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As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (As-Cath) from an alligator (Alligator sinensis) into the target luteinizing hormone (lh) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of As-Cath was instrumental in establishing the lh knockout (LH–_As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of disease-resistant transgenic catfish in an environmentally responsible manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or Alligator sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P ​> ​0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P ​> ​0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

Disease and pest are the main factors causing grape yield reduction. Correct and timely identification of these symptoms are necessary for the vineyard. However, the commonly used CNN models limit their performance on leaf images with complex backgrounds, due to the lack of global receptive field. In this article, we propose an effective and accurate approach based on Ghost-convolution and Transformer networks for diagnosing grape leaf in field. First, a grape leaf disease and pest dataset containing 11 classes and 12,615 images, namely GLDP12k is collected. Ghost network is adopted as the convolutional backbone to generate intermediate feature maps with cheap linear operations. Transformer encoders with multi-head self-attention are integrated behind to extract deep semantic features. Then we get the Ghost enlightened Transformer model, namely GeT. After analyzing five hyper-parameters, the optimized GeT is transfer-learnt from ImageNet which provides a 4.3% accuracy bonus. As the results show, with 180 frame-per-second, 1.16 M weights and 98.14% accuracy, GeT surpasses other models, and is 1.7 times faster and 3.6 times lighter than MobilenetV3_large (97.7%). This study shows that the GeT model is effective and provides an optional benchmark for field grape leaf diagnosis.