We identified a protease specifically expressed in the soldier caste of a “social aphid”, which has a social organization like bees and ants, and demonstrated that the protease is a principal insecticidal component of the “aphid venom” injected by the soldiers into natural enemies. This study was initiated for the purpose of elucidating the molecular mechanisms underlying the differentiation of the solider caste and the molecular basis of biological functions specific to the aphid soldiers, and identified an important soldier-specific gene for the first time. The protease is a bioactive substance derived from a unique, hitherto unexplored organism, and is expected to provide new insights into the origin and evolution of animal sociality, soldier castes, animal venoms, etc.

The results of this study were published in the July 26 on-line edition' of the Proceedings of the National Academy of Science of the United States of America (PNAS).

Kutsukake, M., Shibao, H., Tamura, T., Hoshino, T., Ohgiya, S. and Fukatsu, T. (2004): Venomous protease of aphid soldier for colony defense. Proc. Natl. Acad. Sci. U.S.A., 101(31): 11338-11343.

An amazingly large number of organisms existing on the Earth have acquired specific, diverse and sophisticated biological functions through the evolutionary course. The diverse and exquisite biological functions not only appeal to scientific searching mind and inquisitive spirit, but also offer aesthetic attractiveness, and even great industrial and economic values in the form of bioactive substances and lead compounds for medical drugs. Such aspects of the biodiversity have been referred to as “biological and genetic resources”. However, biological functions thus far explored, investigated and utilized by the mankind no doubt constitute only the very tip of an iceberg, in comparison with the enormous volume of the biodiversity.

Among various bioactive substances, venoms have attracted much attention of researchers because of their drastic biological activities. A variety of organisms have acquired capabilities to synthesize, accumulate and utilize different poisonous substances for attacking enemies, for defending themselves, and for getting rid of competitors. By now, various kinds of organismal venoms have been investigated, such as snake venom, scorpion venom, spider venom, wasp venom, puffer poison, snail venom, and others, but it goes without saying that they constitute another tip of an iceberg.

Some social insects like bees, wasps and ants produce a group of individuals specialized for defending their colony and often armed with venomous substance, which are called “workers” or “soldiers”. In addition to bees, wasps, ants and termites, some aphids also have social organization in their colonies.

Aphids (Homoptera: Aphididae) are tiny insects, sucking plant sap and often forming a large colony on the host plant. Feeble aphids are favorable preys of various carnivorous insects. It is often observed that larvae of ladybird beetles (Coccinellidae), hover flies (Syrphidae) and lacewings (Chrysopidae) are voraciously feeding on aphids. While most of aphids are devoured helplessly, some “social” aphids produce soldier nymphs specialized for attacking enemies. The soldiers remain in nymphal stage without molting and growing, and are capable of attacking predators and defend their colonies [See Fig. 1].

Fig. 1. Soldier nymphs of a social aphid, Tuberaphis styraci, attacking a lacewing larva.

The developmental process of soldier differentiation is of particular interest. Since a female aphid  produce female nymphs without mating (parthenogenesis), an aphid colony consists of clonal individuals of identical genetic constitution. Nevertheless, offspring from a same aphid mother develop into two types of nymphs, namely normal nymphs and soldier nymphs, which are distinctly different from each other in respect to morphology, behavior and reproductive capacity [See Fig. 2]. The phenotypic differences in spite of the identical gene repertory may be attributed to differences in how genes work, that is, gene expression patterns. If so, by identifying genes specifically in soldier nymphs, we would be able to understand the molecular mechanisms involved in the caste differentiation and the molecular basis of the soldier-specific biological functions. Therefore, we selected Tuberaphis styraci with second instar soldier caste as a model species of social aphid, and started to explore soldier-specific genes.

Fig. 2. Comparison between normal nymph and soldier nymph of Tuberaphis styraci

By using a cDNA subtraction technique, we explored genes that are actively expressed in soldier nymphs but not in normal nymphs of T. styraci. As a result, most of clones obtained were derived from a single gene encoding a protease called cathepsin B. The gene was expressed in soldier nymphs at a level 2,000 times higher than in normal nymphs, and the gene expression was localized in the midgut. The cathepsin B protein was also specifically detected in soldier nymphs and localized in the midgut.

Considering the localization in gut, the cathepsin B protease may be thought to be digestive enzyme for food proteins. However, aphid’s food, plant sap, contains very little protein. Moreover, if the protease is used for digestion, it should be needed for normal nymphs that grow and reproduce rather than for soldier nymphs. That is, it is hardly possible that cathepsin B acts as digestive enzyme for proteins, and its abundant expression specific to soldier nymphs suggests its close relationship to a certain biological function peculiar to the soldier caste.

The soldier nymphs of T. straci attack enemies by stinging with piercing mouthparts. Attacked predators wriggle over, are immobilized by paralysis, and finally killed. [See Figs. 1 and 3A] The analysis of proteins from paralyzed moth larva revealed a large amount of the soldier-specific cathepsin B. [See Fig. 3B.], indicating that the soldier nymphs injected the protease into the enemy upon attack. In order to confirm the insecticidal action of the cathepsin B protase, the protein was prepared by using genetically modified microbes. By injecting  the recombinant protease, the moth larvae were killed, proving the insecticidal activity.

On the basis of these results, it was demonstrated that the protease constitutes a major component of the “aphid venom” and plays an important role in colony defense as a primary social function of the soldier caste.

Fig. 3. A soldier nymph of Tuberaphis styraci attacking a larva of wax moth, Galleria mellonella. Detection of soldier-specific cathepsin B protease by immuno-blotting. Proteins extracted from two soldier nymphs, with the soldier-derived protease identified. Proteins extracted from a wax moth larva attacked and paralyzed by 30 soldier nymphs of T. straci, with the injected protease identified. Proteins extracted from a wax moth larva kept with 30 normal nymphs of T. styraci, with no protease detected.