Some Like it HOT: Horizontal Operon Transfer.

While horizontal gene transfer (HGT) is well documented in bacteria, the role and frequency of HGT across eukaryotes remains poorly understood. Kominek et al. identified a horizontal operon transfer (HOT) event, with clear evidence for selection to facilitate gene expression, that has allowed a group of yeasts to scavenge iron using bacterially derived genes.

Distinct epigenomic and transcriptomic modifications associated with Wolbachia-mediated asexuality.

Wolbachia are maternally transmitted intracellular bacteria that induce a range of pathogenic and fitness-altering effects on insect and nematode hosts. In parasitoid wasps of the genus Trichogramma, Wolbachia infection induces asexual production of females, thus increasing transmission of Wolbachia. It has been hypothesized that Wolbachia infection accompanies a modification of the host epigenome. However, to date, data on genome-wide epigenomic changes associated with Wolbachia are limited, and are often confounded by background genetic differences. Here, we took sexually reproducing Trichogramma free of Wolbachia and introgressed their genome into a Wolbachia-infected cytoplasm, converting them to Wolbachia-mediated asexuality. Wolbachia was then cured from replicates of these introgressed lines, allowing us to examine the genome-wide effects of wasps newly converted to asexual reproduction while controlling for genetic background. We thus identified gene expression and DNA methylation changes associated with Wolbachia-infection. We found no overlaps between differentially expressed genes and differentially methylated genes, indicating that Wolbachia-infection associated DNA methylation change does not directly modulate levels of gene expression. Furthermore, genes affected by these mechanisms exhibit distinct evolutionary histories. Genes differentially methylated due to the infection tended to be evolutionarily conserved. In contrast, differentially expressed genes were significantly more likely to be unique to the Trichogramma lineage, suggesting host-specific transcriptomic responses to infection. Nevertheless, we identified several novel aspects of Wolbachia-associated DNA methylation changes. Differentially methylated genes included those involved in oocyte development and chromosome segregation. Interestingly, Wolbachia-infection was associated with higher levels of DNA methylation. Additionally, Wolbachia infection reduced overall variability in gene expression, even after accounting for the effect of DNA methylation. We also identified specific cases where alternative exon usage was associated with DNA methylation changes due to Wolbachia infection. These results begin to reveal distinct genes and molecular pathways subject to Wolbachia induced epigenetic modification and/or host responses to Wolbachia-infection.

Infection Dynamics of Cotransmitted Reproductive Symbionts Are Mediated by Sex, Tissue, and Development.

One of the most prevalent intracellular infections on earth is with Wolbachia, a bacterium in the Rickettsiales that infects a range of insects, crustaceans, chelicerates, and nematodes. Wolbachia is maternally transmitted to offspring and has profound effects on the reproduction and physiology of its hosts, which can result in reproductive isolation, altered vectorial capacity, mitochondrial sweeps, and even host speciation. Some populations stably harbor multiple Wolbachia strains, which can further contribute to reproductive isolation and altered host physiology. However, almost nothing is known about the requirements for multiple intracellular microbes to be stably maintained across generations while they likely compete for space and resources. Here, we use a coinfection of two Wolbachia strains ("wHa" and "wNo") in Drosophila simulans to define the infection and transmission dynamics of an evolutionarily stable double infection. We find that a combination of sex, tissue, and host development contributes to the infection dynamics of the two microbes and that these infections exhibit a degree of niche partitioning across host tissues. wHa is present at a significantly higher titer than wNo in most tissues and developmental stages, but wNo is uniquely dominant in ovaries. Unexpectedly, the ratio of wHa to wNo in embryos does not reflect those observed in the ovaries, indicative of strain-specific transmission dynamics. Understanding how Wolbachia strains interact to establish and maintain stable infections has important implications for the development and effective implementation of Wolbachia-based vector biocontrol strategies, as well as more broadly defining how cooperation and conflict shape intracellular communities. IMPORTANCEWolbachia is a maternally transmitted intracellular bacterium that manipulates the reproduction and physiology of arthropods, resulting in drastic effects on the fitness, evolution, and even speciation of its hosts. Some hosts naturally harbor multiple strains of Wolbachia that are stably transmitted across generations, but almost nothing is known about the factors that limit or promote these coinfections, which can have profound effects on the host's biology and evolution and are under consideration as an insect-management tool. Here, we define the infection dynamics of a known stably transmitted double infection in Drosophila simulans with an eye toward understanding the patterns of infection that might facilitate compatibility between the two microbes. We find that a combination of sex, tissue, and development all contributes to infection dynamics of the coinfection.

Comparative genomics of the miniature wasp and pest control agent Trichogramma pretiosum.

BACKGROUND: Trichogrammatids are minute parasitoid wasps that develop within other insect eggs. They are less than half a millimeter long, smaller than some protozoans. The Trichogrammatidae are one of the earliest branching families of Chalcidoidea: a diverse superfamily of approximately half a million species of parasitoid wasps, proposed to have evolved from a miniaturized ancestor. Trichogramma are frequently used in agriculture, released as biological control agents against major moth and butterfly pests. Additionally, Trichogramma are well known for their symbiotic bacteria that induce asexual reproduction in infected females. Knowledge of the genome sequence of Trichogramma is a major step towards further understanding its biology and potential applications in pest control. RESULTS: We report the 195-Mb genome sequence of Trichogramma pretiosum and uncover signatures of miniaturization and adaptation in Trichogramma and related parasitoids. Comparative analyses reveal relatively rapid evolution of proteins involved in ribosome biogenesis and function, transcriptional regulation, and ploidy regulation. Chalcids also show loss or especially rapid evolution of 285 gene clusters conserved in other Hymenoptera, including many that are involved in signal transduction and embryonic development. Comparisons between sexual and asexual lineages of Trichogramma pretiosum reveal that there is no strong evidence for genome degradation (e.g., gene loss) in the asexual lineage, although it does contain a lower repeat content than the sexual lineage. Trichogramma shows particularly rapid genome evolution compared to other hymenopterans. We speculate these changes reflect adaptations to miniaturization, and to life as a specialized egg parasitoid. CONCLUSIONS: The genomes of Trichogramma and related parasitoids are a valuable resource for future studies of these diverse and economically important insects, including explorations of parasitoid biology, symbiosis, asexuality, biological control, and the evolution of miniaturization. Understanding the molecular determinants of parasitism can also inform mass rearing of Trichogramma and other parasitoids for biological control.

Examining Wolbachia-Induced Parthenogenesis in Hymenoptera.

The maternally transmitted reproductive manipulator Wolbachia can impact sex ratios of its arthropod host by different mechanisms, ultimately promoting the spread of infection across a population. One of these reproductive phenotypes, parthenogenesis induction (PI), is characterized by the asexual production of female offspring, which in many cases results in an entirely female population. Cases of Wolbachia-mediated PI are most common in the order Hymenoptera, specifically in parasitoid wasps. The complex sex determination pathways of hymenopterans, their diverse life histories, the multiple cytogenetic mechanisms of PI, and the lack of males make functional studies of parthenogenesis induction challenging. Here, we describe the mechanisms of PI, outline methods to recognize and cure PI-Wolbachia infection, and note possible complications when working with PI-Wolbachia strains and their parthenogenetic hosts.

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.

Insect-microbe interactions and their influence on organisms and ecosystems.

Microorganisms are important associates of insect and arthropod species. Insect-associated microbes, including bacteria, fungi, and viruses, can drastically impact host physiology, ecology, and fitness, while many microbes still have no known role. Over the past decade, we have increased our knowledge of the taxonomic composition and functional roles of insect-associated microbiomes and viromes. There has been a more recent shift toward examining the complexity of microbial communities, including how they vary in response to different factors (e.g., host genome, microbial strain, environment, and time), and the consequences of this variation for the host and the wider ecological community. We provide an overview of insect-microbe interactions, the variety of associated microbial functions, and the evolutionary ecology of these relationships. We explore the influence of the environment and the interactive effects of insects and their microbiomes across trophic levels. Additionally, we discuss the potential for subsequent synergistic and reciprocal impacts on the associated microbiomes, ecological interactions, and communities. Lastly, we discuss some potential avenues for the future of insect-microbe interactions that include the modification of existing microbial symbionts as well as the construction of synthetic microbial communities.

Spatial and temporal dynamics of soybean gall midge (Resseliella maxima) parasitism by Synopeas maximum.

BACKGROUND: Soybean gall midge, Resseliella maxima Gagné (Diptera: Cecidomyiidae), is a pest that impacts soybean yield in the Midwest United States. While biological control using parasitoids may be a promising approach for managing this pest, it is crucial to have a deep understanding of parasitism dynamics to ensure effective implementation. We investigated R. maxima parasitism using a combination of three methods: rearing of field-collected hosts, dissection, and molecular assays. RESULTS: We confirmed parasitism of R. maxima by the recently described wasp Synopeas maximum Awad & Talamas (Hymenoptera: Platygastridae) and used our combinatorial approach to observe the spatial and temporal relationships between R. maxima and its parasitoid in the field. The number of R. maxima adults was greater in symptomatic plants on field edges than the field interior, but such a pattern was inconsistent for parasitism rates. Parasitism rates were generally highest early and late in the season, and lower in the middle of the season when the number of R. maxima was highest. CONCLUSION: In Minnesota, overall season-long parasitism rates of R. maxima were low (< 2%). To facilitate a wider investigation of the biological control of R. maxima, we designed a protocol for high throughput DNA extraction and quantitative polymerase chain reaction (qPCR) that can be used across a broader geography. Further research should evaluate how parasitism rates by Synopeas maximum could be promoted in production fields. © 2023 Society of Chemical Industry.

Arthropod predator community associated with soybean gall midge, Resseliella maxima (Diptera: Cecidomyiidae), in Minnesotan soybean fields.

The soybean gall midge, Resseliella maxima Gagné (Diptera: Cecidomyiidae), is a pest that injures soybeans in the Midwest United States. Little is known about the natural enemies of R. maxima or the potential for biological control. Therefore, we performed a 2-yr survey in Minnesota to examine the predator community associated with R. maxima infestations. We found that Orius insidiosus (Say) (Heteroptera: Anthocoridae) and Pterostichus melanarius (Illiger) (Coleoptera: Carabidae) were the most common foliar- and ground-foraging predators, respectively. Some of the commonly encountered predator species were tested in laboratory predation experiments. Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) and O. insidiosus represented the foliar-foraging predators tested, and H. axyridis consumed significantly more R. maxima larvae than O. insidiosus. Among the ground-foraging predators, 4 carabids were tested. Poecilus lucublandus (Say) (Coleoptera: Carabidae) and Pt. melanarius consumed significantly more R. maxima larvae than Poecilus chalcites (Say) (Coleoptera: Carabidae) and Bembidion quadrimaculatum oppositum (Say) (Coleoptera: Carabidae). We conclude that Pt. melanarius should receive further attention as a potential biological control agent of R. maxima, due to its high abundance in the soybean fields in this study, temporal overlap with the pest, and high propensity to feed on the pest.

The Hazel Stem Borer, Agrilus pseudocoryli (Coleoptera: Buprestidae), as a Pest of Hybrid Hazelnuts.

Nuts from hybrid hazels (Corylus americana Walter × Corylus avellana L.) are a burgeoning, novel industry for the Great Lakes region of the United States. As such, scant information exists about the pest complex, although stem-boring beetles in the genus Agrilus Curtis (Coleoptera: Buprestidae) have been noted by growers as potential pests. Here, we report results of a comprehensive survey of 186 plants in a hybrid hazel planting consisting of nine cultivars in southeastern Minnesota affected by stem borers. Sixteen percent of the hybrid hazel plants exhibited between 25 and 100% buprestid associated dieback. Morphological and molecular identification of the adult and larval buprestids colonizing hybrid hazel plants revealed that the hazel stem borer, Agrilus pseudocoryli Fisher, was the only species present. Adult A. pseudocoryli were observed on plants between 8 June and 13 July 2021. Plants with higher damage ratings from the hazel stem borer were more likely to exhibit signs of eastern filbert blight (Anisogramma anomala Peck), a fungal pathogen virulent to C. avellana. Some hazel cultivars were more susceptible to damage from stem borers than others. Taken together, our findings suggest that the hazel stem borer has the potential to impact hybrid hazelnut yield. We speculate that a regional drought may have promoted increased populations of the hazel stem borer, as native Agrilus species are often secondary pests that become problematic when environmental stresses decrease host resistance. More research is needed to elucidate interactions between hazels, buprestid pests, and environmental conditions to predict impacts on crop yields.

The Genome of the Soybean Gall Midge ( Resseliella maxima ).

The cecidomyiid fly, soybean gall midge, Resseliella maxima Gagné, is a recently discovered insect that feeds on soybean plants in the Midwest US. Resseliella maxima larvae feed on soybean stems which may induce plant death and can cause considerable yield losses, making it an important agricultural pest. From three pools of 50 adults each, we used long-read nanopore sequencing to assemble a R. maxima reference genome. The final genome assembly is 206 Mb with 64.88X coverage, consisting of 1009 contigs with an N50 size of 714 kb. The assembly is high quality with a BUSCO score of 87.8%. Genome-wide GC level is 31.60% and DNA methylation was measured at 1.07%. The R. maxima genome is comprised of 21.73% repetitive DNA, which is in line with other cecidomyiids. Protein prediction annotated 14,798 coding genes with 89.9% protein BUSCO score. Mitogenome analysis indicated that R. maxima assembly is a single circular contig of 15,301 bp and shares highest identity to the mitogenome of the Asian rice gall midge, Orseolia oryzae (Wood-Mason). The R. maxima genome has one of the highest completeness levels for a cecidomyiid and will provide a resource for research focused on the biology, genetics, and evolution of cecidomyiids, as well as plant-insect interactions in this important agricultural pest.

The genome of the soybean gall midge (Resseliella maxima).

The cecidomyiid fly, soybean gall midge, Resseliella maxima Gagné, is a recently discovered insect that feeds on soybean plants in the Midwestern United States. R. maxima larvae feed on soybean stems that may induce plant death and can cause considerable yield losses, making it an important agricultural pest. From three pools of 50 adults each, we used long-read nanopore sequencing to assemble a R. maxima reference genome. The final genome assembly is 206 Mb with 64.88× coverage, consisting of 1,009 contigs with an N50 size of 714 kb. The assembly is high quality with a Benchmarking Universal Single-Copy Ortholog (BUSCO) score of 87.8%. Genome-wide GC level is 31.60%, and DNA methylation was measured at 1.07%. The R. maxima genome is comprised of 21.73% repetitive DNA, which is in line with other cecidomyiids. Protein prediction annotated 14,798 coding genes with 89.9% protein BUSCO score. Mitogenome analysis indicated that R. maxima assembly is a single circular contig of 15,301 bp and shares highest identity to the mitogenome of the Asian rice gall midge, Orseolia oryzae Wood-Mason. The R. maxima genome has one of the highest completeness levels for a cecidomyiid and will provide a resource for research focused on the biology, genetics, and evolution of cecidomyiids, as well as plant-insect interactions in this important agricultural pest.

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs.

RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.

Wolbachia and Virus Alter the Host Transcriptome at the Interface of Nucleotide Metabolism Pathways.

Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila melanogaster model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show that Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.IMPORTANCE Recently developed arbovirus control strategies leverage the symbiotic bacterium Wolbachia, which spreads in insect populations and blocks viruses from replicating. While this strategy has been successful, details of how this "pathogen blocking" works are limited. Here, we use a combination of virus infections, fly genetics, and transcriptomics to show that Wolbachia and virus interact at host nucleotide metabolism pathways.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Sensing, Signaling, and Secretion: A Review and Analysis of Systems for Regulating Host Interaction in Wolbachia.

Wolbachia (Anaplasmataceae) is an endosymbiont of arthropods and nematodes that resides within host cells and is well known for manipulating host biology to facilitate transmission via the female germline. The effects Wolbachia has on host physiology, combined with reproductive manipulations, make this bacterium a promising candidate for use in biological- and vector-control. While it is becoming increasingly clear that Wolbachia's effects on host biology are numerous and vary according to the host and the environment, we know very little about the molecular mechanisms behind Wolbachia's interactions with its host. Here, I analyze 29 Wolbachia genomes for the presence of systems that are likely central to the ability of Wolbachia to respond to and interface with its host, including proteins for sensing, signaling, gene regulation, and secretion. Second, I review conditions under which Wolbachia alters gene expression in response to changes in its environment and discuss other instances where we might hypothesize Wolbachia to regulate gene expression. Findings will direct mechanistic investigations into gene regulation and host-interaction that will deepen our understanding of intracellular infections and enhance applied management efforts that leverage Wolbachia.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Conflict in the Intracellular Lives of Endosymbionts and Viruses: A Mechanistic Look at Wolbachia-Mediated Pathogen-blocking.

At the forefront of vector control efforts are strategies that leverage host-microbe associations to reduce vectorial capacity. The most promising of these efforts employs Wolbachia, a maternally transmitted endosymbiotic bacterium naturally found in 40% of insects. Wolbachia can spread through a population of insects while simultaneously inhibiting the replication of viruses within its host. Despite successes in using Wolbachia-transfected mosquitoes to limit dengue, Zika, and chikungunya transmission, the mechanisms behind pathogen-blocking have not been fully characterized. Firstly, we discuss how Wolbachia and viruses both require specific host-derived structures, compounds, and processes to initiate and maintain infection. There is significant overlap in these requirements, and infection with either microbe often manifests as cellular stress, which may be a key component of Wolbachia's anti-viral effect. Secondly, we discuss the current understanding of pathogen-blocking through this lens of cellular stress and develop a comprehensive view of how the lives of Wolbachia and viruses are fundamentally in conflict with each other. A thorough understanding of the genetic and cellular determinants of pathogen-blocking will significantly enhance the ability of vector control programs to deploy and maintain effective Wolbachia-mediated control measures.

Evolutionary Genetics of Cytoplasmic Incompatibility Genes cifA and cifB in Prophage WO of Wolbachia.

The bacterial endosymbiont Wolbachia manipulates arthropod reproduction to facilitate its maternal spread through host populations. The most common manipulation is cytoplasmic incompatibility (CI): Wolbachia-infected males produce modified sperm that cause embryonic mortality, unless rescued by embryos harboring the same Wolbachia. The genes underlying CI, cifA and cifB, were recently identified in the eukaryotic association module of Wolbachia's prophage WO. Here, we use transcriptomic and genomic approaches to address three important evolutionary facets of the cif genes. First, we assess whether or not cifA and cifB comprise a classic toxin-antitoxin operon in wMel and show that the two genes exhibit striking, transcriptional differences across host development. They can produce a bicistronic message despite a predicted hairpin termination element in their intergenic region. Second, cifA and cifB strongly coevolve across the diversity of phage WO. Third, we provide new domain and functional predictions across homologs within Wolbachia, and show that amino acid sequences vary substantially across the genus. Finally, we investigate conservation of cifA and cifB and find frequent degradation and loss of the genes in strains that no longer induce CI. Taken together, we demonstrate that cifA and cifB exhibit complex transcriptional regulation in wMel, provide functional annotations that broaden the potential mechanisms of CI induction, and report recurrent erosion of cifA and cifB in non-CI strains, thus expanding our understanding of the most widespread form of reproductive parasitism.

Penetrance of symbiont-mediated parthenogenesis is driven by reproductive rate in a parasitoid wasp.

Trichogramma wasps are tiny parasitoids of lepidopteran eggs, used extensively for biological control. They are often infected with the bacterial symbiont Wolbachia, which converts Trichogramma to an asexual mode of reproduction, whereby females develop from unfertilized eggs. However, this Wolbachia-induced parthenogenesis is not always complete, and previous studies have noted that infected females will produce occasional males in the lab. The conditions that reduce penetrance of the parthenogenesis phenotype are not well understood. We hypothesized that more ecologically relevant conditions of limited host access will sustain female-biased sex ratios. After restricting access to host eggs, we found a strong relationship between reproductive rate and sex ratio. By limiting reproduction to one hour a day, wasps could sustain up to 100% effective parthenogenesis for one week, with no significant impact on total fecundity. Reproductive output in the first 24-hours appears to be critical to the total sex ratio of the entire brood. Limiting oviposition in that period resulted in more effective parthenogenesis after one week, again without any significant impact on total fecundity. Our data suggest that this phenomenon may be due to the depletion of Wolbachia when oviposition occurs continuously, whereas Wolbachia titers may recover when offspring production is limited. In addition to the potential to improve mass rearing of Trichogramma for biological control, findings from this study help elucidate the context-dependent nature of a pervasive symbiotic relationship.