Comparative study of the impact of dietary supplementation with different types of CpG oligodeoxynucleotides (CpG ODNs) on enhancing intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles in Pacific white shrimp (Litopenaeus vannamei).

The CpG oligodeoxynucleotides (CpG ODNs) reportedly possess the capacity to strengthen immunity in mammals. This experiment was conducted to evaluate the impact of dietary supplementation with 17 types of CpG ODNs on intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles of the shrimp Litopenaeus vannamei. Diets including 50 mg kg-1 CpG ODNs wrapped in egg whites were prepared and divided into 17 different groups, with 2 control groups (normal feed and feed with egg whites). These CpG ODNs supplemented diets and the control diets were fed to L. vannamei (5.15 ± 0.54 g) three times daily at 5%-8% shrimp body weight for three weeks. The results of consecutive detection of intestinal microbiota by 16S rDNA sequencing indicated that 11 of the 17 types of CpG ODNs significantly enhanced intestinal microbiota diversity, increased the populations of several probiotic bacteria, and activated possible mechanisms relevant to diseases. The immune-related genes expression and antioxidant capacity in hepatopancreas further demonstrated that the 11 types of CpG ODNs effectively improved the innate immunity of shrimp. Additionally, histology results showed that the CpG ODNs in the experiment did not damage the tissue structure of hepatopancreas. The results suggest that CpG ODNs could be used as a trace supplement to improve the intestinal health and immunity of shrimp.

FOXL2 and DMRT1L Are Yin and Yang Genes for Determining Timing of Sex Differentiation in the Bivalve Mollusk Patinopecten yessoensis.

Sex determination and differentiation have long been a research hotspot in metazoans. However, little is known about when and how sex differentiation occurs in most mollusks. In this study, we conducted a combined morphological and molecular study on sex differentiation in the Yesso scallop Patinopecten yessoensis. Histological examination on gonads from 5- to 13-month-old juveniles revealed that the morphological sex differentiation occurred at 10 months of age. To determine the onset of molecular sex differentiation, molecular markers were screened for early identification of sex. The gonadal expression profiles of eight candidate genes for sex determination or differentiation showed that only two genes displayed sexually dimorphic expression, with FOXL2 being abundant in ovaries and DMRT1L in testes. In situ hybridization revealed that both of them were detected in germ cells and follicle cells. We therefore developed LOG10(DMRT1L/FOXL2) for scallop sex identification and confirmed its feasibility in differentiated individuals. By tracing its changes in 5- to 13-month-old juveniles, molecular sex differentiation time was determined: some scallops differentiate early in September when they are 7 months old, and some do late in December when they are 10 months old. Two kinds of coexpression patterns were found between FOXL2 and DMRT1L: expected antagonism after differentiation and unexpected coordination before differentiation. Our results revealed that scallop sex differentiation co-occurs with the formation of follicles, and molecular sex differentiation is established prior to morphological sex differentiation. Our study will assist in a better understanding of the molecular mechanism underlying bivalve sex differentiation.

cfMSP-1, an extremely acidic matrix protein involved in shell formation of the scallop Chlamys farreri.

Matrix proteins play an important role in biomineralization by mollusks. In this study, we cloned and characterized an acidic protein (pI=3.36) homolog of cfMSP-1 that is highly expressed in the mantle transcriptome of the scallop Chlamys farreri. RT-PCR and in situ hybridization showed that cfMSP-1 is specifically expressed in the outer fold of the mantle edge and pallial part. The expression level of cfMSP-1 remarkably increased and then reduced gradually to a value that is ~2-fold higher than basal levels after shell notching. Knock-down expression of cfMSP-1 in adults via dsRNA injection gave a disordered folia surface. Both shell notching and RNAi experiments indicated that cfMSP-1 plays an essential role in the formation of the folia of C. farreri.

Identification and characterization of four ferritin subunits involved in immune defense of the Yesso scallop (Patinopecten yessoensis).

As a primary iron storage protein, ferritin plays a vital role in iron homeostasis and innate immunity. In this study, four ferritin subunits (PyFer1, PyFer2, PyFer3, and PyFer4) were cloned from the Yesso scallop, Patinopecten yessoensis, by rapid amplification of cDNA ends (RACE) following in silico transcriptome analysis. The full-length cDNAs of the four ferritins are 895, 920, 891, and 1400 bp in length, respectively, and each contains a putative iron response element (IRE) in its 5' UTR. Meanwhile, multiple A+U-destabilizing elements (TATT or ATTTA) are present in the 3' UTRs of PyFer2 and PyFer4. The open reading frames of the four ferritins are 522, 516, 516, and 519 bp, encoding 173, 171, 171, and 172 amino acids, respectively. These proteins have typical ferritin structures, with four long α-helices, one short α-helix and an L-loop. All of the predicted proteins possess both the ferroxidase center of mammalian H ferritins (E25, Y32, E59, E60, H63, E105, and Q139) and the iron nucleation site of mammalian L ferritins (H116, D129, and E132), and the recombinant proteins possess apparent ferroxidase activity. Quantitative real-time PCR analysis revealed that the expression of the four PyFers was significantly elevated at the D-shaped stage and was relatively high in the adult mantle and hepatopancreas. Furthermore, the four PyFers were significantly up-regulated by iron or bacterial challenge, and all four purified recombinant PyFers were able to inhibit the growth of the scallop pathogen Vibrio anguillarum. These results suggest that these PyFers are likely to play important roles in many fundamental biological processes in P. yessoensis, including immune defense, iron homeostasis, and shell development.

Comparative Cytogenetics Analysis of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians with Ct-1 DNA by Fluorescence In Situ Hybridization.

The chromosomes of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians were studied by FISH using C. farreri C(0)t-1 DNA probes. The results showed that C(0)t-1 DNA signals spread on all chromosomes in the three scallops, whereas signal density and intensity were different strikingly. Clustering brighter signals presented in the centromeric and telomeric regions of most C. farreri chromosomes, and in the centromeric or pericentromeric regions of several P. yessoensis chromosomes. Comparative analysis of the mapping indicated a relatively higher homology in the repetitive DNA sequences of the genome between C. farreri and P. yessoensis than that between C. farreri and A. irradians. In addition, FISH showed that the distribution of C(0)t-1 DNA clustering signals in C. farreri displayed completely similar signal bands between homologous chromosomes. Based on the C(0)t-1 DNA fluorescent bands, a more exact karyotype of C. farreri has been obtained. In this study, the comparative analysis based on C(0)t-1 DNA provides a new insight into the chromosomal reconstructions during the evolution process, and it is helpful for understanding an important source of genomic diversity, species relationships, and genome evolution.