Influence of Endogenous Bacteria on Behavioral Responses in Leptocybe invasa: An Analysis of mVOCs.

Microorganisms within insects play a vital role in maintaining the basal physiological functions of the insects, with olfactory signals as critical components of insect survival strategies. Leptocybe invasa (L. invasa), an invasive alien pest inflicting significant damage to eucalyptus trees, harbors a rich and varied bacterial community within its body. However, the impact of its endogenous bacteria and their microbial Volatile Organic Compounds (mVOCs) on the behavioral preferences of L. invasa remains unexplored to date. This study focused on nine cultivable and dominant endogenous bacterial strains within L. invasa. Using a Y-tube olfactometer, we investigated the behavioral responses of female L. invasa to the mVOCs emitted by these bacteria. Concurrently, gas chromatography-mass spectrometry (GC-MS) was employed to quantify the mVOCs produced by these endogenous bacteria. Our findings revealed that Staphylococcus sp. exhibited the highest attractiveness of L. invasa, whereas Microbacterium sp. and E. cloacae exerted the most significant avoidance effects. The analysis of the mVOCs further highlighted the significance of aldehyde compounds, notably 2,3,6-trichlorobenzaldehyde, and alkane compounds, such as eicosane, in mediating the repellency and attraction effects. These results contribute to a deeper understanding of the invasion mechanism of L. invasa and provide a scientific basis for developing novel biopesticides or elicitors.

Differences in Male-Killing Rickettsia Bacteria between Lineages of the Invasive Gall-Causing Pest Leptocybe invasa.

(1) Background: Leptocybe invasa (Hymenoptera: Eulophidae) is a global invasive pest that seriously damages eucalyptus plants and has caused serious harm to forestry production in many countries. Two genotypically distinct lineages of L. invasa have been detected outside of Australia, namely, lineage A and lineage B. However, the composition and abundance of endosymbiotic bacteria in L. invasa are still unclear between lineages. Therefore, the purpose of this study was to compare the bacterial communities in female adults of L. invasa of different lineages distributed in the same domain; (2) Methods: The PacBio Sequel II platform was used to compare bacterial community composition between lineages of L. invasa by sequencing the V1-V9 region of the 16S rRNA gene, and fluorescence quantitative PCR was used to compare the relative expression of Rickettsia between lineages of L. invasa; (3) Results: A total of 437 operational taxonomic units (OTUs) were obtained. These OTUs were subdivided into 20 phyla, 32 classes, 77 orders, 129 families, and 217 genera. At the genus level, the dominant bacteria in lineage A and lineage B were Rickettsia and Bacteroides, respectively. There were differences in the bacterial community of L. invasa between lineages, and the abundance and relative expression of Rickettsia in lineage A were significantly higher than those in lineage B; (4) Conclusions: There were differences in the bacterial community of L. invasa between lineages, and the abundance and relative expression of Rickettsia in lineage A were significantly higher than those in lineage B.

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae).

Leptocybe invasa (Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression of L. invasa's target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes' stability (RPS30, ACTR, 18S rRNA, ACT, RPL18, GAPDH, 28S rRNA, and TUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water, Eucalyptus sap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied that ACT and ACTR were the most accurate when comparing sexes. For analyzing different somites, 28S rRNA and RPL18 were ideal; the 28S rRNA and RRS30 were perfect for analyzing at different temperatures. The combination of ACT and GAPDH helped to analyze gene expression in different diets, and GAPDH and 28S rRNA were suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes from L. invasa for precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.

Genome-wide association study identifies SNP markers and putative candidate genes for terpene traits important for Leptocybe invasa resistance in Eucalyptus grandis.

Terpenes are an important group of plant specialized metabolites influencing, amongst other functions, defence mechanisms against pests. We used a genome-wide association study to identify single nucleotide polymorphism (SNP) markers and putative candidate genes for terpene traits. We tested 15,387 informative SNP markers derived from genotyping 416 Eucalyptus grandis individuals for association with 3 terpene traits, 1,8-cineole, γ-terpinene, and p-cymene. A multilocus mixed model analysis identified 21 SNP markers for 1,8-cineole on chromosomes 2, 4, 6, 7, 8, 9, 10, and 11, that individually explained 3.0%-8.4% and jointly 42.7% of the phenotypic variation. Association analysis of γ-terpinene found 32 significant SNP markers on chromosomes 1, 2, 4, 5, 6, 9, and 11, explaining 3.4-15.5% and jointly 54.5% of phenotypic variation. For p-cymene, 28 significant SNP markers were identified on chromosomes 1, 2, 3, 5, 6, 7, 10, and 11, explaining 3.4-16.1% of the phenotypic variation and jointly 46.9%. Our results show that variation underlying the 3 terpene traits is influenced by a few minor loci in combination with a few major effect loci, suggesting an oligogenic nature of the traits.

Isolation, Identification, and Analysis of Potential Functions of Culturable Bacteria Associated with an Invasive Gall Wasp, Leptocybe invasa.

Symbioses between invasive insects and bacteria are one of the key drivers of insect invasion success. Gall-inducing insects stimulate host plants to produce galls, which affects the normal growth of plants. Leptocybe invasa Fisher et La Salle, an invasive gall-inducing wasp, mainly damages Eucalyptus plantations in Southern China, but little is known about its associated bacteria. The aim of this study was to assess the diversity of bacterial communities at different developmental stages of L. invasa and to identify possible ecological functions of the associated bacteria. Bacteria associated with L. invasa were isolated using culture-dependent methods and their taxonomic statuses were determined by sequencing the 16S rRNA gene. A total of 88 species belonging to four phyla, 27 families, and 44 genera were identified by phylogenetic analysis. The four phyla were Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, mainly from the genera Pantoea, Enterobacter, Pseudomonas, Bacillus, Acinetobacter, Curtobacterium, Sphingobium, Klebsiella, and Rhizobium. Among them, 72 species were isolated in the insect gall stage and 46 species were isolated from the adult stage. The most abundant bacterial species were γ-Proteobacteria. We found significant differences in total bacterial counts and community compositions at different developmental stages, and identified possible ecological roles of L. invasa-associated bacteria. This study is the first to systematically investigate the associated bacteria of L. invasa using culture-dependent methods, and provides a reference for other gall-inducing insects and associated bacteria.

Bacterial diversity of Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) from different geographical conditions in China.

Insects harbor numerous endosymbionts, including bacteria, fungi, yeast, and viruses, which could affect the ecology and behavior of their hosts. However, data regarding the effect of environmental factors on endosymbiotic bacteria of Leptocybe invasa (Hymenoptera: Eulophidae) are quite rare. In this study, we assessed the diversity of endosymbiotic bacteria of L. invasa from 10 different geographic populations collected across China through the Illumina MiSeq platform. A total of 547 OTUs were generated, which were annotated into 19 phyla, 33 classes, 75 orders, 137 families, and 274 genera. The dominant bacteria detected in L. invasa were Rickettsia, and Pantoea, Enterobacter, Pseudomonas, Acinetobacter, and Bacillus were also annotated among each population. Nevertheless, the endosymbiotic bacterial abundance and diversity varied among different populations, which was related to the local climate (annual mean high temperature). The bacterial function prediction analysis showed that these endosymbiotic bacteria were concentrated in metabolism, such as carbohydrate, amino acid, and energy metabolism. Overall, the results provide a comprehensive description of the endosymbiotic bacteria in 10 different populations of an important eucalyptus pest L. invasa, and help to understand the endosymbiotic bacterial diversity and adaptation of various conditions.

Genomic Breeding for Diameter Growth and Tolerance to Leptocybe Gall Wasp and Botryosphaeria/Teratosphaeria Fungal Disease Complex in Eucalyptus grandis.

Eucalyptus grandis is one of the most important species for hardwood plantation forestry around the world. At present, its commercial deployment is in decline because of pests and pathogens such as Leptocybe invasa gall wasp (Lepto), and often co-occurring fungal stem diseases such as Botryosphaeria dothidea and Teratosphaeria zuluensis (BotryoTera). This study analyzed Lepto, BotryoTera, and stem diameter growth in an E. grandis multi-environmental, genetic trial. The study was established in three subtropical environments. Diameter growth and BotryoTera incidence scores were assessed on 3,334 trees, and Lepto incidence was assessed on 4,463 trees from 95 half-sib families. Using the Eucalyptus EUChip60K SNP chip, a subset of 964 trees from 93 half-sib families were genotyped with 14,347 informative SNP markers. We employed single-step genomic BLUP (ssGBLUP) to estimate genetic parameters in the genetic trial. Diameter and Lepto tolerance showed a positive genetic correlation (0.78), while BotryoTera tolerance had a negative genetic correlation with diameter growth (-0.38). The expected genetic gains for diameter growth and Lepto and BotryoTera tolerance were 12.4, 10, and -3.4%, respectively. We propose a genomic selection breeding strategy for E. grandis that addresses some of the present population structure problems.

Insect egg-induced physiological changes and transcriptional reprogramming leading to gall formation.

Gall-inducing insects and their hosts present some of the most intricate plant-herbivore interactions. Oviposition on the host is often the first cue of future herbivory and events at this early time point can affect later life stages. Many gallers are devastating plant pests, yet little information regarding the plant-insect molecular interplay exists, particularly following egg deposition. We studied the physiological and transcriptional responses of Eucalyptus following oviposition by the gall-inducing wasp, Leptocybe invasa, to explore potential mechanisms governing defence responses and gall development. RNA sequencing and microscopy were used to explore a susceptible Eucalyptus-L. invasa interaction. Infested and control material was compared over time (1-3, 7 and 90 days post oviposition) to examine the transcriptional and morphological changes. Oviposition induces accumulation of reactive oxygen species and phenolics which is reflected in the transcriptome analysis. Gene expression supports phytohormones and 10 transcription factor subfamilies as key regulators. The egg and oviposition fluid stimulate cell division resulting in gall development. Eucalyptus responses to oviposition are apparent within 24 hr. Putative defences include the oxidative burst and barrier reinforcement. However, egg and oviposition fluid stimuli may redirect these responses towards gall development.

Comparison of bacterial diversity and abundance between sexes of Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) from China.

BACKGROUND: Insects harbor a myriad of microorganisms, many of which can affect the sex ratio and manipulate the reproduction of the host. Leptocybe invasa is an invasive pest that causes serious damage to eucalyptus plantations, and the thelytokous parthenogenesis, low temperature resistance, protection in galls, generation overlap and small body of L. invasa contribute to its rapid invasion and population growth. However, the endosymbiotic bacterial composition, abundance and sex differences of L. invasa remain unclear. Therefore, this research aimed to identify the bacterial communities in L. invasa adults and compare them between the sexes of L. invasa lineage B. RESULTS: The Illumina MiSeq platform was used to compare bacterial community composition between females and males of L. invasa by sequencing the V3-V4 region of the 16S ribosomal RNA gene. A total of 1,320 operational taxonomic units (OTUs) were obtained. These OTUs were subdivided into 24 phyla, 71 classes, 130 orders, 245 families and 501 genera. At the genus level, the dominant bacteria in females and males were Rickettsia and Rhizobium, respectively. CONCLUSION: The endosymbiotic bacteria of L. invasa females and males were highly diverse. There were differences in the bacterial community of L. invasa between sexes, and the bacterial diversity in male specimens was greater than that in female specimens. This study presents a comprehensive comparison of bacterial communities in L. invasa and these data will provide an overall view of the bacterial community in both sexes of L. invasa with special attention on sex-related bacteria.

Ultrastructure of Female Antennal Sensilla of an Endoparasitoid Wasp, Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae: Tetrastichinae).

The antennal sensilla of female Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae: Tetrastichinae) were observed with scanning and transmission electron microscopy in this study. The antenna of Q. mendeli was geniculate, and the flagellum was composed of seven subsegments. Six distinct types of sensory receptors were observed, including sensilla basiconic capitate peg, sensilla böhm, sensilla chaetica, sensilla campaniformia, sensilla placodea and sensilla trichodea. Sensilla basiconic capitate pegs were found on the flagellomeres, and Böhm sensilla were found on the basal part of scape and the pedicel. Two morphological subtypes of sensilla chaetica were found on the antennae, and sensilla campaniformia were only found on the pedicel. Sensilla placodea were divided into two morphological subtypes that were found on the flagellomeres. Sensilla trichodea were found on the 2nd-6th flagellomere. By comparison to existing antennal sensilla, it was found that sensilla basiconic capitate peg, sensilla chaetica, sensilla placodea and sensilla trichodea were the most common sensilla of the parasitoids of Eulophidae. The external and internal morphology, types, number, distribution, length, and width of these sensilla were described, and their possible functions are discussed in conjunction with the host-detection behavior. Future studies on the host location mechanisms in Q. mendeli will be facilitated by these observations.

Life inside a gall: closeness does not favour horizontal transmission of Rickettsia between a gall wasp and its parasitoid.

The incidence of horizontal transmission as a route for spreading symbiont infections is still being debated, but a common view is that horizontal transfers require intimate between-species relationships. Here we study a system that meets ideal requirements for horizontal transmission: the gall wasp Leptocybe invasa and its parasitoid Quadrastichus mendeli (Hymenoptera: Eulophidae). These wasps belong to the same subfamily, spend most of their lives inside the same minute gall and are both infected by Rickettsia, a maternally inherited endosymbiotic bacteria that infects several arthropods, sometimes manipulating their reproduction, like inducing thelytokous parthenogenesis in L. invasa. Despite intimate contact, close phylogenetic relationship and the parasitoid's host specificity, we show that host and parasitoid do not share the same Rickettsia. We provide indirect evidence that Rickettsia infecting Q. mendeli may be inducing thelytokous parthenogenesis, as the symbiont is densely present in the reproductive apparatus and is vertically transmitted. Phylogenetic analyses based on 16S and gltA placed this symbiont in the leech group. The confirmed and presumed parthenogenesis-inducing Rickettsia discovered so far only infect eulophid wasps, and belong to three different groups, suggesting multiple independent evolution of the parthenogenesis inducing phenotype. We also show some degree of cospeciation between Rickettsia and their eulophid hosts.

Effect of the Gall Wasp Leptocybe invasa on Hydraulic Architecture in Eucalyptus camaldulensis Plants.

The gall wasp, Leptocybe invasa (Hymenoptera; Eulophidae), is a devastating pest of eucalypt plantations in the Middle East, the Mediterranean basin, Africa, India, South-East Asia, and China. Heavy galling causes the leaves to warp and in extreme cases it may stunt the growth of the trees of Eucalyptus camaldulensis. However, the physiological mechanisms underlying how L. invasa inhibits the growth of plants of E. camaldulensis are unclear. Because the growth rate of plants is mainly dependent on photosynthesis that is largely correlated with hydraulic architecture, we speculate that galling of L. invasa depresses hydraulic conductance of stem and leaf. In the present study, we examined the effects of L. invasa galling on hydraulic architecture and photosynthetic parameters in E. camaldulensis plants. We found that galling of L. invasa significantly decreased stem hydraulic conductance (K stem), midday leaf water potential (Ψmd), minor vein density, and stomatal density (SD). Furthermore, the stomatal conductance (g s), chlorophyll content, CO2 assimilation rate (A n) and photosynthetic electron flow were reduced in infected plants. Therefore, the galling of L. invasa not only declined the water supply from stem to leaves, but also restricted water transport within leaf. As a result, galled plants of E. camaldulensis reduced leaf number, leaf area, SD and g s to balance water supply and transpirational demand. Furthermore, galled plants had lower leaf nitrogen content, leading to decreases in chlorophyll content, CO2 assimilation rate and photosynthetic electron flow. These results indicate that the change in hydraulic architecture is responsible for the inhibition of growth rate in galled plants.

The Transcriptome and Terpene Profile of Eucalyptus grandis Reveals Mechanisms of Defense Against the Insect Pest, Leptocybe invasa.

Plants have evolved complex defenses that allow them to protect themselves against pests and pathogens. However, there is relatively little information regarding the Eucalyptus defensome. Leptocybe invasa is one of the most damaging pests in global Eucalyptus forestry, and essentially nothing is known regarding the molecular mechanisms governing the interaction between the pest and host. The aim of the study was to investigate changes in the transcriptional landscape and terpene profile of a resistant and susceptible Eucalyptus genotype in an effort to improve our understanding of this interaction. We used RNA-seqencing to investigate transcriptional changes following L. invasa oviposition. Expression levels were validated using real-time quantitative PCR. Terpene profiles were investigated using gas chromatography coupled to mass spectometry on uninfested and oviposited leaves. We found 698 and 1,115 significantly differentially expressed genes from the resistant and susceptible interactions, respectively. Gene Ontology enrichment and Mapman analyses identified putative defense mechanisms including cell wall reinforcement, protease inhibitors, cell cycle suppression and regulatory hormone signaling pathways. There were significant differences in the mono- and sesquiterpene profiles between genotypes and between control and infested material. A model of the interaction between Eucalyptus and L. invasa was proposed from the transcriptomic and chemical data.