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.

Chromosome-level genome assemblies of two parasitoid biocontrol wasps reveal the parthenogenesis mechanism and an associated novel virus.

BACKGROUND: Biocontrol is a key technology for the control of pest species. Microctonus parasitoid wasps (Hymenoptera: Braconidae) have been released in Aotearoa New Zealand as biocontrol agents, targeting three different pest weevil species. Despite their value as biocontrol agents, no genome assemblies are currently available for these Microctonus wasps, limiting investigations into key biological differences between the different species and strains. METHODS AND FINDINGS: Here we present high-quality genomes for Microctonus hyperodae and Microctonus aethiopoides, assembled with short read sequencing and Hi-C scaffolding. These assemblies have total lengths of 106.7 Mb for M. hyperodae and 129.2 Mb for M. aethiopoides, with scaffold N50 values of 9 Mb and 23 Mb respectively. With these assemblies we investigated differences in reproductive mechanisms, and association with viruses between Microctonus wasps. Meiosis-specific genes are conserved in asexual Microctonus, with in-situ hybridisation validating expression of one of these genes in the ovaries of asexual Microctonus aethiopoides. This implies asexual reproduction in these Microctonus wasps involves meiosis, with the potential for sexual reproduction maintained. Investigation of viral gene content revealed candidate genes that may be involved in virus-like particle production in M. aethiopoides, as well as a novel virus infecting M. hyperodae, for which a complete genome was assembled. CONCLUSION AND SIGNIFICANCE: These are the first published genomes for Microctonus wasps which have been deployed as biocontrol agents, in Aotearoa New Zealand. These assemblies will be valuable resources for continued investigation and monitoring of these biocontrol systems. Understanding the biology underpinning Microctonus biocontrol is crucial if we are to maintain its efficacy, or in the case of M. hyperodae to understand what may have influenced the significant decline of biocontrol efficacy. The potential for sexual reproduction in asexual Microctonus is significant given that empirical modelling suggests this asexual reproduction is likely to have contributed to biocontrol decline. Furthermore the identification of a novel virus in M. hyperodae highlights a previously unknown aspect of this biocontrol system, which may contribute to premature mortality of the host pest. These findings have potential to be exploited in future in attempt to increase the effectiveness of M. hyperodae biocontrol.

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19.

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

Stochastic Reaction-Diffusion Model of the Binding of Monoclonal Antibodies to CD4 Receptors on the Surface of T Cells.

A stochastic reaction-diffusion model was developed to describe the binding of labeled monoclonal antibodies (mAbs) to CD4 receptors on the surface of T cells. The mAbs diffused to, adsorbed on, and underwent monovalent and bivalent binding to CD4 receptors on the cell surface. The model predicted the time-dependent nature of all populations involved in the labeling process. At large time, the populations reached equilibrium values, giving the number of antibodies bound to the T cell (ABC) defined as the sum of monovalently and bivalently bound mAbs. The predicted coefficient of variation (CV%) of the (ABC) values translated directly to a corresponding CV% of the measured mean fluorescence intensity (MFI). The predicted CV% was about 0.2% from the intrinsic fluctuations of the stochastic reaction process, about 5% after inclusion of the known fluctuations in the number of available CD4 receptors, and about 11% when fluctuations in bivalent binding affinity were included. The fluorescence detection process is expected to contribute approximately 7%. The abovementioned contributions to CV% sum up to approximately 13%. Work is underway to reconcile the predicted values and the measured values of 17% to 22%.

Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production.

Pichia pastoris is extensively used to produce various heterologous proteins. Amounts of biopharmaceutical drugs and industrial enzymes have been successfully produced by fed-batch high-cell-density fermentation (HCDF) of this cell factory. High levels of cell mass in defined media can be easily achieved and therefore large quantities of recombinant proteins with enhanced activities and lower costs can be obtained through HCDF technology. A robust HCDF process makes a successful transition to commercial production. Recently, efforts have been made to increase the heterologous protein production and activity by the HCDF of P. pastoris. However, challenges around selecting a suitable HCDF strategy exist. The high-level expression of a specific protein in P. pastoris is still, at least in part, limited by optimizing the methanol feeding strategy. Here, we review the progress in developments and applications of P. pastoris HCDF strategies for enhanced expression of recombinant proteins. We focus on the methanol induction strategies for efficient fed-batch HCDF in bioreactors, mainly focusing on various stat-induction strategies, co-feeding, and the limited induction strategy. These processes control strategies have opened the door for expressing foreign proteins in P. pastoris and are expected to enhance the production of recombinant proteins.

Continuous production process of retroviral vector for adoptive T- cell therapy.

Adoptive T-Cell therapy is being considered as a promising method for cancer treatment. In this approach, patient's T cells are isolated, modified, expanded, and administered back to the patient. Modifications may include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) to the isolated cells by using retroviral vectors. PG13 cells, derivatives of NIH3T3 mouse fibroblasts, are being used to stably produce retroviral vectors that transduce the T cells. PG13 cells are anchorage-dependent cells that grow in roller bottles or cell factories and lately also in fixed bed bioreactors to produce the needed viral vector. To scale up viral vector production, PG13 cells were propagated on microcarriers in a stirred tank bioreactor utilizing an alternating tangential flow perfusion system. Microcarriers are 10 µm - 0.5 mm beads that support the attachment of cells and are suspended in the bioreactor that provides controlled growth conditions. As a result, growth parameters, such as dissolved oxygen concentration, pH, and nutrients are monitored and continuously controlled. There were no detrimental effects on the specific viral vector titer or on the efficacy of the vector in transducing the T cells of several patients. Viral vector titer increased throughout the 11 days perfusion period, a total of 4.8 × 1011 transducing units (TU) were obtained with an average titer of 4.4 × 107 TU/mL and average specific productivity of 10.3 (TU) per cell, suggesting that this method can be an efficient way to produce large quantities of active vector suitable for clinical use.

Genome-Wide High-Throughput RNAi Screening for Identification of Genes Involved in Protein Production.

With an increasing number of blockbuster drugs being recombinant mammalian proteins, protein production platforms that focus on mammalian proteins have had a profound impact in many areas of basic and applied research. Many groups, both academic and industrial, have been focusing on developing cost-effective methods to improve the production of mammalian proteins that would support potential therapeutic applications. As it stands, while a wide range of platforms have been successfully developed for laboratory use, the majority of biologicals are still produced in mammalian cell lines due to the requirement for posttranslational modification and the biosynthetic complexity of target proteins. An unbiased high-throughput RNAi screening approach can be an efficient tool to identify target genes involved in recombinant protein production. Here, we describe the process of optimizing the transfection conditions, performing the genome-wide siRNA screen, the activity and cell viability assays, and the validation transfection to identify genes involved with protein expression.

The venom composition and parthenogenesis mechanism of the parasitoid wasp Microctonus hyperodae, a declining biocontrol agent.

A biocontrol system in New Zealand using the endoparasitoid Microctonus hyperodae is failing, despite once being one of the most successful examples of classical biocontrol worldwide. Though it is of significant economic importance as a control agent, little is known about the genetics of M. hyperodae. In this study, RNA-seq was used to characterise two key traits of M. hyperodae in this system, the venom, critical for the initial success of biocontrol, and the asexual reproduction mode, which influenced biocontrol decline. Expanded characterisation of M. hyperodae venom revealed candidates involved in manipulating the host environment to source nutrition for the parasitoid egg, preventing a host immune response against the egg, as well as two components that may stimulate the host's innate immune system. Notably lacking from the venom-specific expression list was calreticulin, as it also had high expression in the ovaries. In-situ hybridisation revealed this ovarian expression was localised to the follicle cells, which may result in the deposition of calreticulin into the egg exochorion. Investigating the asexual reproduction of M. hyperodae revealed core meiosis-specific genes had conserved expression patterns with the highest expression in the ovaries, suggesting M. hyperodae parthenogenesis involves meiosis and that the potential for sexual reproduction may have been retained. Upregulation of genes involved in endoreduplication provides a potential mechanism for the restoration of diploidy in eggs after meiosis.

Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing.

Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective CRISPR/Cas9-engineered therapies.

Improved protein expression in HEK293 cells by over-expressing miR-22 and knocking-out its target gene, HIPK1.

Stable cell lines can continuously produce a recombinant protein without the need to repeatedly engineer the genome. In a previous study HIPK1, Homeodomain-interacting Protein Kinase 1, was found to be a target of the microRNA miR-22 that, when repressed, improved expression of both an intracellular and a secreted protein. In this report, HEK293 cells stably over-expressing miR-22 were compared with HEK293 with knockout of HIPK1, executed by CRISPR/Cas9, for their ability to improve recombinant protein expression. In this model case of luciferase, over-expression of miR-22 improved overall activity 2.4-fold while the HIPK1 knockout improved overall activity 4.7-fold.

Genetic Diversity in Invasive Populations of Argentine Stem Weevil Associated with Adaptation to Biocontrol.

Modified, agricultural landscapes are susceptible to damage by insect pests. Biological control of pests is typically successful once a control agent has established, but this depends on the agent's capacity to co-evolve with the host. Theoretical studies have shown that different levels of genetic variation between the host and the control agent will lead to rapid evolution of resistance in the host. Although this has been reported in one instance, the underlying genetics have not been studied. To address this, we measured the genetic variation in New Zealand populations of the pasture pest, Argentine stem weevil (Listronotus bonariensis), which is controlled with declining effectiveness by a parasitoid wasp, Microctonus hyperodae. We constructed a draft reference genome of the weevil, collected samples from a geographical survey of 10 sites around New Zealand, and genotyped them using a modified genotyping-by-sequencing approach. New Zealand populations of Argentine stem weevil have high levels of heterozygosity and low population structure, consistent with a large effective population size and frequent gene flow. This implies that Argentine stem weevils were able to evolve more rapidly than their biocontrol agent, which reproduces asexually. These findings show that monitoring genetic diversity in biocontrol agents and their targets is critical for long-term success of biological control.

TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes.

Species of the genus Trichoderma are ubiquitous in the environment and are widely used in agriculture, as biopesticides, and in the industry for the production of plant cell wall-degrading enzymes. Trichoderma represents an important genus of endophytes, and several Trichoderma species have become excellent models for the study of fungal biology and plant-microbe interactions; moreover, are exceptional biotechnological factories for the production of bioactive molecules useful in agriculture and medicine. Next-generation sequencing technology coupled with systematic construction of recombinant DNA molecules provides powerful tools that contribute to the functional analysis of Trichoderma genetics, thus allowing for a better understanding of the underlying factors determining its biology. Here, we present the creation of diverse vectors containing (i) promoter-specific vectors for Trichoderma, (ii) gene deletions (using hygromycin phosphotransferase as selection marker), (iii) protein localization (mCherry and eGFP, which were codon-optimized for Trichoderma), (iv) gene complementation (neomycin phosphotransferase) and (v) overexpression of encoding gene proteins fused to fluorescent markers, by using the Golden Gate cloning technology. Furthermore, we present the design and implementation of a binary vector for Agrobacterium-mediated transformation in Trichoderma to increase the homologous recombination rate and the generation of a novel selection marker based on carboxin resistance.

Ecological Stoichiometry: A Link Between Developmental Speed and Physiological Stress in an Omnivorous Insect.

The elemental composition of organisms belongs to a suite of functional traits that may adaptively respond to fluctuating selection pressures. Life history theory predicts that predation risk and resource limitations impose selection pressures on organisms' developmental time and are further associated with variability in energetic and behavioral traits. Individual differences in developmental speed, behaviors and physiology have been explained using the pace-of-life syndrome (POLS) hypothesis. However, how an organism's developmental speed is linked with elemental body composition, metabolism and behavior is not well understood. We compared elemental body composition, latency to resume activity and resting metabolic rate (RMR) of western stutter-trilling crickets (Gryllus integer) in three selection lines that differ in developmental speed. We found that slowly developing crickets had significantly higher body carbon, lower body nitrogen and higher carbon-to-nitrogen ratio than rapidly developing crickets. Slowly developing crickets had significantly higher RMR than rapidly developing crickets. Male crickets had higher RMR than females. Slowly developing crickets resumed activity faster in an unfamiliar relative to a familiar environment. The rapidly developing crickets did the opposite. The results highlight the tight association between life history, physiology and behavior. This study indicates that traditional methods used in POLS research should be complemented by those used in ecological stoichiometry, resulting in a synthetic approach that potentially advances the whole field of behavioral and physiological ecology.

Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells.

The ability to produce recombinant proteins by utilizing different "cell factories" revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO) cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK), while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

Genome-Wide High-Throughput RNAi Screening for Identification of Genes Involved in Protein Production.

With an increasing number of blockbuster drugs being recombinant mammalian proteins, protein production platforms that focus on mammalian proteins have had a profound impact in many areas of basic and applied research. Many groups, both academic and industrial, have been focusing on developing cost-effective methods to improve the production of mammalian proteins that would support potential therapeutic applications. As it stands, while a wide range of platforms have been successfully developed for laboratory use, the majority of biologicals are still produced in mammalian cell lines due to the requirement for posttranslational modification and the biosynthetic complexity of target proteins. An unbiased high-throughput RNAi screening approach can be an efficient tool to identify target genes involved in recombinant protein production. Here we describe the process of optimizing the transfection conditions, performing the genome-wide siRNA screen, the activity and cell viability assays and the validation transfection to identify genes involved with protein expression.

Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen.

Protein expression from human embryonic kidney cells (HEK 293) is an important tool for structural and clinical studies. It is previously shown that microRNAs (small, noncoding RNAs) are effective means for improved protein expression from these cells, and by conducting a high-throughput screening of the human microRNA library, several microRNAs are identified as potential candidates for improving expression. From these, miR-22-3p is chosen for further study since it increased the expression of luciferase, two membrane proteins and a secreted fusion protein with minimal effect on the cells' growth and viability. Since each microRNA can interact with several gene targets, it is of interest to identify the repressed genes for understanding and exploring the improved expression mechanism for further implementation. Here, the authors describe a novel approach for identification of the target genes by integrating the differential gene expression analysis with information obtained from our previously conducted high-throughput siRNA screening. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. Repressing the target gene, HIPK1, is found to increase luciferase and GPC3 expression 3.3- and 2.2-fold, respectively.

Short-term exposure to predation affects body elemental composition, climbing speed and survival ability in Drosophila melanogaster.

Factors such as temperature, habitat, larval density, food availability and food quality substantially affect organismal development. In addition, risk of predation has a complex impact on the behavioural and morphological life history responses of prey. Responses to predation risk seem to be mediated by physiological stress, which is an adaptation for maintaining homeostasis and improving survivorship during life-threatening situations. We tested whether predator exposure during the larval phase of development has any influence on body elemental composition, energy reserves, body size, climbing speed and survival ability of adult Drosophila melanogaster. Fruit fly larvae were exposed to predation by jumping spiders (Phidippus apacheanus), and the percentage of carbon (C) and nitrogen (N) content, extracted lipids, escape response and survival were measured from predator-exposed and control adult flies. The results revealed predation as an important determinant of adult phenotype formation and survival ability. D. melanogaster reared together with spiders had a higher concentration of body N (but equal body C), a lower body mass and lipid reserves, a higher climbing speed and improved adult survival ability. The results suggest that the potential of predators to affect the development and the adult phenotype of D. melanogaster is high enough to use predators as a more natural stimulus in laboratory experiments when testing, for example, fruit fly memory and learning ability, or when comparing natural populations living under different predation pressures.

A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.

Cotton (Gossypium hirsutum) provides the world's dominant renewable textile fiber, and cotton fiber is valued as a research model because of its extensive elongation and secondary wall thickening. Previously, it was assumed that fibers elongated as individual cells. In contrast, observation by cryo-field emission-scanning electron microscopy of cotton fibers developing in situ within the boll demonstrated that fibers elongate within tissue-like bundles. These bundles were entrained by twisting fiber tips and consolidated by adhesion of a cotton fiber middle lamella (CFML). The fiber bundles consolidated via the CFML ultimately formed a packet of fiber around each seed, which helps explain how thousands of cotton fibers achieve their great length within a confined space. The cell wall nature of the CFML was characterized using transmission electron microscopy, including polymer epitope labeling. Toward the end of elongation, up-regulation occurred in gene expression and enzyme activities related to cell wall hydrolysis, and targeted breakdown of the CFML restored fiber individuality. At the same time, losses occurred in certain cell wall polymer epitopes (as revealed by comprehensive microarray polymer profiling) and sugars within noncellulosic matrix components (as revealed by gas chromatography-mass spectrometry analysis of derivatized neutral and acidic glycosyl residues). Broadly, these data show that adhesion modulated by an outer layer of the primary wall can coordinate the extensive growth of a large group of cells and illustrate dynamic changes in primary wall structure and composition occurring during the differentiation of one cell type that spends only part of its life as a tissue.

Factors controlling sediment denitrification in midwestern streams of varying land use.

We investigated controls on stream sediment denitrification in nine headwater streams in the Kalamazoo River Watershed, Michigan, USA. Factors influencing denitrification were determined by using experimental assays based on the chloramphenicol-amended acetylene inhibition technique. Using a coring technique, we found that sediment denitrification was highest in the top 5 cm of the benthos and was positively related to sediment organic content. To determine the effect of overlying water quality on sediment denitrification, first-order stream sediments were assayed with water from second- and third-order downstream reaches, and often showed higher denitrification rates relative to assays using site-specific water from the first-order stream reach. Denitrification was positively related to nitrate (NO3 ) concentration, suggesting that sediments may have been nutrient-limited. Using stream-incubated inorganic substrata of varying size classes, we found that finer-grained sand showed higher rates of denitrification compared to large pebbles, likely due to increased surface area per volume of substratum. Denitrification was measurable on both inorganic substrata and fine particulate organic matter loosely associated with inorganic particles, and denitrification rates were related to organic content. Using nutrient-amended denitrification assays, we found that sediment denitrification was limited by NO3- or dissolved organic carbon (DOC, as dextrose) variably throughout the year. The frequency and type of limitation differed with land use in the watershed: forested streams were NO3- -limited or colimited by both NO3- and DOC 92% of the time, urban streams were more often NO3- -limited than DOC-limited, whereas agricultural stream sediments were DOC-limited or co-limited but not frequently limited by NO3- alone.

Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase.

Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for homogalacturonan (HG) alpha-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG alpha-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. blast analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.