DOI: 10.5061/dryad.b3m45
title: Data from: An alternative pathway to eusociality: exploring the molecular and functional basis of fortress defense

description: Some animals express a form of eusociality known as 'fortress
 defense', in which defense rather than brood care is the primary
 social act. Aphids are small plant-feeding insects, but like termites,
 some species express division of labor and castes of aggressive juvenile
 'soldiers'. What is the functional basis of fortress defense
 eusociality in aphids? Previous work showed that the acquisition of venoms
 might be a key innovation in aphid social evolution. We show that the
 lethality of aphid soldiers derives in part from the induction of
 exaggerated immune responses in insects they attack. Comparisons between
 closely-related social and non-social species identified a number of
 secreted effector molecules that are candidates for immune modulation,
 including a convergently-recruited protease described in unrelated aphid
 species with venom-like functions. These results suggest that aphids are
 capable of antagonizing conserved features of the insect immune response,
 and provide new insights into the mechanisms underlying the evolution of
 fortress defense eusociality in aphids.
description: Degree of melanziation
 raw dataTo visually evaluate and
 quantify melanization in larvae attacked by aphid soldiers, we used
 double-blind observational assays. We quantified the melanization
 phenotype before and after attack by aphid soldiers by using a subjective
 visual assay of melanization. We collected P. obesinymphae galls in the
 vicinity of Nashville, TN in July 2013. To facilitate observation, galls
 were split in half, and we placed individual Drosophila larvae, a
 surrogate for common Dipertan predators of galling aphids, in galls with
 aphids or in galls in which the aphids had been removed. Larvae remained
 in galls for 1 hr. Each D. melanogaster larvae was photographed before
 attack, immediately following attack, after removing the attacking
 soldiers, and 1 hr following attack. Only Drosophila larvae being attacked
 by at least 10 aphid soldiers were included in the study. Additionally, as
 controls, Drosophila larvae were stabbed either once or multiple times
 with a sterile needle. Larvae were then placed in an empty gall for 1 hr.
 Using a double-blind assay, all Drosophila larvae were assigned a score
 based on the proportion of the body melanized: 1 (no observable
 melanization) to 4 (surface completely melaninized). The sample size for
 each group was greater than or equal to 10.Surviorship assay raw
 dataTo examine if the over
 activation of the melanization pathway affected the survivorship of D.
 melanogaster following attack by aphid soldiers, we compared the
 survivorship of Drosophila mutants deficient in the melanization response
 to wild-type Drosophila. To measure survivorship, we introduced larvae to
 a gall, monitored the larvae every 20 minutes and noted the time to death,
 defined as when the larvae stopped moving.qPCR raw
 dataTo examine which genes
 were differential regulated following attack by aphid soldiers, we used
 quantitative PCR to assess expression levels of multiple immune genes in
 two experimental groups of D. melanogaster larvae: 1) Larvae attacked by
 P. obesinymphae for 1 hr; or 2) Larvae placed in an empty gall for 1 hr.
 One hour was chosen because it was prior to the average observed time of
 death caused by aphid soldiers (Lawson et al. 2014), but enough time to
 observe a melanization response. In order to provide a standardized
 reference and to establish whether our assays with aphids were sensitive
 enough to detect immunological activation, we also measured the expression
 of each these genes in Drosophila that were stabbed with sterile
 needles.Phenoloxidase assay raw
 dataPhenoloxidase levels in
 Drosophila tissues were measured using a simple procedure based on the
 transformation of L-dopa to dopachrome in the presence of phenoloxidase,
 measured as function of optical density, as described by Lario et al.
 (1993). Larvae were singly placed in freshly collected P. obesinymphae
 galls for 45 minutes, and subsequently removed with a small paintbrush. A
 control group of larvae of similar size was not exposed to the aphids and
 was labeled the “unattacked” group. In order to differentiate between
 phenoloxidase activation during mechanical wounding of the epithelia by
 aphid stylets, and circulating phenoloxidase activation in the hemolymph,
 Drosophila hemolymph was extracted from both the attacked larvae and the
 control group using fine needles. After hemolymph extraction, the larval
 midguts were removed using a pair of dissecting tweezers. The remaining
 cuticle was then placed in a 1.5 mL Eppendorf tube and ground by a pestle.
 The tubes were centrifuged briefly, and the liquid portion was removed by
 a pipette for use in the phenoloxidase assay, in addition to the extracted
 hemolymph. Immediately after extraction, the hemolymph was transferred to
 ice-chilled ELISA wells containing 50 μL of 20 mM TRIS buffer, pH 7.0,
 with 1% sodium citrate. The cuticle fluids were transferred by pipette to
 ELISA wells containing the same mixture. After the hemolymph and cuticle
 fluids were collected, 50 μL of 3,4-dihydroxyphenyl-alanine (L-dopa), 5
 mg/mL in TRIS buffer, were added to each well. Mixtures were incubated at
 32°C for 45 min, and the wells were subsequently read in an ELISA
 spectrophotometer at a wavelength of 490 nm. Blank wells containing 50 μL
 TRIS buffer and 50 μL L-dopa in TRIS but no hemolymph or cuticle fluids
 were used as controls.