Identification of testis development-related genes by combining Iso-Seq and RNA-Seq in Zeugodacus tau.

Introduction: Zeugodacus tau (Walker) is an invasive pest. An effective method to control this pest is the sterile insect technique (SIT). To better apply this technique, it is necessary to understand testis development progression. Methods: Differentially expressed genes (DEGs) during testis development were analyzed by PacBio Iso-Seq and RNA-seq. Results: RNA-Seq library of Z. tau testes on day 1, 6, and 11 post eclosion were constructed. We identified 755 and 865 differentially expressed genes in the comparisons of T6 (testes on day 6) vs. T1 and T11 vs. T1, respectively. The KEGG pathway analysis showed that the DEGs were significantly enriched in retinol metabolism, vitamin B6 metabolism, and ascorbate and aldarate metabolism pathways. Knockdown of retinol dehydrogenase 12-like (rdh12-like), pyridoxal kinase (pdxk) and regucalcin (rgn), the representative gene in each of the above 3 pathways, reduced the hatching rate of Z. tau offspring. In addition, we identified 107 Drosophila spermatogenesis-related orthologous genes in Z. tau, of which innexin 2 (inx2) exhibited significantly up-regulated expression throughout testis development, and the knockdown of this gene reduced offspring hatching rate. Discussion: Our data indicated that rdh12-like, pdxk, rgn, and inx2 genes were related to testis development, and they were conserved in tephritid species. These results suggested that this gene might have the same function in tephritid. The findings provide an insight into testis development and spermatogenesis in tephritid species.

Identification and functional verification of Y-chromosome-specific gene typo-gyf in Bactrocera dorsalis.

Genes on the Y chromosome play important roles in male sex determination and development. The identification of Y-chromosome-specific genes not only provides a theoretical basis for the study of male reproductive development, but also offers genetic control targets for agricultural pests. However, Y-chromosome genes are rarely characterized due to their high repeatability and high heterochromatinization, especially in the oriental fruit fly. In this study, 1 011 Y-chromosome-specific candidate sequences were screened from 2 to 4 h Bactrocera dorsalis embryo datasets with the chromosome quotient method, 6 of which were identified as Y-chromosome-specific sequences by polymerase chain reaction, including typo-gyf, a 19 126-bp DNA sequence containing a 575-amino acid open reading frame. Testicular deformation and a significant reduction in sperm number were observed after typo-gyf knockdown with RNA interference in embryos. After typo-gyf knockout with clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 in the embryonic stage, the sex ratio of the emergent adults was unbalanced, with far more females than males. A genotype analysis of these females with the Y-chromosome gene MoY revealed no sex reversal. Typo-gyf knockout led to the death of XY individuals in the embryonic stage. We conclude that typo-gyf is an essential gene for male survival, and is also involved in testicular development and spermatogenesis. The identification of typo-gyf and its functional verification provide insight into the roles of Y-chromosome genes in male development.

CRISPR/Cas9-induced Mutation of Sex Peptide Receptor Gene Bdspr Affects Ovary, Egg Laying, and Female Fecundity in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an invasive and polyphagous pest of horticultural crops, and it can cause huge economic losses in agricultural production. The rapid development of CRISPR/Cas9 gene editing technology has provided new opportunities for the scientific control of agricultural pests. Here, we explore the applicability of the B. dorsalis sex peptide receptor (Bdspr) as a target gene for the CRISPR/Cas9-based sterile insect technique (SIT) in B. dorsalis. We screened two high-efficient single guide RNAs (sgRNAs) for gene editing. The results showed that both mutation efficiency and germline transmission rate were 100% in the surviving G0 females (8/8) from injected embryos, and that 75% of mosaically mutated G0 females (6/8) were sterile. The 50% of heterozygous G1 females (4/8) could not lay eggs; 100% of eggs laid by them could not survive; and 62.5% of individual females (5/8) had abnormal ovaries. These results indicate that Bdspr plays an important role in regulating fertility, egg viability, and ovary development in female B. dorsalis, suggesting that the spr gene can be used for CRISPR/Cas9-based SIT in B. dorsalis.

Early embryonic transcriptomes of Zeugodacus tau provide insight into sex determination and differentiation genes.

Zeugodacus tau (Walker) is an invasive pest. The sterile insect technique is an environment-friendly method for pest control. Understanding the mechanism of sex determination will contribute to improving efficiency of this technique. In this study, we identified the transformer (tra) gene in Z. tau. One female-specific and two male-specific isoforms of tra were found in Z. tau, and the male-specific splicing pattern of tra was found to occur 5 h after egg laying. We performed transcriptome sequencing at 1 h (E1), 5 h (E5), and 9 h (E9) after egg laying and obtained high-quality transcriptome libraries of early embryo development. We identified 13 297 and 11 713 differentially expressed genes (DEGs) from E5 versus E1 and E9 versus E1 comparisons, respectively. To explore the potential functions of the DEGs during embryonic development, Gene Ontology, Clusters of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes analyses were performed. Twenty-six genes potentially involved in sex determination or differentiation, including Maleness-on-the-Y (MoY), were identified in Z. tau. To verify the transcriptome results, 15 genes were selected for quantitative real-time PCR validation. The results were consistent with the transcriptome sequencing results. Moreover, U2 small nuclear riboprotein auxiliary factor (U2AF-50), female lethal d (fl(2)d), and virilizer (vir) were highly expressed at E5, indicating that they may be related to the sex-specific splicing of tra. Further functional analysis is needed to confirm this speculation. Our data provide an insight into the mechanism underlying sex determination and differentiation in tephritid species.

Error Bounds of Imitating Policies and Environments for Reinforcement Learning.

In sequential decision-making, imitation learning (IL) trains a policy efficiently by mimicking expert demonstrations. Various imitation methods were proposed and empirically evaluated, meanwhile, their theoretical understandings need further studies, among which the compounding error in long-horizon decisions is a major issue. In this paper, we first analyze the value gap between the expert policy and imitated policies by two imitation methods, behavioral cloning (BC) and generative adversarial imitation. The results support that generative adversarial imitation can reduce the compounding error compared to BC. Furthermore, we establish the lower bounds of IL under two settings, suggesting the significance of environment interactions in IL. By considering the environment transition model as a dual agent, IL can also be used to learn the environment model. Therefore, based on the bounds of imitating policies, we further analyze the performance of imitating environments. The results show that environment models can be more effectively imitated by generative adversarial imitation than BC. Particularly, we obtain a policy evaluation error that is linear with the effective planning horizon w.r.t. the model bias, suggesting a novel application of adversarial imitation for model-based reinforcement learning (MBRL). We hope these results could inspire future advances in IL and MBRL.

Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai.

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, the full-length cDNA of the cytosolic carbonic anhydrase c isoform of G. przewalskii (GpCAc) was amplified and sequenced; mRNA levels and enzyme activity of GpCAc and blood chemistry were evaluated to understand the compensatory responses as the naked carp returned to the saline-alkaline lake after spawning. We found that GpCAc had a total length of 1400 bp and encodes a peptide of 260 amino acids. Comparison of the deduced amino acid sequences and phylogenetic analysis showed that GpCAc was a member of the cytosolic carbonic anhydrase II-like c family. Cytosolic-carbonic-anhydrase-c-specific primers were used to analyze the tissue distribution of GpCAc mRNA expression. Expression of GpCAc mRNA was found in brain, gill, liver, kidney, gut, and muscle tissues, but primarily in the gill and posterior kidney; however, none was evident in red blood cells. Transferring fish from river water to lake water resulted in a respiratory alkalosis, osmolality, and ion rise in the blood, as well as significant decreases in the expression and enzyme activity of GpCAc in both the gill and kidney within 96 h. These results indicate that GpCAc may play an important role in the acclimation to both high salinity and carbonate alkalinity. Specifically, G. przewalskii decreases cytosolic carbonic anhydrase c expression to compensate for a respiratory alkalosis and to aid in osmoregulation during the transition from river to saline-alkaline lake.