Screening and Identification of Transcription Factors Potentially Regulating Foxl2 Expression in Chlamys farreri Ovary

Foxl2 is an evolutionarily conserved female sex gene, which is specifically expressed in the ovary and mainly involved in oogenesis and ovarian function maintenance. However, little is known about the mechanism that regulates Foxl2 specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of Chlamysfarreri with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 107 CFU, and the length of the inserted fragment was 0.75 Kb~2 Kb, which fully met the demand of yeast library screening. The highly transcriptional activity promoter sequence of C. farreri Foxl2 (Cf-Foxl2) was determined at −1000~−616 bp by dual-luciferase reporter (DLR) assay and was used as bait to screen possible transcription factors from the Y1H library. Eleven candidate factors, including five unannotated factors, were selected based on Y1H as well as their expressional differences between ovaries and testes and were verified for the first time to be involved in the transcriptional regulation of Cf-Foxl2 by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of Foxl2 in the ovary.

OVARY DEVELOPMENT, FSH AND LH GENES EXPRESSION OF INDONESIAN LEAFFISH, Pristolepis grootii (Bleeker, 1852), INJECTED WITH LUTEINIZING HORMONE-RELEASING HORMONE ANALOG

Indonesian leaffish, Pristolepis grootii (Bleeker, 1852), is an undomesticated freshwater fish species native to the rivers, flooded swamps, and tributaries of Indonesia. The fish is mainly captured for consumption. In order to prevent its extinction and supply its growing demands, the artificial breeding of the fish should be developed. The purpose of this study was to determine the optimum dose of luteinizing hormone-releasing hormone analog (LHRHa) for stimulating the female P. grootii gonadal development at a dosage of 0, 1, 10, and 50 µg kg-1 of fish. Female fish (20.0 ± 0.6 g) were adapted for 30 days in the rearing environment and then separated into 12 aquariums with six fish per aquarium. Fish were then reared for another 21 days and fed with Tubifex sp. The LHRHa injection was conducted twice on day-7 and 14. Fish bodyweight, gonadosomatic index, gonad histology, blood estradiol-17â, and FSH-â and LH-â gene expression were evaluated at day 0, 7, 14, and 21. The results showed that the injection of the LHRHa hormone stimulated the development of fish gonads and was better achieved with a higher concentration of LHRHa. The best treatment was observed by the administration of 50 µg kg-1 of LHRHa that produced the fastest development among all treatments. This study demonstrated that the LHRHa induction could potentially stimulate the gonadal development of the newly domesticated fish. To our knowledge, this is the first study that reported the success of the induction of female gonad development in the Indonesian leaffish P. grooti.KEYWORDS:

Attack of the dark clones the genetics of reproductive and color traits of South African honey bees (Apis mellifera spp.)

The traits of two subspecies of western honey bees, Apis mellifera scutellata and A.m. capensis, endemic to the Republic of South Africa (RSA), are of biological and commercial relevance. Nevertheless, the genetic basis of important phenotypes found in these subspecies remains poorly understood. We performed a genome wide association study on three traits of biological relevance in 234 A.m. capensis, 73 A.m. scutellata and 158 hybrid individuals. Thirteen markers were significantly associated to at least one trait (P ≤ 4.28 × 10−6): one for ovariole number, four for scutellar plate and eight for tergite color. We discovered two possible causative variants associated to the respective phenotypes: a deletion in GB46429 or Ebony (NC_007070.3:g.14101325G>del) (R69Efs*85) and a nonsense on GB54634 (NC_007076.3:g.4492792A>G;p.Tyr128*) causing a premature stop, substantially shortening the predicted protein. The mutant genotypes are significantly associated to phenotypes in A.m. capensis. Loss-of-function of Ebony can cause accumulation of circulating dopamine, and increased dopamine levels correlate to ovary development in queenless workers and pheromone production. Allelic association (P = 1.824 x 10−5) of NC_007076.3:g.4492792A>G;p.Tyr128* to ovariole number warrants further investigation into function and expression of the GB54634 gene. Our results highlight genetic components of relevant production/conservation behavioral phenotypes in honey bees.

Histone acetylation regulates the expression of genes involved in worker reproduction in the ant Temnothorax rugatulus

Background In insect societies, queens monopolize reproduction while workers perform tasks such as brood care or foraging. Queen loss leads to ovary development and lifespan extension in workers of many ant species. However, the underlying molecular mechanisms of this phenotypic plasticity remain unclear. Recent studies highlight the importance of epigenetics in regulating plastic traits in social insects. Thus, we investigated the role of histone acetylation in regulating worker reproduction in the ant Temnothorax rugatulus. We removed queens from their colonies to induce worker fecundity, and either fed workers with chemical inhibitors of histone acetylation (C646), deacetylation (TSA), or the solvent (DMSO) as control. We monitored worker number for six weeks after which we assessed ovary development and sequenced fat body mRNA. Results Workers survived better in queenless colonies. They also developed their ovaries after queen removal in control colonies as expected, but not in colonies treated with the chemical inhibitors. Both inhibitors affected gene expression, although the inhibition of histone acetylation using C646 altered the expression of more genes with immunity, fecundity, and longevity functionalities. Interestingly, these C646-treated workers shared many upregulated genes with infertile workers from queenright colonies. We also identified one gene with antioxidant properties commonly downregulated in infertile workers from queenright colonies and both C646 and TSA-treated workers from queenless colonies. Conclusion Our results suggest that histone acetylation is involved in the molecular regulation of worker reproduction, and thus point to an important role of histone modifications in modulating phenotypic plasticity of life history traits in social insects.

A cryptic variation in a member of the Ovate Family Proteins is underlying the melon fruit shape QTL fsqs8.1

Key message The gene underlying the melon fruit shape QTL fsqs8.1 is a member of the Ovate Family Proteins. Variation in fruit morphology is caused by changes in gene expression likely due to a cryptic structural variation in this locus. Abstract Melon cultivars have a wide range of fruit morphologies. Quantitative trait loci (QTL) have been identified underlying such diversity. This research focuses on the fruit shape QTL fsqs8.1, previously detected in a cross between the accession PI 124112 (CALC, producing elongated fruit) and the cultivar ‘Piel de Sapo’ (PS, producing oval fruit). The CALC fsqs8.1 allele induced round fruit shape, being responsible for the transgressive segregation for this trait observed in that population. In fact, the introgression line CALC8-1, carrying the fsqs8.1 locus from CALC into the PS genetic background, produced perfect round fruit. Following a map-based cloning approach, we found that the gene underlying fsqs8.1 is a member of the Ovate Family Proteins (OFP), CmOFP13, likely a homologue of AtOFP1 and SlOFP20 from Arabidopsis thaliana and tomato, respectively. The induction of the round shape was due to the higher expression of the CALC allele at the early ovary development stage. The fsqs8.1 locus showed an important structural variation, being CmOFP13 surrounded by two deletions in the CALC genome. The deletions are present at very low frequency in melon germplasm. Deletions and single nucleotide polymorphisms in the fsqs8.1 locus could not be not associated with variation in fruit shape among different melon accessions, what indicates that other genetic factors should be involved to induce the CALC fsqs8.1 allele effects. Therefore, fsqs8.1 is an example of a cryptic variation that alters gene expression, likely due to structural variation, resulting in phenotypic changes in melon fruit morphology.

The Impact of Exogenous Tetrodotoxin on the piRNAs and mRNAs Profiles of Gonads in Pufferfish Takifugu Flavidus

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism of TTX accumulation in gonads is complex and unclear. Piwi/piRNA complexes are essential for germline specification, gametogenesis, and gonadal development, they also demonstrate sexual dimorphism in teleosts. Hence, the present study investigated the expression of piRNAs and mRNAs by transcriptomics in cultured pufferfish Takifugu flavidus after intramuscular administration of exogenous TTX. The results showed 80 piRNAs were down-regulated and 223 genes were up-regulated in the ovary after TTX administration. By contrast, 286 piRNAs were down-regulated and 445 genes were up-regulated after TTX administration in testis. Functional and pathway analyses indicated that the TTX up-regulated genes were enriched in the Wnt, ErbB and GnRH signaling pathways in ovary, while were enriched in the oocyte meiosis, estrogenesis biosynthesis and cell apoptosis-related pathways in testis. Interestingly, these genes were also the potential target genes of TTX downregulated piRNAs. Amhr2 and cyp19a were also identified as sex-related genes, involved in TTX administration. These results showed a certain level of consistency with the enrichment pathway analysis, which indicated that the TTX could affect the expression of sex-related genes, and work as an inhibitor for the testicular meiosis, but as a promoting factor for ovary development through piRNAs in pufferfish. In addition, TUNEL staining showed that significant apoptosis was detected in the TTX treated testis, and the role of the cell apoptotic pathway was further confirmed. Overall, this research will contribute to an understanding of the piwi function in TTX sexual dimorphism accumulation in pufferfish and provide a basis for further studies.

Qualitative lysine crotonylome analysis in the ovarian tissue of Harmonia axyridis (Pallas)

Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM), which has been studied at the proteomics level in a few species, with the study of Kcr in female fertility and in insect species is still lacking. Harmonia axyridis (Pallas) is a well-known beneficial insect used as a natural biological control agent against aphids in agriculture. Here, global Kcr identification in ovarian tissue of H. axyridis at diapause stage was performed to reveal potential roles for Kcr in H. axyridis ovarian cellular processes, female fertility and diapause regulation. In total, 3084 Kcr sites in 920 proteins were identified. Bioinformatic analyses revealed the distribution of these proteins in multiple subcellular localization categories and their involvement in diverse biological processes and metabolism pathways. Carbohydrate and energy metabolism related cellular processes including citric acid cycle, glycolysis and oxidative phosphorylation appeared be affected by Kcr modification. In addition, regulation of translation and protein biosynthesis may reflect Kcr involvement in diapause in H. axyridis, with Kcr affecting ribosome activities and amino acid metabolism. Moreover, Kcr modulation H. axyridis ovary development regulation may share some common mechanism with Kcr participation in some disease progression. These processes and pathways were uncovered under diapause stage, but possibly not enriched/specific for diapause stage due to limitations of qualitative proteomics experimental design. Our results informs on the potential for Kcr modifications to regulate female fertility and insect physiology.

Distinct gene expression dynamics in germ line and somatic tissue during ovariole morphogenesis in Drosophila melanogaster

The survival and evolution of a species is a function of the number of offspring it can produce. In insects the number of eggs that an ovary can produce is a major determinant of reproductive capacity. Insect ovaries are made up of tubular egg-producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the larval ovary at the beginning, middle and end of terminal filament formation. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development. We found that the number of differentially expressed somatic genes is highest during late stages of terminal filament formation and includes many signaling pathways that govern ovary development. We also show that germ line tissue, in contrast, shows greater differential expression during early stages of terminal filament formation, and highly expressed germ line genes at these stages largely control cell division and DNA repair. We provide a tissue-specific and temporal transcriptomic dataset of gene expression in the developing larval ovary as a resource to study insect reproduction.