Male germ cell proliferation and apoptosis in sexually immature meagre Argyrosomus regius (Asso, 1801) treated with recombinant follicle stimulating hormone

The meagre Argyrosomus regius (Asso, 1801) is a marine fish species that has an increasing aquaculture production in Europe. Lowering the age at maturity of hatchery-produced juveniles would support meagre aquaculture by reducing time between generations in selective breeding programs and reducing industrial costs for broodstock maintenance. The aim of this work was to assess the effects of a treatment with recombinant follicle stimulating hormone (rFsh), produced in ovarian cells of Chinese hamsters, on male germ cell proliferation and apoptosis in sexually immature meagre. The rFsh-treated fish had higher gonadosomatic index, larger seminiferous tubules, more abundant luminal spermatozoa, a lower density of anti-PCNA positive single A spermatogonia, a higher density of anti-PCNA positive spermatocysts and a lower incidence of germ cell apoptosis than control groups. The present study demonstrated the effectiveness of the produced rFsh in stimulating testis development and spermatogenesis in pre-pubertal meagre. Moreover, the rFsh treatment proved to be highly efficient in removing the apoptotic block of spermatogenesis observed in juvenile meagre, allowing spermatogonial survival and progress towards meiosis. The administration of rFsh did not stimulate spermatogonial self-renewal, a process whose control still needs to be elucidated.info:eu-repo/semantics/publishedVersio

Legionella and legionellosis in touristic-recreational facilities. Influence of climate factors and geostatistical analysis in Southern Italy (2001-2017)

Legionella is the causative agent of Legionnaires' disease, a flu-like illness normally acquired following inhalation or aspiration of contaminated water aerosols. Our recent studies revealed that climatic parameters can increase the number of reported cases of community-acquired Legionnaires' disease. Here, we evaluated the presence of Legionella in water networks and the distribution of Legionnaires' disease cases associated with touristic-recreational facilities in the Apulia region (southern Italy) during the period 2001-2017 using geostatistical and climatic analyses. Geostatistical analysis data revealed that the area with the highest concentration of Legionella in water systems also had the greatest number of cases of Legionnaires' disease associated with touristic-recreational facilities. Climatic analysis showed that higher daily temperature excursion (difference between maximum and minimum temperature) on the day of sampling was more often associated with Legionella-positive samples than Legionella-negative samples. In addition, our data highlighted an increased risk of Legionnaires' disease with increases in precipitation and average temperature and with decreases in daily temperature excursion (difference between maximum and minimum temperature over the course of 24 h in the days of incubation period of disease) and minimum temperature. Healthcare professionals should be aware of this phenomenon and be particularly vigilant for cases of community-acquired pneumonia during such climatic conditions and among the tourist population. The innovative geo-statistical approach used in this study could be applied in other contexts when evaluating the effects of climatic conditions on the incidence of Legionella infections

Management of Microbiological Contamination of the Water Network of a Newly Built Hospital Pavilion

The good installation, as well as commissioning plan, of a water network is a crucial step in reducing the risk of waterborne diseases. The aim of this study was to monitor the microbiological quality of water from a newly built pavilion before it commenced operation. Overall, 91 water samples were tested for coliforms, Escherichia coli, enterococci, Pseudomonas aeruginosa and Legionella at three different times: T0 (without any water treatment), T1 (after treatment with hydrogen peroxide and silver ions at initial concentration of 20 mg/L and after flushing of water for 20 min/day for seven successive days) and T2 (15 days later). Coliforms were detected in 47.3% of samples at T0, 36.3% at T1 and 4.4% at T2. E. coli was isolated in 4.4% of the samples only at T1, while enterococci appeared in 12.1% of the samples at T1 and in 2.2% at T2. P. aeruginosa was isolated in 50.5% of the samples at T0, 29.7% at T1 and 1.1% at T2. Legionella pneumophila serogroup 8 was isolated in 80.2% of the samples at T0, 36.3% at T1 and 2.2% at T2. Our results confirmed the need for a water safety plan in new hospital pavilions to prevent the risk of waterborne diseases

Impact of lockdown on the microbiological status of the hospital water network during COVID-19 pandemic

The COVID-19 pandemic started in China in early December 2019, and quickly spread around the world. The epidemic gradually started in Italy at the end of February 2020, and by May 31, 2020, 232,664 cases and 33,340 deaths were confirmed. As a result of this pandemic, the Italian Ministerial Decree issued on March 11, 2020, enforced lockdown; therefore, many social, recreational, and cultural centers remained closed for months. In Apulia (southern Italy), all non-urgent hospital activities were suspended, and some wards were closed, with a consequent reduction in the use of the water network and the formation of stagnant water. This situation could enhance the risk of exposure of people to waterborne diseases, including legionellosis. The purpose of this study was to monitor the microbiological quality of the water network (coliforms, E. coli, Enterococci, P. aeruginosa, and Legionella) in three wards (A, B and C) of a large COVID-19 regional hospital, closed for three months due to the COVID-19 emergency. Our study revealed that all three wards' water network showed higher contamination by Legionella pneumophila sg 1 and sg 6 at T1 (after lockdown) compared to the period before the lockdown (T0). In particular, ward A at T1 showed a median value = 5600 CFU/L (range 0-91,000 CFU/L) vs T0, median value = 75 CFU/L (range 0-5000 CFU/L) (p-value = 0.014); ward B at T1 showed a median value = 200 CFU/L (range 0-4200 CFU/L) vs T0, median value = 0 CFU/L (range 0-300 CFU/L) (p-value = 0.016) and ward C at T1 showed a median value = 175 CFU/L (range 0-22,000 CFU/L) vs T0, median value = 0 CFU/L (range 0-340 CFU/L) (p-value < 0.001). In addition, a statistically significant difference was detected in ward B between the number of positive water samples at T0 vs T1 for L. pneumophila sg 1 and sg 6 (24% vs 80% p-value < 0.001) and for coliforms (0% vs 64% p-value < 0.001). Moreover, a median value of coliform load resulted 3 CFU/100 ml (range 0-14 CFU/100 ml) at T1, showing a statistically significant increase versus T0 (0 CFU/100 ml) (p-value < 0.001). Our results highlight the need to implement a water safety plan that includes staff training and a more rigorous environmental microbiological surveillance in all hospitals before occupying a closed ward for a longer than one week, according to national and international guidelines

Fin Spine Bone Resorption in Atlantic Bluefin Tuna, Thunnus thynnus, and Comparison between Wild and Captive-Reared Specimens

Bone resorption in the first spine of the first dorsal fin of Atlantic bluefin tuna (ABFT) has long been considered for age estimation studies. In the present paper spine bone resorpion was assessed in wild (aged 1 to 13 years) and captive-reared (aged 2 to 11 years) ABFT sampled from the Mediterranean Sea. Total surface (TS), solid surface (SS) and reabsorbed surface (RS) were measured in spine transverse sections in order to obtain proportions of SS and RS. The spine section surface was found to be isometrically correlated to the fish fork length by a power equation. The fraction of solid spine bone progressively decreased according to a logarithmic equation correlating SS/TS to both fish size and age. The values ranged from 57% in the smallest examined individuals to 37% in the largest specimens. This phenomenon was further enhanced in captive-reared ABFT where SS/TS was 22% in the largest measured specimen. The difference between the fraction of SS of wild and captive-reared ABFT was highly significant. In each year class from 1- to 7-year-old wild specimens, the fraction of spine reabsorbed surface was significantly higher in specimens collected from March to May than in those sampled during the rest of the year. In 4-year-old fish the normal SS increase during the summer did not occur, possibly coinciding with their first sexual maturity. According to the correlations between SS/TS and age, the rate of spine bone resorption was significantly higher, even almost double, in captive-reared specimens. This could be attributed to the wider context of systemic dysfunctions occurring in reared ABFT, and may be related to a number of factors, including nutritional deficiencies, alteration of endocrine profile, cortisol-induced stress, and loss of spine functions during locomotion in rearing conditions.Versión del editor4,411

Reliability of melanomacrophage centres as indicators of stress response in teleost fishes

Melanomacrophage centres (MMCs) are aggregates of pigmented phagocytes, characterized by heterogeneous inclusions and located in hemolymphopoietic organs of various non-mammalian vertebrates [1]. In the present work, data from published and ongoing studies on MMCs were analysed in order to get insights on MMC reliability as response biomarkers to stress and environmental pollution. Liver samples from 31 wild and captive reared Atlantic bluefin tuna (Thunnus thynnus) [2, 3] and from 90 wild European anchovies (Engraulis encrasicolus) caught in areas differently exposed to industrial and agricultural pollutants [4], and liver and spleen samples from 47 wild and captive reared greater amberjack (Seriola dumerili) [5, unpublished data] were fixed in 10% buffered formalin and embedded in paraffin wax. Deparaffinized sections were stained with haematoxylin-eosin; Mallory’s basic fucshin (Merck) and Perls VanGieson (Bio-Optica) stainings were used to identify lipofuscin–ceroids and ferric iron respectively; peroxidase detection was performed by a Leukocyte Peroxidase kit (Sigma). The terminal deoxynucleotidyl transferase-mediated d’UTP nick-end labelling (TUNEL) method was used to identify apoptotic cells and the immunohistochemical detection of cytochrome P450 monooxygenase 1A (CYP1A) was performed by means of polyclonal antibodies anti-fish CYP1A (Biosense Laboratories). Lipofuscin–ceroids and ferric iron were detected in MMCs of all the three examined species, whereas peroxidase was mainly detected in free macrophages. In Atlantic bluefin tuna, a high density of MMCs and liver apoptotic cells, and a strong CYP1A immunostaining were observed in young individuals reared in the central Adriatic Sea compared with adults caught from the wild or reared in the western Mediterranean. In European anchovy, a high density of MMCs and a strong CYP1A immunostaining were observed in fish sampled in the Gulf of Gela, a marine area dramatically affected by environmental pollution. In greater amberjack, MMC density was higher in spleen than in liver sections. Confinement in captivity did not affect MMC density, whereas differences were found between males and females and among fish in different reproductive conditions. The present study confirms that MMCs represent a useful biomarker of fish exposure to environmental pollution; however, sex and reproductive state may affect MMC density, possibly leading to data misinterpretations

Proliferation and Apoptosis of Cat (Felis catus) Male Germ Cells during Breeding and Non-Breeding Seasons

The domestic cat (Felis catus) is a seasonal-breeding species whose reproductive period starts when the day length increases. Since the existing information on cat spermatogenesis is limited and somewhat contradictory, in the present study, germ cell proliferation and apoptosis in feral adult tomcats orchiectomized during reproductive (reproductive group, RG; February–July) and non-reproductive (non-reproductive group, NRG; November and December) seasons were compared. Cross-sections taken from the middle third of the left testis were chemically fixed and embedded in paraffin wax. Histological sections were processed for the immunohistochemical detection of proliferating germ cells (PCNA) and for the identification of apoptotic cells (TUNEL method). The percentage of PCNA-positive spermatogonia was higher in the RG than in the NRG. On the contrary, germ cell apoptosis was higher in the NRG than in the RG. Our results confirm that cat spermatogenesis is modulated on a seasonal basis and suggests that spermatogenesis control involves changes in germ cell proliferation and apoptosis according to a common paradigm of seasonally breeding species

OOGENESIS IN WILD AND REARED GREATER AMBERJACK SERIOLA DUMERILI (RISSO, 1810)

Introduction The incorporation of new species in the aquaculture industry necessitates to control the reproductive function in captivity and to produce high numbers of high-quality eggs. Greater amberjack Seriola dumerili (Risso, 1810) caught from the wild and reared in captivity have been shown not to develop further than early vitellogenesis or if they did complete vitellogenesis, they failed to undergo oocyte maturation and required exogenous hormonal therapies to induce ovulation and spawning (Mylonas et al., 2004).The present work represents an overview of the results obtained in a study on the oogenesis of wild and captive-reared greater amberjack carried out within the EU FP7 project Diversify (Zupa et al, 2017; Pousis et al., 2018, 2019). Material and Methods Twenty-one wild and twelve captive-reared greater amberjack females were sampled during 2014, 2015 and 2016 at three different phases of the reproductive cycle: early gametogenesis (EARLY), late April-early May (wild fish = 5; captive-reared fish = 4); advanced gametogenesis (ADVANCED), late May-early June (wild fish = 4; captive-reared fish = 4); spawning (SPAWNING), late June-early July (wild fish = 12; captive-reared fish = 4). Wild fish were sampled on board a professional purse-seine fishing vessel operating around the Pelagie Islands (Sicily, Italy); captive-reared individuals belonged to a broodstock captured as juveniles and moved to a sea cage of Argosaronikos Fishfarming S.A. (Salamina Island, Greece). For each fish, biometric data (fork length, FL, in cm; body mass, BM, in kg; testis mass, TM, in g) were registered and gonadosomatic index (GSI = 100 × TM/BM) was calculated. Liver samples were store at -80°C and subsequently used for the analysis of vitellogenin (vtga, vtgb and vtgc) expression through RT-PCR. Ovary samples were used for histological analysis and for vitellogenin receptor (vtgr and lrp13) expression analysis through RT-PCR. Blood samples were centrifuged and plasma was stored at -20°C for the analysis of testosterone, 17β-estradiol and 17,20β-dihydroxypren-4-en-3-one by ELISA assays. Results and Discussion The GSI and all the sex steroid plasma levels were lower in captive-reared fish. During the EARLY phase, wild and captive-reared fish displayed perinucleolar or early vitellogenesis as the most advanced oocyte stage. During the ADVANCED phase, when the wild greater amberjack breeders were already in spawning condition (Fig. 1a), ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes (Fig. 1b). During the SPAWNING period, all captive-reared fish had regressed ovaries, while wild breeders still displayed oocytes at late vitellogenesis and maturation stages as well as postovulatory follicles. The expression levels of vtga, vtgb and vtgc did not differ significantly between captive-reared and wild females. Ovarian vtgr and lrp13 transcription was more active during early gametogenesis, suggesting that vitellogenin receptor transcripts were synthesized by previtellogenic oocytes and remained in the cellular mRNA pool until oocytes resumed meiosis and entered vitellogenesis. A reduced vtgr and lrp13 transcription was observed in captive-reared compared wild greater amberjack during the EARLY phase. The observed reproductive dysfunction, leading to oocyte atresia and reduced gonadosomatic index, arose during the early phase of oogenesis, when transcription of vitellogenin receptor genes appeared to be reduced, and did not appear to be associated to a lower liver capacity to synthesize the egg yolk precursors. Severe reproductive dysfunctions were observed also in males of the same broodstock and involved low sex steroid plasma concentrations and precocious cessation of spermatogenesis (Zupa et al., 2017). Preliminary data obtained within the H2020 project NewTechAqua indicate that hatchery-produced greater amberjack reared in sea cages in Salamina (Greece) have similar GSI compared with wild fish sampled in the same period of the reproductive cycle (early June 2021). Although further analyses are required, the available data indicate that hatchery-produced individuals might be less affected by captivity-induced stress than wild-caught breeders. Financial grant provided by the European Union ́s Programmes FP7 (GA 603121, DIVERSIFY) and H2020 (GA 862658, NewTechAqua). References Mylonas, C.C., Papandroulakis, N., Smboukis, A., Papadaki, M., Divanach, P. 2004. Induction of spawning of cultured greater amberjack (Seriola dumerili) using GnRHa implants. Aquaculture, 237: 141-154. Pousis, C., Mylonas, C. C., De Virgilio, C., Gadaleta, G., Santamaria, N., Passantino, L., Zupa, R., Papadaki, M., Fakriadis, I., Ferreri, R., Corriero A. 2018. The observed oogenesis impairment in greater amberjack Seriola dumerili (Risso, 1810) reared in captivity is not related to an insufficient liver transcription or oocyte uptake of vitellogenin. Aquaculture Research, 49: 243-252. Pousis, C., Rodríguez, C., De Ruvo, P., De Virgilio, C., Pérez, J.A., Mylonas, C. C., Zupa, R., Passantino, L., Santamaria, N., Valentini, L., Corriero A. 2019. Vitellogenin receptor and fatty acid profiles of individual lipid 1 classes of oocytes from wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle. Theriogenology, 140: 73-83. Zupa, R., Rodríguez, C., Mylonas, C. C., Rosenfeld, H., Fakriadis, I., Papadaki, M., Pérez, J. A., Pousis, C., Basilone, G., Corriero, A. 2017a. Comparative study of reproductive development in wild and captive-reared greater amberjack Seriola dumerili (Risso,1810). PLoS ONE 12(1): e0169645

Spermatogenesis enhancement in hatchery-produced greater amberjack (Seriola dumerili)

INTRODUCTION Greater amberjack Seriola dumerili (Risso, 1810) is a promising emerging aquaculture species thanks to its rapid growth and consumers’ appreciation. Wild-caught greater amberjack males reared in sea cages showed alteration of plasma sex steroid concentrations, high testicular apoptosis, reduced germ cell proliferation and low sperm quality. We report the effects of gonadotropin releasing hormone agonist (GnRHa) and human chorionic gonadotropin (hCG) administration on testis development and male germ cell proliferation in hatchery-produced greater amberjack. METHODS Four-year-old hatchery-produced greater amberjack males (F1 generation) reared in a sea cage in Salamina (Greece) were treated with GnRHa, either through EVAc implants (50 μg kg-1 body weight) or injections (20 μg kg-1 body weight), hCG (1000 IU kg-1 body weight) or were left untreated as controls. Two fish per group were treated in mid-May, when testes were in active spermatogenesis. Two weeks after treatments, fish were sacrificed and i) gonadosomatic index (GSI) was calculated as 100 × testis weight/body weight; ii) testis samples were fixed in Bouin’s solution and destined to histological analysis and to the immunodetection of the proliferating cell nuclear antigen (PCNA). The effects of the treatments on spermatogonial proliferation and germ cell progression towards meiosis was assessed through the count of the number of PCNA-positive single spermatogonia and the number of spermatocysts (PCNA-positive spermatogonial cysts + spermatocyte cysts). RESULTS & DISCUSSION The treatments resulted in an increase of GSI (untreated: 1.0 ± 0.2; GnRHa implant: 2.0 ± 1.4; GnRHa injection: 2.1 ± 0.8; hCG: 2.6 ± 0.7) and seminiferous tubule diameter (untreated: 142.5 ± 23.1 μm; GnRHa implant: 151.0 ± 14.7 μm; GnRHa injection: 196.1 ± 50.8 μm; hCG = 191.4 ± 8.3 μm). According to the subjective histological evaluation, testes of treated fish showed an increase of germinal epithelium height, larger lumina of seminiferous tubules and more abundant luminal spermatozoa compared with untreated controls. Fish treated with hCG showed the most dramatic changes, characterized by confluence of seminiferous tubules in large sperm masses in the internal testicular region. The hormone treatments resulted in both a decrease of proliferating single spermatogonia (untreated: 108.1 ± 1.3; GnRHa implant: 77.9 ± 47.4; GnRHa injection: 37.3 ± 9.2; hCG: 24.7 ± 21.3 cells/mm2) and an increase of PCNA-positive spermatocysts (untreated: 839.4 ± 12.8; GnRHa implant: 959.2 ± 30.8; GnRHa injection: 868.6 ± 379.5; hCG: 2074.0 ± 38.4 spermatocysts/mm2). In conclusion, all the tree treatments were effective in inducing testicular maturation through the stimulation of germ cell progression towards meiosis. Although the injection of hCG showed the most marked overall effects, GnRHa implantation was still able, after two weeks, to support both spermatogonial proliferation and progression towards meiosis, thus suggesting the capacity to sustain spermatogenesis over a longer period compared with the other two treatments. The project received funding from the ERA-NET Cofund BlueBio program (BESTBROOD project)

SPERMATOGENESIS IN WILD AND CAPTIVE-REARED GREATER AMBERJACK Seriola dumerili (RISSO, 1810)

Introduction Greater amberjack Seriola dumerili (Risso, 1810) is an excellent candidate for aquaculture thanks to its rapid growth, excellent flesh quality and worldwide market appreciation. As other captive-reared fishes exhibiting reproductive dysfunctions (Zohar and Mylonas, 2001), greater amberjack males produced reduced sperm volume with a decreased sperm quality. The present work represents an overview of the results obtained in a comparative research study on the spermatogenesis of wild and captive-reared greater amberjack carried out in the framework of the EU FP7 project Diversify (www.diversifyfish.eu) (Zupa et al, 2017a, b). Material and Methods Twelve greater amberjack males caught from the wild and reared in captivity for three years in a sea cage in Salamina Island (Greece) and 14 males caught from the wild around Pelagie Islands (Sicily, Italy), were sampled during three phases of the reproductive cycle: early spermatogenesis (late April-early May), advanced spermatogenesis (late May-early June) and spawning (late June-July). For each fish, biometric data (fork length, FL, in cm; body mass, BM, in kg; testis mass, TM, in g) were registered and gonadosomatic index (GSI = 100 × TM/BM) calculated. Testis samples were chemically fixed and destined to basic histological analysis and to the identification of proliferating and apoptotic germ cells through the immunohistochemical detection of the proliferating cell nuclear antigen (PCNA) and the TUNEL method, respectively. Blood samples were centrifuged and plasma was collected and stored at -20°C for the analysis of 17β-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT) and 17,20β-dihydroxypren-4-en-3-one (17,20β-P) by ELISA assays. Results and Discussion Captive-reared fish showed lower GSI and smaller seminiferous lobules compared to wild fish in all the three phases of the reproductive cycle. Anti-PCNA immunostaining was observed in the nuclei of spermatogonia and primary spermatocytes (Fig. 1a). All the captive-reared and most of the wild greater amberjack showed TUNEL-positive germ cells (Fig. 1b). Individuals reared in captivity showed a gradual decrease of germ cell proliferation throughout the three reproductive phases, which led to a precocious cessation of the spermatogenesis as well as a higher germ cell apoptosis in early spermatogenesis. In all three reproductive phases, captive-reared fish showed lower T, 11-KT and 17,20β-P plasma concentrations compared to wild fish; however, captive-reared fish showed a many-fold higher E2 plasma levels during the early spermatogenesis. The occurrence of a severe endocrine dysfunction was described in captive-reared greater amberjack males, including low T, 11-KT and 17,20β-P plasma levels during all the examined spermatogenesis phases. Abnormally high E2 plasma concentrations were associated to an increased germ cell apoptosis during early spermatogenesis. The observed reproductive dysfunction finally led to a lower sperm concentration and quality (Zupa et al., 2017a). A severe impairment of the reproductive function was observed also in females of the same broodstock and involved low steroid plasma concentrations and extensive atresia of late vitellogenic oocytes (Zupa et al., 2017b). Preliminary data obtained within the H2020 project NewTechAqua indicate that hatchery-produced greater amberjack males reared in sea cages in Salamina (Greece) have similar GSI compared with wild fish sampled in the same period of the reproductive cycle (early June 2021). Although further analyses are required, the available data seem to suggest that the reproductive function might be less affected by captivity-induced stress in hatchery-produced greater amberjack than in wild-caught breeders. Financial grant provided by the European Union ́s Programmes FP7 (GA 603121, DIVERSIFY) and H2020 (GA 862658, NewTechAqua) References Zohar, Y., Mylonas, C. C. 2001. Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197: 99–136. Zupa, R., Fauvel, C., Mylonas, C. C., Pousis, C., Santamaria, N., Papadaki, Μ., Fakriadis, I., Cicirelli, V., Mangano, S., Passantino, L., Lacalandra, G. M., Corriero, A. 2017a. Rearing in captivity affects spermatogenesis and sperm quality in greater amberjack Seriola dumerili (Risso, 1810). Journal of Animal Science, 95: 4085-4100. Zupa, R., Rodríguez, C., Mylonas, C. C., Rosenfeld, H., Fakriadis, I., Papadaki, M., Pérez, J. A., Pousis, C., Basilone, G., Corriero, A. 2017b. Comparative study of reproductive development in wild and captive-reared greater amberjack Seriola dumerili (Risso,1810). PLoS ONE 12(1): e0169645