Conventionally, immunology has focused on molecular and cellular mechanisms against pathogens and parasites to ensure survival of individuals. Recently, the notion of social immunity has emerged, which highlights the mechanisms in social animals to combat against pathogens, parasites, and other enemies to ensure survival of their society as a whole. Conceptually, social immunity is analogous to but distinct from individual immunity. However, we discovered that, in the social aphid Nipponaphis monzeni, molecular and cellular immune components of soldier individuals are extremely up-regulated and massively excreted via “body eruption” upon gall breakage, and the “hyperclotting” body fluid repairs the damaged gall for colony defense, which uncovers unexpected molecular, cellular, and evolutionary commonalities across individual immunity and social immunity.

Social insects often exhibit striking altruistic behaviors, of which the most spectacular ones may be self-destructive defensive behaviors called autothysis, “self-explosion,” or “suicidal bombing.” In the social aphid Nipponaphis monzeni, when enemies damage their plant-made nest called the gall, soldier nymphs erupt to discharge a large amount of body fluid, mix the secretion with their legs, and skillfully plaster it over the plant injury. Dozens of soldiers come out, erupt, mix, and plaster, and the gall breach is promptly sealed with the coagulated body fluid. What molecular and cellular mechanisms underlie the self-sacrificing nest repair with body fluid for the insect society? Here we demonstrate that the body cavity of soldier nymphs is full of highly differentiated large hemocytes that contain huge amounts of lipid droplets and phenoloxidase (PO), whereas their hemolymph accumulates huge amounts of tyrosine and a unique repeat-containing protein (RCP). Upon breakage of the gall, soldiers gather around the breach and massively discharge the body fluid. The large hemocytes rupture and release lipid droplets, which promptly form a lipidic clot, and, concurrently, activated PO converts tyrosine to reactive quinones, which cross-link RCP and other macromolecules to physically reinforce the clot to seal the gall breach. Here, soldiers’ humoral and cellular immune mechanisms for wound sealing are extremely up-regulated and utilized for colony defense, which provides a striking case of direct evolutionary connection between individual immunity and social immunity and highlights the importance of exaggeration and cooption of preexisting traits to create evolutionary novelties.

Animals are always under the risk of infections with bacteria, fungi, and other parasites, and immune systems are therefore essential for their survival in the natural environment. Whereas the Ig-based adaptive immunity is specific to vertebrates, innate immune mechanisms are generally found in diverse animals encompassing vertebrates and invertebrates (1). Upon breakage of surface barrier and invasion of microbes or other nonself entities, body fluid coagulation, hardening, and melanization immediately occur to stop bleeding and to localize the invasion, in which enzymes with protein cross-linking activities such as phenoloxidases (POs) and transglutaminases play important roles (2, 3). Subsequently, specialized hemocytes are recruited to participate in wound sealing as well as phagocytosis and encapsulation of the intruders (4, 5). Finally, antimicrobial peptides and other potent effector molecules are transiently and drastically induced and released into the body fluid to kill the intruders (1, 6).

Conventionally, immunology has focused on molecular and cellular mechanisms against noxious biological agents to ensure survival of individuals. Recently, the notion of social immunity has emerged, which highlights traits and mechanisms of social insects and other group-living animals to combat against pathogens, parasites, and other natural enemies to ensure survival of their colony or society as a whole (7, 8). Although the conceptualization of some fundamental commonalities between individual immunity and social immunity across the organismal hierarchical levels may be insightful in understanding the general aspect of biological systems, the mechanistic bases of social immunity, which are mainly behavioral, physiological, and organizational ones, are, needless to say, distinct from the molecular and cellular immune mechanisms (7, 8).

Here we report a case of social immunity in which innate humoral and cellular immune mechanisms at the individual level are extremely exaggerated and directly utilized for colony defense at the group level in an ecological context. In addition to well-known social insects such as ants, bees, wasps, and termites, some aphids are known to be social, producing morphologically specialized or nonspecialized individuals called soldiers, which perform altruistic tasks including colony defense against predators and cleaning and repairing of their plant-made nests known as galls (9, 10). The social aphid Nipponaphis monzeni forms large galls on the tree Distylium racemosum (Fig. 1A) in which hundreds to thousands of insects proliferate by sucking plant sap from the inner wall (Fig. 1 B and C) (11). In N. monzeni, all nymphs exhibit an extended first instar stage, perform social tasks as soldiers, and then grow and reproduce (11, 12). In spring, young galls of N. monzeni are often attacked by lepidopteran enemies (13), whose larvae tunnel and damage gall tissues and also consume inhabiting aphids. Upon invasion of the predator, monomorphic first-instar soldiers attack the enemy by stinging with their stylets (Fig. 1D). Then, many soldiers gather around the hole on the gall wall and erupt to discharge a large amount of whitish body fluid from their cornicles on the abdominal tip (Fig. 1 E and F and Movie S1). The shriveled soldiers actively mix the secretion with their legs and skillfully plaster it over the plant injury, and the secretion promptly solidifies (Fig. 1G and Movie S1). Dozens of soldiers come out, erupt, mix, and plaster, and the hole is completely plugged by the coagulated body fluid (Fig. 1 H and I and Movie S1) (12, 14). Here, the soldier nymphs of N. monzeni perform not only a soldier-type aggressive task of attacking enemies but also a worker-type housekeeping task of repairing their plant-made nest, which entails a series of unique self-destructive behaviors. The molecular and cellular mechanisms that underlie the self-sacrificing nest repair with the use of body fluid for the insect society is of great interest, which prompted us to investigate the biochemistry, physiology, cell and molecular biology, and developmental and evolutionary aspects of the gall-repairing body fluid produced by soldier nymphs of N. monzeni.

Self-sacrificing gall repair by soldier nymphs of N. monzeni. (A) A gall formed on the tree D. racemosum. (B) An inside view of a gall. (C) A magnified image of gall contents: white arrow, first-instar soldier nymph; black arrow, adult; small white arrow, powdery aggregate consisting of excreted wax; small black arrow, aphid cadaver. (D) Soldier nymphs attacking a moth larva by stinging with their stylet. (E) A gall-repairing soldier nymph discharging body fluid. (F) Scanning EM image of a soldier nymph (ventral view); arrows indicate droplets of body fluid discharged from cornicles. (G) Soldier nymphs plastering their own body fluid onto plant injury ( Movie S1 ). (H) A gall with a naturally repaired hole (arrow). (I–L) An experimentally bored hole filled by body fluid of soldier nymphs at 0 h (I), 1 h (J), 2 h (K), and 6 h after plugging (L). (M) LGCs discharged from a soldier nymph ( Movie S2 ). (N) An enlarged image of LGCs. (O) An abdominal cross-section of a soldier nymph whose body cavity is full of LGCs: bc, bacteriome; gu, midgut; ov, ovary. (P) A thin section of a solidified soldier’s body fluid 3 d after gall repair; white arrows indicate unruptured LGCs and black arrows indicate nuclei of ruptured LGCs.

Results and Discussion

Darkening upon Clotting, Large Globular Cells, and a Few Major Proteins in Body Fluid of Soldier Nymphs. The soldiers’ secreted body fluid turned black as its solidification proceeded (Fig. 1 I–L). Mechanical stimulation of soldier nymphs in a saline solution elicited the body fluid secretion (Fig. 1M and Movie S2), which demonstrated that the body fluid contains numerous peculiar large cells, called the large globular cells (LGCs) (14), that fill up the soldier’s body cavity (Fig. 1 N and O). Histological examination of the hole-filling plugs identified nuclei and granules derived from LGCs (Fig. 1P). SDS/PAGE of the soldiers’ body fluid detected only a few major protein bands (Fig. 2A, Left), suggesting that these proteins may play some roles in the self-sacrificing gall repair by soldier nymphs. Fig. 2. PO and RCP, major proteins in the N. monzeni soldier’s body fluid. (A) SDS/PAGE of soldier’s body fluid: (Left) general protein staining with Coomassie Brilliant Blue (CBB), (Middle) immunoblotting against PO, and (Right) immunoblotting against RCP. Numbers 1–6 show major protein bands. Asterisks indicate nonspecific signals. (B) Measurement of PO activity in soldier’s body fluid. (C) Measurement of PO activity in whole body extract of N. monzeni and allied nipponaphidine aphids N. distyliicola and N. yanonis that are incapable of gall repair with the use of their body fluid. In B and C, letters a and b indicate statistically significant differences (Tukey’s honestly significant difference test, P < 0.05). (D) SDS/PAGE and RCP-targeted immunoblotting of soldier’s body fluid proteins from eight sympatric gall colonies a–h: general staining with CBB (Left) and immunoblotting against RCP (Right). Asterisks indicate nonspecific signals. (E) RT-PCR amplification of RCP cDNA sequences from five sympatric gall colonies i–m. Two sequences were amplified from colonies i, j, and m, whereas only one sequence was obtained from colonies k and l. (F) Schematic diagram showing amino acid sequences of RCP alleles deduced from complete cDNA sequences obtained from colonies i–m. Colored boxes depict seven types of repeat motif, each 8 aa in size: gray, GSGQGSYT; green, EHEQGS[H/Y][T/N/I]; blue, EHGQGS[H/Y][T/N/I]; orange, EPEESGYT; purple, EHKQGSHT; pink, GPGQGS[H/Y][T/N/I]; and yellow, GHEQGS[H/Y][T/N/I]. Daggers indicate repeat motifs in which the first amino acid residue is replaced as indicated. Dashed lines highlight deduced insertions/deletions of the motifs during the evolution of RCP. “S” indicates the N-terminal signal sequence. (G) Attributes and sequence accession numbers for the RCP alleles identified from gall colonies i–m.

Extraordinarily Abundant Tyrosine in Body Fluid of Soldier Nymphs. As depicted in Fig. 4A, tyrosine is the main and starting substrate for the PO-mediated melanization pathway. Notably, tyrosine was extraordinarily predominant among free amino acids in the body fluid of soldier nymphs of N. monzeni, measuring 31.7 ± 5.5 mM and accounting for 75.9% of total free amino acids (Fig. 4B). We found that (i) tyrosine was the most abundant free amino acid in the body fluid of N. monzeni irrespective of developmental stage, (ii) the level of tyrosine was more than twice as high in soldier nymphs than in N. monzeni adults, and (iii) such high tyrosine titers are not observed in nonsocial aphids like A. pisum (19) (Fig. 4 B–D). In soldier nymphs of N. monzeni, tyrosine was detected not only from hemolymph (0.62 ± 0.21 nmol per insect, n = 9) but also from LGCs (0.14 ± 0.08 nmol per insect, n = 9). DOPA was negligible in soldiers’ freshly secreted body fluid (0.02 ± 0.03 mM, n = 7) but became detectable 15 min after secretion (2.3 ± 1.8 mM, n = 7), indicating conversion of tyrosine to DOPA in the solidifying body fluid.

Abundant Lipids in Clotted Body Fluid and LGCs. Here it should be noted that soldiers’ excreted body fluid is full of LGCs (Fig. 1 M–O), and LGCs are full of lipid droplets (Fig. 3G). Chloroform extraction of hole-filling plugs collected from repaired galls revealed that lipids accounted for 82.5 ± 3.1% (n = 12) of their dry weight, indicating that lipids quantitatively constitute the major component of soldiers’ coagulated body fluid. Cornicles are a pair of tube- or pore-like defensive organs specifically found on the abdominal tip of aphids (24, 25). When stimulated or threatened, aphids secrete sticky liquid from the cornicles, which contain waxy substances and alarm pheromones, to deter predatory attacks and to elicit escaping behavior of colony mates (26⇓–28). Previous studies reported that triglycerides are among the major components of the aphid’s cornicle secretion (29⇓–31). Our lipid analysis revealed that (i) the lipids in the secretion were mostly triglycerides whose estimated total concentration was as high as 528 ± 85 mM (n = 7), (ii) the major components were triglycerides consisting of sorboyl and dimyristic acids (C6:2, C14:0, C14:0; 35.0%) and sorboyl, myristic, and palmitoyl acids (C6:2, C14:0, C16:0; 28.7%), (iii) the lipid composition did not change before and after body fluid coagulation, and (iv) the fatty acids separated by alkaline hydrolysis of soldiers’ excreted body fluid were almost the same as those constituting the triglycerides (SI Appendix, Fig. S5).

Two-Step Model for the Mechanism of Gall Repair: Immediate Formation of Lipidic Soft Clot Followed by PO-Mediated Clot Hardening. On the basis of these results, we propose a model for the molecular and cellular mechanisms underlying the self-sacrificing gall repair by soldier nymphs of N. monzeni. In the soldier’s body cavity, inactive PO and lipid droplets are stored in LGCs, whereas tyrosine and RCP are accumulated in hemolymph (Fig. 6A, Left). Upon discharge of the body fluid, LGCs rupture and release lipid droplets, which promptly form a lipidic soft clot, and PO is concurrently activated, presumably by hemolymphal enzymes, and initiates the PO cascade, by which tyrosine is converted to reactive quinones (Fig. 6A, Middle). Subsequently, proteins and other macromolecules are cross-linked by the reactive quinones, and the lipidic clot is physically reinforced and melanized (Fig. 6A, Right). Fig. 6. Hypothetical model for the evolution of self-sacrificing gall repair in N. monzeni. (A) Molecular and cellular mechanisms underlying the body fluid clotting for self-sacrificing gall repair by soldier nymphs of N. monzeni. PO, tyrosine, and triglycerides are stored in LGCs, whereas RCP and tyrosine are accumulated in body fluid. The soldier’s body fluid excretion results in immediate formation of a lipidic soft clot and activation of the PO-mediated melanin synthesis cascade. Activated PO converts tyrosine into reactive quinones, which cross-link RCP and other proteins to physically stabilize and harden the clot. (B) General molecular and cellular mechanisms of body fluid clotting and wound sealing in insects, in which PO activation, body fluid clotting, and hemocyte aggregation play pivotal roles. (C) Hypothetical evolutionary scenario for gall-repairing soldier nymphs of N. monzeni, in which preexisting clotting mechanisms are extremely exaggerated—PO drastically up-regulated, tyrosine overproduced, and specialized LGCs proliferated—to cause massive clotting outside the insect body. (D) Self-sacrificing gall repair by soldier nymphs of N. monzeni, in which body fluid clotting mechanisms at the individual level are coopted for social defense at the colony level.

Exaggeration and Cooption of Wound Sealing and Defense Mechanisms at Individual Level for Social Defense at Colony Level. When an insect is injured, the epidermal breach is promptly sealed by body fluid clotting, in which PO activation, melanization, protein cross-linking, and hemocyte aggregation are involved (2, 3) (Fig. 6B). Here we hypothesize that these biochemical and cellular mechanisms of body fluid clotting at the individual level are extremely up-regulated in soldier nymphs of N. monzeni, and, by recruiting the aphid-specific defensive trait of cornicle secretion behavior, the superclotting body fluid is collectively excreted by soldier nymphs in a highly coordinated manner, thereby promptly accomplishing the gall repair at the colony level in a social context (Fig. 6 C and D).