Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites

Point mutations in the voltage-gated sodium channel gene associated with pyrethroid resistance in Iranian populations of the European red mite Panonychus ulmi

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maracieh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F15381, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field

Molecular and genetic analysis of resistance to METI-I acaricides in Iranian populations of the citrus red mite Panonychus citri

The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found

Lipids revert inert Aβ amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Aβ-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Aβ-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds ‘forward' in a near-irreversible manner from the monomeric Aβ peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Aβ amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Aβ but rather toward soluble amyloid protofibrils. We characterized these ‘backward' Aβ protofibrils generated from mature Aβ fibers and compared them with previously identified ‘forward' Aβ protofibrils obtained from the aggregation of fresh Aβ monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Aβ-aggregates that could readily be activated by exposure to biological lipids

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism

The salivary proteome of Tetranychus urticae : key to its polyphagous nature?

The herbivorous spider mite Tetranychus urticae (Acari: Tetranychidae) is notorious for having an extremely large host plant range. Being this polyphagous is exceptional since plants possess various defensive mechanisms, including the production of toxins. Herbivores need to be able to deal with these defenses in order to thrive on a specific plant. This requires adaptation, often leading to specialization. Nevertheless, T. urticae, as a species, has been recorded on plants belonging to more than 140 families, and must therefore have evolved a variety of mechanisms allowing it to cope with a diverse arsenal of plant defenses. In arthropod herbivores, the saliva has been proposed as an important mediator of digestion, detoxification and defense suppression. In this PhD thesis, the composition of spider mite saliva was investigated using a multidisciplinary approach based on in silico prediction and proteomics analysis, combined with proterosomal expression analysis and confirmation of the salivary origin using whole-mount in situ hybridization. A list of almost one hundred T. urticae salivary proteins is presented, which will provide the foundation for a more detailed understanding of the mite-plant interaction at the molecular level. Some salivary proteins were shown to act as “effectors”, manipulating the host defense response. An OrthoMCL analysis further shed some light on the salivary proteome of the related specialists T. evansi and T. lintearius. Finally, a new spider mite-specific salivary protein family (denominated the SHOT family) is identified, showing a clear link with specialization to certain host plants. It is concluded that the salivary protein complement of T. urticae is key to its polyphagous nature

3D vibro-acoustic simulations of problems with damping layers using the Wave Based Method and its hybrid extensions

Present-day CAE tools for 3D vibro-acoustic simulation are often based on methods such as the Finite Element Method (FEM). However, these methods are in practice limited to low-frequency simulations due to the strongly increasing computational cost with frequency. The family of the Wave Based Method (WBM) and hybrid extensions can effectively lower this computational cost and allows efficient simulations of vibro-acoustic systems at higher frequencies.status: accepte

Modelling of sound damping multi-layers using a hybrid Finite Element - Wave Based Method

To assess and optimise the effect of multi-layered trim for the reduction of noise and vibration transmission, design engineers greatly depend on CAE tools. Mostly, these are based on element based simulation techniques such as the Finite Element Method (FEM). In practise, however, these methods are limited to low-frequency simulations due to the strongly increasing computational cost with frequency. They lose even more of their applicable frequency range when used for coupled acoustic-poro-elastic analysis. With the current simulation technologies, it is very challenging to solve a full FEM-model for the damped vibro-acoustic system into the mid-frequency domain. The Wave Based Method (WBM) is very well suited for this full system modelling, since it has a very good convergence rate as compared to the FEM. On the other hand, the WBM shows its efficiency best for moderately complex geometries, where the domain can be divided into a (small) number of convex subdomains. To overcome this limitation, hybrid Finite Element - Wave Based approaches have been developed and explored for coupling different physical media; acoustic, structural and structural-acoustic couplings can be tackled. This contribution extends this family of hybrid FE-WBM methods towards the coupling of acoustic WBM models with poro-elastic FEM models. In this way, the method benefits from the computational efficiency of the Wave Based Method for the acoustic calculations without losing the Finite Element Method’s ability to model the often layered and complexly shaped poroelastic materials in great detail.status: publishe

A Multi-Level Wave Based Method to predict the dynamic response of 2D poroelastic materials containing inclusions

Poroelastic materials are often applied as eective noise measures. They are, however, most eective at higher frequencies. A lot of research has been performed to increase absorption also at lower frequencies. A promising solution is to add inhomogeneities to the foams, being inclusions or perforations. Recently, a Wave Based Method was developed to predict the dynamic response of poroelastic materials, described by the theory of Biot. This Trefftz approach was shown to be very effective for geometrically simple problems. A sufficient condition for the method to converge is that the considered problem domain is convex. Non-convex domains have to be partitioned into convex subdomains. Consequently, domains with circular inclusions cannot be accurately accounted for with the Wave Based Method; this problem was overcome with the so-called Multi-Level framework, which was introduced for acoustic and structural dynamic problems. This paper extends the Multi-Level approach to efficiently account for inclusions in a poroelastic material. The method is validated through comparison to the Finite Element Method.status: accepte