Surfactant-producing Microorganisms Isolated From The Gut

Oecologia

Oecologia (1988) 77:140-142

9 Springer-Verlag1988

Short communication Surfactant-producing microorganisms isolated from the gut of a EucMyptus-feeding sawfly, Perga affinis affinis C.P. Ohmart 1, J.R. Thomas 1, and B. Bubela 2, ~r 1 CSIRO Division of Forestry and Forest Products, PO Box 4008, Canberra, ACT 2600 Australia 2 BAAS Becking Geobiological Laboratory, Canberra ACT, Australia

Summary. Microorganisms which produce surfactants were isolated from the gut of a eucalypt-feeding sawfly. It is possible that surfactants in the guts of some defoliators may be microbiologically-produced. Key words: Surfactants - Eucalyptus - Perga affinis affinis

It has been suggested that a large number of plant secondary metabolites are deleterious to insect herbivores and, as a result, some general theories have evolved regarding the chemical defenses of plants against herbivores (Feeny 1976; Levin 1976; Rhoades 1979; Rhoades and Cates 1976). Some studies, however, have shown that secondary metabolites have no deleterious effects on certain insect herbivores (Bernays 1981, 1982). Surfactants present in the gut contents of some insect herbivores can reduce the precipitation of dietary proteins by tannins and it has been suggested that the gut of many insect herbivores have detergent properties (Martin and Martin 1984). Further, due to the presence of these surfactants, tannins do not interfere with digestion or lower the nutritive quality of foliage for insect herbivores (Martin et al. 1987). Eucalyptus foliage contains large amounts of terpenes (essential oils) and tannins which potentially reduce the quality of the foliage for insect herbivores. However, eucalypt-feeding insects do not appear to be adversely affected by dietary oils or tannins (Fox and Macauley 1977; Morrow and Fox 1980; Ohmart et al. 1985) and eucalypts have at least as many associated herbivores as do other trees (Morrow 19~77; Ohmart et al. 1983). Physiological mechanisms used by herbivorous insects to process these chemicals have not been determined. It is well known that certain microorganisms produce surfactants (Korsaric 1987). Surfactants produced by bacteria have enabled a microbiological approach to be investigated for enhancing recovery from natural oil reservoirs (Bubela 1985). We hypothesized that some eucalypt-defoliating insects may contain microorganisms in their digestive tracts which produce surfactants. These surfactants may prevent tannins from interferring with digestion by lowering the interfacial tension between essential oils and gut fluids, enhancing the insect's ability to deal with these compounds. * Deceased

Offprint requests to: C.P. Ohmart

Material and methods Twelve groups of Perga affinis affinis Kirby (Hymenoptera: Pergidae) larvae were collected over a 5-month period from colonies located within a 35 km radius of Canberra, Australian Capital Territory. Larvae were anaesthetized with chloroform and the abdomen opened in a laminar flow chamber using sterilized equipment. The hind gut was then opened and a small quantity of gut contents was removed and inoculated onto a sterile agar medium (Table 1). Incubation of initial cultures from the gut was done anaerobically with no success. All subsequent cultures were aerobically incubated. After microbial colonies fon~aed on the agar plates (3-7 days at room temperature) a loop sample of a pure microbial colony was inoculated into a sterile liquid medium (Table 1). After 3-5 days incubation at room temperature, surfactant production was assessed by measuring the reduction in interfacial tension (IT) of the liquid medium against a standard liquid (hexadecane). An unioculated medium was also incubated and its IT measured as a controt. Interracial tension measurements were made using the "drop-weight" method of Harkins and Brown (1919) modified so that the fluid being measured for IT was dispensed into hexadecane from a precision syringe (500 ul), the plunger of which was operated by a micrometer head. The

Table 1. Composition of the medium upon which the gut microflora of P.a. affinis were cultured (Liquid medium is simply the above medium without the addition of agar) 5 g/t K2HPO4 2 g/1 KHzPO4 10 g/1 glucose 5 g/l yeast extract 20 g/1 agar plus 1 ml of the following solutions Solution A 1 g/1 FeC13 0.4 g/1 ZnSO4-7H20 0.1 g/1 CuSO~. 5H20 0.1 g/1 MnC12 "4H20 0.08 g/l CoC12-6HzO 0.024 g/1 NiCI2 -6H20

Solution B 5.3 g/t00 ml CaCIz "2HzO 5 g/100 ml MgCI2-6H20

plus 1 ml of 0.15 g/100 ml solution of EDTA (ethylenediaminetetra-acetate disodium salt)

I41 d r o p was forced t h r o u g h a smooth, circular glass tip, o f a k n o w n radius 1. Results

Surfactant-producing microorganisms were isolated from 10 o f the 12 groups o f larvae collected (Table 2). In 7 o f these groups the isolates p r o d u c e d average drops in IT o f more than 20 millinewtons (Table 2) indicating that the surfactants were very effective. In m a n y cases several different species o f microorganisms, differentiated by colony morphology, were cultured from a single larval gut. Because of the w o r k involved in subculturing and testing a single m i c r o o r g a n i s m for surfactant p r o d u c t i o n it was not possible to test all organisms cultured on the agar plates. Three types o f surfactant-producing microorganisms, differentiated by colony m o r p h o l o g y , were cultured from isolates during the study. Two types were rarely isolated and were not subcultured for identification. M o r e than 95% o f the surfactant-producing microorganisms isolated from the sawfly larvae were a single filamentous yeast species. Whenever this yeast species was isolated from a larva it was the species selected for subculturing and it always p r o d u c e d surfactants. If it was not isolated then a n o t h e r m i c r o o r g a n ism was selected for subculture. It is possible that some larvae did n o t test positive for surfactant-producing microorganisms simply because the wrong m i c r o o r g a n i s m was selected for subculturing. It is possible that surfactants within insect herbivores could result from microbial activity and there are there possible ways by which this could occur. Firstly, surfactants p r o d u c e d by yeasts a n d / o r bacteria resident on leaf surfaces are ingested by insects when the leaf is consumed. Secondly, the insect gut could be operating similarly to a chemostat in relation to the surfactant-producing microorganisms. F o liage u p o n which the microorganisms are resident is consumed by the insect. As this material is passed through the gut the microorgansims release surfactants into the gut. The microorganisms are then passed out o f the gut in faeces. M i c r o o r g a n i s m s are constantly ingested and egested a n d produce surfactants as they pass through the gut. Thirdly, t Detailed explanation of modified drop-weight method available from authors

surfactant-producing microorganisms m a y be contained in special tissues within the insect gut. M a r t i n and K u k o r (1984) suggested that ingested microbes m a y metabolize potential toxins present in insect diets while Jones (1984) discussed several cases where p l a n t allelochemicals inhibited insect-microbial systems. Little work, however, has been done on the gut microbiology o f foliage-feeding insects (M.M. Martin, personal c o m m u nication). A great deal o f w o r k needs to be done to fully explore the possibility that surfactants in the guts o f insect herbivores are microbiologically-produced. Acknowledgements. We thank Dr. R. Milner for valuable help and advice on initial lab work and, along with Dr. K.M. Old, for useful comments on earlier versions of the manuscript. References

Bernays EA (1981) Plant tannins and insect herbivors: an appraisal. Ecol Entomol 6:353-360 Bernays EA (1982) The insect on the plant - a closer look. Proc 5th Symp. Insect-Plant Interactions, Wageningen, pp 3-17 Bubela B (1985) Effects of biological activity on the movements of fluids through porous rocks and sediments and its application to enhanced oil recovery. Geomicrobiol J 4:313-317 Feeny PP (1976) Plant apparency and chemical defenses. Recent Adv Phytochem 10:1-40 Fox LR, Macauley BJ (1977) Insect grazing on Eucalyptus in response to variation in leaf tannins and nitrogen. Oecologia (Berlin) 29:145-162 Harkins WD, Brown FE (1919) The determination of surface tension, and the weight of falling drops: the surface tension of water and benzene by the capillary height method. Am Chem Soc J 41:499-524 Jones CG (1984) Microorganisms as mediators of plant resource exploitation by insect herbivores. In: Price PW, Slobodchikoff CN, Gaud NS (eds) Novel approaches to interactive systems. Wiley, New York, pp 53-99 Kosaric (1987) Biosurfactants and biotechnology. Dekker, New York, pp 344 Levin DA (1976) The chemical defenses of plants to pathogens and herbivores. Annu Rev Ecol Syst 7:121-159 Martin MM, Kukor JJ (1984) Role of mycophagy and bacteriophagy in invertebrate nutrition. In: Klug M J, Reddy CA (eds) Current perspectives in microbial ecology. Amer Soc Microbiol, Washington, DC, pp 257-263 Martin MM, Martin JS (1984) Surfactants: their role in preventing

Table 2. Interracial tension (IT) (_+ 1 s.d.) measurements of microbial cultures from the guts of P. a. affinis larvae Collection site

Wanniassa, ACT Site 1 Wanniassa, ACT Site 2 Yarralumla, ACT Site 1 Fisher, ACT Site 1 Curtin, ACT Yarralumla, ACT Site 2 Braidwood, NSW Bungendore, NSW Macks Reef Rd, Site 1 Macks Reef Rd, Site 2 Fisher, ACT Site 2

Date (1987)

1/1o 28'10 28q0 t3/ll 13'11 13ql 27ql 27'11 27ql 27ql 21q2

No. of larvae examined

5 8

No. of larvae from which surfactant produced

2 8

IT (mN) Control

Inoculated cultures Surfactant producing

Nonsurfactant producing

4;2 (0.8) 12.2 (7.0)

33.1 (2.1) 32.2 (2.4) 32.3 (2.4) 29.6 34.2 31.6 (0.5) 34.6 (1.3) 0 33.6 (0.7) 0

8

3

8,8 (0.8)

9 8 8 8 8

0 7 7 2 5

0 10.7 (1.6) 14.2 (2.1) 22.3 (3.9) 23.7 (3.7)

8

8

11.1 (1.5)

5 5

0 5

0 7.9 (2.8)

0

33.9 33.0 33.0 33.3 33.3 33.3 33.1 33.1 33.1 33.0 32.4

142 the precipitation of proteins by tannins in insect guts. Oecologia (Berlin) 61 : 342-345 Martin MM, Martin JS, Bernays EA (1987) Effects of surfactants, pH, and certain cations on precipitation of proteins by tannins. J Chem Ecol 11:485494 Morrow PA (1977) Host specificity of insects in a community of three co-dominant Eucalyptus species. Aust J Ecol 2:89-106 Morrow PA, Fox LR (1980) Effects of variation in Eucalyptus essential oil yield on insect growth and grazing damage. Oecologia (Berlin) 45: 209-219 Ohmart CP, Stewart LG, Thomas JR (1983) Phytophagous insect communities in the canopies of three Eucalyptus forest types in southeastern Australia. Aust J Ecol 8 : 395-403

Ohmart CP, Stewart LG, Thomas JR (1985) Effects of food quality, particularly nitrogen concentrations, of Eucalyptus blakelyi foliage on the growth of Paropsis atomaria larvae (Coleoptera: Chrysomelidae). Oecologia (Berlin) 65 : 543-549 Rhoades DF (1979) Evolution of plant chemical defense against herbivores. In: Rosenthal GA, Janzen DH (eds) Herbivores: their interaction with secondary plant metabolites. Academic Press, New York, pp 4-55 Rhoades DF, Cates RG (1976) Toward a general theory of plant antiherbivore chemistry. Recent Adv Phytochem 10:168-213 Received April 14, 1988