Cold Spring Harbor protocols最新文献

Olfactory classical conditioning paradigms have been extensively used since the early 1970s to apply genetic approaches to the study of memory in Drosophila. Over the intervening years, advances in genetics have largely changed the focus of studies from the role of single genes in memory to investigation of memory-relevant neuronal circuits. However, the original behavioral paradigms have remained largely unaltered, besides investigators making a few useful tweaks to the training and testing apparatus and modifications to the operating procedures. In this protocol, we provide the reader with a detailed description of the manufacture and assembly of a typical T-maze apparatus, where populations of adult flies can be trained and their odor memory tested later, by giving them a binary choice between the two trained odors. We describe how variations of the training apparatus permit both aversive (odor-shock) and appetitive (odor-sugar) memories to be studied. In addition, we describe a recent modification of the apparatus and protocol that permits study of multisensory (color and odor) aversive and appetitive learning. Control assays for sensory acuity and locomotion are also included.

Memory has been extensively studied in Drosophila since the early 1970s. Straightforward aversive and appetitive conditioning paradigms train populations of flies to associate the pairing of one of two odors with either punishment or reward. After training, the flies show either preferential avoidance or approach behavior, to the appropriate odor, when given a choice between the two odors in a simple T-maze apparatus. These basic experimental approaches have proven useful in understanding the genetic, molecular, cellular, and neuronal network bases of various valence-specific memories in the fly brain. In addition, numerous modifications to these assays have permitted the study of a broad range of memory-related phenomena. Labile short-term avoidance and approach memories can be readily distinguished from more stable "consolidated" long-term memory equivalents. Prior or subsequent experience of the training cues, and manipulations of the flies' condition, have revealed how parallel competing memories and incompatible states can temporarily interfere with memory retrieval, providing insight into mechanisms of forgetting. Recent studies have also modified the training and testing apparatus to allow simultaneous presentation of odors and colors, providing insight into mechanisms of multisensory learning.

Anautogenous female mosquitoes, which ingest a blood meal from warm-blooded vertebrates to produce eggs, have become a valuable model organism for investigating signaling pathways and physiological processes that occur during egg development. Different molecular pathways tightly regulate the initiation of egg development and are governed by a balance among different insect hormones. Gravid (mature egg-carrying) females deposit fully developed eggs at the end of each gonotrophic cycle, which is defined as the time interval between the ingestion of a blood meal to oviposition. An intact eggshell protects the oocyte and embryo inside from external factors such as desiccation, physical damage, etc., and the various eggshell proteins are spatially and temporary deposited during oogenesis. Additionally, follicle resorption (oosorption) during blood meal-induced mosquito ovarian follicle development is an adapted physiological process that optimizes reproductive fitness. Mosquito oocytes grow and mature synchronously throughout oogenesis; however, during the later stages of oogenesis, some oocytes may undergo oosorption if sufficient nutrients are unavailable. This introduction highlights how mosquito egg development can be used to investigate follicular resorption and identify proteins involved in eggshell formation in Aedes aegypti mosquitoes.

The host associations of mosquitoes vary by species, with some species being relative generalists, whereas others specialize, to varying extents, on a particular subset of the available host community. These host associations are driving factors in transmission dynamics of mosquito-vectored pathogens. For this reason, characterizing the host associations of mosquito species is critical for understanding the epidemiology of mosquito-vectored pathogens. Diverse methods have been used to associate mosquito species with their hosts. These typically include collecting mosquitoes that bite a restrained host (bait) or collecting wild blood-engorged mosquitoes and matching their blood meal to reference samples (blood meal analysis). Blood meal analysis refers to a collection of molecular techniques for determining the taxonomic identity of the source of a mosquito blood meal using cytological, serological, or DNA-based characteristics of the blood meal. Blood meal analyses that are based on DNA markers have advantages over cytological and serological methods and are effective for determining species-level identities of hosts from a broad range of potential host taxa. Here, we discuss effective techniques for analyzing blood meals.

Transposon-mediated transgenesis has revolutionized both basic and applied studies of mosquito vectors of disease. Currently, techniques such as enhancer traps and transposon tagging, which rely on remobilizable insertional mutagenesis, are only possible with transposon-based vector systems. Here, we provide general descriptions of methods and applications of transposon-based mosquito transgenesis. The exact procedures must be adapted to each mosquito species and comparisons of some differences among different mosquito species are outlined. A number of excellent publications showing detailed and specific protocols and methods are featured and referenced.

Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes, Anopheles, and Culex The most versatile element, piggyBac, is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as "enhancer traps" to identify endogenous genes with specific expression characteristics.

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.

All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at -20°C or -80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.

In insects, oocyte resorption (oosorption) or follicular atresia is one of the key physiological processes and evolutionary strategies used to optimize reproductive fitness. Mosquitoes are ideal model organisms for studying egg maturation in arthropods, as their follicle development is initiated only following the ingestion of a blood meal, followed by a carefully orchestrated series of hormonally regulated events leading to egg maturation. A cohort of approximately 100 follicles per mosquito ovary begin developing synchronously. However, a significant fraction of follicles ultimately undergo apoptosis and oosorption, especially when available resources from the blood meal are limited. Therefore, simple, rapid, and reliable techniques to accurately evaluate follicular atresia are necessary to understand mechanisms underlying follicle development in insects. This protocol describes how to detect apoptotic follicle cells within the Aedes aegypti mosquito ovaries using a commercially available fluorescent-labeled inhibitor of caspases (FLICA). Caspases are key players in animal apoptosis. In this assay, the FLICA reagent enters the intracellular compartment of follicles in dissected mosquito ovaries and covalently binds to active caspases. The bound reagent remains within the cell and its fluorescent signal can be observed by confocal microscopy. Although this method was specifically developed for visualizing apoptotic ovarian follicles during Ae. aegypti mosquito egg development, it should be applicable to other mosquito tissues that undergo caspase-mediated program cell death in a time-dependent manner.