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

Hibridização In Situ de Fluorescência para Estudo da Espermatogênese de Mosquitos

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.

Autor em destaque: hibridização in situ de fluorescência de montagem total para estudar a espermatogênese no mosquito Anopheles  

Hibridação in situ por fluorescência de montagem total para estudo da espermatogênese no mosquito Anopheles

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  

Geração de sondas de DNA para marcar os cromossomos sexuais de mosquitos anofelinos e preparação da solução de hibridização

Comece usando um kit de extração comercial para extrair o DNA genômico das pupas ou de mosquitos Anopheles machos adultos. Em seguida, prepare a mistura de reação de PCR. Rotular o 18 SR-DNA e o satélite AGY 53B realizando uma reação de PCR.Para a preparação da solução de hibridização, adicione cinco microlitros de sonda de DNA marcado a um tubo de 1,5 mililitro. Em seguida, adicione cinco microlitros de DNA de esperma de salmão. Precipitar a sonda de DNA adicionando dois volumes de etanol a 100% e volume 0,1 volume de acetato de sódio a três molares.Mantenha a menos 20 graus Celsius por pelo menos 2,5 horas. Centrifugar o tubo a 17.000 G durante 20 minutos a quatro graus Celsius. Em seguida, retire o etanol e seque ao ar o pellet em temperatura ambiente no escuro por 20 minutos.Em seguida, prepare o tampão de hibridização em um tubo de 1,5 mililitro. Vórtice a solução por um minuto e deixe o sulfato de dextran dissolver a 37 graus Celsius por 30 minutos. Para obter a solução de hibridização, dissolver a paleta de DNA em 20 a 30 microlitros do tampão de hibridização.Vórtice a solução por um minuto. Dê uma volta rápida e guarde os tubos a 37 graus Celsius no escuro.

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Research

JoVE Journal

Immunology and Infection

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

341 visualizações. Comece usando um kit de extração comercial para extrair o DNA genômico das pupas ou de mosquitos Anopheles machos adultos. Em seguida, prepare a mistura de reação de PCR. Rotular o 18 SR-DNA e o satélite AGY 53B realizando uma reação de PCR.Para a preparação da solução de hibridização, adicione cinco microlitros de sonda de DNA marcado a um tubo de 1,5 mililitro. Em seguida, adicione cinco microlitros de DNA de esperma de salmão. Precipitar a sonda de DNA adicionando doi...

Vídeo: 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 changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying 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 spermatogenesis.

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

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