Zhenguo Qiao

Identification of Transcriptome-Derived Microsatellite Markers and Their Association with the Growth Performance of the Mud Crab (Scylla paramamosain)

Microsatellite markers from a transcriptome sequence library were initially isolated, and their genetic variation was characterized in a wild population of the mud crab (Scylla paramamosain). We then tested the association between these microsatellite markers and the growth performance of S. paramamosain. A total of 129 polymorphic microsatellite markers were identified, with an observed heterozygosity ranging from 0.19 to 1.00 per locus, an expected heterozygosity ranging from 0.23 to 0.96 per locus, and a polymorphism information content (PIC) ranging from 0.21 to 0.95 per locus. Of these microsatellite markers, 30 showed polymorphism in 96 full-sib individuals of a first generation family. Statistical analysis indicated that three microsatellite markers were significantly associated with 12 growth traits of S. paramamosain. Of these three markers, locus Scpa36 was significantly associated with eight growth traits, namely, carapace length, abdomen width (AW), body height (BH), fixed finger length of the claw, fixed finger width of the claw, fixed finger height of the claw, meropodite length of pereiopod 2, and meropodite length of pereiopod 3 (MLP3) (P<0.05). Locus Scpa75 was significantly associated with five growth traits, namely, internal carapace width, AW, carapace width at spine 8, distance between lateral spine 2 (DLS2), and MLP3 (P<0.05). Locus Spm30 was significantly associated with BH, DLS2, and body weight (P<0.05). Further analysis suggested a set of genotypes (BC at Scpa36, BC and BD at Scpa75, and AC at Spm30) that have great potential in the selection of S. paramamosain for growth traits. These findings will facilitate the development of population conservation genetics and molecular marker-assisted selective breeding of S. paramamosain and other closely related species.

ISOLATION AND CHARACTERIZATION OF A FERRITIN cDNA FROM THE MUD CRAB SCYLLA PARAMAMOSAIN

Abstract Ferritin is an important protein for iron storage in cells. A hepatopancreas cDNA library from the mud crab Scylla paramamosain was constructed using the SMART technique. A complete cDNA sequence that showed high identity with the conserved sequence of the ferritin gene was cloned from the cDNA library and subjected to further investigation. The full-length ferritin gene of Scylla paramamosain (SpFer) consists of 767 bp and contains a complete open reading frame of 513 bp and a 26-bp iron-respective element in the 5′-untranslated region. The gene encodes a polypeptide of 170 amino acids, constituting a predicted molecular weight of 19.44 kDa and a theoretical isoelectric point of 5.24. The deduced protein shares 84% identity with the ferritin protein of the crab Eriocheir sinensis. Quantitative real-time PCR analyses revealed that the expression of ferritin was ubiquitous in different organs of S. paramamosain, including muscle, heart, ovary, testis, and hepatopancreas. The highest expression level was found in the heart, while testis tissue showed the lowest level. Ferritin mRNA expression in continuous developmental stages in zoeal phases, including Z1, Z2, Z3, Z4, and Z5, as well as megalopa and juvenile crab I stages, were also examined by quantitative real-time PCR. The expression level of ferritin was highest in the Z1 stage and lowest in the megalopa stage. This study provides useful information regarding the structure and function of ferritin and will play an important role in immunity and resistance research in S. paramamosain.

Isolation of the C-type lectin like-domain cDNAs from the mud crab Scylla paramamosain Estampador, 1949, and its expression profiles in various tissues, during larval development, and under Vibrio challenge

Scylla paramamosain Estampador, 1949 is one of the most precious marine crabs farmed in China. The crab is prone to infection by microbes at various stages during its growth, leading to high mortality. C-type lectin has a characteristic carbohydrate recognition domain and plays an important role in the immunity system. A cDNA library from the mud crab S. paramamosain was constructed using the SMART technique. Two complete cDNA sequences showing high identities with the C-type lectin gene were isolated from the library. The two full-length C-type lectin cDNAs of S. paramamosain (SpLec1 and SpLec2) consist of 746 and 834 bp, respectively. Quantitative realtime PCR analyses revealed that the C-type lectins were expressed mainly in haemocytes, muscle and hepatopancreas, with the highest expression level found in hepatopancreas. The C-type lectin mRNA expression in continuous developmental stages during zoeal phases were also examined by quantitative real-time PCR. After infection with Vibrio parahaemolyticus (Sakazaki, 1963) at a concentration of 2.3 × 10 6 cfu/ml, the temporal expression of SpLec1 and SpLec2 mRNA in the megalopa stage was first increased, reached a maximum, and then dropped to the original level again. The research of C-type lectins in Scylla paramamosain could shed new light on studies on immunity and moulting in the mud crab.

Two transcripts of HMG-CoA reductase related with developmental regulation from Scylla paramamosain: Evidences from cDNA cloning and expression analysis.

3‐Hydroxy‐3‐methylglutaryl coenzyme‐A (HMG‐CoA) reductases (HMGRs), which catalyze the conversion of HMG‐CoA to mevalonate, may have an important role in the synthesis of methyl farnesoate (MF). In this study, we obtained two HMGR cDNA sequences termed Sp‐HMGR1 (membrane‐bound form) and Sp‐HMGR2 (soluble form), which encode 967 and 654 amino acids, respectively. The two cDNAs possess entirely identical sequences except that Sp‐HMGR1 is 1,382 bp, which encodes a sterol‐sensed domain (SSD; a membrane‐bound domain) and was first found in crustacean HMGR, larger than Sp‐HMGR2. Thus, it was deduced that these cDNAs might be derived from a single genomic DNA sequence. Sp‐HMGRs have the typical features of the HMGR class of proteins. However, residue 844 in Sp‐HMGR1, which is usually occupied by a Ser residue in other species, has an unusual Ala substitution. This Ser is thought to be involved in enzyme activity regulation by reversible phosphorylation. A putative “PEST” sequence that, until now, has only been found in crustacean species was also identified in the C‐terminus of both transcripts, and a sterol‐sensing domain, which was first found in crustacean species, was identified in Sp‐HMGR1; these findings suggest that Sp‐HMGR might function in some special regulatory mechanism. Furthermore, the quantitative real‐time polymerase chain reaction results showed that the two transcripts have different expression patterns; Sp‐HMGR2 was mainly expressed in the mandibular organ (MO) of adult crabs, whereas Sp‐HMGR1 was mainly expressed in other tissues and fertilized eggs up until the fourth juvenile crab stage. The fluctuating gene expression seemed to suggest a relationship between Sp‐HMGRs and the development of the crab, especially during the larval stage. Besides, the fluctuation of Sp‐HMGR1 in ovary, brain, and thoracic ganglia during the ovary development seemed to have some correlation with the nutrition accumulation of ovaries, whether the SSD domain evolved in this process deserve further investigation. Moreover, it remains unclear whether the significant variation in ovary, brain, and thoracic ganglia during ovary development suggests that other tissues in addition to the MO could synthesize MF.

Identification of a trypsin gene from Scylla paramamosain and its expression profiling during larval development

) was 881 bp, with a 780-bp open reading frame encoding a polypeptide of 259 amino acid residues. Comparison with genomic DNA revealed that it contained two introns. The deduced monomer of trypsin had a molecular weight of 28.15 KD and the isoelectric point was 4.49. Alignment analysis and structure prediction showed that

Amino Acid, Fatty Acid, and Metal Compositions in Edible Parts of Three Cultured Economic Crabs: Scylla paramamosain, Portunus trituberculatus, and Eriocheir sinensis

Mud crab, swimming crab, and Chinese mitten crab are the most popular economic crabs cultured in China. However, little knowledge is available about the composition of amino acids, fatty acids, and heavy metals in those cultured crabs, despite the large amounts consumed. Scylla paramamosain, Portunus trituberculatus, and Eriocheir sinensis were, respectively, employed for an investigation of these three crabs. The results on the three representative species revealed that muscle, hepatopancreas, and ovary each had its own compositional pattern. The composition of total amino acids in all the crabs was well balanced. The proportion of flavor amino acid in muscles was higher than that in hepatopancreas and ovary. The composition of total fatty acids showed that two functional unsaturated fatty acids, docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), were both abundant in the three crabs. The ratios of n-3/n-6 polyunsaturated fatty acids (PUFAs) in these crabs also showed that they were rich in n-3 PUFA. Among the seven metals explored, zinc, copper, and chromium were the most abundant. It also deserved attention that these crabs were threatened by high accumulation of cadmium in the edible hepatopancreas, which might arise from the pollution of water used for aquaculture. This data could be used as a reference baseline for monitoring the food properties of the three crabs in levels of amino acids, fatty acids, and metals.