Scavenger deterrent factor (SDF) from symbiotic bacteria of entomopathogenic nematodes

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are symbiotically associated with bacteria in the genera Xenorhabdus and Photorhabdus, respectively. The symbiotic bacteria produce a chemical compound(s) that deterred ants from feeding on nematode-killed insects (i.e., cadavers) and has been previously referred to as an Ant Deterrent Factor (ADF). We studied the response of different arthropod scavenger species which included the ant Lepisiota frauenfeldi, cricket Gryllus bimaculatus, wasps Vespa orientalis and Paravespula sp., and calliphorid fly Chrysomya albiceps, to ADF. These scavengers (ants, crickets, and wasps) were exposed to cadavers with and without the nematode/bacterium complex or to Photorhabdus luminescens cultures of different ages on different substrates. The ant, cricket, and wasp species did not feed on nematode-killed insects containing the nematode/bacterium complex that were 2 days old and older but fed on 1-day-old nematode-killed and freeze -killed insects. Crickets consumed 2- to 7-day-old axenic nematode-killed insects, 1-, 4-, and 5-day-old insects killed by the bacterium, Serratia marcescens, and freeze-killed, putrid insects that were up to 10 days old. The crickets only partially consumed 2- and 3-day-old insects killed by S. marcescens which differed significantly from the 1-, 4-, and 5-day-old killed insects by this bacterium. Ants fed only on 5% sucrose solution (control) and 1- to 3- day old cultures of P. luminescens containing 5% sucrose but not on older cultures of P. luminescens. Wasps did not feed on meat treated with P. luminescens supernatant, whereas they fed on meat treated with Escherichia coli supernatant and control meat. Calliphorid flies did not oviposit on meat treated with P. luminescens supernatant but did oviposit on untreated meat. Based on the response of these scavengers, the chemical compound(s) responsible for this deterrent activity should be called "scavenger deterrent factor" (SDF).     

To complete their life cycle, pathogenic nematode-bacteria complexes deter scavengers from feeding on their host cadaver

The life cycle of commercially used molluscicidal rhabditid nematodes Phasmarhabditis hermaphrodita and entomopathogenic steinernematid nematodes is similar: infective stages carry symbiotic bacteria, which kill their host. Nematodes complete their life cycle feeding on the proliferating symbiont and the host tissue. After 1-2 weeks, new infective stages carrying the bacteria leave the host cadaver in search of new hosts. The removal of invertebrate cadavers by scavengers is extremely fast and represents a severe threat to the developing nematodes.Two-choice trials were used to assess prey choice of the generalist predator/scavenger Pterostichus melanarius (Coleoptera: Carabidae) between Deroceras reticulatum (Mollusca: Agriolimacidae) slugs or wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae killed by infection of P. hermaphrodita/Steinernema affine and control killed by freezing. We demonstrate that the presence of either of the two nematodes tested deters the beetles from consuming infected cadavers. As P. hermaprodita cannot infect an insect host, we hypothesise the deterrent effect being an evolutionary adaptation of the nematode/bacteria complex rather than the ability of the beetles to avoid potentially infective cadavers.     

Facultative scavenging as a survival strategy of entomopathogenic nematodes

Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode-bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.     

Impacts of Argentine ants on avian nesting success

Biological invasions can have severe and widespread impacts on ecological communities. A few species of ants have become particularly damaging invaders but quantitative data of their impacts on many taxa is still lacking. We provide experimental evidence using artificial nests baited with quail eggs that the invasive Argentine ant (Linepithema humile) can be a significant avian nest predator – Argentine ants recruited to more nests and in higher abundance than the native ant species they displace. However, at a site invaded by Argentine ants, we monitored over 400 nests of a ground-nesting species, the Dark-eyed Junco (Junco hyemalis), and found that less than 2% of nests failed as a result of Argentine ant predation/infestation. A review of the literature also suggests that Argentine ants may not be a serious threat to bird nests relative to other predators or parasites. However, invasive ants with the capability of overwhelming prey though stinging (specifically the red-imported fire ant, Solenopsis invicta), may have a higher impact on avian nesting success. Received 14 January 2005; revised 28 April 2005; accepted 12 May 2005.     

Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes

Mutual effects between the symbiotic bacteria of entomopathogenic nematodes, Photorhabdus luminescens and Xenorhabdus poinarii, and entomopathogenic fungi were investigated in vitro. A dual culture assay on nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA) medium revealed that P. luminescens is antagonistic to Metarhizium anisopliae, Beauveria bassiana, B. brongniartii and Paecilomyces fumosoroseus by inhibiting their growth and conidial production; the fungal growth was not inhibited by X. poinarii. In a second laboratory experiment, crude extract produced by M. anisopliae was tested for its activity against P. luminescens and X. poinarii. Crude extract from M. anisopliae was antibacterial to P. luminescens and X. poinarii at 1000 g/ml and inhibited their growth on NBTA, but had no effect at 100 or 10 g/ml. The influence of the crude extract of M. anisopliae on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis and Steinernema glaseri was assayed on Sabouraud Dextrose Agar (SDA) plates. Results showed that the crude extract of M. anisopliae had no toxic effects even at highest concentration (1000 g/ml).     

Consumption of introduced prey by native predators: Argentine ants and pit-building ant lions

When populations of native predators are subsidized by numerically dominant introduced species, the structure of food webs can be greatly altered. Surprisingly little is known, however, about the general factors that influence whether or not native predators consume introduced species. To learn more about this issue, we examined how native pit-building ant lions (Myrmeleon) are affected by Argentine ant (Linepithema humile) invasions in coastal southern California. Compared to areas without L. humile, invaded areas contained few native ant species and were deficient in medium-sized and large bodied native ants. Based on these differences, we predicted that Argentine ants would negatively affect ant lion larvae. Contrary to this expectation, observational surveys and laboratory growth rate experiments revealed that Myrmeleon were heavier, had longer mandibles, and grew more quickly when their main ant prey were Argentine ants rather than native ants. Moreover, a field transplant experiment indicated that growth rates and pupal weights were not statistically different for larval ant lions reared in invaded areas compared to those reared in uninvaded areas. Argentine ants were also highly susceptible to capture by larval Myrmeleon. The species-level traits that presumably make Argentine ant workers susceptible to capture by larval ant lions—small size and high activity levels—appear to be the same characteristics that make them unsuitable prey for vertebrate predators, such as horned lizards. These results underscore the difficulties in predicting whether or not numerically dominant introduced species serve as prey for native predators.     

Aggressive interactions between the introduced Argentine ant, Linepithema humile and the native odorous house ant, Tapinoma sessile

The Argentine ant (Linepithema humile) is an invasive species that disrupts the balance of natural ecosystems by displacing indigenous ant species throughout its introduced range. The mechanisms by which Argentine ants effectively compete against native ant species have been previously addressed in field studies that centered on interference and exploitation competition at baits and mainly examined the colony-level performance of Argentine ants. Detailed behavioral observations explaining the basis for the strong competitive ability of L. humile are comparatively rare. To gain a better understanding of the mechanisms by which Argentine ants displace native ants we examined the aggressive interactions between the Argentine ants and the odorous house ant, Tapinoma sessile in four different aggression assays: (1) worker dyad interactions, (2) symmetrical group interactions, (3) intruder introductions into an established resident colony, and (4) a resource competition assay which focused on competition for food and nesting space. Our results demonstrate a clear disparity between worker-level and colony-level fighting ability of Argentine ants and provide behavioral evidence to explain the superior interference ability of Argentine ants in group assays. Argentine ants experienced mixed success in fighting against odorous house ants in dyad interactions, but gradually gained a numerical advantage in symmetrical group interactions by active cooperation among nestmates. Results of the resource competition assay indicate that Argentine ants recruit rapidly, numerically dominate food and nesting sites, and aggressively displace T. sessile from baits. Taken together, the results of these assays allow us to pinpoint the behavioral mechanisms responsible for the remarkable competitive ability of Argentine ants.     

Ant species confer different partner benefits on two neotropical myrmecophytes

The dynamics of mutualistic interactions involving more than a single pair of species depend on the relative costs and benefits of interaction among alternative partners. The neotropical myrmecophytes Cordia nodosa and Duroia hirsuta associate with several species of obligately symbiotic ants. I compared the ant partners of Cordia and Duroia with respect to two benefits known to be important in ant-myrmecophyte interactions: protection against herbivores provided by ants, and protection against encroaching vegetation provided by ants. Azteca spp., Myrmelachista schumanni, and Allomerus octoarticulatus demerarae ants all provide the leaves of Cordia and Duroia some protection against herbivores. However, Azteca and Allomerus provide more protection than does Myrmelachista to the leaves of their host plants. Although Allomerus protects the leaves of its hosts, plants occupied by Allomerus suffer more attacks by herbivores to their stems than do plants occupied by other ants. Relative to Azteca or Allomerus, Myrmelachista ants provide better protection against encroaching vegetation, increasing canopy openness over their host plants. These differences in benefits among the ant partners of Cordia and Duroia are reflected in the effect of each ant species on host plant size, growth rate, and reproduction. The results of this study show how mutualistic ant partners can differ with respect to both the magnitude and type of benefits they provide to the same species of myrmecophytic host.     

Visual and Chemical Cues Used in Host Location and Acceptance by a Dipteran Parasitoid

Locating potential hosts for egg laying is a critical challenge in the life history of many insects. Female insects in several orders have evolved mechanisms to find hosts by using olfactory and visual signals derived from their hosts. We describe visual and chemical cues used by the dipteran parasitoid Apocephalus paraponerae (Diptera: Phoridae) in the location and acceptance of its host ant Paraponera clavata (Hymenoptera: Formicidae: Ponerinae). Our results show that A. paraponerae uses the visual cue of ant body size when locating hosts at short range and that these flies lay more eggs in ants that retain their surface chemicals than in ants with these chemicals removed. We compare the cues used by A. paraponerae with cues used by tephritid fruit flies in location and acceptance of their hosts, and we suggest further avenues for the study of host location, acceptance, and host discrimination of A. paraponerae and other parasitoids of ants.     

Interference competition by Argentine ants displaces native ants: implications for biotic resistance to invasion

The Argentine ant Linepithema humile (Dolichoderinae) is one of the most widespread invasive ant species in the world. Throughout its introduced range, it is associated with the loss or reduced abundance of native ant species. The mechanisms by which these native species are displaced have received limited attention, particularly in Australia. The role of interference competition in the displacement of native ant species by L. humile was examined in coastal vegetation in central Victoria (southeastern Australia). Foragers from laboratory colonies placed in the field consistently and rapidly displaced the tyrant ant Iridomyrmex bicknelli, the big-headed ant Pheidole sp. 2, and the pony ant Rhytidoponera victoriae from baits. Numerical and behavioural dominance enabled Argentine ants to displace these ants in just 20 min; the abundance of native species at baits declined 3.5–24 fold in direct relation to the rapid increase in L. humile. Most precipitous was the decline of I. bicknelli, even though species in this typically dominant genus have been hypothesized to limit invasion of L. humile in Australia. Interspecific aggression contributed strongly to the competitive success of Argentine ants at baits. Fighting occurred in 50–75% of all observed interactions between Argentine and native ants. This study indicates that Argentine ants recruit rapidly, numerically dominate, and aggressively displace from baits a range of Australian native ant species from different subfamilies and functional groups. Such direct displacement is likely to reduce native biodiversity and indirectly alter food web structure and ecosystem processes within invaded areas. Biotic resistance to Argentine ant invasion from native ants in this coastal community in southeastern Australia is not supported in this study.     

Nest raiding by the invasive Argentine ant on colonies of the harvester ant, Pogonomyrmex subnitidus

Invasive ants often displace native ants, and published studies that focus on these interactions usually emphasize interspecific competition for food resources as a key mechanism responsible for the demise of native ants. Although less well documented, nest raiding by invasive ants may also contribute to the extirpation of native ants. In coastal southern California, for example, invasive Argentine ants (Linepithema humile) commonly raid colonies of the harvester ant, Pogonomyrmex subnitidus. On a seasonal basis the frequency and intensity of raids vary, but raids occur only when abiotic conditions are suitable for both species. In the short term these organized attacks cause harvester ants to cease foraging and to plug their nest entrances. In unstaged, one-on-one interactions between P. subnitidus and L. humile workers, Argentine ants behaved aggressively in over two thirds of all pair-wise interactions, despite the much larger size of P. subnitidus. The short-term introduction of experimental Argentine ant colonies outside of P. subnitidus nest entrances stimulated behaviors similar to those observed in raids: P. subnitidus decreased its foraging activity and increased the number of nest entrance workers (many of which labored to plug their nest entrances). Raids are not likely to be the result of competition for food. As expected, P. subnitidus foraged primarily on plant material (85% of food items obtained from returning foragers), but also collected some dead insects (7% of food items). In buffet-style choice tests in which we offered Argentine ants food items obtained from P. subnitidus, L. humile only showed interest in dead insects. In other feeding trials L. humile consistently moved harvester ant brood into their nests (where they were presumably consumed) but showed little interest in freshly dead workers. The raiding behavior described here obscures the distinction between interspecific competition and predation, and may well play an important role in the displacement of native ants, especially those that are ecologically dissimilar to L. humile with respect to diet. Received 15 July 2005; revised 19 October 2005; accepted 26 October 2005.     

Seasonal patterns of persistence and infectivity of Metarhizium anisopliae var. acridum in grasshopper cadavers in the Sahel

Field-based experiments were conducted to evaluate the fate and infectivity of the entomopathogenic fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes) in grasshopper cadavers in the Sahel. Unlike uninfected cadavers, which were rapidly scavenged, those infected with the fungus persisted in the environment for a number of weeks. The environmental factor most associated with cadaver disappearance was rainfall. The high environmental humidity associated with rainfall was also required for sporulation of the fungus on host cadavers, although the likelihood of sporulation differed between microsites. Characteristics of the infection profile from infective cadavers were investigated by the sequential exposure of uninfected hosts to sporulating cadavers in field cages. This experiment revealed that cadavers remained infective for > 30 days, with the net infectivity changing through time. The most likely explanation for these changes is climatic influences on both the fungus and host. High humidity was not required for infection. A measurement of the transmission coefficient between healthy hosts and sporulating cadavers in the field was obtained at a realistic density of infectious cadavers. This revealed a figure of 0.45 m² day^–1. Overall, these experiments show that following host death, M. anisopliae var. acridum can be persistent in the environment, sporulate on host cadavers and reinfect new hosts at a realistically low field density, although at least in arid or semi-arid areas, rainfall may be critical to the horizontal transmission of this pathogen.     

Entomopathogenic symbiotic bacteria,Xenorhabdus and Photorhabdus of nematodes

Xenorhabdus and Photorhabdus species are entomopathogenic bacteria with a wide insect host range, that belong to the family Enterobacteriaceae. Xenorhabdus and Photorhabdus species symbiotically associate with nematodes of the families Steinernematidae and Heterorhabditidae respectively. The factor(s) determining the symbiotic interaction between nematodes and bacteria are yet to be identified. Xenorhabdus and Photorhabdus species exist in two main phenotypic forms, a phenomenon known as phase variation. The phase I (or primary form) varies from phase II (or secondary form) in certain physiological and morphological characteristics. There is no variation in the DNA integrity of phase I and phase II and this supports epigenetic regulatory mechanism in phase variation. Certain pathogenic determinants such as pili, lipopolysaccharides and toxins contribute to the pathogenicity of Xenorhabdus and Photorhabdus species, and both appear to be equally pathogenic to insects. The observed similarity in their virulence to insect hosts may reflect possible in vivo conversion of phase II to phase I, however the host cellular invasion and virulence is yet to be properly understood. The virulence of Xenorhabdus variants varies among insects apparently due to factors which include the feeding habits of the insects. The molecular mechanism and biological significance of phase variation are presently unknown.     

Does scavenging extend the host range of entomopathogenic nematodes (Nematoda: Steinernematidae)?

Living and freeze-killed natural and laboratory hosts, with different susceptibility to entomopathogenic nematodes, were exposed to the larvae of Steinernema affine and Steinernema kraussei in two different experimental arenas (Eppendorf tubes, Petri dishes), and the success of the colonisation and eventual progeny production were observed. Both nematodes were able to colonise both living and dead larvae of Galleria mellonella (Lepidoptera) and adult Blatella germanica (Blattodea) even though the progeny production in dead hosts was lower on average. Living carabid beetles, Poecilus cupreus, and elaterid larvae (Coleoptera) were resistant to the infection, however, both nematodes were able to colonise and multiply in several dead P. cupreus and in a majority of dead elaterid larvae. By scavenging, EPNs can utilise cadavers of insects that are naturally resistant to EPN infection, and so broaden their host range.     

Effects of a novel entomopathogenic nematode-infected host formulation on cadaver integrity, nematode yield, and suppression of Diaprepes abbreviatus and Aethina tumida

An alternative approach to applying entomopathogenic nematodes entails the distribution of nematodes in their infected insect hosts. Protection of the infected host from rupturing, and improving ease of handling, may be necessary to facilitate application. In this study our objective was to test the potential of a new method of formulating the infected hosts, i.e., enclosing the infected host in masking tape. Tenebrio molitor L. cadavers infected with Heterorhabditis indica Poinar, Karunakar and David or Steinernema carpocapsae (Weiser) were wrapped in tape using an automatic packaging machine; the machine was developed to reduce labor and to standardize the final product. The effects of the tape formulation on the ability to protect the cadavers from mechanical damage, nematode yield, and pest control efficacy were tested. After exposure to mechanical agitation at 7-d-post-infection, S. carpocapsae cadavers in tape were more resistant to rupture than cadavers without tape, yet H. indica cadavers 7-d-post-infection were not affected by mechanical agitation (with or without tape), nor was either nematode affected when 4-d-old cadavers were tested. Experiments indicated that infective juvenile yield was not affected by the tape formulation. Laboratory experiments were conducted measuring survival of the root weevil, Diaprepes abbreviatus (L.), or the small hive beetle, Aethina tumida Murray, after the application of two H. indica-infected hosts with or without tape per 15 cm pot (filled with soil). A greenhouse experiment was also conducted in a similar manner measuring survival of D. abbreviatus. In all experiments, both the tape and no-tape treatments caused significant reductions in insect survival relative to the control, and no differences were detected between the nematode treatments. Fifteen days post-application, the infected host treatments caused up to 78% control in A. tumida, 91% control in D. abbreviatus in the lab, and 75% in the greenhouse. These results indicate potential for using the tape-formulation approach for applying nematode infected hosts.     

Natural enemies of natural enemies: the potential top‐down impact of predators on entomopathogenic nematode populations

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Invasive interactions: can Argentine ants indirectly increase the reproductive output of a weed?

The direct and indirect interactions of invasive ants with plants, insect herbivores, and Hemiptera are complex. While ant and Hemiptera interactions with native plants have been well studied, the effects of invasive ant–scale insect mutualisms on the reproductive output of invasive weeds have not. The study system consisted of Argentine ants (Linepithema humile), boneseed (Chrysanthemoides monilifera monilifera), and sap-sucking scale insects (Hemiptera: Saissetia oleae and Parasaissetia nigra), all of which are invasive in New Zealand. We examined the direct and indirect effects of Argentine ants on scale insects and other invertebrates (especially herbivores) and on plant reproductive output. Argentine ants spent one-third of their time specifically associated with scale insects in tending behaviours. The invertebrate community was significantly different between uninfested and infested plants, with fewer predators and herbivores on ant-infested plants. Herbivore damage was significantly reduced on plants with Argentine ants, but sooty mould colonisation was greater where ants were present. Herbivore damage increased when ants were excluded from plants. Boneseed plants infested with Argentine ants produced significantly more fruits than plants without ants. The increase in reproductive output in the presence of ants may be due to increased pollination as the result of pollinators being forced to relocate frequently to avoid attack by ants, resulting in an increase in pollen transfer and higher fruit/seed set. The consequences of Argentine ant invasion can be varied; not only does their invasion have consequences for maintaining biodiversity, ant invasion may also affect weed and pest management strategies.     

Response of ants to a deterrent factor(s) produced by the symbiotic bacteria of entomopathogenic nematodes

The production of an ant-deterrent factor(s) (ADF) by Xenorhabdus nematophila and Photorhabdus luminescens, the symbiotic bacteria of the nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora, respectively, was examined. In addition to an in vivo assay in which bacteria were tested for their ability to produce ADF within insect cadavers (M.E. Baur, H. K. Kaya, and D. R. Strong, Biol. Control 12:231-236, 1998), an in vitro microtiter dish assay was developed to monitor ADF activity produced by bacteria grown in cultures. Using these methods, we show that ADF activity is present in the supernatants of bacterial cultures, is filterable, heat stable, and acid sensitive, and passes through a 10-kDa-pore-size membrane. Thus, ADF appears to be comprised of a small, extracellular, and possibly nonproteinaceous compound(s). The amount of ADF repellency detected depends on the ant species being tested, the sucrose concentration (in vitro assays), and the strain, form, and age of the ADF-producing bacteria. These findings demonstrate that the symbiotic bacteria of some species of entomopathogenic nematodes produce a compound(s) that deters scavengers such as ants and thus could protect nematodes from being eaten during reproduction within insect cadavers.     

Argentine ants displace floral arthropods in a biodiversity hotspot

Argentine ant (Linepithema humile (Mayr)) invasions are often associated with the displacement of ground‐dwelling arthropods. Argentine ant invasions can also exert other effects on the community through interactions with plants and their associated arthropods. For example, carbohydrate resources (e.g. floral or extrafloral nectar) may influence foraging behaviour and interactions among ants and other arthropods. In South Africa's Cape Floristic Region, Argentine ants and some native ant species are attracted to the floral nectar of Leucospermum conocarpodendron Rourke (Proteaceae), a native tree that also has extrafloral nectaries (EFNs). Despite having relatively low abundance in pitfall traps, Argentine ants visited inflorescences more frequently and in higher abundance than the most frequently observed native ants, Camponotus spp., though neither native nor Argentine ant floral foraging was influenced by the EFNs. Non‐metric multidimensional scaling revealed significant dissimilarity in arthropod communities on inflorescences with Argentine ants compared to inflorescences with native or no ants, with Coleoptera, Diptera, Hymenoptera, Arachnida, Orthoptera, and Blattaria all being underrepresented in inflorescences with Argentine ants compared to ant‐excluded inflorescences. Native honeybees (Apis mellifera capensis Eschscholtz) spent 75% less time foraging on inflorescences with Argentine ants than on inflorescences without ants. Neither Argentine ant nor native ant visits to inflorescences had a detectable effect on seed set of Le. conocarpodendron. However, a pollen supplementation experiment revealed that like many other proteas, Le. conocarpodendron is not pollen‐limited. Flower predation was negatively associated with increased ant visit frequency to the inflorescences, but did not differ among inflorescences visited by native and Argentine ants. Displacement of arthropods appears to be a consistent consequence of Argentine ant invasions. The displacement of floral arthropods by Argentine ants may have far‐reaching consequences for this biodiversity hotspot and other regions that are rich in insect‐pollinated plants.