Improved methodology for fixation and preparation of Aedes aegypti embryos.

The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies.

Conserved and novel enhancers in the Aedes aegypti single-minded locus recapitulate embryonic ventral midline gene expression.

Transcriptional cis-regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A. aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves.

Multiple-P450 Gene Co-Up-Regulation in the Development of Permethrin Resistance in the House Fly, Musca domestica.

This paper reports a study conducted at the whole transcriptome level to characterize the P450 genes involved in the development of pyrethroid resistance, utilizing expression profile analyses of 86 cytochrome P450 genes in house fly strains with different levels of resistance to pyrethroids/permethrin. Interactions among the up-regulated P450 genes and possible regulatory factors in different autosomes were examined in house fly lines with different combinations of autosomes from a resistant house fly strain, ALHF. Eleven P450 genes that were significantly up-regulated, with levels > 2-fold those in the resistant ALHF house flies, were in CYP families 4 and 6 and located on autosomes 1, 3 and 5. The expression of these P450 genes was regulated by trans- and/or cis-acting factors, especially on autosomes 1 and 2. An in vivo functional study indicated that the up-regulated P450 genes also conferred permethrin resistance in Drosophila melanogaster transgenic lines. An in vitro functional study confirmed that the up-regulated P450 genes are able to metabolize not only cis- and trans-permethrin, but also two metabolites of permethrin, PBalc and PBald. In silico homology modeling and the molecular docking methodology further support the metabolic capacity of these P450s for permethrin and substrates. Taken together, the findings of this study highlight the important function of multi-up-regulated P450 genes in the development of insecticide resistance in house flies.

Assessing organ-level immunoreactivity in a rat model of sepsis using TSPO PET imaging.

There is current need for new approaches to assess/measure organ-level immunoreactivity and ensuing dysfunction in systemic inflammatory response syndrome (SIRS) and sepsis, in order to protect or recover organ function. Using a rat model of systemic sterile inflammatory shock (intravenous LPS administration), we performed PET imaging with a translocator protein (TSPO) tracer, [18F]DPA-714, as a biomarker for reactive immunoreactive changes in the brain and peripheral organs. In vivo dynamic PET/CT scans showed increased [18F]DPA-714 binding in the brain, lungs, liver and bone marrow, 4 hours after LPS injection. Post-LPS mean standard uptake values (SUVmean) at equilibrium were significantly higher in those organs compared to baseline. Changes in spleen [18F]DPA-714 binding were variable but generally decreased after LPS. SUVmean values in all organs, except the spleen, positively correlated with several serum cytokines/chemokines. In vitro measures of TSPO expression and immunofluorescent staining validated the imaging results. Noninvasive molecular imaging with [18F]DPA-714 PET in a rat model of systemic sterile inflammatory shock, along with in vitro measures of TSPO expression, showed brain, liver and lung inflammation, spleen monocytic efflux/lymphocytic activation and suggested increased bone marrow hematopoiesis. TSPO PET imaging can potentially be used to quantify SIRS and sepsis-associated organ-level immunoreactivity and assess the effectiveness of therapeutic and preventative approaches for associated organ failures, in vivo.

Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, ß2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence.

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1's regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

Eurythenes atacamensis sp. nov. (Crustacea: Amphipoda) exhibits ontogenetic vertical stratification across abyssal and hadal depths in the Atacama Trench, eastern South Pacific Ocean.

Eurythenes S.I. Smith in Scudder, 1882 (Crustacea: Amphipoda) are prevalent scavengers of the benthopelagic community from bathyal to hadal depths. While a well-studied genus, molecular systematic studies have uncovered cryptic speciation and multiple undescribed lineages. Here, we apply an integrative taxonomic approach and describe the tenth species, Eurythenes atacamensis sp. nov., based on specimens from the 2018 Atacamex and RV Sonne SO261 Expeditions to the southern sector of the Peru-Chile Trench, the Atacama Trench (24-⁠21°S). Eurythenes atacamensis sp. nov. is a large species, max. observed length 83.2 mm, possesses diagnostic features, including a short gnathopod 1 palm and a chelate gnathopod 2 palm, and a distinct genetic lineage based on a 16S rRNA and COI phylogeny. This species is a dominant bait-attending fauna with an extensive bathymetric range, spanning from 4974 to 8081 m. The RV Sonne SO261 specimens were recovered along a 10-station transect from abyssal to hadal depths and further examined for demographic and bathymetric-related patterns. Ontogenetic vertical stratification was evident across the trench axis, with only juveniles present at abyssal depths (4974-6025 m). Total length-depth analysis revealed that the size of females was unrelated to depth, whereas juveniles followed a sigmoidal relationship with a step-up in size at depths >7200 m. Thus, these bathymetric trends suggest that juveniles and females employ differing ecological strategies in subduction trench environments. This study highlights that even dominant and ecologically important species are still being discovered within the abyssal and hadal environments. Continued systematic expeditions will lead to an improved understanding of the eco-evolutionary drivers of speciation in the world's largest ecosystem.

Synthesis of 1,1-Diboryl Alkenes Using the Boryl-Heck Reaction.

The synthesis of 1,1-diboryl alkenes from terminal alkenes is reported. 1,1-Regioselective addition is observed for both conjugated and unconjugated alkenes, allowing for a single method to prepare a wide range of 1,1-diboryl alkenes.

Current Effector and Gene-Drive Developments to Engineer Arbovirus-Resistant Aedes aegypti (Diptera: Culicidae) for a Sustainable Population Replacement Strategy in the Field.

Arthropod-borne viruses (arboviruses) such as dengue, Zika, and chikungunya viruses cause morbidity and mortality among human populations living in the tropical regions of the world. Conventional mosquito control efforts based on insecticide treatments and/or the use of bednets and window curtains are currently insufficient to reduce arbovirus prevalence in affected regions. Novel, genetic strategies that are being developed involve the genetic manipulation of mosquitoes for population reduction and population replacement purposes. Population replacement aims at replacing arbovirus-susceptible wild-type mosquitoes in a target region with those that carry a laboratory-engineered antiviral effector to interrupt arboviral transmission in the field. The strategy has been primarily developed for Aedes aegypti (L.), the most important urban arbovirus vector. Antiviral effectors based on long dsRNAs, miRNAs, or ribozymes destroy viral RNA genomes and need to be linked to a robust gene drive to ensure their fixation in the target population. Synthetic gene-drive concepts are based on toxin/antidote, genetic incompatibility, and selfish genetic element principles. The CRISPR/Cas9 gene editing system can be configurated as a homing endonuclease gene (HEG) and HEG-based drives became the preferred choice for mosquitoes. HEGs are highly allele and nucleotide sequence-specific and therefore sensitive to single-nucleotide polymorphisms/resistant allele formation. Current research efforts test new HEG-based gene-drive designs that promise to be less sensitive to resistant allele formation. Safety aspects in conjunction with gene drives are being addressed by developing procedures that would allow a recall or overwriting of gene-drive transgenes once they have been released.

Mycotoxins causing amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) remains a terminal disease without an established etiology for the majority of patients. The dominant theory of ALS before the 1970's was the presence of a poison. One of the primary means of treating patients with a toxic exposure has been plasma exchange, but plasma exchange of ALS patients failed to alter the clinical course. The failure of plasma exchange assumes the patient is no longer exposed to the poison. If the exposure to poison continued, then plasma exchange alone would fail. I found laboratory evidence of a poisoning in every patient with ALS examined. A search for specific poisons found evidence of mycotoxins. Treatment with antifungal agents corrected the laboratory findings. All of the ALS patients had evidence of immune suppression. There is mounting evidence that many mycotoxins cause both neurotoxicity and immune suppression. These mycotoxins may be able to explain the full spectrum of pathology in ALS without a secondaryevent.

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti.

The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015-2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.

Diffusion Tensor Imaging and Chemical Exchange Saturation Transfer MRI Evaluation on the Long-Term Effects of Pulsed Focused Ultrasound and Microbubbles Blood Brain Barrier Opening in the Rat.

Blood-brain barrier opening (BBBO) with pulsed Focused Ultrasound (pFUS) and microbubbles (MB) has received increasing interest as a method for neurotherapeutics of the central nervous system. In general, conventional MRI [i.e., T2w, T2∗w, gadolinium (Gd) enhanced T1w] is used to monitor the effects of pFUS+MB on BBBO and/or assess whether sonication results in parenchymal damage. This study employed multimodal MRI techniques and 18F-Fludeoxyglucose (FDG) PET to evaluate the effects of single and multiple weekly pFUS+MB sessions on morphology and glucose utilization levels in the rat cortex and hippocampus. pFUS was performed with 0.548 MHz transducer with a slow infusion over 1 min of OptisonTM (5-8 × 107 MB) in nine focal points in cortex and four in hippocampus. During pFUS+MB treatment, Gd-T1w was performed at 3 T to confirm BBBO, along with subsequent T2w, T2∗w, DTI and glucose CEST (glucoCEST)-weighted imaging by high field 9.4 T and compared with FDG-PET and immunohistochemistry. Animals receiving a single pFUS+MB exhibited minimal hypointense voxels on T2∗w. Brains receiving multiple pFUS+MB treatments demonstrated persistent T2w and T2∗ abnormalities associated with changes in DTI and glucoCEST when compared to contralateral parenchyma. Decreased glucoCEST contrast was substantiated by FDG-PET in cortex following multiple sonications. Immunohistochemistry showed significantly dilated vessels and decreased neuronal glucose transporter (GLUT3) expression in sonicated cortex and hippocampus without changes in neuronal counts. These results suggest the importance to standardize MRI protocols in concert with advanced imaging techniques when evaluating long term effects of pFUS+MB BBBO in clinical trials for neurological diseases.

A comparison of fatty acid and sensory profiles of raw and roasted pecan cultivars.

Five fatty acids comprise the bulk of the lipid content in pecans: palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Understanding the profiles of these fatty acids and how they relate to sensory characteristics may offer an explanation for flavor and flavor defects that may exist in certain cultivars of pecans. The objective of this study was to examine and compare fatty acid profiles of three cultivars of pecans (Major, Lakota, and Chetopa), over two crop years, under raw and roasted preparation methods, and understand the fatty acids association with sensory attributes. Percentages of palmitic, stearic, oleic, linoleic, and linolenic acids to total fatty acid content were determined using gas chromatography, and sensory profiles were generated using descriptive sensory analysis. Similar trends were seen across samples, with oleic acid comprising the majority of the total fatty acids and linolenic acid comprising the smallest percentage. There were significant differences in fatty acid content among cultivars and between pecans in the first and second crop year. Few associations were found between the fatty acids and sensory attributes, which suggest that combinations of the fatty acids contribute to certain pleasant or undesirable flavor attributes in the pecans. Subtle differences in fatty acid composition may lead to variation in flavor and flavor intensity or draw attention to or from certain attributes during consumption. Differences in crop year indicated that fatty acid content and therefore flavor are variable year to year. PRACTICAL APPLICATION: This study will help understand how fatty acid content of pecans varies from year to year. This should be taken into account when manufacturing products with pecans as the nutritional content of the product may change as the result.

New species of Eurythenes from hadal depths of the Mariana Trench, Pacific Ocean (Crustacea: Amphipoda).

Eurythenes S. I. Smith in Scudder, 1882 are one of the largest scavenging deep-sea amphipods (max. 154 mm) and are found in every ocean across an extensive bathymetric range from the shallow polar waters to hadal depths. Recent systematic studies of the genus have illuminated a cryptic species complex and highlighted the benefits of using a combination of morphological and molecular identification approaches. In this study, we present the ninth species, Eurythenes plasticus sp. nov., which was recovered using baited traps between the depths 6010 and 6949 m in the Mariana Trench (Northwest Pacific Ocean) in 2014. This new Eurythenes species was found to have distinct morphological characteristics and be a well-supported clade based on sequence variation at two mitochondrial regions (16S rDNA and COI). While this species is new to science and lives in the remote hadal zone, it is not exempt from the impacts of anthropogenic pollution. Indeed, one individual was found to have a microplastic fibre, 83.74% similar to polyethylene terephthalate (PET), in its hindgut. As this species has a bathymetric range spanning from abyssal to hadal depths in the Central Pacific Ocean basin, it offers further insights into the biogeography of Eurythenes.

Genetic Variation and Potential for Resistance Development to the tTA Overexpression Lethal System in Insects.

Release of insect pests carrying the dominant lethal tetracycline transactivator (tTA) overexpression system has been proposed as a means for population suppression. High levels of the tTA transcription factor are thought to be toxic due to either transcriptional squelching or interference with protein ubiquitination. Here we utilized the Drosophila melanogaster Genetic Reference Panel (DGRP) to examine the influence of genetic variation on the efficacy of a female-specific tTA overexpression system. The level of female lethality between DGRP lines varied from 11 to 97% with a broad sense heritability of 0.89. A genome-wide association analysis identified 192 allelic variants associated with high or low lethality (P < 10-5), although none were significant when corrected for multiple testing. 151 of the variants fell within 108 genes that were associated with several biological processes including transcription and protein ubiquitination. In four lines with high female lethality, tTA RNA levels were similar or higher than in the parental tTA overexpression strain. In two lines with low lethality, tTA levels were about two fold lower than in the parental strain. However, in two other lines with low lethality, tTA levels were similar or approximately 30% lower. RNAseq analysis identified genes that were up or downregulated in the four low female lethal lines compared to the four high lethal lines. For example, genes associated with RNA processing and rRNA maturation were significantly upregulated in low lethal lines. Our data suggest that standing genetic variation in an insect population could provide multiple mechanisms for resistance to the tTA overexpression system.

Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by Aedes aegypti.

The mosquito vector Aedes aegypti transmits arthropod-borne viruses (arboviruses) of medical importance, including Zika, dengue, and yellow fever viruses. Controlling mosquito populations remains the method of choice to prevent disease transmission. Novel mosquito control strategies based on genetically manipulating mosquitoes are being developed as additional tools to combat arbovirus transmission. Genetic control of mosquitoes includes two basic strategies: population suppression and population replacement. The former aims to eliminate mosquito populations while the latter aims to replace wild populations with engineered, pathogen-resistant mosquitoes. In this review, we outline suppression strategies being applied in the field, as well as current antiviral effector genes that have been characterized and expressed in transgenic Ae. aegypti for population replacement. We discuss cutting-edge gene drive technologies that can be used to enhance the inheritance of effector genes, while highlighting the challenges and opportunities associated with gene drives. Finally, we present currently available models that can estimate mosquito release numbers and time to transgene fixation for several gene drive systems. Based on the recent advances in genetic engineering, we anticipate that antiviral transgenic Ae. aegypti exhibiting gene drive will soon emerge; however, close monitoring in simulated field conditions will be required to demonstrate the efficacy and utility of such transgenic mosquitoes.

Fauna of the Kemp Caldera and its upper bathyal hydrothermal vents (South Sandwich Arc, Antarctica).

Faunal assemblages at hydrothermal vents associated with island-arc volcanism are less well known than those at vents on mid-ocean ridges and back-arc spreading centres. This study characterizes chemosynthetic biotopes at active hydrothermal vents discovered at the Kemp Caldera in the South Sandwich Arc. The caldera hosts sulfur and anhydrite vent chimneys in 1375-1487 m depth, which emit sulfide-rich fluids with temperatures up to 212°C, and the microbial community of water samples in the buoyant plume rising from the vents was dominated by sulfur-oxidizing Gammaproteobacteria. A total of 12 macro- and megafaunal taxa depending on hydrothermal activity were collected in these biotopes, of which seven species were known from the East Scotia Ridge (ESR) vents and three species from vents outside the Southern Ocean. Faunal assemblages were dominated by large vesicomyid clams, actinostolid anemones, Sericosura sea spiders and lepetodrilid and cocculinid limpets, but several taxa abundant at nearby ESR hydrothermal vents were rare such as the stalked barnacle Neolepas scotiaensis. Multivariate analysis of fauna at Kemp Caldera and vents in neighbouring areas indicated that the Kemp Caldera is most similar to vent fields in the previously established Southern Ocean vent biogeographic province, showing that the species composition at island-arc hydrothermal vents can be distinct from nearby seafloor-spreading systems. δ 13C and δ 15N isotope values of megafaunal species analysed from the Kemp Caldera were similar to those of the same or related species at other vent fields, but none of the fauna sampled at Kemp Caldera had δ 13C values, indicating nutritional dependence on Epsilonproteobacteria, unlike fauna at other island-arc hydrothermal vents.

Gold-Catalyzed C-H Functionalization with Aryl Germanes.

The development of orthogonal Csp2 -Csp2 coupling regimes to the omnipresent Pd-catalysis class would enable an additional dimension of modularity in the construction of densely functionalized biaryl motifs. In this context, the identification of potent functional groups for selective transformations is in high demand. Although organogermanium compounds are generally believed to be of low reactivity in homogenous catalysis, this report discloses the highly efficient and orthogonal reactivity of aryl germanes with arenes under gold catalysis. The method is characterized by mildness, the employment of an air- and moisture-stable gold catalyst, and robustness. Our mechanistic studies show that aryl germanes are highly reactive with Au(I) and Au(III). Our computational data suggest that the origin of this reactivity primarily lies in the relatively low bond dissociation energy and as such low distortion energy to reach the key bond activating transition state.