The Drosophila embryonic central nervous system has been used for decades as a model for understanding the genetic regulation of axon guidance and other aspects of neural development. Foundational studies using antibody staining to examine the embryonic ventral nerve cord in wild-type and mutant animals led to the discovery of evolutionarily conserved genes that regulate fundamental aspects of axon guidance, including midline crossing of axons. The development of the regular, segmentally repeating structure of axon pathways in the ventral nerve cord can illustrate basic principles of axon guidance to beginning students and can also be used by expert researchers to characterize new mutants, detect genetic interactions between known genes, and precisely quantify variations in gene function in engineered mutant lines. Here, we describe a protocol for collecting and fixing Drosophila embryos and visualizing axon pathways in the embryonic ventral nerve cord using immunofluorescence or immunohistochemical staining methods. As embryogenesis in Drosophila takes ~24 h to complete, a 1-d collection yields embryos representing all stages of development from newly fertilized through ready-to-hatch larvae, allowing investigation of multiple developmental events within a single batch of collected embryos. The methods described in this protocol should be accessible to introductory laboratory courses as well as seasoned investigators in established research laboratories.

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.

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.

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.

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.

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.

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.

After more than a century of searching, scientists have discovered a gene in snails that may control asymmetries inside many animals

When the National Transportation Safety Board office in Alaska first hears about a plane going down, protocol is to begin gathering information

A Two-Day Festival in the historic Arts & Industries Building brings community, artists and scholars together for a “Culture Lab”

On this episode of Generation Anthropocene, we dive into one of the most underappreciated elements on the periodic table

Food is a focal point for understanding broader environmental problems. In this podcast, we learn how food buyers are influenced in surprising ways.

An unlikely friendship with Guy Burgess, the infamous British double-agent, brought unexpected joy to Stanley Weiss

As The Meg hits theaters, dive into what we really know about this chompy predator

The ancient burial structures, strikingly similar all across Europe and the mediterranean, have puzzled scientists and historians for centuries

By raising her son to be curious about the natural world, this mother helped shape the trajectory of the National Museum of Natural History

The Northwest Territories Power Corporation expects that by the end of March it will have generated 72 per cent of power for the North Slave region using hydro, and 28 per cent from diesel. Last year, about half the region's power was from diesel because of low water levels.

More than 50 Norman Wells residents gathered at the Royal Canadian Legion on Thursday night to voice frustrations over high heating fuel prices.

Tammy Roberts, Home Base's executive director, said before these services launched, there was "no place for youth to go" other than the emergency room or to RCMP cells. 

In opening remarks at the Inuit-Crown partnership committee meeting in Ottawa on Friday, Crown-Indigenous Relations Minister Gary Anandasangaree said the government is preparing to deliver an apology in Nunavik — the Inuit region of northern Quebec.

Former Dawson City Mayor Bill Kendrick has filed his defence against the town's encroachment violation lawsuit.

With president-elect Donald Trump promising to drill in Alaska's Arctic National Wildlife Refuge, the Gwich'in are preparing to fight for the Porcupine caribou herd.

Wanita Johnson, who celebrates her birthday this weekend in Whitehorse, still has a sharp memory for how the Second World War helped shape her life.

Water quality guidelines for mercury are 0.02 micrograms per litre of water. Several results show that levels at four sites are far above that threshold. During the second week of October, one value close to the mine was more than seven times the acceptable level.

Paraglider Ben Lewis remembers spinning around wildly as he got pulled up higher and higher into the violent storm. That was when he realized, about 6,700 metres up in the sky and still rising, that he was not likely to see his family again.

Yukon appeal court Justice Karen Horsman granted a temporary stay of the requirement on Friday, the latest development in an ongoing legal battle over the property between the territorial government, cabin-owner Len Tarka and tenant Eric DeLong. 

Stephanie Yuill spent five weeks visiting First World War sites across Europe on a bike she bought from the grocery store. 

As people across the country mourn the Honourable Justice Murray Sinclair, some in the Northwest Territories justice system are reflecting on his contributions to the country — one of those being Gladue principles. 

A Whitehorse homeowner has been charged in connection with an explosion last year that destroyed his home and killed a man in a neighbouring house. 

Hay River RCMP say they believe two weekend incidents, including one in which a person was shot, are linked to drugs and organized crime.

A group of volunteers have been providing emergency services to West Dawson residents during the periods of freeze up and break up, when there is no reliable access across the Yukon River to town.

John Quirke stayed in the job for 25 years — with a front row seat to Nunavut's politics as he managed the operations of the assembly and acted as the principal adviser for MLAs on parliamentary procedure among other things. 

A man who fled from police, drove onto a walking trail, triggered a shelter-in-place advisory for the Chateau Nova hotel in Yellowknife and subsequently broke into a home was on bail at the time and under house arrest, police say.

In Fort Good Hope, N.W.T., work is underway on a modular home construction centre that local leaders say will help the community meet housing and employment goals.

FIFA Football for Schools (F4S) has come to Canada, with a pilot program in the N.W.T. already underway and offering coach training. 

Historians say that Sarah Emma Edmonds exaggerated many aspects of her wartime experiences. Still, she bravely served in the Union Army, becoming one of hundreds of women who fought in the conflict in secret

Newspaper editor Horace Greeley unsuccessfully ran against incumbent Ulysses S. Grant in November 1872. Twenty-four days later, he died of unknown causes at a private mental health facility

In a new biopic starring Kate Winslet, Miller's many lives—as an artist, model, muse, cook and war correspondent—need little embellishment

In the early 1990s, historians and the public alike questioned how Disney's America would accurately and sensitively document the nation's thorny past

Fifty years ago, on October 5, 1974, David Kunst completed the first verified circumnavigation of the globe on foot. Along the way, he met Princess Grace of Monaco, raised money for UNICEF and lost a brother to bandits

An exhibition at the Huntington Library, Art Museum and Botanical Gardens explores how Western intellectuals viewed the climate crisis between 1780 and 1930

The bronze wreath immortalized the moment when the members of the Honor Guard removed their hats and placed them on the president's grave during his burial

A new film revisits the 90 minutes before the first episode of "Saturday Night Live" premiered on NBC on October 11, 1975

The British explorer named dozens of geographical features and sites in the region, ignoring the traditions of the Indigenous peoples who’d lived there for millennia

As a child, the future president acquired a marine animal's skull, which became the first specimen in his natural history collection

During and after the Civil War, inventive illustrations allowed Democrats and Republicans to turn American ballots into powerful propaganda

When the U.S. Army massacred a Lakota village at Blue Water, dozens of plundered artifacts ended up in the Smithsonian. The unraveling of this long-buried atrocity is forging a path toward reconciliation

With flinty perseverance and a golden touch, Belinda Mulrooney earned an unlikely fortune in the frozen north and reshaped the Canadian frontier

A new exhibition at the British Library explores the public, private and spiritual lives of such figures as Joan of Arc, Christine de Pizan and Hildegard of Bingen

After he was forced off the German stage in 1934 by antisemitic hecklers, Leo Reuss found a daring way to hide in plain sight