The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

The frequency of insecticide/acaricide target‐site resistance is increasing in arthropod pest populations and is typically underpinned by single point mutations that affect the binding strength between the insecticide/acaricide and its target‐site. Theory predicts that although resistance mutations clearly have advantageous effects under the selection pressure of the insecticide/acaricide, they might convey negative pleiotropic effects on other aspects of fitness. If such fitness costs are in place, target‐site resistance is thus likely to disappear in the absence of insecticide/acaricide treatment, a process that would counteract the spread of resistance in agricultural crops. Hence, there is a great need to reliably quantify the various potential pleiotropic effects of target‐site resistance point mutations on arthropod fitness. Here, we used near‐isogenic lines of the spider mite pest Tetranychus urticae that carry well‐characterized acaricide target‐site resistance mutations to quantify potential fitness costs. Specifically, we analyzed P262T in the mitochondrial cytochrome b, the combined G314D and G326E substitutions in the glutamate‐gated chloride channels, L1024V in the voltage‐gated sodium channel, and I1017F in chitin synthase 1. Five fertility life table parameters and nine single‐generation life‐history traits were quantified and compared across a total of 15 mite lines. In addition, we monitored the temporal resistance level dynamics of populations with different starting frequency levels of the chitin synthase resistant allele to further support our findings. Three target‐site resistance mutations, I1017F and the co‐occurring G314D and G326E mutations, were shown to significantly and consistently alter certain fitness parameters in T. urticae. The other two mutations (P262T and L1024V) did not result in any consistent change in a fitness parameter analyzed in our study. Our findings are discussed in the context of the global spread of T. urticae pesticide resistance and integrated pest management.

Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. Furthermore, available BFPs were not systematically characterized for imaging in cultured mammalian cells and common model organisms. Here we introduce a pair of new BFPs, named Electra1 and Electra2, developed through hierarchical screening in bacterial and mammalian cells using a novel dual-expression vector. We performed systematic benchmarking of Electras against state-of-art BFPs in cultured mammalian cells and demonstrated their utility as fluorescent tags for structural proteins. The Electras variants were validated for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice in comparison with one of the best in the class BFP mTagBFP2 using one-photon and two-photon microscopy. The developed BFPs are suitable for multicolor imaging of cultured cells and model organisms in vivo. We believe that the described dual-expression vector has a great potential to be adopted by protein engineers for directed molecular evolution of FPs.

The genus Gnaptor Brullé, 1983 (Blaptini, Gnaptorina) occurs in southeast Europe as well as in Asiatic regions. As regards its taxonomy, four morphological species have been attributed: Gnaptor boryi, G. prolixus, G. spinimanus and G. medvedevi. Here, we use two different mitochondrial genetic markers (16S and cytochrome c oxidase subunit 1 (COI)) in order to investigate the relationships between the populations of the species G. boryi in Greece, compare them with the current taxonomy and conjecture about its biogeographic history. In total, 29 specimens (28 G. boryi and one G. prolixus) were analyzed using maximum likelihood and Bayesian inference methods. Our results clarified the presence of three well-supported lineages: two belongs to G. boryi and one to G. prolixus. The first diversification of these lineages started in the Late Miocene at 9 Mya with the split of G. prolixus from Turkey and the second major split occurred in the Early Pliocene at 3.7 Mya between the two lineages of G. boryi distributed separately in northern Greece and Peloponnesos. According to Statistical Dispersal - Vicariance Analysis and dispersal-extinction-cladogenesis analysis analyses, vicariance seems to be the biogeographic event responsible for the divergence of the two major lineages of G. boryi.

Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.