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 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 ants^6,7. 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 crosses^6,12,13.

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.
   NOTE: To obtain sufficient numbers of sexual individuals during the reproductive season, colonies containing more than 300 individuals are preferred.
2. Transfer the ant specimens from the collected branches to an artificial plaster nest with a glass cover using an aspirator (Figure 2, left).
3. Maintain colonies in the artificial nest at 25 °C under a 16:8 h light/dark cycle. Provide tap water with a wash bottle to wet the plaster.
   1. Add about 100 mg of dry cricket powder wrapped in aluminum foil and a brown sugar water-filled tip (20 µL tip) every other day until new reproductives (F₁ winged-queens and F₁ males) emerge.

2. Experimental Laboratory Crosses

NOTE: New reproductives start to emerge from late summer to autumn (Figure 3). Long-winged queens are produced sexually, and short-winged queens are produced clonally and have the maternal genome (Figure 1). Use long-winged queens and males for inbreeding crosses.

1. To stop individuals from moving, place colonies in a constant environment room at 4 °C for 15 min.
2. Remove the mid-legs of 30 workers using forceps under a stereoscopic microscope and transfer them into new smaller plaster nest (Figure 2, right) for inbreeding crosses.
   NOTE: The legs are removed to distinguish the present workers from the workers that will be produced by the subsequent inbreeding crosses.
3. Add 3-4 larvae or pupae into a plaster nest containing workers.
   NOTE: Workers show exploratory activity in the F₀ queens-less colony. Larvae or pupae can effectively attract these workers and new reproductives in the center of the colony during crossing test. As a result, keep conditions of the experimental colony close to the normal colony.
4. Transfer a long-winged queen and a male into a plaster nest prepared in step 2.3 for inbreeding crosses.
5. Keep colonies at 25 °C under a 16:8 h light/dark cycle with food and water provided as described in 1.3 until the queen lose her wings and lay eggs.
   NOTE: This takes one week to a month.
6. Check the experimental colony everyday under a stereoscopic microscope. After performing inbreeding crosses between the F₁ offspring, eggs can be observed under a stereoscopic microscope.
7. After the F₁ queen starts laying eggs, remove F₁ males and larvae or pupae added in step 2.3 from the nest to avoid mixing of the F₁ generation (males and females used for inbreeding crosses) and the F₂ generation (offspring produced from inbreeding crosses).
   NOTE: If there are few males in the colony, it is possible to induce inbreeding crosses using one male and 1 to 3 queens in the same experimental colony.
8. Keep colonies under the same conditionsas described in 1.3, until F₂ offspring emerge.
   NOTE: Transfer F₁ queen and F₂ offspring into new larger plaster nest (Figure 2, left) for long-term colony keeping.

3. Evaluation of Inbreeding Success

1. DNA extraction and genotyping of the parental generation (F₀)
   1. Remove one leg of a F₀ queen using forceps and transfer the leg to a 1.5 mL microtube containing 100 µL of chelation agent.
   2. Under a stereoscopic microscope, dissect a female abdomen in glass dish filled with 300 µL of ultrapure water using forceps and isolate the spermatheca containing the sperm from mated males.
   3. Peel away the tissue of the spermatheca and isolate the sperm from the tissue of the female using insect pins.
      NOTE: To facilitate sperm extraction from the spermatheca, store female specimens in 100% EtOH for more than one day before dissection.
   4. Using a micropipette, transfer the sperm into a 1.5 mL microtube containing 100 µL of chelation agent.
   5. Incubate samples of F₀ queen and sperms prepared in step 3.1.1 and 3.1.3, respectively, at 95 °C for 20 min. Flash centrifuge the microtube and store at 4 °C.
   6. Genotype all samples using method described elsewhere⁴.
2. DNA extraction from the pair of ants used for inbreeding crosses (F₁)
   1. After confirming egg production by the sib mated F₁ queen, extract the DNA of the queen using her shed wings or one mid-leg and genotype using same method described in section 3.1 above.
   2. Extract DNA of F₁ male using one leg and genotype them using same method described in section 3.1 above.
      NOTE: Samples can be stored in 100% EtOH before DNA extraction, and DNA in chelation agent can be stored for two months at 4 °C.
3. Evaluation of male fertility in males produced from inbreeding crosses
   NOTE: Diploid males produced from inbreeding crosses are often sterile.
   1. Dissect internal reproductive organs in a glass dish with 400 µL of PBS solution using forceps.
   2. Remove PBS and add 4% paraformaldehyde (PFA) using a micropipette.
   3. Fix the tissue by incubating in PFA for 30 min at room temperature (15 - 25 °C).
   4. Wash tissue 5 times with 400 µL of PBS using a micropipette.
   5. Dilute the 4',6-diamidino-2-phenylindole (DAPI) solution to 1 µg/mL in PBS.
   6. Remove PBS and add approximately 300 µL of this dilute DAPI staining solution to tissue.
   7. Incubate 15 min under dark condition at room temperature (15 - 25 °C).
   8. Wash tissue 5 times with 400 µL of PBS, and transfer tissue on center of slide glass using forceps.
   9. Mount tissue on mounting medium containing Tetramethylrhodamine (TRITC)-conjugated phalloidin.
   10. Observe samples by confocal laser scanning microscope using 20X or 63X objective lenses.
   11. Use a 405 nm excitation laser and a hybrid detector at 410-530 nm for DAPI detection.
   12. Use a 561 nm excitation laser and a hybrid detector at 565-650 nm for TRITC detection.
   13. Use a scan speed of 400 Hz (400 lines/s) at a resolution at 1024 × 1024 pixels.
   14. Capture images using a software platform.

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.