Using X-ray Micro-Computed Tomography to Three-Dimensionally Visualize the Foregut of the Glassy-Winged Sharpshooter (Homalodisca vitripennis).

Dissecting the heads of small insects belonging to the order Hemiptera for detailed anatomical investigation with light or scanning electron microscopy is difficult, time-consuming, and destructive, often resulting in sample preparation artifacts. Nevertheless, the structural details of these insects often hold critical information regarding their interactions with bacterial pathogens. For example, the glassy-winged sharpshooter (GWSS) is an efficient vector of the bacterium Xylella fastidiosa, the causal agent of Pierce's disease in grape vines, but the foregut of this species is exceptionally difficult to dissect. Here, we describe a simple, non-destructive method to investigate the structure of the anterior gut of GWSS using high-resolution micro-computed X-ray tomography (microCT). The use of microCT eliminates the need for destructive dissection and reveals the morphology of small insects in three dimensions, allowing the user to virtually dissect the sample. The use of microCT imaging is a promising and powerful tool in the entomological sciences for studying the structures of vector insects, especially for difficult-to-dissect regions such as the foregut.

The cibarial pump of the xylem-feeding froghopper Philaenus spumarius produces negative pressures exceeding 1 MPa.

The xylem sap of vascular plants is an unlikely source of nutrition, being both nutrient poor and held under tensions (negative pressures) that can exceed 1 MPa. But some insects feed on xylem sap exclusively, extracting copious quantities using a muscular cibarial pump. However, neither the strength of the insect's suction, nor the direct energetic cost of xylem ingestion, have ever been quantified. Philaenus spumarius froghoppers were used to address these gaps in our knowledge. Micro-CT scans of its cibarium and measurements of cibarial muscle sarcomere length revealed that P. spumarius can generate a maximum tension of 1.3 ± 0.2 MPa within its cibarium. The energetic cost of xylem extraction was quantified using respirometry to measure the metabolic rate (MR) of P. spumarius while they fed on hydroponically grown legumes, while xylem sap excretion rate and cibarial pumping frequency were simultaneously recorded. Increasing the plants' xylem tensions up to 1.1 MPa by exposing their roots to polyethylene glycol did not reduce the insects' rate of xylem excretion, but significantly increased both MR and pumping frequency. We conclude that P. spumarius can gain energy feeding on xylem sap containing previously reported energy densities and at xylem tensions up to their maximum suction capacity.

Tsetse Fly Transmission Studies of African Trypanosomes.

African trypanosomes are naturally transmitted by bloodsucking tsetse flies in sub-Saharan Africa and these transmission cycles can be reproduced in the laboratory if clean tsetse flies and suitable trypanosomes are available for experiments. Tsetse transmission gives access to more trypanosome developmental stages than are available from in vitro culture, albeit in very small numbers; for example, the sexual stages of Trypanosoma brucei have been isolated from infected tsetse salivary glands, but have not yet been reported from culture. Tsetse transmission also allows for the natural transition between different developmental stages to be studied.Both wild-type and genetically modified trypanosomes have been successfully fly transmitted, and it is possible to manipulate the trypanosome environment inside the fly to some extent, for example, the induction of expression of genes controlled by the Tet repressor by feeding flies with tetracycline.

A Quantum Dot-Immunofluorescent Labeling Method to Investigate the Interactions between a Crinivirus and Its Whitefly Vector.

Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor)-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV) virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs), into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605) for biotin-conjugated anti-LIYV IgG (B-αIgG). IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated approx. seven- to eight-fold slower than that of Alexa Fluor.

Morfología del cibario en subgéneros de Psorophora Robineau-Desvoidy (Diptera: Culicidae) / Cibarial morphology in Psorophora Robineau-Desvoidy subgenera (Diptera: Culicidae)

We describe the cibarial morphology in eight Psorophora species Robineau-Desvoidy: Ps. (Grabhamia) cingulata (Fabricius), Ps. (Gra.) confinnis (Lynch Arribálzaga), Ps. (Janthinosoma) ferox (Humboldt), Ps. (Jan.) albipes (Theobald), Ps. (Jan.) cyanescens (Coquillett), Ps. (Psorophora) lineata (Humboldt), Ps. (Pso.) cilipes (Fabricius), y Ps. (Pso.) ciliata (Fabricius). The species belonging to subgenus Grabhamia Theobald are characterized by palatal papillae in central position and the presence of cibarial armature. The teeth in Ps. cingulata are equal meanwhile in Ps. confinnis the internal row is spatulate with apex denticulate. In Janthinosoma Lynch Arribálzaga we observed six palatal papillae (the fore pair with less size to others) and armature absent, instead we observe small spicules toward posterior plate: abundant in Ps. ferox, and few in Ps. albipes and Ps. cyanescens. The subgenus Psorophora presents four equal size palatal papillae, cibarial armature absent, also distinctive number of trichoid sensilla (12-17), in comparison to other two subgenera (5-10). We suggest to include those diagnostic characters in the future taxonomic and systematic studies in the genus Psorophora.

Se describe la morfología del cibario en ocho especies de Psorophora Robineau-Desvoidy: Ps. (Grabhamia) cingulata (Fabricius), Ps. (Gra.) confinnis (Lynch Arribálzaga), Ps. (Janthinosoma) ferox (Humboldt), Ps. (Jan.) albipes (Theobald), Ps. (Jan.) cyanescens (Coquillett), Ps. (Psorophora) lineata (Humboldt), Ps. (Pso.) cilipes (Fabricius), y Ps. (Pso.) ciliata (Fabricius). Los integrantes del subgénero Grabhamia Theobald se caracterizan por la posición central de las papilas palatales y la presencia de armadura cibarial, en Ps. cingulata los dientes son todos iguales, mientras que en Ps. confinnis la hilera más interna es espatulada con ápices dentados. En Janthinosoma Lynch Arribálzaga se observaron seis papilas palatales, (el par anterior de menor tamaño a las otras), y no presenta armadura, no obstante, en su lugar se aprecian pequeñas espículas hacia la placa posterior, que variaron en Ps. ferox (abundantes sobre placa posterior), Ps. albipes y Ps. cyanescens (pocas sobre la placa posterior). En Psorophora se apreciaron cuatro papilas palatales de igual tamaño, no se presenta armadura cibarial, sin embargo, fueron distintivos el número de sensillas tricoideas (12-17) respecto a los otros subgéneros (5-10). Estas características permiten diferenciar los tres subgéneros, y sugieren la inclusión de dichos caracteres en los estudios taxonómicos o sistemáticos del género.