Neofunctionalization of an ancient domain allows parasites to avoid intraspecific competition by manipulating host behaviour

The study addresses how solitary parasitoid wasps avoid costly superparasitism (multiple eggs laid in the same host), a key facet of intraspecific competition that shapes population dynamics and adaptation. While parasitoids are known to discriminate parasitized from unparasitized hosts—potentially via marking pheromones—the proximate mechanisms and ecological significance of superparasitism avoidance remain debated. Using the Drosophila-parasitizing wasp Leptopilina boulardi (Lb), the authors hypothesize that parasitoids can actively manipulate host behaviour to avoid superparasitism, and seek to identify the molecular effectors and host pathways involved, as well as the evolutionary origins of this strategy.

Prior work documents widespread superparasitism avoidance in solitary parasitoids and its fitness consequences, including reduced offspring size, prolonged development, and increased mortality under superparasitism. Traditional explanations emphasize host discrimination—potentially via external or internal marking pheromones—yet direct mechanisms are unclear. Some contexts may make superparasitism advantageous (e.g., competitor elimination, enhanced immune suppression), and in L. boulardi, a symbiotic virus (LbFV) can increase superparasitism, suggesting manipulation by symbionts rather than parasitoid adaptation. Drosophila–parasitoid systems have provided mechanistic insights into host immune responses and parasitoid virulence, but how parasitoids may manipulate host behaviour to avoid intraspecific competition remained underexplored.

• Study system: Leptopilina boulardi parasitizing Drosophila melanogaster larvae; additional tests on D. hydei, D. mauritiana, D. simulans, and D. yakuba. L. heterotoma used for comparative functional and evolutionary analyses. Lb strain verified free of LbFV infection.
• Behavioural assays: Hosts and wasps cohabited in fly bottles at defined parasitoid:host ratios (e.g., 1:10, 1:20). Escape behaviour quantified as larvae leaving food and crawling on bottle walls; escape index calculated over time (15-min intervals, typically 30–75 min; 4-h total exposure in some assays). Superparasitism quantified by dissecting larvae to count wasp eggs per host.
• Venom sufficiency/necessity tests: Sex-specific induction (female vs male wasps). Direct injection of venom into larvae (e.g., 1:50, 1:100 dilutions) vs PBS controls to assess escape behaviour.
• Multi-omics to define venom proteins: De novo L. boulardi genome assembly (PacBio long reads; 354.8 Mb; N50 = 2.7 Mb). Venom gland transcriptome and venom proteomics used to compile venom protein (VP) catalogue; prioritized highly expressed VPs (Z test P < 0.01), identifying three venom-specialized RhoGAP domain genes.
• Functional genetics in parasitoids: RNAi by dsRNA microinjection into developing wasps to knock down candidate venom genes, including three RhoGAPs (EsGAP1–3). qRT-PCR validated knockdown. Effects on host escape behaviour measured.
• ROS assays in hosts: CNS and other tissues stained with DCFH-DA to quantify ROS levels after parasitization or venom injection, across time points up to ~3 h. Genetic manipulation of ROS via UAS-catalase driven by Actin-GAL4 (ubiquitous), Elav-GAL4 (neuronal), and Hml-GAL4 (hemocytes) to test necessity for escape behaviour.
• Protein localization: Western blot and immunostaining to detect EsGAP1 in parasitized host CNS.
• Evolutionary analyses: Genome-wide scans for RhoGAP domains in L. boulardi (Lb) and L. heterotoma (Lh), phylogenetic analyses to resolve lineages, assess gene architectures (domain composition), and expression specializations (venom vs widespread). RNAi knockdown of top Lh venom RhoGAPs to test conservation of function.

• L. boulardi parasitization induces robust host escape behaviour: over 77% of D. melanogaster larvae left food and crawled onto bottle surfaces within the first hours after wasp introduction; this was not observed without wasps. Non-escaping hosts experienced higher superparasitism (significantly more eggs per host; ANOVA P < 0.0001), and most (74.34%) non-escaped hosts died without yielding adults.
• Venom is necessary and sufficient: Only female wasps (that inject venom during oviposition) induced escape; purified venom injections elicited escape in a dose-dependent manner (e.g., significant increases at 1:100 and 1:50 dilutions; reported P = 0.008 and 0.004, respectively).
• Identification of venom-specialized RhoGAPs: Multi-omics revealed three highly expressed venom proteins with RhoGAP domains (PF00620), with strong venom-specific expression. RNAi knockdown of each (EsGAP1–3) in wasps significantly reduced host escape indices (>50% reduction vs controls), with EsGAP1 showing the strongest effect across assays.
• ROS in host CNS mediates escape: Parasitization rapidly elevated ROS specifically in host CNS (but not broadly in other tissues), peaking during the first ~3 h, paralleling escape behaviour dynamics. Venom injection similarly increased CNS ROS. Neuronal expression of catalase (Elav-GAL4>UAS-catalase) suppressed ROS elevation and significantly reduced escape indices, demonstrating CNS ROS is necessary for behaviour. Ubiquitous catalase (Actin-GAL4) also reduced ROS and escape; hemocyte-specific catalase (Hml-GAL4) did not.
• EsGAP delivery to host CNS: EsGAP1 protein was detected in parasitized larval CNS by Western blot and immunostaining, supporting venom transfer and CNS targeting during parasitization.
• Cross-species generality and fitness outcomes: Knocking down EsGAPs in wasps increased superparasitism across multiple Drosophila hosts (D. hydei, D. mauritiana, D. simulans, D. yakuba), with reported increases in average eggs per host (e.g., ~49%, 54%, 31%, 41% vs controls), indicating EsGAP-driven escape generally aids superparasitism avoidance.
• Evolutionary origin and neofunctionalization: Lb and Lh genomes contain 15 and 14 RhoGAP-domain loci, respectively. Phylogeny revealed two lineages: conserved, multidomain RhoGAPs with broad expression, and lineage-specific expansions consisting of RhoGAP-only genes specialized for venom expression (including EsGAPs). RNAi of top Lh venom RhoGAPs similarly reduced host escape, indicating an ancestral origin of this function predating Lb–Lh divergence and independent expansions with neofunctionalization.

The study demonstrates that solitary parasitoid wasps can actively avoid intraspecific competition by manipulating host behaviour: L. boulardi injects venom RhoGAP proteins (EsGAPs) that elevate ROS in the host CNS, triggering larval escape from food and thus reducing the likelihood of subsequent oviposition in the same host (superparasitism). This mechanism complements or may supersede classical host discrimination via marking pheromones, providing a direct, rapid, and adaptive strategy under competitive conditions. The requirement for CNS ROS ties a defined host physiological pathway to a clear behavioural output, and neuronal catalase experiments establish necessity of ROS signaling for escape. Evolutionarily, EsGAPs exemplify neofunctionalization of an ancient RhoGAP domain into venom-specialized effectors through lineage-specific expansion, with analogous genes and functions present in both specialist (Lb) and generalist (Lh) Leptopilina, suggesting an ancestral innovation prior to their split. This advances understanding of how molecular effectors evolve to manipulate host behaviour and mediate competition outcomes in parasitoid–host systems.

This work identifies a molecular mechanism by which Leptopilina parasitoids avoid superparasitism: venom RhoGAP proteins (EsGAPs) are injected into hosts, induce ROS accumulation in the CNS, and trigger host escape behaviour, thereby reducing intraspecific competition and enhancing parasitoid reproductive efficiency. Multi-omics, functional knockdowns, and cross-species assays establish EsGAP necessity for escape and link behaviour to CNS ROS. Phylogenetic analyses reveal lineage-specific expansions and venom-specialized expression, supporting neofunctionalization of an ancient domain before the divergence of L. boulardi and L. heterotoma. Future work should clarify the precise molecular targets of EsGAPs in host neurons, dissect potential diversification of EsGAP paralogs (e.g., roles in immune suppression vs behaviour), resolve the evolutionary origin (lateral gene transfer vs duplication) of RhoGAP-only venom proteins, and assess ecological dynamics in natural populations beyond laboratory conditions.

• Laboratory-maintained wasp strains and controlled assay conditions may not fully capture ecological variability; selection on competition-related traits could be stronger in the lab than in the wild.
• The evolutionary origin of RhoGAP-only venom genes is unresolved (possible lateral gene transfer vs rapid divergence after duplication) due to phylogenetic uncertainties.
• Some reported statistics are constrained by assay windows (focus on 30–75 min) and may not encompass longer-term behavioural dynamics.
• Functional redundancy among EsGAP paralogs and potential pleiotropy (e.g., immune modulation) were not fully disentangled; off-target effects in RNAi were noted as partial cross-reductions among RhoGAP transcripts.
• Direct neuronal targets and signaling pathways downstream of ROS leading to escape were not identified; causality was established for ROS necessity but not sufficiency at the single-cell or circuit level.