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

Intraspecific competition, a central topic in ecology and evolution, significantly impacts population dynamics and adaptation. Limited resources lead to reduced population size and genetic diversity, increasing extinction risk. Conversely, competition drives natural selection, potentially enabling rapid adaptation and niche expansion. This is particularly relevant in parasitoid wasps, where reproductive success depends heavily on host condition and the female's oviposition strategy. Superparasitism, where multiple eggs are laid in a single host, and its avoidance are key aspects of intraspecific competition in solitary wasps, but the underlying mechanisms and ecological contexts remain largely unexplored. The fitness costs and evolutionary consequences of superparasitism have been debated extensively. While superparasitism generally lowers reproductive efficiency, it may be adaptive under certain conditions, potentially offering advantages in eliminating competitors or enhancing host immune suppression. Recent research suggests that superparasitism can be manipulated by symbionts, highlighting the complexity of this phenomenon. This study addresses the lack of understanding regarding the mechanisms of superparasitism avoidance by focusing on *Leptopilina boulardi*, a solitary parasitoid of *Drosophila*, as a model system.

The literature review section extensively cites previous studies on intraspecific competition, its impact on population dynamics and adaptation in various species, particularly in parasitoid wasps. It highlights the contrasting perspectives on the adaptive value of superparasitism, with some studies suggesting potential benefits under specific circumstances, while others focus on its negative consequences for reproductive success. The role of symbionts in influencing superparasitism is also addressed. Previous research on *Drosophila* parasites and their interactions with hosts is reviewed, setting the stage for the current study's focus on the molecular mechanisms underlying superparasitism avoidance in *L. boulardi*. The limited existing knowledge on this topic underscores the need for further research, particularly at the molecular level.

The study employed a multi-faceted approach combining behavioral observations, genomic analysis, and in vivo functional studies. *Leptopilina boulardi* (Lb), a solitary parasitoid of *Drosophila*, was used as the model organism. First, behavioral assays were conducted to assess the incidence of superparasitism under varying parasite-to-host ratios. The researchers observed a host escape response triggered by Lb parasitism. To identify the molecular basis of this behavior, a high-quality Lb genome was generated using PacBio sequencing, followed by transcriptome and proteome analysis of venom. This comprehensive genomic approach allowed for the identification of candidate genes, particularly three RhoGAP domain-containing genes showing high venom expression, termed EsGAP1-3. RNA interference (RNAi) experiments were employed to knock down the expression of these genes in Lb, followed by behavioral assays to determine their impact on host escape. Reactive oxygen species (ROS) levels in the host central nervous system (CNS) were measured using DCFH-DA staining to investigate the mechanism of host behavior manipulation. Phylogenetic analysis of RhoGAP domain-containing genes in Lb and a related generalist species, *Leptopilina heterotoma* (Lh), was performed to understand the evolutionary history of this mechanism. Additionally, the study investigated the extent of this manipulation across different *Drosophila* species.

The key findings include: 1) Lb females actively avoid superparasitism by inducing host escape behavior. 2) This escape behavior is triggered by venom components, specifically three newly identified RhoGAP domain-containing proteins (EsGAP1-3) that are highly expressed in Lb venom. 3) Knockdown of EsGAP genes significantly reduces host escape behavior, demonstrating their causal role in this manipulation. 4) EsGAP proteins induce a significant increase in reactive oxygen species (ROS) levels in the host CNS, correlating with the observed escape behavior. 5) Phylogenetic analysis suggests that EsGAP genes arose through neofunctionalization and expression specialization of an ancient RhoGAP domain, predating the divergence of specialist and generalist *Leptopilina* species. 6) The host escape strategy is not limited to *Drosophila melanogaster*; it also affects other *Drosophila* species, suggesting a broad effectiveness of this manipulation mechanism. 7) The findings provide direct evidence supporting the adaptive value of superparasitism avoidance for the parasite. 8) The study successfully characterised the genetic basis for the parasitoid-induced host escape behaviour, which is an adaptive strategy for avoiding superparasitism.

This study addresses the long-standing debate regarding the adaptive significance of superparasitism avoidance. While previous hypotheses focused on host discrimination and pheromonal cues, this research reveals a novel mechanism involving direct manipulation of host behavior. The identification of EsGAP genes and their role in inducing ROS accumulation in the host CNS provides a clear molecular basis for this behavior. The phylogenetic analysis demonstrates that this sophisticated manipulation strategy evolved through neofunctionalization of an ancient domain, highlighting the power of evolutionary innovation in parasite-host interactions. The broad effectiveness of this strategy across different Drosophila species suggests its evolutionary importance. The findings highlight the significance of direct host manipulation in parasite adaptation and provide new insights into the ecological and evolutionary dynamics of parasite-host coevolution. The results challenge previous models, emphasizing the proactive and manipulative nature of superparasitism avoidance.

This research provides compelling evidence for a novel mechanism of superparasitism avoidance in the parasitoid wasp *Leptopilina boulardi*: the manipulation of host escape behavior through the injection of EsGAP proteins, which induce ROS accumulation in the host CNS. The evolutionary analysis suggests an ancient origin of the EsGAP genes, with neofunctionalization and expression specialization playing crucial roles. Future research could explore the specific downstream targets of EsGAP proteins in the host CNS, further elucidate the mechanism of ROS-induced escape, and investigate the prevalence of similar mechanisms in other parasite-host systems. This study makes a significant contribution to our understanding of parasite adaptation and the evolutionary arms race between parasites and their hosts.

The study primarily focused on laboratory-reared strains of *Leptopilina boulardi*. While the findings suggest a broad applicability of the host escape mechanism, further investigation is needed to confirm its relevance in natural populations and diverse ecological contexts. The RNAi experiments, while successful in demonstrating the role of EsGAP genes, might have off-target effects that could impact the interpretation of results. Although the study extensively investigated the mechanism in *Drosophila* hosts, examining additional host species could further broaden the understanding of this phenomenon. The precise evolutionary pathway of EsGAP neofunctionalization requires more in-depth analysis.