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Neuron navigator 2 overexpression indicates poor prognosis of colorectal cancer and promotes invasion through the SSH1L/cofilin-1 pathway

Background Neuron navigator 2 (NAV2) encodes a member of the neuron navigator gene family, which plays a role in tumorigenesis and cell migration. However, the prognostic value of NAV2 expression in colorectal cancer (CRC) patients and the potential pathway through which NAV2 promotes migration and invasion in CRC cell lines is poorly understood. Methods The expression level of NAV2 was detected in CRC tissues from two different CRC cohorts by immunohistochemistry, qRT-PCR and Western blotting; the correlation between NAV2 expression and clinicopathological characters was analyzed, and the prognostic value of NAV2 expression was analyzed using a Cox regression model. CRC cell lines with NAV2 knocked out were used to validate the function and potential pathway used by NAV2 to promote CRC cell migration and invasion. Results The results showed that NAV2 was overexpressed in CRC tissues, and it was closely correlated with depth of invasion, and lymph and distant metastasis. Multivariate analysis indicated that high NAV2 expression was a poor prognostic indicator of recurrence-free survival and overall survival in CRC patients. Furthermore, Cox regression analysis revealed that high NAV2 expression integrated with high tumor budding grade was a powerful independent predictive factor of CRC clinical outcome. In vitro and in vivo assays demonstrated that knockdown of NAV2 led to reduced migration and invasion of cancer cells, and the process involved the regulation of F-actin polymerization through the SSH1L/cofilin-1 pathway. Conclusion Based on these findings, NAV2 could serve as both a prognostic biomarker and a potential therapeutic target for patients with NAV2-positive CRC. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0237-3) contains supplementary material, which is available to authorized users.

Background
Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide [1]. Many improvements have been made in screening, detection and adjuvant therapy for CRC in recent years [2,3]. However, the long-term survival associated with this malignant disease is not satisfactory due to tumor recurrence and metastasis [4]. To improve the prognosis of CRC patients, there is a great need to identify efficient new targets for effective disease management.
Neuron navigator 2 (NAV2) is one of three members of the neuron navigator family [5]. NAV2 protein contains various functional domains, including a calponin homology (CH) domain, four coiled-coil (CC) domains, a cytoskeletal interacting domain (CSID), and an AAAdomain [6]. The functional domains were shown to be involved in a variety of cellular processes, including protein degradation, signal transduction, regulation of gene expression, membrane fusion, microtubule dynamics and cell migration. It has been proven that NAV2 affects cell migration via adjusting microtubule dynamics [7,8], and microtubule dynamics induces basement membrane breakdown, actin stress fiber formation, metal matrix protease secretion [9]. NAV2 also regulates the cytoskeleton, which affects cell-cell and cell-matrix adhesion, facilitating tumor cell invasion and metastasis [10].
The actin cytoskeleton plays a central role in cell migration by mediating processes such as cell-substrate adhesion, protrusion, phagocytosis and cytokinesis [11]. Cofilin is an essential regulator of actin dynamics, and researchers have revealed that phosphorylation and inactivation of cofilin-1 was capable of blocking actin polymerization and metastasis in cancers [12,13]. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by the cofilinphosphatase, Slingshot (SSH) [13,14]. Meanwhile, the NAV2 homolog, Sickie, regulates actin dynamics via the (SSH)-cofilin pathway [15]. NAV2 was deemed to be one of target genes of APC/Wnt/β-catenin, which is a signaling pathway that plays an essential role in tumor progression according to studies using the CRC cell line SW480 [16]. Moreover, NAV2 was reported to be highly expressed in neuroblastoma cells and uterine endometrial stromal sarcoma [17,18], as well as in a small group of CRC patients [16].
In sum, we can infer that NAV2 is overexpressed in CRC, and it promotes invasion and metastasis via its impact on actin or microtubule dynamics and its involvement in the SSH1L/cofilin-1 pathway. The objectives of this study were to investigate the mRNA and protein expression levels of NAV2 in CRC, the role of NAV2 in CRC cell migration and invasion, the prognostic value of NAV2 expression in CRC patients, and the potential pathways by which NAV2 exerts its influence on actin dynamics.

Clinical specimens and follow-up
Tumor and paired normal mucosa tissues were removed by surgery from 138 CRC patients (cohort 1) at the XiangYa Hospital (Changsha, China) between January 2007 and December 2008. The samples were then embedded in paraffin. Follow-up of patients in cohort 1 was terminated on March 30, 2015. Recurrencefree survival (RFS) was defined as time to any event, irrespective of cause, except for any second primary cancers. Recurrence of or death from the same cancer and all treatment-related deaths or deaths from other causes are events. Second primary same cancers and other primary cancers are ignored, and loss to follow-up is censored. Overall survival (OS) was defined as time to death, irrespective of cause. Local recurrence, distant metastases, second primary colorectal cancers, and second other primary cancers are ignored. Loss to follow-up is censored [19]. Fresh cancerous colorectal and paired normal mucosa tissues from 76 patients (cohort 2) were procured from the Department of General Surgery of XiangYa Hospital of Central South University between December 2013 and August 2014. Paired tissues were tested by real-time qRT-PCR, and 16 paired tissues were selected randomly for Western blotting analysis. Primary tumor and paired metastatic tumor tissues (lymph node, peritoneal metastasis or liver metastasis) from 16 patients were selected for Western blotting analysis. All procedures were in compliance with the ethical guidelines of the XiangYa Hospital. The normal mucosa was excised 5 cm away from the tumor and was confirmed by pathologists for absence of tumor cells. Tumor stage was classified according to the 7th edition of the AJCC TNM staging system for CRC.

Estimation of tumor budding and immunohistochemical staining
Hematoxylin-eosin (H & E) staining was performed on 138 CRC samples. The degree of tumor budding was classified as low-grade or high-grade, corresponding to 0-9 and ≥10 in one 20× field, respectively [20]. The protocol for immunohistochemical staining was performed according to a previous report [17]. The tissue slides were deparaffinized, and antigen retrieval was performed by immersing the slides into boiling EDTA-Tris buffer (pH 8.2) for 4 min. After incubation with 3 % H 2 O 2 for 15 min, slides were incubated with primary antibody (Rabbit polyclonal to NAV2, Abcam, ab101790, 1:1000) overnight (4°C), followed by incubated with secondary antibody for 30 min at room temperature. DAB (3,3-diaminobenzidine) was used to visualize positive immune reaction. Nuclei were counterstained with hematoxylin [21]. Briefly, the sections were scored using a foutier scale according to percentage of positive cells and staining intensity: (−) or 0, tissue specimens without staining (0-10 %); (+) or 1, tissue specimens with weak staining (10-25 %); (++) or 2, tissue specimens with moderate staining (25-50 %) and (+++) or 3, tissue specimens with strong staining (>50 %). (−) and (+) were defined as low expression, and (++) and (+++) were defined as high expression (or over-expression) [22]. The result of staining was evaluated independently by two board-certified clinical pathologists blinded to the clinical parameters. Any discrepancy between the two evaluators was resolved by re-evaluation and careful discussion until agreement was reached.

Cell culture and antibodies
Cell lines NCM460, HRT-18, Caco2, SW620, SW480, HCT-116, LoVo, A549 and HepG2 were purchased from the Cell Centre of Xiang Ya Medicine College of the Central South University (Changsha, China); AGS was obtained from the Xiang ya Central Experiment Laboratory, Central South University. HT-29 and RKO Cells were a gift from Doctor Duan Q (from the Xiang Ya Hospital of Central South University). All cells were cultured in 1640 (Biological Industries, State of Israel). All media were supplemented with 10 % fetal bovine serum (Biological Industries, State of Israel). Cells were cultured at 37°C in humidified air with 5 % CO 2 . Primary antibodies: (NAV2, SSHIL, cofilin1, p-cofilin1, Abcam; β-actin, CST; p-SSHIL, ECM Biosciences).
shRNA transfection NAV2 shRNA and the matching negative control shRNA were purchased from the GENECHEM Company (Shang Hai, China). The shRNAs were transfected into HCT-116 and LoVo according to the instructions. Cells were seeded in six-well plates at a density of 1 × 10 5 cells/well. The transfection reagent and negative control shRNA -transfected cells were used as control.

In vitro motility and invasion
For the scratch wound healing assay, cells were grown to confluence in a 6-well plate and were wounded using a 20 μl sterile pipette tip. Wound healing within the scraped line was documented daily over 36 h, and repeated at least three times in duplicate. The cells' invasive ability was measured in 24-well transwell chambers (Corning, USA). 10 % FBS was added to the bottom chamber as a chemo-attractant. For the invasion assay, Matrigel (1:4, BD Biosciences, USA) was added to the transwell membrane chambers and incubated at 37°C for 4 h. Cells (1 × 10 5 cells per well) were seeded and incubated for 36 h before analysis. Non-migrating cells on the upper side of the membrane were removed, and the cells that migrated to the lower chamber were fixed with 4 % paraformaldehyde and stained with Wright-Giemsa to assess invasion. The number of invading cells was determined for five randomly selected fields under a microscope. Each experiment was performed on at least three occasions in duplicate.

In vivo tumor metastasis
HCT-116 and LoVo cells transfected with shNAV2 and empty vector were suspended in 1640, then injected into the spleen of athymic nude mice (2 × 10 6 cells/ mouse, 5 mice per group). The spleen was resected 30 min after injection. Tumor volume was calculated according to the following formula: length × (width) 2 /2. Mice were sacrificed at day 42. All animals received humane care according to the Institutional Animal Care and Treatment Committee of Central South University.

Statistical analysis
Statistical analysis of the differences in the expression levels of NAV2 mRNA in paired tissues were analyzed by the paired t-test. Correlation between NAV2 expression and clinicopathological factors was estimated by the chi-square test. RFS and OS curves were plotted according to the Kaplan-Meier method; A Cox regression model was used to perform multivariate analysis, parameters of significance in univariate analysis were included in the multivariate analysis. The statistical between ≥2 groups was analyzed by Student's t-test or one-way analysis of variance. The correlation between NAV2 expression levels and tumor budding grades were determined Spearman's correlation analysis. Statistical P-values < 0.05 were considered significant. The statistical software programs used were Prism 5 (Graph Pad Software Inc., La Jolla, CA, USA) and SPSS 13.0 for Windows (SPSS, Chicago, IL).

Results
NAV2 was overexpressed in CRC tissue and associated with the prognostic outcome of CRC patients We investigated the relationship between NAV2 protein or mRNA expression and clinicopathologic features in cohort 1 or cohort 2 ( Table 1; Additional file 1: Table S1). NAV2 mRNA (Fig. 1b, paired t-test, p < 0.0001) and protein (Fig. 1e, paired t-test, p < 0.0001; Additional file 2: Table S2) expression in tumor tissues were higher than that in paired normal tissues. NAV2 expression was significantly correlated with TNM stages in cohort 1 ( Table 1, chi-square test, p < 0.0001) and cohort 2 (Fig. 1d, Student's t-test p < 0.0001), depth of invasion, lymph node status and distant metastasis. However, no significant correlation was found between NAV2 expression and other clinicopathologic variables studied such as age, gender, tumor location, tumor differentiation, adjuvant therapy and CEA. In addition, we found that NAV2 mRNA (Fig. 1c, paired t-test, p = 0.0142) and protein (Fig. 1f, paired t-test, p = 0.0043) expression in metastatic tumor tissue were higher than in paired primary tumor tissue. Based on these findings, we can conclude that high expression of NAV2 was associated with more aggressive biological behavior. To assess the predictive role of NAV2 for prognosis, Kaplan-Meier curves with a logrank test for RFS and OS were undertaken in cohort 1 patients. There was a significant different RFS between NAV2-positive and NAV2-negative groups after the surgery (Fig. 2a, log-rank test, p = 0.0015). NAV2positive tumors had a significantly lower OS rate than those with NAV2-negative tumors (Fig. 2a, log-rank test, p = 0.0004).
The univariate and multivariate analysis for association of prognostic markers of CRC with RFS and OS are summarized in Table 2 (Table 2). In summary, NAV2 and tumor budding were independent prognostic factors that could affected RFS and OS of CRC patients.

Inhibition of NAV2 reduced migration and invasion of CRC cells and tumor metastasis in vitro and vivo
To investigate the expression level of NAV2 in various cell lines including different metastatic potency CRC cells [23][24][25], NAV2 protein expression levels were tested by Western blotting (Fig. 1h). The result showed that NAV2 was more highly expressed in strong metastatic potency cell lines than weak metastatic potency cell lines, and the expression level of metastatic cell line SW460 was higher than that of primary tumor cell line SW480. The efficacy of NAV2 knockdown was confirmed by real-time PCR and Western blot analyses in HCT-116/ LoVo cell lines (Fig. 3a). In the wound healing assay, NAV2 shRNA HCT-116/LoVo cells migrated a much shorter distance into the wound area than the empty vector cells (Fig. 3b). Consistent with this observation, fewer NAV2-silenced cells invaded into the lower chamber than empty vector cells (Fig. 3c). A liver metastatic assay was performed in nude mice to examine the in vivo metastatic potential of NAV2 shRNA HCT-116/LoVo cells and empty vector cells (Fig. 3d). As we can see from the results, the average tumor volume of NAV2 shRNA HCT-116/LoVo cells was smaller than that of the empty vector cells. The average number of both micrometastases and macrometastases in mice liver following injection of NAV2 shRNA cells was fewer than those in mice injected with the empty vector cells as determined by H & E staining. The extent of silencing by the shRNA vector in vivo was verified by Western blotting. In general, these results show that NAV2 could promote migration and invasion of CRC cells.

Knockdown of NAV2 influenced F-actin polymerization through the SSH1L/cofilin-1 pathway
To elucidate the potential pathway through which NAV2 promotes cell invasion, it was necessary to validate the effects of NAV2 on the downstream phosphorylation of SSH1L and cofilin-1 in CRC cells. Here, we found that knockdown of NAV2 dramatically decreased phosphorylation of SSH1L and increased phosphorylation of cofilin-1 compared with control cell lines (Fig. 4a). Cancer cell migration and invasion were associated with actin polymerization [26]. As demonstrated by the results, F-actin polymerization and stress fiber disassembly were decreased in NAV2 shRNA HCT-116/ LoVo cells (Fig. 4b). The expression of p-SSH1L, SSH1L, p-cofilin-1 and cofilin-1 in CRC tissues with different NAV2 expression levels were detected by Western blotting analysis (Fig. 4c). High NAV2 expression level was accompanied by increased phosphorylation of SSH1L and decreased phosphorylation of cofilin-1 compared with low NAV2 expression level.

Discussion
There have been varying results concerning the roles of NAVs in tumorigenesis and tumor progression [17,27,28]. Deletion of NAV3 was detected in colorectal adenomas, cancer tissue and cell lines, the NAV3 aberrations provided two growth advantages to a subpopulation of microsatellite stability CRC linked to inflammation and cell proliferation (See figure on previous page.) Fig. 1 Overexpression of NAV2 in CRC and was associated with more aggressive tumor stage: a Immunohistochemical staining of NAV2 with increased score ranged from stageIto IV in CRC patients from cohort 1. Magnification: ×400; scale bar, 50 μm. NAV2 mRNA level was detected by qRT-PCR in CRC patients from cohort 2 (B-D): b, Tumor tissues (T) were higher than paired normal tissues (NT) (*P < 0.0001); c, Metastatic sites (MS) were higher than primary tumors (PT) (**P = 0.0142); d, Stage III/IV with higher expression level than satgeI/II (***P < 0.0001). Expression levels were normalized to those of β-actin (2 -ΔΔCT ). Western blotting was used to analysis NAV2 protein expression level in CRC patients from cohort 2 (e, f, g), the dates were shown in the Additional file 3: Figure S1. h NAV2 protein expression level in various metastatic potency CRC cells and other cell lines were tested by Western blotting, NAV2 was high expression in high metastatic potency cells, experiments have been repeated three times pathways [29]. Previous studies have highlighted that NAV2 was abundantly expressed in 16 of 20 colon cancers examined but hardly detectable in corresponding non-cancerous mucosae using semi-quantitative RT-PCR, NAV2 showed 3' to 5' helicase activity and exonuclease activity in vitro as an oncogene. The correlation between NAV2 expression and clinical outcomes in human CRC has not been investigated. Here, we show that NAV2 protein and mRNA are highly expressed in primary CRC, taking advantage of diverse detection methods in two different cohorts. Our results are consistent with previous findings [17,18]. Contemporary, Fig. 2 Kaplan-Meier plots with log rank test of Recurrence-free survival and Overall survival. a NAV2-positive tumors had a significantly lower rate Recurrence-free survival (p = 0.0015) and Overall survival (p = 0.0004) than those with NAV2-negative tumors. b A significant increase tumor budding grade ahead of the invasive front was observed with high NAV2 expression compared to low NAV2 expression. Magnification:×400; scale bar, 50 μm. Tumor budding grade ahead of the invasive front was positive correlation with expression level of NAV2 (r = 0.621, p < 0.0001) NAV2 high expression was closely correlated with invasion depth, and lymph node and distant metastases, suggesting that NAV2 not only plays a key role in tumorigenesis but also functions in CRC progression. Multivariate analysis indicates that NAV2 overexpression is a negative prognostic factor in CRC patients in term of the RFS and OS.
Tumor budding is defined as the presence of single tumor cells or small groups of up to five tumor cells at the invasive tumor front [30]. Previous studies have reported that tumor budding often occurs ahead of the defined invasive edge in CRC tissues [31], and the presence of budding was significantly correlated with a higher rate of metastasis and poor prognosis of patients [32,33]. An association between dynamics of cytoskeletal regulation and tumor budding grade was demonstrated in CRC [34]. Meanwhile, AAA+ protein family members, which share homology with one of NAV2 functional protein domains, have also been shown to have increased expression in CRC tumor budding [35]. According to our findings, NAV2 was overexpressed in high tumor budding grade CRC samples, and its expression level was positively correlated with tumor budding grade. Univariate and multivariate analysis indicated that tumor budding served as an independent prognostic marker for RFS and OS in CRC patients. When evaluating NAV2 and tumor budding in combination, patients in the low risk group achieved the longest RFS and OS, whereas those in the high risk group had the shortest RFS and OS. In sum, NAV2 may promote tumor budding generation, but the specific mechanisms require further research.
CRC cells undergo a series of pathologic events during the metastatic process, including invading deep into the intestinal wall, entering into the circulatory or lymphovascular system, arresting at a remote site, proliferation and induction of angiogenesis [36]. Certainly, lymph node and distant metastasis in CRC patients is a negative prognostic factor [37]. In our study, we found that NAV2 expression in lymph node and liver metastases was much higher than in matched primary CRCs, suggesting that NAV2 might be continuously activated throughout CRC development.
In an in vitro study, we confirmed that NAV2 expression level is correlated with metastatic potency in various cell lines, and depletion of endogenous NAV2 in high metastatic potency cell lines, HCT-116 and LoVo, markedly diminished their migratory and invasive abilities. Subsequently, the result of liver metastatic modeling showed that shNAV2 HCT-116 and LoVo cell lines have a These data, in concert with the present findings, support a critical role of NAV2 in regulating invasion and metastasis of CRC. However, so far, the mechanism by which NAV2 promotes tumor cell invasion and metastasis remains unclear, and further studies are needed to elucidate this. Previous researchers reported that NAV2 may act as a downstream target of APC-β-catenin-TCF signaling and was related to the Myc, APC gene [16,38]. The NAV2 gene was found to be frequently fused to the Wnt pathway TCF7L1 gene as a transcription factors involved in CRC reported by the Cancer Genome Atlas Network [39].
(See figure on previous page.) Fig. 3 Knockdown NAV2 inhibited migration and invasion of CRC cells in vitro and vivo. a Inhibition of NAV2 expression level in HCT-116/ LoVo cells were testified by qRT-PCR (One-way analysis, both P < 0.0001) and Western blotting. Experiments have been repeated three times b Wound-healing assay for NAV2 shRNA HCT-116 (Student's t-test, *P = 0.0002) and shRNA LoVo (Student's t-test, **P < 0.0001) compared with empty vector cells. Photographs were taken at 36 h post wounding. Dashed lines indicated the original wound boundaries. Experiments have been repeated three times. c Cell invasion assays, results were presented as the ratios of migrated NAV2 shRNA cells relative to those of empty vector cells HCT-116 (Student's t-test, *P < 0.0001) and LoVo (Student's t-test, **P = 0.0003). Data were representative of three experiments .Original magnification, ×100. d Representative liver metastatic tumors were shown between shRNA cells and empty vector cells; Quantification of the liver macrometastases and micrometastases: shRNA HCT-116 and empty vector cells (Student's t-test, ***P = 0.0003 *P = 0.0006); shRNA LoVo and empty vector cells (Student's t-test, ****P = 0.0023,**P = 0.0008); The average tumor volume of shRNA and the empty vector cells: HCT-116, (Student's t-test, *P = 0.0012); LoVo, (Student's t-test, **P = 0.0004); The extent of silencing by shRNA in vivo was verified by Western blotting (bottom right) Fig. 4 Loss of NAV2 expression decreased the phosphorylation of SSH1L but increased the phosphorylation of cofilin-1. a The expressions of p-SSH1L, SSH1L, p-cofilin-1 and cofilin-1 were detected by Western blotting analysis. Level of p-SSH1L and p-cofilin-1 protein in shRNA and empty vector HCT-116 (Student's t-test, *P < 0.0001, ***P = 0.0006); Level of p-SSH1L and p-cofilin-1 protein in shRNA and empty vector LoVo cells (Student's t-test, **P < 0.0001 ****P < 0.0001). Experiments have been repeated three times. b F-actin polymerization and stress fiber disassembly decreased in NAV2-depleted HCT-116/LoVo cells. Experiments have been repeated three times. c The expressions level of p-SSH1L, SSH1L, p-cofilin-1 and cofilin-1 in CRC tissues with different NAV2 expression level were detected by Western blotting analysis


An Antioxidant Dietary Supplement Improves Brain-Derived Neurotrophic Factor Levels in Serum of Aged Dogs: Preliminary Results

Biological aging is characterized by a progressive accumulation of oxidative damage and decreased endogenous antioxidant defense mechanisms. The production of oxidants by normal metabolism damages proteins, lipids, and nucleotides, which may contribute to cognitive impairment. In this study 36 dogs were randomly divided into four groups and fed croquettes of different compositions for 6 months. We monitored derivatives of reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP) levels in dogs' plasma samples as well as brain-derived neurotrophic factor (BDNF) serum levels at the beginning and at the end of the dietary regime. Our results showed that a dietary regime, enriched with antioxidants, induced a significant decrease of plasma levels of dROMs (p < 0.005) and a significant increase in BDNF serum levels (p < 0.005) after six months. Thus, we hypothesized a possible role of the diet in modulating pro- and antioxidant species as well as BDNF levels in plasma and serum, respectively. In conclusion the proposed diet enriched with antioxidants might be considered a valid alternative and a valuable strategy to counteract aging-related cognitive decline in elderly dogs.

Introduction
Biological aging is characterized by a progressive accumulation of oxidative damage and decreased endogenous antioxidant defense mechanisms [1]. The production of oxidants by normal metabolism damages proteins, lipids, and nucleotides which may contribute to neurodegeneration and, subsequently, cognitive impairment such as Alzheimer's (AD) and Parkinson's diseases (PD) [2,3]. Although the body normally has sufficient protection producing endogenous antioxidants enzymes, such as catalase, glutathione and superoxide dismutase, an imbalance in the pro-oxidant/antioxidant species could increase the risk for lipid peroxidation DNA and protein damage. Oxidative damage also affects neuron function and may contribute to a cognition decline with age. The excess of reactive oxygen species in neuronal level can lead to irreversible damage of cytoskeleton and the microtubular network [4]. Microtubules, important dynamics polar formations of the cytoskeleton, are abundant in neurons, where they provide a scaffold also for their dendrites. Because of oxidative stress conditions the peroxynitrite may react with tyrosine to form 3-nitro-L-tyrosine (3NT). This final product may be selectively incorporated into the -tubulin, resulting in an irreversible blocking of the characteristic dynamics of microtubules causing morphological changes, neuronal death and consequent onset of neurodegenerative diseases. Several experimental evidences have shown the importance of polyunsaturated fatty acids (PUFAs) in human and animal nutrition and development [5]. Further, an omega-3 and omega-6 deficiency likely correlates with the development of behavioral disorder [6].
Thus we hypothesized that a valid alternative and valuable strategy to counteract aging-related cognitive decline and neurodegeneration in aged dogs might be a dietary supplementation with antioxidants. Recent studies of animal models of neurodegenerative diseases suggest that dietary restriction can increase neurons endurance to age-related and disease-specific stresses [7]. Neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can protect neurons against death, as observed for in vivo and in vitro models of acute (stroke, trauma, and seizures) and chronic (Alzheimer's and Parkinson's diseases) neurodegenerative diseases [8].
Our interest in the neurological effects of age stems from a more general concern with the development of a canine model of human cognitive aging. Dogs show age-related pathology similar to that observed in elderly humans, like learning and development impairments [9]. Although the exact mechanisms have not been established so far, recent evidences indicate that a combination of an antioxidantenriched diet with essential fatty acids, like omega-3 and omega-6, can be used to reduce age-dependent impairments and cognitive decline in aged dogs [10]. We focused on BDNF due to its role in supporting the survival and growth of many neuronal subtypes and in mediating the synaptic efficacy, neuronal connectivity and plasticity [11].
We hypothesized that BDNF serum levels might be modulated by an antioxidant-enriched diet.
In humans, BDNF mRNA and protein are decreased in the cortex and hippocampus in mild cognitive impairment (MCI) and AD [12] and are also decreased in cognitive decline [13]. In animals, decreased BDNF serum levels result in a LTP and memory deficit, thus increasing BDNF availability in the brain may be a viable strategy to counteract cognitive decline with aging.
The canine model provides us with an opportunity to test the relationship between cognitive impairment in aging, related to a decrease in BDNF serum levels, and the effects of non-pharmacological interventions.
The aim of this study was to evaluate the effects of a novel dietary supplement endowed with antioxidant properties as an adjuvant in the prevention of oxidative stress conditions and neurodegenerative disorders in aged dogs. The supplement consisted in a mixed formula of fished or chicken proteins, rice carbohydrates, Grifola frondosa, Curcuma longa, Carica papaya, Punica granatum, Aloe vera, Polygonum cuspidatum, Solanum lycopersicum, Vitis vinifera, Rosmarinus officinalis and an Omega 3/6 ratio of 1 : 0.8.
Grifola Frondosa is a culinary-medicinal mushroom that may play an important role in the prevention of many ageassociated neurological dysfunctions, including Alzheimer's and Parkinson's diseases. The mushroom shows neuroprotective, antioxidant and anti-(neuro)inflammatory effects; in fact it reduces beta amyloid-induced neurotoxicity, neurite outgrowth stimulation, and nerve growth factor (NGF) synthesis [14]. It can be considered a useful therapeutic agent in the management and/or treatment of neurodegenerative diseases [15]. It was suggested that there could be an overall improvement in cognitive abilities of subjects when incorporated in their daily diet [16].
Curcuma Longa is a naturally occurring phytochemical compound endowed with powerful free radical scavenging activity [17]. Farinacci et al. observed that curcumin, polyphenol derived from Curcuma longa, reduced neutrophils adhesion and superoxide production in vitro [18]. Moreover, it resulted beneficially in improving spatial attention and motivation deficits associated with impaired cognition in aging and AD, in both humans and dogs [19]. Head et al. observed that 9 aged beagles provided with a medical food cocktail containing 95% of curcuminoids for 3 months had significantly lower error scores and were more accurate across all distances, suggesting an overall improvement in spatial attention [20]. da Rocha et al. demonstrated that C. longa had neuroprotective effects in vitro and in vivo by targeting biochemical pathways associated with neurodegenerative disorders that include cognitive impairments, energy/fatigue, mood and anxiety [21,22].
Carica papaya is a natural compound with a high phenolic and flavonoid content which explains its free radical scavenging and antioxidant potential [22][23][24]. Mehdipour et al. demonstrated its antioxidant effect in vitro and reported a significant decrease in blood lipid peroxidation, while the blood total antioxidant power resulted significantly increased ( < 0.001) [24].
Punica granatum is a plant containing some species of flavonoids and anthocyanins, for example, delphinidin, cyaniding and pelargonidin, which have been shown to have an antioxidant activity in vitro [25] and in vivo [26]. The antioxidant action of P. granatum is related to its free radical scavenging activity against superoxide ions, mainly due to the presence of anthocyanins and to the ability to form metal chelates [25]. Rehydration effects have been also reported [27].
Aloe vera and its extracts have medicinal properties attributed to the active components such as anthrone, chromone, aloe verasin and hydroxylation [28,29]. Sahu et al. demonstrated that Aloe vera had different degrees of antioxidant activity [30]. The antioxidant properties of this plant may depend on the radical scavenging activity. Moreover, life-long dietary supplementation of A. vera was shown to suppress many age-related consequences in vivo. Due to the presence of phenolic acids, polyphenols, sterols, fatty acids and indoles, A. vera may result to be effective in relieving symptoms associated with or preventing neurodegeneration [31].
Polygonum cuspidatum is an important natural source of resveratrol [32]. Due to its numerous pharmacological activities it has been used as an antioxidant [33]. Antioxidant activities of P. cuspidatum have been reported both in vivo and in vitro study [34]. Moreover, three of its dimers, parthenocissin A, quadrangularing A and pallidol, have shown strong free radical quenching and selective singlet oxygen scavenging activity [35]. Trans-resveratrol has been used as antidepressant in chronically stressed rats, probably by acting on the monoaminergic systems, such as the serotonergic and noradrenergic [33]. In fact, chronic treatment with trans-resveratrol was found to inhibit monoamine oxidase-A (MAO-A) activity in all the four brain regions, particularly in the frontal cortex and hippocampus [36].
Solanum lycopersicum is rich of vitamins C and E, lycopene, beta-carotene, lutein and flavonoids such as quercetin, phenols, ascorbic acid (AsA) and dehydroascorbic acid (DHA). It is considered an important plant able to prevent chronic diseases and improve energy balance and antioxidant activity [37]. Both direct and indirect antioxidant activity, as indicated by reduced malondialdehyde (MDA) and nitric oxide (NO) production and increased glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, support the conclusion that tomatoes containing anthocyanins can potentially provide better protection against oxidative stress related chronic diseases [38].
Vitis vinifera can be considered as a potential source of natural antioxidants, due to the presence of carotenoids such as lutein, beta-carotene and polyphenols [39]. The antioxidant activity of grape extracts is due to their reducing power [40]. The grape seed flavanol/procyanidin compounds may act as similar as reductones by donating electrons and reaching with free radicals to convert them to more stable products and terminating the free radical chain reaction [40]. Antioxidant activities of V. vinifera were also assessed by their capacity to prevent Fe 2+ -induced lipid peroxidation in microsomes and their action on Cu 2+ -induced lipid peroxidation in low-density lipoproteins [41]. Astringin, a stilbenoid present in V. vinifera, is endowed with an important antioxidant effect and a higher radical scavenger activity [41].
Rosmarinus officinalis is known to exert an antiproliferative, antioxidant and antibacterial activity [42]. The crude extract also has shown antioxidant and anti-inflammatory activities inhibiting NO production and reducing proinflammatory cytokines (IL-1 ) and enzymes (COX-2) mRNA expression in LPS-activated cells thus highlighting its chemopreventive potential [43]. Afonso et al. showed that phenolic compounds from R. officinalis protected against hypercholesterolemia-induced oxidative stress, increasing the activities of antioxidant enzymes [44]. Several literature reports have demonstrated that R. officinalis exerted multiple benefits for neuronal system and alleviated mood disorders [45]. In particular its active compounds, luteolin, carnosic acid and rosmarinic acid, exhibited neurotrophic effects by improving cholinergic functions [46] and showed neuroprotective properties by inhibiting amyloid precursor protein synthesis and higher brain-derived neurotrophic factor production in hypothalamus cells [45]. Antidepressant-like effect of R. officinalis may be mediated by an interaction with the dopaminergic system, through the activation of dopamine D1 and D2 receptors [47].
Omega 3/6 fatty acids: a good balance of the Omega 3/6 fatty acids ratio in the food is a basic requirement to improve the inflammatory and neurological background [48]. More in detail, n-3 polyunsaturated fatty acids, usually found in fish oil, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are known to both decrease the production of proinflammatory mediators and inhibit natural killer cell activity [49]. Moreover preclinical studies suggested that low plasma omega-6 and omega-3 fatty acids levels were associated with accelerated decline of peripheral nerve function with aging [50]. Intake of PUFAs, mainly omega-3 and omega-6, was shown to increase BDNF production in brain [50,51]. Docking studies on PUFAs and their

Subjects.
Thirty-six dogs of different breeds were randomly and equally divided into four groups based on age and diet (Table 1). First group, made up of 8 dogs (three males and five females, age 9 ± 0.08, mean ± standard error of the mean), was fed a control diet with organic chicken. Second group, made up of 8 dogs (three males and five females, age 7 ± 0.25, mean ± standard error of the mean), was fed a chicken-based food enriched with natural antioxidants. The third group, made up of 8 dogs (four males and four females, age 9 ± 0.63, mean ± standard error of the mean), was fed a fish-based meal and the fourth one was made up of 8 dogs (three males and five females, age 9 ± 0.94, mean ± standard error of the mean) and was fed a fished-based meal enriched with natural antioxidants. d-ROMs and BAP tests (Free Radical Analytical System FRAS 4, H&D s.r.l., Langhirano PR, Italy) were performed before (T0) and at the end (T1) of the treatment in all animals in order to determine the oxidative stress status. The four diets were administered for a six-month period.

Sample Collection and Analysis.
Blood samples were collected from each dog before the new dietary regime (T0) and at the end of the treatment (T1) after six months from cephalic vein and stored in two tubes, one with heparin and the other without anticoagulant. Heparinized plasma samples and serum samples were obtained by blood centrifugation at 4000 g× 1,5 min at 37 ∘ C.
The derivatives of reactive oxygen metabolites (dROMs) and the biological antioxidant potential (BAP), as indicators of oxidative stress, were measured by portable spectrophotometer (Free Radical Analytical System FRAS 4, H&D s.r.l., Langhirano PR, Italy) on plasma samples. In the dROMs test, reactive oxygen metabolites (primarily hydroperoxides) of the sample, in presence of iron released from plasma proteins by an acidic buffer, generate alkoxyl and peroxyl radicals, according to the Fenton reaction. Such radicals then oxidize an alkyl-substituted aromatic amine (N,Ndietylparaphenylendiamine), thus producing a pink-colored derivative which is photometrically quantified at 505 nm [52]. The dROMs concentration is directly proportional to the colour intensity and is expressed as U.CARR (Carratelli Units). One U.CARR corresponds to 0.8 mg/L hydrogen peroxide. The reference values of dROMS are summarized below: (i) reference value 50-90 U.CARR; (ii) threshold borderline: 92-95 U.CARR; (iii) condition of mild oxidative stress: 100-120 U.CARR; (iv) condition of oxidative stress: 140-200 U.CARR; (v) condition of strong oxidative stress: 220-300 U.CARR; (vi) strong oxidative stress: over 300 U.CARR.
In the BAP test plasma samples were added with a colored solution, obtained by mixing a ferric chloride solution with a thiocyanate derivative solution, which causes a discoloration, whose intensity is measured photometrically at 505 nm and it is proportional to the ability of the plasma to reduce ferric ions. The results are expressed as Mol/L of reduced ferric ions. Both tests were validated for canine species [53]. Range values of BAP are listed below:

BDNF Analysis.
BDNF analysis was performed with BG BDNF ELISA kit (Blue Gene Biotech CO., LTD, Shanghai, China) designed for the quantitative determination of canine BDNF. The ELISA test reaction was performed using Crocodile mini Workstation (Totertek Berthold, Pforzheim, Germany). The kit utilizes a monoclonal anti-BDNF antibody and BDNF-HRP conjugate. The assay sample and buffer are incubated together with BDNF-HRP conjugate in precoated plate for one hour. After the incubation period, the wells are washed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the enzymesubstrate reaction forms a blue colored complex. Finally, a stop solution is added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at 450 nm with a microplate reader. The intensity of the color is inversely proportional to the BDNF concentration since BDNF from samples and BDNF-HRP conjugate compete for the anti-BDNF antibody binding site. Since the number of sites is limited, as more sites are occupied by BDNF from the sample, fewer sites are led to bind BDNF-HRP conjugate. A standard curve is then plotted relating the intensity of the color (O.D) to the concentration of standards. The BDNF concentration of each sample is interpolated from this standard curve.

Statistical Analysis.
All data are presented as the mean ± SEM. An unpaired 2-sample Student's -test was used to compare the differences in plasma dROMs, BAP, and BDNF levels between the four groups. All statistical analyses were performed with GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). < 0.05 was considered significant.

Oxidative Stress Status Evaluation.
We firstly analyzed dROMs and BAP levels as a measure of the oxidative stress status of dogs. Analysis was made on dogs plasma in all of the four groups before starting the new dietary regime (T0) and at the end, after six months (T1).
As shown in Figure 1, a significant decrease in plasma levels of dROMs, after six months of feeding regime, in groups 2 and 4 (food supplemented with antioxidant) was observed ( < 0.005). dROMs levels remained unaltered in the first (control group) and third groups (fish-based meal, without antioxidant addition).
These data indicate that a diet enriched with natural antioxidant might be able to promote a decrease of reactive species in plasma of aged dogs.
Antioxidant influence, evaluated with BAP analysis, remained unchanged in all groups of dogs after the diet (Figure 2).
Natural antioxidants seemed to modulate the balance between pro-and antioxidant species through the decrease of dROMs without increasing natural antioxidant defense.

BDNF Evaluation.
Literature reports have shown a decrease in BDNF serum levels to negatively correlate with cognitive decline and deficits in LTP and memory in dogs [13]. BDNF is a neurotrophic factor that can protect neurons against death supporting the survival and growth of many neuronal subtypes [8]. An increase in BDNF serum levels is one of the factors underlying improvements in learning and memory [8,54]. Increasing BDNF availability in the brain, by diet, may be a viable strategy to counteract cognitive decline with aging [7,55,56].
We reported a significant BDNF serum levels increase in groups of dogs that received a diet enriched with antioxidant, < 0.005 (Figure 3), while in the other groups BDNF serum levels remain unchanged.

Discussion
The purpose of this study was to evaluate the possible potential ability of a long-term dietary antioxidant supplementation in controlling the oxidative stress and the general health status of aged dogs. Moreover we demonstrated a possible modulation of BDNF serum levels by a diet with antioxidant supplementation.
Environmental stress and aging may induce psychological stress that possibly influences also the nutrition of pets. An unbalanced diet deficient in essential nutrients may represent a risk factor of degenerative diseases. Among the several mechanisms by which nutrients influence the health status, the balance of oxidative stress has a relevant role. Constantly, in the animal species, metabolic oxidative reactions take place. The goal of these reactions is to balance free radicals production with antioxidants molecules. The inhibitory activity of antioxidant molecules was observed in in vitro studies using plants derivative compounds such as flavonoids, anthocyanins and other poliphenols evaluating their effects on the converting activities ofamylase, -glucosidase and angiotensin-converting enzyme (ACE). They all have shown an inhibitory activity onamylase, -glucosidase and ACE [57].
Furthermore, a significant reduction in dROMs values in the experimental diet enriched with natural antioxidants was observed. In apparently healthy dogs, serum levels of the dROMs ranged between 50 and 90 U.CARR. These values were in agreement with those reported by Pasquini et al. [53]. The antioxidant supplementation significantly decreased dROMs levels from 155 U.CARR (T0) to 120 U.CARR (T1) in the second group and from 150 U.CARR (T0) to 95 U.CARR (T1) in the forth group. Differently, dogs fed the control diet, deficient in antioxidant nutrients, did not modulate the oxidative stress status. The antioxidant status revealed by BAP test was not affected by nutrition and values were at optimal levels throughout the observational period. Probably antioxidant supplements only affected dROMs species but not the endogenous antioxidant components of dogs, which remained in the initial optimal condition. This might be related to the really efficacy of the experimental diet in modulating the oxidative stress.
All these compounds contain anthocyanins and polyphenols with antioxidant effects [7].
With this study we showed a decrease in dROMs species following the administration of the analyzed antioxidant formulation.
Our study reporting the scavenger activity of an antioxidant supplementation in dogs diet is in agreement with other studies about antioxidant effects of singular active principles included into an antioxidant supplementation [58]. The anthocyanins and polyphenols have antioxidant effects in some pathological conditions, such as metabolic disorders, aging-related diseases, cardiovascular diseases cancer, and inflammatory-related disturbs, as well as carotenoids and flavonoids [39,59]. These compounds are inhibitors of lipid peroxidation probably by interfering with the glutathione activity [60]. Resveratrol (3,5,4 -trihydroxystilbene) is a polyphenol naturally present in grapes, berries, peanuts, and other vegetables [61], with therapeutic and neuroprotective functions [7]. Moreover trans-resveratrol, which is highly presented in Polygonum cuspidatum, could modulate BDNF levels through the monoaminergic system activation [36]. In our study we observed that a diet enriched with natural antioxidants was able to increase BDNF serum levels from 100 ± 0.5 pg/mL to 180 ± 0.8 pg/mL, while in the other groups remained unchanged. As to Grifola frondosa, an improvement in cognitive abilities in aged dogs was observed when adding Curcuma longa and Aloe vera to a daily diet through a stimulation of the BDNF synthesis [16]. According to what was observed by Sasaki et al., luteolin, carnosic acid, and rosmarinic acid, from Rosmarinus officinalis, exerted a neuroprotective activity probably modulating the neurotrophic metabolic pathway in the neuronal system [46]. Finally, Kumar et al. observed that PUFAs, like omega-3 and omega-6, along with their metabolites, had more binding affinity towards BDNF [51]. Further, the serum level increase of both fatty acids might be modulated by these metabolites which in turn could regulate the BDNF production in the brain. Neurotrophic factors, such as BDNF, can protect neurons against death and be a preventive approach in neurodegenerative conditions [7,8].
By means of this new canine model of aging, we showed that providing antioxidants within a specific dietary supplement it was possible to restore the balance between proand antioxidants species, possibly modulating also BDNF serum levels. Future studies in both aged humans and dogs will be more effective if antioxidants combinations will be

Conflict of Interests
None of the authors has any financial or personal relationship that could inappropriately influence or bias the content of the paper.


extfieldsofstudy: Biology Medicine

text:
Pseudorhabdosynochus regius n. sp. (Monogenea, Diplectanidae) from the mottled grouper Mycteroperca rubra (Teleostei) in the Mediterranean Sea and Eastern Atlantic

Pseudorhabdosynochus regius n. sp. is described from the gills of the mottled grouper Mycteroperca rubra caught off Senegal, Tunisia and Libya (type-locality: off Dakar, Senegal). The species is distinguished from its congeners by the structure of its sclerotised vagina (length 26–35 μm), which exhibits a trumpet in continuity with the primary canal, a straight primary canal, and primary and secondary chambers included in a common sclerotised mass along the primary canal. The species is also characterised by small squamodiscs (length 20–40 μm) with 10–11 rows of rodlets. Its closest relatives (based on the structure of the sclerotised vagina) are species mostly found in the Mediterranean Sea and parasites on species of Mycteroperca. A second species of Pseudorhabdosynochus Yamaguti, 1958 is reported from the same host and localities but not described. A list of diplectanids from groupers in the Mediterranean Sea is provided. We point out that a recent article was not compliant with the new Article 8.5.3 of the International Code of Zoological Nomenclature; for this reason, three species, P. nhatrangensis Dang, Bristow, Schander & Berland, 2013, P. vietnamensis Dang et al., 2013 and P. brunei Dang et al., 2013, are invalid.

Introduction
The mottled grouper Mycteroperca rubra inhabits a rocky environment in the Mediterranean Sea and along the coast of the Eastern Atlantic Ocean [8]; it is common off Senegal but rare along the North African coast [8,41]. Groupers generally harbour numerous diplectanid monogenean parasites [18,19] and those from the Mediterranean Sea are no exception (Table 1).
We found two species of Pseudorhabdosynochus Yamaguti, 1958 on the gill filaments of M. rubra from Senegal, Tunisia and Libya; these are the first monogeneans reported from this fish. In this paper, we describe the most abundant of these species. The other species, which was rare, will be described when more material enables a full study.

Materials and methods
Five Mycteroperca rubra were obtained from fish markets, including two specimens at Ouakam, Dakar, Senegal (February 2003), two at Sfax, Tunisia (January 2005) and one at Tripoli, Libya (June 2013). In all cases, the fish were dead, but, although their monogenean parasites were in suboptimal condition, they were considered suitable for study. The specimens collected from the fish gills were examined in Petri dishes containing seawater, using a stereomicroscope with incident light. These monogeneans were prepared according to three methods: (a) mounted in ammonium picrate-glycerine [29] (designated as ''p'' with regard to their measurements; (b) mounted in Berlese (designated ''b''); (c) fixed unflattened in ethanol on the gills, then later rehydrated for examination, dehydrated in an ethanol series, stained with carmine, cleared with clove oil and mounted in Canada balsam (unflattened carmine, designated ''uc'').
Monogeneans were drawn with the aid of an Olympus BH2 microscope equipped with a drawing apparatus and DIC optics. The sclerotised parts were measured and designated according to Figure 1. The measurements of the right-hand haptoral hardparts and left-hand equivalents were pooled. All measurements on the drawings were taken with the help of a custom-made transparent rule and are in micrometres. Measurements of the holotype (h) are given separately. Drawings were scanned and redrawn on a computer using Adobe Illustrator. Table 1. Groupers in the Mediterranean (according to [7,8]) and their diplectanid species. The placement of certain host species in Mycteroperca follows recent molecular results [4,40]; some of these species were previously classified within Epinephelus.      Ovary dextral, loops dorsoventrally around right intestinal caecum. Vitelline follicles lateral, coextensive with intestinal caeca and contiguous posterior to testis. Egg inside genital tract, without filament, length uc 108-130 (n = 2), b 100-145 (n = 10).
Sclerotised vagina (nomenclature of parts according to Justine [15]) comprises anterior trumpet, primary canal, and distal sclerotised part, along primary canal, including both primary and secondary chambers. Trumpet in continuity with primary canal, with diameter slightly greater than canal. Primary canal straight, heavily sclerotised, curves just prior to entering primary chamber. Primary chamber heavily sclerotised, with its entrance posterior (i.e. its orientation is opposite to that of primary canal); secondary canal very short; secondary chamber, anterior to and smaller than primary chamber, with same heavily sclerotised structure. Accessory structure not seen. Sclerotised vagina length (measured from tip of trumpet to exterior of bend of primary canal) h 30, uc 29 (27-31, n = 5), p 31 (28-33, n = 7), b31 (26-35, n = 29).

Differential diagnosis
The identification of species of Pseudorhabdosynochus is primarily based on the sclerotised vagina [14,15,17,30]. However, the quadriloculate organ and the haptoral parts, including the squamodiscs, also provide characters useful for species identification [13,14].
The general structure of the sclerotised vagina of P. regius n. sp. has the following characters: the trumpet in continuity with the linear primary canal, the orientation of the primary chamber opposite that of the primary canal, and the primary and secondary chambers grouped within a single heavily sclerotised structure along the primary canal. Other Pseudorhabdosynochus species which have a vaginal structure similar to P. regius are: Pseudorhabdosynochus sosia Neifar & Euzet, 2007 (from Mycteroperca costae). This species can be differentiated by the shape of its trumpet (diameter similar to primary canal in P. regius vs. well differentiated in P. sosia), the shape of the anterior part of its primary canal (straight in P. regius vs. curved in P. sosia), the shape of its primary chamber and the length of the secondary canal (very short in P. regius vs. long in P. sosia). In addition, the two species have very different squamodiscs (central rows V-shaped in P. regius, circular in P. sosia). Pseudorhabdosynochus beverleyburtonae (Oliver, 1984) Kritsky & Beverley-Burton, 1986 (from M. marginata). This species is close to P. sosia and can, therefore, be differentiated from P. regius based on the same characters. Pseudorhabdosynochus enitsuji Neifar & Euzet, 2007 (from M. costae). Its sclerotised secondary chamber is larger than that in P. regius. In addition, the squamodiscs of P. enitsuji have numerous rows of rodlets (14)(15)(16). Pseudorhabdosynochus sinediscus Neifar & Euzet, 2007 (from M. costae). The general structure is strikingly similar in this species, although the secondary chamber is not differentiated in the original description. P. sinediscus is differentiated from all other species of the genus, including P. regius, by the absence of squamodiscs. Pseudorhabdosynochus riouxi (Oliver, 1986) Santos, Buchmann & Gibson, 2000 (from M. marginata). The general structure seems similar in this species, but the shape of the chambers (redescribed by Yang et al. [45]) is different. In addition, P. riouxi has squamodiscs with numerous rows of rodlets (11-22 in the original description). Pseudorhabdosynochus pai Justine & Vignon, 2009 (from E. tauvina (Forsskål) in French Polynesian waters). The general structure is similar, but the primary canal is shorter in P. regius and the shape of the secondary chamber is different and complex (i.e. several secondary chambers) [24]. In addition, P. pai has a huge quadriloculate organ (72-144 inner length) and larger squamodiscs with numerous rows of rodlets (13)(14). The hosts are also widely separated (Central Pacific vs. Atlantic/Mediterranean).

Discussion
We describe a new species of Pseudorhabdosynochus from Mycteroperca rubra in this paper, but also found an additional species. Identification of the second species, which is close to P. sosia and P. beverleyburtonae, will require more material and the examination of comparative material.
Species of Pseudorhabdosynochus are mainly parasites of members of the family Epinephelidae (groupers), including species of Epinephelus, Cephalopholis, Mycteroperca, Variola and Alphestes [19,30] but not Plectropomus [21]. A few species are parasites of members of the closely related Serranidae, including members of Serranus [44], Paralabrax [2], and of a member of the Polyprionidae [15]. A record on a member of the Chaetodontidae [1], never confirmed, is probably due to a mistake in the fish identification. Species of Pseudorhabdosynochus tend to be strictly host-specific, with species often restricted to a single host fish [19], but a few exceptions are known [17,22,30,39]. An interesting aspect of our comparisons of P. regius with similar species, mainly based on the sclerotised vagina, is that most of the closely comparable species are parasitic on members of Mycteroperca (Table 1). This suggests that a group of Pseudorhabdosynochus species, with common vaginal characteristics, parasitises species of Mycteroperca, a genus which was found to be monophyletic in recent molecular studies [4,40]. In contradiction to this hypothesis, P. capurroi Vidal-Martinez & Mendoza-Franco, 1998 from M. bonaci, P. morrhua Justine, 2008 and P. variabilis Justine, 2008, both from M. morrhua, do not share the same vaginal structure [16,43]. Another hypothesis is that the three species of Mycteroperca with similar species of Pseudorhabdosynochus are all from the Mediterranean Sea (and Eastern Atlantic), suggesting that the close relationships of these parasite species reflect a common geographical origin, with the differentiation of various monogenean species in the groupers from the same area. However, a similar vaginal structure is found in P. pai, a parasite of Epinephelus tauvina in the Pacific [24] (i.e. neither a species of Mycteroperca nor Mediterranean), thus negating the two hypotheses mentioned above. We conclude that the relationships of the various species of Pseudorhabdosynochus in the world are complex and resistant to a simple analysis.
M. rubra is common off Senegal but rare along the North African coast [8,41]. Mediterranean populations of groupers were affected by the last glacial period. The presence of the same Pseudorhabdosynochus species in specimens of M. rubra from the Mediterranean Sea and the Atlantic Ocean suggests a new model, which merits investigation using molecular methods.
According to Table 1, the biodiversity of diplectanids in groupers of the Mediterranean appears lower than that observed in groupers from warmer seas and coral reefs [9,15,23,42], where the number of species can reach up to 12 per fish species. However, not all grouper species of the Mediterranean have been studied in detail in terms of their parasite fauna (Table 1).

Nomenclatural validity of three recently described species of Pseudorhabdosynochus
While comparing our specimens with other species, our attention was drawn to a recent paper [5] describing new species of Pseudorhabdosynochus which we checked in terms of its nomenclatural validity.
The recent amendment [11,12] of the International Code of Zoological Nomenclature [10] concerns electronic publication. According to Article 8.5, ''to be considered published, a work issued and distributed electronically must: The descriptions of three new species of Pseudorhabdosynochus from groupers of Vietnam were published in 2013 [5]. The paper was published in the journal International Journal of Aquatic Sciences. The website of this journal (http://www.journal-aquaticscience.com) claims that it is an electronic publication (expressed as ''e-journal'') and no mention of a printed version appears on the website or on the papers themselves. Therefore, the nomenclatural validity of the species described in this journal depends upon compliance with Article 8.5 of the Code, reproduced above. Articles 8.5.1 (after 2011) and 8.5.2 (date of publication indicated in the work itself, as 27 June 2013) are satisfied. However, Article 8.5.3 is not satisfied: there is no mention of a ZooBank number associated with this work. Article 8.5.3.3. describes the cases in which an error can be admitted: ''An error in stating the evidence of registration does not make a work unavailable, provided that the work can be unambiguously associated with a record created in the Official Register of Zoological Nomenclature before the work was published'' (the emboldened ''before'' is our own). We checked (2 December 2014) and found that neither these species nor the paper is indexed in ZooBank. According to the examples illustrating Article 8.5.3.3., even if the registration in ZooBank was performed now or later, the work would be unavailable.
Furthermore, to have its paper registered in ZooBank, the journal needs to meet the criteria enumerated in Articles 8.5.3.1 and 8.5.3.2. Article 8.5.3.1 requires ''the name and Internet address of an organization other than the publisher that is intended to permanently archive the work in a manner that preserves the content and layout, and is capable of doing so'': we found no such repository mentioned on the journal website. This means that if the publisher closes its website, all papers will be lost. Article 8.5.3.2 requires ''an ISSN for the journal containing the work'': the journal does mention an ISSN (2008-8019) but we could not retrieve this ISSN number from online systems such as WorldCat (http:// www.worldcat.org/). Although these details do not specifically concern the case of the species dealt with in the present work, they show, more generally, that the journal itself (International Journal of Aquatic Sciences) could not publish a valid species according to the new ICZN Article 8.5.
Article 11 of the ICZN lists the criteria that make a name available. ''Article 11. Requirements. To be available, a name or, where relevant, a nomenclatural act must satisfy the following provisions: 11.1. Publication. The name or nomenclatural act must have been published, in the meaning of Article 8, after 1757 [10].'' In other words, a work which does not fulfil the criteria of Article 8 (especially, in the case of an electronic publication, Article 8.5. and its new amendment) does not satisfy Article 11 and thus is not published, and the new names of taxa in this work are not available.
To make things clearer, the paper itself [5] is ''published'' in terms of the general vocabulary used for publications, but the work is not published according to the International Code of Zoological Nomenclature [10]. The names of the three new species mentioned in the paper are unavailable, i.e. have not been published according to the International Code of Zoological Nomenclature. They are not valid and cannot be used in any publication which respects the ICZN -i.e. normally, all scientific journals.
Finally, we give here a list of the three names which are unavailable, for the reasons stated above: Pseudorhabdosynochus

Conflict of Interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http:// publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer review process was handled by an Invited Editor, Dominique Vuitton.