The Development of Immunity in a Social Insect: Evidence for the Group Facilitation of Disease Resistance

The development of immunity in a social insect: Evidence for the group facilitation of disease resistance James F. A. Traniello*, Rebeca B. Rosengaus, and Keely Savoie Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215 Communicated by Bert Ho¨lldobler, University of Wurzburg, Wurzburg, Germany, March 25, 2002 (received for review October 31, 2001) The extraordinary diversity and ecological success of the social absent in naı¨ve termites. The dynamics of the immune response insects has been attributed to their ability to cope with the rich and of Z. angusticollis and its associated hemolymph protein profile often infectious microbial community inhabiting their nests and resemble immunization-related protective changes in the protein feeding sites. Mechanisms of disease control used by eusocial constituents of the phylogenetically related roaches (17). species include antibiotic glandular secretions, mutual grooming, Here we show that the level of immunocompetence attained removal of diseased individuals from the nest, and the innate and by termites depends on association with colony members and adaptive immune responses of colony members. Here we demon- that the disease resistance of individuals that have not experi- strate that after a challenge exposure to the entomopathogenic enced direct contact with a pathogen can be significantly en- fungus Metarhizium anisopliae, dampwood termites Zootermopsis hanced through interactions with immunized nestmates. Our angusticollis have higher survivorship when individuals develop studies of the development of immunity in Z. angustocollis reveal immunity as group members. Furthermore, termites signiﬁcantly novel social mechanisms of infection control. improve their ability to resist infection when they are placed in contact with previously immunized nestmates. This ‘‘social trans- Materials and Methods fer’’ of infection resistance, a previously unrecognized mechanism Development of Immunocompetence in Isolated and Grouped Ter- of disease control in the social insects, could explain how group mites. To determine whether the social environment influences living may improve the survivorship of colony members despite the the development of disease resistance, we conducted a series of increased risks of pathogen transmission that can accompany experiments in which we allowed termites to develop immunity sociality. after receiving a nonlethal dosage of a fungal pathogen when nesting either in isolation or groups, and then measured the dapting to the infection risks from pathogenic bacteria, strength of their immune defense with a challenge exposure to Afungi, and other microbes that thrive in the nest environ- a lethal dose of the same pathogen (Fig. 1). Using nymphs of Z. ment has had primary importance in the remarkable diversifi- angusticollis from two stock colonies recently collected in the cation of the social insects (1, 2). Recent research has begun to field, we exposed individuals (average age approximately 600 ϫ 1 ϫ 2 ϫ 3 ͞ reveal the pervasive impact of disease on the evolution of insect days) to either a 5 10 ,5 10 ,or5 10 spores ml social organization, influencing colony and population genetics, suspension of the entomopathogenic fungus Metarhizium aniso- demography, and mating systems (3, 4), among other attributes. pliae or a sporeless 0.1% Tween 80 suspension medium (con- Although the majority of studies have focused on bees and the trols). The three spore concentrations were chosen to ensure that dead wood and soil-nesting ants (Order Hymenoptera), termites exposure levels would be adequate to induce an immune re- (Order Isoptera) also inhabit decayed wood and soil and appear sponse in both isolated termites, which rely only on their to be highly susceptible to fungi and other parasitic infections physiology to resist infection, as well as in grouped termites, (ref. 5; a table detailing the incidence of pathogen and parasite which can use individual physiological responses in combination infection in termites is posted at http:͞͞people.bu.edu͞ with social behaviors to reduce susceptibility (6). Nymphs were ␮ rrosenga͞table1.htm). In addition to the diversity and pathoge- cold-immobilized and subsequently placed dorsally on a 3- l nicity of the microbial community of a termite colony, the droplet of either a spore solution or the control suspension maintenance of a homeothermic nest and the likelihood of medium at 4°C for 1 h. This procedure provided an effective disease transmission through social exchanges between parents method to control the quantity of inoculum received by each and offspring, among offspring, and between mates can exacer- individual. Chilling was not a significant predictor of mortality ϭ Ͼ bate pathogen-related mortality (6, 7). Termite adaptations that (Wald Statistic 2.1, P 0.1). After exposure, termites were reduce disease susceptibility include mutual grooming scaled transferred to sterile Petri dishes lined with moistened filter in frequency to pathogen prevalence (6, 7), the production of paper (Whatman no. 1, 1 ml sterile water) and immediately antibiotic secretions in exocrine glands and other exudates (8, 9), divided into two treatment groups. In the first group, each ϭ ϭ and the communication of information about the presence of nymph (n 50 for controls and n 50 for each spore pathogens in the nest (10). Additionally, termites significantly concentration) was isolated for 10 days. In the second group, ϭ improve their physiological resistance to infection by mounting nymphs were maintained in groups of 10 (n eight groups of 10 ϭ a humoral immune response after they are exposed to a nonle- termites for controls and n eight groups of 10 termites for each thal inoculum of a bacterial or fungal pathogen (11) and produce spore concentration). To permit the development of an immune antibacterial peptides in their salivary glands (12). In our model response, we allowed 10 days to elapse (11), and each termite termite species, Zootermopsis angusticollis, enhanced survivor- from the control and immunization treatments was then chal- ϫ 4 ͞ ship of immunized individuals is correlated with changes in the lenged with a 5 10 spore ml direct dorsal exposure, using the protein constituents of their hemolymph, suggesting humoral procedure described above. After the challenge exposures, ter- immunity. SDS͞PAGE plasma analyses show that qualitative and quantitative changes in protein banding patterns occur with *To whom reprint requests should be addressed. E-mail: [email protected]. specificity after exposure to fungal spores or bacteria. Immune The publication costs of this article were defrayed in part by page charge payment. This proteins identified in other insects (13–16), visible in the hemo- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. lymph of Z. angusticollis nymphs 3–7 days postimmunization, are §1734 solely to indicate this fact. 6838–6842 ͉ PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.102176599 Downloaded by guest on September 28, 2021 Fig. 1. Schematic representation of methods used in studies designed to test the effect of isolation and grouping on the development of immunity. mites were transferred to new Petri dishes maintaining the same groups composed of either five dyed and five unstained termites social and isolation treatments, and survivorship was recorded exposed to a spore-free Tween 80 suspension (n ϭ five repli- daily for 10 days. In addition to these two treatments, we cates) or five dyed and five unstained nestmate nymphs exposed challenged naı¨ve termites (termites that originated from the for the first time to the same 6.5 ϫ 104 spores͞ml challenge same parent colonies but had no exposure to spores or the Tween suspension (n ϭ eight replicates). A total of 202 termites were 80 suspension medium) with the same 5 ϫ 104 spores͞ml solution challenged (n ϭ 134 control and naı¨ve termites that had no and maintained them in isolation (n ϭ 50 nymphs) or in groups contact with the pathogen and n ϭ 68 experimental exposures). of 10 (n ϭ five groups). Survival censuses were conducted daily and dead termites were removed, surface-sterilized with 5.2% Statistics. Survival parameters used in statistical evaluations sodium hypochlorite, and plated on potato dextrose agar to included the survival distribution, percent survival at the end of confirm that M. anisopliae was the cause of mortality (6). the census period, median survival time (LT50), and the hazard Confirmation rates (18) varied from 52.2% to 96%, indicating ratio of death, using the Cox Proportional Regression analysis to that most termites died as a result of exposure to M. anisopliae. generate the Wald Statistic. Regression models included the Comparisons of the survival parameters of termites in all following variables: nesting treatment (group͞isolation compar- treatments allowed us to evaluate the effect of isolation and isons), naı¨ve͞immunized nestmate association, spore concentra- grouping on disease resistance, as well as to confirm that the tion during immunization, and colony of origin (where applica- Tween 80 suspension medium had no immunizing effect. ble). The hazard function characterized the instantaneous rate of death at a particular time, given that the individuals survived up Social ‘‘Transfer’’ of Immunity. To test the hypothesis that naı¨ve to that point, while controlling for the effect of the other individuals can increase their resistance to infection through variables

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