Xiaojun Zhang

Endogenous viral elements (EVEs) serve as molecular fossils that record the ancient co-evolutionary arms race between viruses and their hosts. In this study, by analyzing 105 host crustacean genomes, we identified 252 infectious hypodermal and haematopoietic necrosis virus-derived EVEs (IHHNV-EVEs), which include 183 ancient and 6 recently inserted EVEs. These IHHNV-EVEs are widely distributed among Decapoda, Thoracica, and Isopoda, with some of them exhibiting a syntenic distribution relative to neighboring host sequences, suggesting that the IHHNV or its ancestor are potential pathogens of these species with a long-time dynamic interaction during the evolutionary history. An expansion of IHHNV-EVEs was observed in decapoda genomes, reflecting a reinforced arm race between decapoda and IHHNV. Notably, we found that nearly all recent IHHNV-EVEs were laboratory contaminants, except for a single authentic integration in Penaeus monodon that persists intact across 16 samples from the 2 populations. These temporal dynamics—ancient genomic stabilization versus modern colonization activity—highlight that EVEs serve as dual archives: historical records of past conflicts and active participants in current evolutionary battles. Our findings redefine viral genomic colonization as a continuum, where ancient EVE fixation coexists with persistent integration processes, providing new insights into host-virus co-evolutionary trajectories.

The crustacean hyperglycemic hormone (CHH) is a unique multifunctional neuroendocrine hormone superfamily in crustaceans, crucial for maintaining physiological homeostasis and stress adaptation. To explore the role of CHHs in shrimp metabolism and growth, we identified LvCHH Ia, a CHH family member who regulates glucose metabolism in the Pacific white shrimp (Litopenaeus vannamei), through CHH family gene classification, phylogenetic analysis, gene structure analysis, and transcription factor binding site (TFBS) prediction. Subsequently, we cloned two alternative splicing variants of this gene, LvCHH Ia-1 and LvCHH Ia-2, both expressed in the nervous system but with different expression levels, and LvCHH Ia-2 exhibiting a broader tissue distribution. Using interference (RNAi)-mediated gene silencing and recombinant protein injection, we investigated the functional similarities and differences between the two variants. Our results show that both variants affect glucose metabolism by modulating the expression of key enzyme genes involved in gluconeogenesis/glycolysis, such as HK, TPI, PCK1, ALD. Specifically, they likely regulate hemolymph glucose levels via the Wnt and PI3K-AKT signaling pathways, with LvCHH Ia-1 exerting a more sustained effect on glucose metabolism compared to LvCHH Ia-2. Furthermore, LvCHH Ia may also act as a molting inhibitory hormone by suppressing the expression of ecdysone synthesis-related genes, where LvCHH Ia-2 plays a more significant role. These findings deepen our understanding of CHH regulatory mechanisms in crustaceans and provide potential applications for shrimp physiological research and aquaculture.

Salinity serves as an important environmental factor in ecosystems, driving the evolution of adaptive strategies in euryhaline species. The Pacific white shrimp, Litopenaeus vannamei, is a representative euryhaline species. However, the molecular mechanisms, particularly the roles of cis-regulatory elements, remain elusive in penaeid shrimp. This study tackles this gap by subjecting L. vannamei to a gradual reduction in salinity from 30‰ to 3‰, and then applying ATAC-seq and RNA-seq techniques to dissect the cis-regulation mechanisms underlying low salinity adaptation. A key finding reveals a positive correlation between chromatin accessibility and gene expression, with 36.8% of differentially expressed genes directly associated with changes in chromatin accessibility. The cis-regulation of many osmoregulation-related pathways, such as betaine synthesis and PI3K-Akt signaling pathways, appeared to be a crucial strategy for salinity adaptation in shrimp. By analyzing differentially accessible regions under low salinity stress, we uncovered two known and seven novel candidate transcription factors (TFs) that may play pivotal roles in salinity adaptation. We further conducted a comprehensive analysis of these TFs, including their functions, expression profiles, consensus TFBS motifs, and the functional enrichment and expression profiles of their targeted genes. This study elucidates a complex cis-regulatory network that enables exceptional salinity tolerance in L. vannamei, which provides a foundation for the refinement of genetic breeding programs and desalination aquaculture for penaeid shrimp.

Growth of the Pacific white shrimp Litopenaeus vannamei, the most important farmed crustacean, has consistently been a focal point for breeders. Over the past decades, some candidate genes for shrimp growth have been identified. However, further research is needed to elucidate the molecular regulatory mechanism of these genes. LvMmd2 was previously identified as a candidate gene that may inhibit the growth of L. vannamei. In this study, we analyzed the genotype and expression of the LvMmd2 gene in a breeding family and indicated its role as a growth-inhibiting gene. We found that LvMmd2 co-localized with its homolog LvPAQR3 at the Golgi apparatus. Using co-immunoprecipitation (Co-IP) and DUAL membrane system yeast two-hybrid (MbY2H), we indicated the interactions between LvMmd2 and LvPAQR3, LvPAQR3 and LvRaf1, as well as LvMmd2 and LvRho. These results suggest that LvMmd2 directly and indirectly regulates the Ras signaling pathway. Furthermore, we show that the LvMmd2 gene may indirectly affect the PI3K/AKT, insulin, and Hippo signaling pathways to regulate cell proliferation and differentiation via LvPAQR3 and LvRaf1. Through transcriptome and MbY2H analyses, we have also revealed the interaction between LvMmd2 and proteins involved in growth, immunity, protein transport, synthesis, and modification. These findings demonstrate the various molecular pathways through which LvMmd2 regulates L. vannamei growth. This study provides insights into the mechanism of shrimp growth regulated by Mmd2, enhances our understanding of LvMmd2 function, and highlights its potential application in shrimp breeding.

Noncoding microRNAs tend to evolve within introns of coding genes that provide them with transcriptional opportunity. As an outcome of natural selection, the intragenic position of microRNAs is crucial for their expression, evolution, and functional cooperation with the host gene. Therefore, understanding the evolution of intragenic microRNA structures may bring novel insights into genetic and phenotypic evolution. However, it remains largely unexplored. Here, by analyzing microRNA genomics in 34 metazoan species, we found that the majority (630/1,154) of microRNA families originated from introns of coding genes that provided them with initial transcriptional capacity. The most rapid expansion of intragenic microRNAs happened at the advent of vertebrates when 21 microRNA families emerged from introns of neural genes and reorganized the gene regulatory network, leading to the rise of vertebrate-specific neural crest cells, which transformed the invertebrate head and enabled the ecological shift from filter feeding to active predation. Intragenic microRNAs gradually gain independence from their host genes, which is accelerated by whole-genome duplications. After a whole-genome duplication, the purging of redundant host genes often set an orphaned microRNA “free” to diversify with the transcriptional elements inherited from the host. Whole-genome duplications facilitated a dramatic microRNA diversification during the initial divergence of vertebrates, as the intragenic status of 12 neural crest-regulating microRNAs was retained in jawed vertebrates but was lost in jawless cyclostomes, which diverged their neural crest development. We propose that coding genes not only facilitate the origination of new microRNAs, but also “sacrifice” themselves to help microRNA diversification.