Department of Entomology, Virginia Tech
Timoshevskiy, V. A., Sharma, A., Sharakhov, I. V., Sharakhova, M. V. Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes. J. Vis. Exp. (67), e4215, doi:10.3791/4215 (2012).
Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.
1. Chromosome Preparation
Mosquito larvae were reared using a standard protocol described in Methods in Anopheles Research available at the website of the Malaria Research and Reference Reagent Resource Center (MR4) 13. The temperatures of mosquito rearing were modified to provide the highest number of chromosomes in imaginal discs and lowest mortality of the larvae. The stages of mosquito larvae development were determined based on the sizes of their head capsules 13.
2. Extraction of Repetitive DNA Fractions
Performing FISH of the BAC clone DNA probe on chromosomes from Ae. aegypti and Cx. quinquefasciatus requires using unlabeled repetitive DNA fractions to block unspecific hybridization of the DNA repeats to the chromosomes. The reassociation of single-strand DNA fragmented into pieces of several hundred bp follows a C0t curve where C0 is the initial concentration of single-stranded DNA and t is the reannealing time. DNA fractions with C0t values equal to 10-4-10-1 or 10°-102 are considered as highly and moderately repetitive, respectively.
3. DNA Probe Labeling
Two different protocols were used for the labeling BAC clone DNA probe and IGS rDNA probe.
3.1 BAC clone labeling using nick-translation
3.2 IGS rDNA labeling using PCR
4. Fluorescent in situ Hybridization
This FISH protocol includes two variations: the first for using BAC clone DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus and the second for using IGS rDNA on mitotic chromosomes of An. gambiae. If using BAC clone DNA probes, skip RNase treatment steps 4.3, 4.4, and simultaneous slide/probe denaturation step 4.19. If using IGS rDNA probe, prepare hybridization mixture without C0t DNA fractions, and skip separate slide/probe denaturing steps 4.10, 4.11, 4.16, and 4.17.
5. Representative Results
Insect IDs are located in each segment of the larva. Depending on the position, they transform into different tissues at the adult stage of the insect. The IDs, which are used for the chromosome preparation in this protocol, develop into legs at the adult stage of the mosquito. These IDs are located at the ventral side of the larval thorax and are clearly visible through the cuticle under the microscope (Figure 1). At the early 4th instar larval stage, IDs have a round shape (Figure 1A). The largest numbers of mitosis, ~175 in one ID 9, are accumulated at a later "oval shaped" stage (Figure 1B), which must be considered the optimal stage for slide preparation. At this time, the intermediate ID splits into two: one transforms into a leg and another one transforms into a wing. We prefer using the large leg IDs at the "oval-shaped" stage for the chromosome slide preparation. Figure 1C represents IDs at the latest stage of 4th instar larva development. At this stage, the IDs are already developed into legs and wings, and contain a significant amount of differentiated tissues and a low number of mitosis. This stage of ID development should be avoided for chromosome slide preparation. We also recommend rearing mosquito larvae at low temperatures: 16 °C for Aedes and Culex and 22 °C for Anopheles. This helps to increase the amount of mitosis in IDs 9.
Figure 2 illustrates ID dissection from the thorax of 4th instar larva. Because the cuticle of a live insect is hard to dissect, we recommend using dissecting scissors instead of the needles commonly used for larva preparation. The most crucial procedure for obtaining high-quality chromosome preparation is the hypotonic solution treatment. For best results, we remove the gut and fat body from the larval thorax before this treatment. Swelling of the ID cells during this procedure helps to spread chromosomes on a slide (Figure 3A). The appropriate quality of the hypotonic solution treatment can be easily recognized by the round shape of cells in the preparations (Figure 3A, B). Cells with an oval shape indicate insufficient hypotonic solution treatment (Figure 3C). To be selected for FISH, chromosome preparation should contain at least 50 high-quality chromosome spreads. Normally, ~90% of the slides prepared using this protocol have sufficient quality for FISH 9.
We present two slightly different FISH protocols: an advanced protocol for FISH using genomic BAC clone DNA probe on mitotic chromosomes of Aedes and Culex and a simple FISH protocol for IGS rDNA probe on mitotic chromosomes of Anopheles. The genomes of Aedes and Culex are highly repetitive because of the overrepresentation of transposable elements 7,8. Thus, performing FISH, which utilizes genomic BAC clone DNA as a probe, requires adding unlabeled repetitive DNA fractions to the probe to block unspecific hybridization of the DNA repeats to chromosomes. For the extraction of the repetitive DNA fractions, genomic DNA is denatured at 120 °C for 2 min. Boiling DNA at a high temperature also helps to obtain DNA in fragments of 200-500 bp. DNA is allowed to reassociate after this treatment. The highly repetitive DNA fragments tend to find their mate for reassociation faster than DNA with unique sequences does. As a result, the reassociation of DNA follows a C0 xt curve where C0 is the initial concentration of single-stranded DNA, and t is the reannealing time. DNA fractions with C0t values equal to 10-4--10-1 or 100-102 are considered highly and moderately repetitive, respectively. The time of the reassociation for different C0t DNA fractions can be calculated using the formula t = C0tX × 4.98/C0 , where t - time of incubation, C0tX - C0t fraction (C0t1=1, C0t2=2, etc.) and C0 - initial DNA concentration in μg/μl15 (Table 1). After reassociation, the single-stranded DNA is digested using S1 nuclease. We prefer using all C0t DNA fractions up to C0t3 together instead of the commonly used C0t1 DNA fraction. These C0t fractions include some of the moderately repetitive DNA sequences and together usually represent 35-50% of the original amount of the genomic DNA in Ae. aegypti. The correct proportion between labeled DNA probe and unlabeled C0t DNA fraction depends on the repetitive DNA component in each particular BAC clone. On average, we use 1:20 probe to C0t DNA fraction proportion for obtaining an acceptable signals/background ratio of the FISH result. Prehybridization of the DNA probe with C0t DNA fractions in a tube for 30 min before the actual hybridization on the slide also helps to reduce background. Labeling, hybridization itself, and washing in this protocol are performed using standard conditions 12.
The FISH results of two differently labeled BAC clone DNA probes on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus are shown in Figures 4A and B, respectively. The BAC clone DNA probes produce strong signals in a single position on the chromosomes. Chromosomes shown in Figure 1 are counterstained with YOYO-1 iodide. This dye produces the best banding patterns on Ae. aegypti chromosomes 9. Alternatively, other fluorescent dyes, such as DAPI or propidium iodide, can be utilized for the chromosome counterstaining. For suppressing photobleaching of the slides, we use Prolong Gold antifade mounting medium. This reagent has good signal preservation abilities and also can be easily removed from the slide by rinsing in 1x PBS if it is necessary to use the same slide for several hybridizations.
A simple version of the FISH protocol is designed for hybridization of IGS rDNA probe on mitotic chromosomes of Anopheles. Ribosomal genes in Anopheles are represented as a polymorphic cluster of genes located on sex chromosomes 16. A DNA probe in this protocol is labeled using standard PCR reaction by adding fluorescently labeled Cy3 or Cy5 dNTPs. Because blocking unspecific hybridization of repetitive DNA in euchromatin is not needed, all steps related to using C0t DNA fractions are omitted. Instead, chromosome preparations are pretreated with RNase for preventing hybridization of the IGS rDNA probe to the nucleolus. Chromosomes and the DNA probe are denatured simultaneously by heating the slide together with a probe in a hybridization system at 75 °C for 5 min. Hybridization and washing in this protocol are also performed using standard conditions for FISH 12. The result of FISH is demonstrated in Figure 4C: the polymorphism of the IGS rDNA hybridization between two X chromosomes is clearly visible.
|DNA concentration μg/μl||Reannealing time, min|
Table 1. DNA concentration and reannealing times for preparation of C0t2 and C0t3 fractions.
Figure 1. Stages of the ID development in 4th instar larva: A) an early "round shape" stage; B) an intermediate "oval shape" stage - optimal for the chromosome preparation; C) a late stage - inappropriate for chromosome preparations. The positions of IDs are indicated by arrows on the ventral side of the larval thorax.
Figure 2. Steps of ID dissection: A) decapitated larva (the direction of cuts are indicated by arrows); B) larvae with dissected gut under hypotonic solution treatment (IDs swell and become almost invisible); C) larva after Carnoy's solution application (IDs become white and clearly visible); D) dissected IDs in Carnoy's solution. Positions of IDs in larva are indicated by asterisks.
Figure 3. Different qualities of the chromosome spreads: A) a perfect chromosome spread - round shape of the cells demonstrates sufficient treatment of the IDs in hypotonic solution; B) a perfect hypotonic treatment - chromosomes are slightly undersquashed; C) a poor chromosome spread - the result of insufficient hypotonic treatment is indicated by oval shape of the cells.
Figure 4. Examples of FISH with BAC clones (A, B) and IGS rDNA (C) in the chromosomes of Ae. aegypti (A), Cx. quinquefasciatus (B), and An. gambiae (C). 1, 2 and 3 - are numbers of chromosomes; X - female sex chromosome in An. gambiae.
Nonfluorescent in situ hybridization on mitotic chromosomes of mosquitoes was performed for the first time in 1990 by A. Kumar and K. Rai 17. In that study, 18S and 28S ribosomal DNA genes, cloned together in one plasmid, were placed to the chromosomes of 20 species of mosquitoes. The DNA probe was radioactively labeled and hybridized to the chromosomes from brain ganglia. Among three mosquito genera, a FISH technique has been developed only for mitotic chromosomes from the cell line of Ae. aegypti 10,18,19 and has never been performed on mitotic chromosomes from live mosquitoes. Recently, we developed a simple, robust technique for obtaining high-quality chromosome preparations from IDs of 4th instar larvae 9. This method allows a high number of chromosomes to be obtained in one slide and can be universally used for all species of mosquitoes. The necessity of using only larval, not pupal or adult stages of mosquitoes, for slide preparation is probably the only limitation of the method. The standard FISH method 12 was optimized for using genomic BAC clone and IGS rDNA as probes for the mitotic chromosomes of Aedes, Culex, and Anopheles.
In addition to these specific applications, the FISH protocols described here can also be used for other purposes. The advanced FISH protocol, which utilizes C0t DNA fractions for blocking unspecific hybridization, can also be applied for the hybridization of BAC clones or any other large DNA fragments in heterochromatic regions of Anopheles. Heterochromatic regions are enriched with transposable elements and other repeats, and probes from these regions normally produce strong background on the chromosomes3. Using unlabeled C0t DNA fractions will help to reduce unspecific hybridization of the probe to the chromosomes. The simple version of the FISH protocol can be used for any rDNA or repetitive DNA probes on mitotic chromosomes of mosquitoes and other insects. In addition, it also can be applied for the hybridization of BAC clone DNA in species with low repetitive DNA content in euchromatic regions such as Anopheles or Drosophila. The protocol proposed here will help to obtain highly-finished chromosome-based genome assemblies for mosquitoes and can be broadly used for various cytogenetic applications in other groups of insects.
No conflicts of interest declared.
We thank Sergei Demin and Tatyana Karamysheva for their help with chromosome preparation and FISH on Anopheles. We also thank David Severson for providing us Aedes and Culex genomic DNA BAC clones and Melissa Wade for editing the text. This work was supported by two grants from the National Institutes of Health: 1R21 AI88035-01 to Maria V. Sharakhova and 1R21 AI094289-01 to Igor V. Sharakhov.
|MZ6 Leica stereomicroscope||Leica||VA-OM-E194-354||A different stereomicroscope can be used|
|Olympus CX41 phase microscope||Olympus||CX41||A different phase microscope can be used|
|Olympus BX61 fluorescent microscope||Olympus||BX61||A different fluorescent microscope can be used|
|ThermoBrite Slide Denaturation/Hybridization System||Abbott Molecular||30-144110||Serves as a heating block and a humid chamber|
|Dissecting needles||Fine ScienceTools||10130-10|
|Needle holders||Fine Science Tools||26018-17|
|Dissecting scissors||Fine Science Tools||15000-03|
|75x25 double frosted micro slides||Corning||2949-75x25|
|22x22 mm microscope coverslips||Fisher Scientific||12-544-10|
|Rubber Cement||Fisher Scientific||50-949-105|
|Acetic acid||Fisher Scientific||A491-212|
|Alcohol 200 Proof||Decon Laboratories||2701|
|Hydrochloric acid||Fisher Scientific||A144-500|
|Sodium citrate dihydrate||Fisher Scientific||S279-500|
|Sodium acetate trihydrate||Fisher Scientific||BP334-500|
|Potassium chloride||Fisher Scientific||BP366-500|
|Tris base||Fisher Scientific||BP152-1|
|10% NBF (neutral buffered formalin)||Sigma-Aldrich||HT501128|
|99% formamide||Fisher Scientific||BP227500|
|Dextran sulfate sodium salt||Sigma-Aldrich||D8906|
|20x SSC buffer||Invitrogen||AM9765|
|1 mM YOYO-1 iodide (491/509) solution||Invitrogen||Y3601|
|Antifade Prolong Gold reagent||Invitrogen||P36930|
|dATP, dCTP, dGTP, dTTP||Fermentas||R0141, R0151, R0161, R0171|
|Cy3-dUTP, Cy5-dUTP||GE Healthcare||PA53022, PA55022|
|DNA Polymerase I||Fermentas||EP0041|
|Taq DNA Polymerase||Invitrogen||18038-042|
|Salmon sperm DNA||Sigma-Aldrich||D7656|
|Nonidet-P40 (NP40)||US Biological||NC9375914|
|Qiagen Blood and Cell Culture Maxikit||Qiagen||13362|
|Qiagen Large Construct Kit||Qiagen||12462|