Insect pest management in the age of synthetic biology

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.     

Methods to account for spatial autocorrelation in the analysis of species distributional data: a review

Species distributional or trait data based on range map (extent‐of‐occurrence) or atlas survey data often display spatial autocorrelation, i.e. locations close to each other exhibit more similar values than those further apart. If this pattern remains present in the residuals of a statistical model based on such data, one of the key assumptions of standard statistical analyses, that residuals are independent and identically distributed (i.i.d), is violated. The violation of the assumption of i.i.d. residuals may bias parameter estimates and can increase type I error rates (falsely rejecting the null hypothesis of no effect). While this is increasingly recognised by researchers analysing species distribution data, there is, to our knowledge, no comprehensive overview of the many available spatial statistical methods to take spatial autocorrelation into account in tests of statistical significance. Here, we describe six different statistical approaches to infer correlates of species’ distributions, for both presence/absence (binary response) and species abundance data (poisson or normally distributed response), while accounting for spatial autocorrelation in model residuals: autocovariate regression; spatial eigenvector mapping; generalised least squares; (conditional and simultaneous) autoregressive models and generalised estimating equations. A comprehensive comparison of the relative merits of these methods is beyond the scope of this paper. To demonstrate each method's implementation, however, we undertook preliminary tests based on simulated data. These preliminary tests verified that most of the spatial modeling techniques we examined showed good type I error control and precise parameter estimates, at least when confronted with simplistic simulated data containing spatial autocorrelation in the errors. However, we found that for presence/absence data the results and conclusions were very variable between the different methods. This is likely due to the low information content of binary maps. Also, in contrast with previous studies, we found that autocovariate methods consistently underestimated the effects of environmental controls of species distributions. Given their widespread use, in particular for the modelling of species presence/absence data (e.g. climate envelope models), we argue that this warrants further study and caution in their use. To aid other ecologists in making use of the methods described, code to implement them in freely available software is provided in an electronic appendix.     

Biological evaluation of newly synthesized quinoline-5,8-quinones as Cdc25B inhibitors

Cdc25B protein phosphatase represents an attractive potential therapeutic target for small molecule intervention because of its central role in positively regulating cyclin dependent kinases and thus cell proliferation, as well as its elevated levels observed in many human tumors. Among the most potent previously identified Cdc25 inhibitors have been quinoline quinones, which have a rich legacy as therapeutic agents but have also been associated with nonspecific interactions. In this study, we have interrogated the structure-activity relationship of a focused series of C2-, C3-, or C4-modified quinoline-5,8-quinones on Cdc25B inhibition in vitro. Substitution at the C3-position in this small chemical series were slightly superior to substitutions at the C3-position. For all compounds, recombinant human Cdc25B was approximately 5-fold more sensitive compared to recombinant human PTP1B. Two compounds inhibited HeLa cell growth with IC50 values of approximately 2 microM. Consistent with other para-quinones, some members of this series generated intracellular reactive oxygen species and the in vitro enzyme inhibition was mitigated by addition of reductants or catalase. These results indicate that chemical modifications on the pyridine core are tolerated, providing additional sites for future structural modification of this biologically active pharmacophore.     

Carriers of Loss-of-Function Mutations in EXT Display Impaired Pancreatic Beta-Cell Reserve Due to Smaller Pancreas Volume

Exotosin (EXT) proteins are involved in the chain elongation step of heparan sulfate (HS) biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF) in EXT1 and EXT2 result in hereditary exostoses (HME). Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME) affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT) followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS) in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI). OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46–1.16] vs. controls 1.53 [0.69–3.36] nmol·l⁻¹·min⁻¹, p<0.05). Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22–10.5] vs. controls 10.2 [7.91–12.70] nmol·l⁻¹·min⁻¹ p<0.05), indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm³ p = 0.04). In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.     

Cestode systematics and phylogeny move forward

This paper represents a meeting report for the Fifth International Workshop on Cestode Systematics and Phylogeny held at the Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budějovice, 18–22 July 2005. The major topics discussed included (i) the progress in cestode systematics during 2002–2005, (ii) the use of the life-cycle data in phylogenetic studies, (iii) the utilisation of new morphological and molecular characters in cestode systematics and phylogeny, and (iv) the ongoing work on the completion of the Global Cestode Database.     

A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.     

Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

A protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.     

Optimization and simulation of continuous affinity-recycle extraction (care).

Simulation and optimization of continuous affinity recycle extraction (CARE), a protein purification unit operation based on protein adsorption to solid phase adsorbents, is described in this paper. Rather than packing conventional adsorbent particles in a fixed bed (column), solid/liquid contact is carried out in well-mixed reactors. Continuous operation is achieved by recirculation of the adsorbent particles between two or more contactors. The feasibility of this purification scheme was established with the recovery and isolation of the enzyme beta-galactosidase from E.coli, using the affinity support PABTG/Agarose. A mathematical model describing system performance was developed. The mathematical model was used to optimize several facets of the system design and operation. The base two-stage contractor design was modified by the addition of an intermediate wash stage as well as the incorporation of multiple adsorption stages. These design modifications serve to increase purification, concentration and recovery while utilizing the same amount of adsorbent. The methodology for defining and optimizing objective functions was developed and experimentally validated. Finally, optimum system start-up protocols, minimizing the time required to reach steady-state operation, were developed and experimentally validated. The impact of early introduction of adsorptive purification in a downstream processing sequence, with CARE, was evaluated and is described. Through the early introduction of a highly specific adsorptive step, significant purification is achieved simultaneously with clarification and concentration. In addition, purification performance in CARE was contrasted with that achievable in conventional column chromatography.     

The Influence of Antimicrobial Peptides and Mucolytics on the Integrity of Biofilms Consisting of Bacteria and Yeasts as Affecting Voice Prosthetic Air Flow Resistances

The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech. Biofilm integrity is ensured by extracellular polymeric substances (EPS). This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms. Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan). Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses after biofilm formation. Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance. Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.