Chromosome-level genome assembly of the bethylid ectoparasitoid wasp Sclerodermus sp. 'alternatusi'.

The Bethylidae are the most diverse of Hymenoptera chrysidoid families. As external parasitoids, the bethylids have been widely adopted as biocontrol agents to control insect pests worldwide. Thus far, the genomic information of the family Bethylidae has not been reported yet. In this study, we crystallized into a high-quality chromosome-level genome of ant-like bethylid wasps Sclerodermus sp. 'alternatusi' (Hymenoptera: Bethylidae) using PacBio sequencing as well as Hi-C technology. The assembled S. alternatusi genome was 162.30 Mb in size with a contig N50 size of 3.83 Mb and scaffold N50 size of 11.10 Mb. Totally, 92.85% assembled sequences anchored to 15 pseudo-chromosomes. A total of 10,204 protein-coding genes were annotated, and 23.01 Mb repetitive sequences occupying 14.17% of genome were pinpointed. The BUSCO results showed that 97.9% of the complete core Insecta genes were identified in the genome, while 97.1% in the gene sets. The high-quality genome of S. alternatusi will not only provide valuable genomic information, but also show insights into parasitoid wasp evolution and bio-control application in future studies.

Population genomics of the invasive Northern Giant Hornet Vespa mandarinia in North America and across its native range.

The northern giant hornet Vespa mandarinia (NGH) is a voracious predator of other insect species, including honey bees. NGH's native range spans subtropical and temperate regions across much of east and southeast Asia and, in 2019, exotic populations of the species were discovered in North America. Despite this broad range and invasive potential, investigation of the population genomic structure of NGH across its native and introduced ranges has thus far been limited to a small number of mitochondrial samples. Here, we present analyses of genomic data from NGH individuals collected across the species' native range and from exotic individuals collected in North America. We provide the first survey of whole-genome population variation for any hornet species, covering this species' native and invasive ranges, and in doing so confirm likely origins in Japan and South Korea for the two introductions. We additionally show that, while this introduced population exhibited strongly elevated levels of inbreeding, these signatures of inbreeding are also present in some long-standing native populations, which may indicate that inbreeding depression alone is insufficient to prevent the persistence of NGH populations. As well as highlighting the importance of ongoing monitoring and eradication efforts to limit the spread of this species outside of its natural range, our data will serve as a foundational database for future genomic studies into introduced hornet populations.

Mitogenome architecture supports the non-monophyly of the cosmopolitan parasitoid wasp subfamily Doryctinae (Hymenoptera: Braconidae) recovered by nuclear and mitochondrial phylogenomics.

Mitochondrial DNA gene organisation is an important source of phylogenetic information for various metazoan taxa at different evolutionary timescales, though this has not been broadly tested for all insect groups nor within a phylogenetic context. The cosmopolitan subfamily Doryctinae is a highly diverse group of braconid wasps mainly represented by ectoparasitoids of xylophagous beetle larvae. Previous molecular studies based on Sanger and genome-wide (ultraconserved elements, UCE; and mitochondrial genomes) sequence data have recovered a non-monophyletic Doryctinae, though the relationships involved have always been weakly supported. We characterised doryctine mitogenomes and conducted separate phylogenetic analyses based on mitogenome and UCE sequence data of ~100 representative doryctine genera to assess the monophyly and higher-level classification of the subfamily. We identified rearrangements of mitochondrial transfer RNAs (tRNAs) that support a non-monophyletic Doryctinae consisting of two separate non-related clades with strong geographic structure ('New World' and 'Old World' clades). This geographic structure was also consistently supported by the phylogenetic analyses preformed with mitogenome and UCE sequence data. These results highlight the utility of the mitogenome gene rearrangements as a potential source of phylogenetic information at different evolutionary timescales.

Identification of a viral gene essential for the genome replication of a domesticated endogenous virus in ichneumonid parasitoid wasps.

Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor "domesticated endogenous viruses" (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly for Hyposoter didymator that harbors H. didymator ichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene, U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown of U16 by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicated U16 knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis.

Exploring the ageing methylome in the model insect, Nasonia vitripennis.

BACKGROUND: The ageing process is a multifaceted phenomenon marked by the gradual deterioration of cellular and organismal functions, accompanied by an elevated susceptibility to diseases. The intricate interplay between genetic and environmental factors complicates research, particularly in complex mammalian models. In this context, simple invertebrate organisms have been pivotal, but the current models lack detectable DNA methylation limiting the exploration of this critical epigenetic ageing mechanism. This study introduces Nasonia vitripennis, the jewel wasp, as an innovative invertebrate model for investigating the epigenetics of ageing. Leveraging its advantages as a model organism and possessing a functional DNA methylation system, Nasonia emerges as a valuable addition to ageing research. RESULTS: Whole-genome bisulfite sequencing unveiled dynamic alterations in DNA methylation, with differentially methylated CpGs between distinct time points in both male and female wasps. These changes were associated with numerous genes, enriching for functions related to telomere maintenance, histone methylation, and mRNA catabolic processes. Additionally, other CpGs were found to be variably methylated at each timepoint. Sex-specific effects on epigenetic entropy were observed, indicating differential patterns in the loss of epigenetic stability over time. Constructing an epigenetic clock containing 19 CpGs revealed a robust correlation between epigenetic age and chronological age. CONCLUSIONS: Nasonia vitripennis emerges as a promising model for investigating the epigenetics of ageing, shedding light on the intricate dynamics of DNA methylation and their implications for age-related processes. This research not only expands the repertoire of ageing models but also opens avenues for deeper exploration of epigenetic mechanisms in the context of ageing.

Immune system modulation & virus transmission during parasitism identified by multi-species transcriptomics of a declining insect biocontrol system.

BACKGROUND: The Argentine stem weevil (ASW, Listronotus bonariensis) is a significant pasture pest in Aotearoa New Zealand, primarily controlled by the parasitoid biocontrol agent Microctonus hyperodae. Despite providing effective control of ASW soon after release, M. hyperodae parasitism rates have since declined significantly, with ASW hypothesised to have evolved resistance to its biocontrol agent. While the parasitism arsenal of M. hyperodae has previously been investigated, revealing many venom components and an exogenous novel DNA virus Microctonus hyperodae filamentous virus (MhFV), the effects of said arsenal on gene expression in ASW during parasitism have not been examined. In this study, we performed a multi-species transcriptomic analysis to investigate the biology of ASW parasitism by M. hyperodae, as well as the decline in efficacy of this biocontrol system. RESULTS: The transcriptomic response of ASW to parasitism by M. hyperodae involves modulation of the weevil's innate immune system, flight muscle components, and lipid and glucose metabolism. The multispecies approach also revealed continued expression of venom components in parasitised ASW, as well as the transmission of MhFV to weevils during parasitism and some interrupted parasitism attempts. Transcriptomics did not detect a clear indication of parasitoid avoidance or other mechanisms to explain biocontrol decline. CONCLUSIONS: This study has expanded our understanding of interactions between M. hyperodae and ASW in a biocontrol system of critical importance to Aotearoa-New Zealand's agricultural economy. Transmission of MhFV to ASW during successful and interrupted parasitism attempts may link to a premature mortality phenomenon in ASW, hypothesised to be a result of a toxin-antitoxin system. Further research into MhFV and its potential role in ASW premature mortality is required to explore whether manipulation of this viral infection has the potential to increase biocontrol efficacy in future.

Identification of Parthenogenesis-Inducing Effector Proteins in Wolbachia.

Bacteria in the genus Wolbachia have evolved numerous strategies to manipulate arthropod sex, including the conversion of would-be male offspring to asexually reproducing females. This so-called "parthenogenesis induction" phenotype can be found in a number of Wolbachia strains that infect arthropods with haplodiploid sex determination systems, including parasitoid wasps. Despite the discovery of microbe-mediated parthenogenesis more than 30 yr ago, the underlying genetic mechanisms have remained elusive. We used a suite of genomic, computational, and molecular tools to identify and characterize two proteins that are uniquely found in parthenogenesis-inducing Wolbachia and have strong signatures of host-associated bacterial effector proteins. These putative parthenogenesis-inducing proteins have structural homology to eukaryotic protein domains including nucleoporins, the key insect sex determining factor Transformer, and a eukaryotic-like serine-threonine kinase with leucine-rich repeats. Furthermore, these proteins significantly impact eukaryotic cell biology in the model Saccharomyces cerevisiae. We suggest that these proteins are parthenogenesis-inducing factors and our results indicate that this would be made possible by a novel mechanism of bacterial-host interaction.

Mitochondrial perturbation in immune cells enhances cell-mediated innate immunity in Drosophila.

BACKGROUND: Mitochondria participate in various cellular processes including energy metabolism, apoptosis, autophagy, production of reactive oxygen species, stress responses, inflammation and immunity. However, the role of mitochondrial metabolism in immune cells and tissues shaping the innate immune responses are not yet fully understood. We investigated the effects of tissue-specific mitochondrial perturbation on the immune responses at the organismal level. Genes for oxidative phosphorylation (OXPHOS) complexes cI-cV were knocked down in the fruit fly Drosophila melanogaster, targeting the two main immune tissues, the fat body and the immune cells (hemocytes). RESULTS: While OXPHOS perturbation in the fat body was detrimental, hemocyte-specific perturbation led to an enhanced immunocompetence. This was accompanied by the formation of melanized hemocyte aggregates (melanotic nodules), a sign of activation of cell-mediated innate immunity. Furthermore, the hemocyte-specific OXPHOS perturbation induced immune activation of hemocytes, resulting in an infection-like hemocyte profile and an enhanced immune response against parasitoid wasp infection. In addition, OXPHOS perturbation in hemocytes resulted in mitochondrial membrane depolarization and upregulation of genes associated with the mitochondrial unfolded protein response. CONCLUSIONS: Overall, we show that while the effects of mitochondrial perturbation on immune responses are highly tissue-specific, mild mitochondrial dysfunction can be beneficial in immune-challenged individuals and contributes to variation in infection outcomes among individuals.

Taxonomic revision of the Stenodynerus fastidiosissimus species-group in Western Europe and North Africa (Hymenoptera: Vespidae: Eumeninae).

The fastidiosissimus species-group of Stenodynerus de Saussure, 1863 is revised in Western Europe and North Africa, combining morphological data and DNA barcoding. Six species are recognized: S. difficilis (Morawitz, 1867) stat. resurr. (= S. fastidiosissimus auct.), S. fastidiosissimus (de Saussure, 1855), S. laborans (Costa, 1882) stat. resurr., S. montanus Selis, sp. nov., S. muelleri (Dusmet, 1917) (= Stenodynerus gusenleitneri Giordani Soika, 1986 syn. nov.), and S. rufescens Giordani Soika, 1977 stat. nov. Lectotypes are designated for Odynerus fastidiosissimus de Saussure, 1855 and Odynerus insularis Andr, 1883 non Smith, 1859. A key for the identification of members of this species-group is provided. DNA barcodes are published for every species, representing the first available sequences for the fastidiosissimus species-group.

Functional Characterization of the Niemann-Pick C2 Protein BdioNPC2b in the Parasitic Wasp Baryscapus dioryctriae (Chalcidodea: Eulophidae).

Reverse chemical ecology has been widely applied for the functional characterization of olfactory proteins in various arthropods, but few related studies have focused on parasitic wasps. Here, the odorant carrier Niemann-Pick C2 protein of Baryscapus dioryctriae (BdioNPC2b) was studied in vitro and in vivo. Ligand binding analysis revealed that BdioNPC2b most strongly bound to 2-butyl-2-octenal and which compound could elicit an EAG response and attracted B. dioryctriae adults. Moreover, this odorant attractant significantly improved the reproductive efficiency of B. dioryctriae compared to that of the control. Then, the relationship between BdioNPC2b and 2-butyl-2-octenal was validated by RNAi, and site-directed mutagenesis revealed the involvement of three key residues of BdioNPC2b in binding to 2-butyl-2-octenal through hydrogen bonding. Our findings provide not only a deeper understanding of the olfactory function of NPC2 in wasps but also useful information for improving the performance of the parasitoid B. dioryctriae as a biological control agent.

Identification of candidate genes associated with host-seeking behavior in the parasitoid wasp Diachasmimorpha longicaudata.

BACKGROUND: Diachasmimorpha longicaudata is a hymenopteran fruit fly endoparasitoid. Females of this species find their hosts for oviposition by using complex sensorial mechanisms in response to physical and chemical stimuli associated with the host and host habitat. Ecological and behavioral aspects related to host-seeking behavior for oviposition have been extensively studied in D. longicaudata, including the identification of volatile organic compounds acting as attractants to females. In this sense, molecular mechanisms of chemoreception have been explored in this species, including a preliminary characterization of odorant-binding proteins (OBPs), chemosensory proteins (CSPs) and odorant receptors (ORs), among other proteins. Functional assays on OBP and CSP have been conducted as a first approach to identify molecular mechanisms associated with the female host-seeking behavior for oviposition. The aims of the present study were to identify the D. longicaudata sensory gene repertoire expressed in the antenna of sexually mature and mated individuals of both sexes, and subsequently, characterize transcripts differentially expressed in the antennae of females to identify candidate genes associated with the female host-seeking behavior for oviposition. RESULTS: A total of 33,745 predicted protein-coding sequences were obtained from a de novo antennal transcriptome assembly. Ten sensory-related gene families were annotated as follows: 222 ORs, 44 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 9 CSPs, 13 OBPs, 2 ammonium transporters (AMTs), 8 pickpocket (PPKs) receptors, 16 transient receptor potential (TRP) channels, 12 CD36/SNMPs and 3 Niemann-Pick type C2 like proteins (NPC2-like). The differential expression analysis revealed 237 and 151 transcripts up- and downregulated, respectively, between the female and male antennae. Ninety-seven differentially expressed transcripts corresponded to sensory-related genes including 88 transcripts being upregulated (87 ORs and one TRP) and nine downregulated (six ORs, two CSPs and one OBP) in females compared to males. CONCLUSIONS: The sensory gene repertoire of D. longicaudata was similar to that of other taxonomically related parasitoid wasps. We identified a high number of ORs upregulated in the female antenna. These results may indicate that this gene family has a central role in the chemoreception of sexually mature females during the search for hosts and host habitats for reproductive purposes.

PSRs: Selfish chromosomes that manipulate reproductive development.

B chromosomes are intriguing "selfish" genetic elements, many of which exhibit higher-than-Mendelian transmission. This perspective highlights a group of B chromosomes known as Paternal Sex Ratio chromosomes (PSRs), which are found in several insects with haplo-diploid reproduction. PSRs harshly alter the organism's reproduction to facilitate their own inheritance. A manifestation of this effect is the conversion of female destined individuals into males. Key to this conversion is the mysterious ability of PSRs to cause elimination of the sperm-inherited half of the genome during zygote formation. Here we discuss how PSRs were discovered, what is known about how they alter paternal chromatin dynamics to cause sex conversion, and how PSR-induced genome elimination is different from other forms of programmed genome elimination in different insects. PSRs also stand out because their DNA sequence compositions differ in remarkable ways from their insect's essential chromosomes, a characteristic suggestive of interspecies origins. Broadly, we also highlight poorly understood aspects of PSR dynamics that need to be investigated.

Natural selection and genetic diversity maintenance in a parasitic wasp during continuous biological control application.

Aphidius gifuensis is a parasitoid wasp and primary endoparasitoid enemy of the peach potato aphid, Myzus persicae. Artificially reared, captive wasps of this species have been extensively and effectively used to control populations of aphids and limit crop loss. However, the consequences of large-scale releasing of captive A. gifuensis, such as genetic erosion and reduced fitness in wild populations of this species, remains unclear. Here, we sequence the genomes of 542 A. gifuensis individuals collected across China, including 265 wild and 277 human-intervened samples. Population genetic analyses on wild individuals recovered Yunnan populations as the ancestral group with the most complex genetic structure. We also find genetic signature of environmental adaptation during the dispersal of wild populations from Yunnan to other regions. While comparative genomic analyses of captive wasps revealed a decrease in genetic diversity during long-term rearing, population genomic analyses revealed signatures of natural selection by several biotic (host plants) or abiotic (climate) factors, which support maintenance of the gene pool of wild populations in spite of the introduction of captive wasps. Therefore, the impact of large-scale release is reduced. Our study suggests that A. gifuensis is a good system for exploring the genetic and evolutionary effects of mass rearing and release on species commonly used as biocontrol agents.

High specificity of symbiont-conferred resistance in an aphid-parasitoid field community.

Host-parasite coevolution is mediated by genetic interactions between the antagonists and may lead to reciprocal adaptation. In the black bean aphid, Aphis fabae fabae, resistance to parasitoids can be conferred by the heritable bacterial endosymbiont Hamiltonella defensa. H. defensa has been shown to be variably protective against different parasitoid species, and different genotypes of the black bean aphid's main parasitoid Lysiphlebus fabarum. However, these results were obtained using haphazard combinations of laboratory-reared insect lines with different origins, making it unclear how representative they are of natural, locally (co)adapted communities. We therefore comprehensively sampled the parasitoids of a natural A. f. fabae population and measured the ability of the five most abundant species to parasitize aphids carrying the locally prevalent H. defensa haplotypes. H. defensa provided resistance only against the dominant parasitoid L. fabarum (70% of all parasitoids), but not against less abundant parasitoids, and resistance to L. fabarum acted in a genotype-specific manner (G × G interactions between H. defensa and L. fabarum). These results confirm that strong species- and genotype-specificity of symbiont-conferred resistance is indeed a hallmark of wild A. f. fabae populations, and they are consistent with symbiont-mediated adaptation of aphids to the parasitoids posing the highest risk.

Uncovering floral composition of paper wasp nests (Hymenoptera: Vespidae: Polistes) through DNA metabarcoding.

As the social organism, Polistes wasps build a communal nest using woody fibers with saliva for sustaining brood and adult population throughout the season. Limited information exists regarding the identification specific plant materials employed in wasp nest building. Thus, we firstly tested if the DNA metabarcoding approach utilizing rbcL and trnL molecular markers could identify the plant species quantitatively and qualitatively inform the mixed-origin woody samples. A threshold of 0.01 proportion of reads was applied for rbcL and trnL molecular markers, while this threshold for median proportion was 0.0025. In assessing taxa richness, the median proportion demonstrated superior performance, exhibiting higher taxa detection power, however, rbcL marker outperformed in quantitative analysis. Subsequently, we applied DNA metabarcoding to identify the plant materials from the nests of two Polistes species, P. mandarinus and P. rothneyi. The results showed that higher preference of Quercus and Robinia as the major nest building materials regardless of the surrounding plant communities, by two wasp species. Material diversity was higher for P. rothneyi than P. mandarinus, which may explain the abundance of this species possibly with heightened adaptive capacities in their nesting behavior. This study demonstrated that DNA metabarcoding could identify the complex nest-building plant materials of paper wasps and provide insights into their ecological interactions in the natural ecosystem.

Conventional cytogenetics and microsatellite chromosomal distribution in social wasp Mischocyttarus cassununga (Ihering, 1903) (Vespidae, Polistinae, Mischocyttarini).

Cytogenetics has allowed the investigation of chromosomal diversity and repetitive genomic content in wasps. In this study, we characterized the karyotype of the social wasp Mischocyttarus cassununga using conventional cytogenetics and chromosomal mapping of repetitive sequences. This study was undertaken to extend our understanding of the genomic organization of repetitive DNA in social wasps and is the first molecular cytogenetic insight into the genus Mischocyttarus. The karyotype of M. cassununga had a chromosome number of 2n = 64 for females and n = 32 for males. Constitutive heterochromatin exhibited three distribution patterns: centromeric and pericentromeric regions along the smaller arms and extending almost the entire chromosome. The major ribosomal DNA sites were located on chromosome pair in females and one chromosome in males. Positive signals for the microsatellite probes (GA)n and (GAG)n were observed in the euchromatic regions of all chromosomes. The microsatellites, (CGG)n, (TAT)n, (TTAGG)n, and (TCAGG)n were not observed in any region of the chromosomes. Our results contrast with those previously obtained for Polybia fastidiosuscula, which showed that the microsatellites (GAG)n, (CGG)n, (TAT)n, (TTAGG)n, and (TCAGG)n are located predominantly in constitutive heterochromatin. This suggests variations in the diversity and chromosomal organization of repetitive sequences in the genomes of social wasps.

The Centrosomes of Haploid and Diploid Cells Have an Equal Number of Centrioles in the Parasitoid Wasp Anisopteromalus Calandrae.

BACKGROUND: The centrosome is the main center of the organization of microtubules (MT) in the cell, the origin for the formation of flagella and cilia, as well as the site of many regulatory intracellular processes. In diploid cells, the centrosome includes two centrioles connected to some additional structures and surrounded by pericentriolar material. METHODS: The ultrastructure of the cells was studied using transmission electron microscopy on serial ultrathin sections. RESULTS: Here, using transmission electron microscopy on a complete series of ultrathin sections of the centrosome region, we studied the relation between the number of centrioles and ploidy in diploid cells of female wasps and haploid cells of male in the parasitoid wasp Anisopteromalus calandrae (Hymenoptera). It showed that the haploid cells of the male insect have the same number of centrioles as the diploid cells of the female. CONCLUSIONS: It can be concluded that there is no strict correlation between the number of chromosome sets (ploidy) and the number of centrioles in haplodiploid insects.

Odorant-Binding and Chemosensory Proteins in Fig Wasps: Evolutionary Insights From Comparative Studies.

Fig wasps (Agaonidae; Hymenoptera) are the only pollinating insects of fig trees (Ficus; Moraceae), forming the most closely and highly specific mutualism with the host. We used transcriptome sequences of 25 fig wasps from six genera to explore the evolution of key molecular components of fig wasp chemosensory genes: odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). We identified a total 321 OBPs and 240 CSPs, with each species recording from 6 to 27 OBP genes and 6-19 CSP genes. 318 OBP genes are clustered into 17 orthologous groups and can be divided into two groups: PBP sensitive to pheromone and GOBP sensitive to general odor molecules, such as alcohols, esters, acids, ketones, and terpenoids. 240 CSP genes are clustered into 12 orthologous groups, which can be divided into three major groups and have functions, such as olfactory, tissue formation and/or regeneration, developmental, and some specific and unknown function. The gene sequences of most orthologous groups vary greatly among species and are consistent with the phylogenetic relationships between fig wasps. Strong purifying selection of both OBP and CSP genes was detected, as shown by low ω values. A positive selection was detected in one locus in CSP1. In conclusion, the evolution of chemosensory proteins OBPs and CSPs in fig wasps is relatively conservative, and they play an indispensable role in the life activities of fig wasps. Our results provide a starting point for understanding the molecular basis of the chemosensory systems of fig wasps.

The evolution of constitutively active humoral immune defenses in Drosophila populations under high parasite pressure.

Both constitutive and inducible immune mechanisms are employed by hosts for defense against infection. Constitutive immunity allows for a faster response, but it comes with an associated cost that is always present. This trade-off between speed and fitness costs leads to the theoretical prediction that constitutive immunity will be favored where parasite exposure is frequent. We selected populations of Drosophila melanogaster under high parasite pressure from the parasitoid wasp Leptopilina boulardi. With RNA sequencing, we found the evolution of resistance in these populations was associated with them developing constitutively active humoral immunity, mediated by the larval fat body. Furthermore, these evolved populations were also able to induce gene expression in response to infection to a greater level, which indicates an overall more activated humoral immune response to parasitization. The anti-parasitoid immune response also relies on the JAK/STAT signaling pathway being activated in muscles following infection, and this induced response was only seen in populations that had evolved under high parasite pressure. We found that the cytokine Upd3, which induces this JAK/STAT response, is being expressed by immature lamellocytes. Furthermore, these immune cells became constitutively present when populations evolved resistance, potentially explaining why they gained the ability to activate JAK/STAT signaling. Thus, under intense parasitism, populations evolved resistance by increasing both constitutive and induced immune defenses, and there is likely an interplay between these two forms of immunity.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa.

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.