Molecular characterization and phylogenetic analyses of MetAP2 gene and protein of Nosema bombycis isolated from Guangdong, China.

Background: Pebrine, caused by microsporidium Nosema bombycis, is a devastating disease that causes serious economic damages to the sericulture industry. Studies on development of therapeutic and diagnostic options for managing pebrine in silkworms are very limited. Methionine aminopeptidase type 2 (MetAP2) of microsporidia is an essential gene for their survival and has been exploited as the cellular target of drugs such as fumagillin and its analogues in several microsporidia spp., including Nosema of honeybees. Methods: In the present study, using molecular and bioinformatics tools, we performed in-depth characterization and phylogenetic analyses of MetAP2 of Nosema bombycis isolated from Guangdong province of China. Results: The full length of MetAP2 gene sequence of Nosema bombycis (Guangdong isolate) was found to be 1278 base pairs (bp), including an open reading frame of 1,077 bp, encoding a total of 358 amino acids. The bioinformatics analyses predicted the presence of typical alpha-helix structural elements, and absence of transmembrane domains and signal peptides. Additionally, other characteristics of a stable protein were also predicted. The homology-based 3D models of MetAP2 of Nosema bombycis (Guangdong isolate) with high accuracy and reliability were developed. The MetAP2 protein was expressed and purified. The observed molecular weight of MetAP2 protein was found to be ~43-45 kDa. The phylogenetic analyses showed that MetAP2 gene and amino acids sequences of Nosema bombycis (Guangdong isolate) shared a close evolutionary relationship with Nosema spp. of wild silkworms, but it was divergent from microsporidian spp. of other insects, Aspergillus spp., Saccharomyces cerevisiae, and higher animals including humans. These analyses indicated that the conservation and evolutionary relationships of MetAP2 are closely linked to the species relationships. Conclusion: This study provides solid foundational information that could be helpful in optimization and development of diagnostic and treatment options for managing the threat of Nosema bombycis infection in sericulture industry of China.

Impact of Dietary Selenium on Modulation of Expression of Several Non-Selenoprotein Genes Related to Key Ovarian Functions, Female Fertility, and Proteostasis: a Transcriptome-Based Analysis of the Aging Mice Ovaries.

Female reproductive (ovarian) aging is characterized by a marked decline in quantity and quality of follicles and oocytes, as well as alterations in the surrounding ovarian stroma. In our previous report, we have shown that dietary selenium (Se) insufficiency and supplementation differentially impacted the reproductive efficiency in aging mice; however, the precise understanding of such modulation is still incomplete. In the present study, we sought to determine the impact of low (mildly low level) and moderately high (medium level) Se diets on expression profile of non-selenoprotein genes in the ovaries of aging mice. For this purpose, the aged mice were divided in two groups and fed either a low Se (Se-L; 0.08 mg Se/kg) diet or a moderately high Se (Se-M; 0.33 mg Se/kg) diet. RNA-seq analysis revealed that a total of 168 genes were differentially expressed between the two groups. From these, 72 and 96 differentially expressed genes (DEGs) were found to be upregulated and downregulated, respectively. Gene Ontology (GO) and pathways enrichment (KEGG) analyses revealed that these DEGs were enriched in several key GO terms and biological pathways including PI3K-Akt signaling pathway, steroid hormone biosynthesis, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ovarian steroidogenesis, and Wnt signaling pathway. Further filtering of RNA-seq data revealed that several DEGs such as Star, Hsd3b6, Scd1, Bmp7, Aqp8, Gas1, Fzd1, and Wwc1 were implicated in key ovarian- and fertility-related functions. In addition, some of the DEGs were related to ER homeostasis and/or proteostasis. These results highlight that dietary low and moderately high (medium level) Se diets, in addition to modulation of selenoproteins, can also have an impact on expression of several non-selenoprotein genes in the ovaries of aging mice. To sum up, these findings add more value to our understanding of Se modulation of ovarian functions and female fertility and will pave a way for the focused mechanistic and functional studies in this domain.

Dietary selenium deficiency and supplementation differentially modulate the expression of two ER-resident selenoproteins (selenoprotein K and selenoprotein M) in the ovaries of aged mice: Preliminary data.

In the present report, we determined the impact of dietary selenium (Se) deficiency and supplementation on the expression of two ER-resident selenoproteins i.e., Selenok and Selenom in the ovaries of aging mice. The mRNA expression of Selenok and Selenom (RT-qPCR) was significantly higher in the ovaries of mice fed diets supplemented with inorganic (ISe-S: 0.33 mg Se/kg) and organic (OSe-S: 0.33 mg Se/kg) Se compared to those fed a Se-deficient (Se-D: 0.08 mg Se/kg) diet and both Se-adequate (ISe-A: 0.15 mg Se/kg and OSe-A: 0.15 mg Se/kg) diets. Similarly, the protein signals of SELENOK (immunofluorescence assay) were also significantly higher in the Se-supplemented groups compared to those fed Se-D and Se-adequate (ISe-A and OSe-A) diets. Meanwhile, the rate of in vitro-produced blastocysts developing from MII oocytes was also evaluated and it was revealed that this rate was significantly higher in the Se-supplemented mice compared to those fed a Se-D diet. Altogether, the dietary Se supplementation increased the expression of Selenok (also its protein expression) and Selenom in the ovaries of aging mice, potentially contributing to an improved developmental potential of in vitro-matured M II oocytes.

Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai.

Eimeria stiedai is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 (EsMIC1) and microneme protein 3 (EsMIC3) from E. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 and EsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages of E. stiedai were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of EsMIC1 (711 bp) and EsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 and EsMIC3 showed the highest mRNA expression levels in the merozoites stage of E. stiedai. Western blotting analysis revealed that both recombinant proteins were recognized by E. stiedai positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post E. stiedai infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both EsMIC1 and EsMIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by E. stiedai.

Efficacy of a chlorocresol-based disinfectant product on Toxocara canis eggs.

Toxocara canis is a common parasite of dogs and can cause zoonotic toxocariasis in humans. As a part of control programs for this agent, optimized hygiene including chemical disinfection is considered essential in the prevention and control of zoonotic toxocariasis in humans. However, commonly used disinfectants at present mostly fail to inhibit the embryogenesis and viability of T. canis eggs. To this effect, the present study was designed to evaluate the effect of a chlorocresol-based disinfectant product Neopredisan®135-1 (NP) on embryonic development of T. canis eggs in vitro and to investigate the infectivity of exposed eggs by assessing larval establishment in a mouse model. Under in vitro conditions, NP at a final concentration of 0.25, 0.50, 1, 2, or 4% all exhibited significant killing effect on T. canis embryogenesis compared with the control eggs (P < 0.05), regardless of contact times (30, 60, 90, or 120 min). Such killing activity increased in a concentration- and time-dependent manner, with a maximum killing efficacy of 95.81% at 4% concentration and 120 min exposure time. Comparisons between low and high concentrations and between short and long contact times concluded that a protocol using the 1% concentration of NP with a 90-min contact could be the most suitable for practical application. Additionally, the lower larval recovery in mice inoculated with eggs treated by either 0.25 or 0.5% NP than that from their corresponding controls (P < 0.05) verified once again that NP had an adverse impact on the larval development of T. canis eggs even at a low concentration. To the best of our knowledge, this is the first study to report the effect of the chlorocresol-based disinfectant NP on the embryonation and larval development of T. canis eggs, and the results presented here would contribute to environmental clearance and control of toxocariasis by providing an alternative disinfectant resource. However, it is highlighted that the clearance of the novel and existing sources of infection including larvated eggs in places treated with NP is not guaranteed and therefore continuous monitoring and additional disinfection are still required.

Molecular and Functional Characterization of Inhibitor of Apoptosis Proteins (IAP, BIRP) in Echinococcus granulosus.

The larval stage of Echinococcus granulosus sensu lato, resulting in cystic echinococcosis, a parasitic zoonosis, causes huge economic losses to the livestock industry and poses a threat to public health. Inhibitor of apoptosis proteins (IAPs) is a class of endogenous anti-apoptotic family, which plays a significant functional role in the regulation of organism's development. Herein, to explore potential functions of IAPs in E. granulosus, two members of IAPs from E. granulosus (Eg-IAP and Eg-BIRP) were cloned, expressed, and molecularly characterized. Eg-IAP and Eg-BIRP encoded putative 331 and 168 residue proteins, respectively. Bioinformatic analysis showed that both proteins contained a type II BIR domain-the essential functional domain of IAPs. Fluorescence immunohistochemistry revealed that both proteins were ubiquitously localized in all life-cycle stages of E. granulosus. Our fluorescent quantitative PCR (RT-qPCR) results revealed relatively higher transcription levels of two Eg-IAPs in protoscoleces (PSCs) compared to the 18-day strobilated worms. We further used different concentrations of LCL161, a Smac-mimetic pan-IAPs inhibitor, to induce the apoptosis in PSCs in vitro, and revealed that the survival rate of PSCs and transcription levels of both genes were negatively correlated with the concentration of LCL161. While the results of light microscopy, transmission electron microscopy (TEM), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay also showed a higher apoptotic rate in PSCs with the increasing concentrations of LCL161. Taken together, our findings provide the reasonable evidence that both Eg-IAP and Eg-BIRP have potential implication in critical anti-apoptotic roles during the development of E. granulosus.

miR-26a is Involved in Glycometabolism and Affects Boar Sperm Viability by Targeting PDHX.

miR-26a is associated with sperm metabolism and can affect sperm motility and apoptosis. However, how miR-26a affects sperm motility remains largely unknown. Our previous study indicated that the PDHX gene is predicted to be a potential target of miR-26a, which is responsible for pyruvate oxidative decarboxylation which is considered as a key step for connecting glycolysis with oxidative phosphorylation. In this study, we first reported a potential relationship between miR-26a and PDHX and their expressions in fresh, frozen-thawed, and epididymal boar sperm. Then, sperm viability and survival were determined after transfection of miR-26a. mRNA and protein expression level of PDHX in the liquid-preserved boar sperm after transfection were also determined by RT-qPCR and Western Blot (WB). Our results showed that expression level of PDHX was significantly increased during sperm transit from epididymal caput to corpus and cauda. Similarly, expression of PDHX was significantly higher (P < 0.05) in fresh sperm as compared to epididymal cauda and frozen-thawed sperm. However, the expression of miR-26a in epididymal corpus sperm was significantly higher (P < 0.05) than that of caput and cauda sperm. Furthermore, after transfection of boar sperm with miR-26a mimic and inhibitor under liquid storage, the lowest and highest sperm viability was observed in miR-26a mimic and inhibitor treatment (P < 0.05), respectively. The protein levels of PDHX, after 24 and 48 h of transfection of miR-26a mimics and inhibitor, were notably decreased and increased (P < 0.05), respectively, as compared to negative control (NC) group. In conclusion, the novel and enticing findings of our study provide a reasonable evidence that miR-26a via PDHX, a link between glycolysis and oxidative phosphorylation, could regulate the glycometabolic pathway which eventually affect boar sperm viability and survival.

Dietary Selenium Supplementation Ameliorates Female Reproductive Efficiency in Aging Mice.

Female reproductive (ovarian) aging is distinctively characterized by a markedly reduced reproductive function due to a remarkable decline in quality and quantity of follicles and oocytes. Selenium (Se) has been implicated in playing many important biological roles in male fertility and reproduction; however, its potential roles in female reproduction, particularly in aging subjects, remain poorly elucidated. Therefore, in the current study we used a murine model of female reproductive aging and elucidated how different Se-levels might affect the reproductive efficiency in aging females. Our results showed that at the end of an 8-week dietary trial, whole-blood Se concentration and blood total antioxidant capacity (TAOC) were significantly reduced in Se-deficient (0.08 mg Se/kg; Se-D) mice, whereas both of these biomarkers were significantly higher in inorganic (0.33 mg/kg; ISe-S) and organic (0.33 mg/kg; OSe-S) Se-supplemented groups. Similarly, compared to the Se-D group, Se supplementation significantly ameliorated the maintenance of follicles and reduced the rate of apoptosis in ovaries. Meanwhile, the rate of in vitro-produced embryos resulting from germinal vesicle (GV) oocytes was also significantly improved in Se-supplemented (ISe-S and OSe-S) groups compared to the Se-D mice, in which none of the embryos developed to the hatched blastocyst stage. RT-qPCR results revealed that mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, p21, and Bcl-2 genes in ovaries of aging mice was differentially modulated by dietary Se levels. A considerably higher mRNA expression of Gpx1, Gpx3, Gpx4, and Selenof was observed in Se-supplemented groups compared to the Se-D group. Similarly, mRNA expression of Bcl-2 and p21 was significantly lower in Se-supplemented groups. Immunohistochemical assay also revealed a significantly higher expression of GPX4 in Se-supplemented mice. Our results reasonably indicate that Se deficiency (or marginal levels) can negatively impact the fertility and reproduction in females, particularly those of an advancing age, and that the Se supplementation (inorganic and organic) can substantiate ovarian function and overall reproductive efficiency in aging females.

Transcriptome-based analysis of putative allergens of Chorioptes texanus.

BACKGROUND: Mites of the genus Chorioptes are non-burrowing and cause mange in a wide range of domestic and wild animals including cattle, horses, sheep, goats, panda, moose, camelids, mydaus and alpacas. Molecular biology and host-parasite interactions of Chorioptes texanus are poorly understood, and only a few C. texanus genes and transcript sequences are available in public databases including the allergen genes. METHODS: Chorioptes texanus RNA was isolated from mites, and the transcriptome of C. texanus was analyzed using bioinformatics tools. Chorioptes texanus unigenes were compared with the allergen protein sequences from the mite allergen database website to predict the potential allergens. Chorioptes texanus putative allergen unigenes were compared with hydrolase genes by building a C. texanus hydrolase gene library with the best match of the homologous sequences. Three allergen genes were cloned and expressed, their recombinant proteins were purified and their allergenic activities were preliminarily investigated. RESULTS: Transcriptome sequencing (RNA-Seq) of C. texanus was analyzed and results demonstrated that 33,138 unigenes were assembled with an average length of 751 bp. A total of 15,130 unigenes were annotated and 5598 unigenes were enriched in 262 KEGG signaling pathways. We obtained 209 putative allergen genes and 34 putative allergen-hydrolase genes. Three recombinant allergen proteins were observed to induce different degrees of allergic reactions on rabbit skin. CONCLUSIONS: The present transcriptome data provide a useful basis for understanding the host-parasite interaction and molecular biology of the C. texanus mite. The allergenic activities of recombinant Euroglyphus maynei 1-like (Eur m 1-like) protein, Dermatophagoides ptreronyssinus 1-like (Der p 1-like) protein and Dermatophagoides ptreronyssinus 7-like (Der p 7-like) protein were preliminarily investigated by intradermal skin test. Meanwhile, differences in eosinophil counts were observed in different injected sites of the skin. The identification of putative allergen genes and hydrolase genes offers opportunities for the development of new diagnostic, prevention and treatment methods.

Comparative analysis of host resistance to Sarcoptes scabiei var. cuniculi in two different rabbit breeds.

BACKGROUND: Scabies, caused by infestation of the mite Sarcoptes scabiei, is one of the most severe ectoparasitic diseases in rabbits. Scabies seriously affects the commercial rabbit breeding, causing severe economic losses. Host resistance to S. scabiei is an important factor in further development of the rabbit industry. In the present study, we compared the host resistance to S. scabiei var. cuniculi of a new breed of domestic rabbit propagated by the Sichuan Animal Sciences Academy (QiXing rabbit, QX) compared with that of a traditional rabbit breed in the domestic rabbit industry (IRA rabbit, IRA). METHODS: Both QX and IRA rabbits were experimentally infested with live S. scabiei var. cuniculi mites for 48 h. Then, during the course of four-week experimental infestation period, the body weight of rabbits was recorded every two weeks for calculating body-weight variations in comparison to the non-infested control rabbits. Skin lesions in the foot area were assessed on weekly basis and serum samples were tested weekly for the estimation of changes in the total antibody levels (IgG, IgE and IgM). Moreover, DNA extracted from the blood samples was amplified for analysis of the genetic diversity in the major histocompatibility complex, class II, DQ Alpha (MHC-DQA) gene. RESULTS: Compared to the IRA rabbits, the QX rabbits showed a significantly higher (P < 0.05) relative body weight gain compared to the non-infested control rabbits and significantly lower (P < 0.05) scores for foot skin lesions and higher levels of IgG, IgE and IgM at weeks 1 to 4, week 2 and week 1 post-infestation, respectively. Furthermore, a polymorphism site at position 103 bp of exon two of MHC-DQA gene and a different gene frequency were found between two rabbit breeds, suggesting the genetic basis for the differential host resistance to the S. scabiei var. cuniculi between two rabbit breeds. CONCLUSIONS: The QX rabbits showed higher host resistance to S. scabiei var. cuniculi compared to the IRA rabbits at the clinical, immunological and genetic levels. These results provide a reference for the breeding of rabbits with adequately improved and sustained host resistance to scabies in the domestic rabbit industry.

Implication of Polyhistidine, a Novel Apoptosis Inhibitor, in Inhibiting Lipopolysaccharide-Induced Apoptosis in Boar Sperm.

Lipopolysaccharide (LPS) released from Gram-negative bacteria binds to toll-like receptor 4 (TLR4) and induces boar sperm apoptosis. Similarly, polyhistidine (pHis), a TLR4 agonist, can also bind to TLR4. We hypothesized that pHis could inhibit LPS-induced sperm apoptosis by competitively binding to TLR4 to then improve sperm quality. Therefore, the objective of this study was to examine whether pHis can inhibit LPS-induced sperm apoptosis and affect sperm quality. The results showed that the concentrations of bacterial colonies were significantly increased from 36 to 120 h under liquid storage conditions (p < 0.05); however, concentrations of LPS in boar semen showed a relatively constant trend (4.98 ± 1.55 EU/mL) following 120 h storage. The addition of 100 µg/mL pHis in the BTS extender significantly improved boar sperm motility and viability at 37 °C, and it significantly (p < 0.05) inhibited boar sperm apoptosis under liquid storage (17 °C) and at 37 °C incubation conditions. The co-treatment of LPS and pHis further confirmed that pHis played its role in inhibiting LPS-induced sperm apoptosis. In conclusion, our preliminary findings provide reasonable evidence that pHis could act as an inhibitor of LPS-induced apoptosis in boar sperm stored for longer periods of time. pHis might inhibit LPS-induced sperm apoptosis by competitively binding to TLR4. Nevertheless, further mechanistic studies are awaited to fully elucidate its potential implication in inhibiting LSP-induced apoptosis.

Role of Selenium and Selenoproteins in Male Reproductive Function: A Review of Past and Present Evidences.

Selenium (Se) is an important trace mineral having many essential roles at the cellular and organismal levels in animal and human health. The biological effects of Se are mainly carried out by selenoproteins (encoded by 25 genes in humans and 24 in mice). As an essential component of selenoproteins, Se performs structural and enzymic roles; in the latter context it is well known for its catalytic and antioxidative functions. Studies involving different animal models have added great value to our understanding regarding the potential implications of Se and selenoproteins in mammalian fertility and reproduction. In this review, we highlight the implications of selenoproteins in male fertility and reproduction followed by the characteristic biological functions of Se and selenoproteins associated with overall male reproductive function. It is evident from observations of past studies (both animal and human) that Se is essentially required for spermatogenesis and male fertility, presumably because of its vital role in modulation of antioxidant defense mechanisms and other essential biological pathways and redox sensitive transcription factors. However, bearing in mind the evidences from mainstream literature, it is also advisable to perform more studies focusing on the elucidation of additional roles played by the peculiar and canonical selenoproteins i.e., glutathione peroxidase 4 (GPX4) and selenoprotein P (SELENOP) in the male reproductive functions. Nevertheless, search for the elucidation of additional putative mechanisms potentially modulated by other biologically relevant selenoproteins should also be included in the scope of future studies. However, as for the implication of Se in fertility and reproduction in men, though a few clinical trials explore the effects of Se supplementation on male fertility, due to inconsistencies in the recruitment of subjects and heterogeneity of designs, the comparison of such studies is still complicated and less clear. Therefore, further research focused on the roles of Se and selenoproteins is awaited for validating the evidences at hand and outlining any therapeutic schemes intended for improving male fertility. As such, new dimensions could be added to the subject of male fertility and Se supplementation.

Exploration of miRNA and mRNA Profiles in Fresh and Frozen-Thawed Boar Sperm by Transcriptome and Small RNA Sequencing.

Due to lower farrowing rate and reduced litter size with frozen-thawed semen, over 90% of artificial insemination (AI) is conducted using liquid stored boar semen. Although substantial progress has been made towards optimizing the cryopreservation protocols for boar sperm, the influencing factors and underlying mechanisms related to cryoinjury and freeze tolerance of boar sperm remain largely unknown. In this study, we report the differential expression of mRNAs and miRNAs between fresh and frozen-thawed boar sperm using high-throughput RNA sequencing. Our results showed that 567 mRNAs and 135 miRNAs were differentially expressed (DE) in fresh and frozen-thawed boar sperm. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the majority of DE mRNAs were enriched in environmental information processing such as cytokine-cytokine receptor interactions, PI3K-Akt signaling, cell adhesion, MAPK, and calcium signaling pathways. Moreover, the targets of DE miRNAs were enriched in significant GO terms such as cell process, protein binding, and response to stimuli. In conclusion, we speculate that DE mRNAs and miRNAs are heavily involved in boar sperm response to environment stimuli, apoptosis, and metabolic activities. The differences in expression also reflect the various structural and functional changes in sperm during cryopreservation.

Selenium, Selenoproteins, and Female Reproduction: A Review.

Selenium (Se) is an essential micronutrient that has several important functions in animal and human health. The biological functions of Se are carried out by selenoproteins (encoded by twenty-five genes in human and twenty-four in mice), which are reportedly present in all three domains of life. As a component of selenoproteins, Se has structural and enzymatic functions; in the latter context it is best recognized for its catalytic and antioxidant activities. In this review, we highlight the biological functions of Se and selenoproteins followed by an elaborated review of the relationship between Se and female reproductive function. Data pertaining to Se status and female fertility and reproduction are sparse, with most such studies focusing on the role of Se in pregnancy. Only recently has some light been shed on its potential role in ovarian physiology. The exact underlying molecular and biochemical mechanisms through which Se or selenoproteins modulate female reproduction are largely unknown; their role in human pregnancy and related complications is not yet sufficiently understood. Properly powered, randomized, controlled trials (intervention vs. control) in populations of relatively low Se status will be essential to clarify their role. In the meantime, studies elucidating the potential effect of Se supplementation and selenoproteins (i.e., GPX1, SELENOP, and SELENOS) in ovarian function and overall female reproductive efficiency would be of great value.

Molecular Characterization of Annexin B2, B3 and B12 in Taenia multiceps.

Coenurus cerebralis, the metacestode of Taenia multiceps, causes coenurosis, a disease severely affecting goat, sheep, cattle and yak farming and resulting in huge economic losses annually. Annexins bind calcium ions and play an important role in flatworm parasite development. To explore potential functions of annexins in T. multiceps, three homologous genes, namely, TmAnxB2, TmAnxB3 and TmAnxB12, were screened from the transcriptome dataset, amplified from C. cerebralis cDNA and subjected to bioinformatics analysis. Then, polyclonal antibodies recognizing the recombinant TmAnxB2 (rTmAnxB2) and rTmAnxB3 were prepared for localization of TmAnxB2 and TmAnxB3 in different tissues and developmental stages by immunofluorescence. The transcription of all three genes was also measured by relative fluorescent quantitative PCR. The sizes of rTmAnxB2, rTmAnxB3 and rTmAnxB12 were 58.00, 53.06 and 53.51 kDa, respectively, and rTmAnxB12 was unstable. Both rTmAnxB2 and rTmAnxB3 were recognized by goat-positive T. multiceps sera in Western blots. Immunofluorescence revealed that TmAnxB2 and TmAnxB3 were localized in the protoscolex and cyst wall and TmAnxB3 was also detected in adult cortex. TmAnxB2 and TmAnxB12 mRNA levels were determined to be highest in oncospheres and protoscolex, whereas transcription of TmAnxB3 was highest in scolex and immature segments. Taken together, these findings indicate that TmAnxB2 and TmAnxB12 may play critical roles in T. multiceps larvae, while TmAnxB3 may have important functions in adults. These results will lay the foundation for functional research of annexins in T. multiceps.