Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Summary

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

Abstract

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Introduction

Aphids are small, hemimetabolous insects that colonize on diverse plant families worldwide. They are distinctive for several features, most notably their complex life cycles involving cyclical parthenogenesis and discrete polyphenisms, and their obligate nutritional symbioses with bacterial or yeast endosymbionts that supply nutrients missing from their diet of plant sap¹. While most aphids are host plant specialists, some generalist species are important crop pests, inflicting considerable economic damage on crops either directly or via the pathogens and viruses they vector². The publication of the first aphid genome in....

Protocol

1. Plant Cultures

1. Purchase seeds from any commercial vendor or collect from mature plants in the field.
   NOTE: This protocol is suitable for most commercially available milkweed species (e.g., Asclepias incarnata, A. syriaca, A. curassavica, Gomphocarpus physocarpus). Some seeds may need to be cold-stratified, and instructions from the seed supplier should be checked.
2. Plant seeds in a fine germinating soil (60–70% fine peat moss, perlite, vermiculite, limestone).
   1. Fill a standard seedling tray with germination mix soil; ensuring that the soil reaches the top of the wells.....

Results

Plant cultures: Seeds will take approximately two to four weeks, depending on the season, to grow large enough to be re-potted (Figure 1A). Re-potted seedlings will take another two to four weeks to grow to an optimal size for aphid cultures (Figure 1B).

Aphid cultures: Adult A. nerii are disti.......

Discussion

It has long been recognized that the aposematic A. nerii can provide insights into the patterns and mechanisms of resistance to plant defenses and particularly chemical sequestration^18,37. A number of genomic resources have recently emerged for A. nerii¹⁰, offering new opportunities for ecological and functional genomic studies that use A. nerii as a model. We outline basic protocols in aphid and plant culture, a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sun Gro Fafard Germination Mix	Hummert International	10-0952-2
Sun Gro Fafard 3B/ Metro Mix	Hummert International	10-0951-2
2" x 4" Round Standard Pot	Anderson Pots	1503
DreamTaq DNA Polymerase	ThermoFisher Scientific	EP0701
Trizol	ThermoFisher Scientific	15596026
SuperScript® III First-Strand Synthesis kit	ThermoFisher Scientific	18080051
Power SYBR Green PCR Master Mix	ThermoFisher Scientific	4367659