Assessing phenotypic variation and plasticity of endemic gastropods from thermal water refugia using complex morphometric techniques: A case study of Lake Pețea melanopsids.

Understanding the underlying reasons for phenotypic plasticity and resulting morphological disparity is one of the key topics of evolutionary research. The phenotypic plasticity of extant and fossil melanopsids has been widely documented. Yet millennial-resolution, well-dated records from small aquatic habitats harboring endemics are scarce. The thermal spring-fed Lake Pețea is an ice age refugia harboring a unique endemic warm-water fauna. Subfossil melanopsids display incredible morphological variability from smooth to keeled, elongated to ribbed, shouldered forms. Numerous morphotypes have been considered as individual taxa with a fluent succession from the smooth elongated to the ribbed, shouldered types. This study presents an extensive morphometric analysis of subfossil melanopsids (ca. 3500 specimens) derived from stratified samples with an independent chronology. The aim was to separate morphotypes for investigations of temporal morphological disparity. Our results challenge the widely accepted hypothesis that proposes the evolution of shouldered, compressed, ribbed shells through a two-step process from smooth elongated spindle-shaped shells. Instead, it suggests that the subfossil shells belong to two distinct taxa present throughout the available stratigraphic data. The main components of shape variation, shape globularity, and shell coiling seem allometry-related. Ribs, striation, and keels appear randomly. High-spired spindle-shaped forms were considered to represent specimens of Microcolpia daudebartii hazayi. Bulkier low-spired and shouldered specimens represent phenotypes of Mi. parreyssii parreyssii. The collective and random distribution of morphotypes from the early stages of the lake's history also refutes the idea of a continuous transformation of the elongated forms into compressed, shouldered ones. Rather points to multiple events and environmental stimuli triggering development. Melanopsids appear in Late Glacial horizons, with Theodoxus prevostianus preferring temperatures above 23°C which may indicate the subordinate presence of hot water microhabitats in cooler waters.

Removal of dead fish eggs by Asellus aquaticus as a potential biological control in aquaculture.

The objective of the present experimental study was to gain a better understanding of the foraging activity of Asellus aquaticus during fish egg incubation. A. aquaticus were introduced into experimental setups of dead eggs, viable eggs and hatched larvae of zebrafish (Danio rerio), a commonly used model organism. The amount of A. aquaticus and the duration of their exposure to the eggs significantly affected the proportion of consumed dead eggs in each experimental cycle. A. aquaticus belongs to the group of aquatic detritivores, and no predatory behavior was observed during the experiments. These crustaceans could distinguish between the dead eggs and those containing living embryos. Furthermore, zebrafish larvae remained unharmed by A. aquaticus, even in the absence of alternative food source. These findings underscore the potential sanitary role of these crustaceans in natural waters and offer new perspectives on their possible use as biological control organisms in aquaculture hatcheries. Additionally, our results suggest a potential application of A. aquaticus in combating pathogens by reducing the growth substrates for bacteria and fungi.

Uncommon life history and winter spawning of common carp (Cyprinus carpio) in a natural thermal spring, under temperate climate.

Common carp female generally matures at age 4-5 years old and spawns between April and July under the temperate climate. Contrary to a range of 0-28 °C of temperate freshwaters, the water temperature of Lake Hévíz (Hungary, Central Europe), the largest natural bathable thermal lake in the world, varies between 26 and 35 °C seasonally. The specific environmental conditions (continuously warm water and its individual chemical composition, special nutrient base, lack of natural lakeside spawning substrate compared to usual spawning grounds, continuous high human disturbance, etc.) suggest that the carp population here may also differ in reproductive characteristics from their counterparts in surrounding waters. Our findings suggest that the self-sustaining dwarf common carp population of Lake Hévíz matures 2 to 4 years earlier (at the age of one) and spawns 1 to 3 months before (between February and April, at 27-30 °C water temperature) than carp typically do in the temperate zone (16-20 °C). Successful winter spawning was verified by rearing larvae from the collected eggs and in situ induced propagation.

Review of Central-Eastern European Propagation and Larvae Nursing Method for Common Carp (Cyprinus carpio L.).

Common carp (Cyprinus carpio L.) as a cultivated fish species has huge importance all over the world. According to FAO statistics, carp is the third most widely bred freshwater pond fish species; only two other Cyprinids (silver carp and grass carp) are bred in higher amounts. Carp is native all over Asia and in a large part of Europe. As a result of human intervention, at present, carp are widespread all over the world, except for the Arctic region. Carp breeding was launched in the antique period, in the ancient Chinese Empire and the Roman Empire. The presently applied method of breeding of common carp has a long evolution. From the effectiveness point of view, the propagation and early-life nursing are crucial parts of carp production, as they provide seed stocks for the further growing section. Without effective propagation, there is no intensive carp production. Nowadays, more advanced propagation methods are available all over the world; however, in the current review, only the main milestones and production efficiency of the propagation and nursing method used in the ponds of Eastern Central Europe are discussed. In the historical overview of carp reproduction, first the natural reproduction, then the semi-extensive and intensive hatchery propagation are presented and investigated in detail. The analysis focuses on the advantages and disadvantages of the method. In particular, the different important milestones of the advanced hatchery method are shown and explained. The effectiveness is proven even with practical calculations. Not only the reproduction, but the pond nursing method is also presented and discussed, concentrating on the management of evolutionarily adapted natural feeds (Zooplankton) and their effect on the survival of fish larvae.

Physiological Response of Grower African Catfish to Dietary Black Soldier Fly and Mealworm Meal.

A six-week experiment was carried out to test the effects of total (100%) and partial (50%) replacement of fish meal in the diet of African catfish growers with black soldier fly (B) meal, yellow mealworm (M) meal, and a 1:1 combination of both (BM) on the production and health of fish. A total of 420 fish with an average initial body weight of 200 ± 0.5 g were randomly distributed in triplicate to seven diet groups (C, B50, B100, M50, M100, BM50, and BM100, respectively). The growth performance and feed utilization of fish fed with partial or total replacement levels of FM with B were not significantly affected (p > 0.05) during the 6 weeks of feeding. In contrast, significant differences were observed between the groups fed with a diet where FM was totally replaced with M meal and the control in terms of final body weight, specific growth rate, feed conversion ratio, protein efficiency ratio, and protein productive value. Among the blood plasma biochemistry parameters, total cholesterol exhibited a significant difference (p = 0.007) between the M treatments and the control diet. The fatty acid profile of the liver was changed with respect to the long-chain polyunsaturated fatty acid content in all experimental groups. Parallel with this, the upregulation of elovl5 and fas genes in liver was found in all experimental groups compared to the control. Overall, this study shows that fish meal cannot be substituted with yellow mealworm meal in the practical diet of African catfish without compromising the growth, health and feed utilization parameters.

Carp Breeding in the Carpathian Basin with a Sustainable Utilization of Renewable Natural Resources.

In the Central European region, there is a long tradition of breeding fish in artificially constructed ponds. As the area belongs to the temperate zone, farmed fish need to survive cold winter periods. Common carp (Cyprinus carpio L.), which is an omnivorous, bioturbating species, is well adapted to warm and cold periods and the alluvial water environment. Since the Middle Ages, a large scale, efficient carp farming methodology has been developed in the region, where production is based on natural resources (protein and fatty acid sources) of renewable water ecosystems. This summary aims to present this well-developed breedi:ng method through discussing aspects of hydrobiology and energy transfer through the food chain as well. Capabilities and effects of agro-technical treatments such as liming and organic manuring, zooplankton management and possible supplementary feedings are also reviewed. Analysing chemical processes of waters uncovers that biological production has no carbon footprint; no carbon dioxide is released into the atmosphere. In contrast, gaseous carbon dioxide diffuses into pond water containing calcium and/or magnesium, then it accumulates in algae production and, through energy migration to upper trophic levels, increases carp production. Thus, it can be declared that pond-farmed carp provides an environmentally friendly, delicious meat among products of animal origin.

Ovarian inseminated sperm impacts spawning success in zebrafish, Danio rerio (Hamilton, 1822) even in the absence of a male stimulus.

Reproductive obstacles have led scientists to develop novel techniques/technologies for artificial reproduction. We aimed to investigate the possibility of propagating zebrafish females using sperm ovarian lavage with and without presence of male stimulus. This experiment consisted of several treatments: traditional spawning approaches with females and wild-type males (AB♀ × AB♂); no males present with non-manipulated females (AB♀); no males present with females inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)]; no males present with females inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)]; non-manipulated females kept separately from wild-type males (AB♀|AB♂); females kept separately from wild-type males and inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)|AB♂]; and females kept separately from wild-type males and inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)|AB♂]. There were no released eggs in both negative control treatments (AB♀ and AB♀|AB♂). Egg production increased (ranged from 0 to 28.5 eggs/female) when females were injected in the presence [AB♀ (inj.NaCl) |AB♂] or absence of male stimulus [AB♀ (inj.NaCl) and (AB♀(inj.Tg♂)]. A further increase in egg production [relative to AB♀, AB♀ (inj.NaCl), and AB♀|AB♂] was detected when females were inseminated with pooled sperm from transgenic males in the presence of male stimulus [AB♀(inj.Tg♂)|AB♂; ranged from 2.5 to 55 eggs/female] or when using traditional spawning approaches (AB♀ × AB♂; ranged from 25 to 131 eggs/female). Females inseminated with sperm produced embryos, although no differences were detected when females were inseminated with pooled sperm from transgenic males in presence (11.8 ± 16.3%) or absence (average = 12.6 ± 9.2%) of male stimulus. Traditional spawning approaches produced the most eggs (81.2 ± 42.3 per female) and highest fertilization rate (81.3 ± 10.4).

A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells.

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me2SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN2 at -80 °C displayed the highest OSC viability. Different cooling rates ranging from -1 to -40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me2SO) with VS3 (3 M PG and 3 M Me2SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.

Development and characterization of 49 novel microsatellite markers in the African catfish, Clarias gariepinus (Burchell, 1822).

The African catfish or sharp tooth catfish (Clarias gariepinus) is one of the important species (due to its high environmental tolerance and easily controllable breeding habits) that can significantly contribute to reducing hunger in many countries. It is farmed in numerous African, Asian, and European countries. Moreover, during the last decades its production has grown significantly worldwide. Currently, following the carp, this species is produced in the second largest volume in Hungary. Despite its economic importance, the stocks have been maintained without genetic control or guided breeding. Molecular genetic data on bred populations or strains are very limited. In order to investigate the genetic structure of the stocks, 49 new microsatellite markers were characterized and tested on 32 individuals from a Hungarian farmed stock. All these markers were polymorph. The number of alleles per locus ranged from 2 to 11. The observed and expected overall heterozygosities were between 0.519 and 0.544 respectively and the overall inbreeding coefficient (Fis: 0.063) does not reveal the presence of inbreeding. However, 63% of the markers showed significant deviations from HWE. The results suggest that the maintenance of genetic variation within the stock require high attention in closed bred populations. These new markers provide a useful tool for population and conservation genetics of natural and bred African catfish populations.

Eel sperm cryopreservation: An overview.

The eels are teleost fishes from the order Anguilliformes that includes several species with high commercial value. Due to the high interest for aquaculture production of some eel species and for the need to restore eel species that are endangered, several research groups have directed their research toward developing protocols to cryopreserve the spermatozoa of Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla). In this review, we provide an overview on the different protocols that have been developed so far. The first developed protocols used DMSO as cryoprotectant in both species with good success, obtaining sperm motilities of over 45% in Japanese eel and over 35% in European eel. Moreover, sperm cryopreserved using DMSO was successfully used in fertilization trials, although with low fertilization rates. However, recent studies show that DMSO produce epigenetic changes in eel sperm and therefore, the last developed protocols used methanol as cryoprotectant instead. Cryopreservation protocols using methanol as cryoprotectant, showed improved motility values in both Japanese and European eel. In addition, the latest protocols have been adapted to cryopreserve larger volumes of sperm of up to 5 mL, which is useful for larger scale fertilization trials. The present study introduces the state of the art and future perspectives of the eel sperm cryopreservation to be applied in aquaculture and biological conservation programs.

Artificial insemination of African catfish (Clarias gariepinus) using cryopreserved sperm.

In this study, we aimed to develop a practical protocol for using cryopreserved sperm for induced/wild/tank spawning of fish species with external fertilization. Experiments were carried out on African catfish (Clarias gariepinus) as a model species. Sperm was collected for cryopreservation and diluted with the cryomedium (266 mM fructose, 20% methanol) at a ratio of 1:1 with a final methanol concentration of 2.47 M pH7.73. Diluted sperm was loaded into 0.5-ml straws and cryopreserved by conventional protocol. Samples were prepared for insemination 24 h later, by thawing for 13 s in a 40 °C water bath, and centrifuged at 500 × g for 10 min at 20 °C. The seminal plasma, extender and external cryoprotectant were removed from the concentrated spermatozoa. The pellet was then resuspended in common carp (Cyprinus carpio) seminal plasma to reconstitute the lost volume. Sperm samples were then injected by a catheter into the ovarian cavity through the oviduct of the experimental females by the so-called ovarian lavage method in parallel with the intramuscular hormonal administration (5 mg carp pituitary/kg bw). Inseminated females (n = 9) were monitored for 10 h and ovulated eggs and spermatozoa stored in in the ovary were stripped. Stripped gamete samples were divided into two batches: (1) the first batch contained only the previously injected spermatozoa and was activated by aerated water (WA) immediately after stripping; (2) in case of the second batch additional, freshly stripped sperm was added as positive control to the stripped eggs before water activation (PC). Furthermore, five females were propagated by using the dry fertilization method (in vitro fertilization) as negative control (NC). All sperm and hormone injected females produced fertilised eggs with a hatching rate of 17.7 ±â€¯13.2%, 12.5 ±â€¯9.3%, and 61 ±â€¯11.5% for WA, PC and NC respectively. These results indicate that artificial insemination based on using cryopreserved sperm with ovarian lavage can be a viable alternative to in vitro fertilization in a catfish species. Thus, we describe a proof of principle for a practical protocol for the induced/wild/tank spawning of an externally fertilising fish species with economical importance and propose that the protocol could be also applied to endangered marine or fresh fish species.

Testing cryopreserved European eel sperm for hybridization (A. japonica × A. anguilla).

The objective of this study was to assess impact of cryopreserved European eel sperm and Japanese eel native sperm on early fertilization, hatch, survival, and malformation rates of larvae, as well as develop molecular techniques to distinguish different eel species. Eggs from Japanese eel females (Anguilla japonica) were artificially fertilized with sperm of Japanese eel males and cryopreserved sperm from European eel (A. anguilla, extender was modified Tanaka solution and methanol as cryoprotectant). There were no statistical differences (p > 0.05) among the measured parameters such as fertilization, hatch and survival after 10 days post-hatch rates due to large individual differences. The malformation rate of larvae compared to the hatching rate was higher in cryopreserved groups than in the control indicating that the methodology needs further refinement. Genetic analyses (PCR-RFLP, PCR-HRM) proved a clear result in the detection of paternal contribution in hybridization between the Japanese and the European eel and applied PCR-HRM method is a quick and cost effective tool to identify illegally imported A. anguilla at the glass eel stage, which can be transported from Europe to Asia.

Fusiform vateritic inclusions observed in European eel (Anguilla anguilla L.) sagittae.

Microscopic inclusions have been observed in 7 out of 106 European eel (Anguilla anguilla L.) sagittae using polarizing microscope and scanning electron microscope meanwhile the annual increments were studied to characterize the age structure of the population living in Lake Balaton. The presence of vaterite, a rare calcium carbonate polymorph was observed in these inclusions using Raman spectroscopy. Vateritic sagittae in wild fish are usually considered as symptom of physiological stress. The observed fusiform inclusions represent a new morphological type of vaterite inclusions in eel otolith. Two alternatives are hypothesized to explain their formation: 1) metabolic disorder, such as erroneous protein synthesis; 2) introduction of an alien protein into the eel's inner ear. The origin and physiological significance of this new morphological type of vateritic inclusions is still an open question. Same as whether it can be found in other species or specific only to eel otoliths.

Species-specific markers provide molecular genetic evidence for natural introgression of bullhead catfishes in Hungary.

Since three bullhead catfish species were introduced to Europe in the late 19th century, they have spread to most European countries. In Hungary, the brown bullhead (Ameiurus nebulosus) was more widespread in the 1970s-1980s, but the black bullhead (Ameiurus melas) has gradually supplanted since their second introduction in 1980. The introgressive hybridization of the two species has been presumed based on morphological examinations, but it has not previously been supported by genetic evidence. In this study, 11 different Hungarian habitats were screened with a new species-specific nuclear genetic, duplex PCR based, marker system to distinguish the introduced catfish species, Ameiurus nebulosus, Ameiurus melas, and Ameiurus natalis, as well as the hybrids of the first two. More than 460 specimens were analyzed using the above markers and additional mitochondrial sequence analyses were also conducted on >25% of the individuals from each habitat sampled. The results showed that only 7.9% of the specimens from two habitats belonged to Ameiurus nebulosus, and 92.1% were classified as Ameiurus melas of all habitats, whereas the presence of Ameiurus natalis was not detected. Two specimens (>0.4%) showed the presence of both nuclear genomes and they were identified as hybrids of Ameiurus melas and Ameiurus nebulosus. An additional two individuals showed contradicting results from the nuclear and mitochondrial assays as a sign of a possible footprint of introgressive hybridization that might have happened two or more generations before. Surprisingly, the level of hybridization was much smaller than expected based on the analyses of the North American continent's indigenous stock from the hybrid zones. This phenomenon has been observed in several invasive fish species and it is regarded as an added level of complexity in the management of their rapid adaptation.

Full genome sequence of a novel circo-like virus detected in an adult European eel Anguilla anguilla showing signs of cauliflower disease.

An adult European eel Anguilla anguilla, showing typical signs of the so-called cauliflower disease, was subjected to pathological and molecular virological examinations. Samples taken from internal organs and the polypoid proliferative tissue from the mouth were examined by PCR for the detection of several viruses. Positive results were obtained with a nested PCR targeting the rep gene of circoviruses. Analysis of the partial rep sequence indicated the presence of a putative novel circovirus, but attempts to isolate it remained unsuccessful. The missing part of the genome was acquired by an inverse nested PCR with 2 specific primer pairs, designed from the newly determined rep sequence, followed by genome walking. The circular full genome was found to consist of 1378 nt (GenBank accession no. KC469701). Two oppositely oriented open reading frames (ORFs) were present, of which one was unambiguously identified as a circoviral rep gene. However, the predicted product of the other ORF, though it is a clear positional counterpart of the cap genes, showed no obvious homology to any known circoviral capsid proteins. A stem-loop-like element in the intergenic region between the 5' ends of the ORFs was also found. Phylogenetic calculations indicated that the novel virus belongs to the genus Circovirus in the family Circoviridae. The relative amount of the viral DNA in the organ samples was estimated by quantitative real-time PCR. The results suggested that the examined fish was caught in an active viremic state, although the role of this circovirus in the etiology of the cauliflower diseases could not be ascertained.

Re-visiting phylogenetic and taxonomic relationships in the genus Saga (Insecta: Orthoptera).

Twelve of the 13 bushcricket species of the Saga genus are bisexuals and diploids, except the parthenogenetic and tetraploid bush cricket, Saga pedo. Despite a continuous research effort stretching through the 1900s, the taxonomic relationships of the Saga species are still disputed. In this study, our primary aim was to reveal natural relationships of the European Saga species and three of their Asian relatives, with special attention to the problematic taxonomy of two subspecies: S. campbelli campbelli and S. c. gracilis. Following a phylogenetic analysis of eight species, a comprehensive study was carried out on the above three taxa by using acoustic and morphometric approaches in parallel. Our phylogenetic data showed that European Saga species evolved from a monophyletic lineage. The geographical transitional species S. cappadocica was positioned between European and Asian lineages supporting the idea that the European Saga lineage originated phylogeographically from the Asian clade. The above results showed better agreement with the morphological data than with earlier ones based either on karyology or acoustic information only. After reviewing our data, we concluded that Saga pedo has most likely evolved from S. c. gracilis and not from S. rammei or S. ephippigera, as proposed by earlier studies. S. c. gracilis shares the same ITS2 haplotype with S. pedo, indicating that the latter could have evolved from populations of the former, probably through whole genome duplication. Based on acoustic and morphometric differences, we propose to elevate the two subspecies, S. campbelli campbelli and S. c. gracilis, to species level status, as Saga gracilis Kis 1962, and Saga campbelli Uvarov 1921. The present work sets the stage for future genetic and experimental investigations of Saginae and highlights the need for additional comprehensive analysis involving more Asian Saga species.

Intraovarian transplantation of stage I-II follicles results in viable zebrafish embryos.

Maternal gene products drive early embryogenesis almost exclusively until the mid blastula transition (MBT) in many animal models including fish. However, the maternal contribution to embryogenesis does not stop at MBT, but continues to be an essential regulator of key developmental processes. The extent to which maternal effects contribute to embryonic and larval development is hard to estimate due to the technical difficulty of interfering with maternal gene products by conventional forward and reverse genetic tools. Therefore, novel methods to manipulate maternal factors in oocytes need to be developed. Here, we provide a proof of principle protocol for transplanting stage I-II zebrafish follicles into recipient mothers where donor stage I oocytes can develop to stage IV in 2 weeks and in 3 weeks they develop into mature eggs and produce viable offspring. Moreover, we show that simple microinjection of stage I-II follicles with RNA results in reporter gene expression in oocytes and paves the way for developing tools for interfering with maternal gene activity. This early stage oocyte transplantation protocol provides a means to study cellular and molecular aspects of oocyte development in the zebrafish.