Response of the macadamia seed weevil Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) to Metarhizium anisopliae and Beauveria bassiana in laboratory bioassays.

Macadamia seed weevil, Kuschelorhynchus macadamiae Jennings and Oberprieler, is a major pest of macadamia in eastern Australia, causing yield losses of up to 15%. Current control methods involve two applications of acephate per season but more recently have moved to a single application of indoxacarb, combined with the collection and destruction of fallen nuts that contain developing larvae. As a first step towards reducing the dependence of the industry on synthetic insecticides, we tested six isolates of M. anisopliae, six isolates of B. bassiana and one commercial B. bassiana product (Velifer® biological insecticide) against adult macadamia seed weevil under laboratory conditions. All isolates were pathogenic against adult weevils with M. anisopliae accession ECS1/BRIP 70272 and B. bassiana accession B27/BRIP 70267 causing 97.5% and 92.5% mortality 12 days after being treated at 1 × 107 conidia/mL. Isolates ECS1/BRIP 70272 and B27/BRIP 70267 had the shortest LT50 values of 5.13 days and 5.37 days respectively. The median lethal concentrations (LC50) for ECS1/BRIP 70272 and B27/BRIP 70267 were 1.48 × 105 and 1.65 × 105 conidia/mL respectively. Results of this study indicate that M. anisopliae accession ECS1/BRIP 70272 and B. bassiana accession B27/BRIP 70267 have considerable potential for K. macadamiae control, and should be developed into biological insecticides for integration into macadamia pest management programs.

Sweetpotato weevil, Cylas formicarius (Fab.) (Coleoptera: Brentidae) avoids its host plant when a virulent Metarhizium anisopliae isolate is present.

Metarhizium anisopliae has a wide range of coleopteran hosts, including weevils. Some susceptible insects are known to modify their behavior to prevent infection, typically detecting virulent strains by olfaction, and avoiding physical contact with sources of infection. Laboratory olfactometer assays were conducted on the sweetpotato weevil Cylas formicarius to test the hypothesis that insects would avoid a more virulent strain of M. anisopliae when presented with a strain of low virulence or an untreated control. When adult weevils were allowed to choose between paired test arenas containing sweetpotato roots and M. anisopliae isolates on agar cores, weevils avoided arenas with the highly virulent isolate QS155, showing a preference for either roots with uninoculated agar cores or cores with the low virulence isolate QS002-3. When roots or whole sweetpotato plants were inoculated with M. anisopliae, the preferences of weevils remained broadly similar; weevils were repelled by the highly virulent isolate QS155 when tested against either QS002-3 or uninoculated roots and plants, however weevils were not repelled by the low virulence isolate QS002-3 tested against uninoculated controls. When single-sex groups of weevils were tested separately in the olfactometer using uninoculated whole plants and plants treated with isolate QS155, males and females responded similarly and statistically identical preferences were found for the untreated plants. When weevils were released singly at different times of the day the response time for males was significantly shorter in the afternoon compared to the morning. Males were always significantly faster to respond to olfactory stimuli than females. Understanding factors that may lead to avoidance of virulent M. anisopliae strains by C. formicarius will be an essential part of developing an 'attract-and-infect' strategy for the management of C. formicarius.

Cellular Mass Response to Therapy Correlates With Clinical Response for a Range of Malignancies.

PURPOSE: Cancer patients with advanced-stage disease have poor prognosis, typically having limited options for efficacious treatment, and genomics-based therapy guidance continues to benefit only a fraction of patients. Next-generation ex vivo approaches, such as cell mass-based response testing (MRT), offer an alternative precision medicine approach for a broader population of patients with cancer, but validation of clinical feasibility and potential impact remain necessary. MATERIALS AND METHODS: We evaluated the clinical feasibility and accuracy of using live-cell MRT to predict patient drug sensitivity. Using a unified measurement workflow with a 48-hour result turnaround time, samples were subjected to MRT after treatment with a panel of drugs in vitro. After completion of therapeutic course, clinical response data were correlated with MRT-based predictions of outcome. Specimens were collected from 104 patients with solid (n = 69) and hematologic (n = 35) malignancies, using tissue formats including needle biopsies, malignant fluids, bone marrow aspirates, and blood samples. Of the 81 (78%) specimens qualified for MRT, 41 (51%) patients receiving physician-selected therapies had treatments matched to MRT. RESULTS: MRT demonstrated high concordance with clinical responses with an odds ratio (OR) of 14.80 (P = .0003 [95% CI, 2.83 to 102.9]). This performance held for both solid and hematologic malignances with ORs of 20.67 (P = .0128 [95% CI, 1.45 to 1,375.57]) and 8.20 (P = .045 [95% CI, 0.77 to 133.56]), respectively. Overall, these results had a predictive accuracy of 80% (P = .0026 [95% CI, 65 to 91]). CONCLUSION: MRT showed highly significant correlation with clinical response to therapy. Routine clinical use is technically feasible and broadly applicable to a wide range of samples and malignancy types, supporting the need for future validation studies.

Mutation and cell state compatibility is required and targetable in Ph+ acute lymphoblastic leukemia minimal residual disease.

Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples.

Evaluation of a commercial Bacillus thuringiensis var. israelensis formulation for the control of chironomid midge larvae (Diptera: Chironomidae) in establishing rice crops in south-eastern Australia.

A commercial formulation of Bacillus thuringiensis var. israelensis (B.t.i.) was evaluated for its potential to control chironomid midge larvae in newly sown rice crops in south-eastern Australia. Two replicated small-plot field trials were conducted using product application rates of 0.5-6 kg/ha. In trial 1 application rates between 2 and 6 kg product/ha all significantly (P<0.05) reduced populations of target Chironominae/Orthocladiinae by between 71% and 93% over the 19 day post-treatment monitoring period. Trial 2 was conducted using lower application rates (0.5-2 kg product/ha) and only the 2 kg product/ha rate significantly (P<0.05) reduced numbers of the target group (81% reduction) despite lower application rates resulting in target group suppression of 38-62%. Identification of larvae to species level from selected samples indicated that populations of Chironomus tepperi, the principal pest species that attacks the roots of rice seedlings, were reduced at all application rates; elimination of C. tepperi was achieved in trial 1 at an application rate of 2 kg/ha. Consistent with other studies, non-target Tanypodinae were not adversely affected by B.t.i., and in some treatments populations of Tanypodinae exceeded control levels by up to 73%. In the first trial, which was conducted under relatively high pest pressure, plant establishment was significantly (P<0.05) increased (120-157%) by Vectobac® WDG application rates of 2-6 kg/ha. No significant increase in plant establishment relative to the controls was identified in the second trial, when pest pressure was substantially lower and minimal damage occurred in the control bays. Overall, our results demonstrate that B.t.i. may be an economically viable alternative to broad-spectrum synthetic pesticides for the control of phytophagous midge larvae in establishing rice crops where members of the Chironominae, the group most susceptible to B.t.i., are the principal species of concern. The high specificity of B.t.i. for nematoceran Diptera should lead to reduced impacts on non-target organisms.

Biosecurity and Management Strategies for Economically Important Exotic Tephritid Fruit Fly Species in Australia.

Exotic tephritid incursions are of high concern to Australia's biosecurity and its horticultural industries. It is vital that Australia remains ready to respond to incursions as they arise, as an incursion of tephritid fruit fly species will result in significant economic losses. In this review, we compared Australian incursion management strategies for fruit flies with global management strategies and identified possible areas where improvements could be made in an Australian context. Overall, Australia has a good understanding of the main tephritid threats, of which Bactrocera species from across the Torres Strait (northern Australia) are of most concern. Effective tools for tephritid detection and early warning surveillance at points of entry are in place at ports and in horticultural areas Australia-wide and provide the basis for initiating biosecurity responses in the event of an incursion. Area-wide control measures used in successful eradication attempts globally are available for use in Australia. However, a specific tephritid emergency response plan identifying suitable response measures and control options for species of concern is not yet available. We have identified that Australia has the policies and management tools available to respond to an exotic tephritid incursion, but the speed at which this could be accomplished would be greatly improved by the development of species-specific emergency response plans.

Topical and dietary toxicity of emamectin benzoate, chlorantraniliprole, cyantraniliprole and indoxacarb to larvae of the common armyworm Mythimna convecta (Lepidoptera: Noctuidae).

BACKGROUND: The common armyworm Mythimna convecta is an important pest of pastures and graminaceous crops in Australia, but materials currently registered for its control are limited to broad-spectrum compounds incompatible with integrated pest management (IPM) systems. In this study we assessed the response of M. convecta larvae to four alternative compounds using topical and dietary bioassays. RESULTS: Emamectin benzoate [LC50 (lethal concentration for 50% of insects tested) values 2.69 µg mL-1 topical, 0.017 µg active ingredient (AI) g-1 dietary] and chlorantraniliprole (LC50 values 4.87 µg mL-1  topical, 0.080 µg AI g-1 dietary) were significantly more active than either indoxacarb or cyantraniliprole. Our results showed strong parallels with data on the more extensively studied Australian strains of Helicoverpa armigera, with the most notable differences being the higher contact toxicity of emamectin benzoate to M. convecta and the lower acute dietary activity of formulated cyantraniliprole to this species, which was linked to feeding deterrence. Cyantraniliprole at dietary concentrations of ≥0.02 µg AI g-1 significantly reduced the weight of surviving larvae and frass production (an indirect measure of food consumption) over the seven-day exposure period. There was also some evidence of chlorantraniliprole deterring larval feeding, although to a much more limited extent. CONCLUSIONS: Both emamectin benzoate and chlorantraniliprole are suitable for use against M. convecta. The decision as to which of these compounds should be prioritized for further development should be based on their potential effects on beneficial species once their optimal field rates have been determined.

Triple MAPK inhibition salvaged a relapsed post-BCMA CAR-T cell therapy multiple myeloma patient with a BRAF V600E subclonal mutation.

BACKGROUND: Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite promising response rates achieved with anti-BCMA CAR-T cell therapy, patients may still relapse and there are currently no clear therapeutic options in post-CAR-T settings. In this report, we present a case of a post-BCMA CAR-T relapsed/refractory (RR) MM patient with skin extramedullary disease (EMD) in which a novel MAPK inhibition combinatorial strategy was implemented based on next-generation sequencing and in vitro experiments. CASE PRESENTATION: A 61-year-old male with penta-refractory MM penta- (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CAR-T cell therapy, achieving a best response of VGPR. He progressed after 6 months and was salvaged for a short period with autologous stem cell transplantation. Eventually, he progressed with extramedullary disease manifested as subcutaneous nodules. Based on whole-exome sequencing, we identified a BRAF (V600E) dominant subclone in both bone marrow and cutaneous plasmacytoma. Following in vitro experiments, and according to our previous studies, we implemented a triple MAPK inhibition strategy under which the patient achieved a very good partial response for 110 days, which allowed to bridge him to subsequent clinical trials and eventually achieve a stringent complete response (sCR). CONCLUSION: Here, we show the applicability, effectiveness, and tolerability the triple MAPK inhibition strategy in the context of post-BCMA CAR-T failure in specific subset of patients. The triple therapy could bridge our hospice bound RRMM patient with BRAF (V600E) to further therapeutic options where sCR was achieved. We will further evaluate triple MAPK inhibition in patients with BRAF V600E in a precision medicine clinical trial launching soon.

A pipeline for malignancy and therapy agnostic assessment of cancer drug response using cell mass measurements.

Functional precision medicine offers a promising complement to genomics-based cancer therapy guidance by testing drug efficacy directly on a patient's tumor cells. Here, we describe a workflow that utilizes single-cell mass measurements with inline brightfield imaging and machine-learning based image classification to broaden the clinical utility of such functional testing for cancer. Using these image-curated mass measurements, we characterize mass response signals for 60 different drugs with various mechanisms of action across twelve different cell types, demonstrating an improved ability to detect response for several slow acting drugs as compared with standard cell viability assays. Furthermore, we use this workflow to assess drug responses for various primary tumor specimen formats including blood, bone marrow, fine needle aspirates (FNA), and malignant fluids, all with reports generated within two days and with results consistent with patient clinical responses. The combination of high-resolution measurement, broad drug and malignancy applicability, and rapid return of results offered by this workflow suggests that it is well-suited to performing clinically relevant functional assessment of cancer drug response.

Toxicity of the insecticide terbufos, its oxidation metabolites, and the herbicide atrazine in binary mixtures to Ceriodaphnia cf dubia.

The acute toxicity of terbufos and its major metabolites, tested alone, in binary mixtures or in combination with atrazine were evaluated using neonates of the cladoceran Ceriodaphnia cf dubia. Terbufos, terbufos sulfoxide, and terbufos sulfone tested individually were highly toxic to C. cf dubia, with mean 96-h EC(50) values of 0.08, 0.36, and 0.19 µg/l, respectively. The addition of atrazine (10 µg/l) significantly increased the toxicity of terbufos. The toxicity of terbufos sulfone was unaffected by atrazine, whereas the results for terbufos sulfoxide were equivocal. Equitoxic mixtures of the metabolites showed additive toxicity to C. cf dubia. The high toxicities of terbufos and its environmentally persistent oxidative metabolites suggest that contamination of aquatic systems with this insecticide mixture and the coapplied herbicide atrazine might pose a greater hazard to some biota than their individual toxicities.

Compatibility of Metarhizium anisopliae and Beauveria bassiana with insecticides and fungicides used in macadamia production in Australia.

BACKGROUND: Integrating fungal biocontrol agents into crop protection programs dominated by synthetic pesticides is an important first step towards developing an integrated pest management (IPM) program; however, their successful integration relies on an understanding of how their performance may be impacted by the remaining agrochemicals deployed for managing other pests and diseases. In this study we tested 10 formulated pesticides used in macadamia production at different concentrations to determine their effects on the germination, mycelial growth and sporulation of Metarhizium anisopliae and Beauveria bassiana in vitro. Further tests with laboratory-grade actives of the noncompatible pesticides were conducted to determine whether any antagonistic effects were caused by the active constituent or by formulation additives. RESULTS: At their registered concentrations, formulated trichlorfon, acephate and indoxacarb were compatible with M. anisopliae, whereas B. bassiana showed compatibility with formulated trichlorfon, acephate, indoxacarb, sulfoxaflor and spinetoram. Bioassays using laboratory-grade active constituents indicated that the adverse impact of formulated beta-cyfluthrin on both fungal species and that of formulated methidathion on B. bassiana is probably due to components of the emulsifiable concentrate formulations rather than their active constituents. Diazinon was the only insecticidal active that showed high toxicity to both fungal species. The two fungicides, carbendazim and pyraclostrobin, were toxic to both fungal species at all tested concentrations. CONCLUSION: Our results identify which pesticides used on macadamias in Australia are compatible and incompatible with entomopathogenic fungi. Future studies on pesticide degradation rates will help define the spray intervals required to eliminate these adverse effects.

Transmission of Metarhizium anisopliae and Beauveria bassiana to adults of Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) from infected adults and conidiated cadavers.

Kuschelorhynchus macadamiae is a major pest of macadamias in Australia, causing yield losses of up to 15%. Our previous studies have shown the weevil is susceptible to Beauveria bassiana and Metarhizium anisopliae. The aim of this study was to investigate horizontal transmission of both fungal species to healthy weevils from both infected adults and weevil cadavers. In a confined environment the mortality of healthy adults caused by the transmission of conidia from live fungus-infected adults was < 50%. Under similar experimental conditions, the mortality of healthy adults reached 100% when exposed to conidiated cadavers. However, when conidiated cadavers were used in more spacious environments (insect cages), the mortality of adults was < 80%. Using scanning electron microscopy, it was observed that all healthy adults had conidia attached to all external parts of the body. This suggests that although the conidia were readily transferred to the adults, the lower mortality in the larger insect cages could be the result of an unfavourable environmental factor such as low humidity. The presence of conidia attached to all the adults indicated that they did not show any discriminatory behaviour such as avoidance of conidiated cadavers infected by these two fungal species. The results from this study show that there is potential for enhanced control of adult K. macadamiae via transmission from either fungus-infected adults or conidiated cadavers and this could strengthen sustainable pest management in macadamias.

Solubility Limits of Cholesterol, Lanosterol, Ergosterol, Stigmasterol, and ß-Sitosterol in Electroformed Lipid Vesicles.

Here we use nuclear magnetic resonance to measure the solubility limit of several biologically relevant sterols in electroformed giant unilamellar vesicle membranes containing phosphatidylcholine (PC) lipids in ratios of 1:1:X DOPC:DPPC:sterol. We find solubility limits of cholesterol, lanosterol, ergosterol, stigmasterol, and ß-sitosterol to be 65-70%, ~35%, 30-35%, 20-25%, and ~40%, respectively. The low solubilities of stigmasterol and ß-sitosterol, which differ from cholesterol only in their alkyl tails, show that subtle differences in tail structure can strongly affect sterol solubility. Below the solubility limits, the fraction of sterol to PC-lipid in electroformed vesicles linearly reflects the fraction in the original stock solutions used in the electroformation process.

A low concentration of atrazine does not influence the acute toxicity of the insecticide terbufos or its breakdown products to Chironomus tepperi.

The acute toxicities of the insecticide terbufos and its major breakdown products individually, as binary mixtures, and in combination with the co-applied herbicide atrazine were evaluated using final instar larvae of the midge Chironomus tepperi. Terbufos, terbufos sulfoxide and terbufos sulfone were highly toxic to C. tepperi with mean 96-h EC50 values of 2.13, 3.64 and 2.59 µg/l, respectively. No interaction was observed between atrazine (25 µg/l) and terbufos or its breakdown products while the binary mixture of terbufos sulfoxide and terbufos sulfone exhibited additive toxicity. The high toxicities of terbufos and its environmentally persistent oxidation products suggest that contamination of aquatic systems with this insecticide pose a threat to aquatic organisms whether or not atrazine is also present.

Integration of Entomopathogenic Fungi into IPM Programs: Studies Involving Weevils (Coleoptera: Curculionoidea) Affecting Horticultural Crops.

Weevils are significant pests of horticultural crops and are largely managed with insecticides. In response to concerns about negative impacts of synthetic insecticides on humans and the environment, entomopathogenic fungi (EPF) have been developed as an alternative method of control, and as such appear to be "ready-made" components of integrated pest management (IPM) programs. As the success of pest control requires a thorough knowledge of the biology of the pests, this review summarises our current knowledge of weevil biology on nut trees, fruit crops, plant storage roots, and palm trees. In addition, three groups of life cycles are defined based on weevil developmental habitats, and together with information from studies of EPF activity on these groups, we discuss the tactics for integrating EPF into IPM programs. Finally, we highlight the gaps in the research required to optimise the performance of EPF and provide recommendations for the improvement of EPF efficacy for the management of key weevils of horticultural crops.

Author Correction: Cooperative nutrient accumulation sustains growth of mammalian cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Influence of imidacloprid seed treatments on rice germination and early seedling growth.

BACKGROUND: Seed treatments with the chloronicotinyl insecticide imidacloprid (Gaucho 600 FS) were evaluated to determine whether differences in concentration and exposure regime influence the germination and early growth of rice. RESULTS: Continuous exposure to imidacloprid (4 days at 2000 mg AI L(-1)) significantly (P < 0.001) reduced normal germination by an average of 18% across the 15 cultivars examined. Nine days after sowing, plants showed no adverse effects from continuous imidacloprid treatment during germination, with shoot lengths and root system dry weights equalling, or occasionally exceeding (P < 0.05), those of untreated plants. Short-term imidacloprid exposure (2 h at 2000 mg L(-1)) at initial seed wetting did not affect germination (P > 0.05), and short-term (1 h) exposure of 48 h pregerminated seed to imidacloprid (2000 mg L(-1)) similarly had no significant effect on early subsequent growth. Plants arising from 48 h pregerminated seed exposed to imidacloprid (1 h) at concentrations up to 4000 mg L(-1) immediately before sowing were not significantly different from control plants at either 9 or 25 days post-sowing. CONCLUSION: Results show that imidacloprid will have no adverse effects on plant growth if applied to pregerminated rice shortly before sowing. Continuous exposure of seed during germination had more pronounced effects, and the initial response of different cultivars was highly variable. Cultivars with high levels of sensitivity (such as IR72) require further testing before continuous exposure to imidacloprid during germination can be recommended.

Microfluidic active loading of single cells enables analysis of complex clinical specimens.

A fundamental trade-off between flow rate and measurement precision limits performance of many single-cell detection strategies, especially for applications that require biophysical measurements from living cells within complex and low-input samples. To address this, we introduce 'active loading', an automated, optically-triggered fluidic system that improves measurement throughput and robustness by controlling entry of individual cells into a measurement channel. We apply active loading to samples over a range of concentrations (1-1000 particles µL-1), demonstrate that measurement time can be decreased by up to 20-fold, and show theoretically that performance of some types of existing single-cell microfluidic devices can be improved by implementing active loading. Finally, we demonstrate how active loading improves clinical feasibility for acute, single-cell drug sensitivity measurements by deploying it to a preclinical setting where we assess patient samples from normal brain, primary and metastatic brain cancers containing a complex, difficult-to-measure mixture of confounding biological debris.

Linking single-cell measurements of mass, growth rate, and gene expression.

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.