© 2006 The Authors

Entomologia Experimentalis et Applicata

118

: 1–10, 2006

Journal compilation © 2006 The Netherlands Entomological Society

1

Blackwell Publishing Ltd

MINI REV IEW

Nutritional and functional biology of exudate-feeding

ants

Steven C. Cook* & Diane W. Davidson

University of Utah, Department of Biology, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA

Accepted: 11 October 2005

Key words:

digestive anatomy, ecological stoichiometry, endosymbiont, Malpighian tubules, nitrogen isotope, nitrogen recycling, peritrophic membrane, proventriculus, Hymenoptera, Formicidae

Abstract

Feeding extensively on plant exudates and honeydews, many tropical arboreal ant species exhibit

δ

15

Nvalues characteristic of herbivores. Consistent with hypothesized herbivory, these taxa behave infeeding assays as though more N-deprived than are strictly carnivorous ants. However, to an as yetuncertain degree, relationships with N-upgrading and/or recycling microsymbionts may lowerisotopic ratios, making ants appear to be more herbivorous than they actually are. Nutritional (N)contributions from microsymbionts have been inferred for a variety of ant taxa based on intracellularor extracellular associations between ants and bacteria. However, stronger and more specific inferencesare possible when variability in microsymbiont locations within the digestive system is considered inthe context of taxonomic variability in ant diets and digestive anatomy. Diets of exudate feeders mayvary predictably in ratios of usable carbohydrates (CHOs) to N, depending on the extent to whichthey tend melezitose-producing Homoptera. Status of the peritrophic membrane, proventricularstructure, and number and placement of Malpighian tubules can be interpreted as traits contributingto supply of N and/or CHOs to microsymbionts. In general, a more integrative understanding of antdiets, digestive anatomy, and associated microsymbionts helps to set out specific hypotheses to be

tested experimentally and (where possible) in a phylogenetic context.

Introduction

The nascent fields of ecological stoichiometry (Sterner &Elser, 2002) and related geometric framework (e.g., Rauben-heimer & Simpson, 2004) explicitly recognize mass balanceconsiderations of energy and nutrient flows as essentialto a full understanding of both the functional biology ofindividual organisms and higher level processes in popu-lations, communities, and ecosystems. Fundamental tenetsof these fields are that, food storage aside, consumers oftenact to maintain elemental homeostasis in body compositionwithin limited ranges, and that they respond develop-mentally, physiologically, behaviourally, ecologically,and evolutionarily to different resource ratios. Individualorganisms can be thought of as harvesting energy andmatter from their environments, converting some ofthese resources to biomass, and then returning energy andmaterials to the surroundings. Evolution has likely moldedall of these activities by producing regulatory mechanisms

that both optimize gain and use of limiting macronutrientsand prevent nutrient excesses from reaching body tissues(Raubenheimer & Simpson, 1998, 2004). Organisms con-suming nutritionally unbalanced diets are under the strongestselection to evolve regulation that intervenes between nutrientintake and allocation to body tissues (Raubenheimer &Simpson, 2004).

Ants (Hymenoptera: Formicidae) provide a compellingsystem for studying such regulation because, across generaand subfamilies, diets range from comparatively balanced,relative to elemental composition of body tissues, toextremely unbalanced in nutrient content (Davidson, 2005).Recently, based on isotopic evidence, many tropical ant taxahave been hypothesized to feed as ‘cryptic herbivores’[Hunt’s (2003) review of Davidson et al., 2003, see alsoBlüthgen et al., 2003], evolving under the same nitrogen(N) deprivation faced by many other herbivores (White,1993). Here, we consider these claims in the context ofknown and hypothesized microsymbiont associations ofants, and we review anatomical traits that may modulatemismatches between diets and body composition. Our aim

*

Correspondence: E-mail: cook@biology.utah.edu

eea_374.fm Page 1 Friday, December 9, 2005 1:38 PM

2

Cook & Davidson

is to summarize current research in ways that both raisenew questions and guide future studies in fruitful direc-tions. Although this approach involves some conjecture,we endeavour throughout to use language that distinguishesfacts from hypotheses.

‘Cryptic herbivores’ or N-upgraders/recyclers?

Nitrogen isotope ratios (as

δ

15

N) have recently been deter-mined for a broad range of ant taxa in the lowland tropicalrain forests of Amazonia, Borneo, and Australia, andused in attempts to assess the trophic levels at which theseinsects feed (Blüthgen et al., 2003; Davidson et al., 2003).During N metabolism, fractionation steps preferentiallyeliminate the lighter isotope (Gaebler et al., 1966), causing

δ

15

N values to increase with trophic level at approximately3.4‰ per level (e.g., Schoeninger & DeNiro, 1984), or some-what less between the producer and herbivore trophiclevels (e.g., Van der Zanden & Rasmussen, 2001). For ants,some of the isotopic results merely confirm natural historyobservations (e.g., carnivory in Ponerinae and Ecitoninae),but other findings are more surprising. Thus,

δ

15

N valuesoverlapping those of herbivorous insects, or even plants,were reported for a number of free-living, arboreal speciesin the genera

Camponotus

,

Echinopla

, and

Polyrhachis

(Formicinae),

Dolichoderus

and

Azteca

(Dolichoderinae),

Cephalotes

,

Crematogaster

, and

Cataulacus

(Myrmicinae),and

Pseudomyrmex

and

Tetraponera

(Pseudomyrmecinae)(Blüthgen et al., 2003; Davidson et al., 2003). These ‘her-bivorous’ or (mostly) omnivorous ant taxa do not directlyconsume plant tissues, but feed on abundant, carbohydrate(CHO)-rich, N-poor liquids such as extrafloral nectar(EFN) (Hölldobler & Wilson, 1990; Rico-Gray, 1993; Blüthgenet al., 2000), plant wound secretions, and insect ‘honeydew’(Delabie, 2001; Blüthgen & Fiedler, 2002; Davidson et al.,2004).

Providing CHOs over and above the amounts that canbe combined with available N for the primary metabolismfunding growth and reproduction, such stoichiometricallyimbalanced diets could be nutritionally complete if ‘excessCHOs’ are diverted to functions that increase access tolimiting N (Davidson, 1997). However, relative to taxa fee-ding on more balanced diets, exudate-feeding ants have beeninterpreted to be more N-deprived in their responses tofeeding assays than are more carnivorous taxa (Davidson,2005). Thus, the exchange ratio (ER) of minimum accep-table sucrose (CHO) concentration to minimum accepta-ble amino acid (N) concentration tends to be greater forexudate foragers than for carnivores (Davidson, 2005).This ratio, similar to that of the marginal values of tworesources (sensu Charnov, 1976, see also Kay, 2002), gaugestheir values in the same currency. On an evolutionary

timescale, ants and other organisms have likely evolved todefend optimal intake targets for CHO and N (Simpson &Raubenheimer, 1996), and such adaptations might also beexpected to influence proximate food choices as reflectedin measured ER values. However, decoupling of resourceretrieval from consumption (by larvae) in social insectsmay correlate with little worker knowledge of whole colonyreserves vs. needs, so that foraging decisions are basedprincipally on comparisons of resource quality withbackground levels of availability in the environment (Kay,2002).

To cope with stoichiometrically imbalanced diets, othersap-feeding insects (e.g., homoptera) harbour obligatemicrosymbionts, contributing to N nutrition (e.g., Douglas,1998; Moran et al., 2003), and the same has been suggestedfor a number of exudate-dependent ant taxa. As a group,these ants associate with phylogenetically diverse microsym-bionts inhabiting different parts of the digestive system.Thus, at least one

Tetraponera

species (

T. binghami

,Pseudomyrmecinae) houses several genera of putativelyN-recycling bacteria, related to N-fixers (

Rhizobium

,

Methylobacterium

,

Burkholderia

, and

Pseudomonas

species),as well as Flavobacteria (van Borm et al., 2002), in anelaborate pouch jutting from the anterior ileum andserviced by a network of Malpighian tubules (MTs) andtracheae (Billen & Buschinger, 2000; van Borm et al., 2002;Figure 1). Among myrmicines, closely related

Cataulacus

and

Cephalotes

species (Bolton, 2003) harbour masses ofbacteria in the posterior midgut and a dilated region ofthe anterior ileum (e.g., Caetano & Cruz-Landim, 1985;Caetano et al., 1994; Roche & Wheeler, 1997; Figure 1).Within the Formicinae, certain

Formica

species housebacteria inside cubical cells, forming a unicellular layer,and loosely arranged inside the perimeter of the midgut.

Figure 1 Generalized, composite ant digestive system depicting structures mentioned in text: TH, thorax; G, gaster; es, esophagus; cr, crop; pv, proventriculus; mg, midgut; mt, Malpighian tubules; bp, bacterial pouch; il, ileum; re, rectum.

eea_374.fm Page 2 Friday, December 9, 2005 1:38 PM

Nutritional and functional biology of exudate-feeding ants

3

Additionally, camponotines carry endosymbionts, assignedto the genus

Blochmannia

, in bacteriocytes intercalated withmidgut epithelial cells (Buchner, 1965; Schröder et al., 1996;Sauer et al., 2002). These bacteria show stasis in genomicarchitecture, relative to free-living enteric bacteria (Degnanet al., 2005), and may be closely related to homopteranendosymbionts (Spaulding & von Dohlen, 1998; Moranet al., 2003; Thao & Baumann, 2004; but see Wernegreenet al., 2003), whereas those of

Formica

species are phyloge-netically distinct from both of these groups (Sameshima et al.,1999; Wernegreen et al., 2003). Further screening will likelyreveal microsymbiont associations in additional ant taxa.

Parallels between sap-feeding homoptera and exudate-feeding ants suggest mechanisms possibly contributingto low

δ

15

N in both groups. First, free, plant-derived (low

δ

15

N), non-essential amino acids may be integrated intolarval and worker tissues without the fractionation stepinherent to protein catabolism. Second, like the genome ofendosymbiotic

Buchnera

in aphids (Nakabachi & Ishikawa,1998; Shigenobu et al., 2000), that of

Blochmannia

containsgenes for transaminases (Gil et al., 2003), i.e., genes thatcould mediate the synthesis of essential amino acids bytransferring low

δ

15

N amide groups from relatively abundant,non-essential amino acids (e.g., glutamine, glutamate,and asparagine) to new carbon (C) skeletons made fromdietary sucrose (Febvay et al., 1999). Preferential transferof lighter waste N (

14

N) to form essential amino acids(Macko et al., 1986) should lead to declines in biomass

δ

15

N. The potential importance of this process in manyorganisms housing bacterial microsymbionts is seen in thefact that shuttling of

α

-amino groups by N-free precursors(e.g., citric acid intermediates oxaloacetate and

α

-ketoglutarate) to form glutamate and glutamine, twoN-assimilation intermediates (Stryer, 1995), provides theamide group for 88% and 12% of all cellular N-containingcompounds, respectively (Ikeda et al., 1996). The twoprocesses described here perhaps also contribute toreducing

δ

15

N values in sap-feeding homoptera relative tochewing herbivores (Davidson et al., 2003).

Unlike sap-feeding homoptera, the vast majority of antspecies feed at least occasionally on hunted or scavengedprey. This near universality of omnivory in ants makes itmore remarkable that a subset of ant taxa have

δ

15

N valuesoverlapping those of sap-feeding homoptera and plants.Therefore, additional mechanisms or isotopic fractiona-tion steps must be involved in keeping isotopic ratios low.Although

Blochmannia

could theoretically share

Buch-nera

’s capacity to recycle ammonia-derived glutamine intoother amino acids (Sasaki & Ishikawa, 1993), it also con-tains genes for the operation of the urea cycle (Gil et al.,2003), which transforms waste N into usable forms (e.g.,glutamine). If putative N recycling by formicines were to

use ‘light’ amide groups (from glutamine) preferentially,neogenic amino acids produced by the processes of urearecycling and transamination would be isotopically lighterafter each episode of recycling. A similar argument mightbe made for other ant taxa hypothesized to recycle uricacid N (see succeeding discussion).

Conservation of N through recycling should helpreduce N-deprivation in exudate-feeders, and except fortwo species with specialized diets, camponotines exhibitjust moderate N-deprivation in feeding assays (Davidson,2005). Unexpectedly, however, members of the genus

Dolichoderus (

from the related subfamily Dolichoderinae;Baroni-Urbani et al., 1992; Shattuck, 1992) are no more N-deprived than were camponotines. Spanning a size rangesimilar to that in camponotines,

Dolichoderus

species arenot known to associate with endosymbionts, but repor-tedly do house microbial masses in the gut lumen (Caetanoet al., 1989a,b; Figure 1). The diets of rainforest

Dolichode-rus

and camponotines also differ markedly on average.

Dolichoderus

are dedicated trophobiont tenders, primarilyof Membracoidea and Pseudococcidae (Blüthgen et al., 2000;Delabie, 2001; Dill et al., 2002; Davidson et al., 2004). Mostcamponotines are not, but rather (operationally) widelyranging ‘leaf foragers’ (

sensu

Davidson et al., 2004). Mem-bers of the latter foraging functional group scour leaflaminae for dispersed resources that include species-typicalcombinations of EFN, plant wound secretions, discardedhoneydew from untended sap feeders, fungal secretions,and/or particulate matter (e.g., pollen and fungal spores,Wheeler & Bailey, 1920; Baroni-Urbani & Andrade, 1997).The amino acid profiles of trophobiont ‘honeydews’ are oftenunbalanced in favor of two non-essential amino acids,glutamine and arginine (Douglas, 1993; Blüthgen et al.,2003), and are augmented with trophobiont-synthesizedoligosaccharides, e.g., melezitose in aphids (Mittler, 1958;Wilkinson et al., 1997). Although EFN and wound secre-tions also contain mainly non-essential amino acids, theylack the disproportionate representation of glutamine andarginine and contain predominantly simple sugars [sucrose,fructose, and glucose (Engel et al., 2001; Blüthgen et al.,2004)]. Dietary differences between

Dolichoderus

speciesand camponotines could have selected for different mecha-nisms for maintaining macronutrient homeostasis (seesucceeding discussion).

Why do

Dolichoderus

species not show more evidenceof CHO:N nutrient imbalances? We can advance fourhypotheses. First, although

Dolichoderus

brood areapparently incapable of feeding on solid foods (Wheeler& Wheeler, 1976), amino acids from haemolymph ofharvested trophobionts and other insects (Dill et al., 2002)might contribute to a more nutritionally complete larvalfood supply. Second, these ants could benefit from amino

eea_374.fm Page 3 Friday, December 9, 2005 1:38 PM

4

Cook & Davidson

acid upgrading by bacterial associates of trophobionts(yet to be demonstrated for Membracidae and Coccidae);however, preliminary evidence from aphids shows fewupgraded amino acids incorporated into honeydew(Sandstrom & Moran, 2001). The remaining two hypo-theses emerge from a more detailed consideration of thecomposition of honeydew and are perhaps more readilytestable. First, honeydews are rich in melezitose and otheroligosaccharides that would not be absorbed across the gutwall unless first broken down by saccharide hydrolases(Chapman, 1998). However, invertase is either lacking orpresent at low concentrations in honeydew-feeding ants(Ayre, 1967), and other oligosaccaride-hydrolyzing enzymesare believed to be absent or in low concentrations in manyants (Boevé & Wäckers, 2003) and bees (Ferreira et al., 1998).Therefore, much of the energy present in honeydews maynot be accessible to

Dolichoderus

species; if not, C:N dietaryratios would be more nearly balanced.

Finally,

Dolichoderus

species may recycle N with the aidof microsymbionts in the gut lumen. Non-essential aminoacids present in excess of the insects’ needs should con-stitute waste N to be excreted or stored for disposal whenworkers die. Not surprisingly then, the haemocoels and/orfat bodies of all

Dolichoderus

workers are replete with uratecrystals, in far greater densities than found in

Camponotus

fat body (S Cook, unpubl.), perhaps because efficientammonia recycling by

Blochmannia

prevents significantbuild-up of nitrogenous wastes. In most of the species wehave examined,

Dolichoderus

urate stores also far exceedaccumulations in more predatory ant taxa, suggesting thatthe role of urate storage in this genus could extend beyondsimple waste sequestration to N-recycling by microsym-bionts in the gut lumen (see, e.g., Potrikus & Breznak, 1981;Hongoh & Ishikawa, 1997; Zhuzhikov, 2001, for termites,cockroaches, and plant hoppers, respectively). Consistentwith this hypothesis, workers of some large-bodied

Dolichoderus

species engage in both autocoprophagy andanal trophallaxis with nestmates (D Davidson, pers. obs.for

D. attelaboides

and

D. decollatus

). Zhuzhikov (2001) hassummarized evidence that proctodeal trophallaxis may haveevolved from autocoprophagy and that both are associatedwith microbial symbionts in the posterior gut.

In summary, we conjecture that low availability of usableCHOs, coupled with N-recycling by hindgut microbes andingestion of insect haemolymph, might together explainwhy

Dolichoderus

species resemble most camponotines inbeing just moderately N-deprived. If

Dolichoderus

speciesdo associate with N-recycling microbes, and if light (waste)N were to be recycled preferentially, such factors couldcontribute to the relatively low

δ

15

N values measured inthese species (overlapping those of some sap-feeding andchewing herbivores; Davidson et al., 2003).

Key digestive system components in relation to N

nutrition

Together, the Formicinae and Dolichoderinae are the mostexudate-dependent subfamilies of ants. The digestive systemsof these taxa might therefore be anticipated to be among themost highly specialized for dealing with high-CHO, low-N, liquid foods. Here, we compare key components of theant digestive system across these and other ant subfamilies,with particular attention to the roles of these traits inregulating access to CHO and N resources.

Peritrophic membrane

First, a brief mention should be made of the peritrophicmembrane (PM). Where present in ants and other insects,it consists of a network of proteins and chitinous micro-fibrils excreted from columnar cells of the midgut epithelium(Richards & Richards, 1977) and enveloping food boluses.The network is thought to protect the delicate midgutepithelium from abrasion, to guard against microbialinvasion of host tissues, to help retain food in the midgutfor digestion, and also to compartmentalize the midgutinto regions with different enzymatic functions (reviewedin Richards & Richards, 1977; Chapman, 1998). Absenceof the PM has been documented in various insect taxaspecializing in low molecular weight foods that do notrequire luminal digestion (Terra, 2001). Studied in just afew ant taxa, it is easily detected in carnivorous ponerines,but reportedly absent or reduced (i.e., detectable only byelectron microscopy) where investigated for camponotinesand a single dolichoderine (Waterhouse, 1953; Lehane, 1997).In general, it may be unnecessary in taxa specializing in liquidfoods with low molecular weight solutes. Additionally, absenceof the PM might permit quick absorption of low molecularweight food molecules (amino acids and monosaccharides)in such groups. Given its proposed role in preventing bacterialinvasion of gut cells (Richards & Richards, 1977), its absencecould have facilitated early establishment of associationswith endosymbiotic bacteria in camponotines by allowingrepeated and intimate interactions of bacteria with midgutcells (e.g., Fukatsu, 1994; Harada et al., 1996, for homo-pterans). However, lack of the PM does not ensure acquisi-tion of endosymbionts, given other potential obstacles toformation of stable symbioses (e.g., host cell defenses andevolution of mechanisms for vertical transmission).

Proventriculus

The proventriculus is a cuticular, muscular valve betweenthe worker crop (the colony’s ‘social stomach’) and themidgut (Hölldobler & Wilson, 1990; Figure 1). Previous

eea_374.fm Page 4 Friday, December 9, 2005 1:38 PM

Nutritional and functional biology of exudate-feeding ants

5

studies of this organ show a high degree of anatomicalvariation across the ants (Eisner, 1957; DeMoss, 1973),with the most derived structures appearing in taxa withlarge, body size-adjusted crop capacities (camponotines,cephalotines, and small-bodied dolichoderines withapomorphic proventriculi, Davidson et al., 2004; Figure2A, B). Sclerotized and lacking key muscle groups, thesederived proventriculi ‘passively dam’ the posterior efflux ofcrop contents, allowing for large liquid volumes to accumulateanteriorly in the distended crop, and facilitating food sharingwith nestmates through oral trophallaxis (Eisner, 1957).In the subset of taxa where damming is most complete,sclerotized structures are bypassed by canals (in so-called‘sepalous formicines’) or vestibular sinuses [

Iridomyrmex

,

Ochetellus

, and

Papyrius

species, dolichoderines with themost derived proventriculus; see Eisner (1957) and genericrevisions of Shattuck (1995)]. These channels transmit liquidsslowly into the proventricular bulb, which then pumps themto the midgut. For these taxa, a controlled, gradual influx ofcrop contents to the midgut may assure continuity of resourcesupply to the host and (in formicines) to endosymbionts.To the extent that enzymatic function is retained in themidguts of these exudate feeders, slow influx could alsoguard against both dilution of midgut enzymes (Eisner,1957) and abrupt pH changes that would compromiseenzyme function (Chapman, 1998).

In contrast to derived proventriculi of other dolichode-rines and of sepalous formicines, that of

Dolichoderus

species differs little from homologous organs in basal ant taxathat are not specialized exudate feeders (e.g., Myrmiciinae,Eisner, 1957; Figure 2C). Not surprisingly then,

Dolichoderus

species collect small, body size-adjusted crop loads (David-son et al., 2004). Moreover, feeding rates do not increasewith body size in dolichoderines as they do in formicines,myrmicines, and ponerines, largely because the four largest-bodied

Dolichoderus

species tested (

Do. attelaboides

and

Do. bidens

species groups; see MacKay, 1993) imbibeliquids extraordinarily slowly during individual feedingbouts. Unable to staunch posterior flow of stored cropcontents into the midgut,

Dolichoderus

species may carryand store large quantities of liquids mainly in the hindgut(ileum plus rectum; Figure 1) and provide them to nestmatesonly via anal trophallaxis (authors’ observations, andsee Jaffe et al., 2001, and Roche & Wheeler, 1997, for analtrophallaxis in cephalotines). Exceptionally slow drinkingrates, particularly relative to body size, may reflect thehindgut being filled to capacity with microbes and theliquid resources they are processing. Condemned by theirplesiomorphic proventriculi to feed slowly,

Dolichoderus

species are likely poor exploitative competitors for thediffusely distributed and unpredictable foods utilized bymany formicines (Davidson et al., 2004). However, they arewell suited to feeding on gradually produced honeydews(Tjallingii, 1995; Yao & Akimoto, 2002) and, with nestmatesnearby to assist them, can cooperate in driving leaf-foragingformicines from rich resources within their relatively con-fined feeding areas (Davidson & Cook, in press).

Overall then, formicines and dolichoderines both appearto possess regulatory mechanisms for processing largequantities of CHO-rich liquid foods for the little N theycontain. In sepalous formicines and small-bodied doli-choderine genera with the derived proventriculus, controlis invested in the anterior proventriculus, which enablescollection and sharing of liquid foods through oraltrophallaxis with nestmates (see also Eisner, 1957). Includedamong those nestmates are growing larvae that, in cam-ponotines, have especially high bacteriocyte densities intheir guts (Sauer et al., 2002, for

Camponotus floridanus

).Furthermore,

Blochmannia

in those bacteriacytes mayhave the capacity to convert glutamine to glutamate, fromwhich essential amino acids can be made via transaminationreactions. In

Dolichoderus

species, which lack the anterior

Figure 2 Representative proventricular structures mentioned in the text (from Eisner, 1957). (A) Sepalous formicine proventriculus (here, Camponotus), (B) derived proventriculus of small-bodied dolichoderines (here, Iridomyrmex), and (C) plesiomorphic proventriculus of Dolichoderus species. Proventricular parts: b, bulb; bc, bulbar canals; c, cupola; cr, crop; p, portal; s, sinus; sc, sepalar canals; se, sepals; sv, stomodeal valve.

eea_374.fm Page 5 Friday, December 9, 2005 1:38 PM

6

Cook & Davidson

control point for passive damming, processing of liquidfoods has shifted posteriorly. Lacking the capacity toconvert glutamine to glutamate (Stryer, 1995, for animalsgenerally),

Dolichoderus

species cannot take advantage ofdietary excesses of glutamine (see previous discussion).An option open to large-bodied species is to convert excessnon-essential amino acids to urate, store this in fat body,and later mobilize and transport it (or derived nitrogenouscompounds) to the hindgut via the MTs. It is possible thatextracellular gut microorganisms there can utilize suchcompounds in the biosynthesis of neogenic amino acids.Any resultant essential amino acids would be available tonestmates through anal trophallaxis. If this scenario isconfirmed, it could be limited to relatively large-bodiedspecies with correlated large fat body and hindgut capacities.We have not observed anal trophallaxis in small speciesof

Dolichoderus

or in other small-bodied dolichoderines(

Azteca

,

Technomyrmex

, and

Forelius

species).

Malpighian tubules

A third anatomical feature appearing to correlate withant diets is the number and placement of MTs (Figure 1),which funnel cellular metabolic wastes to the ileum (anteriorhindgut). DeMoss (1973) compared these traits in anttaxa (mainly myrmicines) as potential characters forphylogenetic reconstruction, and we supplement hisdata here with results of our own dissections of specimenspreserved in phosphate-buffered saline (PBS) (Table 1). Both

the number and placement of MTs varied substantially atthe generic level, even among similarly sized taxa. Generally,MTs radiate from attachment sites near the anterior ileumand may appear to extend randomly within the haemocoelor be more closely associated with organs such as the fat body(DeMoss, 1973; Chapman, 1998). Additionally, in someinsects, MTs attach directly to other elements of the digestivesystem (i.e., cryptonephry). Camponotines studied todate are distinctive in both numbers and location of MTs(DeMoss, 1973). Larger MT numbers in camponotines(see succeeding discussion) than in Dolichoderinae, andespecially Ponerinae and Myrmicinae, suggest the needfor faster processing of metabolic wastes and are consistentwith higher metabolism in this group (see previousdiscussion, Table 1). Closely associated with fat body, asin other taxa, camponotine MTs are also distinctive in theirfurther attachment (via membranous fasteners) to the outersurface of the midgut (DeMoss, 1973). Bacteriocytes of

Camponotus

species rest on the midgut’s basementmembrane (Buchner, 1965) and should be in direct contactwith MTs. It is therefore possible that highly concentratedsmall waste molecules exit MTs and pass through thebasal membrane to supply putatively N-recycling midgutendosymbionts. This unusual placement of MTs is at leastsuggestive, and it parallels the close proximity of thesestructures to sites proposed for N-recycling in

Tetraponera

(Billen & Buschinger, 2000) and

Cephalotes

species (Roche& Wheeler, 1997). MT numbers are also relatively highin

T. attenuata

(Table 1), where they may increase rates

Table 1 For ants discussed in the text, summary of associations with microbes and related morphological and physiological traits

Taxon (SF)a

Associations with microbesc,d Proventricular form

Malpighian tubule numberf

Peritrophicmembraneg Respiratory rateh

Camponotus species (F) YIM Sepalouse 16–21x A/R 6.91*,**; 1.93†,¶

Dolichoderus species (D) YD Plesiomorphic 12–17+ A/R? 1.04*,‡

Iridomyrmex cladeb (D) ? Derived, reflexede 4–7x A 2.38†,§

Cephalotes species (M) YHE Sieve-like shielde 5–7+ ? ?Cataulacus species (M) YHE Plesiomorphic ? ? ?Tetraponera species (Ps) YHE Plesiomorphic ∼15+ ? ?

aSF (subfamily): F, Formicinae; D, Dolichoderinae; M, Myrmicinae; and Ps, Pseudomyrmecinae.bFor purposes of this paper, we broadly define the Iridomyrmex clade to include the genus Forelius (Chiotis et al., 2000).cMicrobial associations: Y, yes (confirmed); ?, presence or absence not confirmed.dLocation of microsymbionts (superscripts): E, extracellular; D, digestive lumen; H, hindgut (including ileum); I, intracellular; and M, midgut.eIndicates presence of proventricular canals in some species (see text).f+, Where data were unavailable, we provide supplemental data from dissections of ≥ three individuals of each species examined. PBS-preserved specimens were dissected under a stereoscope and Malpighian tubules counted. Species included: Do. attelaboides, Do. bispinosus, Do. quadentriculatus, Ce. atratus, and Te. attenuata. x, Data taken from DeMoss (1973).gA, absent; R, greatly reduced; and ?, uncertain.h*, Jensen (1978), measured as (µl O2 h

−1) at 20 °C; †, Nielsen (1986), measured as (µl O2 mg dry weight−1 h−1) at 25 °C. ‡, Do.taschenbergi; §, Forelius foetidus; ¶, average of four species; and **, average for two species.

eea_374.fm Page 6 Friday, December 9, 2005 1:38 PM

Nutritional and functional biology of exudate-feeding ants 7

of delivery of N wastes to microbes in the ileum pouch (seeprevious discussion; Figure 1). Among other ant taxa withextracellular microsymbionts, large-bodied Dolichoderusspecies may regularly have fewer MTs than do camponotinesand may process waste N at slower rates than do Blochmannia.Small-bodied Forelius foetidus has far fewer MTs thandoes its larger dolichoderine counterpart (Table 1), andas indicated previously, tiny dolichoderines may lackthe capacity necessary for recycling N in the hindgut.In cephalotines, MT numbers and arrangements havenot been studied in detail, but at least in Cephalotesatratus, the number does not approach that for similar-sized formicines (Table 1). Additionally, ingestion ofuric acid from bird droppings (Roche & Wheeler, 1997,and authors’ observations for Ce. atratus) would placeany putative N source internally, and potentially in directcontact with bacteria in the anterior ileum; such placementwould obviate the need to acquire the source from thehaemocoel.

Implications, unanswered questions, and future

directions

In the ecological literature, exudate-feeding ant taxa arefrequently treated as ‘black boxes’, or ecological equivalents,differing principally in colony size and worker body size.However, incipient studies of the digestive anatomy, andnutritional biology of major ant groups, are beginning toreveal suites of traits that help both account for disparitiesin foraging functional groups (Davidson et al., 2004) andelucidate roles of various ant taxa in ecosystems (Davidson& Cook, in press). The anatomical structures and physio-logical processes reviewed here provide a mechanisticdescription of how ants may extract and conserve N fromN-poor sources via the processing of large volumes of N-poor foods, and/or the biochemical activities of symbioticmicrobes. Although these mechanisms appear to parallelthose used by other insects with N-poor diets (Loeb et al.,1991; Douglas, 1998; Sakaguchi, 2003), substantial variationin digestive anatomy across ants aids in inferring howsuch variation affects the implementation of the proposedmechanisms.

Clearly, many uncertainties remain about the mecha-nisms by which exudate-feeding ants extract and conserveN from their N-poor diets, and studies in at least two areaswould be particularly helpful in the future. First, moreexudate-feeding ant taxa must be screened rigorously formicrosymbionts, including secondary endosymbiontsand extracellular microbes in the gut lumen. To date, mostattention has centred on Blochmannia species, which occurin all sampled Camponotus species and appear to cospeciatewith their hosts (Dasch, 1975; Sameshima et al., 1999; Degnan

et al., 2004). Genomic studies of Blochmannia strains infect-ing related Camponotus hosts show conservation ofgene regions whose products putatively contribute to hostnutrition, but show variability in genes that code forproducts (e.g., cofactors) that may alter host-symbiontnutritional exchanges. Such differences may be useful todetermine how host ecology affects the architecture ofendosymbiotic genomes (Degnan et al., 2005). Poorly studiedsecondary microsymbionts could also vary in interestingways across diets differing by species and even seasonally(Rico-Gray, 1993). Another formicine genus, Formica,presents unusual research opportunities (Sameshima et al.,1999) due to both infrageneric dietary diversity (Hölldobler& Wilson, 1990) and variable occurrence of primary endo-symbionts within species and across closely related species(Buchner, 1965; Dasch, 1975). Furthermore, if recent revi-sions of formicine tribes are correct in showing multipleorigins and losses of the sepalous proventriculus (Bolton,2003), the putative relationship between sepalous canalsand the presence of midgut endosymbionts could be testedby surveys across formicine tribes comprised of memberswith and without sepalous proventriculi. Additionally, if canalsbypassing tightly dammed proventriculi are important tothe continuity in the nutritional input to endosymbionts,microbial associations might also be suspected in doli-choderines with derived proventriculi and vestibularsinuses (see previous discussion and Eisner, 1957). Finally,in other subfamilies, studies of extracellular microsym-bionts are so far restricted to small samples of Cephalotes,Cataulacus, and Tetraponera species, where high microbialdiversity has been documented (Caetano & Cruz-Landim,1985; Caetano et al., 1994; Billen & Buschinger, 2000),and they have yet to identify microsymbionts to taxa inDolichoderus species (Caetano et al., 1989a,b). Expansionof these surveys to additional species could be especiallyproductive if framed to sample dietary and geographicvariation within the respective genera.

Second, other investigations might focus on con-tributions of micosymbionts to ant nutrition. To date, theimportance of microbial contributions to ant nutritioncan only be inferred from genomic studies of the endosym-biont’s metabolic capacity (e.g., Gil et al., 2003) and fromstudying quantity and distribution of endosymbiontsthrough host ontogeny and reproductive stages (e.g.,Sauer et al., 2002; Wolschin et al., 2004). However, it mayeventually be possible to determine them directly bylaboratory experiments with aposymbiotic ants (reviewedin Douglas, 1998 for aphids). Unexpectedly, cleansing withantibiotics of Ca. floridanus workers of their midgut endo-symbionts collapses their midgut bacteriocytes, withoutaffecting worker survivorship (Sauer et al., 2002). Togetherwith well-designed studies using radioactive or stable

eea_374.fm Page 7 Friday, December 9, 2005 1:38 PM

8 Cook & Davidson

isotopes, similar investigations focusing on larvae insteadof workers might achieve greater success.

All of these investigations, as well as those correlatingant diets with variation in the digestive system, would bemost useful if set in a phylogenetic context. Unfortunately,however, phylogenies often emerge piecemeal and currentlyfail to incorporate many key ant groups. Perhaps the ideasand hypotheses presented here will stimulate future studiesto include such groups.

Acknowledgements

Our studies were supported by the U.S. National ScienceFoundation (award no. IBN-9707932 to D.W.D), and aUniversity of Utah Graduate Research Fellowship (to S.C.C).For granting or facilitation permissions to study insidenational parks, we thank Peru’s directorate for ÁreasNacionales Protegidas (ANP, INRENA) and officials of theParque Nacional Manu, Universidad Nacional Agrariade La Molina, the Universiti Brunei Darussalam, and theKuala Belalong Field Studies Centre. Figure 1 artwork byD. Hilton.

References

Ayre GL (1967) The relationship between food and digestiveenzymes in five species of ants (Hymenoptera: Formicidae).The Canadian Entomologist 99: 408–411.

Baroni-Urbani C & Andrade ML (1997) Pollen eating, storing,and spitting in ants. Naturwissenschaften 84: 256–258.

Baroni-Urbani C, Bolton B & Ward PS (1992) The internalphylogeny of ants (Hymenoptera: Formicidae). SystematicEntomology 17: 301–329.

Billen J & Buschinger A (2000) Morphology and ultrastructure ofa specialized bacterial pouch in the digestive tract of Tetraponeraants (Formicidae, Pseudomyrmecinae). Arthropod Structureand Development 29: 259–266.

Blüthgen N & Fiedler K (2002) Interactions between weaver antsOecophylla smaragdina, homopterans, trees and lianas in anAustralian rain forest canopy. Journal of Animal Ecology 71:793–801.

Blüthgen N, Gebauer G & Fiedler K (2003) Disentangling arainforest food web using stable isotopes: dietary diversity ina species-rich ant community. Oecologia 137: 426–435.

Blüthgen N, Gottsberger G & Fiedler K (2004) Sugar and aminoacid composition of ant-attended nectar and honeydew sourcesfrom an Australian rainforest. Austral Ecology 29: 418–429.

Blüthgen N, Verhaagh M, Goitía W, Jaffé K, Morawetz W &Barthlott W (2000) How plants shape the ant community inthe Amazonian rainforest canopy: the key role of extrafloralnectaries and homopteran honeydew. Oecologia 125: 229–240.

Boevé J & Wäckers FL (2003) Gustatory perception and metabolicutilization of sugars by Myrmica rubra ant workers. Oecologia136: 508–514.

Bolton B (2003) Synopsis and classification of Formicidae.Memoirs of the American Entomological Institute 71: 1–370.

van Borm S, Buschinger A, Boomsma JJ & Billen J (2002)Tetraponera ants have gut symbionts related to nitrogen-fixingroot-nodule bacteria. Proceedings of the Royal Society ofLondon. Biological Sciencees B 269: 2023–2027.

Buchner P (1965) Endosymbiosis of Animals with Plant Micro-organisms. John Wiley and Sons, New York.

Caetano FH, Camargo-Mathias MI, Pimentel MAL & TomotakeMEM (1989a) Tipos de microrganismos presentes no ventriculode Dolichoderus attelabioides. XII Coloquio da SociedadeBrasileira de Microscopia Electronica, Caxambu, MG, pp. 111–112.

Caetano FH & da Cruz-Landim C (1985) Presence of microor-ganisms in the alimentary canal of ants of the tribe Cephalotini(Myrmicinae): location and relationship with intestinal struc-tures. Naturalia, São Paulo, 10: 37–47.

Caetano FH, Jaffe K & Crewe RW (1994) The digestive tract ofCataulacus ants: presence of microorganisms in the ileum.Les Insectes Sociaux (ed. by A Lenoir, G Arnold & M Lepage),p. 391. Université Paris Nord, Paris, France.

Caetano FH, Pimentel MAL, Tomotake MEM & Camargo-Mathias MI (1989b) Aspectos ultraestructurais do ventriculode Dolichoderus attelabioides com relaçião a presença de micro-organismos. XII Coloquio da Sociedade Brasileira de Micro-scopia Electronica, Caxambu, MG, pp. 159–160.

Chapman RF (1998) The Insects: Structure and Function, 4thedn. Cambridge University Press, Cambridge, UK.

Charnov EL (1976) Optimal foraging: the marginal value theo-rem. Theoretical Population Biology 9: 129–136.

Chiotis M, Jermiin LS & Crozier RH (2000) A molecular frame-work for the phylogeny of the ant subfamily Dolichoderinae.Molecular Phylogenetics and Evolution 17: 108–116.

Dasch GA (1975) Morphological and Molecular Studies on Intra-cellular Bacterial Endosymbionts of Insects. Yale UniversityPress, New Haven, CT, USA.

Davidson DW (1997) The role of resource imbalances in the evo-lutionary ecology of tropical arboreal ants. Biological Journalof the Linnean Society 61: 153–181.

Davidson DW (2005) Elemental stoichiometry of ants in a NewWorld rain forest. Oecologia 142: 221–231.

Davidson DW & Cook SC (in press) Tropical arboreal ants: link-ing nutrition to roles in rainforest ecosystems. Tropical ForestCommunity Ecology (ed. by WP Carsen & SA Schnitzer).Blackwell Science Ltd, London.

Davidson DW, Cook SC & Snelling RR (2004) Liquid-feedingperformances of ants (Formicidae): ecological and evolu-tionary implications. Oecologia 139: 255–266.

Davidson DW, Cook SC, Snelling RR & Chua TH (2003) Explain-ing the abundance of ants in lowland tropical rainforest cano-pies. Science 300: 969–972.

Delabie JHC (2001) Trophobiosis between Formicidae andHemiptera (Sternorrhyncha and Auchenorrhyncha): an over-view. Neotropical Entomology 30: 501–511.

DeMoss GL (1973) Phylogenetic relationships among selectedgenera of North American Myrmicinae as evidenced bycomparative morphological studies of proventriculi and

eea_374.fm Page 8 Friday, December 9, 2005 1:38 PM

Nutritional and functional biology of exudate-feeding ants 9

Malpighian tubules. PhD Dissertation, University of Tennes-see, Knoxville, TN, USA.

Degnan PH, Lazarus AB, Brock CD & Wernegreen JJ (2004)Host-symbiont stability and fast evolutionary rates in an ant–bacterium association: cospeciation of Camponotus speciesand their endosymbionts, Candidatus Blochmannia. Syste-matic Biology 53: 95–110.

Degnan PH, Lazarus AB & Wernegreen JJ (2005) Genomesequence of Blochmannia pennsylvanicus indicates parallelevolutionary trends among bacterial mutualists of insects.Genomic Research 15: 1023–1033.

Dill M, Williams DJ & Maschwitz U (2002) Herdsmen ants andtheir mealybug partners. Abhandlungen de Senckenber-gischen Naturforschenden Gesellschaft. Frankfurt Am Main557: 1–373.

Douglas AE (1993) The nutritional quality of phloem sap utilizedby natural aphid populations. Ecological Entomology 18: 31–38.

Douglas AE (1998) Nutritional interactions in insect-microbialsymbioses: aphids and their symbiotic bacteria Buchnera.Annual Review of Entomology 43: 17–37.

Eisner T (1957) A comparative morphological study of the pro-ventriculus of ants (Hymenoptera: Formicidae). Bulletin of theMuseum of Comparative Zoology 16: 441–489.

Engel V, Fischer MK & Wäckers FL (2001) Interactions betweenextrafloral nectaries, aphids and ants: are there competitioneffects between plant and homopteran sugar sources? Oecolo-gia 129: 577–584.

Febvay G, Rahbé Y, Rynkiewiecz M, Guillaud J & Bonnot G(1999) Fate of dietary sucrose and neosynthesis of amino acidsin the pea ahpid, Acyrthosiphon pisum, reared on differentdiets. Journal of Experimental Biology 202: 2639–2652.

Ferreira C, Torres BB & Terra WR (1998) Substrate specificities ofmidgut beta-glycosidases from insects of different orders.Comparative Biochemistry and Physiology 119B: 219–225.

Fukatsu T (1994) Endosymbiosis of aphids with microorganisms:a model case of dynamic endosymbiotic evolution. PlantSpecies Biology 9: 145–154.

Gaebler OH, Vitti TG & Vumirovich R (1966) Isotope effects inmetabolism of 15N and 14N from unlabeled dietary proteins.Canadian Journal of Biochemistry 44: 1049–1057.

Gil R, Silva FJ, Zientz E, Delamotte F & González-Candelas Fet al. (2003) The genome sequence of Blochmannia floridanus:comparative analysis of reduced genomes. Proceedings of theNational Academy of Sciences, USA 100: 9388–9393.

Harada H, Oyaizu H & Ishikawa H (1996) A consideration aboutthe origin of aphid intracellular symbiont in connection withgut bacterial flora. Journal of Genetics and Applied Microbiol-ogy 42: 17–26.

Hölldobler B & Wilson EO (1990) The Ants. Belknap Press,Harvard University, Cambridge, MA, USA.

Hongoh Y & Ishikawa H (1997) Uric acid as a nitrogen resourcefor the brown planthopper, Nilaparvata lugens: studies withsynthetic diets and aposymbiontic insects. Zoological Science14: 581–586.

Hunt JH (2003) Cryptic herbivores of the rain forest canopy.Science 300: 916–917.

Ikeda TP, Shauger AE & Kustu S (1996) Salmonella typhimuriumapparently perceives external nitrogen limitation as internalglutamine limitation. Journal of Molecular Biology 259: 589–607.

Jaffe K, Caetano FH, Sánchez P, Hernández JV & Caraballo Let al. (2001) Sensitivity of ant (Cephalotes) colonies and indivi-duals to antibiotics implies feeding symbiosis with gut micro-organisms. Canadian Journal of Zoology 79: 1120–1124.

Jensen TF (1978) Annual production and respiration in antpopulations. Oikos 31: 207–213.

Kay A (2002) Applying optimal foraging theory to assess nutrientavailability ratios for ants. Ecology 83: 1933–1944.

Lehane MJ (1997) Peritrophic matrix structure and function.Annual Review of Entomology 42: 525–550.

Loeb SC, Schwab RG & Demment MW (1991) Response ofpocket gophers (Thomomys bottae) to changes in diet quality.Oecologia 86: 542–551.

MacKay WP (1993) A review of the New World ants of the genusDolichoderus (Hymenoptera: Formicidae). Sociobiology 22: 1–148.

Macko SA, Fogel-Estep ML, Engel MH & Hare PE (1986) Kineticfractionation of stable nitrogen isotopes during amino acidtransamination. Geochimica et Cosmochimica Acta 50: 2143–2146.

Mittler TE (1958) Studies on the feeding and nutrition of Tubero-lachnus salignus (Gmelin) (Homoptera, Aphididae). II. Thenitrogen and sugar composition of ingested phloem sap andexcreted honeydew. Journal of Experimental Biology 35: 74–84.

Moran NA, Dale C, Dunbar H, Smith WA & Ochman H (2003)Intracellular symbionts of sharpshooters (Insecta: Hemiptera:Cicadellinae) form a distinct clade with a small genome. Envi-ronmental Microbiology 5: 116–126.

Nakabachi A & Ishikawa H (1998) Differential display of mRNAsrelated to amino acid metabolism in the endosymbiotic systemof aphids. Insect Biochemstry and Molecular Biology 27:1057–1062.

Nielsen MG (1986) Respiratory rates of ants from differentclimatic areas. Journal of Insect Physiology 32: 125–131.

Potrikus CJ & Breznak JA (1981) Gut bacteria recycle uric acidnitrogen in termites: a strategy for nutrient conservation. Pro-ceedings of the National Academy of Sciences, USA 78: 4601–4605.

Raubenheimer D & Simpson SJ (1998) Nutrient transfer func-tions: the site of integration between feeding behaviour andnutritional physiology. Chemoecology 8: 61–68.

Raubenheimer D & Simpson SJ (2004) Organismal stoichiome-try: quantifying non-independence among food components.Ecology 85: 1203–1216.

Richards AG & Richards PA (1977) The peritrophic membranesof insects. Annual Review of Entomology 22: 219–240.

Rico-Gray V (1993) Use of plant-derived food resources by antsin the dry tropical lowlands of coastal Veracruz. Mexico. Bio-tropica 25: 301–315.

Roche RK & Wheeler DE (1997) Morphological specializations ofthe digestive tract of Zacryptocerus rohweri (Hymenoptera:Formicidae). Journal of Morphology 234: 253–262.

eea_374.fm Page 9 Friday, December 9, 2005 1:38 PM

10 Cook & Davidson

Sakaguchi E (2003) Digestive strategies of small hindgut fer-menters. Journal of Animal Science 74: 327–337.

Sameshima S, Hasegawa E, Kitade O, Minaka N & Matsumoto T(1999) Phylogenetic comparisons of endosymbionts with theirhost ants based on molecular evidence. Zoological Science 16:993–1000.

Sandstrom JP & Moran NA (2001) Amino acid budgets in threeaphid species using the same host plant. Physiological Ento-mology 26: 202–211.

Sasaki T & Ishikawa H (1993) Nitrogen recycling in the endosym-biotic system of the pea aphid, Acyrthosiphon pisum. Zoologi-cal Science 10: 779–785.

Sauer C, Dudaczek D, Hölldobler B & Gross R (2002) Tissuelocalization of the endosymbiotic bacterium ‘CandidatusBlochmannia floridanus’ in adults and larvae of the carpenterant Camponotus floridanus. Applied and Environmental Micro-biology 68: 4187–4193.

Schoeninger MJ & DeNiro MJ (1984) Nitrogen and carbonisotope composition of bone collagen from marine and terrestrialanimals. Geochimica et Cosmochimica Acta 48: 625–639.

Schröder D, Deppisch H, Obermayer M, Krohne G & Stacke-brandt E et al. (1996) Intracellular endosymbiotic bacteria ofCamponotus species (carpenter ants): systematics, evolutionand ultrastructural characterization. Molecular Microbiology21: 479–489.

Shattuck SO (1992) Higher classification of the ant subfamiliesAneuretinae, Dolichoderinae and Formicinae (Hymenoptera:Formicidae). Systematic Entomology 17: 199–206.

Shattuck SO (1995) Generic-level relationships within the antsubfamily Dolichoderinae (Hymenoptera, Formicidae). Sys-tematic Entomology 20: 217–228.

Shigenobu S, Watanabe H, Hattori M, Sakaki Y & Ishikawa H(2000) Genome sequence of the endocellular bacterial sym-biont of aphids Buchnera sp. Nature 407: 81–86.

Simpson SJ & Raubenheimer D (1996) Feeding behaviour,sensory physiology and nutrient feedback: a unifying model.Entomologia Experimentalis et Applicata 80: 55–64.

Spaulding AW & von Dohlen CD (1998) Phylogenetic characteri-zation and molecular evolution of bacterial endosymbionts inPsyllids (Hemiptera: Sternorrhyncha). Molecular Biology andEvolution 15: 1506–1513.

Sterner RW & Elser JJ (2002) Ecological Stoichiometry: the Bio-logy of Elements from Molecules to the Biosphere. PrincetonUniversity Press, Princeton, NJ, USA.

Stryer L (1995) Biochemistry, 4th edn. W.H. Freeman, NY, USA.Terra WR (2001) The origin and functions of the insect peritrophic

membrane and peritrophic gel. Archives of Insect Biochemistryand Physiology 47: 47–61.

Thao ML & Baumann P (2004) Evolutionary relationships ofprimary prokaryotic endosymbionts of whiteflies and theirhosts. Applied and Environmental Microbiology 70: 3401–3406.

Tjallingii WF (1995) Regulation of phloem sap feeding by aphids.Regulatory Mechanisms in Insect Feeding (ed. by RF Chap-man & G de Boer), pp. 120–209. Chapman & Hall, New York.

Van der Zanden MJ & Rasmussen JB (2001) Variation in δ15N andδ13C trophic fractionation: implications for aquatic food webstudies. Limnology and Oceanography 46: 2061–2066.

Waterhouse DF (1953) Occurrence and endodermal origin of theperitrophic membrane in some insects. Nature 172: 676.

Wernegreen JJ, Degnan PH, Lazarus AB, Palacios C & Borden-stein SR (2003) Genome evolution in an insect cell: distinctfeatures of an ant-bacterial partnership. Biological Bulletin204: 221–231.

Wheeler WM & Bailey IW (1920) The feeding habits of pseu-domymecine and other ants. Transactions of the AmericanPhilosophical Society 22: 253–279.

Wheeler GC & Wheeler J (1976) Ant larvae: review and synthesis.Memoirs of the Entomological Society of Washington, number7, 1–108. The Entomological Society of Washington DC.

White TCR (1993) The Inadequate Environment: Nitrogen andthe Abundance of Animals. Springer-Verlag, New York.

Wilkinson TL, Ashford DA, Pritchard J & Douglas AE (1997)Honeydew sugars and osmoregulation in the pea aphidAcyrthosiphon pisum. Journal of Experimental Biology 200:2137–2143.

Wolschin F, Hölldobler B, Gross R & Zientz E (2004) Replicationof the endosymbiotic bacterium Blochmannia floridanus iscorrelated with the developemental and reproductive stages ofits ant host. Applied and Environmental Microbiology 70:4096–4102.

Yao I & Akimoto SI (2002) Flexibility in the composition andconcentration of amino acids in honeydew of the drepa-nosiphid aphid Tuberculatus quercicola. Ecological Entomo-logy 27: 745–752.

Zhuzhikov DP (2001) Autocoprophagy is a precursor of procto-deal trophallaxis in cockroaches and termites. Zoologicheskii-Zhurnal 80: 403–411.

eea_374.fm Page 10 Friday, December 9, 2005 1:38 PM