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      A stationary tweezer platform for high throughput dissections of minute arthropods and extirpation of their minute organs

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          Abstract

          A homemade platform satisfied the need for fast, efficient dissection of minute arthropods and extirpation of their key organs, such as salivary glands and midguts, involved in agricultural disease transmission pathways. With its implementation, ~200 organs could be extirpated per 8 h workday while the subjects are submerged in protein or transcript protectant. A vacuum wand is used to capture insects and position them in the field of view. Two stationary tweezers are positioned on an adjustable scaffold that spans the microscope stage transversely such that their tips, and the insects they immobilize, can be submerged in select dissection media. High tensile strength fishlines are attached to the stationary tweezers for opening and closing with the 5th fingers while hand-held dissection tweezers load insects from the wand to their tines, then extirpate the target organs. Organs are lifted out with glass splints or plastic toothpicks into a final tube of select preservation media for freezing at session end.

          • Constructed from common retail materials

          • Adjustable design fits many microscopes

          • Can also be used in a wide variety of applications, including materials science

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          Most cited references13

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          Transcriptomic Analysis of the Salivary Glands of an Invasive Whitefly

          Background Some species of the whitefly Bemisia tabaci complex cause tremendous losses to crops worldwide through feeding directly and virus transmission indirectly. The primary salivary glands of whiteflies are critical for their feeding and virus transmission. However, partly due to their tiny size, research on whitefly salivary glands is limited and our knowledge on these glands is scarce. Methodology/Principal Findings We sequenced the transcriptome of the primary salivary glands of the Mediterranean species of B. tabaci complex using an effective cDNA amplification method in combination with short read sequencing (Illumina). In a single run, we obtained 13,615 unigenes. The quantity of the unigenes obtained from the salivary glands of the whitefly is at least four folds of the salivary gland genes from other plant-sucking insects. To reveal the functions of the primary glands, sequence similarity search and comparisons with the whole transcriptome of the whitefly were performed. The results demonstrated that the genes related to metabolism and transport were significantly enriched in the primary salivary glands. Furthermore, we found that a number of highly expressed genes in the salivary glands might be involved in secretory protein processing, secretion and virus transmission. To identify potential proteins of whitefly saliva, the translated unigenes were put into secretory protein prediction. Finally, 295 genes were predicted to encode secretory proteins and some of them might play important roles in whitefly feeding. Conclusions/Significance: The combined method of cDNA amplification, Illumina sequencing and de novo assembly is suitable for transcriptomic analysis of tiny organs in insects. Through analysis of the transcriptome, genomic features of the primary salivary glands were dissected and biologically important proteins, especially secreted proteins, were predicted. Our findings provide substantial sequence information for the primary salivary glands of whiteflies and will be the basis for future studies on whitefly-plant interactions and virus transmission.
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            Comparative transcriptomics and proteomics of three different aphid species identifies core and diverse effector sets

            Background Aphids are phloem-feeding insects that cause significant economic losses to agriculture worldwide. While feeding and probing these insects deliver molecules, called effectors, inside their host to enable infestation. The identification and characterization of these effectors from different species that vary in their host range is an important step in understanding the infestation success of aphids and aphid host range variation. This study employs a multi-disciplinary approach based on transcriptome sequencing and proteomics to identify and compare effector candidates from the broad host range aphid Myzus persicae (green peach aphid) (genotypes O, J and F), and narrow host range aphids Myzus cerasi (black cherry aphid) and Rhopalosiphum padi (bird-cherry oat aphid). Results Using a combination of aphid transcriptome sequencing on libraries derived from head versus body tissues as well as saliva proteomics we were able to predict candidate effectors repertoires from the different aphid species and genotypes. Among the identified conserved or core effector sets, we identified a significant number of previously identified aphid candidate effectors indicating these proteins may be involved in general infestation strategies. Moreover, we identified aphid candidate effector sequences that were specific to one species, which are interesting candidates for further validation and characterization with regards to species-specific functions during infestation. We assessed our candidate effector repertoires for evidence of positive selection, and identified 49 candidates with DN/DS ratios >1. We noted higher rates of DN/DS ratios in predicted aphid effectors than non-effectors. Whether this reflects positive selection due to co-evolution with host plants, or increased neofunctionalization upon gene duplication remains to be investigated. Conclusion Our work provides a comprehensive overview of the candidate effector repertoires from three different aphid species with varying host ranges. Comparative analyses revealed candidate effectors that are most likely are involved in general aspects of infestation, whereas others, that are highly divergent, may be involved in specific processes important for certain aphid species. Insights into the overlap and differences in aphid effector repertoires are important in understanding how different species successfully infest different ranges of plant species. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2496-6) contains supplementary material, which is available to authorized users.
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              Localization of Candidatus Liberibacter asiaticus, Associated with Citrus Huanglongbing Disease, in its Psyllid Vector using Fluorescence in situ Hybridization

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                Author and article information

                Contributors
                Journal
                MethodsX
                MethodsX
                MethodsX
                Elsevier
                2215-0161
                25 March 2021
                2021
                25 March 2021
                : 8
                : 101317
                Affiliations
                [0001]University of Arizona, Tucson, United States
                Author notes
                [* ]Corresponding author. jmc6@ 123456email.arizona.edu
                Article
                S2215-0161(21)00110-2 101317
                10.1016/j.mex.2021.101317
                8374364
                34430230
                04658267-c9c4-4476-a7f7-2d349673696b
                © 2021 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 9 October 2020
                : 18 March 2021
                Categories
                Method Article

                minute arthropods,mass dissection,proteomics,transcriptomics,micromanipulation,salivary glands,midguts

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