The Osiris family was first described in the fruit fly Drosophila melanogaster, where twenty-five genes were identified, twenty-two of them clustered within the dosage-sensitive Triplo-lethal locus on chromosome region 83D-E. The proteins encoded by these genes all carry a domain of unknown function, designated DUF1676, along with an N-terminal signal peptide, a pair of conserved cysteine residues, a transmembrane region and a C-terminal AQXLAY motif. Despite nearly two decades of study, most functional work has been confined to Drosophila and the silkworm Bombyx mori, both holometabolous insects that undergo complete metamorphosis. Whether the same genes play comparable roles in insects with incomplete metamorphosis, whose nymphs resemble miniature adults, was an open question with real agricultural implications.

By searching the genome and transcriptome databases of the brown planthopper, the team, led by Cui Zhang, Xinyi He and corresponding authors Xingxing Shen and Yanyuan Bao, identified twenty Osiris genes. Compared with the fruit fly, the planthopper lacks the Osiris1, Osiris4, Osiris5 and Osiris13 genes, hinting that those members may perform species-specific functions in Drosophila. To place the newly found genes in an evolutionary framework, the researchers constructed a maximum likelihood phylogenetic tree using Osiris protein sequences from twenty-three insect species spanning eight orders, from damselflies and grasshoppers to bees, beetles, butterflies, lacewings and mosquitoes. Each planthopper Osiris protein clustered closely with its fruit fly and silkworm counterparts, confirming a high degree of sequence conservation across hundreds of millions of years of insect evolution.

The genomic architecture told an equally striking story. Seventeen of the twenty NlOsiris genes sit together on chromosome 1, while NlOsiris21, NlOsiris22 and NlOsiris23 occupy chromosomes 3, X and 8 respectively. When the team mapped Osiris gene locations across all twenty-three insect species, they detected the same syntenic clustering in every genome examined, with more closely related species showing more similar gene arrangements. This pattern strongly suggests that the entire family arose through tandem gene duplication early in insect evolution and has remained physically linked ever since. The predicted proteins ranged from 179 to 680 amino acids in length, and all contained the signature DUF1676 domain. NlOsiris16 turned out to be unusual in carrying two copies of this domain, while NlOsiris9 lacks a signal peptide and NlOsiris21 lacks a transmembrane region, small deviations that may underpin functional specialization.

Expression analysis revealed a rhythmic, developmentally choreographed pattern. Transcript levels of most NlOsiris genes were almost undetectable in adults and in newly hatched nymphs of each instar, but surged to peak levels in the middle of every nymphal stage, around thirty-six hours after molting for the first through fourth instars and forty-eight to sixty hours for fifth-instar nymphs. In eggs, transcripts were absent at twenty-four and seventy-two hours after laying but appeared at high levels at 144 hours, just before hatching. Tissue profiling showed that most genes were highly expressed in the wing buds, integument and fat body of fifth-instar nymphs, with a few notable exceptions: NlOsiris12 was detected only in wing buds, NlOsiris23 only in the gut, and NlOsiris11 exclusively in fifth-instar female nymphs, a pattern that foreshadowed its peculiar role in female development.

To probe function, the researchers turned to RNA interference, injecting double-stranded RNAs targeting each gene into fourth-instar nymphs and newly emerged adult females. The phenotypes were dramatic and diverse. Silencing nine of the genes, including NlOsiris2, NlOsiris7, NlOsiris9, NlOsiris11, NlOsiris14, NlOsiris16, NlOsiris17, NlOsiris19 and NlOsiris24, significantly reduced survival, with mortality climbing steadily over thirteen days after treatment. Knockdown of NlOsiris7, NlOsiris11 or NlOsiris14 produced shriveled abdomens and abnormally small bodies in fifth-instar nymphs, while adults that survived emerged with flat, deflated abdomens instead of the swollen, stretched abdomens seen in controls. Critically, these females carried deficient ovaries lacking mature oocytes, directly linking three Osiris genes to reproductive organ development.

Other genes sculpted entirely different structures. When NlOsiris19 or NlOsiris24 was silenced, roughly ninety percent of treated insects reached adulthood but bore small, irregularly shaped forewings, in contrast to the normal wings of more than ninety-five percent of control insects. Silencing NlOsiris17 left female adults with transparent, soft ovipositors instead of the brown, hardened structures typical of healthy insects. The flattened abdomens of NlOsiris2-, NlOsiris11- and NlOsiris14-deficient insects prompted a feeding assay on an artificial diet sealed between Parafilm membranes. Control insects produced more than ten salivary sheaths per square centimeter within two days, rising to forty by day seven, while Osiris-silenced insects produced fewer than ten throughout the entire week. The team hypothesizes that silencing these genes altered mouthpart structure, crippling the insect’s ability to feed, a finding with obvious appeal for pest control strategies.

Perhaps the most intriguing results concerned the next generation. Because NlOsiris transcripts spiked in eggs at 144 hours after laying, the researchers injected double-stranded RNAs into newly eclosed females and tracked the fate of their offspring. Silencing eight genes, NlOsiris7, NlOsiris11, NlOsiris14, NlOsiris17, NlOsiris18, NlOsiris19, NlOsiris20 or NlOsiris24, slashed hatching rates to between thirty and fifty-five percent, compared with ninety percent in controls. Dissection of eggs just before hatching showed that more than seventy percent still displayed the dark red eye spots that mark viable, fertilized eggs, ruling out failed fertilization as the cause. Instead, close observation revealed that embryos from NlOsiris17- and NlOsiris24-silenced females developed into first-instar nymphs that became stuck to rice stems and died without escaping the eggshell, indicating that these genes are essential for the final transition from egg to free-living nymph.

The study also exposed how the same gene family can diverge in function across insect lineages. In the silkworm, Osiris9a is expressed specifically in the silk gland and contributes to silk fiber formation, whereas its planthopper counterpart, when silenced, causes lethality. In Drosophila, Osiris7 knockdown traps larvae at the prepupal stage, while in the planthopper the homologous gene governs nymphal body size and egg hatching, reflecting the fundamental difference between complete and incomplete metamorphosis. Meanwhile, nine planthopper Osiris genes produced no obvious individual phenotypes when silenced, but the authors caution that redundancy among similarly expressed family members may mask their importance, a possibility well documented for the tracheal genes Osiris9, Osiris15 and Osiris19 in Drosophila.

The researchers propose that NlOsiris genes are intimately tied to cuticle formation, noting that their expression peaks coincide with those of chitin-binding genes, although direct biochemical evidence of that link remains to be established. Future work, they suggest, should employ comparative transcriptomics to trace the downstream pathways governed by Osiris expression and CRISPR/Cas9 gene editing to confirm the roles inferred from RNA interference. Because the family is absent from all non-insect animals, Osiris genes represent an attractive and highly specific target for novel pest management approaches, potentially enabling genetic control strategies that suppress planthopper survival, feeding or reproduction without the collateral damage associated with broad-spectrum insecticides. For a pest that drains the phloem sap of rice across millions of hectares, a gene family that governs its mouthparts, wings, ovaries and egg hatching may prove to be its most vulnerable achilles heel.

Subject of Research: Functional analysis of the insect-specific Osiris gene family in the brown planthopper Nilaparvata lugens

Article Title: Functional diversity of the Osiris gene family in the brown planthopper

Article References: Zhang, C., He, X., Ma, Y., Liu, Y., Shen, X., & Bao, Y. (2025). Functional diversity of the Osiris gene family in the brown planthopper. Crop Health, 3(1), Article 7. https://doi.org/10.1007/s44297-025-00045-4

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00045-4

Keywords: Osiris gene family, brown planthopper, Nilaparvata lugens, RNA interference, insect development, cuticle formation, egg hatching, gene duplication, phylogenetics, rice pest, pest management, hemimetabolous insects