Name: induction and evaluation of inbreeding crosses using the ant, vollenhovia emeryi
Uploaded: 2018-10-05T15:00:00
Description: Watch this Scientific Journal Video about Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi at JoVE.com

We thank Mr. Taku Shimada, the delegate of AntRoom, Tokyo, Japan, for providing us with his photograph of V. emeryi reproductives. This project was funded by the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists (16J00011), and Grant in Aid for Young Scientists (B)(16K18626).

1. Field Collection and Maintenance of V. Emeryi Colonies in the Laboratory
NOTE: Nests of V. emeryi are found in rotting logs and fallen decaying tree branchesin secondary forests throughout Japan. This species shows two types of colonies, i.e., (1) colonies producing only long-winged queens and (2) colonies mainly producing short-winged queens in addition to small number of long-winged queens8,14. In this pro...

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This article demonstrates protocols that can be used to induce inbreeding crosses and evaluate the occurrence of inbreeding in the ant V. emeryi. In the experiments, genotyping of the individuals used for crosses is necessary to ensure that inbreeding crosses were successful. However, the effectiveness of these crossing tests is clearly apparent as diploid males can be produced throughout the year, while haploid males can only be produced in autumn in both the field and the laboratory6. S...

Eusocial Hymenopteran taxa, such as ants and bees, have evolved a haplodiploid sex-determination system in which individuals that are heterozygous at one or more complementary sex determination (CSD) loci become females, while those that are homo- or hemizygous become males (Figure 1A)1.
Genetic and molecular components involved in the sex determination cascade have been well studied in the honeybee, Apis mellifera, a hymenopteran model organism2,3,4. Recent comparative genomics investigations suggest that ants and honeybees share many putative homologs in the sex determination pathway, such as the initial sex determination gene, csd5. However, evidence for the functional conservation of these homologs is still lacking in ants.
To address this problem, inbreeding lines need to be developed as they are essential for genetic mapping and molecular studies. However, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory because of the complex nature of the life cycles that have evolved.
Here, we use Vollenhovia emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ants6,7. The inbreeding lines of this species were developed previously for linkage mapping of quantitative trait loci (QTL) for traits related to sex determination for the first time in ants6. In addition, the molecular sex-determination cascade has been investigated7. This species has evolved an unusual reproduction system that employs both gynogenesis and androgenesis (Figure 1B)8,9. Most new queens and males are clonally produced from the maternal and paternal genomes, respectively. In addition, workers and some queens are produced sexually8. This reproduction system is particularly well suited to genetic studies because the inbreeding crosses produced using sexually produced queens and males are genetically equivalent to a classic backcross. Since sexually produced queens differ morphologically from queens produced from maternal genomes10 (Figure 1B), conducting and evaluating inbreeding crosses is greatly simplified using this method.
In this article, the methods for establishment of laboratory colonies for crossing test, application of inbreeding crosses using full-sib pairs, and evaluating the success of those crosses using genotyping of colony members and dissection of male offspring genitalia are described in V. emeryi.
Regardless of the reproduction system employed, application of inbreeding crosses is often the essential first step in any investigation of sex determination systems in the Hymenoptera. For example in Cardiocondyla obscurior, the almost complete absence of diploid males after 10 generations of full-sib mating in the laboratory demonstrates absence of CSD locus11. It is possible to predict the number of CSD loci from the ratio of males produced in inbreeding crosses6,12,13.

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			<td height="45" style="height:45px;width:392px;">Plaster powder</td>
			<td style="width:239px;">N/A</td>
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			<td>Any brand can be used</td>
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			<td height="45" style="height:45px;width:392px;">Dry Cricket diet</td>
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			<td style="width:239px;">AS ONE</td>
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			<td>Size varies by number of ants</td>
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			<td height="45" style="height:45px;width:392px;">Incbator</td>
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induction and evaluation of inbreeding crosses using the ant, vollenhovia emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Results of microsatellite analysis using F0 and F1 generations showed that inbreeding crosses were produced successfully (Figure 4)6. As a result of inbreeding crosses, mated queens were obtained within one month of establishing the experimental crossing colonies. A quarter (27.1 &#177; 8.91% SD) of all offspring (F2) from the inbreeding crosses was male, while the remainder was female (workers and a qu...

Watch this Scientific Journal Video about Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi at JoVE.com

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

This method can help answer key questions in the molecular biology and genetic spheres of Hymenoptera, such as sex determination systems. The main advantage of this technique is that we can easily induce inbreeding lines over the ant Vollenhovia Emery, which are essential for genetic mapping and the molecular status. So, this mission can provide insight into the sex determination system of a specific ant and it may also be applied to other ants and other Hymenoptera species, such as wasps.To begin the protocol, collect V.Emeryi colonies during early summer. Transfer the ant specimens from the collected branches to an artificial plaster nest with a glass cover, using an aspirator. Maintain the colonies in the artificial nest at 25 degrees C, under a 16 to eight hour light-dark cycle.Provide tap water with a wash bottle to keep the plaster moist every other day at the same time. Next, add about 100 micrograms of dry cricket powder wrapped in aluminum foil and a brown sugar water-filled tip to the colony, using a 20 microliter tip every other day until new reproductive ants emerge. It is important to collect micro colonies from the fields and to provide them enough food to gain new queens and males.First, stop individuals from moving by placing colonies in a room with a controlled environment set at four degrees Celsius for 15 minutes. Remove the mid-legs of 30 worker ants using forceps under a stereoscope to distinguish the F-zero generation from the workers produced in subsequent inbreeding crosses. Then transfer the ants into a new smaller plaster nest for inbreeding crosses.Next, add three to four larvae or pube into a plaster nest containing workers. Transfer a long winged queen and a male into a previously prepared plaster nest for inbreeding crosses. For this step, it is important to maintain experimental crossing colony at the same state as that of a normal colony.It is difficult to induce inbreeding just by placing males and females together. Keep the colonies at 25 degrees Celsius under a 16 to eight hour light, dark cycle with food and water provided until the queen loses her wings and lays eggs. Check the experimental colony everyday under a stereo microscope.After setting up the inbreeding crosses between the F-one offspring, eggs can be observed under a stereoscope. After the F-one queen begins to lay eggs, remove the F-one males and larvae or pube from the nest to prevent the F-one generation and the F-two generation from mixing. Keep the colonies under the same conditions until the F-two offspring emerge.Remove one leg of an F-zero queen using forceps. Transfer the leg to a 1.5 milliliter micro tube containing 100 microliters of a chill agent. Under a stereoscope dissect a female abdomen in a glass dish filled with 300 microliters of ultra pure water using forceps.Dissimilate the spermatica containing the sperm from mated males. Peel away the tissue of the spermatica and isolate the sperm from the tissue of the female using insect pins. Using a micro pie pad, transfer the sperm into a 1.5 milliliter micro tube containing 100 microliters of a chill agent.Incubate the samples from the F-zero queen and sperm at 95 degrees Celsius for 20 minutes. Flash centro fuse the micro tube and store the samples at four degrees Celsius. After confirming egg production by the siv-mated F-one queen, extract the DNA of the queen using the shed wings or one mid leg and genotype.Next, extract the DNA of an F-one male by genotyping one leg. To evaluate the fertility of the male ants from the inbreeding crosses, dissect internal reproductive organs in a glass dish with 400 microliters of PBS solution using forceps. Then remove the PBS and replace it with four percent paraformaldihyde using a micro pie pad.Fix the tissue by incubating the samples in PFA for forty minutes in room temperature. After fixation, wash the tissues five times with 400 microliters of PBS using a micro pie pad. Dilute DAPI solution to one milligram per milliliter in PBS.Remove the PBS and add approximately 300 microliters of the diluted DAPI staining solution to the tissue. Incubate the tissue for 15 minutes under dark conditions a room temperature. Following the incubation, wash the tissue five times with 400 microliters of PBS and transfer the tissue to the center of a glass slide using forceps.Then mount the tissue on mounting medium containing TRITC tetramethylrhodamine. Finally observe the samples with a confocal laser scanning microscope using the 20 or 60 3x objective lenses. Microscopic images of testes of Haploid male v Emeryi show healthy fibrous tissue or sperm which are absent in Diploid male v Emeryi, which did not show healthy fibrous tissue.The male reproductive organs of Diploid male v Emeryi did not produce sperm and their testes did not develop, suggesting that males produced in inbreeding crosses are sterile. After its development, this technique paved the way for us to explore sex myatony in a constant in Vollenhovia Emeryi for the fast timing ant species.

Research

JoVE Journal

Genetics

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...