From the Cover: Formicine ants: An arthropod source for the pumiliotoxin alkaloids of dendrobatid poison frogs (2024)

From the Cover

Abstract

A remarkable diversity of bioactive lipophilic alkaloids is present in the skin of poison frogs and toads worldwide. Originally discovered in neotropical dendrobatid frogs, these alkaloids are now known from mantellid frogs of Madagascar, certain myobatrachid frogs of Australia, and certain bufonid toads of South America. Presumably serving as a passive chemical defense, these alkaloids appear to be sequestered from a variety of alkaloid-containing arthropods. The pumiliotoxins represent a major, widespread, group of alkaloids that are found in virtually all anurans that are chemically defended by the presence of lipophilic alkaloids. Identifying an arthropod source for these alkaloids has been a considerable challenge for chemical ecologists. However, an extensive collection of neotropical forest arthropods has now revealed a putative arthropod source of the pumiliotoxins. Here we report on the presence of pumiliotoxins in formicine ants of the genera Brachymyrmex and Paratrechina, as well as the presence of these ants in the stomach contents of the microsympatric pumiliotoxin-containing dendrobatid frog, Dendrobates pumilio. These pumiliotoxins are major alkaloids in D. pumilio, and Brachymyrmex and Paratrechina ants now represent the only known dietary sources of these toxic alkaloids. These findings further support the significance of ant-specialization and alkaloid sequestration in the evolution of bright warning coloration in poison frogs and toads.

Keywords: allomones, allopumiliotoxins, cardiotonic activity, chemical defense, myrmicine ants

More than 500 alkaloids have been detected in skin extracts of anurans, and most of these have been assigned to 24 different structural classes (1). Presumably serving as a passive chemical defense against predation and/or microorganisms, these alkaloids originally were thought to be a product of anuran metabolism (2). Evidence now indicates that poison frogs merely have an efficient system that accumulates alkaloids from dietary alkaloid-containing arthropods (3–8). Dendrobatid (Dendrobates, Epipedobates, and Phyllobates) and mantellid (Mantella) frogs raised in captivity do not have detectable alkaloids, yet they possess the ability to selectively accumulate alkaloids provided to them through diet (8, 9). It seems likely that the bufonids (Melanophryniscus) will also have a similar dietary uptake system (10). Myobatrachid (Pseudophryne) frogs of Australia appear to synthesize their indolic pseudophrynamine alkaloids but sequester pumiliotoxins from a dietary source (11).

Representatives of many of the structural classes of alkaloids found in poison frogs and toads are known to occur in specific arthropods and thus such arthropods represent likely dietary sources. Some of these alkaloids, shared by arthropods and poison frogs and toads, are depicted in Fig. 1. Ants of the subfamily Myrmicinae appear to be the source for the 2,5-disubstituted pyrrolidines, the 2,6-disubstituted piperidines, the 3,5-disubstituted pyrrolizidines, the 3,5-disubstituted indolizidines, the 4,6-disubstituted quinolizidines, and the 2,5-disubstituted decahydroquinolines (3, 4). The 3,5-disubstituted lehmizidines (12), the histrionicotoxins (1), and the gephyrotoxins (1) represent alkaloid classes (Fig. 2) that share certain structural features with those of known ant alkaloids, and it is expected that they will prove to be of myrmicine ant origin as well. All of the alkaloids in frog and toad skin that appear to originate by sequestration from myrmicine ants contain unbranched carbon skeletons. Coccinellid beetles appear to be a dietary source for the coccinellines and some of the structurally related tricyclic alkaloids (5). Siphonotid millipedes are the putative dietary source for the spiropyrrolizidine oximes and nitropolyzonamines (7). Although several classes of frog skin alkaloids have been identified in arthropods, certain major structural classes (e.g., the steroidal batrachotoxins, the cardiotonic pumiliotoxins, and the analgetic epibatidine) have not yet been identified from a specific arthropod source. A pumiliotoxin and an allopumiliotoxin were detected in mixed collections of small arthropods from Panama (6). Such alkaloids have a branched carbon skeleton with apparent isoprenoid moieties and therefore were not expected to be of myrmicine ant origin.

In an attempt to identify an arthropod source for the pumiliotoxin family of alkaloids, we made several extensive arthropod collections on Isla Bastimentos, Bocas del Toro Province, Panama. This area was chosen based on the high levels of pumiliotoxins A and B (307A and 323A, respectively; Fig. 3) present in populations of the dendrobatid frog Dendrobates pumilio, and on the previous detection of 307A in mixed leaf-litter collections of arthropods from the same location (6). The pumiliotoxins and most other alkaloids from anuran skin have been assigned code names, which consist of a boldfaced number corresponding to the nominal mass and a boldfaced letter for identification of individual alkaloids (1).

Materials and Methods

Arthropod Collections. We collected arthropods from the following locations on Isla Bastimentos during the dry and wet seasons (February 2–8 and August 20–23, 2003, respectively). Site 1: northwest coast, secondary forest; leaf litter, abundant; 9°21.618′N, 82°12.074′W. Site 2: northwest coast, numerous Cyclanthus and cacao; leaf litter, abundant; 9°21.250′N, 82°12.519′W. Site 3: northwest coast, numerous Heliconia; leaf litter, sparse; 9°21.169′N, 82°12.627′W. Site 4: northwest coast, numerous Heliconia; leaf litter, abundant; 9°21.123′N, 82°12.620′W. Site 5: northwest coast, numerous palms; leaf litter, abundant; 9°20.996′N, 82°12.726′W. Site 6: south coast, secondary forest; leaf litter, abundant; 9°20.364′N, 82°10.807′W. Site 7: northwest coast, numerous Cyclanthus; leaf litter, abundant; 9°21.021′N, 82°12.704′W. Site 8: inland, secondary forest; leaf litter, abundant; 9°20.490′N, 82°10.486′W. All site descriptions are based on the dominant plant type(s) or forest type at each site. The amount of leaf litter at each site was ranked as either sparse or abundant. Mixed arthropod collections were made at sites 1–3, 5, and 6 previously (6).

We collected all arthropod samples with forceps, from leaf litter (6, 7) or directly from vegetation, and placed them in taxon-specific 1.5-ml plastic vials containing methanol. The forceps were cleaned with methanol between each sampling event. Only small arthropods (<10 mm), suitable as prey for D. pumilio, were collected from these sites. After arthropod collections, we collected five frogs from each site during both seasons for analysis of skin alkaloids. We also stomach-flushed 180 frogs during each season to obtain dietary information from four sites (1, 3, 5, and 6). All voucher specimens are located in the herpetological collection at Florida International University.

Analysis of Alkaloid Extracts. Methanol extracts of arthropods were analyzed by using gas chromatography mass spectral analysis (GC-MS), conducted with a Finnigan GCQ instrument with a 30 m × 0.25 mm i.d. DB-1 fused silica column with a temperature program from 100 to 280°C at a rate of 10°C/min. All ant extracts were concentrated to a volume of 10 μl before analysis. The identification of 307A and 323A was based on the comparison of GC mass spectra to authentic standard samples of these compounds.

Methanolic alkaloid fractions were prepared and partitioned for all frog skins as described in ref. 3. Major and minor alkaloids were identified by using a flame-ionization detector after GC on a 6-foot 1.5% OV-1 packed column (2 mm i.d.), programmed from 150 to 280°C at a rate of 10°C/min. An injection volume of 2 μl was used for each frog alkaloid fraction, corresponding to 2 mg of wet weight frog skin (3).

Results

GC-MS analyses of >500 arthropod samples (255 samples during the dry season and 257 samples during the wet season) led to the detection of 307A and 323A in formicine ants of the genera Brachymyrmex (Brachymyrmex longicornis and Brachymyrmex cf. depilis; Fig. 4A) and Paratrechina (Paratrechina steinheili; Fig. 4B), representing two different ant tribes, Myrmelachistiani and Prenolepidini, respectively. With the exception of pyrazines, which are widespread as pheromones in the family Formicidae (13), alkaloids have never been detected in ants of the subfamily Formicinae, which are characterized by the presence of formic acid in poison glands (14). Pyrazines were not detected in either Brachymyrmex or Paratrechina extracts, but were detected in other ant extracts. During the wet season, all Brachymyrmex samples were collected from plants of the genus Heliconia. Pumiliotoxins 307A and 323A were detected in Brachymyrmex samples from sites 5 and 7, whereas only 307A was detected in Brachymyrmex samples from sites 3, 4, and 8. Not all Brachymyrmex samples collected from these sites contained pumiliotoxins. Brachymyrmex also were collected from site 1; however, 307A and 323A were not detected. During the dry season, Brachymyrmex were only collected from site 7 from the leaf litter and no pumiliotoxins were detected. Pumiliotoxins 307A and 323A were detected in P. steinheili samples collected from the leaf litter in site 5 during the wet season. Brachymyrmex cf. depilis was identified in the stomach contents of three frogs from site 6 and one frog from site 5, and P. steinheili was identified in the stomach contents of one frog from site 5. Pumiliotoxins 307A and 323A were detected in skin extracts of frogs from the sites at which the pumiliotoxins were detected in ants (data not shown). The presence of 307A and 323A in ants of the genera Brachymyrmex and Paratrechina as well as in skin extracts of the dendrobatid frog D. pumilio, coupled with the presence of these ants in stomach contents of D. pumilio, strongly suggests that these ants represent a dietary source for pumiliotoxins in these populations of frogs. Pumiliotoxins 307A and 323A were not detected in any of the other several hundred arthropod extracts examined in this study.

Discussion

The pumiliotoxin family of alkaloids consists of >80 alkaloids, which are divided into three major classes: the pumiliotoxins, the allopumiliotoxins, and the hom*opumiliotoxins (1). The pumiliotoxins and allopumiliotoxins are found in all anuran genera known to contain lipophilic alkaloids, which include frogs and toads from a variety of locations worldwide (2, 10, 11, 15). Therefore, it is expected that the dietary source(s) of the pumiliotoxins will share a similar distribution. Pumiliotoxins 307A and 323A occur frequently as major and minor alkaloids in dendrobatid frogs of the genus Dendrobates, occur less frequently in Minyobates, and are absent in Epipedobates and Phyllobates (Table 1). They also occur as major or minor alkaloids in frogs of the genus Mantella, although a dihydroanalog (309A; Fig. 3) is more common (Table 2). Neither 307A nor 323A occur as major or minor alkaloids in toads of the genus Melanophryniscus, where a simpler alkaloid, pumiliotoxin 251D, is often a major alkaloid (Table 2). Pumiliotoxin 323A, but not 307A, occurs in frogs of the genus Pseudophryne, where pumiliotoxin 267C (Fig. 3) and allopumiliotoxin 323B are the only other alkaloids of the pumiliotoxin family to be detected as major or minor alkaloids (Table 2). Pumiliotoxin 267C occurs only rarely in neotropical frogs of the family Dendrobatidae (Table 1). Other members of the pumiliotoxin family, most commonly 251D and 267A (Fig. 3), are present as major or minor alkaloids in all genera of dendrobatids, except Phyllobates (Table 1). Of the two, only 251D occurs in the genera Mantella and Melanophryniscus. Neither 251D nor 267A occurs in Pseudophryne. Some of the allopumiliotoxin 267A found in dendrobatid extracts may have been formed by hydroxylation of dietary pumiliotoxin 251D, as has been shown specifically for frogs of the genus Dendrobates (19).

The genus Brachymyrmex is endemic to the American tropics and subtropics, with ≈40 described species (20, 21). A few species are found in the Old World and are generally restricted to synanthropic habitats. The genus Paratrechina is found in the tropics and subtropics throughout the world (22). Ants of the genus Paratrechina are very common in leaf litter of lowland wet forests, and P. steinheili is one of the most abundant ant species in the neotropics. Therefore, ants in the genera Brachymyrmex and Paratrechina are expected to be coextensive with pumiliotoxin-containing dendrobatid frogs and bufonid (Melanophryniscus) toads (see Tables ​Tables11 and ​and22 for distribution of pumiliotoxins in dendrobatid frogs and bufonid toads, respectively), and it seems probable that species in these ant genera serve as a source for the pumiliotoxins. Ants in the genus Paratrechina may serve as a source for pumiliotoxins in Madagascan mantellid (Mantella) frogs and Australian myobatrachid (Pseudophryne) frogs (see Table 2 for distribution of pumiliotoxins in Old World frogs).

Not all of the Brachymyrmex samples examined in this study contained pumiliotoxins. Pumiliotoxins were found in extracts from five of the six sites in which Brachymyrmex were collected but not in all Brachymyrmex samples from these sites. These data suggest that there is both spatial and temporal variation in the presence of pumiliotoxins among Brachymyrmex ants. Caste-specific alkaloid production is known among myrmicine ants of the genus Solenopsis and may occur in ants of the genus Brachymyrmex (23, 24).

Ants of the subfamily Formicinae are well known for the use of formic acid as a chemical defense against predation. Alkaloids other than the widely distributed pyrazines, which serve as pheromones in many ants (13), have never been detected in ants of the subfamily Formicinae. Pumiliotoxins and allopumiliotoxins are highly toxic (6, 25) and thus presumably also serve as a chemical defense in Brachymyrmex and Paratrechina, along with formic acid. Pumiliotoxins are potent cardiotonics (26, 27) and are therefore of some interest as pharmacological probes (28, 29) and even as potential therapeutics. Certain pumiliotoxins are potent insecticides (30). The present discovery of an ant source of the pumiliotoxins may permit the identification of the gene cluster involved in synthesis of these structurally unique alkaloids.

Despite the large number and diversity of alkaloids previously isolated from myrmicine and pseudomyrmicine ants (13), only two reports document the biosynthesis of alkaloids in such ants (31, 32). Alkaloid sequestration from plants has been well documented among Lepidopteran and Coleopteran arthropods (33) but has not been reported for ants. A role for a microsymbiont in production of ant alkaloids cannot be precluded.

Dendrobatid frogs of the genera Dendrobates, Epipedobates, and Phyllobates consume a high proportion of ants as part of their diet in the wild (34–36). The term “ant-specialist” has been used to describe frogs in the genus Dendrobates and some members of the genus Epipedobates (Epipedobates trivittatus, Epipedobates petersi, and Epipedobates pictus), based on the fact that they consume ants in higher proportions than they are available in their environment (35, 36). However, only Epipedobates erythromos, Epipedobates espinosai, Epipedobates silverstonei, and Epipedobates tricolor of 11 Epipedobates species contained minor or major amounts of pumiliotoxins (Table 1). Most, including E. trivittatus, E. petersi, and E. pictus, contained mainly decahydroquinolines and histrionicotoxins (2), presumably sequestered from dietary myrmicine ants. It should be noted that alkaloids of the pumiliotoxin class (Fig. 3) are much more toxic than the “izidine” alkaloids, decahydroquinolines, and histrionicotoxins (Figs. ​(Figs.11 and ​and2).2). Although not as extensively studied, alkaloid-containing frogs in non-dendrobatid genera (Mantella, Melanophryniscus, and Pseudophryne) have also been shown to consume large numbers of ants (37–39). Currently, 7 of the 24 structural classes of anuran alkaloids are known to also occur in ant extracts. An additional 8 alkaloid classes now are suspected to be of ant origin. Thus, myrmecophagy in dendrobatid frogs has likely played a major role in the evolution of alkaloid sequestration and aposematism (35–37, 40, 41). Recent evidence supporting at least two origins of “diet specialization” and aposematism in dendrobatids appears to support this claim (41). The discovery of pumiliotoxins in formicine ants provides further evidence for the importance of myrmecophagy and alkaloid sequestration in the evolution of aposematic coloration in poison frogs and toads.

Acknowledgments

We thank the Autoridad Nacional del Ambiente and the Republic of Panama for permission to collect and export the samples used in this study (permits SEX/A-15-03 and SEX/A-45-03); the Smithsonian Tropical Research Institute, especially Orelis Arosem*na and Maria Leone, for assistance in obtaining these permits; Kim Arce for assistance with stomach-content identification; and the Florida International University Herpetology Group (K. Arce, C. Duffoo, C. Ugarte, E. Verdon, and S. Whitfield) and J. Snyder for valuable comments on the manuscript. This work was supported in part by Environmental Protection Agency Fellowship U-91608001-0, the Explorers Club, National Science Foundation Grants DBI-0215820 and DEB-0072702, and a Courtesy Associate appointment given by the National Institute of Diabetes and Digestive and Kidney Diseases.

Notes

See Commentary on page 7841.

References