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.     

The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.     

Synthesis and molecular mechanical studies of antiviral copper(II) complexes with morpholinebiguanide drug

The synthesis and characterization of new Cu(II) complexes with morpholinebiguanide drug are described. The complexes formed have been characterized by elemental analysis, IR, UV-VIS, conductance measurements, EPR spectra and magnetic susceptibility measurements. Their biological activity is supported by the experimental data and enhanced by the theoretical ones. The morpholinebiguanide ligand and their copper(II)-complexes were studied by molecular mechanics program.     

Multiple gene phylogenies support the monophyly of cryptomonad and haptophyte host lineages

Cryptomonad algae acquired their plastids by the secondary endosymbiotic uptake of a eukaryotic red alga. Several other algal lineages acquired plastids through such an event [1], but cryptomonads are distinguished by the retention of a relic red algal nucleus, the nucleomorph [2]. The nucleomorph (and its absence in other lineages) can reveal a great deal about the process and history of endosymbiosis, but only if we know the relationship between cryptomonads and other algae, and this has been controversial. Several recent analyses have suggested a relationship between plastids of cryptomonads and some or all other red alga-containing lineages [3-6], but we must also know whether host nuclear genes mirror this relationship to determine the number of endosymbiotic events, and this has not been demonstrated. We have carried out an expressed sequence tag (EST) survey of the cryptomonad Guillardia theta. Phylogenetic analyses of 102 orthologous nucleus-encoded proteins (18,425 amino acid alignment positions) show a robust sister-group relationship between cryptomonads and the haptophyte algae, which also have a red secondary plastid. This relationship demonstrates that loss of nucleomorphs must have taken place in haptophytes independently of any other red alga-containing lineages and that the ancestor of both already contained a red algal endosymbiont.     

Reduced mitochondrial Ca2+ transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca²⁺ homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca²⁺ uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca²⁺ homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.Mitochondrial disorders include a wide range of pathological conditions characterized by defects in organelle homoeostasis and energy metabolism, in particular in the electron transport chain (ETC) complexes. They are mostly caused by mutations in nuclear- or mtDNA-encoded genes of the respiratory chain complexes leading to a variety of clinical manifestations, ranging from lesions in specific tissues, such as in Leber''s hereditary optic neuropathy, to complex multisystem syndromes, such as myoclonic epilepsy with ragged-red fibers, Leigh syndrome or the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS).^{1, 2} Despite the detailed knowledge of the molecular defects in these diseases, their pathogenesis remains poorly understood. The heterogeneity of signs and symptoms depends on the diversity of the genetic background and on patient-specific compensatory mechanisms. Several studies investigated the consequences of nuclear DNA mutations on intracellular organelle physiology and Ca²⁺ homeostasis.^{3, 4} Here we analyzed a cohort of patients with mutations in the mtDNA-encoded ND5 subunit of NADH dehydrogenase in order to correlate the clinical phenotype with relevant intracellular parameters involved in mitochondrial physiology, such as the rate of autophagy and mitophagy fluxes, mitochondrial Ca²⁺ dynamics, mitochondrial membrane potential and their functional relationship.Mitochondrial Ca²⁺ is a key regulator of organelle physiology, and impairment of cation homeostasis is a general feature of many pathological conditions, including mitochondrial diseases.⁵ In addition, Ca²⁺ uptake in this organelle has recently been demonstrated to be a fundamental regulator of autophagy.^{6, 7} Autophagy is involved in physiological organelle turnover and in the removal of damaged or non-functional mitochondria by autophagy (called ‘mitophagy'')^{8, 9, 10, 11} and is critical for organelle quality control. Given the pivotal role of mitochondrial Ca²⁺ in the adaptation of adenosine triphosphate (ATP) production to cellular energy demand, the recent identification of the channel responsible for Ca²⁺ entry into the organelle, the mitochondrial Ca²⁺ uniporter (MCU), is instrumental for the understanding of the regulation of mitochondrial Ca²⁺ transport in both physiological and pathological conditions. MCU was identified in 2011,^{12, 13} and in the following years, molecular insight on its complex regulatory mechanism was obtained. The pore region is composed of MCU, its isoform MCUb¹⁴ and essential MCU regulator (EMRE).¹⁵ The channel is gated by the Ca²⁺-sensitive proteins mitochondrial Ca²⁺ uptake 1 (MICU1) and MICU2^{16, 17, 18, 19} and further regulated by the SLC25A23 protein.²⁰ As to its cellular function, mitochondrial Ca²⁺ has been shown to stimulate ATP production by positive regulation of three key dehydrogenases of the tricarboxylic acid cycle²¹ and of the ETC.²² In parallel, unregulated and sustained organelle Ca²⁺ overload can also lead to the opening of the mitochondrial permeability transition pore,^{23, 24} with consequent dissipation of mitochondrial membrane potential (ΔΨ_mt), release of caspase cofactors and activation of the apoptotic cascade.⁵ Despite the significant molecular understanding of all these cellular processes, their role in the pathogenesis of mitochondrial diseases is still poorly understood. Here we investigated the interplay of these pathways and the possibility of their contribution to determine the severity of the pathology in a cellular model consisting of fibroblasts from patients carrying mutations in the mitochondrial ND5 gene.     

A tertiary plastid uses genes from two endosymbionts

The origin and subsequent spread of plastids by endosymbiosis had a major environmental impact and altered the course of a great proportion of eukaryotic biodiversity. The ancestor of dinoflagellates contained a secondary plastid that was acquired in an ancient endosymbiotic event, where a eukaryotic cell engulfed a red alga. This is known as secondary endosymbiosis and has happened several times in eukaryotic evolution. Certain dinoflagellates, however, are unique in having replaced this secondary plastid in an additional (tertiary) round of endosymbiosis. Most plastid proteins are encoded in the nucleus of the host and are targeted to the organelle. When secondary or tertiary endosymbiosis takes place, it is thought that these genes move from nucleus to nucleus, so the plastid retains the same proteome. We have conducted large-scale expressed sequence tag (EST) surveys from Karlodinium micrum, a dinoflagellate with a tertiary haptophyte-derived plastid, and two haptophytes, Isochrysis galbana and Pavlova lutheri. We have identified all plastid-targeted proteins, analysed the phylogenetic origin of each protein, and compared their plastid-targeting transit peptides. Many plastid-targeted genes in the Karlodinium nucleus are indeed of haptophyte origin, but some genes were also retained from the original plastid (showing the two plastids likely co-existed in the same cell), in other cases multiple isoforms of different origins exist. We analysed plastid-targeting sequences and found the transit peptides in K.micrum are different from those found in either dinoflagellates or haptophytes, pointing to a plastid with an evolutionarily chimeric proteome, and a massive remodelling of protein trafficking during plastid replacement.     

Activity of natural toxins against the silverleaf whitefly, Bemisia argentifolii, using a novel feeding bioassay system

An assay system was developed for the adult silverleaf whitefly, Bemisia argentifolii Bellows & Perring (Homoptera: Aleyrodidae). This practical device was constructed from standard disposable laboratory materials. Whiteflies were harvested directly from the leaf and into a collection vial by vacuum aspiration, minimizing physical damage to the insect. Insects were fed through a cellulose mixed-ester membrane on a diet of 20–27% sucrose alone or sucrose in an extract of zucchini (Curcurbita moschata Duchense). Mortality and honeydew production were scored. At 22–25°C and 50–55% relative humidity, control mortality generally remained at or below 15% during a 48 h assay period. The bioassay system was first tested using the insecticide, Imidacloprid, then used to screen a number of natural products with potential insecticidal activity against the whitefly. Destruxins extracted from the entomopathogenic fungus, Metarhizium anisopliae, and the natural insecticide/nematicide, Ivermectin, as well as bee venom and two of its components, melittin and phospholipase A₂, were found to be toxic to B. argentifolii. Five lectins, Bacillus thuringiensis toxins, gossypol, an extract of Paeciliomyces fumosoroseus, wasp and scorpion venom, and a trypsin inhibitor were not found to be insecticidal to adult B. argentifolii.     

Improving Managers’ Psychophysical Well-Being: Effectiveness of Respiratory Sinus Arrhythmia Biofeedback

High work stress has been consistently associated with disturbed autonomic balance, specifically, lowered vagal cardiac control and increased sympathetic activity, which may lead to increased cardiovascular risk. Stress management procedures have been proposed to reduce autonomic dysfunctions related to work stress in different categories of workers exposed to heightened work demands, while a limited number of studies addressed this issue in managers. The present study was aimed at evaluating the effectiveness of a respiratory sinus arrhythmia (RSA) biofeedback (BF) intervention on psychological and physiological outcomes, in managers with high-level work responsibilities. Thirty-one managers leading outstanding private or public companies were randomly assigned to either a RSA-BF training (RSA-BF; N = 16) or a control group (N = 15). The RSA-BF training consisted of five weekly 45 min sessions, designed to increase RSA, whereas controls had to provide a daily stress diary once a week. After the training, managers in both groups reported reduced heart rate at rest, lower anxiety levels and improvement in health-related quality of life. More importantly, managers in the RSA-BF group showed increased vagal control (as indexed by increased RSA), decreased sympathetic arousal (as indexed by reduced skin conductance and systolic blood pressure) and lower emotional interferences, compared to managers in the control group. Results from this study showed that RSA-BF training was effective in improving cardiac autonomic balance at rest. Moreover, findings from this study underline the effectiveness of biofeedback in reducing psychophysiological negative outcomes associated with stress in managers.     

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

Background  

Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.     

COMMON EVOLUTIONARY ORIGIN OF STARCH BIOSYNTHETIC ENZYMES IN GREEN AND RED ALGAE1

Plastidic starch synthesis in green algae and plants occurs via ADP‐glucose in likeness to prokaryotes from which plastids have evolved. In contrast, floridean starch synthesis in red algae proceeds via uridine diphosphate‐glucose in semblance to eukaryotic glycogen synthesis and occurs in the cytosol rather than the plastid. Given the monophyletic origin of all plastids, we investigated the origin of the enzymes of the plastid and cytosolic starch synthetic pathways to determine whether their location reflects their origin—either from the cyanobacterial endosymbiont or from the eukaryotic host. We report that, despite the compartmentalization of starch synthesis differing in green and red lineages, all but one of the enzymes of the synthetic pathways shares a common origin. Overall, the pathway of starch synthesis in both lineages represents a chimera of the host and endosymbiont glycogen synthesis pathways. Moreover, host‐derived proteins function in the plastid in green algae, whereas endosymbiont‐derived proteins function in the cytosol in red algae. This complexity demonstrates the impacts of integrating pathways of host with those of both primary and secondary endosymbionts during plastid evolution.