© 2001 by The Society for Integrative and Comparative Biology
Chemical Interactions in the Cactus-Microorganism-Drosophila Model System of the Sonoran Desert1
1 Department of Biological Sciences, University of Denver, Denver, Colorado 80208
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 SYNOPSIS THE CACTUS-MICROORGANISM...ALLELOCHEMISTRY OF COLUMNAR...SUBSTRATE UTILIZATIONCHEMICAL INTERACTIONS INVOLVING...THE EFFECT OF CACTUS...PROSPECTS FOR INTEGRATIVE...MOLECULAR BIOLOGY OF PLANT...EVOLUTION OF PLANT-ANIMAL...FUTURE DIRECTIONSReferences |
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The Cactus-Microorganism-Drosophila Model System of the Sonoran Desert represents an excellent paradigm of the role of chemistry in plant-animal interactions. In this system, four species of endemic Drosophila feed and reproduce in necrotic tissue of five species of columnar cacti. Studies over the past 35 yr have characterized a myriad of interactions between the three major components of the model system. The cacti contain a variety of allelochemicals which are primarily responsible for the highly specific pattern of host plant utilization exhibited by the desert Drosophila. Plant chemistry, through its effect on the microbially produced volatile patterns, is further involved in host specificity because the flies use the volatile pattern to cue in on necroses in the appropriate species of cactus. The metabolic activities of microorganisms (bacteria and yeasts) living in the necrosis can affect the substrate chemistry in both positive and negative ways (i.e., acting to increase or to decrease the toxicity of the substrate). Finally, cactus chemistry may affect drosophilid mating behavior since larval rearing substrate has been shown to influence adult hydrocarbon epicuticular composition. In D. mojavensis, adult hydrocarbon profile has been implicated as a determinant of mate choice leading to premating isolation between geographically isolated populations that use chemically different cactus substrates. Current research is focused on the evolution and regulation of genes whose products (cytochrome P450 enzymes) are involved in the specific insect-host plant relationships which exist between the Drosophila species and the cactus species.
There are many reasons why investigators choose to focus their research efforts on what are referred to as "model systems." Typically included among these would be the idea that model systems are easier to study because they are less complex than other scientific situations. At the same time, model systems should be representative of more complex, natural systems so that information that is obtained from their study is broadly applicable. For almost a century, the fruit fly, Drosophila melanogaster, has served as a model organism for the study of genetics. As a genetic paradigm, Drosophila is more tractable to scientific investigation than most organisms and has provided important insights into a wide variety of human maladies from alcohol abuse to neurological brain disorders (Bellen, 1998
). Similarly, the interrelationships of the columnar cacti and the cactophilic Drosophila species of the Sonoran Desert have, for the past 35 yr, provided an excellent model system with which to study relevant questions in evolution, ecological genetics, and chemical ecology. The intent of this article is to briefly review and characterize the chemical interactions between the plants (cacti) and animals (Drosophila) of this model system, and, in addition, provide some thoughts on possible future directions for integrative approaches in this research area.
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THE CACTUS-MICROORGANISM-DROSOPHILA MODEL SYSTEM |
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SYNOPSISTHE CACTUS-MICROORGANISM...ALLELOCHEMISTRY OF COLUMNAR...SUBSTRATE UTILIZATIONCHEMICAL INTERACTIONS INVOLVING...THE EFFECT OF CACTUS...PROSPECTS FOR INTEGRATIVE...MOLECULAR BIOLOGY OF PLANT...EVOLUTION OF PLANT-ANIMAL...FUTURE DIRECTIONSReferences |
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With few exceptions, Drosophila are saphrophagous organisms, feeding on the microorganisms that reside in plant or fungal exudates. In the xeric environment of the Sonoran Desert, only the succulent cacti contain enough water to provide an adequate larval substrate for Drosophila. The association of species of Drosophila found in the Sonoran Desert with cacti was made back in the early 1940s, when several of the species were first described (Patterson and Crow, 1940
; Patterson and Wheeler, 1942). The specific relationships between fly species and cactus species and the ecology of the system in general were further characterized in the 1960s through the work of Bill Heed (and his students and colleagues) at the University of Arizona (see Fogleman, 1990 for a published biography of William Heed).
The major components of the system include 5 species of columnar cacti, and 4 species of cactophilic Drosophila. All stages of the life cycle of these flies are associated with necrosis of the stems of the cacti (Heed, 1978). Physical injury, age, and general health of the plant can all contribute to the formation of a "rot pocket" in a cactus stem. The cactus tissue then provides a substrate for the growth of bacteria and yeasts, which represent the microorganismic components of the model system. The bacterial community is diverse with some cactus-specific forms (Foster and Fogleman, 1993), while the yeasts are relatively limited in number (about a dozen species) and almost all are cactus specific (Starmer et al., 1990). The fact that the columnar cacti are large plants and necrotic stems are relatively obvious substrates has made the study of the chemical ecology of these desert drosophilids easier compared to most other Drosophila species, whose substrates are far more cryptic.
The Sonoran Desert occupies a considerable portion of northwestern mainland Mexico and the Baja Peninsula (Fig. 1). The geographical distributions of several of the cacti make the separation of the desert into mainland and Baja portions relevant to the ecology of the drosophilids. Table 1 presents the insect-host plant relationships between cacti and Drosophila species in the model system. Three of the four endemic drosophilids exhibit host plant shifts between the Baja Peninsula and the mainland. In two of the cases, it is due to the geographic distributions of saguaro and cardón, which are almost mutually exclusive. The third case (D. mojavensis) is due to the distribution of the preferred host plant, agria, which is essentially limited to the Baja Peninsula. Drosophila mettleri is an unusual species in that it utilizes soaked-soil as a breeding substrate (Heed, 1977), and, since saguaro and cardón are the larger cacti (with a greater potential for making soaked soil substrate), this species mirrors the host plant shift of D. nigrospiracula. The four Drosophila species are sympatric over most of the desert, and yet, extensive collecting and rearing records show that there is very little, if any, overlap in substrate usage (Fellows and Heed, 1972). Thus, in any given area, there is essentially a one-to-one relationship between an individual species of cactophilic Drosophila and the species of columnar cacti that serves as its natural substrate. Toxicity, nutritional deficiency, and competition with resident species are the primary causal factors in their host plant separation. The first two of these factors represent chemical interactions between the cacti and the insects.
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ALLELOCHEMISTRY OF COLUMNAR CACTI |
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Although many investigators have contributed to our knowledge of the chemistry of columnar cacti, two individuals stand out: Carl Djerassi and Henry Kircher. Djerassi's work in the 1950s and 60s includes some of the original chemical descriptions of cactus alkaloids and triterpenes (Djerassi and Lippman, 1955
; Djerassi et al., 1953a, b, 1954a, b, 1955, 1957, 1958, 1962). Kircher was a collaborator of Bill Heed and was specifically involved in the chemistry of the cactus-microorganism-Drosophila model system from its inception in the early 1960s (Kircher, 1977, 1980, 1982 and references therein; Kircher and Bird, 1982). The allelochemicals that are salient to the model system are given in Table 2. Examples of the structures of various classes of allelochemicals are shown in Figure 2.
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The columnar cacti contain some representative allelochemicals that are fairly common in insect-host plant relationships. For example, terpenoids (e.g., triterpenes) are generally known to be feeding deterrents (Brattsten, 1986
) and isoquinoline alkaloids (e.g., nicotine) are generally known to interfere with neurotransmission (Berenbaum, 1986). Thus, the triterpene glycosides of agria and organ pipe (Kircher, 1977 and 1980) and the isoquinoline alkaloids of saguaro, cardón, and senita (Djersassi et al., 1962; Brown et al., 1972) cannot be considered particularly unusual. The same cannot be said of the fatty acids and sterols in the columnar cacti. The fatty acids of most higher plants have an even number of carbon atoms and chains that are 14–22 carbon atoms long; with C16 and C18 as the most abundant forms (Fogleman and Kircher, 1986). The fatty acids of agria and organ pipe are primarily capric (C10) and lauric (C12) acids, and these chain lengths are rare among vegetable oils. The lipids of these two cacti are also unusual in that they contain atypical sterols. Normal sterols for higher plants (i.e., campesterol, sitosterol, and stigmasterol) have a single hydroxyl group, but the sterols of agria and organ pipe are primarily 3ß, 6
-dihydroxysterols (Kircher and Bird, 1982). Senita cactus also has unusual sterols due to putative interruptions in the pathways of sterol biosynthesis leading to the accumulation of intermediate forms (Campbell and Kircher, 1980). As might be expected in columnar cacti, these allelochemicals are concentrated in the photosynthetic layer of the stem which is located directly under the skin of the cactus. In this region, the concentrations can be significantly higher than those given in Table 2 which are averages for fresh tissue. However, from casual observations, it appears that after the necrosis has progressed beyond the initial stages, the action of insect larvae (and possibly other factors) serve to homogenize the necrotic tissue.
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SUBSTRATE UTILIZATION |
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The alkaloids, medium chain fatty acids, sterol diols, and triterpene glycosides have all been shown to have toxic effects on various species of the cactophilic Drosophila in such a manner as to partially explain the patterns of host plant utilization (reviewed in Fogleman and Heed, 1989
). Reported in a large number of separate experiments, the toxic effects of these compounds were typically characterized by extracting the compound of interest from fresh tissue, adding the compound to otherwise innocuous medium in ecologically realistic concentrations, and measuring the effect on larval viability, larval development time, adult longevity, and/or fecundity. Often the effects are drastic, suggesting that substrate mistakes made in nature would result in death of the insect.
The classification of senita sterols as allelochemicals is due to the unusual and absolute nutritional requirement of the resident species, D. pachea, for 
7-sterols. Two of the sterols in senita, 7-cholestenol and 7-campestenol, fulfill this unique sterol requirement, and are not found in any other cactus in the model system (Heed and Kircher, 1965). Thus, D. pachea is restricted to utilizing necrotic senita because all the other cacti are nutritionally deficient.
Utilization of senita as a feeding substrate, however, is only possible because D. pachea is able to tolerate the very high concentrations (up to 23% dry weight) of toxic isoquinoline alkaloids (lophocereine and pilocereine) present in senita tissue (Kircher et al., 1967). Similarly, the association of D. nigrospiracula with saguaro and cardón cacti is ultimately dependent on the ability of this species to resist the relatively low, yet demonstratively toxic, levels of secondary metabolites (isoquinoline alkaloids at up to 1.7% dry weight) found in these plants. Conversely, the high concentrations of alkaloids (in senita) and medium chain fatty acids, sterol diols and triterpene glycosides (in agria and organ pipe) exclude D. nigrospiracula from these substrates (Kircher et al., 1967; Fogleman et al., 1986). Still another desert species, D. mettleri, prefers to feed and breed in rot exudate-soaked soils at the base of saguaro and cardón cacti but can only do so because it is resistant to alkaloid concentrations that may be up to twenty-five fold greater than that in fresh cactus tissue (Fogleman et al., 1982; Meyer and Fogleman, 1987). Finally, D. mojavensis, is the only moderately polyphagous desert drosophilid owing to its resistance to the medium-chain fatty acids, sterol diols and high levels of triterpene glycosides found in organ pipe and agria cacti (its primary host plants) and the isoquinoline alkaloids present in the less frequently utilized saguaro and cardón cacti.
In summary, while insect behavior does play a role in several of the cactus species-Drosophila species associations, cactus allelochemistry plays the predominant role in determining the patterns of host utilization. The ultimate determinant of whether or not a particular Sonoran drosophilid is capable of utilizing a specific cactus is the ability of each fly species to circumvent those cactus allelochemicals which are toxic to nonresident species.
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CHEMICAL INTERACTIONS INVOLVING MICROORGANISMS |
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Aside from their obvious nutritional value, microorganisms are important components of the model system for several reasons. First, through their deliquescence of the tissue, they create a physically hospitable environment for larvae and adults. Second, by fermenting the carbohydrates that are present in the cactus, they produce volatile compounds that attract the Drosophila to the developing necrosis. The fact that drosophilids are attracted to alcohols has been known for almost a century (Barrows, 1907
). An analysis of the carbohydrates in cactus tissue that are potentially available for microorganism growth and metabolism demonstrates significant differences between cactus species for both types and concentrations of sugars (Fogleman and Abril, 1990). Due to their high triterpene glycoside content, agria and organ pipe cacti have the highest sugar content. Evidence that the sugars of these glycosides are consumed during the rotting process has been presented by Kircher (1982). Saguaro and senita have the lowest sugar content. As a direct result, the volatile patterns, including alcohols, esters, and volatile fatty acids (e.g., acetic acid), which are typical of necrotic tissue of each of the cactus species, are significantly different (Fogleman and Abril, 1990). A relatively limited number of experiments, primarily with D. mojavensis, have, however, provided convincing evidence supporting the contention that the host specificity of the desert Drosophila is based on the response to a cactus species-specific volatile pattern (Fogleman, 1982; Fogleman and Abril, 1990; Newby and Etges, 1998). Upon entering a volatile plume in nature, it appears that cactophilic Drosophila make a choice to respond or not to respond (and keep flying through it). If they find the volatile pattern to be sufficiently attractive, they turn in the plume to equalize the stimulation of their antennae and fly up the concentration gradient to the point source. Since collection records show that the desert Drosophila seldom make substrate mistakes (i.e., they are not found on or around cactus necroses other than those of their normal host plant), it is reasonable to suggest that they only respond to the volatile pattern that is indicative of the proper cactus species. Thus, microorganisms, through their natural metabolic processes, play an integral role in host plant response by the drosophilids of the model system.
Finally, microorganisms play a role in the chemical interactions between cactus and Drosophila via their direct metabolic activity on the cactus allelochemicals. In some cases, they reduce the toxicity of the necrosis by utilizing cactus allelochemicals as energy sources. For example, the medium-chain fatty acids in organ pipe and the volatiles, 2-propanol and acetone (which are toxic in moderate to high concentration), in both agria and organ pipe can be utilized by certain cactus microbes and are thereby reduced in concentration, which makes the substrate less toxic (Starmer and Aberdeen, 1990). In other cases, their metabolic activity can release toxic allelochemicals from complexed forms that are not toxic. For example, the medium chain fatty acids and sterol diols of agria and organ pipe are normally bound together as esters and are not toxic in this form (Fogleman et al., 1986). Bacterial degradation of the esterified complex releases the sub-units and increases the toxicity of the substrate (Starmer and Aberdeen, 1990). At best, one is left with the conclusions that the role of microorganisms in the model system is dynamic.
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THE EFFECT OF CACTUS CHEMISTRY ON MATING BEHAVIOR IN D. MOJAVENSIS |
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SYNOPSISTHE CACTUS-MICROORGANISM...ALLELOCHEMISTRY OF COLUMNAR...SUBSTRATE UTILIZATIONCHEMICAL INTERACTIONS INVOLVING...THE EFFECT OF CACTUS...PROSPECTS FOR INTEGRATIVE...MOLECULAR BIOLOGY OF PLANT...EVOLUTION OF PLANT-ANIMAL...FUTURE DIRECTIONSReferences |
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Premating reproductive isolation among Baja and mainland populations of D. mojavensis is an interesting and well-studied phenomenon (Wasserman and Koepfer, 1977
; Zouros and D'Entremont, 1980). In choice tests, mainland females discriminate against males from the Baja Peninsula, and this situation has been viewed as a case of incipient speciation. Drosophila are known to utilize visual, auditory, and chemical cues during courtship, and, among the latter, epicuticular hydrocarbons of adults have been shown to be active contact pheromones (Antony and Jallon, 1982). All four cactophilic Drosophila have higher relative proportions of long chain epicuticular hydrocarbons than do drosophilids that are not adapted to the desert (Markow and Toolson, 1990), and this may be related to desiccation tolerance. Even more interesting, however, is the fact that the relative abundance of C35:2 and C37:2 alkadienes in the epicuticular hydrocarbon profile is correlated with male mating success in D. mojavensis. While this story is tantilizingly incomplete, studies have shown that epicuticular hydrocarbon expression and facets of mating behavior in D. mojavensis are influenced by larval rearing substrate (Etges, 1992 and 1998; Brazner and Etges, 1993; Stennett and Etges, 1997). Since mainland and Baja populations utilize different substrates (which differ chemically), the chemical interactions between the cacti and drosophilids has been recently extended to include a direct effect on mating behavior. Further investigation will help resolve the causes of incipient speciation among populations of this species. |
PROSPECTS FOR INTEGRATIVE APPROACHES TO PLANT-ANIMAL INTERACTIONS: RESEARCH AT THE INTERFACE OF ECOLOGY, BIOCHEMISTRY AND MOLECULAR GENETICS |
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Traditional ecological approaches to the study of plant-animal interactions have been largely observational. Accordingly, more than a century's worth of meticulous field research on the behavior and interactions among organisms in their natural ecological niches has provided a wealth of information especially on the host plant associations of thousands of insect species. In fact, few ecological phenomena have been the focus of as much scientific interest as the evolution of highly specialized feeding behaviors that enable many insect species to utilize host plants which contain allelochemicals that are toxic to nonresident species. This remarkable degree of specialization, however, almost always extends beyond simple behavioral adaptations to include changes in an organism's physiology and/or biochemistry. These changes may render target tissues insensitive to the action of a given toxin, reduce a toxin's residency time in an organisms digestive tract or enable the direct metabolic inactivation and elimination of toxin molecules. Thus, while it has long been axiomatic that ecologists be intimately familiar with an organism's habitat, a comprehensive understanding of ecological interactions can really only be gained through a multidisciplinary approach. This must be an approach that fully integrates ecosystem level observations and population genetics with underlying molecular and biochemical mechanisms. Properly employed, this approach has yielded some of the most elegantly complete chapters in modern ecology.
Among the most rapidly growing sectors of interdisciplinary ecological research is molecular ecology, a discipline that that really only coalesced a decade ago, but has since been growing at an exponential rate. This growth is fueled, in part, by the relative ease with which the continuing development of new molecular and analytical techniques has made it possible to address an ever-broader range of organismal questions. Certainly, chemical ecologists and ecological biochemists have long recognized the important role that chemical substances and secondary metabolites play as mediators of biological interactions. Similarly, molecular ecologists are beginning to take advantage of the depth of understanding that can be attained by inclusion of a molecular genetic perspective in addressing such areas as the molecular basis of adaptation, the evolutionary dynamics of quantitative traits, and the role of the gene-environment interface in organismal-level interactions.
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MOLECULAR BIOLOGY OF PLANT-ANIMAL INTERACTIONS |
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Numerous strategies have evolved which enable insects to tolerate the toxicity of many non-nutrient metabolites present in the plants they eat. These can be broadly grouped into three categories: behavioral adaptations, modified physiological pathways and biochemical resistance. The simplest of these, behavioral avoidance strategies, generally involve the spatial and/or temporal isolation of an insect from host plant toxins (Feeney, 1970
; Hargrove and Crossley, 1985). Such strategies, however, often restrict herbivores to nutrient-poor food resources or require the expenditure of valuable time and energy on elaborate foraging behaviors (Schultz, 1983). Alternatively, physiological modifications, which may include accelerated excretion rates, sequestration of toxic allelochemicals and target site insensitivity, enable phytophagous insects to ingest toxic but nutrient-rich plant tissues while minimizing the waste of energy associated with avoidance strategies. As important as behavioral and physiological alterations are as a means of circumventing toxins, direct metabolic detoxication is arguably the strategy which is the most effective and most commonly employed by phytophagous insects (Brattsten, 1988; Gonzalez and Nebert, 1990). Metabolism of the majority of natural and synthetic xenobiotics generally involves at least one of four major categories of enzymes, namely, reductases, hydrolases, transferases and oxidases, especially the polysubstrate monooxygenases.
Better known as cytochrome P450s, these monooxygenase enzymes comprise an ancient superfamily of mostly microsomal heme-thiolate proteins named for their unique spectral absorbance peak at 450 nm (Omura and Sato, 1964). Cytochrome P450s are remarkable for their astounding diversity, both in terms of the reactions that they catalyze and the range of chemically dissimilar endogenous (steroids and prostaglandins) and exogenous (pesticides and plant allelochemicals) substrates upon which they act. Multiple cytochrome P450 genes are typically expressed simultaneously and various isoforms have been shown to be involved in oxidative, peroxidative and reductive metabolism (Nelson et al., 1993).
Several lines of evidence point to the involvement of cytochome P450s in the resistance of desert Drosophila to the isoquinoline alkaloids present in most of the columnar cacti that serve as their host plants (Frank and Fogleman, 1992). First, exposure to alkaloid-containing cactus tissue results in significantly increased total P450 content in desert Drosophila exposed to ecologically relevant concentrations of cactus alkaloids. Second, larval survival through eclosion on cactus substrates is significantly reduced by addition of the P450 inhibitor, piperonyl butoxide (PBO). Similarly, addition of PBO inhibits in vitro alkaloid metabolism by isolated microsomes. Conversely, in vitro alkaloid metabolism by isolated microsomes can be significantly induced by exposure of larvae to either alkaloid-containing cactus tissue or phenobarbital, a broad-spectrum transcriptional inducer of cytochrome P450 genes.
While there is clear evidence pointing to the central role of cytochrome P450-mediated metabolism in the insect-host plant relationships of the cactus-Drosophila-microorganism model system, much remains to be learned. For example, little is known about the mechanisms which underlie the evolution and maintenance of these relationships. Is alkaloid metabolism a function of the action of individual cytochrome P450 isoforms or is it the product of the collective activity of multiple P450s? Is the observed induction of alkaloid metabolizing activity a result of increased mRNA transcription or possibly post-translational modification of the protein itself? Is alkaloid metabolism carried out by the same cytochrome P450 isoform in each cactophilic drosophilid or have multiple forms evolved by convergent evolution? Here, the cactus- microorganism-Drosophila model system provides an excellent context in which to develop a comprehensive understanding of an insect-host plant relationship using a combination of phylogenetic and molecular biological approaches.
Two major concerns arise when investigating the molecular biology and evolution of multigene families like the cytochrome P450s. First, is genetic multiplicity which presents a hurdle to the accurate assignment of orthologous relationships. Such assignment is an essential first step for any phylogenetic analysis. Cytochrome P450 multiplicity in insects has already been well demonstrated through the isolation of part or all of 14 transcriptionally expressed cytochrome P450s from the Mediterranean fruit fly (Danielson et al., 1999). The second major area of concern centers on the ability to accurately assess differences in the activity of individual isozymes, particularly those which may have overlapping catalytic activities. Evidence of cytochrome P450 involvement in a given chemical reaction has traditionally been based on the results of in vitro metabolism assays where isolated microsomes are used to demonstrate NADPH-dependent substrate turnover, the hallmark of P450 involvement. Assessment of isoenzymic substrate specificity is also complicated by genetic multiplicity since metabolism may reflect the activity of one or more gene products.
Considering the difficulties typically associated with purification of individual P450 isoforms, while preserving catalytic activity, the use of a molecular approach to clone cDNA sequences offers several significant advantages. First and foremost, is the ability to isolate multiple target genes in a fraction of the time required for protein purification (Frohman et al., 1988; Zhao and Joho, 1990; Wilkie and Simon, 1991). Once isolated, even partial cDNAs can be used in Northern analysis to assess basal gene expression levels as well as transcriptional induction by xenobiotics. Additionally, the ability to obtain multiple P450 sequences from each of several drosophilid species makes it possible to expedite the identification of orthologous relationships. To this end, a polymerase chain reaction (PCR) based cloning strategy has been used to isolate more than 100 cytochrome P450 cDNAs from four species of alkaloid-resistant cactophilic Drosophila and their nine closest phylogenetic relatives. Based on sequence similarity, the vast majority of these sequences (89%) were categorized as members of the CYP4, CYP6 or CYP9 families, i.e., cytochrome P450s which have already been implicated in xenobiotic resistance in other insect species.
Based on studies of monooxygenase-mediated toxin resistance in other insects and the previously demonstrated induction of in vitro alkaloid metabolizing activity (Frank and Fogleman, 1992), cytochrome P450s likely to be involved in alkaloid detoxication should display transcriptional induction on northern blots. As expected, Northern screens of the PCR-amplified cytochrome P450s from cactophilic Drosophila clearly show that a subset of P450 genes are transcriptionally responsive to one or more cactus alkaloids. The level of transcriptional responsiveness can be broadly classified as either "no significant induction" (i.e., less than a two-fold difference relative to uninduced controls), "moderate induction" (i.e., showing an increase of three-ten fold over controls) or "strong induction" (i.e., greater than a ten-fold increase relative to uninduced controls). Drosophila pachea was excluded from studies of transcriptional responsiveness because of the difficulty of obtaining "baseline data" from organisms raised in the absence of cactus tissue. This stems from the absolute dietary dependence of D. pachea on 7 sterols which necessitates inclusion of senita cactus tissue (and, therefore, alkaloids) in all culture media.
The transcriptional induction of desert drosophilid cytochrome P450s by the broad-spectrum inducer, phenobarbital, provided the first evidence that the underlying patterns of transcriptional regulation might directly account for many of the insect-host plant associations reported by field ecologists. For example, all of the P450s cloned from D. nigrospiracula displayed moderate transcriptional induction with phenobarbital. However, no gene was induced over nine fold in this species. With D. mojavensis, there were three genes that were unresponsive to phenobarbital, two genes that were moderately induced and only one which showed greater than ten-fold induction. In D. mettleri, two of the P450s were not inducible by phenobarbital, four genes were moderately induced and eight displayed strong induction. Thus, there seems to be a general trend toward increasing responsiveness to phenobarbital with D. nigrospiracula P450 genes being the least responsive and those of D. mettleri being the most responsive. This trend is also seen at the organismal level where total P450 content and in vitro alkaloid-metabolizing capacity in the desert Drosophila both show a trend toward increasing responsiveness to phenobarbital with the smallest increases being seen in D. nigrospiracula and the largest in D. mettleri.
More ecologically relevant, however, are the levels and patterns of cytochrome P450 induction seen in response to exposure to isoquinoline alkaloids naturally present in host columnar cacti. Here, only one cytochrome P450 gene from D. nigrospiracula showed any induction while four genes from D. mojavensis and seven from D. mettleri were moderately induced following exposure to saguaro alkaloids. In response to senita alkaloids, moderate induction was seen for three D. mojavensis and seven D. mettleri genes. Interestingly, many of the cytochrome P450 genes that are only moderately responsive to cactus xenobiotics are usually cross-inducible, responding to both saguaro and senita alkaloids. This is in agreement with the results of in vitro alkaloid metabolism assays showing that saguaro alkaloid metabolism is induced to a similar degree by senita and saguaro alkaloids at equivalent concentrations (Frank and Fogleman, 1992; Danielson et al., 1994).
Strong induction by saguaro alkaloids was noted for only one D. mettleri and one D. mojavensis cytochrome P450. Likewise, only one gene from D. mettleri was strongly induced following exposure to senita alkaloids. Interestingly, the most strongly induced genes, seem to respond to a specific subset of cactus allelochemicals (i.e., saguaro alkaloids versus senita alkaloids) but were also even more highly induced by phenobarbital. This corroborates the results of in vitro alkaloid metabolism studies which have demonstrated that saguaro alkaloid metabolism is more strongly induced by PB than by any of the cactus alkaloids.
Taken together, these patterns of transcriptional induction are reflected in the ecology of these organisms. On the basis of both in vivo larval viability and in vitro alkaloid metabolism analyses, D. nigrospiracula was found to be the least alkaloid-tolerant of the Sonoran Desert drosophilids. Appearing to account for this at the molecular level is the observation that the P450 genes of D. nigrospiracula are, for the most part, transcriptionally unresponsive to saguaro alkaloids. On the other hand, both D. mojavensis and D. mettleri are known to be much more tolerant of these same alkaloids and both of these species possess at least one strongly responsive and several moderately responsive P450 sequences. As for senita alkaloids, only D. mettleri has a single gene that is strongly inducible. Again, this is directly reflected at the organismal level since, of the species studied, only D. mettleri is capable of utilizing senita as a host plant. These strongly but more narrowly responsive genes may be the best candidates to account for the pattern of insect host cactus associations observed by field biologists working on the Sonoran Desert system. This is not to suggest that the moderately inducible genes play no role in alkaloid resistance. It merely appears unlikely that they alone can produce in vivo tolerance. For example, D. mojavensis possesses multiple P450s that are moderately responsive to senita alkaloids at the ecologically relevant concentrations found in senita tissue. Even so, D. mojavensis cannot utilize senita as a host plant (Fogleman and Abril, 1990). Here again, the fusion of molecular, biochemical and ecological data provide greater insight than could be obtained using any single approach in isolation.
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EVOLUTION OF PLANT-ANIMAL INTERACTIONS |
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SYNOPSISTHE CACTUS-MICROORGANISM...ALLELOCHEMISTRY OF COLUMNAR...SUBSTRATE UTILIZATIONCHEMICAL INTERACTIONS INVOLVING...THE EFFECT OF CACTUS...PROSPECTS FOR INTEGRATIVE...MOLECULAR BIOLOGY OF PLANT...EVOLUTION OF PLANT-ANIMAL...FUTURE DIRECTIONSReferences |
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Evolution of insect host plant interactions is still another area where the advantages of a multidisciplinary approach that combines natural history with molecular genetics is self-evident. Addressing evolutionary questions within the context of a multigene family, however, necessitates the resolution of the orthologous relationships among the various members. Orthologous genes arise only as products of speciation events and thus divergence between orthologous sequences should be a function of the evolutionary history of a species. By contrast, paralogous genes are those that arise only as a result of gene duplication events. Thus, all members of a multigene family within any genome are paralogously related to each other with the divergence between paralogous sequences being a reflection of the evolutionary history of the gene family rather than that of the species.
Fortunately, it is possible, with reasonable confidence, to identify orthologous relationships among many of the Drosophila cytochrome P450 cDNAs that have been isolated from the Sonoran Desert endemics. This has been facilitated by the fact that the partial cDNAs which comprise most of the desert system sequence database include both a region of rigorous conservation immediately adjacent to the heme-binding peptide and a region in which sequence conservation decreases steadily as one moves toward the stop codon. This provides enough conservation for alignment purposes and enough variability to differentiate orthologs in closely related species.
Phylogenetic analyses of the cytochrome P450 cDNA sequences identified as likely to be responsible for cactus alkaloid resistance in Sonoran Desert Drosophila reveal some interesting possible trends with respect to the fundamental mechanisms underlying the development of P450-mediated biochemical resistance and the evolution of multigene families in general. First, none of the most allelochemical-responsive cytochrome P450s are orthologs of each other. For example, the two genes which are most strongly responsive to saguaro alkaloid exposure (designated CYP4D10 and DU4mb) are not orthologously related, and are not even members of the same cytochrome P450 family—CYP4D10 being in the CYP4 family and DU4mb being in the CYP6 family. The only strongly senita alkaloid-inducible gene, CYP28A1, is a member of still another phylogenetically distinct cytochrome P450 family (Danielson et al., 1997).
A wealth of data on the biogeography and natural history of the Sonoran Desert Drosophila and their closest non-desert relatives, suggest that each species of cactophilic Drosophila has evolved independently into its current xeric niche and thus its present host plant association. When combined with biochemical and molecular phylogenetic data, it appears that this evolution has been achieved through the independent recruitment of other than closely related cytochrome P450 genes. More importantly, however, the application of a fully integrative multidisciplinary approach to an organismally relevant issue in modern ecology has demonstrated that phenomena at the molecular genetic level recapitulate patterns and phenomena at the organismal level.
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FUTURE DIRECTIONS |
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SYNOPSISTHE CACTUS-MICROORGANISM...ALLELOCHEMISTRY OF COLUMNAR...SUBSTRATE UTILIZATIONCHEMICAL INTERACTIONS INVOLVING...THE EFFECT OF CACTUS...PROSPECTS FOR INTEGRATIVE...MOLECULAR BIOLOGY OF PLANT...EVOLUTION OF PLANT-ANIMAL...FUTURE DIRECTIONSReferences |
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While the application of biochemistry and molecular biology to ecosystem-level questions can yield elegant results in the field of modern ecology, much remains to be learned. For example, what role might cytochrome P450s play in resistance to other, non-alkaloid cactus alleochemicals including the sterol diols and triterpene glycosides which are abundant in the tissues of the agria and organ pipe cacti that are the preferred host plant of D. mojavensis? What are the relative contributions of the structural and regulatory regions of cytochrome P450 genes to monooxygenase-mediated host plant utilization? What role do trans-acting regulatory elements fill? Although research to date has focussed on the role of insect biochemistry in host plant utilization, there is an abundance of corresponding questions that could be asked with regard to the biosynthesis of the alkaloids and other toxic allelochemicals present in cacti. Studies of other isoquinoline alkaloids like berberine which is produced in Coptis and Berberis species, has revealed that cytochrome P450s are involved in alkaloid biogenesis as well (Hashimoto and Yamada, 1994
; Kutchen, 1995). The implications and applicability of what is learned from studies of interactions among plants and animals in natural environments cannot be overstated. Plant-animal interactions, in general, and cytochrome P450-associated xenobiotic resistance, in particular, are recurring themes in pest management efforts from agriculture through the control of arthropod vectors of disease. It is, therefore, essential to understand the mechanism by which insects evolve and regulate xenobiotic resistance from the organismal through the genetic level.
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ACKNOWLEDGMENTS |
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This work was supported by NSF grant IBN-9806888 to J.C.F. and P.B.D.
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1 From the Symposium An Integrative Approach to the Study of Terrestrial Plant-Animal Interactions presented at the annual Meeting of the Society for Comparative and Integrative Biology, 5–8 January 2000, at Atlanta, Georgia.
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