The fruit fly. This particular species is commonly used by scientists as a model organism.

To learn more about why Drosophila have become so commonly used in genetics research, check out this profile in The Guardian. https://www.theguardian.com/science/2016/sep/25/in-praise-of-the-humble-fruit-fly-genetics-research

In a mutagenesis experiment in the late 1970's, scientists found and XXY male. This rare finding spurred the thought that is only takes a single Y to become male as long as X dosage can be overcome by other mutations.

In many of the genetically-modified mosquito-control efforts, it is necessary to only release males into the environment. Male mosquitoes do not bite and may or may not pass their genetic modifications to the next generation (depending on the design of the intervention). The authors propose that by over-expressing Yob in laboratory raised mosquitoes, scientists will be able to produce male-only populations without the labor-intensive process of sexing of pupae.

A lot of laboratory technologies derive from natural phenomena (PCR, restriction enzymes, viral vectors, etc.). The discovery of Yob is just the beginning of unlocking the power of what lies within. In an interview with The Scientist, the authors suggest pairing this new knowledge of the mosquito sex determination system with the use of CRISPR gene editing technologies to create mosquitoes that can express Yob on the autosomes. This would allow female-lethality to be passed on to subsequent generations.

Read more in The Scientist: https://www.the-scientist.com/news-opinion/maleness-gene-found-in-malaria-mosquito-33283

There are many scientific efforts afoot to control mosquito populations (especially those species who are vectors for disease) through the use of genetic methods. One such method used in California in 2017 released sterile A. aegypti mosquito males (sterile due to a bacterial infection) into the wild in efforts to reduce the local population.

Read more at NPR: https://www.npr.org/sections/thetwo-way/2017/07/21/538470321/to-shrink-the-mosquito-population-scientists-are-releasing-20-million-of-them

Conversely to the previous experiment where the authors added more Yob and saw evidence of female death, the authors reduced Yob expression in this experiment and saw evidence of male death as survivors had XX genotypes, likely due to under-expression of genes on the male's single X chromosome.

The female-lethality phenomenon seen in the last experiment has been observed before in fruit flies when upstream genes are misregulated and there is an imbalance of gene products between the sex chromosomes and autosomes.

All embryos that underwent successful injection of Yob (as indicated by expression of the fluorescent marker) had the appearance of male larvae and had an XY genotype. The authors think that expression of Yob in XX embryos may have killed them.<br> Embryos that were not successfully injected had approximately 50/50 male to female ratios, as expected.

In this experiment, the authors injected normal Yob transcripts into normal embryos, along with a fluorescent marker to let them know that injection was successful, and allowed the embryos to grow. Once at the larval stage, they could see the fluorescent injection marker and sorted the larvae based on expression of the marker. At the pupal state, the authors were able to sex the mosquitoes.

The Yob transcripts with changes in them, did not change the splicing pattern of dsx like the normal transcript does. Since the changes they made in the transcripts render the transcript non-function, they can assume that the normal Yob transcript is translated into a protein.

Based on what the authors found by analyzing other species, they hypothesize that Yob actually codes for a protein. They made new lab-generated transcripts of Yob with sequence changes that would result in no protein product (either removing the start codon, or adding in a premature stop codon) and transfected those transcripts in to cells to monitor dsx splicing.

In one portion of the Yob region, the authors found evidence of evolutionary selection through the amount, types and patterns of changes in sequence. Other areas of the gene likely had changes between species as well, but they may appear more random in their pattern.

The authors isolated sequences from closely related mosquito species and compared similarities and differences in those sequences computationally.

If a mutation occurs within a gene, it is more likely that it will code for the same amino acid than a different one. This is due to redundancy in the amino acid code and due to evolutionary and selective pressures on organisms to produce functional proteins.

There is less female-form dsx in cells that were transfected with Yob than in cells not transfected, or those transfected with non-functional Yob. They found the same results whether the transfection was done in cell culture or done in live mosquito larvae. The authors still can not tell if Yob is directly interacting with dsx, but they now know that Yob does have an upstream impact dsx splicing.

The authors made mRNA in the lab. The mRNA has the same sequence of what they think is the shortest, but functional Yob transcript. They are testing whether Yob has a direct effect on dsx.

As seen in Figure 1, they are analyzing Yob mRNA through use of a gel, not sequencing, in this experiment. The authors find that transcription of this male factor begins very early after fertilization, at the same time as other genetic markers of zygotic expression.

Simply by evaluating timing of expression, the authors conclude that Yob must be upstream of dsx. However, this statement is also a hypothesis, as they have not found a direct connection between the two genes. This statement will serve as motivation for their next experiments.

They find from an mRNA gel that both males and females express a female form of a downstream sex determination gene (dsx) at 1 hour after fertilization. From the previous experiment, the authors know that Yob expression begins at about 2.5 hours after fertilization. In this experiment, the authors see that at 4 hours post fertilization, the female form of dsx begins to disappear in males but not females. By 8 hours, males start expressing the male form of dsx and the female form is completely absent. In order for the male to start becoming a genetic male, female form transcripts from the mother must be removed before male specific expression can begin.

Designation of where genes or genetic elements are in genomic sequences. This is by far the most difficult task for scientists and is constantly changing as we learn more about important features of the genome. Often, uncommon model organisms (like the mosquito) suffer from lack of annotation because scientists are more focused on other organisms like the human, mouse, or fruit fly.

Introns (noncoding regions) that are not excised—that is, cut out—during pre-mRNA processing.

Of the ~500,000 mRNA sequences returned, a very small portion of them (only 21) were found to derive from the Y chromosome. While the authors didn't explicitly say this, it is assumed that these sequences were not found in the female embryos.

An experimental method developed in the 1980s that amplifies a specific, targeted region of the genome, generally 100-5,000 base pairs (bp) in length.

To learn more, check out this video animation from Cold Spring Harbor Laboratory.

Because mosquito embryos look identical whether male or female, the authors had to use molecular techniques to find out the sex of each embryo. The embryos were separated based on their sexual identity (male or female) and mRNA was extracted and sequenced from the collective male or female embryos.

A genetic sequence on the Y chromosome that is a signal for maleness (hence the M).

Flies, including Ceratitis capitata (Mediterranean fruit fly), Lucilia cuprina (Australian blow fly), and Musca domestica (housefly), and the (red flour beetle) Tribolium castaneum.

In the "default" mode of sex determination, a male fly will be produced. In this case Sxl, tra and dsx are expressed, but pre-mRNA processing results in non-functional SXL and TRA proteins as they both contain premature stop codons. The exons that contain these stop codons are spliced out in the female mRNA isoforms. Both males and females express DSX protein, but each have a different version due to sex-specific splicing. Check out this figure from Chromosomal Sex Determination in Drosophila

Lowercase tra is the gene, either in DNA or RNA form; all-caps TRA is the protein.

Downstream genes; those that are expressed/utilized later in the pathway.

Bioinformatic software programs or algorithms that can compare genomic sequences within, between, and across species to detect similarities and differences in DNA sequence.

Easily changed through evolution.

Most genes have several coding regions (exons) interrupted by noncoding regions (introns). When a gene is translated into pre-mRNA, the molecule will go through processing to remove the introns and create a mature mRNA molecule for translation. During this process, exons or parts of exons can be removed as well, leading to several possible mRNA versions (also called isoforms) of the same gene. In this case, males will express one version of the gene and females will express a different version of the same gene.

The process by which and organism becomes male or female, in both appearance and reproductive capabilities. In most species this is genetic, as discussed here. However, in other species, sex can be determined by external environmental factors and can sometimes switch during the organism's life.

Here, they mean a process determined by genetics.

Malaria is a flu-like disease caused by a parasite. The parasite infects and kills blood cells while also releasing toxins. If left untreated, this infection can result in organ failure and death. It is transmitted by certain species of mosquitoes.<br>

Mosquito larvae live in the water and feast on microorganisms. They go through four growth spurts before becoming a pupa.

The pupal stage is the time at which the mosquito transforms from a water-inhabiting larvae to a wing-bearing adult. Much like butterfly metamorphosis, the mosquito is encased in a hard but thin shell-like structure.

This is a denotation for a nucleotide change. This is ribonucleotide 1 changed from an A to a C.

A technique to deliver nucleic acids to eukaryotic cells through membranes. There are many ways to do this, but two common ones are through chemicals or electricity.

A collective sample. In this case, mRNA samples from multiple female mosquito embryos of the same age were combined before being sequenced. The same was done across different time points and for male mosquitoes. Because mosquitoes are small organisms, there is not enough cells/RNA to perform a quality sequencing reaction. Thus, several or maybe even hundreds of organisms must be combined in order to have enough material. When looking at sequencing data from "pools", you are actually observing the "average", but since these are laboratory raised mosquitoes, it is assumed that they are genetically identical. So the "average" is likely true for all organisms in the population.

Changes in the mosquito genome have made them no longer susceptible or at least not as sensitive to insecticides. Much the same as bacteria developing genetic resistance to antibiotics.

In an abnormal place. In this case, nucleic acids are injected into mosquito larvae leading to expression of a gene that is not normally there.

The female egg cell is a rather large cell compared to the fertilizing male sperm cell. This large egg cell has more mRNA in the cytoplasm (as well as things like mitochondria) than the sperm cell. When the zygote is formed, there is a large maternal contribution of cytoplasmic goodies.

A process by which the expression of genes on the autosomes (all the other chromosomes that are not sex chromosomes) and sex chromosomes (in both sexes) are balanced through either inactivation of chromosomes or increased gene expression from a chromosome.

A particular version of an expressed gene (mRNA), may or may not have all of the exons encoded by the gene.

An organism that has some amount of genetic material from another unrelated organism.

Exposure to radioactive compounds.

Referring to chromosomes.

Having three sets of chromosomes. Most animals are diploid, having two sets of chromosomes, one from mom, one from dad.

Reduced expression of a gene, not complete removal.

Having two different sex chromosomes. In humans, males are the heterogametic sex.

Having two of the same sex chromosomes. In humans, females are the homogametic sex.

Order of butterflies and moths.

Increased expression of genes.

Any change in DNA that results in a non-functional protein. Many different types of mutations can accomplish this, nonsense mutation and large deletions are the first thing that come to mind. They can render a protein non-functional by it being completely absent. Even missense mutations that change a single amino acid can render a protein non-functional if it hits a functional domain, even if the protein itself is present.

A visualization of the number and kinds of chromosomes present in an organism. This term is used loosely here, as XY males were determined through PCR, not the traditional means of visualizing chromosomes under a microscope.

A mobile DNA sequence that contains a gene. In genetic engineering, these sequences can be moved between plasmids or other useful vectors through restriction or recombination sites.

A protein derived from jellyfish that emits a neon green light when excited by UV or blue light.

A continuous circular molecule of DNA. Originally derived from bacteria, plasmids have proven to be a useful delivery system of DNA in molecular biology.

Injecting two or more things (in this case nucleic acids) at once.

This level of structure in a protein is when the amino acid chain begins to have three-dimensional shape. Side chains of the amino acids fold and interact through chemical forces like hydrogen bonds, van der Waals interactions and hydrophobic interactions. The most common secondary structures are helices, sheets, loops and turns.

This type of secondary structure in an amino acid chain is comprised of two alpha helices connected by a loop. This type of structure is indicative of the protein's ability to bind DNA as a transcription factor.

A stop codon (UAA, UAG or UGA (RNA)) that occurs earlier in the sequence than intended, usually due to a change in the DNA. This results in a truncated (not full length) protein.

Start codon (ATG (DNA) AUG (RNA))

Selection against deleterious traits (negative selection).

A stretch of DNA sequence that has the ability to be translated into protein (exons only). An ORF usually begins with a start codon (ATG) and ends with a stop codon (TAA, TAG or TGA).

Changes in DNA that end up coding for a different amino acid in the protein product.

Changes in DNA that ends up coding for the same amino acid in the protein product.

Similar to PCR, RT-PCR is a method that first converts mRNA in to DNA by use of a RT polymerase (enzymes like this are used in viruses like HIV). Once the RNA is in DNA form, the traditional PCR method can be used to amplify transcripts of interest.

in vivo inside of a living organism. In this case an experiment that was done in a cell line (in vitro) and worked, was then done in a living organism to see if it would also work in the context of the whole animal.

A population of cells derived from a single cell isolated from an organism. Thus, all cells in the culture are genetically identical. Often different cell lines have unique experimental qualities to them. In this case, this line lacks the Y chromosome, which may have been genetically engineered or may have occurred by chance.

mRNA molecules that were made in a tube (in vitro outside of a living organism).

A control that helps to ensure that the same amount of RNA is added from each sample, so that if lower or higher levels of RNA are seen in different samples you can ensure it's due to biological differences and not just human loading error.

There is a maternal to zygotic transition (MZT) in the zygote after blastoderm formation but before gastrulation. During this process maternally deposited RNAs from the egg are degraded and the new genome of the zygote is activated and begins transcription and translation of genes. The zygote is now on its own, without the support of good old mom.

Similar genes in different species that derived from a common ancestor.

A single layer of cells (about 4,000) that surround the outside of the zygote. In the fruit fly, this happens about 3 hours after fertilization.

The process of laying eggs in a fly.

A section of genomic DNA sequence. As genome sequences are assembled computationally, scientists first call long sections of sequence a scaffold. They can experimentally determine where this section comes from (in this case the Y chromosome) but know that it is not all the sequences on the Y chromosome. In the mosquito, the Y chromosome is approximately 150 kilobases (kb), but this scaffold only represents 1/3 of the sequence (48 kb).

A small segment of DNA sequence, approximately 100 bp in length. Before being sequenced, DNA (in this case RNA is converted back to DNA before sequencing) is chopped up into small fragments. Each of those small fragments are "read" before being computationally aligned to a reference genome sequence to determine where it comes from in the genome.

The entirety of a cell, organ, organism's gene expression profile. Just like genomics (looking at the whole genome, instead of one gene at a time), transcriptomics looks at all of the transcripts (mRNAs) at once instead of one molecule at a time.

In most animal species, biological males and females have different outward appearances in addition to the different reproductive organs. Birds are some of the most obvious examples. Male peacocks have long and brightly colored tail feathers while female are a dull brown/green.

DNA sequences on the X chromosome that "signal" the presence of the X chromosome

If you think of genetic and signaling pathways like a river, the genes at the very beginning of the pathway are the originating headwaters, or upstream. Whereas genes involved at the end of the pathway are downstream. What is upstream or downstream of you is relative to where you are in the pathway.

Not all sex determination pathways are like humans using the X and Y chromosomes. Over the course of evolution, organisms have developed different ways of genetically indicating male and female.

This is a genetic signal that is at the beginning of the sex determination pathway.