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In most Japanese populations of dojo loach (Misgurnus anguillicaudatus), gonochoristic diploids of genetically diversified groups (A and B, further subdivided into B1 and B2) are present, whereas unisexual clonal lineages inhabit certain localities in the Hokkaido and Ishikawa Prefectures in Japan. Through a series of genetic studies including DNA markers, the clonal loaches were deemed to originate from a hybridization event(s) between the A and B1 groups. However, combined analyses with other DNA markers are needed to identify each genetic group. In this study, we improved the PCR-restriction fragment length polymorphism (RFLP) analysis of the recombination activating gene 1 (RAG1) gene using digestion with two restriction enzymes, PvuII and StuI. The improved RAG1-RFLP analysis showed different fragment patterns for each group: two fragments (245 and 198 bp) for group A, three fragments (198, 147, and 98 bp) for group B1, and a single fragment (443 bp) for group B2. The clonal loaches exhibited four fragments (245, 198, 147, and 98 bp) derived from both groups A and B1. Moreover, the DNA markers were able to detect two different hybrid genotypes (A x B2 and B1 x B2). Thus, the improved RAG1-RFLP markers allowed for quick and accurate group identification of the dojo loaches.

Salmonids possess straying abilities that allows them to exploit open territory and establish new populations. Atlantic salmon (Salmo salar) are expanding their distribution primarily as first-generation escapes from aquaculture, whereas Pacific salmon (Oncorhynchus spp.) are expanding their range in polar and temperate regions due to both anthropogenic and natural influences. Here we utilize artificial intergeneric reciprocal crosses to assess the ability of seven species of Pacific salmon to hybridize with Atlantic salmon. Most cross types were found to produce low numbers of hatched embryos, but none survived to sexual maturation. Survivors consisted of diploids and triploids containing both Atlantic and Pacific salmon parental genomes. Thus, introgression of DNA between Pacific and Atlantic salmon may occur to form F1 hybrids, but transmission to subsequent generations is expected to be rare and occur only over evolutionary time scales. Further, the low viability observed for the most part at early stages of development and in both reciprocal crosses indicates that intergeneric crosses in nature between Atlantic and Pacific salmon are expected to have severe fitness consequences for both dams and sires.

There are 2 genetically divergent groups in the dojo loach Misgurnus anguillicaudatus: A and B. Although most wild-type diploids reproduce sexually, clonal diploids (clonal loach) reproduce gynogenetically in certain areas. Clonal loaches produce unreduced isogenic eggs by premeiotic endomitosis, and such diploid eggs develop gynogenetically following activation by the sperm of sympatric wild-type diploids. These clonal loaches have presumably arisen from past hybridization events between 2 different ancestors. The genomic differences between these 2 groups have not been completely elucidated. Thus, new genetic and cytogenetic markers are required to distinguish between these 2 groups. Here, we compared the 5S rDNA region to develop markers for the identification of different dojo loach groups. The nontranscribed sequence (NTS) of the 5S rDNA was highly polymorphic and group-specific. NTS sequences were found in clades of 2 different groups in clonal loaches. In contrast, we did not find any group-specific sequences in the coding region of the 5S rRNA gene. Sequences were located near the centromere of the short arm of the largest submetacentric chromosomes in groups A and B and clonal loaches. Thus, the 5S rDNA of the dojo loach is conserved at the chromosomal location. Whereas, the sequences of the NTS regions evolved group-specifically in the dojo loach, with the sequences of both groups being conserved in clonal loaches.

The zebrafish is a valuable model organism that is widely used in studies of vertebrate development. In the laboratory, zebrafish embryonic development is normally carried out at 28.5°C. In this study, we sought to determine whether it was possible to modify the speed of embryonic development through the use of short- and long-term variations in incubation temperature. After incubation at 20°C-32°C, most early-stage embryos survived to the epiboly stage, whereas more than half of the embryos died at20°C or32°C. The rate of development differed between embryos incubated at the lowest (18°C) and highest (34°C) temperatures: a difference of 60 min was observed at the 2-cell stage and 290 min at the 1k-cell stage. When blastulae that had developed at 28°C were transferred to a temperature lower than 18°C for one or more hours, they developed normally after being returned to the original 28°C. Analyses using green fluorescent protein

In this letter, the design and experimental validation of polarization-agile circular polarized antenna are presented. The antenna is comprised of a patch antenna with truncated slits and two proximity feedlines. The antenna produces a vertical linear polarized wave when it is fed by a proximity feedline. Horizontal linear polarized wave is then generated by applying a potential difference between two proximity feedlines. The quadrature phase difference between two orthogonal polarized waves is created by adding truncated slits into the patch antenna. By swapping the polarity of the proximity feedlines, polarization sense can be changed. The distributed circuit model is presented to describe the working mechanism of the antenna. Patch antenna with circular polarization agility is then validated through passive-type and active-type switching circuits. The measured axial ratio bandwidth is 4.085% (100 MHz), and the fractional impedance bandwidth is greater than 7% for the left-handed and right-handed circular polarized radiations.

Herein, we investigate the system spectral efficiency (SSE) and system energy efficiency (SEE) for massive two-way multiple-input-multiple-output (MIMO) relaying networks while considering imperfect channel state information (CSI) with $K$ -pair users in the presence of the hybrid relaying processing. First, we obtain the general expression of the signal-to-interference-and-noise-ratio (SINR) for the considered system when the antenna number of the relay station (RS) $M$ tends to be significantly large. Next, we consider the power scaling scheme (PSS) and achieve asymptotic SSE and SEE. We set the pilot sequence transmit power $P_{P}$ to be fixed $P_{P}$ and variable $P_{P}$ . For a nonvanishing SINR, we achieve different power scaling cases under fixed and variable $P_{P}$ . For a fixed $P_{P}$ value, the optimal PSS of the SSE can be obtained when the transmit power of each user can be scaled by $M$ , whereas that of the SEE can be achieved when the transmit power of both the RS and each user terminal can be reduced by $M$ . Based on the variable value of $P_{P}$ , the optimal PSS’ of both the SSE and the SEE can be used when the transmit power of both RS and each user can be scaled by $M^{1/3}$ . Thus, we conclude that the required SSE and SEE performances can be attained in multi-pair massive MIMO two-way relaying networks with a considerably low transmit power at RS and/or the transmit power of each user. The analytical results for the same are validated via simulation.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

SummaryThe cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.

Triple-arm crossed dipole antenna (CPDA) backed by 3D printed cavity exhibits high gain over wideband axial ratio for sub-6 GHz applications is presented. To improve peak gain and axial ratio bandwidth performance, multiple radiating arms are introduced. The arms are cascaded using vacant quarter rings to obtain circular polarization. Unlike the conventional CPDAs that yield single circular polarized (CP) wave, two CP waves produced by double-arm or triple-arm CPDAs enhances overall gain performance across wideband axial ratio. The length and orientation of the multiple arms in the CPDA determine the maximum current generated on the crossed dipole antenna, resulting in an improved gain since two CP waves are combined in the free space. To further increase boresight gain, 3D printed cavity is placed beneath the triple-arm CPDA. Unbalance impedance line presented in this work extends impedance bandwidth of the triple-arm CPDA to cover 3-dB axial ratio bandwidth. For validation purpose, a prototype was fabricated and measured. The measurement results demonstrate that the triple-arm CPDA has a boresight gain of 8.91–11.7 dBic within 3-dB axial ratio bandwidth of 51.4% (3.25–5.5 GHz). The proposed antenna produces a stable gain within sub-6 GHz bands., AEU - International Journal of Electronics and Communications, 158, art. no. 154442; 2022

Most wild types of dojo loach (Misgurnus anguillicaudatus) are gonochoristic diploids that are genetically diversified groups (A and B, further subdivided into B1 and B2), while clonal lineages inhabit certain localities in Japan. Through a series of genetic studies including DNA markers, the clonal loaches were deemed to originate from a hybridization event(s) between the A and B1 groups. However, combined analyses with other DNA markers are needed to identify each genetic group. In this study, we improved the PCR-restriction fragment length polymorphism (RFLP) analysis of the recombination activating gene 1 (RAG1) gene using digestion with two restriction enzymes, PvuII and StuI. The improved RAG1-RFLP analysis showed different fragment patterns for each group: two fragments (245 and 198 bp) for group A, three fragments (198, 147, and 98 bp) for group B1, and a single fragment (443 bp) for group B2. The clonal loaches exhibited four fragments (245, 198, 147, and 98 bp) derived from both groups A and B1. Moreover, the DNA markers were able to detect two different hybrid genotypes (A × B2 and B1 × B2). Thus, the improved RAG1-RFLP markers allowed for quick and accurate group identification of the dojo loaches.

Made available in DSpace on 2021-06-25T15:04:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 AES Tiete/ANEEL project Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Rivulidae comprises a family of fish largely distributed in Brazil that includes 201 species, of which 125 are considered endangered. This fact emphasizes the need for development of conservation strategies including studies on genetics and reproduction. In this paper, we describe aspects of biology and reproduction of the rivuliid species Hypsolebias sertanejo. We outline the reproductive behaviour of this species under laboratory conditions, analyze ploidy status by flow cytometry, describe reproductive behaviour and performance and test dry and wet incubation of eggs. Although H. sertanejo showed well known patterns of reproductive behaviour, we verified many peculiarities inherent to its reproductive biology. As expected, most individuals were diploid (87.71%), however 14.29% were considered mosaics. Although no sterility was observed within mosaics, infertility of these fish was not fully evaluated. Hatching rate of the eggs collected was very low following both dry and wet incubation (5.04 and 3.79%, respectively). These results provide interesting information regarding the reproductive success of this species, and suggest that chromosomal abnormalities described may reduce the survival of H. sertanejo under natural conditions, limiting the perpetuation of this species, and emphasizing the need for more preservation efforts, including artificial propagation and gene banking. Sao Paulo State Univ, Aquaculture Ctr, Jaboticabal, SP, Brazil SAA SP, Fishery Inst, Sao Paulo, SP, Brazil Univ South & Southern Para Unifesspa, Biol Fac FACBIO, Grp Studies Reprod Amazon Fish GERPA LANEC, Maraba, Para, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Pirassununga, SP, Brazil Hokkaido Univ, Fac Fisheries Sci, Hakodate, Hokkaido, Japan Sao Paulo State Univ, Aquaculture Ctr, Jaboticabal, SP, Brazil AES Tiete/ANEEL project: 4690000174 FAPESP: 2010/17429-1 FAPESP: 2011/11664-1

A single fed circular polarized microstrip patch antenna excited through near field coupling is proposed. L-shape near-field feed line is used to feed the microstrip patch antenna as it is useful to control the direction of the current, and change the polarization sense of the circularly polarized wave. By using near field coupling technique, the size of the microstrip patch antenna is reduced to be less than a half-wavelength of the guided wave. Along with the size reduction, slits must be inserted into the antenna to achieve good axial ratio. Its length has been optimized numerically to generate axial ratio that cover 2.4 GHz ISM band. Experiment results shows that the proposed antenna possesses an axial ratio bandwidth of 4.1% and 10-dB return loss covers 2.35 to 2.54 GHz. The proposed antenna is eligible for a communication network that uses right- and left-handed circularly polarized wave spatially.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

Rivulidae comprises a family of fish largely distributed in Brazil that includes 201 species, of which 125 are considered endangered. This fact emphasizes the need for development of conservation strategies including studies on genetics and reproduction. In this paper, we describe aspects of biology and reproduction of the rivuliid species Hypsolebias sertanejo. We outline the reproductive behaviour of this species under laboratory conditions, analyze ploidy status by flow cytometry, describe reproductive behaviour and performance and test dry and wet incubation of eggs. Although H. sertanejo showed well known patterns of reproductive behaviour, we verified many peculiarities inherent to its reproductive biology. As expected, most individuals were diploid (87.71%), however 14.29% were considered mosaics. Although no sterility was observed within mosaics, infertility of these fish was not fully evaluated. Hatching rate of the eggs collected was very low following both dry and wet incubation (5.04 and 3.79%, respectively). These results provide interesting information regarding the reproductive success of this species, and suggest that chromosomal abnormalities described may reduce the survival of H. sertanejo under natural conditions, limiting the perpetuation of this species, and emphasizing the need for more preservation efforts, including artificial propagation and gene banking.

Gonochoristic wild-type dojo loaches (Misgurnus anguillicaudatus) are diploid (2n = 50) and reproduce bisexually. However, sympatric clonal diploids generate unreduced diploid isogenic eggs that develop gynogenetically. Clone-origin triploidy arises following the incorporation of a haploid wild-type sperm nucleus into the diploid egg. Triploid females produce fertile haploid eggs by meiotic hybridogenesis, while triploid males are sterile. Clonal loaches arose from past hybridization event(s) between genetically diverse groups, A and B. Artificial hybrid females between the 2 groups produce unreduced and/or aneuploid eggs, but the hybrid males are sterile. In this study using FISH, we analyzed chromosome pairing in meiotic cells of clone-origin triploid and inter-group hybrid males to clarify the cytogenetic mechanisms underlying the male-specific sterility. We used a repetitive sequence probe to identify group B-derived chromosomes and a 5.8S + 28S rDNA probe to identify pairs of homologous chromosomes. We found that asynapsis and irregular synapsis occur in triploid and hybrid males containing 2 different genomes and that this may cause the formation of sterile germ cells. These results will help us to understand hybrid sterility from the viewpoint of synapsis behavior.

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3’UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.

Made available in DSpace on 2020-12-10T19:50:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra Astyanax altiparanae, triploids present sterility and increased carcass yield, suggesting the generation of tetraploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40 degrees for 2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 degrees C were compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping, and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 +/- 9.6%), 24 (16.0 +/- 9.1%), and 26 mpf (10.3 +/- 6.2%) in comparison with the control group (62.2 +/- 7.7%). The flow cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf presented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat shock treatment at 26 mpf (40 degrees C for 2 min) is optimum for tetraploid induction in the A. altiparanae. Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in basic and applied sciences. Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Univ Fed Rural Rio de Janeiro, BR-465 Km 7, BR-23897000 Seropedica, RJ, Brazil Hokkaido Univ, Fac Fisheries Sci, 3-1-1 Minato Cho, Hakodate, Hokkaido 0418611, Japan Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 FAPESP: 2016/12383-0 CNPq: 471140/2012-0

The goldfish (Carassius auratus auratus) is a useful species for embryonic micromanipulations because of its large egg size and wide temperature tolerance. Here, we describe in detail the rate of development and morphological characteristics of goldfish embryos incubated at temperatures between 10 °C and 30 °C. The cleavage speed increased rapidly as temperature increased. Synchronized cell divisions occurred at 131 min intervals at 10ºC, at 33 min intervals at 20 °C, and at 19 min intervals at 30 °C during the cleavage period. The rate of hatched abnormal embryos significantly increased at temperatures of 26 °C and above, while there was no change in the number of abnormal embryos at temperatures less than 24 °C. Moreover, the blastomeres around the center of the blastodisc rose in the direction of the animal pole at temperatures less than 14 °C. At the lower temperatures, clusters of maternally-supplied germplasm were visualized both at the ends of the first three cleavage furrows and at the border between the lower and upper tiers at the 16- to 32-cell stage, with injection of artificial mRNA and vasa in situ hybridization. This study showed that temperature affects not only developmental speed but also the shape of the blastodisc and the distribution of maternally-supplied materials in the blastodisc. By controlling the temperature, it is possible for researchers to prepare many stages of embryos and shapes of the blastodisc from a single batch of eggs.