is expressed as necrosis of the hepatopancreatic tubule epithelium that proceeds from the proximal (older) portion of the tubules, in the center of the organ, to the peripheral (younger) tubule tips. A marked intertubular hemocytic inflammation followed by encapsulation and fibrosis of affected tubules follows in subacute aflatoxicosis, but is not as developed as in acute aflatoxicosis. The mandibular organ in aflatoxicosis displays a necrosis of the peripheral epithelial cells of the cords within the gland that progresses proximally to the central vein. Only a slight hemocytic inflammation accompanies the degenerative changes in this latter organ.     

Use of Polymerase Chain Reaction for the Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus in Penaeid Shrimp

A rapid and reliable polymerase chain reaction (PCR) method was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. The oligonucleotide primers amplify a 1681-bp fragment of IHHNV, which encompasses the coding sequence for one of the viral coat proteins. The PCR method detects IHHNV in hemolymph and homogenized tissue obtained from the cephalothorax or pleopods of infected shrimp. The technique was also successfully applied to tissue samples preserved in 70% ethanol. The correct size fragment was amplified using IHHNV obtained from six different geographic regions in three different species of penaeid shrimp. No DNA extraction method was necessary for this technique. The use of hemolymph or pleopods provides a nondestructive screening method by which to test juveniles and adult broodstock for the presence of IHHNV. Received September 21, 1999; accepted January 21, 2000     

The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes

An expressional lack of fibroblast growth factor 9 (FGF9) would cause male-to-female sex reversal in the mouse, implying the essential role of FGF9 in testicular organogenesis and maturation. However, the temporal expression of FGF9 and its receptors during testicular development remains elusive. In this study, immunohistochemistry was used to identify the localization of FGF9 and its receptors at different embryonic and postnatal stages in mice testes. Results showed that FGF9 continuously expressed in the testis during development. FGF9 had highest expression in the interstitial region at 17–18 d post coitum (dpc) and in the spermatocytes, spermatids and Leydig cell on postnatal days (pnd) 35–65. Regarding receptor expression, FGFR1 and FGFR4 were evenly expressed in the whole testis during the embryonic and postnatal stages. However, FGFR2 and FGFR3 were widely expressed during the embryonic testis development with higher FGFR2 expression in seminiferous tubules at 16–18 dpc and higher FGFR3 expression in interstitial region at 17–18 dpc. In postnatal stage, FGFR2 extensively expressed with higher expression at spermatids and Leydig cells on 35–65 pnd and FGFR3 widely expressed in the whole testis. Taken together, these results strongly suggest that FGF9 is correlated with the temporal expression profiles of FGFR2 and FGFR3 and possibly associated with testis development.     

中国明对虾基因组小卫星重复序列分析

通过对中国明对虾基因组随机DNA片断的测序 ,我们获得了总长度约 6 4 10 0 0个碱基的基因组DNA序列 ,从中共找到 172 0个重复序列。其中 ,小卫星序列的数目为 398个 ,占重复序列总数目的 2 3 14 %。这些小卫星序列的重复单位长度为 7- 16 5个碱基 ,集中分布于 7- 2 1个碱基范围内 ,其中以重复单位长度为 12个碱基的重复序列数目最多 ,为 5 8个 ,占小卫星重复序列总数目的 14 5 7%。不同拷贝数目所对应的重复序列的数目情况为 :拷贝数目为 2的重复单位所组成的重复序列数目最多 ,为 137个 ;其次是拷贝数目为 3的重复序列 ,为12 2个 ,且随着拷贝数目的增加 ,由其所组成的重复序列的数目呈递减的趋势。其中一部分序列见GeneBank数据库 ,登录号为AY6 990 72 -AY6 990 76。 398个重复序列分别由 398种重复单位所组成 ,因而小卫星重复序列的类型很多 ,我们初步分成三类 :两种碱基组成类别、三种碱基组成类别和四种碱基组成类别 ,并进一步根据各个重复序列中所含有的碱基种类的数量从大到小排列这些碱基而分成若干小类。从这些分类中可以看出 ,中国明对虾基因组中的小卫星整体上是富含A T的重复序列 ,并具有一定的“等级制度” ,揭示了其与微卫星重复序列之间的关系 ,即一部分小卫星重复序列可能起源于微卫星     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

Cloning and expression pattern of vat-1 homolog gene in zebrafish

The VAT-1 protein is present in the electric organ of marine rays where it is suggested to play a central role in nerve signal transmission. VAT-1 homolog protein was also identified in mouse and human but its function remains to be determined. We have investigated VAT-1 homolog in zebrafish Danio rerio since it is an excellent model amenable to the combination of genetic, molecular and embryological studies. Amino acid sequence analysis shows that the zebrafish VAT-1 homolog shares approximately 51-61% identity with the electric ray, mouse, and human counterparts. By in situ hybridization, vat-1 homolog mRNA is first observed in the trigeminal nuclei at the 8-somite stage. At 20-somite stage, vat-1 homolog is detected in the brain, namely in primary clusters of neurons, in the epiphysis and in the hindbrain. vat-1 homolog is also present in the neural tube but this expression disappears after 72 h post-fertilization. At 24 h post-fertilization, vat-1 homolog starts to be expressed in the developing gut. At later stages, vat-1 homolog is present throughout the brain, appears in the maturing retina and the pharyngeal cavity.     

Temporal distribution and abundance of shrimp postlarvae and juveniles in the mangroves of Muthupet,Tamilnadu, India

The temporal distribution patterns of the predominantly occurring postlarvae and juvenile shrimps in the mangrove and associated habitats of Muthupet, India were investigated for two years from February 1984 to January 1986. Among the eight commercially important species recorded, Penaeus indicus H. Milne Edwards, P. merguiensis De Man, P. monodon Fabricus and Metapenaeus dobsoni (Miers) were predominant. The postlarval recruitment size variedwith species: P. indicus and P. merguiensis recruited at the size of 9–11 mm total length (TL), P. monodon at 12–14 mm TL and M. dobsoni at 4–6 mm TL. The species P. indicus, P. merguiensis and M. dobsoni were observed continuously throughout the study period with maximum abundance occurring from July to September in 1984–85 and from August through October in 1985–86. P. monodon occurred seasonally from November to January in both years. Postlarvae and juvenile catches were low during low salinity and high salinity periods and a higher density was observed in the months of moderate water salinity. Large numbers of P. indicus, P. merguiensis and M. dobsoni clearly showed the preference to the detritus rich muddy substrate, whereas P. monodon did not show any preference and was equally abundant over different substrate types.     

Cloning and expression of a cDNA for the human homolog of mouse T cell and mast cell growth factor P40

A cDNA encoding the human homolog of mouse T-cell and mast cell growth factor P40 was derived from peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin and phorbol myristate acetate. Sequence analysis of the cDNA predicted a precursor protein of 144 amino acids including a signal peptide of 18 residues, a structure identical with that of mouse P40. The homology between the mouse and human proteins is 55% with a perfect conservation of the 10 cysteine residues present in the mature polypeptide. Expression of the cDNA for human P40 in a baculovirus vector yielded a protein capable of enhancing in vitro survival of human T cell lines.