Ralph W. Howard, C. A. McDaniel, and Gary J. Blomquist.
Chemical Mimicry as an Integrating Mechanism for Three Termitophiles Associated with Reticulitermes virginicus (Banks).
Psyche 89(1-2):157-167, 1982.

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CHEMICAL MIMICRY AS AN INTEGRATING MECHANISM FOR THREE TERMITOPHILES ASSOCIATED WITH
RETICULITERMES VIRGIMCUS (BANKS)'.'
The mechanisms by which termitophiles integrate themselves into the social life of termite colonies have long intrigued entomologists (Kistner, 1979). Various authors have suggested that plausible inte- gration mechanisms might include the using of "appeasement chem- icals" (Wilson, 1971), passing as morphological mimics (Kistner, 1968). or engaging in behavioral mimicry (Kistner, 1979). We recently reported (Howard et al., 1980a) that the host-specific, highly integrated termitophile Trichopsenius frosti Seevers asso- ciated with Reticulitermesflavipes (Kollar) possesses the same com- plex mixture of cuticular hydrocarbons as its termite host. We suggested that this was an example of chemical mimicry which func- tioned to integrate this beetle into the termite society. Reticulitermes virginicus (Banks) is sympatric with R. flavipes throughout much of its range and, as predicted (Howard et al., 1978; Blomquist et al., 1979), the two species possess distinctly dif- ferent cuticular hydrocarbons which function as species recognition cues (Howard et al., 1982). They also have different termitophilous cohorts. Thus, T. frosti is associated only with R. flavipes whereas T. depressus Le Conte, Xenistusa hexagonalis Seevers (both Sta- phylinidae: Trichopseniinae), and Philotermes howardi Kistner and Gut (Staphylinidae: Aleocharinae) are associated only with R. vir- ginicus. We now report that the three R. virginicus staphylinids also appear to use chemical mimicry as an integrating mechanism; i.e., -
'Manuscript received h.1' the editor June 3, 1982. ~1,soptera: Rh;noterrnilitidae.
3Forestry Sciences Laboratory, Southern Forest Experiment Station, P. 0. Box 2008 GMF, Gulfljort. MS 39503.
4Author 10 whom correspondence .should he addressei/. ~Na/ional Monitoring and Residue Analysis Laboratory. USDA Animal and Plant Health Inspection Service. P. 0. Box 3209. G~dfport, MS 39503. 6Department ofBiochemistry, University of Nevada- Reno, Reno, N V 89557



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158 Psyche [VOI. 89
they have the same complex mixture of cuticular hydrocarbons as their host termite. In addition, we report that at least one of these beetles (X. hexagonalis) biosynthesizes its hydrocarbons. Portions of several colonies of R. virginicus were collected in 1979 from pine logs in Harrison, Jackson, and Stone Counties, Missis- sippi. The beetles were separated from the termites, counted by species, and stored at -20å¡ until used. A total of 230 beetles was collected: 10 P. howardi, 140 T. depressus, and 80 X. hexagonalis. Cuticular hydrocarbons from pooled samples (by species) were iso- lated and separated as previously described (Howard et al., 1978). Hydrocarbons were characterized by gas-liquid chromatography (GC) retention times and by electron impact (El) and chemical ioni- zation (CI) mass spectrometry (Howard et al., 1980b; Jackson and Blomquist, 1976). Double bond stereochemistries were determined by comparison with standards using argentation thin-layer chroma- tography (AgNOs-TLC) (Kates, 1972).
In vitro biosynthesis experiments were conducted as previously described (Howard et a/., 1980a) using 60 X. hexagonalis collected from a single colony of R. virginicus in September 1979. Radioactivity was assayed by liquid scintillation counting for 10 minutes at about 85 percent counting efficiency. All counting was done with a standard deviation of less than 5 percent. A portion of the isolated hydrocarbons was assayed for total radioactivity. The remainder of the material was separated by AgN03-TLC into satu- rated, monounsaturated, and diunsaturated components, which then were assayed for radioactivity.
The retention times of all peaks present in the GC profile of cuticular hydrocarbons from R. virginicus (Fig. 1) match those from the GC profile of the cuticular hydrocarbons of P. howardi (Fig. 2), T. depressus (Fig. 3), and X. hexagonalis (Fig. 4). Confirmation of the chemical identity for each of the hydrocarbon components in most of the GC peaks was obtained by El and CI mass spectrometry (MS). In every instance, the GC-MS retention times and mass spec- tra of the beetle hydrocarbon components were identical to those



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Fig. 1.
GC trace of total cuticular hydrocarbons of Retitwlilermes virginicus. GC conditions: 1.83 m X 3 mm i.d. Stainless steel column packed with 3 percent (w/w) SP-2 100 on 1001 120 mesh Supelcoport: temperature programmed from 150' to 325OC at 5O C/ min.
Fig. 2.
GC trace of total cuticular hydrocarbons of Philotert~es howardi. GC conditions same as for Fig. 1.




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19821 Howard, McDaniel, Blomquist - Three Termitophiles 161 Fig. 3.
GC trace of total cuticular hydrocarbons of Trichopsenius depressus. GC conditions same as for Fig. 1.
Fig. 4 GC trace of total cuticular hydrocarbons of Xenisiusa hexagonalis. GC conditions same as for Fig. 1.




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162 Psyche [Val. 89
termite host is strongly suggestive for their role as integrating fac- tors. It also supports our earlier hypothesis that cuticular hydrocar- bons may serve as species recognition cues (Howard et al., 1978; Blomquist et al., 1979; Howard et al., 1980a; Howard et al., 1982). Behavioral evidence for this interpretation comes from the finding (Howard, unpublished observations) that live T. depressus placed into laboratory colonies of R. flavipes were killed by the termites within a 24-hour period (five observations). Similarly, the placing of live T.frosti into laboratory colonies of R. virginicus results in their being killed (five observations). Beetles can be freely exchanged among different colonies of their hosts however. These two Tri- chopsenius spp. are nearly identical morphologically and behavior- ally, but differ markedly with respect to cuticular hydrocarbons. Similar transplants of workers or soldiers of R.flavipes or R. virgi- nicus into colonies of the other species also resulted in the death of the alien individual (five observations). Transplants of conspecific termites into different colonies did not produce agonistic interac- tions (five observations). As with the beetles, the two termite species are morphologically and behaviorally quite similar. We have shown that R. virginicus workers are antagonistic towards neutral, critical- point dried (CPD) conspecific workers treated with R. flavipes cuticular hydrocarbons (Howard et al., 1982), but are not aggressive toward CPD workers treated with R. virginicus cuticular hydro- carbons. While we cannot exclude the possibility of other biochemi- cal differences among either the beetles or their host termites, GC comparisons of total body extracts revealed none. The termitophiles associated with R. virginicus (in common with other termitophiles) possess many epidermal glands (Kistner, 1979) which have often been postulated to be a source of chemicals which in some manner aids in the integration of the beetles into the termite society. While we cannot rule out this interpretation, we would like to suggest an alternative hypothesis for the function of these glandu- lar products. Termitophiles are never found in great abundance (Wilson, 197 1; Kistner, 1979), and the nature of termite nest-galley systems is such as to present substantial problems in the location and recognition of conspecifics. Perhaps these glands are producing pheromones directed at conspecifics rather than kairomones directed at their host. Since pheromones are usually produced in extremely



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19821 Howard, McDaniel, Blomquist - Three Termitophiles 163 small amounts, such an interpretation would explain the lack of GC evidence to date for beetle derived biochemicals different from those of their termite host. An experimental test of this hypothesis must await the development of suitable bioassays. Reticulitermes virginicus and its termitophiles have been co- evolving for a long period of time (Kistner, 1968, 1979). The beetles are totally integrated into the social life of the colony and appear to be chemically indistinguishable from the termites (chemical mim- icry) vis-a-vis their cuticular hydrocarbons. Most known termite- termitophile associations, however, occur within the family Termiti- dae (Kistner, 1979). These associations are characterized by termito- philes ranging in status from nonintegrated to totally integrated. If our hypothesis is correct regarding the integrating role of cuticular hydrocarbons then a corresponding spectrum of congruences of hydrocarbon profiles would be predicted among the termitophiles of these communities. We are presently testing this hypothesis. Many species of ants are known to have inquilines associated with them, but unlike termitophiles, these myrmecophiles are seldom host specific (Wilson, 1971). In addition, myrmecophiles seem to show a wider range of integration (or lack thereof) than do termito- philes. A correspondingly greater range of integrating mechanisms might therefore be expected, and have been found. These include body color, appeasement substances, trichomes, unicellular epi- dermal glands, physogastry, exudatoria and grandular antennae. All have been superbly reviewed by Wilson (1971) and Kistner (1979). The most recent addition to this plethora of mechanisms is the finding that the scarab beetle M.~*rrnecaphodius excavaticollis (Blanchard) associated with various So1enopsi.s spp. ("fire ants") has a cuticular hydrocarbon composition which closely mimics that of its current ant host (Van der Meer, personal communication in Howard and Blomquist, 1982). The mechanism by which the beetles achieve this is unknown. Each of the four ant hosts that the scarab beetles is found with, however, has a unique hydrocarbon profile. Perhaps ants, like subterranean termites, also use cuticular hydro- carbons as species-recognition cues. Clearly a great deal remains to be learned before we achieve an adequate understanding of the diversity of relationships between social insects and their guests.



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Table 1. Continued
GC
Carbon
Peak1 Component number2 Diagnostic MS ions3 See Figures 1 to 4.
'~etermined from CI-MS where (M - I)* is always the base peak. EI-MS.
'El Z-6,9-C23 indicates E/ Z-6,9-tricosadiene, where the slash indicates that one double bond is cis (Z) and one is trans (E), but which is which, is unknown. The diene in peak 11 is named correspondingly.



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[Vol. 89
The three highly integrated staphylinid termitophiles (Philo- termes howardi Kistner and Gut, Trichopensius depressus Le Conte, and Xenistusa hexagonalis Seevers) associated with Reticuli- termes virginicus (Banks), possess the same cuticular hydrocarbons as their host. This congruence is hypothesized to be a form of chem- ical mimicry and is postulated to function as a major way these beetles achieve integration into the termite society. G. J. Blomquist acknowledges the support of the Science and Education Administration of the U.S. ~&artment of Agriculture under grant 7801 064 from the Competitive Research Grant Office. BLOMQUIST, G. J., R. W. HOWARD, AND C. A. MCDANIEL. 1979.
Structures of the cuticular hydrocarbons of the termite Zootermopsis angusticollis (Hagen). Insect. Biochem. 9: 365-370. BLOMQUIST, G. J., R. W. HOWARD, C. A. MCDANIEL, S. REMALEY, L. A. DWYER, AND D. R. NELSON.
1980. Application of methoxymercuration-demercuration followed by mass spectrometry as a convenient microanalytical technique for double-bond location in insect-derived alkenes. J. Chem. Ecol. 6(1): 257-269. HOWARD, R. W.
1978.
Proctodeal feeding by termitophilous Staphylinidae associated with Re- ticulitermes virginicus (Banks). Science 201: 541 -543. HOWARD, R. W., AND G. J. BLOMQUIST.
1982.
Chemical ecology and biochemistry of insect hydrocarbons. Annu. Rev. Entomol. 27: 149-172.
HOWARD, R. W., C. A. MCDANIEL, AND G. J. BLOMQUIST. 1978.
Cuticular hydrocarbons of the eastern subterranean termite, Reticuli- termes flavipes (Kollar) (Isoptera: Rhinotermitidae). J. Chem. Ecol. 4(2): 233-245.
HOWARD, R. W., C. A. MCDANIEL, AND G. J. BLOMQUIST. 1980a. Chemical mimicry as an integrating mechanism: cuticular hydrocar- bons of a termitophile and its host. Science 210: 431-433. HOWARD, R. W., C. A. MCDANIEL, D. R. NELSON, AND G. J. BLOMQUIST. 1980b.
Chemical ionization mass spectrometry: application to insect-derived cuticular alkanes. J. Chem. Ecol. 6(3): 609-623.



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19821 Howard, MeDaniel, Blomquist - Three Termitophiles 167 HOWARD, R. W., C. A. MCDANIEL, D. R. NELSON, G. J. BLOMQUIST, L. T. GELBAUM AND L. H. ZALKOW.
1982. Cuticular hydrocarbons of Reticulitern~es virginicus (Banks)' and their role as potential species- and caste-recognition cues. J. Chem. Ecol. 8: 1227- 1239.
JACKSON, L. L., AND G. J. BLOMQUIST.
1976.
Insect waxes. P. 201-233. In Chemistry and Biochemistry of Natural Waxes. P. E. Kolattukudy (ed.). Elsevier, Amsterdam, Oxford, and New York. 459 p.
KATES, M.
1972.
Techniques of Lipidology: Isolation, Analysis and Identification of Lip- ids. North-Holland Publishing Company, Amsterdam, and American Elsevier Publishing Company, New York. 610 p. KISTNER, D. H.
1968.
Revision of the African species of the termitophilous tribe Corotocini (Coleoptera: Staphylinidae). 1. A new genus and species from Ovambo- land and its zoogeographic significance. J. N. Y. Entomol. Soc. 76: 213-221.
KISTNER, D. H.
1979. Social and evolutionary significance of social insect symbionts. P. 339-413. In Social Insects. Vol. 1. H. R. Hermann fed.). Academic Press, New York, San Francisco, and London. 437 p. WILSON, E. 0.
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The Insect Societies. The Belknap Press of Harvard University Press, Cambridge, Mass. 548 p.




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