Production and progeny testing of androgenetic rosy barb Puntius conchonius

Protocol for androgenetic cloning of the rosy barb, Puntius conchonius, with contrasting gray and golden strains is described. At the intensity of 4.2 W/m<SUP>2</SUP>, UV irradiation for 3.0 min inactivates the maternal genome in eggs of the gray barb. Following activation by the golden barb sperm, 24-min old eggs are shocked at 41&#176;C for 2 min to restore diploidy. Maternal genomic inactivation is confirmed by the (i) golden body color, (ii) karyotyping, and (iii) progeny testing of F<SUB>1</SUB>-F<SUB>3</SUB> progenies. Estimates of stage-specific mortality of haploid and diploid androgenotes indicate no change in the time scale or developmental sequence, when sperm of related strain is used for activation, and when haploid genome regulates the development. Survival of androgenetic clones remains constant for the F<SUB>1</SUB>, F<SUB>2</SUB>, and F<SUB>3</SUB> progenies and is about 15% and 7% at hatching and sexual maturity, respectively. Homozygosity of the androgenotes is shown to inflict greater mortality. Between F<SUB>1</SUB> and F<SUB>3</SUB> generations, the heterozygosity of the androgenetic clone is decreased, as evidenced by reduction in size hierarchy. Though the reproductive performance of the F<SUB>1</SUB>, F<SUB>2</SUB>, and F<SUB>3</SUB> supermales is superior to the normal ones, the realized fecundity remains equal around 80 progenies per brood. The 92 crosses involving 16 supermales and 10 normal dams yield 75-100% male progenies, confirming the possible operation of XX&#x2640;:XY&#x2642; sex determination system. The frequency of unexpected occurrence of female progenies is about 8%, the causes for which are discussed

Production of androgenetic tiger barb, Puntius tetrazona

The protocol for successful induction of androgenetic cloning of the tiger barb, Puntius tetrazona, with contrasting gray and blond strains is described. At the intensity of 4.2 W/m<SUP>2</SUP>, the UV irradiation for 3.5 min totally inactivates the maternal genome in eggs of the gray barb. Following activation by the blond barb sperm, the 24-min-old eggs are shocked at 41&#176;C for 2 min to restore diploidy. Maternal genomic inactivation is confirmed by (i) expression of green fluorescent protein (GFP) gene in 16-h-old haploid embryo; (ii) blond body colour in the diploid fry and adult; and (iii) progeny testing. Androgenetic males and females were reared to sexual maturity and their reproductive performance was evaluated. The performance of the supermales (YY) is superior to normal ones (XY), but that of androgenetic females (X<SUP>2</SUP>X<SUP>2</SUP>) is inferior to normal ones (X<SUP>1</SUP>X<SUP>2</SUP>). Fifteen crosses involving five supermales and eight normal dams sire 97-100% male progenies, confirming the operation of XX&#x2640;:XY&#x2642; sex determination system in the tiger barb. The unexpected occurrence of female progeny is a ubiquitous feature of supermales of at least half a dozen fish species

Effect on growth and reproduction of hormone immersed and masculinized fighting fish Betta splendens

To produce all-male progenies in the fighting fish, Betta splendens, six groups of fry were subjected to discrete immersion treatment at different 17α-methyltestosterone (MT) doses (viz. 100, 200, 500, 700, 900, and 1,000 μg/l) for a constant duration (3 hr/day) and frequency (second, fifth, and eighth day after hatching). The treatment at 900 μg/l led to 98% masculinization and 71% survival at sexual maturity. Treated groups, which showed significant deviation from the 1:1 sex ratio, were classified into two different series: S1 and S2. The groups that showed nearly cent-percent masculinization were classified as S1, and the other groups were classified as S2. The S1 males showed remarkably slower growth and attained 3.5 cm total length compared to 6.0 cm attained by a normal male. The S2 males attained 5.4 cm total length. Apart from these morphological defects, both S1 and S2 males suffered functional (decreased sperm count and sperm motility) and behavioral defects (incomplete embracing during mating) in their reproductive ability, leading to approximately 50% and 30% reduction in fecundity per mating, respectively. The cumulative fecundity loss suffered by the S1 male during its active reproductive phase is discussed. When normal and sex-reversed males were presented, a female preferred the former. Progeny testing of the sex-reversed males showed the occurrence of 12.75% males, indicating the possible role of autosomal genes in the sex determination mechanism of this species. Discrete immersion treatment at optimal/super-optimal doses ensured not only a higher percentage of masculinization, but also a higher frequency of homogametic males (XX)

Growth enhancement and food conversion efficiency of transgenic fish Labeo rohita

Three family lines of fast growing transgenic rohu Labeo rohita (rohu) were generated by electroporated-sperm-mediated transfer of the vectors harboring CMV promoter or grass carp β-actin promoter fused to endogenous rohu GH (rGH) cDNA. The gene transfer efficiency was 25%. The transgenic rohu (family line 1) with CMV promoter showed a growth enhancement of four times normal size, whereas those (family lines 2 and 3) generated with β-actin promoter grew 4.5 and 5.8 times faster than their respective control siblings. Southern analysis confirmed the transgene extrachromosomal (Te) persistence until the 60th week in family 1. The individuals of family lines 2 and 3, however, showed integration (Ti), as well as persistence as extarchromosomal copies (Te) until the age of 30 weeks. Mosaicism of the transgene was shown at the levels of its presence and expression. The ectopic expression of rGH mRNA was confirmed by RT-PCR. Feeding experiments revealed that the transgenic rohu ate food at a lower rate but grew more efficiently than their control siblings

Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway

International audienceThe habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits alpha 2 and beta 4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain

Transition Metal Ion (Mn²⁺, Fe²⁺, Co²⁺, and Ni²⁺)‑Doped Carbon Dots Synthesized via Microwave-Assisted Pyrolysis: A Potential Nanoprobe for Magneto-fluorescent Dual-Modality Bioimaging

Heteroatom-doped carbon dots (C-dots) have captured widespread research interest owing to high fluorescence and biocompatibility for multimodal bioimaging applications. Here, we exemplify a rapid, facile synthesis of ethylenediamine (EDA)-functionalized transition metal ion (Mn²⁺, Fe²⁺, Co²⁺, and Ni²⁺)-doped C-dots via one-pot microwave (MW)-assisted pyrolysis at 800 W within 6 min using <i>Citrus limon</i> (lemon) extract as a carbon source. During MW pyrolysis, the precursor extract undergoes simultaneous carbonization and doping of metal ions onto C-dot surfaces in the presence of EDA. The EDA-functionalized transition metal ion-doped C-dots (i.e., Mn/C, Fe/C, Co/C, and Ni/C-dots) are collectively termed as TMCDs. The water-soluble TMCDs exhibited a size of 3.2 ± 0.485 nm and were enriched with amino and oxo functionalities and corresponding metal-oxide traces on the surfaces, as revealed from Fourier transfer infrared and X-ray photoelectron spectroscopy analyses. Interestingly, TMCDs demonstrated excitation-wavelength-dependent emission with brighter photoluminescence (PL) at 460 nm. Compared to pristine C-dots with a PL quantum yield (QY) of 48.31% and a fluorescence lifetime of 3.6 ns, the synthesized Mn/C, Fe/C, Co/C, and Ni/C-dots exhibited PL QY values of 35.71, 41.72, 75.07, and 50.84% as well as enhanced fluorescence lifetimes (τ_av) of 9.4, 8.6, 9.2, and 8.9 ns, respectively. The TMCDs significantly exhibited enhanced biocompatibility in human colon cancer cells (SW480) for fluorescence bioimaging and showed ferromagnetic and superparamagnetic behavior with vibrant <i>T</i>₁-contrast ability. Interestingly, the maximum longitudinal (<i>r</i>₁) relaxivity of 0.341 mM^–1 s^–1 was observed for Mn/C-dots in comparison to that of 3.1–3.5 mM^–1 s^–1 of clinically used Gd-DTPA magnetic resonance (MR)-contrast agent in vitro (1.5 T). Similarly, the maximum longitudinal relaxivity (<i>r</i>₁) of 0.356 mM^–1 s^–1 was observed for Ni/C-dots (1.5 T) with respect to 4.16 ± 0.02 mM^–1 s^–1 attained for Gd-DTPA in vivo (8.45 T). Thus, the rapid, energy-efficient MW-assisted pyrolysis presents lemon extract derived, EDA-functionalized TMCDs with enhanced PL and efficient <i>T</i>₁ contrast as potential magneto-fluorescent nanoprobes for dual-modality bioimaging applications