Cold Spring Harbor Protocols

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...

Understanding how female mosquitoes find a prospective host is crucial to developing means that can interfere with this process. Many methods are available to researchers studying cues and orientation mechanisms that modulate female mosquito attraction to hosts. Behaviors that can be monitored with these assays include activation, taking flight, upwind flight along an odor plume (optomotor anemotaxis), close approach to the stimulus (including hovering), and landing. Video recording can three-dimensionally document flight tracks and can correlate overall distribution patterns and moment-to-moment movements with odor contact and the presence of nearby cues such as a visual target...

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...

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins...

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues...

Immunohistochemistry (IHC) is a powerful technique used for visualizing cellular components and determining the presence and/or location of proteins or other macromolecules in tissue samples. The classical IHC process involves the detection of epitopes using a highly specific primary antibody. This is followed by a secondary antibody that is coupled to a reporter molecule or fluorophore and capable of binding to the primary antibody and allowing for protein immunodetection. Although IHC does not routinely provide quantitative results compared to an enzyme-linked immunoassay or western blotting, it permits the localization of the proteins in intact tissues...

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in the Aedes aegypti mosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs...

The female mosquito is endowed with the responsibility of gene transfer from one generation to another through careful delivery of her eggs to an appropriate environment. To guarantee a successful reproduction process, female mosquitoes use an evaluative approach to select an oviposition site that will ensure that their progeny successfully hatch and develop from larvae to pupa to adult. Specific conditions must be met during oviposition site selection before gravid females deposit eggs, including but not limited to appropriate temperature, salinity, and pH; the presence of nutrition; and a low risk of predation...

This introduction reviews techniques used to examine the distribution and expression of gene transcripts and proteins in a variety of tissues/organs in the medically important global disease vector mosquito, Aedes aegypti Specifically, these methods allow the detection of cell-specific transcript expression by fluorescent in situ hybridization; facilitate immunohistochemical mapping of a protein of interest in whole-mount small tissue/organ samples; examine the subcellular localization of proteins, such as membrane transporters, through sectioning of paraffin-embedded tissue/organ samples; and finally, enable the efficient separation of cytosolic and membrane proteins for western blot analysis without the need for specialized equipment (e...

Egg-laying behavior in insects has a dramatic impact on their fitness, controlling their extent and distribution across ecosystems. Thus, understanding the cues that guide mosquito egg laying is an important goal. Measuring mosquito egg-laying preference is critically important for understanding the sensory and neural mechanisms guiding adaptive breeding decisions. Here, we present a method for ascertaining the egg-laying preference of individual Aedes aegypti mosquitoes in small chambers with two distinct oviposition substrate choices...

Egg-laying preferences of mosquitoes can reveal key neurosensory mechanisms informing the decision-making process for this critical behavior. A single blood meal results in a gravid female Aedes aegypti mosquito laying more than 100 eggs. Therefore, egg counting represents a potentially time-consuming and laborious component to behavioral assays, such as those that measure oviposition preference or fecundity. Automated algorithms that count eggs from images can dramatically reduce the time required for this step of data processing and analysis and increase reproducibility associated with having multiple human observers count the eggs...

Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells...

Genetic background can have subtle or profound effects on mutant phenotypes, providing additional information regarding the function of the gene. If your mutation is maintained on one genetic background but you wish to analyze it on another, it is a simple matter to transfer the mutation to a recipient strain background by repeated backcrossing (introgression) as detailed in this protocol. The resulting strain is called a congenic strain, defined as a strain carrying the mutation within a segment of chromosome from the donor strain with the remainder of the genome from the recipient strain...

Preimplantation embryo culture is a valuable approach to investigate a preimplantation lethal phenotype. Standard culture methods have been perfected such that the entire preimplantation period and the process of implantation can be followed in vitro. This protocol provides modifications for the analysis of clutches of embryos from heterozygous matings specifically for the purpose of distinguishing a preimplantation phenotype in homozygous mutants.

Histological analysis is an informative part of any mutational analysis. Interpretation of histological sections is much easier, and comparisons of mutant and wild-type embryos are more reliable if the plane of sectioning is precisely controlled. This protocol provides methods for obtaining sections with reproducible orientation for embryos between E4.5 and E9.5 still in the uterus and for dissected embryos E9.5 and older.

Preimplantation development covers a period of ∼4.5 d from fertilization to implantation in the uterus. If a homozygous mutant phenotype causes the death of embryos during this period, simple culture methods are available that support preimplantation development to allow a thorough morphological assessment. Embryos are recovered from the oviducts or uterus and examined for gross morphology, cell number, and progression through cleavage stages. Blastocysts can also be cultured over the implantation period and undergo a process analogous to implantation in vitro...

If homozygous mutant mice survive to adulthood, are fertile, and have no visible phenotypes attributable to mutation of the relevant gene, there are a number of possible reasons why an effect of the mutation is not evident. Technical errors that might have occurred during gene targeting or genotyping must first be eliminated. Variable penetrance of the mutation should be considered as well as the possibility of age-related or late-onset phenotypes, such as tumor formation or other pathologies. The gene expression pattern and nature of the protein product of the gene could provide clues...

Viable homozygous mutant newborn mice may show effects of a mutation at any time during their development by exhibiting abnormal structure, function, or lethality. This overview guides the analysis of postnatal mice through gross anatomical assessment and the detection of visible phenotypes prior to weaning such as altered growth patterns, neurological problems, or abnormalities in movement or coordination. Advice on marking pups for identification purposes and providing adequate nutrition in the event of eating problems is given...

Once a recessive mutation has been established in a mouse strain in the heterozygous state, the task of phenotypic analysis of the homozygous mutants can begin. This overview leads you through a sequence of steps to determine whether the homozygous mutants are present at birth or whether the mutation causes prenatal lethality. In the case of a prenatal lethality, the time of death of the mutants, which could occur at any time during pre- or postimplanation development, must be firmly established before further phenotypic analysis...

Following the production of chimeras from targeted embryonic stem (ES) cells or obtaining founders from CRISPR-Cas gene editing in preimplantation embryos, the desired targeted mutation must be recovered and established in the heterozygous state in a strain or stock of mice for further study. The breeding schemes for ES chimeras and CRISPR-Cas founders differ. For ES cell chimeras, we discuss the relative benefits of breeding from male or female chimeras. We discuss the importance of genetic background and provide practical advice for putting the mutation on inbred or outbred backgrounds or producing a coisogenic strain...