roles of peritrophic membranes in protecting herbivorous insects from ingested plant allelochemicals

86 BarbehennArchives of Insect Biochemistry and Physiology 47:86–99 (2001)

© 2001 Wiley-Liss, Inc.

Roles of Peritrophic Membranes in ProtectingHerbivorous Insects From Ingested Plant Allelochemicals

Raymond V. Barbehenn*

Department of Biology, University of Michigan, Ann Arbor

Four mechanisms by which peritrophic membranes (PMs) po-tentially protect herbivorous insects from ingested allelo-chemicals are reviewed: adsorption, ultrafiltration, polyanionexclusion, and the capacity of PMs to act as antioxidants. Mostof the research on the protective roles of PMs against ingestedallelochemicals has focused on their impermeability to tannins.Adsorption of tannins by the PMs in grasshoppers may limittheir permeability, but ultrafiltration of tannin complexes inthe caeca is an alternative explanation. Polyanion exclusiondoes not explain the impermeability of caterpillar PMs totannins (polyphenolate anions). Ultrafiltration remains themost likely mechanism by which tannins, and other testedallelochemicals, are retained in the endoperitrophic space. Al-though the pores in PMs are too large to impede the passageof most free allelochemicals, large allelochemical complexes areretained. Such complexes form in the gut fluid of caterpillarsbetween tannic acid, proteins, lipids, and polyvalent metal cat-ions, and also in the gut fluid of grasshoppers (Melanoplussanguinipes) between some amphiphilic allelochemicals (digi-toxin) and surfactant micelles. Further work is needed to ex-amine the role of PMs as antioxidants in vivo, such as theirpotential to bind catalytically-active metal ions. Arch. InsectBiochem. Physiol. 46:86–99, 2001. © 2001 Wiley-Liss, Inc.

Contract grant sponsor: NSF; Contract grant number: IBN-997483.

*Correspondence to: Raymond V. Barbehenn, Departmentof Biology, University of Michigan, Ann Arbor, MI 48109.E-mail: [email protected]

Received 31 October 2000; Accepted in revised form 12 Feb-ruary 2001

INTRODUCTION

Herbivorous insects avoid the potentially toxiceffects of ingested plant allelochemicals by fourgeneral mechanisms: biochemical detoxification,rapid excretion, storage excretion, and non-absorp-tion. Biochemical detoxification mechanisms, es-pecially those present in midgut tissues, have beenmost thoroughly studied. These include chemicaltransformations catalyzed by enzymes such as cy-tochrome P450 monooxygenases, esterases, andhydrolases (Lindroth, 1991). Less well understoodare mechanisms that reduce the absorption of in-gested allelochemicals, and thereby reduce the sub-sequent need for detoxification.

The passive absorption of a chemical throughthe gut epithelium can generally be predicted onthe basis of properties that affect the solubilityof the chemical in the phospholipid membranes(e.g., hydrophobicity, size, charge) (Shah andGuthrie, 1970; Vander et al., 1980). The physico-

PMs as Barriers to Ingested Allelochemicals 87

chemical properties that enable a compound todiffuse across cell membranes would not be ex-pected to resemble those limiting permeabilityacross peritrophic membranes (PMs). PMs formthin sheaths (or “peritrophic envelopes”) aroundthe contents of the midgut lumen. They are ex-tracellular matrices composed of a meshwork ofchitin microfibrils, attached to which is a gel-likemixture of proteins, glycoproteins, and proteogly-cans. In cases involving allelochemicals or allelo-chemical complexes that do not readily permeatePMs, PMs potentially function as a first line ofdefense, reducing or eliminating the absorptionof certain allelochemicals. Examples of known andpotential cases in which PMs limit allelochemicalabsorption are presented in Table 1. Non-absorp-

TABLE 1. Non-Absorption of Plant Allelochemicals Ingested by Herbivorous Insects: Potential Role ofPeritrophic Membranes (PMs)*

PermeabilityChemical class Chemical (MW) Insect species (Order) of PMs Reference

Chromene Encecalin (232) Trirhabda geminata (C) Unknown Kunze et al. (1996)Melanoplus sanguinipes (O) Unknown Berenbaum and Isman (1989)

Thiophene α-Terthienyl (248) Melanoplus sanguinipes (O) Unknown Berenbaum and Isman (1989)Sesquiterpene Parthenin and Melanoplus sanguinipes (O) Unknown Isman (1985)

Lactone others (262–322)Alkaloid Cocaine (303) Eloria noyesi (L) Unknown Blum et al. (1981)

SenecionineN-oxide (350) Melanoplus sanguinipes (O) Unknown Ehmke et al. (1989)

Phenol Chlorogenic acid Helicoverpa zea (L) Low Barbehenn (unpublished data),(354) Isman and Duffey (1983)

Manduca sexta (L) Low Barbehenn (unpublished data)Cardenolide Ouabain (585) Schistocerca gregaria (O) Unknown Scudder and Meredith (1982)

Digitoxin (765) Manduca sexta (L) Low Barbehenn (unpublished data)Schistocerca gregaria (O) Unknown Isman and Duffey (1983)Melanoplus sanguinipes (O) Low Barbehenn (1999), Smirle and

Isman (1992)Flavonoid Rutin (610) Helicoverpa zea (L) Low Barbehenn (unpublished data),

Isman and Duffey (1983)Polyphenol Tannic acid Orgyia leucostigma (L) Impermeable Barbehenn and Martin (1992)

(789–1027) Malacosoma disstria (L) Impermeable Barbehenn and Martin (1994)Helicoverpa zea (L) Impermeable Barbehenn (unpublished data)Papilio glaucus (L) Impermeable Barbehenn (unpublished data)Schistocerca gregaria (O) Impermeable Bernays et al. (1980)Anacridium melanorhodon (O) Impermeable Bernays et al. (1980)Melanoplus sanguinipes (O) Imperm.-Perma Barbehenn et al. (1996),

Bernays et al. (1980)Phoetaliotes nebrascensis (O) Imperm.-Perm.a Barbehenn et al. (1996)

Quebracho Locusta migratoria (O) Impermeable Bernays et al. (1981)(1000–3000) Schistocerca gregaria (O) Impermeable Bernays et al. (1981)

Chortoicetes terminifera (O) Impermeable Bernays et al. (1981)Zonocerus variegatus (O) Impermeable Bernays et al. (1981)

*C = Coleoptera; L = Lepidoptera; O = Orthoptera. “Low” permeability is arbitrarily defined as less than 5% of the intro-duced chemical permeating the PE in vivo or in in situ assays.aPMs in M. sanguinipes and P. nebrascensis were impermeable to pentagalloyl glucose and hexagalloyl glucose, respec-tively. See Figure 1 and Barbehenn et al. (1996) for permeabilities of other tannins.

tion in cases labeled “unknown” in Table 1 couldresult either from the PMs, the midgut epithe-lium, or both structures. In these cases, the pos-sibility that PMs are an effective barrier toallelochemical absorption has not been examined.

Work on PMs in herbivorous insects has fo-cused primarily on grasshoppers (Orthoptera) andcaterpillars (Lepidoptera). PMs in grasshoppersare formed sequentially by the midgut epithelium,producing multiple layers (Type 1 PMs). Grass-hoppers, such as Locusta migratoria, form a newPM every 15 min, maintaining an average of 16PMs while feeding (Baines, 1978). The formationof PMs in caterpillars varies between Type 1 andType 2, the latter of which is characterized by asingle PM secreted by the anterior midgut (Pe-

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ters, 1992; Ryerse et al., 1992). Orgyia pseudo-tsugata caterpillars maintain three PMs (Brandtet al., 1978). The few herbivorous beetles thathave been examined, such as Diabrotica undecim-punctata, form Type 1 PMs (Peters, 1992; Ryerseet al., 1994).

Most of the work on the protective roles ofPMs has focused on their defense against ingestedtannins. The impermeability of PMs in tannin-tolerant insects was commonly believed to be akey mechanism that allows them to consumetannins without incurring damage to their mid-gut epithelia. Feeny (1970) first suggested thatthe PMs in a caterpillar (Operophtera brumata)retain ingested tannins in the endoperitrophicspace: no staining from condensed or hydrolyz-able tannins was observed on the surface of themidgut epithelium in histological sections fromthe guts of larvae, fed oak leaves. Based on ex-tensive microscopical and chemical studies ongrasshoppers, Bernays and colleagues (Bernays,1978; Bernays et al., 1980, 1981) concluded thatthe PMs of graminivorous species are permeableto hydrolyzable tannins (but impermeable to con-densed tannins), whereas PMs in polyphagousspecies are impermeable to condensed and hydro-lyzable tannins. Microscopical studies made bySteinly and Berenbaum (1985), showed that tan-nin-sensitive Papilio polyxenes caterpillars de-velop midgut lesions after ingesting a mixture ofcondensed and hydrolyzable tulip tree tannins.By comparison, the tannin-tolerant larvae ofPapilio glaucus developed few, if any, lesions af-ter ingesting tulip tree tannins (Steinly andBerenbaum, 1985). These results are commonlyinterpreted as reflecting differences in the perme-abilities of PMs in these species, since tanninswere typically believed to exert their effects by a“non-specific precipitant action” on componentsof the epithelium (Bernays, 1978). Direct mea-surements of the permeability of PMs to tannicacid in several caterpillar species showed thatthey are impermeable (Table 1) (Barbehenn andMartin, 1992, 1994). However, similar studies ontwo polyphagous grasshopper species (Melanoplussanguinipes and Phoetaliotes nebrascensis) showedthat their PMs are permeable to some componentsof tannic acid (Fig. 1) (Barbehenn et al., 1996).Only a small amount of work has been done on

Fig. 1. Permeability of PMs to tannins in Melanoplussanguinipes (top) and Phoetaliotes nebrascensis (bottom)grasshoppers. Three components of tannic acid were mea-sured: tetragalloyl glucose (4GG), pentagalloyl glucose (5GG),and hexagalloyl glucose (6GG). Endoperitrophic tannin lev-els were measured 1.5–2 h after their introduction into thisspace in ex vivo gut preparations. Exoperitrophic tanninspermeated the PMs into an incubating solution through asmall opening cut through the midgut wall. Modified fromBarbehenn et al. (1996).

the permeability of PMs to allelochemicals otherthan tannins. Recent work suggests that PMs inM. sanguinipes and Manduca sexta reduce thepermeation of some amphiphilic allelochemicals,such as digitoxin (Table 1) (Barbehenn, 1999).


The primary question in this review is “Towhat extent and by what mechanisms do PMsprotect herbivorous insects from ingested allelo-chemicals?” The general belief has been thatPMs—structures that are permeable to a varietyof digestive enzymes and nutrients—would notbe expected to restrict the permeation of mostplant allelochemicals. The small amount of infor-mation that is currently available is consistentwith this idea. However, there are at least twogeneral types of allelochemicals that are knownto be retained to a significant extent by PMs, andan ability to predict other compounds that havelow permeability through PMs may be facilitatedbased on an understanding of the mechanismsthat limit PM permeability. Three potentialmechanisms for limiting the permeability of PMsare adsorption, ultrafiltration, and anion exclu-sion. Recently, it has also been hypothesized thatPMs function as antioxidant defenses in the mid-gut lumen (Felton and Summers, 1995).

PROTECTIVE MECHANISMSAdsorption

Adsorption processes include a variety ofnon-covalent associations between molecules andsurfaces. Adsorption of ingested hydrolyzableand condensed tannins has been observed on thePMs of grasshoppers (Bernays and Chamberlain1980; Bernays et al., 1981). In Schistocercagregaria (polyphagous), the PMs contained anaverage of 49% of their dry weight in tannic acid(hydrolyzable tannin) when insects fed on foli-age treated with 16 or 20% (dry weight) tannin.This doubling in weight of the PMs is all themore striking because three-fourths of the in-gested tannic acid was hydrolyzed in the gut lu-men (Bernays, 1978). In S. gregaria, the mostthoroughly studied species, adsorption may befacilitated by the collection of tannins in numer-ous small caecal pockets (Bernays, 1981). Be-cause of the rapid flux of water through thecaeca, bulk flow of particles and tannins into thecaeca results in their collection in this region.Large amounts of mucopolysaccharides (proteo-glycans) were identified in the pockets of the cae-cal PMs in S. gregaria, and Bernays (1981)hypothesized that these substances might be re-sponsible for tannin adsorption.

Large amounts of condensed tannin werealso associated with the PMs of both polyphagousand graminivorous grasshoppers (Bernays, 1978;Bernays et al., 1981), suggesting that adsorptionis widespread among grasshoppers, irrespectiveof the presence of caecal pockets (Bernays andChapman, 2000). However, tannic acid was notextracted from the PMs of the polyphagous grass-hopper M. sanguinipes in any significant amount(Barbehenn et al., 1996). Nor was any significantadsorption of tannic acid observed on the PMs ofa polyphagous caterpillar (Orgyia leucostigma)(Barbehenn and Martin, 1992). Further work isneeded to determine whether tannins are adsorbedon PMs throughout the length of the guts of grass-hoppers, or whether the high degree of tannin “ad-sorption” in grasshopper PMs is the result of thesieving of tannin complexes in the caeca.

The adsorption of compounds other thantannins has not been examined in the PMs ofherbivorous insects. Although not an herbivor-ous insect, Aedes aegypti larvae egest DDT andDDE in their PMs (Abedi and Brown, 1961). Fi-nally, although not an allelochemical that isusually produced in leaves, lectins bind to siteson the endoperitrophic face of PMs, reducingthe permeability of PMs (Peters, 1992; Eise-mann et al., 1994).

Ultrafiltration

Permeability of PMs . PMs are often likenedto ultrafilters, membranes that limit the perme-ation of molecules and colloidal particles in thesize range of 1–100 nm. For PMs to effectivelylimit the permeation of free allelochemicals, poresizes in the matrix would have to be at the lowend of this size range. How big are the pores inthe PMs of herbivorous insects?

Dextrans have been widely used as sizemarkers in vivo in permeability studies of verte-brate extracellular matrices (e.g., Caulfield andFarquhar, 1974; Chang et al., 1975a). Methodsusing fluorescein isothiocyanate (FITC)-labeleddextrans have also been adapted for permeabilitystudies of PMs in grasshoppers and caterpillars(Barbehenn and Martin, 1995). This study showedthat as Orgyia caterpillars consumed larger sizesof FITC-dextrans, the flux of these markersthrough the PMs decreased logarithmically (Fig.2). The flux of dextrans decreased to cut-off points

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(0 flux) of 32.5 nm for O. leucostigma (Fig. 2),and 33.4 nm for O. pseudotsugata. A comparisonof the average sizes of dextrans ingested (endo-peritrophic) with those that diffused through PMs(exoperitrophic) of the two Orgyia species demon-strated that there is little restriction in the sizeof dextrans permeating PMs up to approximately20 nm (Fig. 3). The polydisperse FITC-dextranmarkers greater than this size were fractionated;only the smaller molecules were able to permeatethe PMs, producing an asymptotic relationshipthat underestimates (but roughly approximates)maximum pore size (e.g., 25.6 nm for O. leucos-tigma, and 21.3 nm for O. pseudotsugata) (Fig.3). When a single polydisperse marker (2,000 kDanominal M– w; 42 or 47 nm average diameter) wasingested by four species of caterpillars, the diam-eters of permeating dextrans ranged from 21–29nm. Similarly, in three species of polyphagousgrasshoppers, the average diameters of permeat-ing dextrans ranged from 24–36 nm (Barbehennand Martin, 1995).

Lehane (1997) points out that the abovemethod, based on diffusion through PMs, wouldbe expected to favor measurement of the smallerfraction of FITC-dextrans that are able to per-

meate PMs, whereas larger molecules would beforced through the gel-like matrix more rapidlyby bulk flow (e.g., gut peristalsis). This would sug-gest that results based on diffusion through PMsare conservative estimates of the sizes of perme-ating markers, and also underestimates of fluxin vivo. Maximum flux measured in O. leuco-stigma (Fig. 2) represents 1% of the FITC-dext-ran in the lumen permeating per hour. Althoughnot a study on herbivorous insects, similar results(>17-nm-diameter pores) have been obtained invivo in larval mosquitos using ingested FITC-dex-trans (Edwards and Jacobs-Lorena, 2000). A sec-ond method to measure pore sizes in the PMs ofherbivorous insects, the size distribution of diges-tive enzymes, has produced estimates of 7–8 nm(Santos and Terra, 1986; Fereira et al., 1994). Pos-sible reasons for differences between these meth-ods have been discussed previously (Barbehennand Martin, 1995).

Based on the permeability of PMs in herbivor-ous insects to high molecular weight FITC-dex-trans and digestive enzymes, free allelochemicalswould not be expected to be retained by PMs inthe endoperitrophic space as a result of ultrafil-tration. Allelochemicals with very high molecu-

Fig. 2. Ingested FITC-dex-trans permeating the PMs ofOrgyia leucostigma caterpil-lars. The amounts and sizes ofthese markers permeatinginto an incubating solutionthrough the PMs of ex vivogut preparations were mea-sured with high-performancesize exclusion chromatogra-phy. Reproduced from Barbe-henn and Martin (1995) withpermission of the publisher.


lar weights (large diameters) make up only asmall fraction of the wide array of compounds in-gested by herbivorous insects. Pentagalloyl glu-cose, a hydrolyzable tannin that is completelyretained by PMs in a variety of caterpillar spe-cies, is among the larger allelochemicals (MW 940Da) (Table 1), but has a hydrated diameter of only2.2 nm (Barbehenn and Martin, 1997). Therefore,non-absorption is most likely to occur in cases inwhich ingested allelochemicals aggregate to formhigh molecular weight complexes in the gut lu-men (or where the midgut epithelium acts as aneffective site of detoxification). Allelochemicals canbe grouped into two categories based on their par-tition coefficients between the predominantlyaqueous phase and the lipid phase of gut fluid:hydrophilic allelochemicals and lipophilic oramphiphilic allelochemicals.

Hydrophilic allelochemicals. Among the hy-drophilic allelochemicals, tannins have been beststudied in insects. These polyphenols are wellknown to form insoluble precipitates or solublecomplexes with proteins in vitro or in vivo (Hager-man and Klucher, 1986; Takechi and Tanaka,1987; Haslam et al., 1992; Stern et al., 1996;Baxter et al., 1997). In addition, tannins form com-

plexes with polysaccharides, lipids, and polyvalentmetal ions (Cai et al., 1989, 1990; Haslam et al.,1992; Takechi and Tanaka, 1987; DeVeau andSchultz, 1992; Ikeda et al., 1992; Murdiati et al.,1991; Slabbert, 1992; McDonald et al., 1996).

In the wide range of physicochemical condi-tions found in the gut fluids of herbivorous in-sects (Appel, 1993), the chemistry of complexformation will vary. For instance, in the high pHgut fluid of many caterpillars (pH 9–11), tanninsform polyphenolate anions. The loss of proteinprecipitating capacity of tannins at high pH isnow legendary (e.g., Feeny, 1970; Berenbaum,1980). This capacity is also lost in the presenceof surface-active phospholipids (surfactants) overa wide range of pHs (Martin et al., 1987). Yetpolyphenolate anions will complex with certainproteins (Martin et al., 1985; Hagerman andKlucher, 1986; Haslam et al., 1992; Stern et al.,1996), as well as with some lipids (DeVeau andSchultz, 1992; Barbehenn and Martin, 1998), andpolyvalent metal ions (Martin, et al., 1985;Murdiati et al., 1991; Haslam et al., 1992; Slab-bert, 1992; McDonald et al., 1996). By contrast,in the neutral to weakly acidic midgut pH ofgrasshoppers, tannins may hydrogen-bond or form

Fig. 3. Ultrafiltration of ingestedFITC-dextrans by the PMs of Orgyialeucostigma and Orygia pseudo-tsugata caterpillars. The asymptoticrelationship between the sizes of in-gested and permeating dextransdemonstrates how high molecularweight dextrans can be used to ap-proximate maximum pore diametersin PMs. Modified from Barbehennand Martin (1995).

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hydrophobic interactions with a wide variety ofsubstances, potentially including the componentsof PMs (Hagerman, 1989; Hagerman et al., 1998).

Much of the work on tannin complexation hasbeen done in vitro in simple buffers. The pairwiseinteractions between tannins and specific solutesdo not necessarily model interactions in gut fluid.For instance, whereas tannic acid is completely pre-cipitated in a pH 10 buffer containing physiologicalconcentrations of divalent metal cations, little pre-cipitation occurs in gut fluid containing similar lev-els of these cations (Fig. 4) (Barbehenn and Martin,1998). Each of three groups of substances in thegut fluid of Manduca sexta caterpillars (proteins,lipids, and divalent metal cations) plays a role inthe formation of tannin complexes (Fig. 4). Ultra-filtration of these multicomponent tannin complexesdemonstrated that most of the hydrolyzable tanninforms a high molecular weight colloidal suspension,particles that are believed to be retained by PMs incaterpillars (Barbehenn and Martin, 1998). The re-maining fraction of tannins formed precipitates,which presumably also have low permeabilitythrough PMs. Complex formation in grasshoppergut fluid has not been studied, but is likely to in-volve many of the same types of interactions (Mar-

tin et al., 1987; Hagerman, 1989). Tannin-bindingproteins have been found in the saliva of vertebrateherbivores (Luck et al., 1994; McArthur et al., 1995,Baxter et al., 1997), but no comparable examina-tion of insect saliva or PM proteins has been made.The sequestration of tannins by tannin-binding pro-teins in large (90-nm diameter) micelles in vitro sug-gests that such complexes would have the potentialto retard the permeation of tannins through thePMs of herbivorous insects (Luck et al., 1994).

Finally, non-absorption of some hydrophilicallelochemicals may result from covalent bondingwith other compounds in the lumen. Quinones,formed from phenol oxidation, bond to nucleophilicgroups in proteins or form melanin-like substancesin the guts of some caterpillars and grasshoppers(Felton et al., 1989; Appel, 1993; Barbehenn andMartin, 1994; Barbehenn et al., 1996). Like quin-ones, sesquiterpene lactones and pyrrolizidine al-kaloids also alkylate sulfhydryl groups in proteins(Felton, 1996). The possibility that reactive allelo-chemicals might be scavenged by nucleophilic aminoacid side groups (e.g., -SH, -NH2) in proteins in thematrix of PMs has not been examined.

Lipophilic and amphiphilic allelochemicals.The non-aqueous phase of gut fluid in herbivo-rous insects is composed primarily of micelles,along with a much smaller concentration of lipiddroplets (Martin and Martin, 1984; Turunen andChippendale, 1989). Micelles form during the di-gestive process from the aggregation of lysophos-pholipids (surfactants), as well as a variety ofother amphiphilic and lipophilic compounds(Hawke, 1973; Turunen and Chippendale, 1989).Ingested lipophilic and amphiphilic allelochem-icals are unlikely to exist as monomers in gutfluid. Rather, they would partition into the lipidphase as they move from plant tissues to gutfluid. Alternatively, some hydrophobic polycyclicallelochemicals, such as xanthotoxin, appear toaggregate with one another in stacked plate ar-rangements (Atwood, 1983; Barbehenn, 1999).Pure surfactant micelles range from 3–12 nm indiameter, whereas mixed micelles may be hun-dreds of nanometers in diameter (Vander et al.,1980, Mimms et al., 1981; Furth et al., 1984; Joneset al., 1987). To the extent that allelochemicals inthe gut fluid are associated with large mixed mi-celles, their rate of permeation through PMswould be reduced. The process of retaining seques-

Fig. 4. Effect of removing certain substances from gut fluidon tannin precipitation. Manduca sexta caterpillar gut fluidwas collected from larvae fed tomato foliage. The percent oftannic acid remaining suspended in the supernatant layerfollowing centrifugation of treated and untreated gut fluidwas measured with HPLC. Reproduced from Barbehenn andMartin (1998).


tered lipophilic and amphiphilic chemicals withan ultrafilter has been termed extractive ultrafil-tration (Watters et al., 1989).

Evidence for extractive ultrafiltration in vivois limited, but explains the reduced permeationof digitoxin in M. sanguinipes and M. sexta (Table1), and possibly in S. gregaria (Isman and Duffey,1983). PMs may also reduce the permeability ofouabain in M. sanguinipes (Fig. 5). However, thelack of association between this more polarcardenolide and lysolecithin micelles, and its rela-tively high permeability through PMs, suggeststhat a mechanism other than extractive ultrafil-tration is at work (Barbehenn, 1999). These re-sults also suggest that non-absorption of ouabainin S. gregaria (Scudder and Meredith, 1982) islargely the result of efficient detoxification in themidgut epithelium, although PMs may reduce therate of absorption into the epithelium. Anotherexample that is consistent with extractive ultra-filtration is found in H. zea that ingest rutin(amphiphilic) in artificial diets (Isman and Duffey,1983; Barbehenn, unpublished data).

Saponins and cardenolides are amphiphilic

allelochemicals that can complex with sterols(Roddick, 1979; Harmatha et al., 1987; Bloem etal., 1989). The stability of these complexes in gutfluid is suggested by the alleviation of saponin tox-icity when artificial diets contain mixtures of cer-tain sterols and saponins (Harmatha et al., 1987;Bloem et al., 1989). Clearly, the precipitation of sa-ponins in the endoperitrophic space would greatlyreduce their permeability through PMs. The fate ofingested saponins has also been predicted to be af-fected by gut pH and saponin ionization (Duffey andStout, 1996). For instance, tomatine complexationwith cholesterol is greatly favored at a pH above8.5 (charged form predominant). Molecular chargecould potentially affect the degree of complexationand subsequent permeability of saponins throughthe PMs of caterpillars (high pH) or grasshoppersand beetles (acidic gut pH) (Duffey and Stout, 1996).However, digitoxin permeability through the PMsof M. sanguinipes (midgut pH 6.8) and M. sexta(midgut pH 8.5–11.3) were similarly low (Table1) (Dow, 1984; Appel and Martin, 1990; Barbe-henn et al., 1996).

Variation in the lipid composition of in-gested foliage (or artificial diet) has the poten-tial to affect the complexation of lipophilic andamphiphilic allelochemicals in gut fluid (Hawke,1973; Turunen and Chippendale, 1989; Bernays,1992). For instance, the use of artificial diets,which typically contain droplet-forming triglyc-erides (≥1,000 nm diameter), could increase theefficiency of extractive ultrafiltration of lipophilicallelochemicals by PMs. In the milkweed bug,digitoxin is sequestered in the body in lipid drop-lets (Duffey et al., 1978), suggesting that suchallelochemicals could also be retained in theendoperitrophic space in lipid droplets. Finally,it has been hypothesized that phytosterol com-position may affect saponin precipitation in thegut lumen (Duffey and Stout, 1996).

Polyanion Exclusion

Several observations suggest that the ma-trix of PMs might be far less permeable to nega-tively charged molecules than to those that areneutral or positively charged. First, there arestructural and biochemical similarities betweenthe basement membranes of kidney glomeruli andPMs (Miller and Lehane, 1993; Summers andFelton, 1996). Both are secreted extracellular ma-

Fig. 5. Role of PMs in limiting lipophilic and amphiphilicallelochemical permeability through Melanoplus sanguinipesgut preparations. An asterisk denotes a significant (P < 0.05)decrease in allelochemical permeability when PMs werepresent. The association of digitoxin with surfactant micellesand its low permeability through PMs are consistent withextractive ultrafiltration. Permeability coefficients have unitsof 10-3 cm-3 h-1. Modified from Barbehenn (1999).

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trices, and both contain proteoglycans. Proteo-glycans, over the wide range of pHs found in thegut lumens of herbivorous insects, have numer-ous negatively charged sites (Chang et al., 1975b;Farquhar, 1991). Charge exclusion by proteo-glycans restricts the permeability of glomerularbasement membranes to negatively charged pro-teins (7.2 nm diameter) and to polyanionic mark-ers, such as dextran sulfate (1.8–3.6 nm diameter)(Chang et al., 1975b). Finally, PMs in tsetse fliesexhibit an increase in in vitro permeability to theanionic protein alkaline phosphatase when theconcentration of Ca2+ is increased from 0 to 50mM (Miller and Lehane, 1993). Based on theseobservations, it was hypothesized that charge ex-clusion might greatly reduce the permeability ofPMs in caterpillars to tannins (Barbehenn andMartin, 1994). The attractiveness of the polyanionexclusion hypothesis is its substantial physiologi-cal and biochemical basis, and its explanation ofthe retention of relatively small polyphenolateanions (approximately 2 nm diameter) in theendoperitrophic space. In addition, the permeabil-ity of tannins (primarily tetragalloyl glucose)through the PMs of two grasshopper species (Fig.1) would also be consistent with this hypothesis,since tannins would not be ionized in the mildlyacidic to neutral gut pH of these species.

Comparisons of the permeabilities of PMs toFITC-dextran (monoanionic) and dextran sulfate(polyanionic) showed no support for the polyanionexclusion hypothesis (Barbehenn and Martin, 1997).The permeability of the PMs of a caterpillar (O.leucostigma) and a grasshopper (M. sanguinipes) topolyanions was not reduced, contrary to the predic-tion. The proteoglycan content of the matrix of PMsin the test species may be too low to produce aneffective electrostatic barrier to polyanions. In H.zea PMs, the proteoglycan content is only 8.4%(Summers and Felton, 1996). In addition, proteo-glycans in herbivorous insects are believed to beuniformly distributed within the PMs, rather thanconcentrated in outer layers, as in Diptera (Peters,1992; Miller and Lehane, 1993).

Antioxidant Potential

The potential for PMs to function as anti-oxidant defenses that protect the midgut epithe-lium was first proposed by Felton and Summers(1995). According to this hypothesis, PMs perform

a function analogous to that of the mucous layerthat protects vertebrate gastric mucosal cells fromreactive oxygen species (ROS) (Hiraishi et al.,1991). Substances in PMs scavenge hydroxyl radi-cals (HO·) and reduce hydroperoxide formationin vitro in midgut tissue homogenates (Summersand Felton, 1996). However, since biological mol-ecules are all subject to attack by HO· (Halliwelland Gutteridge, 1999), the question should beasked “What properties of PMs make them morecompetitive sinks for HO· than the numerousother substances in the midgut lumens of her-bivorous insects?” The close proximity of PMs tothe midgut epithelium, and their function assemipermeable barriers to all substances in theendoperitrophic space, are both properties of PMsthat might give them functions analogous to mu-cous layers. However, the presence of PMs wouldnot be expected to reduce the damage caused byHO· formation in the majority of the volume ofthe endoperitrophic space. Nor would the prox-imity of PMs to the midgut epithelium necessar-ily reduce HO· damage to the epithelium if HO·is generated in the ectoperitrophic space. SinceHO· is believed to be produced by the Fe2+-cata-lyzed breakdown of hydrogen peroxide (H2O2)(Fenton reaction), any molecule that binds ironwill be subject to the site-specific attack of HO·(Halliwell and Gutteridge, 1990). Antioxidantsthat function by binding catalytically-active iron,such as serum albumin, are termed suicide anti-oxidants (Halliwell and Gutteridge, 1990). Thepossibility that PMs might function as suicide an-tioxidants was implied by Felton and Summers(1996), although no work has been done to exam-ine this possibility. The basis for this function liesin the potential for both negatively charged(proteoglycan) and uncharged (chitin) componentsof PMs to chelate transition metal cations, suchas iron and copper (Koshijima et al., 1973). Bind-ing of these ions by PMs before they can bindwith epithelial membranes would reduce the dam-age of HO· to epithelial membranes.

Similarly, PMs could have an antioxidantfunction if they reduce the absorption of allelo-chemicals that redox cycle, such as many quin-ones (Smith, 1985; Gant et al., 1988). If suchallelochemicals are alkylated by the nucleophilicside chains of proteins in the matrix of PMs, PMscould reduce oxidative stress (via O2

– generation)


in the midgut epithelium. The potential for thisprotective role has not been examined.

Finally, the pattern of thickness observed ingrasshopper PMs (i.e., thicker PMs in more polypha-gous species that consume greater amounts ofphenols) (Bernays and Simpson, 1989) is consistentwith an antioxidant function, in addition to ultra-filtration (Barbehenn et al., 1996). Thicker PMsmight protect the gut epithelium more effectively,such as by a “suicide antioxidant” mechanism. Phe-nol oxidation, which generates ROS, has been ob-served in some grasshoppers (Barbehenn et al.,1996), but further work is needed both on oxida-tive stress and defense mechanisms in grasshop-pers to examine this idea.

CONCLUSION

There is a need for a model that integratesthe defensive mechanisms of PMs towards a widerange of allelochemicals and across a variety ofdifferent types of herbivorous insects. Currentlythere is insufficient information to state a cohe-sive model. However, there is sufficient informa-tion to begin this process and to suggest researchneeds. Of four potential mechanisms of defenseprovided by PMs against ingested allelochemicals,ultrafiltration remains the most important (basedon current knowledge). In most cases, ultrafiltra-tion of allelochemicals requires complexation ofthe allelochemicals in the endoperitrophic spaceto form high molecular weight aggregates. Adsorp-tion of tannins may occur on the PMs of polypha-gous and graminivorous grasshoppers, but analternative, that tannins form high molecularweight complexes in gut fluid that are filtered inthe caecal PMs, is possible. Combinations ofmechanisms (e.g., adsorption and ultrafiltration)might also function together.

A number of studies have shown that tissuelesions are produced if certain allelochemicals per-meate PMs and are absorbed into the midgut epi-thelia of non-adapted species (e.g., Lindroth et al.,1988; Lindroth and Peterson, 1988; Thiboldeauxet al., 1998). A second cause of tissue damage isexposure to micromolar levels of H2O2 (or otherROS), which can cause DNA damage and cell death(Hanham et al., 1983; Imlay et al., 1988; Summersand Felton, 1994). The oxidation of phenols in thegut lumen (Felton et al., 1989; Appel, 1993,

Barbehenn and Martin, 1994; Barbehenn et al.,1996) produces H2O2, potentially causing tissuedamage despite the retention of ingested phenolsin the endoperitrophic space (Barbehenn et al.,2001). Therefore, the presence or absence of epi-thelial tissue damage is not sufficient to distin-guish PM permeability from impermeability;damage is potentially confounded by the detoxi-fication capacity of the gut epithelium. It is pos-sible that degeneration of the cells that secretePMs leads to an accumulation of allelochemicals(e.g., tannins) in gut tissues as an aftereffect oftoxicity, rather than as a cause. To examine therole of PMs in the non-absorption of ingestedallelochemicals, direct measurement of the per-meability of PMs is needed. To examine the modeof action of ingested phenolics and other oxidiz-able allelochemicals, determination of oxidationin the gut lumen is needed. Without these datait is difficult to determine the mechanism(s) in-volved in sensitivity or tolerance of herbivorousinsects to specific allelochemicals.

The hypotheses posed above point to the nu-merous areas of research on PMs that remainuntouched. For instance, are thicker PMs in poly-phagous grasshoppers the result of higher ratesof secretion (more laminae) or thicker individualPMs (more matrix material)? Are there similarpatterns in caterpillars related to dietary expo-sure to toxins and/or prooxidants? It would be ofinterest to know whether the secretion of PMscan be induced by the ingestion of toxic and/orprooxidant allelochemicals. Such a pattern of PMproduction would parallel the pattern of elevatedmucin secretion in vertebrate gut mucosal cellsin the presence of ROS (HO· and H2O2) (Hiraishiet al., 1991). Such a pattern would also parallelthe genetic variation in rates of secretion of PMsby A. aegypti, a trait that is related to DDT re-sistance (Abedi and Brown, 1961).

PMs that reduce the permeability of toxicallelochemicals work together with other secreteddefenses and the midgut epithelium to protectherbivorous insects. The benefit to insects of non-absorption can result from reduced toxin burdenson the detoxification systems in the midgut epi-thelium and/or reduced oxidative stress. Con-versely, in the absence of fully functional PMs areinsects more readily intoxicated by allelochemicaldefenses? The ability to disrupt the structure of

96 Barbehenn

PMs is an old idea that has recently gainedground in research on genetically engineeredplants and chemical antagonists of chitin forma-tion (Clarke et al, 1977; Wang and Granados,2000; Gongora et al., 2001). These developmentsmay provide a tool to better understand the rolesof PMs in vivo.

ACKNOWLEDGMENTS

I thank Professor Michael M. Martin for im-provements made to the manuscript, and formany years of collaboration. Professor ElizabethBernays and an anonymous reviewer made sug-gestions for additional important improvements.This work was supported by NSF grant IBN-997483 to RVB.

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