Insect. Soc. 51 (2004) 339 – 341 0020-1812/04/040339-03 DOI 10.1007/s00040-004-0751-3 © Birkhäuser Verlag, Basel, 2004
Insectes Sociaux
Research article
Antibacterial activity of larval saliva of the European paper wasp Polistes dominulus (Hymenoptera, Vespidae) S. Turillazzi 1, B. Perito 1, L. Pazzagli 2, B. Pantera 2, S. Gorfer 1 and M. Tancredi 1 1 2
Dip. Biologia Animale e Genetica, Università di Firenze, via Romana 17, 50125 Firenze, Italy, e-mail:
[email protected] Dip. Scienze Biochimiche, Università di Firenze, Firenze, Italy
Received 7 November 2003; revised 17 March 2004; accepted 29 March 2004.
Summary. Microbiological tests demonstrated antibacterial activity (against Bacillus subtilis, Gram +, and Escherichia coli, Gram –) of larval salivary secretions of Polistes dominulus but failed to demonstrate its antifungal activity. Key words: Paper wasps, larvae, salivary secretions, antibacterial, Bacillus subtilis.
Introduction Groups of individuals can be particularly vulnerable to attacks by pathogens due to the promiscuity of their members. Indeed sociality is associated with significant costs due to the increased risk of disease transmission (Brockmann, 1984). In a recent book on the argument, P. Schmid-Hempel (1998) observed that social insects represent ‘a study subject that has an enormous potential to understand how parasites co-evolve with their hosts’. Insects have an individual immune system with a humoral and a cellular component but it does not appear to be as sophisticated as that of Vertebrates (see SchmidHempel, 1998). However social insects can also produce various substances with antimicrobial activity. In fact they are gland factories (Billen and Morgan, 1998) and several of their secretions have been shown to be antiseptic. A striking example is the metapleural glands of ants which have antiseptic and antifungal activity (references in Hölldobler and Wilson, 1990). In leaf cutter ants these glands produce a secretion consisting of various substances active against specific microorganisms; this secretion protects the ants themselves and their clonal mutualistic fungus (Bot et al., 2002). Several components of the venom of various ants, wasps and bees also present antimicrobial activity (Park et al., 1995; Orivel et al., 2001; Storey et al., 1991; Phisalix, 1922) while some termites have soldiers which produce antibacterial secretions from their frontal glands (Rosengaus et al., 2000).
There is very little information about antimicrobial secretions in social wasps. In pioneering research on antimicrobial substances produced by various common insects, Pavan (1952) found some activity in alcoholic body extracts of workers of Vespa crabro L., while Park et al. (1995) demonstrated the activity of Mastoparan-B, a protein component of the venom of V. affinis L., and Gambino (1993) reported antibiotic activity of the saliva of Vespula germanica (F.) larvae. In social wasps, larval saliva is an important component of nutritional flow in a colony (references in Hunt, 1991). Larvae have enormously developed salivary glands which open near the mouth. The abundant saliva is sucked by the adults which actively solicit its emission by stimulating the larva’s head with their mouthparts (Cummings et al., 1999). Indeed larval saliva is thought to have various social roles (Hunt, 1991). Polistes dominulus (Christ) is a European paper wasp which is fairly common in human settlements and has recently been introduced into the United States. It is the best known species of Polistes and is a model for sociobiological studies (Turillazzi and West-Eberhard, 1996). The larvae of P. dominulus, like those of all other vespids, grow in paper cells and are tended continuously by adults until they pupate. They are fed by the adults with chewed prey and have an impervious gut where they store food remnants throughout larval life. Just after cocoon spinning and before initiation of the pupal stage the larvae discard all the remnants (meconium) enveloped in the gut peritrophic membrane and press them to the bottom of their cells. We wished to investigate if the novel function that larval saliva seems to have in Vespula germanica is also present in other wasp genera. Therefore, we checked for antimicrobial activity of the saliva of larvae of the paper wasp Polistes dominulus.
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Antibacterial activity of paper wasp larval saliva
Materials and methods
Results
Collection of saliva
The results are shown in Table 1. Saliva of P. dominulus larvae showed antibacterial activity against B. subtilis and E. coli. We could not demonstrate activity against C. albicans, C. parapsilosis or C. krusei.
We milked salivary secretions from larvae of field and laboratory colonies. Field colonies were directly collected from natural sites or transplanted into small cages kept open in the field and brought to the laboratory only for saliva milking. Colonies reared in the laboratory were kept in glass cages (15 ¥ 15 ¥ 15 cm) where the wasps were supplied with water, sugar and fly maggots ad libitum. We used small capillaries to stimulate the mouthparts of the larvae and to collect the drops of saliva. The saliva was immediately tested or transferred to Eppendorf vials maintained in a –24°C freezer until microbiological assays. The same larvae could be milked various times with an interval of 3 or more days.
Discussion and conclusions Salivary secretion is mainly composed of carbohydrates, proteins and free amino acids (in a concentration 50 times more than in the richest floral nectars) and is obviously a very attractive food for the adults (Hunt, 1991); according to this author, larval saliva serves mainly as an appeasement to prevent cannibalism by adults. The two-way, larva-adult trophallaxis in social wasps (adults supply the larvae with chewed food and regurgitated liquid) has been considered even by older authors as the most important factor favoring the origin and evolution of sociality in these insects (see Hunt, 1991 for references and discussion). As in Vespula germanica the salivary secretions of Polistes dominulus larvae contain substances with antibiotic activity against Gram + and Gram – bacteria. However we could not demonstrate antifungal activity on the fungal species we tested. The main function of these substances is not yet clear but they probably provide both individual and social protection against diseases. However, experiments to confirm a possible higher resistance against pathogens in colonies without the support of larval saliva are made difficult by the important nutritional function this secretion has in vespid colonies. A common problem for all vespid and bee larvae reared in closed cells is to control the spread of pathogens that can develop in food remnants stored in the gut throughout larval life. This problem can extend into pupal life if fecal pellets in the discharged peritrophic sac are not eliminated by adults, as in the subfamily Stenogastrinae and in the polistine tribe Ropalidiini. The finding of antimicrobial activity in the larval saliva of a vespine species (Gambino, 1993) and a polistine species (this paper) may provide some answers to the question of how immature brood in these wasps counteract the spread of pathogens in colonies raised by the stocking of large quantities of fecal pellets in the bottom of the pupal cells (cfr. Rosengaus et al., 1998).
Microbial strains We used the following microbial strains: Escherichia coli JM109, Bacillus subtilis ATCC (American Type Culture Collection) 6633, Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258. Agar diffusion test We assayed microbial growth inhibition with the agar diffusion method. The Gram + strain, B. subtilis, and the Gram – strain, E. coli, were used as bacterial indicators. Petri dishes with PY solid medium (Antibiotic Medium N. 3, Oxoid, plus 15 g/l agar) were plated with 105 E. coli cells from an overnight culture in PY medium. Petri dishes with Nutrient Agar (Oxoid) medium were overlain with 7 ml of NA inoculated with 106 B. subtilis spores. Saliva was spotted on the plates (Table 1). Penicillin G and Tetracycline were used as standards for Gram + and Gram – bacteria, respectively. They were spotted on the plates as 1 ml of solution (12.5 mg/ml for Penicellin G and 100 mg/ml for Tetracycline). Plates were incubated for 24 h at 37°C. C. albicans, C. parapsilosis and C. krusei were used as fungal indicators. Petri dishes with YPD solid medium (1% yeast extract, 2% peptone, 2% glucose, 20 g/l agar) were plated with 104 cells of each yeast from an overnight culture in YPD. Saliva was spotted on the plates (Table 1). Nystatin was used as standard. Filter paper discs imbibed with 25 ml of nystatin solution (100 mg/ml), were put on the surface of the solidified medium. Plates were incubated for 24 h at 30°C. Antimicrobial activity was indicated by clear zones of growth inhibition on the plates.
Table 1. Results of the agar diffusion test with samples of saliva from P. dominulus larvae Microorganism
Antimicrobial standard 1
B. subtilis
Penicillin G [11.5]
E. coli
Tetracycline [6.0]
C. parapsilosis C. albicans C. krusei
Nystatin [13.5] Nystatin [11.5] Nystatin [11.0]
1 2
Antimicrobial volume (ml)
Volume of saliva (ml)
No. tests performed
No. positive tests of saliva 2
1
5
11
1
5
8
5 5 5
10 10 10
11 [5.09 + – 0.93] 8 [7.88 + – 4.02] 0 0 0
25 (in disk) 25 (in disk) 25 (in disk)
Antimicrobial standard were chosen as positive controls. In brackets average diameter (in mm) of inhibiton zone of two tests. In brackets average diameter (in mm) of inhibition zone + – SD.
Insect. Soc.
Vol. 51, 2004
At present we do not know the mode of action of the active substances and their flow among colony members. It may be that larvae swallow part of their saliva (J.H. Hunt observed that larvae of some American species readily accept and swallow saliva when it is supplied by the experimenter – pers. comm.) or that it returns to them when nourished by the adults, or both. This would explain the occasional finding that the regurgitates of adults have slight antimicrobial activity (unpubl. obs.). In any case, social wasp larvae, with their enlarged salivary glands, are not the socially inert, packed-in-cells, immature brood we might think they are; they represent an important component of a social wasp colony (Hunt, 1991). This possible additional function of their saliva opens a new and interesting line of research.
Acknowledgments We thank C. Cotoneschi, L. Carresi, C. Barberio, G. Mastromei and C. Indorato (University of Florence) for their assistance and J.H. Hunt for a revision and comments on the manuscript as well as two anonymous referees. Research was funded by University of Florence.
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