Comparative transcriptome analysis of trout skin pigment cells

Background Enormous variability in skin colour and patterning is a characteristic of teleost fish, including Salmonidae fishes, which present themselves as a suitable model for studying mechanisms of pigment patterning. In order to screen for candidate genes potentially involved in the specific skin pigment pattern in marble trout (labyrinthine skin pattern) and brown trout (spotted skin pattern), we conducted comparative transcriptome analysis between differently pigmented dermis sections of the adult skin of the two species. Results Differentially expressed genes (DEGs) possibly associated with skin pigment pattern were identified. The expression profile of 27 DEGs was further tested with quantitative real-time PCR on a larger number of samples. Expression of a subset of ten of these genes was analysed in hybrid (marble x brown) trout individuals and compared with the complexity of their skin pigment pattern. A correlation between the phenotype and the expression profile assessed for hybrid individuals was detected for four (gja5, clcn2, cdkn1a and tjp1) of the ten candidate genes tested. The potential role of these genes in skin pigment pattern maintenance is discussed. Conclusions Our results indicate that the maintenance of different pigment patterns in trout is dependent upon specific communication—involving gap junctions, tight junctions and ion channels—between chromatophores present in differentially pigmented skin regions.publishedVersio

Comparative transcriptome analysis of trout skin pigment cells

Background Enormous variability in skin colour and patterning is a characteristic of teleost fish, including Salmonidae fishes, which present themselves as a suitable model for studying mechanisms of pigment patterning. In order to screen for candidate genes potentially involved in the specific skin pigment pattern in marble trout (labyrinthine skin pattern) and brown trout (spotted skin pattern), we conducted comparative transcriptome analysis between differently pigmented dermis sections of the adult skin of the two species. Results Differentially expressed genes (DEGs) possibly associated with skin pigment pattern were identified. The expression profile of 27 DEGs was further tested with quantitative real-time PCR on a larger number of samples. Expression of a subset of ten of these genes was analysed in hybrid (marble x brown) trout individuals and compared with the complexity of their skin pigment pattern. A correlation between the phenotype and the expression profile assessed for hybrid individuals was detected for four (gja5, clcn2, cdkn1a and tjp1) of the ten candidate genes tested. The potential role of these genes in skin pigment pattern maintenance is discussed. Conclusions Our results indicate that the maintenance of different pigment patterns in trout is dependent upon specific communication—involving gap junctions, tight junctions and ion channels—between chromatophores present in differentially pigmented skin regions

Primena krioprezervacije sperme na gajenje i očuvanje salmonidnih vrsta: saradnja Slovenije i Mađarske

U svrhu očuvanja dve salmonidne vrste koje su autohtone u slivu reke Soče u Sloveniji, pastrmske glavatice (Salmo marmoratus) i lipljena Jadranskog porekla (Thymallus thymallus) primenjena je krioprezervacija sperme. Populacije ovih vrsta ozbiljno su ugrožene hibridizacijom i introgresijom sa alohtonim vrstama: potočnom pastrmkom (Salmo trutta m. fario) i lipljenom Dunavskog porekla koji su unešeni u sliv reke Soče u dvadesetom veku. Ribolovački klub Tolmin, koji upravlja jednim delom reke Soče, razvio je akcioni plan za očuvanje genetskih resursa i restauraciju autohtonih vrsta u njihova primarna staništa. Ovaj akcioni plan podrazumeva prestanak poribljavanja alohtonim salmonidnim vrstama koje mogu da naprave hibride sa lokalnim vrstama, identifikaciju postojećih “čistih” vrsta, stvaranje matica od onih jedinki koji pripadaju čistim vrstama, poribljavanje potomcima čistih vrsta, stvaranje “utočišta” za postojeće čiste vrste i stalni monitoring populacija riba u njihovim vodotokovima. Genetske analize ovih populacija izvršili su naučnici sa Odeljenja za Nauke o životinjama Univerziteta u Ljubljani. Krioprezervacija sperme Jadranskog lipljana i pastrmske glavatice čini sastavni deo akcionog plana za očuvanje od 2009. godine. Kada je reč o lipljanu, ne postoji više ni jedna čista populacija, stoga je cilj programa očuvanja povećanje udela Jadranskog genotipa kod matica. Sperma i uzorak peraja uzorkovani su od divljih mužjaka na mestu mresta. Sperma je krioprezervirana i sačuvana dok genetske analize nisu završene za svaki od uzoraka (2-3 nedelje). Krioprezervirana sperma koja je sadržala veću količinu Jadranskog genotipa nego što je to na početku definisano je otopljena i iskorišćena za fertilizaciju jaja takozvanih ‘Jadranskih’ ženki. Potomstvo iz jaja oplođenih krioprezerviranom spermom je odgajeno do matica i trenutno 70-80% matica lokalnog lipljana potiče iz krioprezervirane sperme. Kada je reč o pastrmskoj glavatici, krioprezervirana sperma se koristi za stvaranje ‘utočišta’. Sperma se sakuplja od divljih mužjaka čiste populacije pre sezone mresta (rani novembar) a zatim se krioprezervira. Sperma se čuva u tečnom azotu do sezone mresta (decembar-januar) kada dolazi do oplodnje jaja ženki koje pripadaju identičnoj populaciji. Oplodnja se obavlja krioprezerviranom supermom. Jaja u stadijumu očne mrlje se zatim nasade u veštačka gnezda u pripremljenom potoku koji predstavlja ‘utočište’. Stoga veliki broj mužjaka iz čiste populacije učestvuje u stvaranju novih populacija, dok u isto vreme nije potrebno ukloniti mužjake iz originalne populacije, a nasađivanje jaja u stadijumu očne mrlje obezbeđuje obeležavanje nove teritorije. Isti protokol se koristi za krioprezervaciju obe vrste: sperma se meša u odnosu 1:1 sa ekstenderom koji sadrži 200 mM glukoze, 40 mM KCl, 30 mM trisa (pH iznosi 8.0 sa cc. HCl) a metanol se koristi kao krioprotektant u finalnoj koncentraciji od 10% v/v . Cevčice od 0.5-ml se pune rastvorenom spermom koja se zatim zamrzava u pari tečnog azota na 3cm iznad nivoa azota u trajanju od 3 minuta. Pošto se sačuva u tečnom azotu, uzorci se tope u vodenoj kupatilu na 40 °C u trajanju od 13 sekundi. Ovako razvijeni protokol rezultira u 50-70% izmrešćenih jedinki kod obe vrste.Sperm cryopreservation was applied to the conservation efforts of two salmonid species autochthonous to the drainage of the Soča river in Slovenia, the marble trout (Salmo marmoratus) and the Adriatic lineage of the grayling (Thymallus thymallus). Populations of these species were seriously compromised by hybridization and introgression with allochthonous species: the brown trout (Salmo trutta m. fario) and the Danubian lineage of the grayling that were introduced to the Soča drainage during the 20th century. The Angling club of Tolmin, that manages a part of the Soča system has developed an action plan for the conservation of the genetic resources and restoration of the autochthonous species in their original habitats. This action plan includes the cessation of the stocking of any allochthonous salmonids that could hybridize with the local species, identification of existing pure populations, establishment of broodstocks of non-introgressed individuals, stocking the rivers with the progeny of the broodstocks, creation of „sanctuary” streams for the existing pure populations and continuous monitoring of fish populations in their watercourses. Genetic analyses of the populations are conducted by scientists from the Department of Animal Science of the University of Ljubljana. Cryopreservation of sperm from the Adriatic grayling and the marble trout has constituted an integral part of the conservation activities since 2009. In case of the grayling, no pure populations remain, thus, the objective of the conservation program is to increase the proportion of Adriatic genotype in the broodstock. Sperm and fin clips were collected from wild males on the spawning grounds. Sperm was cryopreserved and stored until the genetic analysis was completed on each sample (2-3 weeks). Cryopreserved sperm of individuals containing higher than a pre-defined proportion of Adriatic genotype was thawed and used for fertilization of eggs known „Adriatic” females. Progeny hatching from eggs fertilized with cryopreserved sperm was grown to broodstock and currently 70-80% of the local grayling broodstock originates from cryopreserved sperm. In case of the marble trout, sperm cryopreservation is used in the creation of „sanctuary” streams. Sperm is collected from wild males of a given pure population prior to the spawning season (early November) and cryopreserved. Sperm is stored in liquid nitrogen until the spawning season (December-January) when eggs of females from the identical population are fertilized with the cryopreserved sperm. Eyed eggs are then stocked into artificially created nests in the prepared „sanctuary” stream. Thus, a high number of males of the given pure population participates in the creation of the new population, removal of males from the original population is unnecessary, and stocking of eyed eggs ensures imprinting at the new location. The same protocol is used for cryopreservation of both species: sperm is mixed at a ratio of 1:1 with an extender containing 200 mM glucose, 40 mM KCl, 30 mM Tris (pH set to 8.0 with cc. HCl) and methanol is used as a cryoprotectant at 10% v/v final concentration. Diluted sperm is loaded into 0.5-ml straws and frozen in the vapor of liquid nitrogen at 3 cm above the level of nitrogen for 3 minutes. Following storage in liquid nitrogen, samples are thawed at a 40 °C water bath for 13 sec. The developed protocol results in 50-70% hatch in both species