Name: Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution
Uploaded: 2023-05-26T12:25:00
Duration: 1 min 49 s
Description: Watch this Scientific Journal Video about Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution at JoVE.com

generating dna probes to label the sex chromosomes of anopheles mosquitoes and preparing the hybridization solution

Fluorescens in situ-hybridisering for at studere myggespermatogenese

Malaria imposes an enormous burden on the health and well-being of the global human population. In 2021, the World Health Organization (WHO) estimated that malaria caused 619,000 deaths, of which 96% occurred in Sub-Saharan Africa1. The disease is transmitted by mosquitoes belonging to the Anopheles genus, and in Sub-Saharan Africa, three species, namely Anopheles gambiae (An. gambiae), Anopheles coluzzi (An, coluzzi) and Anopheles arabiensis (An. Arabiensis) have a disproportionately large role in malaria transmission, accounting for 95% of malaria cases globally. Control programs relying on traditional methods such as insecticides and antimalarial drugs have saved millions of lives; however, in recent years, the rising resistance to these control methods has challenged their efficacy1,2. In addition, restrictions imposed by the COVID-19 pandemic have affected the availability of key malaria control interventions, which, according to the 2022 WHO World Malaria Report, has increased malaria incidence1. In the last two decades, novel genetic control methods have been developed in laboratory settings to target&#160;Anopheles mosquitoes3,4,5,6,7,8,9,10. Among these strategies, those based on gene drive systems (GDSs) and synthetic sex ratio distorters (SDs) seem promising. GDSs rely on the possibility of transmitting, at a very high frequency, a genetic modification that affects female fertility or impairs the parasite life cycle in the mosquito5,11,12. SDs, instead, act by skewing the sex ratio of a mosquito progeny toward males, which leads, over time, to the collapse of a target population due to a lack of females4,6,13. The main components of these genetic systems act primarily on the reproductive organs of the mosquitoes, where the gametes, eggs, and sperm are produced following meiotic division14.
In this protocol, advances in cytogenetic techniques are employed to explore spermatogenesis in An. gambiae focusing on the chromosomes&#39; behavior in situ. The structure of the mosquito testis and the biological processes that take place within it have been previously investigated using a number of cytological methods, such as immunofluorescence, fluorescent reporter transgenes, and DNA and RNA fluorescence in situ hybridization (FISH)15,16,17,18,19,20; the organs show a spindle-like shape, in which the lower pole is attached to a deferent duct connected to the male accessory glands. In the upper pole, the germline stem cells niche proliferates and differentiates into spermatogonia cells embedded inside spermatocysts formed by somatic cells. Following multiple rounds of mitotic division, the spermatogonia differentiate into spermatocytes, which enter meiosis. At prophase, autosome and sex chromosomes pair with their homologs, and crossing over takes place. After the meiotic divisions, round haploid spermatids are generated and enter spermiogenesis, and this process leads to the formation of mature haploid spermatozoa in which the cytoplasm has been removed, the nuclear chromatin is condensed, and flagella emerge at the basal part of the nuclei21,22 (Figure 1 and Figure 2).
In general, spermiogenesis starts around the mid-pupal stage, and mature spermatozoa can be detected in the late pupal stage in the sperm reservoir23. The maturation process of the spermatocysts continues during adult life23,24,25. In Anopheles testes, each step of spermatogenesis can be easily identified by looking at the nuclear morphology of the cells in each spermatocyst (Figure 2). Whole-mount fluorescence in situ hybridization (WFISH), described in this protocol, allows researchers to specifically label a chromosomal region and track it during spermatogenesis while preserving the native structure of the organ and cell nuclei position; this represents an advantage when compared to the standard DNA FISH protocol in which the organ is usually squashed, leading to tissue damage19. In the current protocol, fluorescent probes are used to stain repetitive sequences on the sex chromosomes and, thus, track their behavior during spermatogenesis, from diploid dividing cells to mature haploid spermatozoa. WFISH can be particularly useful for studying sex chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis4,19,26,27.
Given their role as malaria vectors, Anopheles mosquitoes are the target of an increasing number of genetic vector control strategies, which often act in the reproductive organs of these organisms. Several mosquito mutants and cytological phenotypes have been generated that require novel cytological techniques to be investigated26,27,28,29. The method described in this study sheds light on the understanding of spermatogenesis, as well as the cytological mechanisms behind genetic strategies that have the potential to control malaria-transmitting mosquitoes.

Forfatter Spotlight: Helmonteret fluorescens in situ-hybridisering for at studere spermatogenese i Anopheles-myggen  

Helmonteret fluorescens in situ-hybridisering for at studere spermatogenese i Anopheles-myggen

The scope of our research is to develop and share cost-effective and sustainable genetic strategies that aim at reducing the number of malaria-transmitting mosquitoes in South Saharan Africa. In the last few years, we have demonstrated the power of gene drive technologies in suppressing caged mosquito populations. We are currently running a number of experiments to challenge these technologies in more complex experimental setup and to answer the regulatory requirements that are absolutely necessary for the potential implementation of these technologies in the field.FISH is a molecular cytogenetic technique that allows for the staining of a specific DNA sequence. It typically involves the squashing of the organ of interest, which can results in the loss of information about the spatial arrangement of cells. Wellman FISH allows for the preservation of the native cytological structure of the organs.Wellman FISH can be used to study chromosome behavior in mosquito reproductive organs. Reproductive organs are the target of several genetic strategies for vector control. Wellmann FISH has the potential to shed light on the mechanism that take place in the reproductive organs of well-time mosquito, as well as in any genetic strain of interest.

Watch this Scientific Journal Video about Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution at JoVE.com

Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution  

Generering af DNA-sonder til mærkning af kønskromosomer af Anopheles-myg og forberedelse af hybridiseringsopløsningen

Begynd med at bruge et kommercielt ekstraktionssæt til at ekstrahere det genomiske DNA fra pupperne eller fra voksne mandlige Anopheles-myg. Forbered derefter PCR-reaktionsblandingen. Mærk 18 SR-DNA og satellitten AGY 53B ved at udføre en PCR-reaktion.Til fremstilling af hybridiseringsopløsningen tilsættes fem mikroliter mærket DNA-sonde til et 1,5 ml rør. Derefter tilsættes fem mikroliter laksesæd-DNA. Bunds DNA-sonden ved at tilsætte to volumener 100% ethanol og 0,1 volumen af tre molære natriumacetat.Opbevares ved minus 20 grader Celsius i mindst 2,5 timer. Centrifuger røret ved 17.000 G i 20 minutter ved fire grader Celsius. Fjern derefter ethanolen og lufttør pelleten ved stuetemperatur i mørke i 20 minutter.Forbered derefter hybridiseringsbufferen i et 1,5 ml rør. Vortex opløsningen i et minut og lad dextransulfatet opløses ved 37 grader Celsius i 30 minutter. For at opnå hybridiseringsopløsningen opløses DNA-paletten i 20 til 30 mikroliter hybridiseringsbuffer.Vortex opløsningen i et minut. Udfør et hurtigt spin, og opbevar rørene ved 37 grader Celsius i mørke.

Research

JoVE Journal

Immunology and Infection

Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...