Microbial changes and associated metabolic responses modify host plant adaptation in Stephanitis nashi.

Symbiotic microorganisms are essential for the physiological processes of herbivorous pests, including the pear lace bug Stephanitis nashi, which is known for causing extensive damage to garden plants and fruit trees due to its exceptional adaptability to diverse host plants. However, the specific functional effects of the microbiome on the adaptation of S. nashi to its host plants remains unclear. Here, we identified significant microbial changes in S. nashi on 2 different host plants, crabapple and cherry blossom, characterized by the differences in fungal diversity as well as bacterial and fungal community structures, with abundant correlations between bacteria or fungi. Consistent with the microbiome changes, S. nashi that fed on cherry blossom demonstrated decreased metabolites and downregulated key metabolic pathways, such as the arginine and mitogen-activated protein kinase signaling pathway, which were crucial for host plant adaptation. Furthermore, correlation analysis unveiled numerous correlations between differential microorganisms and differential metabolites, which were influenced by the interactions between bacteria or fungi. These differential bacteria, fungi, and associated metabolites may modify the key metabolic pathways in S. nashi, aiding its adaptation to different host plants. These results provide valuable insights into the alteration in microbiome and function of S. nashi adapted to different host plants, contributing to a better understanding of pest invasion and dispersal from a microbial perspective.

Comparing the Performance of Hyphantria cunea (Lepidoptera: Arctiidae) on Artificial and Natural Diets: Feasibility of Mass-Rearing on Artificial Diets.

In China, Hyphantria cunea (Drury) is an invasive phytophagous pest; it attacks nearly all species of defoliated trees. To develop integrated pest management programs (IPM) for H. cunea, we need to ensure the availability of insects by mass-rearing them on artificial diets under laboratory conditions. This study compared the growth characteristics, nutritional indices, growth indices, and digestive enzyme activity of insects reared on Pterocarya stenoptera C.DC (Fagales: Juglandaceae), the Chinese wingnut, and an artificial diet. We also investigated the correlation between diet components and growth indices using principal components analysis and Pearson correlation analysis. We found that mass-rearing of H. cunea on an artificial diet was feasible. It led to a shorter developmental period, with heavier larvae and pupae than natural diets. The principal components analysis indicated that the growth indices and α-Amylase were significantly positively associated with PC1, which explained 82.45% of the total data variability. Pearson correlation analysis showed a significant correlation between digestion, absorption parameters, and growth. Developing a mass-rearing program to produce H. cunea on an artificial diet will be valuable for improving IPM strategies. Understanding the mechanism of the responses of phytophagous insect populations to anthropogenic diet regulation can provide new ideas and methods for pest control.

iTRAQ Proteomic Analysis of Interactions Between 20E and Phospholipase C in Apolygus lucorum (Meyer-Dür).

Polyphagous Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. Hormone 20-hydroxyecdysone (20E) regulates multiple insect development and physiology events. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes and related proteins that can be affected by PLC still remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblotting techniques were used to compare differentially expressed proteins (DEPs) in A. lucorum in response to the treatment of 20E and the PLC inhibitor U73122 as well as their combination. A total of 1,624 non-redundant proteins and 97, 248, 266 DEPs were identified in the 20E/control, U73122/control, and 20E + U73122/control groups, respectively. Only 8 DEPs, including pathogenesis-related protein 5-like, cuticle protein 19.8, trans-sialidase, larval cuticle protein A2B-like, cathepsin L1, hemolymph juvenile hormone-binding protein, ATP-dependent RNA helicase p62-like, and myosin-9 isoform X1, were detected in all three groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEPs were involved in diverse signaling pathways. The results were validated by immunoblotting, which highlighted the reliability of proteomics analysis. These findings provided novel insights into the function of PLC in 20E signaling pathway in A. lucorum.

Insights into chemosensory genes of Pagiophloeus tsushimanus adults using transcriptome and qRT-PCR analysis.

Pagiophloeus tsushimanus is a new, destructive, and monophagous weevil pest that thrives on Cinnamomum camphora, found in Shanghai. The functions of chemosensory genes involved in the host location and intraspecific communication of P. tsushimanus remain unknown. The male-female transcriptomes of P. tsushimanus adults were assembled using Illumina sequencing, and we focused on all chemosensory genes in transcriptomes. In general, 58,088 unigenes with a mean length of 1018.19 bp were obtained. In total, 39 odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 22 olfactory receptors (ORs), 16 gustatory receptors (GRs), eight ionotropic receptors (IRs), and five sensory neuron membrane proteins (SNMPs) were identified. PtsuOBPs comprised four subfamilies (20 Minus-C, one Plus-C, two Dimer, and 15 Classic). Both PtsuOBPs and PtsuCSPs contained a highly conserved sequence motif of cysteine residues. PtsuORs including one olfactory receptor co-receptors (Ptsu/Orco) comprised seven predicted transmembrane domains. Phylogenetic analysis revealed that PtsuOBPs, PtsuCSPs, and PtsuORs in P. tsushimanus exhibited low homology compared to other insect species. The results of tissue- and sex-specific expression patterns indicated that PtsuOBPs and PtsuORs were highly abundant in the antennae; whereas, PtsuCSPs were not only highly abundant in antennae, but also abdominal apexes, wings, and legs. In conclusion, these results enrich the gene database of P. tsushimanus, which may serve as a basis for identifying novel targets to disrupt olfactory key genes and may provide a reverse validation method to identify attractants for formulating potential eco-friendly control strategies for this pest.

Phospholipase C gamma (PLCγ) regulates soluble trehalase in the 20E-induced fecundity of Apolygus lucorum.

Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis (Bt) cotton in China. Additionally, 20-hydroxyecdysone (20E) has important functions in many biological processes, including insect reproduction. Phospholipase C (PLC), which is an essential enzyme for phosphoinositide metabolism, is involved in 20E signal transduction, but its function in 20E-mediated reproduction in A. lucorum remains unclear. In this study, 20E increased AlPLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis. The 20E treatment also induced the considerable accumulation of two second messengers, inositol triphosphate and diacylglycerol. The expression levels of genes encoding vitellogenin (AlVg) and soluble trehalase (AlTre-1) were similar to those of AlPLCγ, and were upregulated in response to 20E. The silencing of AlPLCγ resulted in downregulated expression of AlTre-1 and AlVg. However, the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression. Moreover, the silencing of AlVg did not alter AlTre-1 expression. Furthermore, an examination of the insect specimens indicated that AlPLCγ is required for female adult reproduction, and that downregulated expression of this gene is associated with decreases in fecundity, adult longevity, and egg hatching rate as well as delayed oocyte maturation. We propose that 20E regulates AlTre-1 expression via AlPLCγ and affects Vg expression as well as ovary development to facilitate the reproductive activities of A. lucorum females.

Life history of aggression in Anastatus disparis (Hymenoptera: Eupelmidae) with extreme male-male combat.

Aggressive behaviour is common in animals and typically has lifetime consequences. As younger males have higher residual reproductive value than older males and lose more from injuries than older males do, the propensity for fighting tends to increase with age in many empirical reports and species. However, fighting patterns in those empirical reports cannot confirm the hypothesis that individuals cannot readily inflict injuries on their opponents. To address this shortcoming, a parasitoid wasp species, Anastatus disparis (Hymenoptera: Eupelmidae), was used as an experimental model to explore the characteristics of aggression from a life-history perspective; this wasp exhibits extreme fighting, resulting in contestants experiencing injury and death. Results showed that the energetic costs of fighting to injury significantly shortened life and caused the loss of most mating ability. Inconsistent with general predictions, the frequency and intensity of fighting in A. disparis significantly decreased with male age. Further study results showed significantly more young males were received by and successfully mated with virgin females, and most genes related to energy metabolism were downregulated in aged males. Our study provided supporting evidence that young A. disparis males show more aggression likely because of their resource holding potential and sexual attractiveness decline with age.

Sexual dimorphism and sex-biased gene expression in an egg parasitoid species, Anastatus disparis.

BACKGROUND: Differences in the expression of genes present in both sexes are assumed to contribute to sex differences including behavioural, physiological and morphological dimorphisms. For enriching our knowledge of gender differences in an important egg parasitoid wasp, Anastatus disparis (Hymenoptera: Eupelmidae), sex-biased differences in gene expression were investigated using Illumina-based transcriptomic analysis. RESULTS: A total of 15,812 resulting unigenes were annotated, and a large set of genes accounting for 50.09% of the total showed sex-biased expression and included 630 sex-specific genes. Gene Ontology (GO) enrichment analyses showed that the functional categories associated with sex-biased genes were mainly related to reproduction. In addition, the transcriptome data provided evidence that sex pheromones in A. disparis are produced by the female, and activity of Δ12-desaturases appear to have been replaced by Δ9-desaturases playing roles in sex pheromone production. The large set of sex-biased genes identified in this study provide a molecular background for sexually dimorphic traits such as flyability, longevity, and aggression in this species and suggests candidate venom proteins expressed only in females that could be used for biological control. CONCLUSIONS: This study provides comprehensive insight into sexually dimorphic traits of a parasitoid wasp and can inform future research into the molecular mechanisms underlying such traits and the application of parasitoids to the biological control of pest species.

Effect of Winning Experience on Aggression Involving Dangerous Fighting Behavior in Anastatus disparis (Hymenoptera: Eupelmidae).

Aggressive behavior is widely observed in animal species for acquiring important resources and usually includes both dangerous and nondangerous fighting patterns. Only a few species show dangerous fighting patterns that are defined by fights ending with contestants being severely injured or killed. Prior experience, an important factor in many species, has been demonstrated to affect a contestant's subsequent fighting behavior. Few studies have focused on the effect of experience on aggression involving dangerous fighting patterns. Here, an egg parasitoid wasp, Anastatus disparis, which shows extreme and dangerous fighting behavior to acquire mating opportunities, was used as an experimental model. Our results showed that the fighting intensity of the winning males significantly decreased subsequent fighting behavior, which was inconsistent with general predictions. Transcriptomic analyses showed that many genes related to energy metabolism were downregulated in winners, and winners increased their fighting intensity after dietary supplementation. Our study suggested that fighting in A. disparis is a tremendous drain on energy. Thus, although males won at combat, significant reductions in available energy constrained the intensity of subsequent fights and influenced strategic decisions. In addition, winners might improve their fighting skills and abilities from previous contests, and their fighting intensity after dietary supplementation was significantly higher than that of males without any fighting experience. Generally, in A. disparis, although winners increased their fighting ability with previous experience, the available energy in winners was likely to be a crucial factor affecting the intensity and strategic decisions in subsequent fights.

Effect of variation in objective resource value on extreme male combat in a quasi-gregarious species, Anastatus disparis.

BACKGROUND: Aggressive behaviour is widely observed in animal kingdom, which compete for resources such as territory, food and mates. Resource value is the most important non-strategic factor influencing fighting behaviour, and may vary among contests and contestants. Usually, contestants adjust their fighting behaviour when the resource value changes, and as potentially damaging and energetically costly, individuals of most species usually avoid conflict escalation. However, in a quasi-gregarious egg parasitoid, Anastatus disparis (Hymenoptera: Eupelmidae), mates are valuable resources and females mate only once; thus, males engage in frequently extreme combat behaviour to acquire mating opportunities, even in the absence of females. In this study, we attempted to test whether males of this species have the ability to adjust their fighting behaviour in response to changes in the objective value of female. RESULTS: Our results suggested that objective resource value in A. disparis is likely to be influenced by female mating status rather than by fecundity. Consistent with a number of empirical studies, A. disparis males adjusted their fighting behaviour according to the value of the contested resources: males significantly increased their fighting intensity to acquire mating opportunities with virgin females but decreased their fighting intensity for mated females. We also found that rather than chemical cues, visual cues and physical sexual contact appear to play a role in determining males' ability to detect variation in female mating status. CONCLUSIONS: Our study suggested that although in this species, males have evolved extreme fighting behaviour and females are valuable resources, males do not always escalate fighting behaviour in competition for mating with a female. Valuable resources and variation in resource value were detected and estimated by A. disparis males, which then adjusted their fighting behaviour accordingly and to some extent avoided incoming fighting costs.

Sexual Transcription Differences in Brachymeria lasus (Hymenoptera: Chalcididae), a Pupal Parasitoid Species of Lymantria dispar (Lepidoptera: Lymantriidae).

Sex differences in gene expression have been extensively documented, but little is known about these differences in parasitoid species that are widely applied to control pests. Brachymeria lasus is a solitary parasitoid species and has been evaluated as a potential candidate for release to control Lymantria dispar. In this study, gender differences in B. lasus were investigated using Illumina-based transcriptomic analysis. The resulting 37,453 unigene annotations provided a large amount of useful data for molecular studies of B. lasus. A total of 1416 differentially expressed genes were identified between females and males, and the majority of the sex-biased genes were female biased. Gene Ontology (GO) and Pathway enrichment analyses showed that (1) the functional categories DNA replication, fatty acid biosynthesis, and metabolism were enhanced in females and that (2) the only pathway enriched in males was phototransduction, while the GO subcategories enriched in males were those involved in membrane and ion transport. In addition, thirteen genes involving transient receptor potential (TRP) channels were annotated in B. lasus. We further explored and discussed the functions of TRPs in sensory signaling of light and temperature. In general, this study provides new molecular insights into the biological and sexually dimorphic traits of parasitoids, which may improve the application of these insects to the biological control of pests.

Behavioural and transcriptional changes in post-mating females of an egg parasitoid wasp species.

In many animals, mating is essential for the production of offspring by females; however, mating seems to not be necessary in Hymenoptera insects. Virgin females can produce offspring, although the sex of the offspring is all male. Usually, behavioural and physiological changes are induced by mating in female insects, including parasitoid wasps. However, very little is known about the resulting changes in gene expression that contribute to the post-mating response in females; thus, we studied this aspect in the egg parasitoid wasp species Anastatus disparis (Hymenoptera: Eupelmidae) by transcriptional analysis. A total of 55 differentially expressed genes were identified in post-mating females, and most of the genes (90.9%) were downregulated. Upregulated genes encoded products that were mainly involved in fatty acid synthesis and pyrimidine metabolism, while the downregulated genes were mainly involved in substance transport and metabolism. In addition, post-mating A. disparis females exhibited a tendency to accelerate egg maturation and became unreceptive to further mating. Based on the transcriptional data, we discuss how specific genes mediate these behavioural and physiological changes. Overall, our study provided new and comprehensive insights into post-mating changes in females and provided a basis for future mechanistic studies.

Male-male lethal combat in the quasi-gregarious parasitoid Anastatus disparis (Hymenoptera: Eupelmidae).

Most animals employ aggressive behaviours to acquire resources such as food, territory and mates. Although mating is important for males, which typically exhibit competitive behaviours to gain mating opportunities, they generally tend to avoid conflict escalation; while extreme combat also occurs in some species and results in death. In this study, male-male lethal combat behaviour in Anastatus disparis was examined (Hymenoptera: Eupelmidae) by investigating the characteristics of fighting and the factors that influence fighting intensity in this species. Male fight intensity in A. disparis increased with both competitor density and female presence, while it was not influenced by the relatedness among male competitors. By comparing the frequency of received attacks between injured and non-injured males, we found that the former were more vulnerable to attack. In contrast to death due to lethal attack, death that occurs as a result of A. disparis combat may be the cumulative effect of injuries sustained over repeated competitive encounters. Combined with the biological characters of A. disparis, we discuss potential factors contributing to the evolution of fatal conflict in this species.