Comprehensive Transcriptome Analysis of Artemia franciscana Across Developmental Stages Using Illumina and Nanopore Sequencin

The brine shrimp Artemia franciscana is a halophilic crustacean that adaptively produces diapause embryos (cysts) in response to adverse environmental stressors. These embryos can survive complete dehydration (anhydrobiosis) and freezing, which is related to the accumulation of high amounts of trehalose, and to a lesser extent of glycerol, in diapause cysts. To gain insight into the molecular basis of water and solute transport during the entry and exit of diapause in A. franciscana, here we have sequenced the transcriptome of this species and isolated the genomic repertoire of aquaporins. The results indicate that A. franciscana has one entomoglyceroporin (EglpL), three classical aquaglyceroporins Glp1, Glp2 and another expressed as two splice forms (Glp3_v1 and Glp3_v2), one unorthodox aquaporin related to the vertebrate Aqp12, and one cation permeating big brain channel (Bib). Functional characterization of these channels in frog oocytes indicated that EglpL and the three Glps transport water and glycerol, whereas Aqp12 remained intracellular. Further RNAi experiments showed that when the expression of both EglpL and Glp3_v1 is knocked down the levels of glycerol in diapause embryos are drastically reduced, and embryo survival during desiccation is compromised. These data suggest that EglpL and Glp3_v1 are involved in the acquisition of stress tolerance in A. franciscana diapause embryos. The brine shrimp Artemia franciscana serves as a valuable model organism in developmental biology and aquaculture research due to its unique ability to survive extreme environmental conditions. To gain a comprehensive understanding of its gene expression across developmental stages, we performed a transcriptomic analysis of A. franciscana using a combination of short-read (Illumina) and long-read (Oxford Nanopore Technologies, ONT) sequencing technologies. Total RNA was extracted from a pool of adult males, females, nauplii, and desiccated cysts. The RNA integrity was assessed, with RNA Integrity Number (RIN) values ranging from 7.20 (nauplii) to 9.20 (adults), ensuring high-quality RNA for sequencing. We generated over 50 million paired-end reads per sample using the NovaSeq 6000 platform (Illumina), with a read length of 2 × 76 bp, alongside long-read sequencing performed on a GridION Mk1 platform (ONT). Transcriptome assembly was achieved by combining Illumina and ONT reads using RNABloom, followed by error correction using Ratatosk. The assembled transcripts were clustered with Rapclust to determine which sequences belonged to the same biological transcript or gene. Functional annotation of the assembled transcripts was performed using Transdecoder, followed by Diamond Blastp searches against the nr database, and protein domain detection via Interproscan. Additionally, Daphnia magna protein sequences were aligned with A. franciscana proteins to provide functional annotations for transcripts lacking clear assignments. This multi-layered annotation approach enabled a more complete understanding of gene function across developmental stages. Our study provides the first comprehensive transcriptome resource for A. franciscana, offering valuable insights into the molecular mechanisms underlying its development and resilience. These findings will not only support further investigations into the biology of A. franciscana but also contribute to broader research in evolutionary biology, stress response mechanisms, and aquaculture.