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.
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.
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 2010, the pea aphid Acyrthosiphon pisum3, marked an important milestone in the study of aphid biology because it provided the genomic resources for addressing questions about the insect's adaptations to the herbivorous lifestyles, including those that might lead to a better control strategies4. Since that time, additional genomic resources have accumulated with the publication of an annotated genome for the soybean aphid Aphis glycines5, and publicly-available whole genome resources for another three-aphid species (Myzus cerasi (black cherry aphid), Myzus persicae (peach-potato aphid), Rhopalosiphum padi (bird cherry-oat aphid)6. Valuable de novo transcriptomic resources are available as well for a number of other aphid species (e.g.,Aphis gossypii (cotton aphid)7, Sitobion avenae (grain aphid)8, Cinara pinitabulaeformis (pine aphid)9, Aphis nerii (milkweed-oleander aphid)10).
Aphids have also made lasting contributions to our understanding of the plant-insect interactions and the ecology of the life on plants11. One area where aphids have made particularly important contributions is in our understanding of the chemical ecology of the host plant interactions. Herbivorous insects express diverse adaptations for overcoming plant defenses, and some even co-opt plant defenses for their own benefit12,13,14. For example, the milkweed-oleander aphid, Aphis nerii, is a bright yellow, invasive aphid found in temperate and tropical regions worldwide that colonizes on plants in the milkweed family (Apocynaceae). Plants in the family Apocynaceae have evolved diverse chemical defenses, including milky latex and cardiac glycosides known as cardenolides, that bind the cation carrier Na,K-ATPase and are effective deterrents to generalist herbivores15,16. Milkweed specialists express various modes of resistance to cardenolides, and some selectively or passively accumulate or modify cardenolides in their tissues as a means to deter predation or for other benefits17. A. nerii is thought to sequester cardenolides in this way, although the mechanisms and functional benefits remain unclear10,18.
Given the genomic resources at hand, A. nerii provides an excellent experimental model for the study of the molecular and genetic mechanisms involved in the chemo-ecological interactions between toxic host plants and their specialist herbivores. It is worth noting that, while some of the earliest studies of A. nerii focused on sequestration of cardenolides19, since that time, studies of A. nerii have provided insights into a broad set of evolutionary and ecological questions, including the genetic structure of invasive insects20 and the interplay between bottom-up and top-down regulation on the herbivore density21. A. nerii is thus a good candidate as an experimental model for an especially broad set of studies of the insect-plant interactions. Critical to the success of any study with A. nerii is the careful culture of aphid populations, which includes the culture of plants on which the aphids depend, as well as an efficient generation of high-quality -omic data. Our goal is to guide the reader through both. Outlined below are 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. While these methods are written for A. nerii, the general culturing, extraction, and analysis methods can extend to a variety of aphid species.
1. Plant Cultures
2. Aphid Cultures
3. DNA Extraction
4. Microsatellite PCR and Sequencing for Aphid Genotyping
5. RNA Extraction
6. RNAseq De Novo Transcriptome Assembly, Annotation, and Differential Expression Analysis
7. qPCR Verification of Differentially Expressed Genes
NOTE: If users are interested in differentially expressed genes from their RNAseq experiments, the following protocol can be used to verify patterns of differential expression.
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 distinguished by some darkened cauda and may be unwinged (apterous, Figure 3A, B) or winged (alate, Figure 3C, D). Developing wing pads become visible when nymphs reach the third instar (Figure 3E, F). Stock cultures are best maintained by transferring one to three mid-instar and one adult-aged unwinged aphids; this ensures a healthy, mixed age population. Populations to be used for experiments should be cultured using unwinged aphids as described above (2.4). One A. nerii adult can produce 3-10 offspring per day, dependent on the host plant and age of the aphid10.
DNA and RNA extractions: Single, adult A. nerii will yield approximately 100–200 ng/µL DNA (80 µL elution; Figure 4A) and 150–300 ng/µL RNA (30 µL elution; Figure 4B). Representative microsatellite peaks are shown in Figure 5. Representative relative expression of a candidate gene under three conditions (control, Treatment 1, Treatment 2) are calculated in Table 2 and shown in Figure 6.
Figure 1: Representative plants for aphid cultures. (A) Seedlings can be re-potted after they have developed their first full set of true leaves. (B) Plants can be used for aphid cultures when they have developed 3-4 sets of true leaves. Please click here to view a larger version of this figure.
Figure 2: Examples of tools used for culturing aphids. (A) Mouth pipettes can be created using 3/16" ID x 1/4" OD plastic tubing, a 1,000 µL pipette tip, and a 200 µL pipette tip. (B, C) Use cup cages (clear plastic cups with the top cut off and secured with fine mesh) to securely fit over the top of 4 in. pots used for aphid cultures. This allows for ample light and ventilation to create a suitable environment for the aphids and plant, and keeps the aphids contained. Please click here to view a larger version of this figure.
Figure 3: Representative adult and nymph Aphis nerii. (A, B) Apterous (unwinged) adult A. nerii are identified by darkened cauda at their posterior end. (C, D) Alate (winged) adults are identified by fully developed wings and darkened cauda at their posterior. (E, F) Developing A. nerii nymphs go through four instar stages and developing wing pads become apparent during the third instar stage. Please click here to view a larger version of this figure.
Figure 4: Representative gels. (A) DNA extractions (1kb ladder). Seven A. nerii DNA extractions are visualized in lanes 3-9. Negative control is in lane 10. (B) RNA extractions. Eleven A. nerii RNA extractions are visualized in lanes 3-13. Please click here to view a larger version of this figure.
Figure 5: Representative microsatellite peaks. 6-FAM-tagged peaks are visualized in blue. LIZ-500 ladder is shown in orange. Please click here to view a larger version of this figure.
Figure 6: qPCR verification of a differentially expressed gene. Representative mRNA relative quantity (RQ) expression (calculated using the ΔΔCt method, Table 2) shown for a candidate gene of interest under three conditions: control, treatment 1, treatment 2. Graph shows decreased expression of candidate gene under treatments 1 and 2 compared to the control (Table 2). Please click here to view a larger version of this figure.
Primer Name | Direction | Sequence (5'-3') |
Ago24_F | forward | TTTTCCCGGCACACCGAGT |
Ago24_R | reverse | GCCAAACTTTACACCCCGC |
Ago 53_F | forward | TGACGAACGTGGTTAGTCGT |
Ago 53_R | reverse | GGCATAACGTCCTAGTCACA |
Ago 59_F | forward | GCGAGTGGTATTCGCTTAGT |
Ago 59_R | reverse | GTTACCCTCGACGATTGCGT |
Ago 66_F | forward | TCGGTTTGGCAACGTCGGGC |
Ago 66_R | reverse | GACTAGGGAGATGCCGGCGA |
Ago 69_F | forward | CGACTCAGCCCCGAGATTT |
Ago 69_R | reverse | ATACAAGCAAACATAGACGGAA |
Ago 84_F | forward | GACAGTGGTGAGGTTTCAA |
Ago 84_R | reverse | ACTGGCGTTACCTTGTCTA |
Ago 89_F | forward | GAACAGTGCTCGCAGTCTAT |
Ago 89_R | reverse | GACAGCGTAAACATCGCGGT |
Ago 126_F | forward | GGTACATTCGTGTCGATTT |
Ago 126_R | reverse | TAAACGAAAAAACCACGTAC |
Table 1: Microsatellite primer sequences used to genotype Aphis nerii20.
Target | Sample | Ct Mean | Ct Std. Dev | ΔCt | avg. ΔCt | ΔΔCt | RQ=2^(-ΔΔCt) | RQ SEM |
ef1a | 1.1 | 22.59 | 0 | |||||
ef1a | 1.2 | 20.31 | 0 | |||||
ef1a | 1.3 | 20.36 | 0.226 | |||||
ef1a | 1.4 | 20.27 | 0.036 | |||||
ef1a | 1.5 | 20.55 | 0.003 | |||||
ef1a | 1.6 | 20.52 | 0.245 | |||||
ef1a | 2.1 | 20.49 | 0.082 | |||||
ef1a | 2.2 | 19.86 | 0.033 | |||||
ef1a | 2.3 | 20.19 | 0.037 | |||||
ef1a | 2.4 | 19.67 | 0.058 | |||||
ef1a | 2.5 | 20.25 | 0.188 | |||||
ef1a | 2.6 | 18.16 | 0.089 | |||||
ef1a | 3.1 | 20.93 | 0.157 | |||||
ef1a | 3.2 | 20.22 | 0.003 | |||||
ef1a | 3.3 | 20.44 | 0.039 | |||||
ef1a | 3.4 | 20.91 | 0.559 | |||||
ef1a | 3.5 | 20.63 | 0.017 | |||||
ef1a | 3.6 | 20.3 | 0.135 | |||||
gene of interest | 1.1 | 24.6 | 0.173 | 2.01 | 0 | 1 | ||
gene of interest | 1.2 | 24.25 | 0.019 | 3.94 | 2.975 | 0 | 1 | 0 |
gene of interest | 1.3 | 24.79 | 0.04 | 4.43 | 0 | 1 | ||
gene of interest | 1.4 | 25.23 | 0.285 | 4.96 | 4.695 | 0 | 1 | 0 |
gene of interest | 1.5 | 24.6 | 0.103 | 4.05 | 0 | 1 | ||
gene of interest | 1.6 | 25.08 | 0.033 | 4.56 | 4.305 | 0 | 1 | 0 |
gene of interest | 2.1 | 27.52 | 0.155 | 7.03 | 5.019033762 | 0.03084042 | ||
gene of interest | 2.2 | 27.23 | 0.061 | 7.37 | 7.2 | 3.428355679 | 0.092888533 | 0.031024057 |
gene of interest | 2.3 | 27.18 | 0.058 | 6.99 | 2.56158174 | 0.169389724 | ||
gene of interest | 2.4 | 27.45 | 0 | 7.78 | 7.385 | 2.820764967 | 0.141535419 | 0.013927153 |
gene of interest | 2.5 | 27.44 | 0.032 | 7.19 | 3.138956897 | 0.113521944 | ||
gene of interest | 2.6 | 28 | 0 | 9.84 | 8.515 | 5.284272079 | 0.025661119 | 0.043930413 |
gene of interest | 3.1 | 27.23 | 0.143 | 6.3 | 4.292437371 | 0.051032588 | ||
gene of interest | 3.2 | 27.05 | 0.088 | 6.83 | 6.565 | 2.891234282 | 0.134788164 | 0.041877788 |
gene of interest | 3.3 | 27.45 | 0.109 | 7.01 | 2.578145722 | 0.167456035 | ||
gene of interest | 3.4 | 27.58 | 0.019 | 6.67 | 6.84 | 1.709038085 | 0.305863936 | 0.069203951 |
gene of interest | 3.5 | 27.06 | 0.067 | 6.43 | 2.384498984 | 0.191511246 | ||
gene of interest | 3.6 | 27.36 | 0 | 7.06 | 6.745 | 2.513723938 | 0.175103043 | 0.008204101 |
Table 2: Calculations for qPCR ΔΔCt verification of candidate gene. Candidate gene expression is calculated relative to ef1a (Figure 6). Samples 1.1-1.6 represent six biological replicates under the control treatment; samples 2.1-2.6 represent six biological replicates under Treatment 1; samples 3.1-3.6 represent six biological replicates under Treatment 2. Ct Std. Dev. is calculated from three technical replicates.
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 sequestration18,37. 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, and molecular/genomic techniques, with the assumption that future work on this species will likely involve studies that utilize genomic and functional ecological approaches. Many open questions remain about the mechanisms and significance of cardenolide detoxification and sequestration in A. nerii. Techniques such as RNAi for expression knockdown or gene editing approaches will prove valuable in this regard.
One of the challenges in culturing aphids is in their prodigious capacities for the reproduction and dispersal. These traits, which directly relate to why they are serious crop pests, means that aphid cultures require almost daily attention, as well as extreme care if isogenic lines are required for experiments. The techniques described above, including those for generating data for the analysis of gene expression, while similar to general protocols for aphid rearing and molecular analysis, provide a specific step-by-step guide to generating sufficient biological material for A. nerii for a diverse set of molecular and ecological applications.
To this end, if functional or ecological genomic studies are on the horizon for A. nerii, these will need to be coupled with live cultures to fully capitalize on the experimental opportunities they offer. Insect herbivores live in complex communities on their host plants, and both intraspecific interactions38,39 as well as interspecific interactions40 shape the ultimate response of A. nerii to their host plants. The host plants, A. nerii specialize on, represent a diverse set of plants that express divergent life history strategies15,21, underscoring the importance of coupling purely genomic or physiological approaches with experimental manipulations that account for naturally-occurring variation in A. nerii communities. The methods outlined here are starting points for a functional and ecological genomic perspective on A. nerii and its interactions with toxic host plants.
The authors have nothing to disclose.
We would like to thank Michelle Moon (Vanderbilt University) for assistance with photography. Vanderbilt University provided support to PA and SSLB is supported by DGE-1445197.
Sun Gro Fafard Germination Mix | Hummert International | 10-0952-2 | |
Sun Gro Fafard 3B/ Metro Mix | Hummert International | 10-0951-2 | |
2x 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 |