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7-18-2016 Dichotomous Key to ( pisum) Apterous Parthenogenic Instars Bates College Department of Biology

Daisy Diamond Bates College, [email protected]

Daniel Levitis Bates College, [email protected]

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Citation Information Bates College Department of Biology; Diamond, Daisy; and Levitis, Daniel, "Dichotomous Key to Pea Aphid (Acyrthosiphon pisum) Apterous Parthenogenic Instars" (2016). SCARAB Data Repository. 1. http://scarab.bates.edu/datasets/1

This Open Access is brought to you for free and open access by SCARAB. It has been accepted for inclusion in SCARAB Data Repository by an authorized administrator of SCARAB. For more information, please contact [email protected]. Key to the apterous parthenogenetic instars of Pea (Acyrthosiphon pisum)

Julia Daisy Diamond1,2, Daniel A. Levitis1

1Bates College, Department of Biology 2 contact: [email protected]

Summary We provide a dichotomous key, with photographs and illustrations, for distinguishing between instars of the pea aphid (Acyrthosiphon pisum) in the developmental pathway leading to the apterous parthenogenetic adult. Lengths of body, antenna and cauda are provided for a sample of each instar.

Keywords aphid development, nymphal stages, identification key, wingless aphid instars

Introduction The pea aphid (Acyrthosiphon pisum) is widely studied as both a laboratory organism and agricultural pest and has been identified as an emerging model species (Brisson and Stern 2006). Identifying the particular instar of an individual can be challenging for students and other investigators new to this species. Like many aphids, A. pisum has numerous developmental pathways. While a key to the instars has been published (in Italian) for the pathway leading to a winged female (Digilio 1995), many laboratory studies focus on the apterous (wingless) individuals. As such, we here provide a dichotomous key with illustrations (Figure 1) and photos (Figures 2 and 3) of the four nymph stages and resulting adult of apterous parthenogenetic female pea aphids. This key is based on information from Digilio (1995) and a laboratory population of green morph pea aphids (see Methods below). A fuller description of the developmental pathways and stages of this species can be found in Schmidtberg and Vilcinskas (2016).

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Figure 1: Line drawings of five instars of parthenogenetic wingless pea aphids Line drawings of nymph instars (a) I, (b) II, (c) III, (d) IV, and (e) the apterous parthenogenetic female they develop into. Key features for distinguishing instars include antennal segment number, and cauda length and shape. Each drawing is on a slightly different scale but each scale bar is 1 mm. Illustrations by J.D. Diamond, 2016.

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Dichotomous Key

1. Antennae with 5 sections (2) (Figures 2.a, 2.b) Antennae with 6 sections (3) (Figures 2.c, 2.d, 2.e) 2. Antennal segment 5 has dark pigment mostly only near the tip (Nymph I) (Figure 2.a) Antennal segment 5 has dark pigment mostly along its entire length (Nymph II) (Figure 2.b) 3. Cauda is wider than it is long or roughly equilateral (Nymph III) (Figure 3.c) Cauda is longer than it is wide (4) (Figures 3.d, 3.e) 4. Cauda is up to twice as long as it is wide and has relatively short setae (Nymph IV) (Figure 3.d) Cauda is clearly more than twice as long as it is wide and has relatively long setae (Adult) (Figure 3.e)

3 Figure 2: Photomicrographs of antennae of five instars of parthenogenetic wingless pea aphids. Instars I and II have five segments in each antenna, all others have six. This can be harder to distinguish visually than caudal shape. a. b.

c. d.

4 Figure 2 (continued): Photomicrographs of antennae of five instars of parthenogenetic wingless pea aphids. e.

Figure 3: Photomicrographs of cauda of five instars of parthenogenetic wingless pea aphids. The tail-like cauda becomes longer and pointier with each instar, particularly Nymph III through Adult. a. b.

5 Figure 3 (continued): Photomicrographs of cauda of five instars of parthenogenetic wingless pea aphids. c. d.

e.

6 Methods Study Population A single wild green Acrythosiphon pisum was obtained from a bean plant at Fresh Start Farms, an organic farm in Lisbon, Maine and allowed to reproduce parthenogenetically in the laboratory on (fava bean plants) grown from seed (Jung Seed Company, Randolph WI). The population was founded from one individual reproducing clonally. We kept the population at 22±3°C. We placed the plants 20 cm below fluorescent greenhouse lights with a 14/10 light/dark cycle. All focal individuals in our study were apterous parthogenetic females. They were gathered from our stock population by aspirator. For taking images (Figures 2 and 3) and analyzing aphids, we used a SMZ 1500 Stereoscope. A previous guide for identifying nymph stages was used a source to find appropriate characteristics (Digilio 1995). To find the mean body length (tip of head to base of cauda), caudal length and antennal length of each instar, measurements were taken based on image analysis in Adobe Photoshop (Table 1). Object lengths in photographs were calculated based on 5.545 micrometers per pixel.

Table 1. Mean size of instars, antennae and cauda of the pea aphid (Acyrthosiphon pisum) from our population. N = 5 at each instar. Stage Body Standard Cauda Standard Antenna Standard Segment Deviation of Length Deviation Length Deviation of Length Body Segment (mm) of Cauda (mm) Antenna (mm) Length Length Length Nymph I 1.7 0.2 0.15 0.06 2.1 0.1 Nymph II 2.5 0.3 0.21 0.03 3.3 0.5 Nymph III 3.1 0.4 0.33 0.06 4.0 0.6 Nymph IV 3.3 0.3 0.41 0.09 5.1 0.5 Adult 4.1 0.2 0.84 0.01 6.4 0.6

Conclusion

Antennal segment number and caudal shape are sufficient for distinguishing all instars except between Nymph I and Nymph II. In our population, as well as that studied by (Digilio 1995), pigmentation of the antennae can be used to distinguish these stages. While Nymph I is on the average considerably smaller than Nymph II, particularly in antennal length, individual size varies considerably, and even antennal length may not be a useful discriminant character.

Funding

This work was supported by a Bates Faculty Development Fund grant to D. L. Portions of this work were completed in the Bates College Imaging and Computing Center which was established with support from the National Institute of General Medical Sciences of the National Institutes of Health (P20 GM-103423).

7 References

Brisson, J. A. and D. L. Stern (2006). "The pea aphid, Acyrthosiphon pisum: an emerging genomic model system for ecological, developmental and evolutionary studies." BioEssays 28(7): 747-755.

Digilio, M. (1995). "Rapid identification of the nymphal stages of Acyrthosiphon pisum (Harris)(Homoptera Aphidoidea)." Bollettino di Zoologia Agraria e di Bachicoltura 27(1): 111- 116.

Schmidtberg, H. and A. Vilcinskas (2016). The Ontogenesis of the Pea Aphid Acyrthosiphon pisum. Biology and Ecology of Aphids. A. Vilcinskas, CRC Press: 14-51.

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