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>
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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 ...

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