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

Wolbachia is famous among biologists for its extraordinary reproductive tricks. Because it is transmitted only from mothers to offspring through the egg cytoplasm, the bacterium has evolved an arsenal of strategies to favor infected females at the expense of males and uninfected individuals. It can kill infected males outright, convert genetically male embryos into females, induce females to reproduce without mating, and, most famously, trigger cytoplasmic incompatibility, or CI. In CI, when a Wolbachia-infected male mates with an uninfected female, or with a female carrying a different Wolbachia strain, the fertilized eggs die during embryonic development. If, however, the female carries the same strain, her eggs can rescue the paternal chromosomes and development proceeds normally. The net effect is that infected females enjoy a reproductive advantage, and the bacterium sweeps through populations on the back of this manipulation.

The review’s central insight is that the three rice planthopper species host strikingly different Wolbachia strains with very different effects. In the brown planthopper, the native strain wLug infects fewer than half of individuals in the field, and rather than inducing CI it appears to boost the fecundity of its host. In the small brown planthopper, by contrast, the strain wStri infects roughly ninety-nine percent of wild populations and induces complete cytoplasmic incompatibility while simultaneously enhancing reproduction. The white-backed planthopper’s strain, wSfur, infects about ninety percent of individuals but shows weak or no CI and only minimal effects on fecundity. These strain-specific differences matter enormously for population dynamics, because the reproductive effects of the bacterium directly alter the base population size of the insects and therefore influence the scale of planthopper infestations in the field.

One of the most intriguing findings concerns what happens when a Wolbachia strain is moved into a new host species. Researchers successfully transfected wStri from the small brown planthopper into the brown planthopper, where the bacterium adopted a tissue distribution similar to that in its native host but reached significantly higher densities than the native wLug strain. Although wStri still induced cytoplasmic incompatibility in its new home, the intensity of the effect was notably reduced. This observation carries an important message: the strength of CI is shaped not only by the symbiont itself but also by the genetic background of the host. The transfection also had a practical payoff, because the introduced wStri inhibited infection and transmission of rice ragged stunt virus in the brown planthopper, hinting at significant pest control potential.

At the molecular level, the mechanism of cytoplasmic incompatibility has only recently come into focus. Because Wolbachia cannot be cultured outside living cells, identifying the bacterial factors behind CI long remained a stubborn challenge. The breakthrough came with the discovery of two adjacent genes, cifA and cifB, encoding proteins that appear to be the key effectors. In transgenic experiments in fruit flies, expressing CifB and CifA in uninfected males was sufficient to reproduce the CI phenotype, with embryos dying after mating with uninfected females. Conversely, expressing CifA alone in uninfected females rescued embryos from Wolbachia-infected males. Mutations in the PD-(D/E)XK nuclease, deubiquitinase, and peptidase domains of CifB impair the function of these factors, and the three-dimensional structures of the two proteins have now been resolved, revealing that their interaction depends on charge attraction across three interfacial regions.

Two competing models attempt to explain how these proteins work. The toxin-antidote model proposes that CifB is delivered to the embryo, where it acts as a toxin, while Wolbachia in the egg synthesizes CifA, which binds CifB and neutralizes it, allowing normal development. The host modification model instead holds that Cif proteins modify targets within the gametes themselves: modifications to sperm induce CI, while modifications to female gametes reverse the damage and rescue embryos. Recent functional studies lend weight to the modification model. CifA has been detected in sperm nuclei during spermatogenesis, where it causes retention of histones and deficiency of protamines in mature sperm, and both CifA and CifB have been shown to act as nucleases that degrade long non-coding RNAs and damage DNA during late sperm formation. Notably, the rules differ across strains: in some systems CifB alone suffices to induce CI, while in others both proteins must be expressed together, underscoring how diverse these mechanisms remain.

The wStri genome adds another layer of complexity. It contains three copies of the cifA-cifB gene pair, and phylogenetic analysis shows that the known CI factor groups have expanded from five to ten, with the wStri factors belonging to newly identified groups VI and VIII whose functions remain unknown. On the host side, proteomic and RNA interference studies in the small brown planthopper have identified candidate host factors. The cytoplasmic aminopeptidase-like protein, or CAL, appears to be associated with CI lethality, while the gene iLvE, involved in branched-chain amino acid biosynthesis, was the most significantly downregulated gene in infected insects, and knocking it down in uninfected males reduced fertility in a way that could be restored by mating with infected females. Another candidate, NDUFA8, which encodes a subunit of a mitochondrial enzyme complex, was upregulated in infected females, and suppressing it reproduced CI-like embryo mortality, suggesting a role in the rescue phenotype.

Beyond incompatibility, Wolbachia acts as a reproductive booster for its planthopper hosts. Removing the bacterium from brown planthoppers significantly shortens oviposition and reduces female fecundity, and wStri similarly promotes egg production in the small brown planthopper. Comparative genomics and metabolomics show that both wStri and wLug carry complete pathways for synthesizing B vitamins, including biotin and riboflavin, nutrients that play crucial roles in host reproduction and that the insects apparently cannot fully supply themselves. At the physiological level, the bacterium upregulates vitellogenin, the yolk precursor protein essential for egg formation, increases mitotic division of germ cells, and accelerates the breakdown of nurse cells during peak oviposition, thereby channeling more nutrients into egg production. Wolbachia also alters microRNA expression in its host, reshaping the expression of fecundity-related genes, and preliminary evidence suggests changes in mitochondrial energy metabolism may contribute as well.

The picture is further complicated by interactions with other symbionts. In the white-backed planthopper, Wolbachia coexists with another intracellular bacterium, Cardinium, and lines carrying both symbionts show distinct microbiomes and metabolite profiles. Remarkably, double-infected lines exhibit lower fecundity than uninfected ones, indicating that combined symbiont effects can be more consequential than either bacterium alone. For pest management, the implications are considerable. The CI principle underlies incompatible insect technique programs already deployed against mosquitoes, in which males carrying specific Wolbachia strains are released to mate with wild females, causing their fertilized eggs to die and suppressing target populations. Whether similar strategies can be scaled up for rice planthoppers remains an open question, and the review’s authors caution that field deployment would require careful risk assessment, including the possibility of resistance evolution, ecological side effects, and horizontal transmission of Wolbachia to non-target species. Still, as planthopper outbreaks intensify and pesticide resistance grows, a bacterium that can both sabotage and supercharge insect reproduction may prove to be one of the most valuable allies rice farmers have.

Subject of Research: Wolbachia-mediated reproductive manipulation, including cytoplasmic incompatibility and fecundity enhancement, in three rice planthopper pest species

Article Title: Wolbachia-mediated reproductive manipulation in rice planthoppers

Article References: Niu, Y.-D., Wang, M.-K., Yan, Z.-C., Bing, X.-L., & Hong, X.-Y. (2025). Wolbachia-mediated reproductive manipulation in rice planthoppers. Crop Health, 3(1), Article 20. https://doi.org/10.1007/s44297-025-00059-y

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00059-y

Keywords: Wolbachia, rice planthoppers, cytoplasmic incompatibility, endosymbiont, Nilaparvata lugens, Laodelphax striatellus, Sogatella furcifera, cif genes, fecundity, pest control, symbiosis, B vitamins