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

Summary

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.

Abstract

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.

Introduction

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 organism^2,3,4. Recent comparative....

Protocol

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 queens^8,14. In this protocol, we collected the latter type of colonies in Ishikawa prefecture, Japan.

1. Collect V. emeryi colonies during early summer.<....

Results

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 ± 8.91% SD) of all offspring (F₂) from the inbreeding crosses was male, while the remainder was female (workers and a qu.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Plaster powder	N/A	N/A	Any brand can be used
Charcoal, Activated, Powder	Wako	033-02117,037-02115
Slide glass	N/A	N/A	Any brand can be used
Dry Cricket diet	N/A	N/A	Any brand can be used
Brown shuger	N/A	N/A	Any brand can be used
Styrene Square-Shaped Case	AS ONE	Any size	Size varies by number of ants
Incbator			Any brand can be used
Aluminum block bath Dry thermo unit DTU-1B	TAITEC	0014035-000
1.5mL Hyper Microtube,Clear, Round bottom	WATSON	131-715CS
Ethanol (99.5)	Wako	054-07225
Stereoscopic microscope	N/A	N/A	Any brand can be used
Forseps	DUMONT	0108-5-PO
Chelex 100 sodium form	SIGMA	11139-85-8
Phosphate Buffer Saline (PBS) Tablets, pH7.4	TaKaRa	T9181
Paraformaldehyde	Wako	162-16065
-Cellstain- DAPI solution	Dojindo Molecular Technologies	D523
VECTASHIELD Hard・Set Mounting Medium with TRITC-Phalloidin	Vector Laboratories	H-1600
ABI 3100xl Genetic Analyzer	Applied Biosystems		Directly contact the constructor formore informations.
Confocal laser scanning microscope Leica TCS SP8	Leica		Directly contact the constructor formore informations.
HC PL APO CS2 20x/0.75 IMM	Leica		Directly contact the constructor formore informations.
HC PL APO CS2 63x/1.20 WATER	Leica		Directly contact the constructor formore informations.
Leica HyDTM	Leica		Directly contact the constructor formore informations.
Leica Application Suite X (LAS X)	Leica		Directly contact the constructor formore informations.