Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review

Protocols for the cryopreservation of fish gametes have been developed for many different fish species, in special, freshwater salmonids and cyprinids. Methods for sperm freezing have progressed during the last decades due to the increasing number of potential applications: aquaculture (genetic improvement programs, broodstock management, helping with species having reproductive problems), biotechnology studies using model fish species (preservation of transgenic or mutant lines), cryobanking of genetic resources from endangered species, etc. This mini-review tries to give an overview of the present situation of this area of research, identifying the main challenges and perspectives, redirecting the reader to more in-depth reviews and papers. (C) 2016 Elsevier Inc. All rights reserved.European Training Network IMPRESS (Marie Sklodowska-Curie Actions) from PROMAR programme [642893]COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) from PROMAR programmeResearch Centre of Excellence from PROMAR programme [9878-3/2016/FEKUT, KMR_12-1-2012-0436]NKFIH (OTKA) from PROMAR programme [109847]KLING from PROMAR programme [31-03-05FEP-73]CRIOBIV from PROMAR programme [31-03-05-FEP-59]REPLING from PROMAR programme [31-03-05-FEP-69]info:eu-repo/semantics/publishedVersio

Reproductive anatomy of Chondrichthyans: notes on specimen handling and sperm extraction. II. Sharks and chimaeras

[EN] Sperm extraction and artificial insemination may serve ex situ conservation initiatives for threatened sharks and related species. A comparison of the reproductive anatomy of eight chondrichthyans is presented in this study, emphasizing the important differences when performing these reproductive techniques. Additionally, we show how to obtain sperm samples from both living and dead specimens using cannulation, abdominal massage, or oviducal gland extraction. These tools can improve the success of breeding programs developed in aquaria and research facilities. The chondrichthyan fishes, which comprise sharks, rays, and chimaeras, are one of the most threatened groups of vertebrates on the planet. Given this situation, an additional strategy for the protection of these species could be the ex situ conservation projects developed in public aquaria and research centers. Nevertheless, to increase sustainability and to develop properly in situ reintroduction strategies, captive breeding techniques, such as sperm extraction and artificial insemination, should be developed. These techniques are commonly used in other threatened species and could be also used in chondrichthyans. However, the different reproductive morphologies found in this group can complicate both processes. Therefore, a comparison of the reproductive anatomy of eight distinct chondrichthyans, with an emphasis on those important differences when performing sperm extraction or artificial insemination, is carried out herein. Sharks and chimaeras belonging to the Scyliorhinidae, Carcharhinidae, Centrophoridae, Etmopteridae, Hexanchidae, and Chimaeridae families were obtained from commercial fisheries, public aquaria, and stranding events. In addition, the process of obtaining viable sperm samples through cannulation, abdominal massage, and oviducal gland extraction is described in detail for both living and dead animals.This research was partially funded by the Fundacion Biodiversidad (PRCV00683). P.G.-S. has a PhD contract from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ACIF 2018 (ACIF/2018/147). V.G. has a postdoc contract from the MICIU, Programa Juan de la Cierva-Incorporacion (IJCI-2017-34200).García-Salinas, P.; Gallego Albiach, V.; Asturiano, JF. (2021). Reproductive anatomy of Chondrichthyans: notes on specimen handling and sperm extraction. II. Sharks and chimaeras. Animals. 11(8):1-20. https://doi.org/10.3390/ani11082191S12011

Reproductive Anatomy of Chondrichthyans: Notes on Specimen Handling and Sperm Extraction. I. Rays and Skates

[EN] Many species of rays and skates are endangered, and ex situ conservation programs developed by research centers and public aquaria could improve this situation. To reproduce these species in captivity, scientists need to know how to extract their sperm and how to conduct their artificial insemination; however, the anatomical diversity of the reproductive organs of this group of animals is a handicap. A comparison of the reproductive anatomies of 11 distinct species is presented here, emphasizing the important differences between the species when performing sperm extraction or artificial insemination. In addition, the process of obtaining sperm samples from both live and dead males is described in detail, using both cannulation and abdominal massage. The superorder Batoidea (rays, skates, and relatives), constitutes one of the most threatened group of vertebrates. Strengthening ex situ conservation programs developed in research centers and public aquaria could be a way of addressing this situation. However, captive breeding programs must be improved to prevent the capture of wild animals and to develop proper in situ reintroduction strategies. Sperm extraction and artificial insemination are two techniques commonly used in other threatened species, which could also be used in rays and the like. However, the different reproductive morphologies present within this group of animals may hamper both processes. Here, we present a comparison of the reproductive anatomies of 11 distinct batoid species, emphasizing the important differences between the species when performing sperm extraction or artificial insemination. Both male and female animals, belonging to the Rajidae, Dasyatidae, Torpedinidae and Myliobatidae families, from the Mediterranean Sea were studied. In addition, we describe the procedure to extract sperm using both cannulation and abdominal massage, either from live or dead batoids Finally, the obtention of motile sperm recovered from the oviducal gland of females is described. These techniques generate a new range of possibilities for the conservation of these threatened species.This research was partially funded by the Fundacion Biodiversidad (PRCV00683). PGS has a PhD contract from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ACIF 2018 (ACIF/2018/147). VG has a postdoc contract from the MICIU, Programa Juan de la Cierva-Incorporacion (IJCI-2017-34200).García-Salinas, P.; Gallego Albiach, V.; Asturiano, JF. (2021). Reproductive Anatomy of Chondrichthyans: Notes on Specimen Handling and Sperm Extraction. I. Rays and Skates. Animals. 11(7):1-16. https://doi.org/10.3390/ani11071888S11611

Intracellular pH regulation and sperm motility in the European eel

[EN] Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH(i)) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4-7.6. The natural pH(i) range of sperm samples in the quiescent stage was 7.4-8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH(e)), as well as between the pH(i) and the pH of the diluent. The change post-activation in seawater (ASW) depended on the initial pH(e) of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH(e) 8, and no pH(i) variation when pH(e) was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH(i) changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation. (C) 2020 Elsevier Inc. All rights reserved.Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS). Victor Gallego has a postdoc grant from the MICIU (Juan de la Cierva-Incorporacion; IJCI-2017-34200Pérez Igualada, LM.; Gallego Albiach, V.; Asturiano, JF. (2020). Intracellular pH regulation and sperm motility in the European eel. Theriogenology. 145:48-58. https://doi.org/10.1016/j.theriogenology.2020.01.026S4858145Nishigaki, T., José, O., González-Cota, A. L., Romero, F., Treviño, C. L., & Darszon, A. (2014). Intracellular pH in sperm physiology. Biochemical and Biophysical Research Communications, 450(3), 1149-1158. doi:10.1016/j.bbrc.2014.05.100Ohta, H., Ikeda, K., & Izawa, T. (1997). Increases in concentrations of potassium and bicarbonate ions promote acquisition of motility in vitro by Japanese eel spermatozoa. The Journal of Experimental Zoology, 277(2), 171-180. doi:10.1002/(sici)1097-010x(19970201)277:23.0.co;2-mOda, S., & Morisawa, M. (1993). Rises of intracellular Ca2+ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts. Cell Motility and the Cytoskeleton, 25(2), 171-178. doi:10.1002/cm.970250206TANAKA, S., UTOH, T., YAMADA, Y., HORIE, N., OKAMURA, A., AKAZAWA, A., … KUROKURA, H. (2004). Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel. Fisheries Science, 70(5), 780-787. doi:10.1111/j.1444-2906.2004.00871.xGallego, V., Martínez-Pastor, F., Mazzeo, I., Peñaranda, D. S., Herráez, M. P., Asturiano, J. F., & Pérez, L. (2014). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results. Aquaculture, 418-419, 155-158. doi:10.1016/j.aquaculture.2013.10.022Vílchez, M. C., Morini, M., Peñaranda, D. S., Gallego, V., Asturiano, J. F., & Pérez, L. (2017). Role of potassium and pH on the initiation of sperm motility in the European eel. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 203, 210-219. doi:10.1016/j.cbpa.2016.09.024M�ri�n, T., Krasznai, Z., Balkay, L., Emri, M., & Tr�n, L. (1997). Role of extracellular and intracellular pH in carp sperm motility and modifications by hyperosmosis of regulation of the Na+/H+ exchanger. Cytometry, 27(4), 374-382. doi:10.1002/(sici)1097-0320(19970401)27:43.0.co;2-cPerez, L., Aturiano, J. F., Tomas, A., Zegrari, S., Barrera, R., Espinos, F. J., … Jover, M. (2000). Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. Journal of Fish Biology, 57(6), 1488-1504. doi:10.1111/j.1095-8649.2000.tb02227.xGallego, V., Carneiro, P. C. F., Mazzeo, I., Vílchez, M. C., Peñaranda, D. S., Soler, C., … Asturiano, J. F. (2013). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology, 79(7), 1034-1040. doi:10.1016/j.theriogenology.2013.01.019Mylonas, C. C., Duncan, N. J., & Asturiano, J. F. (2017). Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality. Aquaculture, 472, 21-44. doi:10.1016/j.aquaculture.2016.04.021Asturiano, J. F., Pérez, L., Garzón, D. L., Marco-Jiménez, F., Peñaranda, D. S., Vicente, J. S., & Jover, M. (2004). Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm. Fish Physiology and Biochemistry, 30(3-4), 283-293. doi:10.1007/s10695-005-1553-xThomas, J. A., Buchsbaum, R. N., Zimniak, A., & Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry, 18(11), 2210-2218. doi:10.1021/bi00578a012Krasznai, Z. (2003). Role of ion channels and membrane potential in the initiation of carp sperm motility. Aquatic Living Resources, 16(5), 445-449. doi:10.1016/s0990-7440(03)00054-8Miura, T., & Miura, C. I. (2003). Molecular control mechanisms of fish spermatogenesis. Fish Physiology and Biochemistry, 28(1-4), 181-186. doi:10.1023/b:fish.0000030522.71779.47Fechner, S., Alvarez, L., Bönigk, W., Müller, A., Berger, T. K., Pascal, R., … Kaupp, U. B. (2015). A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm. eLife, 4. doi:10.7554/elife.07624Peñaranda, D., Pérez, L., Gallego, V., Barrera, R., Jover, M., & Asturiano, J. (2008). European Eel Sperm Diluent for Short-term Storage. Reproduction in Domestic Animals, 45(3), 407-415. doi:10.1111/j.1439-0531.2008.01206.xChauvaud, L., Cosson, J., Suquet, M., & Billard, R. (1995). Sperm motility in turbot, Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics. Environmental Biology of Fishes, 43(4), 341-349. doi:10.1007/bf00001167Billard, R., Cosson, J., & Crim, L. W. (1993). Motility of fresh and aged halibut sperm. Aquatic Living Resources, 6(1), 67-75. doi:10.1051/alr:1993008Effer, B., Figueroa, E., Augsburger, A., & Valdebenito, I. (2013). Sperm biology of Merluccius australis: Sperm structure, semen characteristics and effects of pH, temperature and osmolality on sperm motility. Aquaculture, 408-409, 147-151. doi:10.1016/j.aquaculture.2013.05.040Inaba, K., Dréanno, C., & Cosson, J. (2003). Control of flatfish sperm motility by CO2and carbonic anhydrase. Cell Motility and the Cytoskeleton, 55(3), 174-187. doi:10.1002/cm.10119Lee, H. cheung, Johnson, C., & Epel, D. (1983). Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm. Developmental Biology, 95(1), 31-45. doi:10.1016/0012-1606(83)90004-0Babcock, D. F., Rufo, G. A., & Lardy, H. A. (1983). Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proceedings of the National Academy of Sciences, 80(5), 1327-1331. doi:10.1073/pnas.80.5.132

Ionic control of sperm motility and trials for the improvement of pufferfish (Takifugu alboplumbeus) sperm extenders

[EN] Seminal plasma characteristics, such as ionic composition and pH, are important for sperm maturation and further motility. In this work, a first batch of experiments evaluated the effect of the absence of several ions (Na+, K+, Mg2+) in the artificial seminal plasma, as well as the effect of artificial seminal plasma pH and seawater pH, on the sperm activation in the pufferfish (Takifugu alboplumbeus, named before Takifugu niphobles). In all the experiments, total motility, progressive motility, curvilinear velocity, straight line velocity, and average path velocity were measured. The absence of Na+, K+ or Mg2+ ions did not reduce the sperm motility in comparison with the control. In contrast, artificial seminal plasma pH had a strong effect on sperm motility. Samples diluted in artificial seminal plasma at pH 6.5 showed a high reduction of sperm motility in comparison with higher pHs (7.5, 8.5 and 9.5), and this inhibition was reversible. Where the effect of the pH of artificial seawater in sperm motility (used as activation medium) was concerned, there were no significant differences between the control (pH 8.2) and pH 6.5, indicating that pufferfish spermatozoa can swim in acidic environments. Finally, nigericin (that make equal the intracellular pH and extracellular pH) was added, and the highest sperm motility was found at pH 7.0, which suggested that the optimal intracellular pH of pufferfish for sperm motility is 7.0. Our second batch of experiments was developed to optimize the pufferfish sperm extenders to achieve longer preservation under refrigeration, assessing the addition of several concentrations of magnesium and NaHCO3 (used in extenders for other fish species), as well as different pHs. From the extender experiments it was observed that bicarbonate reduced the sperm motility after 7 days of incubation, and it can be concluded that the use of magnesium 2 mM as part of the extender composition, in combination with the pH measured in the pufferfish sperm (7.5), seems to be the best option for the short-medium term storage of the sperm of this species.Generalitat Valenciana funded the stay in Japan of JFA, LPI and VG (BEST 2018/093,/112 and/124, respectively). VG has a postdoctoral grant from the Spanish Ministry of Science, Innovation and Universities (MICIU; Programa Juan de la Cierva-Incorporacion; IJCI-2017-34200).Pérez Igualada, LM.; Asturiano, JF.; Yoshida, M.; Gallego Albiach, V. (2022). Ionic control of sperm motility and trials for the improvement of pufferfish (Takifugu alboplumbeus) sperm extenders. Aquaculture. 554:1-11. https://doi.org/10.1016/j.aquaculture.2022.73814611155

Sperm quality parameters of Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica): new conservation tools from a gamete perspective

[EN] The sensitive state of conservation of several endemic fish species such as Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica) has led governments to consider the implementation of conservation measures to preserve their populations. However, limited knowledge about the reproductive biology of these species makes it necessary to investigate different aspects of their reproductive cycle. In this sense, the main objectives of this work were i) to advance knowledge of the breeding biology of both species, and ii) to develop protocols for the conservation of gametes for the future management and conservation. During the spring of 2019 a temporal series of samplings were carried out in different places in the Comunitat Valenciana. Sperm samples were collected and sperm motion parameters were assessed for the first time in both species. Kinetic patterns were similar showing high motility and velocity values during the first 30 s, and a rapid decrease from that point. At the same time, an in-depth morphometric analysis was carried out using computer-assisted sperm analysis software. Spermatozoa from A. iberus and V. hispanica showed similar sizes and shapes to other external fertilizers belonging to Cyprinodontiformes, with small spherical heads, uniflagellated and without acrosomes. In addition, a new cryopreservation protocol was designed for cryobanking the sperm of these threatened species. Cryopreserved samples showed lower motility than fresh samples but reaching acceptable percentages of motile cells after thawing of around 20 and 25% (A. iberus and V. hispanica, respectively). This study is the first of its kind to successfully achieve gamete cryopreservation of these two endemic and endangered species from the Iberian Peninsula, providing new and useful tools to complement the management and conservation programs that are being developed for both species.This project has received funding from the Generalitat Valenciana (GV; Valencia, Spain) under the "Subvenciones para la realizacion de proyectos de I+D+I desarrollados por grupos de investigacion emergentes (GV/2019/130)". VG has a postdoc grant from the Ministerio de Economia, Industria y Competitividad (MINECO; Madrid, Spain) under the project IJCI-2017-34200. We would like to thank all the technicians of the CCEDCV for their work and effort during the samplings, specially to J. Velazquez, J. Hernandez and A. Pradillo.Blanes-García, M.; Risueño, P.; Pérez Igualada, LM.; Asturiano, JF.; Gallego Albiach, V. (2021). Sperm quality parameters of Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica): new conservation tools from a gamete perspective. Aquaculture. 530:1-9. https://doi.org/10.1016/j.aquaculture.2020.735819S1953

Identification and stable expression of vitellogenin receptor (VTGR) through vitellogenesis in the European eel

[EN] In teleosts, vitellogenin (Vtg) is a phospholipoglycoprotein synthesized by the liver, released into the blood circulation and incorporated into the oocytes via endocytosis mediated by the Vtg receptor (VTGR) to form the yolk granules. The VTGR is crucial for oocyte growth in egg-laying animals but is also present in non-oviparous vertebrates, such as human. The VTGR belongs to the low-density lipoprotein receptor superfamily (LDLR) and is also named very-low-density lipoprotein receptor (VLDLR). In this study, we identified and phylogenetically positioned the VTGR of a basal teleost, the European eel, Anguilla anguilla. We developed quantitative real-time PCR (qRT-PCR) and investigated the tissue distribution of vtgr transcripts. We compared by qRT-PCR the ovarian expression levels of vtgr in juvenile yellow eels and pre-pubertal silver eels. We also analyzed the regulation of ovarian vtgr expression throughout vitellogenesis in experimentally matured eels. The Vtg plasma level was measured by homologous ELISA experimental maturation. Our in silico search and phylogenetical analysis revealed a single vtgr in the European eel, orthologous to other vertebrate vtgr. The qRT-PCR studies revealed that vtgr is mainly expressed in the ovary and also detected in various other tissues such as brain, pituitary, gill, fat, heart, and testis, suggesting some extra-ovarian functions of VTGR. We showed that vtgr is expressed in ovaries of juvenile yellow eels with no higher expression in pre-pubertal silver eels nor in experimentally matured eels. This suggests that vtgr transcription already occurs during early pre-vitellogenesis of immature eels and is not further activated in vitellogenic oocytes. European eel Vtg plasma level increased throughout experimental maturation in agreement with previous studies. Taken together, these results suggest that vtgr transcript levels may not be a limiting step for the uptake of Vtg by the oocyte in the European eel.M.M., A.G.L. and S.D. were granted with Short Term Scientific Missions by the COST Office (COST Action FA1205: AQUAGAMETE). S.D. was also awarded with a grant from the UPV's School of Doctorate (Accion para la Internacionalizacion de los Programas de Doctorado) in 2017. We thank E. Feunteun and colleagues, MNHN, Dinard, for the ovarian samples from eels of the Fremur, France.Morini, M.; Lafont, AG.; Maugars, G.; Baloche, S.; Dufour, S.; Asturiano, JF.; Pérez Igualada, LM. (2020). Identification and stable expression of vitellogenin receptor (VTGR) through vitellogenesis in the European eel. Animal. 14(6):1213-1222. https://doi.org/10.1017/S1751731119003355S12131222146Abascal, F., Zardoya, R., & Posada, D. (2005). ProtTest: selection of best-fit models of protein evolution. Bioinformatics, 21(9), 2104-2105. doi:10.1093/bioinformatics/bti263Ali, B. R., Silhavy, J. L., Gleeson, M. J., Gleeson, J. G., & Al-Gazali, L. (2012). A missense founder mutation in VLDLR is associated with Dysequilibrium Syndrome without quadrupedal locomotion. BMC Medical Genetics, 13(1). doi:10.1186/1471-2350-13-80Andersen, Ø., Xu, C., Timmerhaus, G., Kirste, K. H., Naeve, I., Mommens, M., & Tveiten, H. (2017). Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar ): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes. Molecular Reproduction and Development, 84(11), 1191-1202. doi:10.1002/mrd.22881Bidwell, C., & Carlson, D. (1995). Characterization of vitellogenin from white sturgeon, Acipenser transmontanus. Journal of Molecular Evolution, 41(1). doi:10.1007/bf00174046Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., … Schneider, W. J. (1994). Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family. The EMBO Journal, 13(21), 5165-5175. doi:10.1002/j.1460-2075.1994.tb06847.xBurzawa-Gerard, E., & Dumas-Vidal, A. (1991). Effects of 17β-estradiol and carp gonadotropin on vitellogenesis in normal and hypophysectomized European silver female eel (Anguilla anguilla L.) employing a homologous radioimmunoassay for vitellogenin. General and Comparative Endocrinology, 84(2), 264-276. doi:10.1016/0016-6480(91)90049-cChen, J.-N., López, J. A., Lavoué, S., Miya, M., & Chen, W.-J. (2014). Phylogeny of the Elopomorpha (Teleostei): Evidence from six nuclear and mitochondrial markers. Molecular Phylogenetics and Evolution, 70, 152-161. doi:10.1016/j.ympev.2013.09.002Clelland, E. S., & Kelly, S. P. (2010). Tight junction proteins in zebrafish ovarian follicles: Stage specific mRNA abundance and response to 17β-estradiol, human chorionic gonadotropin, and maturation inducing hormone. General and Comparative Endocrinology, 168(3), 388-400. doi:10.1016/j.ygcen.2010.05.011Damsteegt, E. L., Mizuta, H., Hiramatsu, N., & Lokman, P. M. (2015). How do eggs get fat? Insights into ovarian fatty acid accumulation in the shortfinned eel, Anguilla australis. General and Comparative Endocrinology, 221, 94-100. doi:10.1016/j.ygcen.2014.12.019Dufour S, Burzawa-Gerard E, Le Belle N, Sbaihi M and Vidal B 2003. Reproductive endocrinology of the European eel, Anguilla anguilla. In Eel biology (eds. K Aida, K Tsukamoto and K Yamauchi ), pp 373–383. Springer-Verlag, Tokyo, Japan.Dufour, S., Lopez, E., Le Menn, F., Le Belle, N., Baloche, S., & Fontaine, Y. A. (1988). Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol. General and Comparative Endocrinology, 70(1), 20-30. doi:10.1016/0016-6480(88)90090-1Henkel, C. V., Burgerhout, E., de Wijze, D. L., Dirks, R. P., Minegishi, Y., Jansen, H. J., … van den Thillart, G. E. E. J. M. (2012). Primitive Duplicate Hox Clusters in the European Eel’s Genome. PLoS ONE, 7(2), e32231. doi:10.1371/journal.pone.0032231Henkel, C. V., Dirks, R. P., de Wijze, D. L., Minegishi, Y., Aoyama, J., Jansen, H. J., … van den Thillart, G. E. E. J. M. (2012). First draft genome sequence of the Japanese eel, Anguilla japonica. Gene, 511(2), 195-201. doi:10.1016/j.gene.2012.09.064Herz, J., & Bock, H. H. (2002). Lipoprotein Receptors in the Nervous System. Annual Review of Biochemistry, 71(1), 405-434. doi:10.1146/annurev.biochem.71.110601.135342Hiramatsu, N., Luo, W., Reading, B. J., Sullivan, C. V., Mizuta, H., Ryu, Y.-W., … Hara, A. (2012). Multiple ovarian lipoprotein receptors in teleosts. Fish Physiology and Biochemistry, 39(1), 29-32. doi:10.1007/s10695-012-9612-6Hiramatsu, N., Todo, T., Sullivan, C. V., Schilling, J., Reading, B. J., Matsubara, T., … Hara, A. (2015). Ovarian yolk formation in fishes: Molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins. General and Comparative Endocrinology, 221, 9-15. doi:10.1016/j.ygcen.2015.01.025Hummel, S., Lynn, E. G., Osanger, A., Hirayama, S., Nimpf, J., & Schneider, W. J. (2003). Molecular characterization of the first avian LDL receptor. Journal of Lipid Research, 44(9), 1633-1642. doi:10.1194/jlr.m300014-jlr200Jéhannet P, Kruijt L, Damsteegt EL, Swinkels W, Heinsbroek LTN, Lokman PM and Palstra AP. A mechanistic model for studying the initiation of anguillid vitellogenesis by comparing the European eel (Anguilla anguilla) and the shortfinned eel (A. australis). General and Comparative Endocrinology (in press). https://doi.org/10.1016/j.ygcen.2019.02.018Lafont, A.-G., Rousseau, K., Tomkiewicz, J., & Dufour, S. (2016). Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation. General and Comparative Endocrinology, 235, 177-191. doi:10.1016/j.ygcen.2015.11.021Mazzeo, I., Peñaranda, D. S., Gallego, V., Baloche, S., Nourizadeh-Lillabadi, R., Tveiten, H., … Pérez, L. (2014). Temperature modulates the progression of vitellogenesis in the European eel. Aquaculture, 434, 38-47. doi:10.1016/j.aquaculture.2014.07.020Mizuta, H., Luo, W., Ito, Y., Mushirobira, Y., Todo, T., Hara, A., … Hiramatsu, N. (2013). Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 166(1), 81-90. doi:10.1016/j.cbpb.2013.07.005Mizuta, H., Mushirobira, Y., Nagata, J., Todo, T., Hara, A., Reading, B. J., … Hiramatsu, N. (2017). Ovarian expression and localization of clathrin (Cltc) components in cutthroat trout, Oncorhynchus clarki: Evidence for Cltc involvement in endocytosis of vitellogenin during oocyte growth. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 212, 24-34. doi:10.1016/j.cbpa.2017.06.021Morini, M., Peñaranda, D. S., Vílchez, M. C., Gallego, V., Nourizadeh-Lillabadi, R., Asturiano, J. F., … Pérez, L. (2015). Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCζ1) increase through induced spermatogenesis in European eel. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 187, 168-176. doi:10.1016/j.cbpa.2015.05.028Morini, M., Peñaranda, D. S., Vílchez, M. C., Nourizadeh-Lillabadi, R., Lafont, A.-G., Dufour, S., … Pérez, L. (2017). Nuclear and membrane progestin receptors in the European eel: Characterization and expression in vivo through spermatogenesis. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 207, 79-92. doi:10.1016/j.cbpa.2017.02.009Nader, N., Dib, M., Courjaret, R., Hodeify, R., Machaca, R., Graumann, J., & Machaca, K. (2018). VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling. Journal of Cell Science. doi:10.1242/jcs.212522Okabayashi, K., Shoji, H., Nakamura, T., Hashimoto, O., Asashima, M., & Sugino, H. (1996). cDNA Cloning and Expression of theXenopus laevisVitellogenin Receptor. Biochemical and Biophysical Research Communications, 224(2), 406-413. doi:10.1006/bbrc.1996.1040Palstra, A. P., & van den Thillart, G. E. E. J. M. (2010). Swimming physiology of European silver eels (Anguilla anguilla L.): energetic costs and effects on sexual maturation and reproduction. Fish Physiology and Biochemistry, 36(3), 297-322. doi:10.1007/s10695-010-9397-4Pankhurst, N. W. (1982). Relation of visual changes to the onset of sexual maturation in the European eel Anguilla anguilla (L.). Journal of Fish Biology, 21(2), 127-140. doi:10.1111/j.1095-8649.1982.tb03994.xPasquier, J., Lafont, A.-G., Jeng, S.-R., Morini, M., Dirks, R., van den Thillart, G., … Dufour, S. (2012). Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family. PLoS ONE, 7(11), e48931. doi:10.1371/journal.pone.0048931Perez, L., Aturiano, J. F., Tomas, A., Zegrari, S., Barrera, R., Espinos, F. J., … Jover, M. (2000). Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. Journal of Fish Biology, 57(6), 1488-1504. doi:10.1111/j.1095-8649.2000.tb02227.xPérez L, Vílchez MC, Gallego V, Mazzeo I, Peñaranda DS, Weltzien FA, Dufour S and Asturiano JF 2012. Trying to reproduce the European eel (Anguilla anguilla) under captivity: experiments with females. In Proceeding of the Domestication in Finfish Aquaculture, October 2012, Olsztyn, Poland, pp. 11–15.Prat, F., Coward, K., Sumpter, J. P., & Tyler, C. R. (1998). Molecular Characterization and Expression of two Ovarian Lipoprotein Receptors in the Rainbow Trout, Oncorhynchus mykiss 1. Biology of Reproduction, 58(5), 1146-1153. doi:10.1095/biolreprod58.5.1146Reading, B. J., Hiramatsu, N., Schilling, J., Molloy, K. T., Glassbrook, N., Mizuta, H., … Sullivan, C. V. (2014). Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes. Journal of Lipid Research, 55(11), 2287-2295. doi:10.1194/jlr.m050286Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312-1313. doi:10.1093/bioinformatics/btu033Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., & Yamamoto, T. (1992). Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity. Proceedings of the National Academy of Sciences, 89(19), 9252-9256. doi:10.1073/pnas.89.19.9252Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680. doi:10.1093/nar/22.22.4673Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., … Herz, J. (1999). Reeler/Disabled-like Disruption of Neuronal Migration in Knockout Mice Lacking the VLDL Receptor and ApoE Receptor 2. Cell, 97(6), 689-701. doi:10.1016/s0092-8674(00)80782-5Tyler, C. R., Pottinger, T. G., Coward, K., Prat, F., Beresford, N., & Maddix, S. (1997). Salmonid Follicle-Stimulating Hormone (GtH I) Mediates Vitellogenic Development of Oocytes in the Rainbow Trout, Oncorhynchus mykiss 1. Biology of Reproduction, 57(5), 1238-1244. doi:10.1095/biolreprod57.5.1238Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.01

De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages

[EN] Paralogues pairs are more frequently observed in eels (Anguilla sp.) than in other teleosts. The paralogues often show low phylogenetic distances; however, they have been assigned to the third round of whole genome duplication (WGD), shared by all teleosts (3R), due to their conserved synteny. The apparent contradiction of low phylogenetic difference and 3R conserved synteny led us to study the duplicated gene complement of the freshwater eels. With this aim, we assembled de novo transcriptomes of two highly relevant freshwater eel species: The European (Anguilla anguilla) and the Japanese eel (Anguilla japonica). The duplicated gene complement was analysed in these transcriptomes, and in the genomes and transcriptomes of other Actinopterygii species. The study included an assessment of neutral genetic divergence (4dTv), synteny, and the phylogenetic origins and relationships of the duplicated gene complements. The analyses indicated a high accumulation of duplications (1217 paralogue pairs) among freshwater eel genes, which may have originated in a WGD event after the Elopomorpha lineage diverged from the remaining teleosts, and thus not at the 3R. However, very similar results were observed in the basal Osteoglossomorpha and Clupeocephala branches, indicating that the specific genomic regions of these paralogues may still have been under tetrasomic inheritance at the split of the teleost lineages. Therefore, two potential hypotheses may explain the results: i) The freshwater eel lineage experienced an additional WGD to 3R, and ii) Some duplicated genomic regions experienced lineage specific rediploidization after 3R in the ancestor to freshwater eels. The supporting/opposing evidence for both hypotheses is discussed.This study received funding from the project REPRO-TEMP (AGL2013-41646-R) funded by the Spanish Ministry of Economy and Competitiveness, and from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), which also included the predoctoral contracts of CR and JGHJ. VG has a postdoctoral grant from the Spanish Ministry of Science, Innovation and Universities (MICIU; Programa Juan de la Cierva-Incorporacion; IJCI-2017-34200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Rozenfeld, C.; Blanca Postigo, JM.; Gallego Albiach, V.; García-Carpintero, V.; Herranz-Jusdado, JG.; Pérez Igualada, LM.; Asturiano, JF.... (2019). De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages. PLoS ONE. 14(6):1-25. https://doi.org/10.1371/journal.pone.0218085S125146Gu, X., Wang, Y., & Gu, J. (2002). Age distribution of human gene families shows significant roles of both large- and small-scale duplications in vertebrate evolution. Nature Genetics, 31(2), 205-209. doi:10.1038/ng902Cañestro, C., Albalat, R., Irimia, M., & Garcia-Fernàndez, J. (2013). Impact of gene gains, losses and duplication modes on the origin and diversification of vertebrates. Seminars in Cell & Developmental Biology, 24(2), 83-94. doi:10.1016/j.semcdb.2012.12.008Llorente, B., Malpertuy, A., Neuvéglise, C., de Montigny, J., Aigle, M., Artiguenave, F., … Dujon, B. (2000). Genomic Exploration of the Hemiascomycetous Yeasts: 18. Comparative analysis of chromosome maps and synteny withSaccharomyces cerevisiae. FEBS Letters, 487(1), 101-112. doi:10.1016/s0014-5793(00)02289-4Colbourne, J. K., Pfrender, M. E., Gilbert, D., Thomas, W. K., Tucker, A., Oakley, T. H., … Basu, M. K. (2011). The Ecoresponsive Genome of Daphnia pulex. Science, 331(6017), 555-561. doi:10.1126/science.1197761Bailey, J. A., Gu, Z., Clark, R. A., Reinert, K., Samonte, R. V., Schwartz, S., … Eichler, E. E. (2002). Recent Segmental Duplications in the Human Genome. Science, 297(5583), 1003-1007. doi:10.1126/science.1072047Samonte, R. V., & Eichler, E. E. (2002). Segmental duplications and the evolution of the primate genome. Nature Reviews Genetics, 3(1), 65-72. doi:10.1038/nrg705David, L. (2003). Recent Duplication of the Common Carp (Cyprinus carpio L.) Genome as Revealed by Analyses of Microsatellite Loci. Molecular Biology and Evolution, 20(9), 1425-1434. doi:10.1093/molbev/msg173Jaillon, O., Aury, J.-M., Brunet, F., Petit, J.-L., Stange-Thomann, N., Mauceli, E., … Bernot, A. (2004). Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature, 431(7011), 946-957. doi:10.1038/nature03025Rondeau, E. B., Minkley, D. R., Leong, J. S., Messmer, A. M., Jantzen, J. R., von Schalburg, K. R., … Koop, B. F. (2014). The Genome and Linkage Map of the Northern Pike (Esox lucius): Conserved Synteny Revealed between the Salmonid Sister Group and the Neoteleostei. PLoS ONE, 9(7), e102089. doi:10.1371/journal.pone.0102089Albalat, R., & Cañestro, C. (2016). Evolution by gene loss. Nature Reviews Genetics, 17(7), 379-391. doi:10.1038/nrg.2016.39Hafeez, M., Shabbir, M., Altaf, F., & Abbasi, A. A. (2016). Phylogenomic analysis reveals ancient segmental duplications in the human genome. Molecular Phylogenetics and Evolution, 94, 95-100. doi:10.1016/j.ympev.2015.08.019Chain, F. J. J., Feulner, P. G. D., Panchal, M., Eizaguirre, C., Samonte, I. E., Kalbe, M., … Reusch, T. B. H. (2014). Extensive Copy-Number Variation of Young Genes across Stickleback Populations. PLoS Genetics, 10(12), e1004830. doi:10.1371/journal.pgen.1004830MABLE, B. K. (2004). ‘Why polyploidy is rarer in animals than in plants’: myths and mechanisms. Biological Journal of the Linnean Society, 82(4), 453-466. doi:10.1111/j.1095-8312.2004.00332.xOtto, S. P., & Whitton, J. (2000). POLYPLOID INCIDENCE AND EVOLUTION. Annual Review of Genetics, 34(1), 401-437. doi:10.1146/annurev.genet.34.1.401Albertin, W., & Marullo, P. (2012). Polyploidy in fungi: evolution after whole-genome duplication. Proceedings of the Royal Society B: Biological Sciences, 279(1738), 2497-2509. doi:10.1098/rspb.2012.0434Schmutz, J., Cannon, S. B., Schlueter, J., Ma, J., Mitros, T., Nelson, W., … Jackson, S. A. (2010). Genome sequence of the palaeopolyploid soybean. Nature, 463(7278), 178-183. doi:10.1038/nature08670Del Pozo, J. C., & Ramirez-Parra, E. (2015). Whole genome duplications in plants: an overview fromArabidopsis. Journal of Experimental Botany, 66(22), 6991-7003. doi:10.1093/jxb/erv432Soltis, D. E., Visger, C. J., & Soltis, P. S. (2014). The polyploidy revolution then...and now: Stebbins revisited. American Journal of Botany, 101(7), 1057-1078. doi:10.3732/ajb.1400178Masterson, J. (1994). Stomatal Size in Fossil Plants: Evidence for Polyploidy in Majority of Angiosperms. Science, 264(5157), 421-424. doi:10.1126/science.264.5157.421Parisod, C., Holderegger, R., & Brochmann, C. (2010). Evolutionary consequences of autopolyploidy. New Phytologist, 186(1), 5-17. doi:10.1111/j.1469-8137.2009.03142.xBlanc, G., & Wolfe, K. H. (2004). Widespread Paleopolyploidy in Model Plant Species Inferred from Age Distributions of Duplicate Genes[W]. The Plant Cell, 16(7), 1667-1678. doi:10.1105/tpc.021345Sémon, M., & Wolfe, K. H. (2007). Consequences of genome duplication. Current Opinion in Genetics & Development, 17(6), 505-512. doi:10.1016/j.gde.2007.09.007Inoue, J., Sato, Y., Sinclair, R., Tsukamoto, K., & Nishida, M. (2015). Rapid genome reshaping by multiple-gene loss after whole-genome duplication in teleost fish suggested by mathematical modeling. Proceedings of the National Academy of Sciences, 112(48), 14918-14923. doi:10.1073/pnas.1507669112Wolfe, K. H. (2001). Yesterday’s polyploids and the mystery of diploidization. Nature Reviews Genetics, 2(5), 333-341. doi:10.1038/35072009Kassahn, K. S., Dang, V. T., Wilkins, S. J., Perkins, A. C., & Ragan, M. A. (2009). Evolution of gene function and regulatory control after whole-genome duplication: Comparative analyses in vertebrates. Genome Research, 19(8), 1404-1418. doi:10.1101/gr.086827.108Wang, X., Jin, D., Wang, Z., Guo, H., Zhang, L., Wang, L., … Paterson, A. H. (2014). Telomere‐centric genome repatterning determines recurring chromosome number reductions during the evolution of eukaryotes. New Phytologist, 205(1), 378-389. doi:10.1111/nph.12985Glasauer, S. M. K., & Neuhauss, S. C. F. (2014). Whole-genome duplication in teleost fishes and its evolutionary consequences. Molecular Genetics and Genomics, 289(6), 1045-1060. doi:10.1007/s00438-014-0889-2Chester, M., Gallagher, J. P., Symonds, V. V., Cruz da Silva, A. V., Mavrodiev, E. V., Leitch, A. R., … Soltis, D. E. (2012). Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus (Asteraceae). Proceedings of the National Academy of Sciences, 109(4), 1176-1181. doi:10.1073/pnas.1112041109Gordon, J. L., Byrne, K. P., & Wolfe, K. H. (2011). Mechanisms of Chromosome Number Evolution in Yeast. PLoS Genetics, 7(7), e1002190. doi:10.1371/journal.pgen.1002190Dehal, P., & Boore, J. L. (2005). Two Rounds of Whole Genome Duplication in the Ancestral Vertebrate. PLoS Biology, 3(10), e314. doi:10.1371/journal.pbio.0030314Christoffels, A., Koh, E. G. L., Chia, J., Brenner, S., Aparicio, S., & Venkatesh, B. (2004). Fugu Genome Analysis Provides Evidence for a Whole-Genome Duplication Early During the Evolution of Ray-Finned Fishes. Molecular Biology and Evolution, 21(6), 1146-1151. doi:10.1093/molbev/msh114Vandepoele, K., De Vos, W., Taylor, J. S., Meyer, A., & Van de Peer, Y. (2004). Major events in the genome evolution of vertebrates: Paranome age and size differ considerably between ray-finned fishes and land vertebrates. Proceedings of the National Academy of Sciences, 101(6), 1638-1643. doi:10.1073/pnas.0307968100Leggatt, R. A., & Iwama, G. K. (2003). Occurrence of polyploidy in the fishes. Reviews in Fish Biology and Fisheries, 13(3), 237-246. doi:10.1023/b:rfbf.0000033049.00668.feCOMBER, S. C. L., & SMITH, C. (2004). Polyploidy in fishes: patterns and processes. Biological Journal of the Linnean Society, 82(4), 431-442. doi:10.1111/j.1095-8312.2004.00330.xBraasch, I., Gehrke, A. R., Smith, J. J., Kawasaki, K., Manousaki, T., Pasquier, J., … Catchen, J. (2016). The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons. Nature Genetics, 48(4), 427-437. doi:10.1038/ng.3526Bian, C., Hu, Y., Ravi, V., Kuznetsova, I. S., Shen, X., Mu, X., … Li, X. (2016). The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts. Scientific Reports, 6(1). doi:10.1038/srep24501Lien, S., Koop, B. F., Sandve, S. R., Miller, J. R., Kent, M. P., Nome, T., … Davidson, W. S. (2016). The Atlantic salmon genome provides insights into rediploidization. Nature, 533(7602), 200-205. doi:10.1038/nature17164Blischak, P. D., Mabry, M. E., Conant, G. C., & Pires, J. C. (2018). Integrating Networks, Phylogenomics, and Population Genomics for the Study of Polyploidy. Annual Review of Ecology, Evolution, and Systematics, 49(1), 253-278. doi:10.1146/annurev-ecolsys-121415-032302Robertson, F. M., Gundappa, M. K., Grammes, F., Hvidsten, T. R., Redmond, A. K., Lien, S., … Macqueen, D. J. (2017). Lineage-specific rediploidization is a mechanism to explain time-lags between genome duplication and evolutionary diversification. Genome Biology, 18(1). doi:10.1186/s13059-017-1241-zDUFOUR, S., WELTZIEN, F.-A., SEBERT, M.-E., LE BELLE, N., VIDAL, B., VERNIER, P., & PASQUALINI, C. (2005). Dopaminergic Inhibition of Reproduction in Teleost Fishes: Ecophysiological and Evolutionary Implications. Annals of the New York Academy of Sciences, 1040(1), 9-21. doi:10.1196/annals.1327.002Henkel, C. V., Burgerhout, E., de Wijze, D. L., Dirks, R. P., Minegishi, Y., Jansen, H. J., … van den Thillart, G. E. E. J. M. (2012). Primitive Duplicate Hox Clusters in the European Eel’s Genome. PLoS ONE, 7(2), e32231. doi:10.1371/journal.pone.0032231Lafont, A.-G., Rousseau, K., Tomkiewicz, J., & Dufour, S. (2016). Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation. General and Comparative Endocrinology, 235, 177-191. doi:10.1016/j.ygcen.2015.11.021Maugars, G., & Dufour, S. (2015). Demonstration of the Coexistence of Duplicated LH Receptors in Teleosts, and Their Origin in Ancestral Actinopterygians. PLOS ONE, 10(8), e0135184. doi:10.1371/journal.pone.0135184Morini, M., Pasquier, J., Dirks, R., van den Thillart, G., Tomkiewicz, J., Rousseau, K., … Lafont, A.-G. (2015). Duplicated Leptin Receptors in Two Species of Eel Bring New Insights into the Evolution of the Leptin System in Vertebrates. PLOS ONE, 10(5), e0126008. doi:10.1371/journal.pone.0126008Pasqualini, C., Weltzien, F.-A., Vidal, B., Baloche, S., Rouget, C., Gilles, N., … Dufour, S. (2009). Two Distinct Dopamine D2 Receptor Genes in the European Eel: Molecular Characterization, Tissue-Specific Transcription, and Regulation by Sex Steroids. Endocrinology, 150(3), 1377-1392. doi:10.1210/en.2008-0578Pasquier, J., Lafont, A.-G., Jeng, S.-R., Morini, M., Dirks, R., van den Thillart, G., … Dufour, S. (2012). Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family. PLoS ONE, 7(11), e48931. doi:10.1371/journal.pone.0048931Rozenfeld, C., Butts, I. A. E., Tomkiewicz, J., Zambonino-Infante, J.-L., & Mazurais, D. (2016). Abundance of specific mRNA transcripts impacts hatching success in European eel, Anguilla anguilla L. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 191, 59-65. doi:10.1016/j.cbpa.2015.09.011Morini, M., Peñaranda, D. S., Vílchez, M. C., Nourizadeh-Lillabadi, R., Lafont, A.-G., Dufour, S., … Pérez, L. (2017). Nuclear and membrane progestin receptors in the European eel: Characterization and expression in vivo through spermatogenesis. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 207, 79-92. doi:10.1016/j.cbpa.2017.02.009Ravi, V., & Venkatesh, B. (2018). The Divergent Genomes of Teleosts. Annual Review of Animal Biosciences, 6(1), 47-68. doi:10.1146/annurev-animal-030117-014821Peña-Llopis, S., & Brugarolas, J. (2013). Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications. Nature Protocols, 8(11), 2240-2255. doi:10.1038/nprot.2013.141Andrews S. FastQC: A quality control tool for high throughput sequence data. 2010. p. http://www.bioinformatics.babraham.ac.uk/projects/Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. doi:10.1093/bioinformatics/btu170Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, J., … Regev, A. (2013). De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols, 8(8), 1494-1512. doi:10.1038/nprot.2013.084Howe, K., Clark, M. D., Torroja, C. F., Torrance, J., Berthelot, C., Muffato, M., … Matthews, L. (2013). The zebrafish reference genome sequence and its relationship to the human genome. Nature, 496(7446), 498-503. doi:10.1038/nature12111Kai, W., Kikuchi, K., Tohari, S., Chew, A. K., Tay, A., Fujiwara, A., … Venkatesh, B. (2011). Integration of the Genetic Map and Genome Assembly of Fugu Facilitates Insights into Distinct Features of Genome Evolution in Teleosts and Mammals. Genome Biology and Evolution, 3, 424-442. doi:10.1093/gbe/evr041Schartl, M., Walter, R. B., Shen, Y., Garcia, T., Catchen, J., Amores, A., … Warren, W. C. (2013). The genome of the platyfish, Xiphophorus maculatus, provides insights into evolutionary adaptation and several complex traits. Nature Genetics, 45(5), 567-572. doi:10.1038/ng.2604Nomura, K., Fujiwara, A., Iwasaki, Y., Nishiki, I., Matsuura, A., Ozaki, A., … Tanaka, H. (2018). Genetic parameters and quantitative trait loci analysis associated with body size and timing at metamorphosis into glass eels in captive-bred Japanese eels (Anguilla japonica). PLOS ONE, 13(8), e0201784. doi:10.1371/journal.pone.0201784Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., & Zdobnov, E. M. (2015). BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics, 31(19), 3210-3212. doi:10.1093/bioinformatics/btv351Pertea, M., Kim, D., Pertea, G. M., Leek, J. T., & Salzberg, S. L. (2016). Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nature Protocols, 11(9), 1650-1667. doi:10.1038/nprot.2016.095Li, H., & Durbin, R. (2010). Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics, 26(5), 589-595. doi:10.1093/bioinformatics/btp698Li, L. (2003). OrthoMCL: Identification of Ortholog Groups for Eukaryotic Genomes. Genome Research, 13(9), 2178-2189. doi:10.1101/gr.1224503Capella-Gutierrez, S., Silla-Martinez, J. M., & Gabaldon, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25(15), 1972-1973. doi:10.1093/bioinformatics/btp348Lartillot, N., Lepage, T., & Blanquart, S. (2009). PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics, 25(17), 2286-2288. doi:10.1093/bioinformatics/btp368Huerta-Cepas, J., Szklarczyk, D., Forslund, K., Cook, H., Heller, D., Walter, M. C., … Bork, P. (2015). eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Research, 44(D1), D286-D293. doi:10.1093/nar/gkv1248Finn, R. D., Clements, J., & Eddy, S. R. (2011). HMMER web server: interactive sequence similarity searching. Nucleic Acids Research, 39(suppl), W29-W37. doi:10.1093/nar/gkr367Alexa A, Rahnenfuhrer J. topGO: Enrichment analysis for gene ontology. 2016. p. R package version 2.29.0.Kanehisa, M., Sato, Y., & Morishima, K. (2016). BlastKOALA and GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome Sequences. Journal of Molecular Biology, 428(4), 726-731. doi:10.1016/j.jmb.2015.11.006Burgerhout, E., Minegishi, Y., Brittijn, S. A., de Wijze, D. L., Henkel, C. V., Jansen, H. J., … van den Thillart, G. E. E. J. M. (2016). Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel ( Anguilla anguilla ); Biomarkers for broodstock selection. General and Comparative Endocrinology, 225, 185-196. doi:10.1016/j.ygcen.2015.08.006Ager-Wick, E., Dirks, R. P., Burgerhout, E., Nourizadeh-Lillabadi, R., de Wijze, D. L., Spaink, H. P., … Henkel, C. V. (2013). The Pituitary Gland of the European Eel Reveals Massive Expression of Genes Involved in the Melanocortin System. PLoS ONE, 8(10), e77396. doi:10.1371/journal.pone.0077396Minegishi, Y., Aoyama, J., Inoue, J. G., Miya, M., Nishida, M., & Tsukamoto, K. (2005). Molecular phylogeny and evolution of the freshwater eels genus Anguilla based on the whole mitochondrial genome sequences. Molecular Phylogenetics and Evolution, 34(1), 134-146. doi:10.1016/j.ympev.2004.09.00

Impact of dietary fatty acids on muscle composition, liver lipids, milt composition and sperm performance in European eel

[EN] In order for European eel aquaculture to be sustainable, the life cycle should be completed in captivity. Development of broodstock diets may improve the species' reproductive success in captivity, through the production of high-quality gametes. Here, our aim was to evaluate the influence of dietary regime on muscle composition, and liver lipids prior to induced maturation, and the resulting sperm composition and performance. To accomplish this fish were reared on three "enhanced" diets and one commercial diet, each with different levels of fatty acids, arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Neutral lipids from the muscle and liver incorporated the majority of the fatty acid profile, while phospholipids incorporated only certain fatty acids. Diet had an effect on the majority of sperm fatty acids, on the total volume of extractable milt, and on the percentage of motile sperm. Here, our results suggest that the total volume of extractable milt is a DHA-dependent process, as we found the diets with the highest DHA levels induced the most milt while the diet with the lowest DHA level induced the least amount of milt The diet with the highest level of ARA induced medium milt volumes but had the highest sperm motility. EPA also seems important for sperm quality parameters since diets with higher EPA percentages had a higher volume of milt and higher sperm motility. In conclusion, dietary fatty acids had an influence on fatty acids in the tissues of male eel and this impacted sperm performance. (C) 2015 Elsevier Inc All rights reserved.This study relates to the project: Reproduction of European Eel: Towards a Self-sustained Aquaculture (PRO-EEL) funded by the European Commission 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology" (Grant Agreement no. 245257). JFA and LP had a grant to stay in Denmark from the Universitat Politecnica de Valencia (PAID-00-11). Special thanks to P. Lauesen (Billund Aquaculture Service), and C. Graver (Danish Aquaculture Organisation) for help during experimentation, and Lars Holst, BioMar A/S, who took part in sourcing and feed production. IAEB, RB, LP, JFA and JT received travel grants from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).Butts, IAE.; Baeza Ariño, R.; Stottrup, JG.; Kruger-Johnsen, M.; Jacobsen, C.; Pérez Igualada, LM.; Asturiano Nemesio, JF.... (2015). Impact of dietary fatty acids on muscle composition, liver lipids, milt composition and sperm performance in European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 183:87-96. https://doi.org/10.1016/j.cbpa.2015.01.015S879618

Effect of the probiotic Lactobacillus rhamnosus on the expression of genes involved in European eel spermatogenesis

[EN] Positive effects of probiotics on fish reproduction have been reported in several species. In the present study, 40 male European eels were weekly treated with recombinant hCG for 9 weeks and with three different concentrations (10(3), 10(5), and 10(6) CFU/mL) of probiotic Lactobacillus rhamnosus IMC 501 (Sinbyotec, Italy). The probiotics were daily added to the water from the sixth week of the hCG treatment. Males from the treated and control groups were sacrificed after 1, 2, and 3 weeks of probiotic treatment (seventh ninth weeks of hCG treatment); at this point, sperm and testis samples were also collected. Sperm volume was estimated, and motility was analyzed by computer-assisted sperm analysis software. Alternations in transcription of specific genes involved in reproductive process such as activin, androgen receptors alpha and beta (ar alpha and ar beta), progesterone receptor 1 (pr1), bone morphogenetic protein 15 (bmp15), and FSH receptor (fshr) were analyzed in the testis. After 2 weeks of probiotic treatment, sperm production and sperm motility parameters (percentage of motile cells and percentage of straight-swimming spermatozoa) were increased in the European eel treated with 105 CFU/mL compared to controls or to the other probiotic doses. These changes were associated with increases in messenger RNA expression of activin, ar alpha, ar beta, pr1, and fshr. Conversely, after 3 weeks, activin and pr1 expression decreased. No significant changes were observed on bmp15 expression throughout the duration of the treatment with 10(5) CFU/mL dose. The lowest and highest probiotic dose (10(3) and 10(6) CFU/mL, respectively) inhibited the transcription of all genes along all the experiment, except for ar alpha and ar beta after 1 week of probiotic treatment when compared to controls. The changes observed by transcriptomic analysis and the sperm parameters suggest that a treatment with L rhamnosus at 10(5) CFU/mL for 2 weeks could improve spermatogenesis process in Anguilla anguilla. (C) 2015 Elsevier Inc. All rights reserved.This study was funded by the European Community’s 7th FP (grant agreement no. 245257, PRO-EEL) and COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) Victor Gallego and M. Carmen Vilchez have predoctoral grants from MINISTERIO DE ECONOMIA Y COMPETITIVIDAD (BES-2009-020310) and UNIVERSIDAD POLITECNICA DE VALENCIA PAID Program (2011-S2-02-6521), respectively. Fondo Ateneo 2012 to Oliana Carnevali.Vilchez Olivencia, MC.; Santangeli, S.; Maradonna, F.; Gioacchini, G.; Verdenelli, C.; Gallego Albiach, V.; Peñaranda, D.... (2015). Effect of the probiotic Lactobacillus rhamnosus on the expression of genes involved in European eel spermatogenesis. Theriogenology. 84(8):1321-1331. https://doi.org/10.1016/j.theriogenology.2015.07.011S1321133184