The suitability assessment on site selection for bottom seeding scallop culture based on analytic hierarchy process

Scallop culture is an important way of bottom-seeding marine ranching,which is of great significance to improve the current situation of fishery resources.However,there are some problems in site-selection evaluation of marine ranching,such as imperfect criteria system,complex structure,untargeted criteria quantification,etc.In addition,no site-selection evaluation method of bottom-seeding culture areas for scallops is available.Therefore,we established a hierarchy structure model according to the analytic hierarchy process(AHP)theory,in which social,physical,chemical,and biological environments are used as main criteria,and marine functional zonation,water depth,current,water temperature,salinity,substrate type,water quality,sediment quality,red tide,phytoplankton,and zooplankton are used as sub-criteria,on which a multi-parameter evaluation system is set up.Meanwhile,the dualism method,assignment method,and membership function method were used to quantify sub-criteria,and a quantitative evaluation for the entire criteria was added,including the evaluation and analysis of two types of unsuitable environmental situations.By overall consideration in scallop yield,quality,and marine ranching construction objectives,the weight of the main criteria could be determined.Five grades in the suitability corresponding to the evaluation result were divided,and the Python language was used to create an evaluation system for efficient calculation and intuitive presentation of the evaluation outcome.Eight marine cases were simulated based on existing survey data,and the results prove that the method is feasible for evaluating and analyzing the site selection of bottom-seeding culture areas for scallops under various environmental situations.The proposed evaluation method can be promoted for the site selection of bottom-seeding marine ranching.This study provided theoretical and methodological references for the site selection evaluation of other types of marine ranching.

Effects of Temperature on Haemocyte and Granulocyte Counts and Expressions of Immunity-Related Genes in Haemocytes of Scallop Chlamys farreri After Vibrio anguillarum Infection

Bivalve live in aquatic environment and the water temperature can affect their immunity directly.In this research,the scallop Chlamys farreri was injected with 10^4 or 10^7 CFU mL^-1 Vibrio anguillarum and cultured at 11℃,17℃,23℃,and 28℃,respectively.For the control scallop,only phosphate-buffered saline(PBS)was injected.Then total haemocytes and granulocytes were measured by ELISA using monoclonal antibodies.In the meantime,expressions of six immunity-related genes,including lipopolysaccharide andβ-1,3-glucan binding protein(CfLGBP),C-type lectin(CfLec-2),Toll-like receptor(Cf TLR),Lysozyme(CfLYZ),superoxide dismutase(SOD),and phenoloxidase(CfPO)in haemocytes were measured using quantitative real-time PCR.The results showed that total haemocytes counts in 10^4 CFU mL^-1 injection groups showed no differences compared to the control group at all temperatures.However,they varied significantly in 10^7 CFU mL^-1 injection groups at 3 h at 11℃,6–12 h at 17℃,3–48 h at 23℃,and 12–48 h at 28℃.Granulocytes counts in 10^4 CFU mL^-1 injection groups showed no variance compared to the control group at all temperatures,except for 12 h at 23℃,and 24–36 h at 28℃.They were significantly decreased in 10^7 CFU mL^-1 injection groups during 6–48 h at 11℃,12–48 h at 17℃,3–48 h at 23℃,and 3–72 h at 28℃.The expression levels of six immunity-related genes in haemocytes of 10^7 CFU mL^-1 injection groups were significantly higher than those of control group and 10^4 CFU mL^-1 injection groups at all temperatures.The results indicated that infected with high concentration of vibrios,haemocyte counts,granulocyte counts and the expressions of immunity-related genes in scallop C.farreri were significantly affected by environmental temperature.

Effects of hypoxia on survival,behavior,and metabolism of Zhikong scallop Chlamys farreri Jones et Preston 1904

Hypoxia,a frequent occurring threat in coastal regions,often results in mass mortalities of marine organisms and brings a serious ecological problem.The commercially important Zhikong scallop Chlamys farreri is being under such a threat as the risks of eutrophication and hypoxia have risen in their culture areas.However,little information has been known concerning their tolerance to hypoxia and their strategy for survival.In the present study,a 20-day experiment was conducted to determine the ef fects of hypoxia on the survival,behavior,and metabolism of Zhikong scallop.With the LC_(50)for dissolved oxygen(DO)being estimated as 1.8 mg/L,the survival of Zhikong scallop can be greatly challenged even under the moderate hypoxic condition of around 2.0 mg/L DO.The survival rate ranged from 69%to 59%when DO dropped from 3.0 to 2.0 mg/L,and it was further reduced to 47%at 1.5 mg/L DO.In hypoxic conditions,the scallops became significantly active,which may be explained as escape attempts to avoid hypoxic water.To save energy,Zhikong scallop would depress their respiration.However,when DO dropped from 3.0 to 2.0 mg/L,the oxygen consumption rate hardly changed.The upregulation of lactate dehydrogenase activity and the unrepressed phosphofructokinase activity,which often result in the unbalanced cellular homeostasis and energy budget,may account for the observed increase in the mortality rate of Zhikong scallops.In general,Zhikong scallop is sensitive to hypoxia events,though possible escape attempts,depressed respiration,and oxaloacetate-pathway may increase their survival chance.

Mapping Toll-Like Receptor Signaling Pathway Genes of Zhikong Scallop(Chlamys farreri) with FISH

Toll-like receptor(TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop(Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes(Cf TLR, Cf Myd88, Cf TRAF6, Cf NFκB, and Cf IκB) containing bacterial artificial chromosomes(BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species.

POLYCULTURE OF SCALLOP CHLAMYS FARRERI AND KELP LAMINRIA JAPONICA IN SUNGO BAY

Several polyculture models of scallop Chlamys farreri and kelp Laminaria japonica currently employedin Sungo Bay and other parts of northern China are described in this paper. Economic benefits of differ-ent polyculture models are analysed based on the growth rate, culture density and market price. In addi-tion, site selection, critical environmental conditions and polyculture problems are discussed in detail.

Genetic diversity and specific markers in four scallop species, Patinopecten yessoensis, Argopecten irradians, Chlamys nobilis and C.farreri

The AFLP （amplified fragrnent length polymorphism）technique was used to analyse the genetic diversity in four scallop species, Patinopecten yessoensis, A rgopecten irradians, Chlamys nobilis and C. farreri. The genetic similarity indexes of these four species are 0.8415, 0.7863, 0.7190 and 0.6731, while Shannon diversity indexes are 43.52, 58.87, 80.16 and 92.83, respectively. As analyzed, the genetic diversities in two native species, i.e., C, farreri and C. nobilis, are higher than those in other two introduced species, A. irradians and P. yessoensis. The results also showed that C. nobilis and C, farreri shared the most common loci. The genetic distance indicated that C. nobilis and C. farreri are closely related. Moreover, out of 510 AFLP markers, 21 specific bands are found to distinguish the four species scallops and these markers may be applied to the specific germplasm characterization and molecular assistant classification in scallops.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

TETRAPLOID INDUCTION BY INHIBITING MITOSIS I IN SCALLOP CHLAMYS FARRERI

Tetraploid induction was carried out by inhibiting mitosis I in fertilized eggs of Chlamys farreri . Mitosis I was blocked with cold shock (5 7℃), Cytochalasin B (0.75 mg/L) and 6 dimethylaminopurine (6 DMAP) (60 75 mg/L) when 60% fertilized eggs released polar body II at 20℃. At 4 cells embryo stage, the ploidy was determined by counting chromosome number. In control groups, most embryos were diploids (72.22%) and aneuploids (24.78%). In Cytochalasin B, cold shock and 6 DMAP treated groups, tetraploids were respectively 10.51%, 4.08%, and 13.34%; aneuploids were 43.10%, 35.93% and 29.16%,and triploids were 7.84%, 8.52% and 18.33%. At D larva stage, ploidy was determined by flow cytometry (FCM). The ploidy analysis of day 2 larvae showed diploids in control group and also in three treated groups. Juvenile scallops (0.2 0.3cm) which were harvested in two control groups and two CB treated groups were all diploids through checking ploidy individually by FCM.

Effect of Fouling Organisms on Food Uptake and Nutrient Release of Scallop (Chlamys nobilis, Reeve) Cultured in Daya Bay

Biofouling is an important factor that affects the bivalve farming industry. Fouling organisms may reduce growth and survival rate of the cultured species. Fouler are often filter feeders, so they are potential competitors for food resource with the cul- tured species. The present study was conducted to measure the impact of fouling on food uptake and nutrient release in April and June, 2006 in Daya Bay near Guangzhou, China. Results showed that fouling organisms had significant effect on food uptake and nutrient release. The chlorophyll a uptake rate of fouled scallops was 7.53 Lh-1 ± 1.416 Lh-1 and 11.94 Lh-1± 2.497 Lh-1 in April and June, respectively, significantly higher than those of cleaned scallops, i.e., 4.23 Lh-1 ± 2.744 Lh-1 and 2.57 Lh-1 ± 1.832 Lh-1 respectively. The consumption of total particulate matter by fouled scallops in April and June was 5.52 Lh-1 ± 0.818 Lh-1 and 3.07 Lh-1 ± 0.971 Lh-1, respectively; the corresponding results for cleaned scallops are 2.49 Lh-1 ± 0.614 Lh-1 and 2.37 ± 1.214 Lh-1, respectively. Fouling in- creased ammonia release significantly. The ammonia release rate of fouled scallops was 33.81 Lh-1±7.699 Lh-1 and 76.39 Lh-1 ± 9.251 Lh-1 in April and June, while cleaned scallops released 2.46 Lh-1 ± 0.511 Lh-1 and 7.23 Lh-1 ± 1.026 Lh-1 ammonia, respectively. Phos- phate release of fouled scallops was 22.72 Lh-1 ± 9.978 Lh-1 in June and cleaned scallops released phosphate 6.01 Lh-1 ± 0.876 Lh-1 in April. Therefore, fouling contributed much to food reduction and concentration increase of ammonia and phosphate in water.

Size fraction of phytoplankton and the contribution of natural plankton to the carbon source of Zhikong scallop Chlamys farreri in mariculture ecosystem of the Sanggou Bay

The biomass and size fraction of phytoplankton in terms of chlorophyll a（Chl a） was measured during four cruises conducted in April, July, October 2013 and January 2014 in mariculture area, the Sanggou Bay, China.Results show that total Chl a levels in the surface seawater of the Sanggou Bay generally range from 0.10 to 20.46μg/L, with an average value of 2.13 μg/L. Nano-phytoplankton was the most important size-fraction and accounted for about 65.1% of total Chl a. In order to evaluate the importance of the ＂protozoan trophic link＂ for energy transfer from the microbial loop to filter-feeding feeders, Zhikong scallop Chlamys farreri was then offered a natural planktonic community as potential prey. Results show that scallops obtained carbon source from natural plankton with the rate of 11 033.05 μg/（g·d）. Protists（nanoflagellates and ciliates） were the dominant source of carbon retained by scallop（48.78%）. The microbial loop provided 58.45% of the carbon source for farmed scallops. These results indicate that the microbial loop represent a valuable trophic resource in mariculture system of the Sanggou Bay.

EFFECTS OF DIFFERENT COMBINATIONS OF CRYOPROTECTANTS ON SPERMATOZOON VITALITY OF CHINESE SCALLOP,CHLAMYS FARRERI,DURING LOW TEMPERATURE EQUILIBRIUM

The effects of membrane penetrating anti-freezing agents(MPAAs), DMSO(dimethyl sulfoxide),glycerol,EG(ethylene glycol) and methanol in combination with different cryoprotective additives suchas carbohydrates,macromolecules and inorganic compounds on the spermatozoon vitality of Chinesescallop,Chlamys farreri,during 1 h 0℃ equilibrium were investigated.When only MPAAs existed,the detrimental effects of different MPAAs ranked in the following order:DMSO【methanol【EG【glycerol.When carbohydrates were added into MPAAs solution,5% glucose caused larger decrease ofspermatozoon vitality than 2.4% lactose.5% glucose or 2.4% lactose in 7.5% glycerol caused com-plete damage.10% yolk was best in maintaining the spermatozoon vitality except when used incombination with 10% methanol.10% milk significantly decreased spermatozoon vitality in EG andmethanol and enhanced its vitality in glycerol,but did not significantly influence it in DMSO.Glycinewas apparently detrimental to spermatozoon vitality.The average

Association of myostatin Variants with Growth Traits of Zhikong Scallop(Chlamys farreri)

Scallop is a popular sea food and an important aquaculture shellfish.Identification of genes and genetic variants relating to scallop growth could benefit high-yielding scallop breeding.Myostatin(MSTN) is a conservative regulator of muscle growth,and has become one of the most important target genes for genetic improvement of the production of farmed animals.In this study,four single nucleotide polymorphisms(SNPs) were identified in the 5' flanking region of MSTN gene(Cf MSTN) in Zhikong scallop(Chlamys farreri).The association of these SNPs with scallop growth traits,including shell length,shell height,body weight and striated muscle weight was analyzed.The SNP g-1162G>T was found to associate with shell length,shell height,and striated muscle weight.The TT type scallops showed significantly higher trait values than those of GT type,and the GG type individuals exhibited median values.On the contrary,significantly more Cf MSTN transcripts were detected in the striated muscle of GT type scallops than in those of TT and GG type ones.Our results suggested that Cf MSTN might regulate the scallop muscle growth negatively,and SNP g-1162G>T can be used as a candidate marker for the selective breeding of high-yielding scallop.

Purification and Identification of Antioxidant Peptides from the Scallop Skirts of Mimachlamys Nobilis

The processes of scallop meats can produce a large amount of by-products like scallop skirts,which are rich in nutrients. This work aimed to obtain the information about antioxidant peptides derived from scallop skirts of Mimachlamys nobilis. The hydro-lysates obtained by trypsin exhibited the highest antioxidant activity( 75.6%,79.5% and 76.7% using DPPH,hydroxyl and superoxide anion radical scavenging activity assays,respectively). The antioxidant peptides( MNAP) were separated and purified using UF membranes,GFC processes and RP-HPLC,and the antioxidant activity of MNAP was identified to be 85.7% using DPPH radical scavenging activity assay. The peptides( MNAP) were composed of two peptides with their molecular weights2 855.6 Da and 3 007.2 Da using MALDI-TOF-MS,respectively. The results suggested that the scallop skirts of Mimachlamys nobilis could be explored as a source of antioxidant peptides.

Characterization, Expression and Function Analysis of DAX1 Gene of Scallop(Chlamys farreri Jones and Preston 1904) During Its Gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop(Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at m RNA and protein levels. The c DNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame(ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher(P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.

Development of 101 Novel EST-Derived Single Nucleotide Polymorphism Markers for Zhikong Scallop (Chlamys farreri)

Zhikong scallop(Chlamys farreri) is an important maricultured species in China.Many researches on this species,such as population genetics and QTL fine-mapping,need a large number of molecular markers.In this study,based on the expressed sequence tags(EST),a total of 300 putative single nucleotide polymorphisms(SNPs) were selected and validated using high resolution melting(HRM) technology with unlabeled probe.Of them,101(33.7%) were found to be polymorphic in 48 individuals from 4 populations.Further evaluation with 48 individuals from Qingdao population showed that all the polymorphic loci had two alleles with the minor allele frequency ranged from 0.046 to 0.500.The observed and expected heterozygosities ranged from 0.000 to 0.925 and from 0.089 to 0.505,respectively.Fifteen loci deviated significantly from Hardy-Weinberg equilibrium and significant linkage disequilibrate was detected in one pair of markers.BLASTx gave significant hits for 72 of the 101 polymorphic SNP-containing ESTs.Thirty four polymorphic SNP loci were predicted to be non-synonymous substitutions as they caused either the change of codons(33 SNPs) or pretermination of translation(1 SNP).The markers developed can be used for the population studies and genetic improvement on Zhikong scallop.

Ecological effects of co-culturing sea cucumber Apostichopus japonicus (Selenka) with scallop Chlamys farreri in earthen ponds

Monthly changes in sedimentation and sediment properties were studied for three different culture treatments: sea cucumber monoculture (Mc), sea cucumber and scallop polyculture (Ps-c) and scallop monoculture (Ms). Results indicated that the survival rate of sea cucumber was significantly higher in Ps-c cultures than in Mc cultures. Sea cucumber yield was 69.6% higher in Ps-c culture than in Mc culture. No significant differences in body weight and scallop shell length were found between Ps-c and Ms cultures. The mean sedimentation rate of total particulate matter (TPM) was 72.2 g/(m2 d) in Ps-c cultures, with a maximum of 119.7 g/(m2 d), which was markedly higher than that of Mc (mean value). Sedimentation rates of organic matter (OM), total organic carbon (TOC), total nitrogen (TN) and total phosphorus (TP) in Ps-c cultures were also significantly higher than those in Mc cultures. TOC and TN contents of sediment increased rapidly in the first 5 months in Ms cultures and remained at a high level. TOC and TN contents in Mc and Ps-c cultures decreased during sea cucumber feeding seasons and increased during sea cucumber dormancy periods (summer and winter). The study demonstrates that co-culture of sea cucumber and scallop in earthen ponds is an alternative way to alleviate nutrient loads and improve water quality in coastal aquaculture systems. Moreover, it provides the additional benefit of an increased sea cucumber yield.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

Comparative transcriptomics reveals genes involved in metabolic and immune pathways in the digestive gland of scallop Chlamysfarreri following cadmium exposure

Chlamys farreri is an economically important mollusk that can accumulate excessive amounts of cadmium(Cd). Studying the molecular mechanism of Cd accumulation in bivalves is difficult because of the lack of genome background. Transcriptomic analysis based on high-throughput RNA sequencing has been shown to be an efficient and powerful method for the discovery of relevant genes in non-model and genome reference-free organisms. Here, we constructed two c DNA libraries(control and Cd exposure groups) from the digestive gland of C. farreri and compared the transcriptomic data between them. A total of 227 673 transcripts were assembled into 105 071 unigenes, most of which shared high similarity with sequences in the NCBI non-redundant protein database. For functional classification, 24 493 unigenes were assigned to Gene Ontology terms. Additionally, Eu Karyotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes analyses assigned 12 028 unigenes to 26 categories and 7 849 unigenes to five pathways, respectively. Comparative transcriptomics analysis identified 3 800 unigenes that were differentially expressed in the Cd-treated group compared with the control group. Among them, genes associated with heavy metal accumulation were screened, including metallothionein, divalent metal transporter, and metal tolerance protein. The functional genes and predicted pathways identified in our study will contribute to a better understanding of the metabolic and immune system in the digestive gland of C. farreri. In addition, the transcriptomic data will provide a comprehensive resource that may contribute to the understanding of molecular mechanisms that respond to marine pollutants in bivalves.

Immunohistochemical identification of dynorphin A and Kappa opioid receptor-1 in the digestive system of scallop Chlamys farreri

Little is known about the roles of dynorphin and Kappa opioid receptor(KOR) in mollusks. In this study, we aim to determine the distribution of dynorphin A and KOR-1 in the digestive system of the scallop Chlamys farreri. Using immunohistochemical staining, we confirmed the expression of dynorphin A and KOR-1 in the digestive system of C. farreri. Dynorphin A immunopositive cells were identified in intestine and hepatopancreas. In intestine, small and spherical dynorphin A immunopositive cells(4–9 μm in diameter) were scattered among the long columnar epithelial cells(ECs). In hepatopancreas, cells containing masses(5–14 μm in diameter) of dynorphin A immunopositive products were observed in epithelium of acinis. These immunopositive cells may be synthetic and/or secretory cells of dynorphin A. Dynorphin A immunoreactive products were commonly observed in epithelium and connective tissue(CT) of labial palps, mouth labia and stomach, which presented in forms of grains, fibers or flakes. KOR-1 immunoreactive material was observed in ECs and CTs of labial palps, mouth labia and stomach, intestine and hepatopancreas. The distribution of both dynorphin A and KOR-1 in the digestive organs suggests an involvement of dynorphin via KOR-1 in the functional regulation of the digestive system of C. farreri.

Expression Pattern of phb2 and Its Potential Function in Spermatogenesis of Scallop(Chlamys farreri)

Prohibitin(PHB) participates in several biological processes including apoptosis, transcription regulation and suppression of cell proliferation in mammals. In this study, we cloned the full-length c DNA of prohibitin 2(Cf-phb2) from the testis of scallop(Chlamys farreri). The deduced amino acid sequence presented a characteristic of PHB family with the PHB domain, and clustered with PHB2 of other species. Temporal and spatial expression of Cf-phb2 in testis during the reproductive cycle was detected by quantitative real-time PCR(q RT-PCR) and in situ hybridization. The expression of Cf-phb2 in the testis increased when testis developed from the resting stage to mature stage. The m RNA abundance of Cf-phb2 was the highest at mature stage, which was about 15-fold higher than that at proliferative stage. The expression of Cf-phb2 could be detected by in situ hybridization in all types of germ cells in testis, including spermatogonia, spermatocytes, spermatids and spermatozoa. The intensity of the signal increased with the spermatogenesis and was the highest in spermatids, which suggested that CF-PHB2 might affect the spermatogenesis of C. farreri.