Linoleic acid as corpse recognition signal in a social aphid.

Social insect colonies constantly produce dead insects, which cause sanitary problems and potentially foster deadly pathogens and parasites. Hence, many social insects have evolved a variety of hygienic behaviors to remove cadavers from the colonies. To that end, they have to discriminate dead insects from live ones, where chemical cues should play important roles. In ants, bees and termites, such corpse recognition signals, also referred to as "death pheromones" or "necromones", have been identified as fatty acids, specifically oleic acid and/or linoleic acid. Meanwhile, there has been no such report on social aphids. Here we attempted to identify the "death pheromone" of a gall-forming social aphid with second instar soldiers, Tuberaphis styraci, by making use of an artificial diet rearing system developed for this species. On the artificial diet plates, soldiers exhibited the typical cleaning behavior, pushing colony wastes with their heads continuously, against dead aphids but not against live aphids. GC-MS and GC-FID analyses revealed a remarkable increase of linoleic acid on the body surface of the dead aphids in comparison with the live aphids. When glass beads coated with either linoleic acid or body surface extract of the dead aphids were placed on the artificial diet plates, soldiers exhibited the cleaning behavior against the glass beads. A series of behavioral assays showed that (i) soldiers exhibit the cleaning behavior more frequently than non-soldiers, (ii) young soldiers perform the cleaning behavior more frequently than old soldiers, and (iii) the higher the concentration of linoleic acid is, the more active cleaning behavior is induced. Analysis of the lipids extracted from the aphids revealed that linoleic acid is mainly derived from phospholipids that constitute the cell membranes. In conclusion, we identified linoleic acid as the corpse recognition factor of the social aphid T. styraci. The commonality of the death pheromones across the divergent social insect groups (Hymenoptera, Blattodea and Hemiptera) highlights that these unsaturated fatty acids are generally produced by enzymatic autolysis of cell membranes after death and therefore amenable to utilization as a reliable signal of dead insects.

Temporal division of labor in an aphid social system.

Temporal division of labor, or age polyethism, in which altruistic caste individuals change their tasks with aging, is widely found in bees and ants (Hymenoptera) and also in other social insects. Here we report the discovery of elaborate age polyethism in a social aphid (Hemiptera). Tuberaphis styraci is a gall-forming aphid in which monomorphic first instar nymphs differentiate into normal nymphs and soldiers upon second instar molt. Soldiers neither grow nor reproduce but perform gall cleaning and colony defense. Using an artificial diet rearing system, we collected age-defined groups of soldiers and monitored their social behaviors. We observed that young soldiers tend to clean whereas old soldiers preferentially attack, thereby verifying age-dependent task switching from housekeeping to defense. Strategic sampling, age estimation and behavioral observation of soldiers from natural galls revealed that (1) young cleaning soldiers tend to inhabit upper gall regions with adult insects, (2) old attacking soldiers tend to be distributed in lower gall regions, particularly around the gall openings, and (3) the gall structure is linked to intra-nest movement, aging and task switching of soldiers in an adaptive manner. These results highlight an evolutionary parallelism comparable to the sophisticated temporal division of labor observed in honeybee colonies.

Plant Manipulation by Gall-Forming Social Aphids for Waste Management.

Many social aphids form spectacular galls on their host plants, in which hundreds to thousands of aphids thrive for several months or even for over a year. Here, in addition to colony defense against natural enemies, waste disposal is an important task for the gall dwellers to sustain their social life. In open galls, soldier nymphs actively clean colony wastes such as honeydew droplets, cast-off skins, and cadavers by pushing them with their head out of the gall opening. In the gall, the excreted honeydew is coated with aphid-derived powdery wax to form "honeydew balls," which prevents the aphids from wetting and drowning with their own excretion. How the aphids deal with the accumulated honeydew in closed galls has been a mystery. Here, we report a novel gall-cleaning mechanism: the gall inner surface absorbs and removes the liquid waste through the plant vascular system. Such a plant-mediated water-absorbing property is commonly found in aphids forming closed galls, which must have evolved at least three times independently. By contrast, the inner surface of open galls is wax-coated and water-repelling, and in some cases, the inner surface is covered with dense trichomes, which further enhance the water repellency. In conclusion, gall-forming aphids induce novel plant phenotypes to manage the waste problems by manipulating plant morphogenesis and physiology for their own sake. This review describes our recent studies on waste management strategies by gall-forming social aphids and discusses future directions of this research topic.

Exaggeration and cooption of innate immunity for social defense.

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.

Water-repellent plant surface structure induced by gall-forming insects for waste management.

Many animals and plants have evolved elaborate water-repellent microstructures on their surface, which often play important roles in their ecological adaptation. Here, we report a unique type of water-repellent structure on a plant surface, which develops as an insect-induced plant morphology in a social context. Some social aphids form galls on their host plant, in which they produce large amounts of hydrophobic wax. Excreted honeydew is coated by the powdery wax to form 'honeydew balls', which are actively disposed by soldier nymphs through an opening on their gall. These activities are enabled by a highly water-repellent inner gall surface, and we discovered that this surface is covered with dense trichomes that are not found on normal plant surfaces. The trichomes are coated by fine particles of the insect-produced wax, thereby realizing a high water repellency with a cooperative interaction between aphids and plants. The plant leaves on which the gall is formed often exhibit patchy areas with dense trichomes, representing an ectopic expression of the insect-induced plant morphology. In the pouch-shaped closed galls of a related social aphid species, by contrast, the inner surface was not covered with trichomes. Our findings provide a convincing example of how the extended phenotype of an animal, expressed in a plant, plays a pivotal role in maintaining sociality.

An insect-induced novel plant phenotype for sustaining social life in a closed system.

Foraging, defense and waste disposal are essential for sustaining social insect colonies. Hence, their nest generally has an open structure, wherein specialized castes called workers and soldiers perform these tasks. However, some social aphids form completely closed galls, wherein hundreds to thousands of insects grow and reproduce for several months in isolation. Why these social aphids are not drowned by accumulated honeydew has been an enigma. Here we report a sophisticated biological solution to the waste problem in the closed system: the gall inner surface is specialized for absorbing water, whereby honeydew is promptly removed via the plant vascular system. The water-absorbing closed galls have evolved at least twice independently among social aphids. The plant-mediated waste removal, which entails insect's manipulation of plant morphogenesis and physiology, comprises a previously unknown mechanism of nest cleaning, which can be regarded as 'extended phenotype' and 'indirect social behavior' of the social aphids.

Altruistic colony defense by menopausal female insects.

Recent studies have suggested that an extended postreproductive life span, such as life after menopause in human females, will evolve when the indirect (kin-selected) fitness benefits from altruistic behavior are greater than the direct fitness benefits from continuing reproduction. Under some conditions in which postreproductive altruism is more beneficial and/or continuing reproduction is more costly, the postreproductive life span can be shaped by natural selection. However, indirect fitness benefits during postreproductive survival have been documented mainly in intelligent mammals such as humans and cetaceans, in which elder females possess enhanced social knowledge through learning. Here we show that postreproductive females of the gall-forming aphid Quadrartus yoshinomiyai (Nipponaphidini) can gain indirect fitness benefits through their altruistic colony defense. These females cease reproduction around the time of gall opening and defend the colony by sticking themselves to intruding predators with a waxy secretion that is accumulated in their body with aging. Our results suggest that the presence of an age-related trait for altruistic behavior promotes the evolution of postreproductive altruism in this social insect via kin selection under natural selection imposed by predators.

Mechanisms regulating caste differentiation in an aphid social system.

For evolution and maintenance of the social systems of insect colonies, caste production should be controlled in response to external cues so that caste ratio in the colony is kept at an optimal range. Recent developments using artificial diet rearing techniques have revealed an underlying mechanism for adaptive control of caste production in a social aphid, Tuberaphis styraci, which has a sterile soldier caste in the 2(nd) instar. Aphid density was the proximate cue that acts on 1(st) instar nymphs and embryos to induce soldier differentiation. The final determination of soldier differentiation occurred postnatally, probably at a late 1(st) instar stage. Direct contact stimuli from live non-soldier aphids mediated the density effect. While coexisting non-soldiers facilitated soldier differentiation in 1(st) instar nymphs, coexisting soldiers acted to suppress such differentiation. These results suggest that caste production in aphid colonies is controlled by positive and negative feedback mechanisms consisting of density-dependent induction and suppression of soldier differentiation. Here, we demonstrate the mechanisms that coordinate aphid society, and provide a striking case of clonal superorganism system where simple responses of colony members to local extrinsic stimuli are integrated into a highly organized regulation of the whole colony.

Scab formation and wound healing of plant tissue by soldier aphid.

In the social aphid Nipponaphis monzeni, a unique gall-repairing behaviour has been known: when a hole is made on the gall, many soldier nymphs discharge body fluid on the breach, which promptly solidifies and plugs the hole. Here, we experimentally investigated the subsequent fate of repaired galls and their inhabitants. Irrespective of natural repair by soldier nymphs or artificial repair with adhesive, repaired galls survived significantly better than non-repaired galls. Within a month after repair, the plant tissue around the hole proliferated and sealed up the hole. Many soldier nymphs were localized at the hole area and extermination of inhabiting aphids by insecticides aborted the gall regeneration, indicating that the gall regeneration requires inhabiting aphids, wherein soldier nymphs are likely to play a major role. This study provides an unprecedented case of scab formation and wound healing, which occurs at an animal-plant interface: scab derived from insect body fluid promptly plugs damaged plant tissue and subsequently the insects actively stimulate regeneration of the plant tissue, whereby the compromised plant tissue recovers. We suggest that the novel system may have evolved in the aphid lineage through enhancement and recruitment of the pre-existing capabilities of haemolymph coagulation and gall formation.

Evolution of soldier-specific venomous protease in social aphids.

In social aphids of the genus Tuberaphis a cysteine protease gene of the family cathepsin B exhibits soldier-specific expression and intestinal protease production. The product is orally excreted and injected by soldier nymphs into natural enemies, thereby exerting an insecticidal activity. In an attempt to gain insights into when and how the novel venomous protease for the altruistic caste has evolved, we investigated the soldier-specific type (S-type) and nonspecific type (N-type) cathepsin B genes from social and nonsocial aphids. All the social aphids examined, representing the genera Tuberaphis, Astegopteryx, and Cerataphis, possessed both the S-type and N-type genes. Phylogenetically distant nonsocial aphids also possessed cathepsin B genes allied to the S-type and the N-type, indicating the evolutionary origin of these genes in the common ancestor of extant aphids. In Tuberaphis species the S-type genes exhibited significant soldier-specific expression and accelerated molecular evolution whereas the N-type genes did not. In Astegopteryx and Cerataphis species, meanwhile, both the S-type and N-type genes exhibited neither remarkable soldier-specific expression nor accelerated molecular evolution. These results suggest that the S-type gene acquired the soldier-specific expression and the venom function after divergence of the genus Tuberaphis. On the structural model of the S-type protease of Tuberaphis styraci the accelerated molecular evolution was associated with the molecular surface rather than the catalytic cleft, suggesting that the venom activity was probably acquired by relatively minor modifications on the molecular surface rather than by generation of a novel active site. In Cerataphis jamuritsu the S-type gene was, although containing a stop codon, structurally almost intact and still transcribed, suggesting recent pseudogenization of the gene copy and possible relevance to relaxed functional constraint in the highly multiplied protease gene family. On the basis of these results we suggest that the massive amplification in aphid cathepsin B genes might have predisposed the evolution of venomous protease in the social aphid lineage and argue that gene duplication, accelerated molecular evolution, and acquisition of novel gene function must have played considerable roles in the evolution of complex biological systems including insect sociality.

Large gene family expansion and variable selective pressures for cathepsin B in aphids.

Aphids exclusively feed on plant phloem sap that contains much sugar and some nonessential amino acids but is poor in lipids and proteins. Conventionally, it has been believed that aphids substantially have no intestinal digestion of proteins. However, we here report an unexpected finding that cysteine protease genes of the family cathepsin B are massively amplified in the lineage of aphids and that many of the protease genes exhibit gut-specific overexpression. By making use of expressed sequence tag data, sequenced cDNAs, and genomic trace sequences of the pea aphid Acyrthosiphon pisum, we identified a total of 28 cathepsin B-like gene copies in the genome of A. pisum. Phylogenetic analyses of all the cathepsin B genes in aphids revealed that genic expansion has continuously proceeded with basal, intermediary, and recent duplications. Estimation of molecular evolutionary rates indicated that major alterations of the rates often occurred after duplications. For example, a gene copy ("348") was shown to be slow evolving and close to genes of other insects like Drosophila melanogaster, whereas the other gene copies appeared to have evolved faster with higher ratios of nonsynonymous to synonymous substitutions. We identified a number of gene copies (16 in A. pisum) that contained a replacement at the site required for catalytic activity of the protease. Among these, 2 copies were pseudogenes, whereas the remaining copies were structurally intact and possibly acquired new functions. For example, a cluster of such gene copies ("1674") has been subjected to positive selection. Quantitative reverse transcriptase-polymerase chain reaction analyses revealed that the more conserved gene copy ("348") showed a constitutive expression, whereas 5 other forms ("84," "16," "16D," "1874," and "2744") were preferentially expressed in the gut of A. pisum. Putative biological roles of the diversified cathepsin B-like gene copies in aphids are discussed in relation to their nutritional physiology specialized for plant sap feeding lifestyle.

Density-dependent induction and suppression of soldier differentiation in an aphid social system.

We investigated the mechanism underlying the control of soldier production in colonies of a social aphid, Tuberaphis styraci, which has a sterile soldier caste in the second instar. High aphid density was shown to induce soldier production in T. styraci. Analysis of natural colonies revealed that the soldier proportion tended to increase with aphid density but reached a plateau. Artificial diet experiments identified a similar plateau of soldier proportion under high-density conditions. In order to gain insights into the controlling mechanism of soldier production, the effect of soldiers on reproducing adult aphids was examined using the artificial diet system. It was experimentally demonstrated that soldier production was suppressed by coexisting soldiers, whereas coexisting non-soldiers facilitated soldier production. These results suggested that caste ratio in the colony of T. styraci is controlled by positive and negative feedbacks consisting of density-dependent induction and suppression of soldier differentiation.

Venomous protease of aphid soldier for colony defense.

In social aphids, morphological, behavioral, and physiological differences between soldiers and normal insects are attributed to differences in gene expression between them, because they are clonal offspring parthenogenetically produced by the same mothers. By using cDNA subtraction, we identified a soldier-specific cysteine protease of the family cathepsin B in a social aphid, Tuberaphis styraci, with a second-instar soldier caste. The cathepsin B gene was specifically expressed in soldiers and first-instar nymphs destined to be soldiers. The cathepsin B protein was preferentially produced in soldiers and showed a protease activity typical of cathepsin B. The cathepsin B mRNA and protein were localized in the midgut of soldiers. For colony defense, soldiers attack enemies with their stylet, which causes paralysis and death of the victims. Notably, after soldiers attacked moth larvae, the cathepsin B protein was detected from the paralyzed larvae. Injection of purified recombinant cathepsin B protein certainly killed the recipient moth larvae. From these results, we concluded that the cathepsin B protein is a major component of the aphid venom produced by soldiers of T. styraci. Soldier-specific expression of the cathepsin B gene was found in other social aphids of the genus Tuberaphis. The soldier-specific cathepsin B gene showed an accelerated molecular evolution probably caused by the action of positive selection, which had been also known from venomous proteins of other animals.

Density triggers soldier production in a social aphid.

For evolution and maintenance of the social system of insect colonies, investment in the sterile caste should be adequately controlled in response to environmental cues. Recent developments using artificial diet rearing techniques have revealed an underlying mechanism of caste control in a gall-forming aphid, Tuberaphis styraci, which has a soldier caste in the second instar. Statistical analyses of field-collected galls detected a significant positive correlation between aphid density and soldier proportion in the natural colonies of T. styraci. Artificial diet experiments showed that soldiers are produced under crowded conditions. Detailed experiments demonstrated that soldiers are produced in a density-dependent manner rather than in a colony size-dependent manner. From these results, it was concluded that aphid density is the crucial cue that triggers soldier production in T. styraci. This study provides, to our knowledge, the first experimental demonstration of an environmental factor involved in aphid soldier differentiation.

The proximate cue of density-dependent soldier production in a social aphid.

Tuberaphis styraci is a social aphid that produces 2nd instar soldiers with morphological, behavioral and reproductive division. High aphid density was shown to induce soldier production in T. styraci, although direct cue of soldier induction associated with high density has been unknown. In order to identify the proximate environmental cue underlying the density-dependent soldier production, a series of experiments was conducted using an artificial diet rearing technique. When adult aphids were reared with live normal nymphs, live soldiers, dead normal nymphs, shed skins, honeydew globules and excreted wax, only live normal nymphs effectively induced soldier production. In order to gain insights into the nature of soldier-inducing cue associated with normal aphids, we performed artificial diet experiments using partitioned and non-partitioned chambers, in which direct contact between aphids was either inhibited or allowed. Induction of soldiers was observed only when direct contact was allowed. Therefore, it was shown that the soldier-inducing cue is neither volatile in the air nor diffusible through the diet, but is transmitted between normal non-soldier aphids via direct contact. On the basis of these results, we suggest that the soldier-inducing cue might be physical stimulus combined with non-volatile surface chemicals whose properties differ between normal aphids and soldiers.

Aphid soldier differentiation: density acts on both embryos and newborn nymphs.

The mechanism of caste differentiation in a social aphid Tuberaphis styraci, which has a sterile soldier caste in the 2nd instar, was investigated using an artificial diet rearing system. High aphid density induced soldier production. Combinatorial prenatal and postnatal density treatments revealed that (1) either prenatal high density or postnatal high density is sufficient for soldier induction; (2) thus, embryos in the maternal body and newborn 1st instar nymphs are both responsive to high density; (3) the combination of prenatal high density and postnatal high density enhances soldier differentiation in a synergistic manner; and (4) the final determination of soldier differentiation occurs postnatally, probably at a late 1st instar stage. This study first throws light on the developmental aspects of caste differentiation in a social aphid.

Hidden from the host: Spiroplasma bacteria infecting Drosophila do not cause an immune response, but are suppressed by ectopic immune activation.

Insects and other arthropods have an effective innate immune system that can clear infections with bacteria and other microorganisms. Despite this ability, one group of bacteria, the spiroplasmas, survive unharmed within the haemolymph of a wide range of arthropod hosts. We investigated the interaction between one member of this clade, a relative of Spiroplasma poulsonii, and the immune system of its Drosophila host. Expression of antimicrobial genes in spiroplasma-infected flies did not differ from wild-type controls either in the naturally infected state, nor after septic shock. We therefore concluded that spiroplasma infection did not induce an immune response in its host, but that this absence of response was unlikely to be because the bacterium inhibited response. Further experiments revealed immune reactions induced ectopically did reduce parasite titre. We therefore conclude that this bacterium has a novel form of interaction with its host, being hidden from the host immune system, but potentially suppressible by it.

A trace amine, tyramine, functions as a neuromodulator in Drosophila melanogaster.

The tyramine receptor (TyrR) is a G protein-coupled receptor for trace amines, cloned in Drosophila melanogaster, and claimed to be either an octopamine receptor or a tyramine receptor. We previously reported that in the larval neuromuscular junctions, the modulatory effect on the excitatory junction potentials of tyramine is distinctly different from that of octopamine. The effect of tyramine but not of octopamine was selectively abolished in the TyrR mutant hono, suggesting that this gene encodes a receptor for tyramine, and not for octopamine. We examined whether there was a gene-dosage effect of this tyramine modulation using combinations of hono, deficiency (Df) and wild-type alleles. The tyramine effect was observed in hono heterozygotes (+/hono), which showed intermediate levels of response, but was not seen in +/Df or hono/Df hemizygotes. While these further suggest that tyramine is the true ligand, it is possible that the gene-dosage effect is only evident above some threshold of gene expression levels. Immunohistochemical staining using an anti-tyramine antibody identified tyramine-containing neurons in the larval central nervous system, some of which were distinct from the octopamine-containing neurons. Taken together, these results strongly suggest that tyramine functions as a neuromodulator.