Control of pest ants by pathogenic fungi: state of the art.

Pest ants are known for their damage to biodiversity, harm to agriculture, and negative impact on human welfare. Ants thrive when environmental opportunities arise, becoming pests and/or invading non-native areas. As social insects, they are extremely difficult to control using sustainable methods like biological control. The latter, although safer to the environment, acts slowly allowing the ants to use their individual and social defenses. Among biocontrol agents, fungal pathogens were proposed as promising, however, it is difficult to ascertain their success when the bibliography has not been reviewed and condensed. Therefore, this paper is the first in performing such task by analyzing publications mainly from 2000 to 2022 about the control of pest ants by fungi. From 85 publications selected, 77% corresponded to laboratory studies. Beauveria and Metarhizium were the genera most used in laboratory and field studies. Most of them included Acromyrmex and Atta leaf-cutter ants (LCA), and Solenopsis fire ants. From laboratory experiments, we evaluated how ant net mortality was affected by ant and fungal species, and also by origin, concentration, and inoculation technique of the fungal strains tested. Beauveria bassiana and Metarhizium anisopliae produced the greatest mortality, along with the inoculation spray technique and fungal strains collected from ants. There was a positive relationship between ant mortality and fungal concentration only for those studies which evaluated more than one concentration. Twenty field experimental studies were found, covering 13 pest species, mainly LCA and Solenopsis invicta. Only B. bassiana was tested on Solenopsis, M. anisopliae was mostly used for Acromyrmex, and M. anisopliae or Trichoderma were mainly used with Atta species. The median control field efficiency varied from 20% to 85% for different fungi and ant genera. When grouping all fungal species together, the median control efficiency seemed to be better for Acromyrmex (67%) than for Atta and Solenopsis (both 43%). Our review shows that, at this stage of knowledge, it is very difficult to extrapolate any result. We offer suggestions to improve and standardize laboratory and field experimental studies in order to advance more efficiently in the fungal control of pest ants.

Conidiobolus lunulus, a newly discovered entomophthoralean species, pathogenic and specific to leaf-cutter ants.

Conidiobolus lunulus is a recently described entomophthoralean species isolated from leaf-cutter ants. This fungus discharges not only primary but also secondary conidia and microconidia of different shapes. Because nothing was known about the biology of the fungus, and its interactions with hosts, we first evaluated if its pathogenicity against leaf-cutter ants changes with the fungal age (time grown in vitro), and if it is related to the conidial structures produced. Afterwards, we tested its virulence at three combinations of temperature and relative humidity. In addition, we noted all visible causes of death by recovering different microorganisms from the dead, non-sterilized, ants to evaluate C. lunulus virulence when pathogens carried naturally by the ants were present. Finally, we used the conditions that lead to the highest mortality to evaluate fungal virulence to other host species, including non-leaf-cutter ants. Results indicated that C. lunulus was pathogenic from a culture age of 1 to 5 days, with a peak at 2-days-old, from which we registered median lethal times of 1-2 days and 85% of the cadavers with fungal conidiation. Our results suggest that primary conidia and moon-shaped microconidia were infective. Evaluations of mortality using 2-days-old cultures on several leaf-cutter ant colonies showed 1) significantly faster mortality of C. lunulus inoculated ants in comparison to controls, 2) significantly greater and faster mortality at 23.7 °C than at 21.2 °C, 3) significantly higher and faster mortality at 88% than at 57% RH, and 4) a significant reduction of other pathogens in C. lunulus inoculated ants in comparison to controls. C. lunulus was highly specific to leaf-cutter ants, as hardly any increase in mortality was observed on inoculated ants, and no conidia were recorded on cadavers of the other three non-leaf-cutter ant species tested. Our results highlight that C. lunulus is a very promising biological control agent against leaf-cutter ants.

Ritualized aggressive behavior reveals distinct social structures in native and introduced range tawny crazy ants.

How workers within an ant colony perceive and enforce colony boundaries is a defining biological feature of an ant species. Ants fall along a spectrum of social organizations ranging from single-queen, single nest societies to species with multi-queen societies in which workers exhibit colony-specific, altruistic behaviors towards non-nestmate workers from distant locations. Defining where an ant species falls along this spectrum is critical for understanding its basic ecology. Herein we quantify queen numbers, describe intraspecific aggression, and characterize the distribution of colony sizes for tawny crazy ant (Nylanderia fulva) populations in native range areas in South America as well as in their introduced range in the Southeastern United States. In both ranges, multi-queen nests are common. In the introduced range, aggressive behaviors are absent at all spatial scales tested, indicating that within the population in the Southeastern United States N. fulva is unicolonial. However, this contrasts strongly with intraspecific aggression in its South American native range. In the native range, intraspecific aggression between ants from different nests is common and ritualized. Aggression is typically one-sided and follows a stereotyped sequence of escalating behaviors that stops before actual fighting occurs. Spatial patterns of non-aggressive nest aggregation and the transitivity of non-aggressive interactions demonstrate that results of neutral arena assays usefully delineate colony boundaries. In the native range, both the spatial extent of colonies and the average number of queens encountered per nest differ between sites. This intercontinental comparison presents the first description of intraspecific aggressive behavior for this invasive ant and characterizes the variation in colony organization in the native-range, a pre-requisite to a full understanding of the origins of unicoloniality in its introduced range.

Pseudacteon notocaudatus and Pseudacteon obtusitus (Diptera: Phoridae), two new species of fire ant parasitoids from South America.

Ongoing studies in South America of phorid flies of the genus Pseudacteon Coquillett 1907 have revealed two further new species in this genus that are described here: P. obtusitus and P. notocaudatus. Both species are parasitoids of Solenopsis (F.) fire ants.

Natural enemies of Atta vollenweideri (Hymenoptera: Formicidae) leaf-cutter ants negatively affected by synthetic pesticides, chlorpyrifos and fipronil.

In southern South America, Ada vollenweideri Forel (Hymenoptera: Formicidae) is a significant pest of several crops and forestry, also considered to reduce the carrying capacity of pastures. The most usual control method used in Latin America is the application of synthetic pesticides, mainly chlorpyrifos and fipronil. However, no studies have assessed the effects of these agrochemicals on natural enemies of ants. We aimed to evaluate the efficiency of these pesticides on leaf-cutter ants' control and to test their effect on phorid fly parasitoids. Chlorpyrifos failed to exert complete control over ant colonies in the field and was gravely detrimental to specific parasitoids, reducing their percentage of parasitism, pupal survivorship, and adult longevity. Fipronil, however, exerted complete control over the treated colonies. Laboratory tests using both pesticides, either on ants from foraging trails or on pupariae, showed that chlorpyrifos and fipronil decreased larval and pupal survivorship, as well as adult longevity of parasitoids, in comparison to controls. In conclusion, these pesticides will likely affect parasitoids with regard to their reproductive capacity, leading to the decreased levels of natural parasitism observed in the field after treatments. We discuss why neither pesticide should be taken into account for integrated pest management programs.

Optimal conditions to rear phorid parasitoids (Diptera: Phoridae) of Atta vollenweideri and Acromyrmex lundii (Hymenoptera: Formicidae).

Phorid flies have been considered viable options for biological control of leaf-cutter ants because they are highly specific to these hosts, producing direct mortality and also affecting the normal functioning of colonies. Designing protocols for mass rearing of these insects requires knowing the temperature and humidity conditions that optimize their development in terms of duration, survivorship and longevity of adults. We reared Apocephalus setitarsus Brown, Eibesfeldtphora trilobata Disney, and Myrmosicarius brandaoi Disney, which are specific leaf-cutter ant parasitoids of Atta vollenweideri Forel, and Apocephalus neivai Borgmeier and Myrmosicarius catharinensis Borgmeier, which are parasitoids of Acromyrmex lundii Guérin-Méneville. Phorids were maintained under one of five different rearing conditions, 20°C high humidity (20HH), 24°C low and high humidity (24LH and 24HH), and 28°C low and high humidity (28LH and 28HH). Flies from all species could complete their development under all the conditions tested. As expected, the 20HH treatment significantly lengthened all developmental periods in all species, whereas the shortest duration was achieved under 28HH. Although pupal survivorship was highest at 24HH, the greatest longevity of adults was achieved at 20HH. Percentage of pupae obtained and pupal survivorship also exhibited the highest values at 24HH. The rearing efficiency index was highest for all species at 24HH. Therefore, we suggest this temperature and humidity combination as the most suitable for rearing these species, whereas the 20HH treatment is ideal for maintaining adults for longer periods, which would be advantageous for synchronizing matings and mass releases of adults in the field.

Pseudacteon tricuspis: its behavior and development according to the social form of its host and the role of interference competition among females.

We studied how the behavior and performance of Pseudacteon tricuspis Borgmeier varies with the social form of its host Solenopsis invicta Buren, in its native range in Argentina where monogyne colonies are more abundant than polygynes (approximately 75 vs. 25%). Female, P. tricuspis took 44% less time (50 vs. 89 s) to attack monogyne than polygyne ants, but oviposition attempts were similar (23 vs. 18 attacks). The presence of the parasitoid affected the average size of foragers on the trail, with the proportion of minor workers increasing on both social forms. In the laboratory, P. tricuspis selected similar host sizes, although pupal survival was 25% higher on monogynes than on polygynes. Developmental times of both genders were similar (33-35 d), although larger females emerged from bigger hosts. The sex ratio of P. tricuspis was more male biased when exploiting polygyne ants. Intraspecific competition significantly affected parasitoid reproductive success, being significantly higher for a solitary female than when three females were present, although the size of workers selected did not vary. The male:female ratio also changed, being 1:1 without competition but 2:1 with competition. We demonstrated for the first time the consequences of interference competition among P. tricuspis females, a common behavior observed in others parasitoids. We discuss why P. tricuspis sex ratios are always biased toward males in both social forms and suggest that similar studies of interference competition within and between already naturalized Pseudacteon species in the United States could help predict establishment patterns.

Life-history traits and parasitism rates of four phorid species (Diptera: Phoridae), parasitoids of Atta vollenweideri (Hymenoptera: Formicidae) in Argentina.

Leaf-cutting ants in the genus Atta F. (Formicidae, Attini) are among the most important pest arthropods in Central and South America, consuming more vegetation than any other animal group. Among the organisms attacking ants in nature, flies of the family Phoridae have been proposed as the most promising biocontrol agents for pest ants. Four phorid species, Apocephalus setitarsus Brown, Myrmosicarius brandaoi Disney, Myrmosicarius gonzalezae Disney, and Eibesfeldtphora trilobata Disney, were reared from ants collected at Atta vollenweideri Forel nests and off foraging trails in Santa Fe province in Argentina. E. trilobata attacked larger ants and had bigger adults than the other species, also exhibiting the longest developmental time. Correlations between size of hosts and size of adults, as well as between size of adults and developmental times, could be established only in some cases. No differences were found between the sizes of the hosts from which males and females emerged. The natural percentage of parasitism varied throughout the seasons and seemed to be influenced by the extreme drought affecting the study site. We discuss why all four species would be suitable candidates for integrating an assemblage of biocontrol agents against A. vollenweideri.

Behavior and development of Pseudacteon curvatus (Diptera: Phoridae) varies according to the social form of its host Solenopsis invicta (Hymenoptera: Formicidae) in its native range.

We assessed the performance of Pseudacteon curvatus Borgmeier with respect to the social form of Solenopsis invicta Buren in Argentina In the field, we studied the effect the parasitoid on size and proportion of ant foragers. In the laboratory, we evaluated P. curvatus oviposition preferences; host size elected; developmental periods; and sexual size dimorphism, sex ratio, and parasitoid survivorship. P. curvatus affected the average size of foraging workers on both social forms diminishing the proportion of big and increasing the proportion of minor workers. P. curvatus required a shorter orientation time and exhibited a greater number of attacks when ovipositing on monogynes workers. In the laboratory, host sizes elected by P. curvatus were similar between social forms. However, attacks on polygyne colonies were more variable, increasing the number of unviable offspring. Developmental times of females and males of P. curvatus were similar for both social forms, but total developmental periods were shorter for males from monogyne colonies. We did not find differences between sexes in emerging adults' size by social form and the female: male sex ratio was 1:1 for both social forms. P. curvatus pupae survival and adult emergence per trial from monogyne colonies were greater than from polygyne colonies. The rarity of polygyne S. invicta in its native range may prevent this phorid from adjusting its life history to that social form. Consequences of applying this phorid in biological control are discussed.

Distributional patterns of Pseudacteon associated with the Solenopsis saevissima complex in South America.

Classical biological control efforts against imported fire ants have largely involved the use of Pseudacteon parasitoids. To facilitate further exploration for species and population biotypes a database of collection records for Pseudacteon species was organized, including those from the literature and other sources. These data were then used to map the geographical ranges of species associated with the imported fire ants in their native range in South America. In addition, we found geographical range metrics for all species in the genus and related these metrics to latitude and host use. Approximately equal numbers of Pseudacteon species were found in temperate and tropical regions, though the majority of taxa found only in temperate areas were found in the Northern Hemisphere. No significant differences in sizes of geographical ranges were found between Pseudacteon associated with the different host complexes of fire ants despite the much larger and systemic collection effort associated with the S. saevissima host group. The geographical range of the flies was loosely associated with both the number of hosts and the geographical range of their hosts. Pseudacteon with the most extensive ranges had either multiple hosts or hosts with broad distributions. Mean species richnesses of Pseudacteon in locality species assemblages associated with S. saevissima complex ants was 2.8 species, but intensively sampled locations were usually much higher. Possible factors are discussed related to variation in the size of geographical range, and areas in southern South America are outlined that are likely to have been under-explored for Pseudacteon associated with imported fire ants.

Cytogenetic analysis of three species of Pseudacteon (Diptera, Phoridae) parasitoids of the fire ants using standard and molecular techniques.

Pseudacteon flies, parasitoids of worker ants, are being intensively studied as potentially effective agents in the biological control of the invasive pest fire ant genus Solenopsis (Hymenoptera: Formicidae). This is the first attempt to describe the karyotype of P. curvatus Borgmeier, P. nocens Borgmeier and P. tricuspis Borgmeier. The three species possess 2n = 6; chromosomes I and II were metacentric in the three species, but chromosome pair III was subtelocentric in P. curvatus and P. tricuspis, and telocentric in P. nocens. All three species possess a C positive band in chromosome II, lack C positive heterochromatin on chromosome I, and are mostly differentiated with respect to chromosome III. P. curvatus and P. tricuspis possess a C positive band, but at different locations, whereas this band is absent in P. nocens. Heterochromatic bands are neither AT nor GC rich as revealed by fluorescent banding. In situ hybridization with an 18S rDNA probe revealed a signal on chromosome II in a similar location to the C positive band in the three species. The apparent lack of morphologically distinct sex chromosomes is consistent with proposals of environmental sex determination in the genus. Small differences detected in chromosome length and morphology suggests that chromosomes have been highly conserved during the evolutionary radiation of Pseudacteon. Possible mechanisms of karyotype evolution in the three species are suggested.

Cytogenetic analysis of three species of Pseudacteon (Diptera, Phoridae) parasitoids of the fire ants using standard and molecular techniques

Pseudacteon flies, parasitoids of worker ants, are being intensively studied as potentially effective agents in the biological control of the invasive pest fire ant genus Solenopsis (Hymenoptera: Formicidae). This is the first attempt to describe the karyotype of P. curvatus Borgmeier, P. nocens Borgmeier and P. tricuspis Borgmeier. The three species possess 2n = 6; chromosomes I and II were metacentric in the three species, but chromosome pair III was subtelocentric in P. curvatus and P. tricuspis, and telocentric in P. nocens. All three species possess a C positive band in chromosome II, lack C positive heterochromatin on chromosome I, and are mostly differentiated with respect to chromosome III. P. curvatus and P. tricuspis possess a C positive band, but at different locations, whereas this band is absent in P. nocens. Heterochromatic bands are neither AT nor GC rich as revealed by fluorescent banding. In situ hybridization with an 18S rDNA probe revealed a signal on chromosome II in a similar location to the C positive band in the three species. The apparent lack of morphologically distinct sex chromosomes is consistent with proposals of environmental sex determination in the genus. Small differences detected in chromosome length and morphology suggests that chromosomes have been highly conserved during the evolutionary radiation of Pseudacteon. Possible mechanisms of karyotype evolution in the three species are suggested.

Associations of fire ant phorids and microhabitats.

We examined flight activity patterns for a guild of fire ant parasitoids in western Argentina in relationship to their host's location (mound/foraging trail) and light condition (full sun/partial sun/full shade) at different scales, from the individually sampled mound to the full day's summation for each species. We asked first whether taxa showed preferences among these conditions, and second, whether certain species and sexes might be found together more frequently than expected to by chance. All species, except the P. obtusus species complex, were significantly more likely to be found attacking ants at disturbed mounds than at paired foraging trails. The P. nocens complex and P. litoralis were more likely to be in the shade when temperatures were above the overall mean of the study (28.3 degrees C), whereas others, such as the P. obtusus complex and P. tricuspis, were more likely to be in full sun under these same conditions. Our analyses indicated that a limited set of species, particularly P. nocens with P. litoralis, and males with female P. obtusus and P. tricuspis, were more likely to be found together than expected. We also found decreasing proportions of males with increasing time of analysis. We discuss the implications of host location, metereological conditions, and sex ratios in relationship to ongoing classical biological control efforts using species of these phorids.

Development of Pseudacteon nocens (Diptera: Phoridae) on Solenopsis invicta and Solenopsis richteri fire ants (Hymenoptera: Formicidae).

We studied the developmental performance of the large morph of Pseudacteon nocens Borgmeier (Diptera: Phoridae), a prospective biological control agent of imported fire ants (Hymenoptera: Formicidae). We measured selected life history traits of this parasitoid as a function of 1) host species (Solenopsis invicta Buren versus Solenopsis richteri Forel), 2) temperature (22 versus 28 degrees C), 3) source population of the fly (Corrientes and Santiago del Estero, Argentina), and 4) varied size distributions of offered host ants. Developmental periods were influenced by host species, although the populations responded in opposing manners. Developmental times, however, were most strongly influenced by temperature with total developmental periods lengthened by 17-32% at 22 degrees C. Pupal mortality was also significantly lower at this temperature. Although numbers of progeny per female were significantly higher for the Corrientes population, we found no significant differences in progeny per female according to host species. Interestingly, we found that females were larger than males, and flies from Corrientes were larger than those from Santiago del Estero, even after statistical adjustments for host size. The modal frequency of host size elected in all treatment combinations tested was identical (0.6 mm), a size that represented the apparent threshold for producing female progeny. These laboratory and additional field observations demonstrate considerable interpopulational variation in P. nocens and lend further support to the applied approach focusing at the population, as opposed to the species level, with respect to both source and target areas for classical biological control introductions of Pseudacteon flies.

Developmental rates and host specificity for Pseudacteon parasitoids (Diptera: Phoridae) of fire ants (Hymenoptera: Formicidae) in Argentina.

This study extends our comparative knowledge of Pseudacteon interactions with Solenopsis fire ant workers. Reported in this work are development times for seven Argentinean parasitoid species reared on two hosts, Solenopsis richteri Forel and Solenopsis invicta Buren, under laboratory temperature regimes comparable with those of the climatic zones occupied by these host species. Developmental times spanned 31-66 d across phorid species, and in general did not differ between genders or host species, but were longer at lower temperatures. The size distribution of flies reared was bimodal, with a group of large (Pseudacteon borgmeieri, Pseudacteon nocens, Pseudacteon obtusus and Pseudacteon tricuspis) and small (Pseudacteon cultellatus, Pseudacteon curvatus, and Pseudacteon nudicornis) species. P. borgmeieri was exceptional with respect to length of developmental time. Also reported are results of initial oviposition and developmental studies of some of these phorid species on other Argentinean Solenopsis ant species; P. curvatus was the only species able to complete its development on nonhost fire ants. These results support the concept of incorporating several complementary species of Pseudacteon in the biological control of pest fire ants.

Myrmecophytic Cecropia: antiherbivore defenses under different nutrient treatments.

In greenhouse experiments, we compared putative biotic, chemical, physical and phenological defenses of six myrmecophytic Cecropia species cultivated under high and low nutrient regimes. We tested the intraspecific predictions of the C:N balance hypothesis for a broader range of defenses than included in other studies to date. Treatment effects on defenses appear to depend on the nutrient constituents of those defenses. Only strictly carbon-based defenses such as tannins and phenolics reached higher concentrations at the lower nutrient level. The production of glycogen-rich and membrane-bound Müllerian body ant rewards (MBs) increased with greater levels of both nutrients (this study) and light (Folgarait and Davidson 1994). In contrast, lipid- and amino acid-rich pearl body food rewards (PBs) were produced in greater numbers under conditions of high nutrient levels (this study) and low light (Folgarait and Davidson 1994), both of which should have contributed to a relative excess of nitrogen. Nutrient effects on toughness and leaf expansion rates (perhaps serving as phenological defenses) were inconsistent with the predictions of the C:N balance hypothesis. Mature leaves are protected principally by chemical and physical defenses, and new leaves, by biotic defenses. As in a previous study, interspecific comparisons agreed with the resource availability theory of plant defense. Plant investment in immobile defenses (tannins and phenolics, and leaf toughness), and in a defense with high initial construction costs (trichilia differentiated to produce MBs) were greater in each of three comparatively slow-growing "gap" Cecropia typical of small openings in primary forest, than in closely related and fast-growing "pioneer" species of large-scale disturbances (riparian edge and land slips). In contrast, both production of PBs (with negligible initial construction costs) and leaf expansion rates were greater in pioneers than in gap species. Rapid onset of biotic defenses during new leaf development (earlier in pioneers) may reduce new leaf herbivory in all species.