Results

Alkaloid profiles of the beetles surveyed are presented in Table 1. Characteristic or major mass spectral peaks, [M+H]⁺, represented are the rearranged BTX-A acetate (m/z 460), batrachotoxinin A (BTX-A) (m/z 418), and the BTX-A crotonate (m/z 486), in an ≈4:2:1 ratio in most collections. The BTX-A crotonate usually emerged as two overlapping peaks of varying proportions, corresponding to the trans and cis BTX-A O-crotonate esters with the trans usually equivalent to or slightly predominating over cis. Minor peaks were present for BTX (m/z 539) and h-BTX (m/z 553). Structures of these alkaloids are shown in Fig. 1. In addition, several unknown BTX alkaloids were detected.

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Fig. 1.

Structures of the most common batrachotoxins found in Choresine beetles.

Of the 391 beetles present in collections, 85% were Choresine pulchra (Pic) (CP) and 14% were Choresine semiopaca (Wittmer) (CS). There were also four Choresine sp. A (CA) and one Choresine rugiceps (Wittmer) (CR). Beetles were collected from May to September 2001 (mainly August) and from May to November 2002 (mainly November). All were collected in or within 2 km of the village of Herowana (elevation, ≈1,400 m). Some collections were made within the village, and others were made along the nearby Fio River. There is no discernable difference in collections made in 2001 or 2002 in the proportions of CS in the collections. A total of 40 separate collections were analyzed. A summary of the alkaloid data for the following collection types (I–VI) is presented in Table 1 (collections were segregated as types by species present in the beetle mixtures, although no evident pattern emerged in BTXs detected): type I, 29 separate collections of CP beetles; type II, 5 separate collections of CP+CS; type III, 3 separate collections of CP and CA or CR; type IV, one 3-beetle collection of CS (which originated within the village); type V, one single-beetle collection of CP; type VI, one 17-beetle collection (not positively identified as to species but likely all CP). One single-beetle collection of CP from November 2002 of type I had no detectable BTXs, whereas the single-beetle collection of CP (type V) from July 2001 had major amounts of the crotonate ester of BTX-A. Alkaloids were characterized by their protonated molecular ions [M+H]⁺, fragmentations, and liquid chromatography-retention times. Many had not been detected in prior studies of passerine birds of Papua New Guinea (13, 14).

Several previously uncharacterized BTX alkaloids are reported in Table 1 that had not been seen either in frog or bird: a molecular weight (MW) 524 alkaloid that lacks the N-methyl from the homomorpholine ring of the BTX nucleus; a MW 435/437 chloro analog of BTX-A, where the 20-hydroxyl group is replaced by chlorine; a MW 499 material, likely a homocrotonate of BTX-A, fragmenting to m/z 400; and two alkaloids of MW 513, one giving an m/z 400 fragment and the other giving a fragment at m/z 428 (perhaps an ethyl ketal of the O-crotonate of BTX-A) and perhaps a pyrrole-hydroxylated BTX (MW 564) and pyrrole-ethoxylated BTX (MW 582) or h-BTX (MW 596). Additional characterization will be required. Some of these alkaloids are likely to be artifacts.

It initially appeared that C. semiopaca (collection type IV) might be a richer source of batrachotoxin than other Choresine species. In a collection of type II where C. semiopaca was mixed with C. pulchra (25 CS and 28 CP, August 2001), BTX was a major alkaloid and h-BTX was also detected. Another mixed collection of type II (32 CS and 9 CP, September 2001), however, had no detectable BTX or h-BTX.

Discussion

Whereas the birds always had h-BTX >> BTX, the situation is reversed in the beetles, where h-BTX is scarcely detectable or absent. In frogs of the genus Phyllobates, BTX levels exceed those of h-BTX by ≈2-fold. The level of BTX in one collection of three C. semiopaca was found to be 1.8 μg per beetle or roughly one-tenth the amount present in a complete skin of either P. bicolor or P. aurotaenia but far less than that found in a P. terribilis skin, which has ca. 1 mg. The amounts of BTX in beetle collections were, however, extremely variable. Our atmospheric pressure chemical ionization mass spectrometer was sufficiently sensitive, even in the full scan mode (m/z 50–900 atomic mass units), to detect BTXs in a single beetle.

Beetles are not known to synthesize steroidal skeletons, such as that found in the BTXs, although they do use the mevalonate pathway to form terpenes. Instead, steroids and triterpenoids in beetles are presumed to originate from plant phytosterols (cholesterol, stigmasterol, sitosterol, ergosterol, etc.), which are modified by the beetle or possibly by a symbiont or collection of symbionts (19). The possible role of symbionts in the production of beetle BTXs is being investigated.

The four species of Choresine beetles in our collections range in body length from ≈5.4 to 6.2 mm and in body breadth (across elytra) from 2.25 to 2.65 mm and average, in dried specimens, ≈5.7 mm in length and 2.3 mm in breadth. Each of these species has a series of glandular vesicles that may protrude from the lateral margins of abdominal segments 1–6 (Fig. 2), plus additional large vesicles from the prothorax (anterior and lateral margins) and metathorax (lateral margins). The elytra are shaped such that vesicles can evert even when the elytra are closed. Such vesicles are characteristic of Melyrids of the subfamily Malachiinae to which Choresine belongs. The systematics of this group for the Pacific area, treated mainly by the late Walter Wittmer, now remain a challenge for the relatively few contemporary entomologists studying Melyrid beetle taxonomy. To our knowledge, there have been no reports of secondary, bioactive compounds from beetles of the family Melyridae.

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Fig. 2.

A C. pulchra beetle with an insert showing vesicles.

Little is known about the natural history of these Choresine beetles. Males and females are distinguishable only with a dissecting microscope. Choresine beetles, although apparently not brightly colored, do display a metallic blue-violaceous colored elytra that contrasts strongly with the yellow and blackish coloration of the pronotum (Fig. 2). Contrasting colors have some aposematic value, and this contrast is probably more conspicuous in sunlight. In Herowana, where the local people captured and preserved hundreds, most beetles were caught flying early in the morning or were found under leaves of garden plants. It was uncommon for a collector to catch more than ≈10–15 beetles per day. No adult Choresine beetles were found aggregated in groups. Local naturalists were unable to provide any information about larvae, host plants, or other habits, other than that adults were more commonly found in or around fresh thatch bunches used for roofing material. Local men warned that when working in fields or with thatch, one should take precautions to prevent these beetles from alighting in an eye or in facial perspiration because the beetles cause a burning sensation that can be severe.

Collections in museums of the continental United States offered few additional clues. These beetles are often among insects collected when fogging canopies; however, it is rare to collect more than a few Choresine beetles per tree. Other collections found at the California Academy of Sciences contained specimens obtained by netting individuals in grass of Madang Province (D. Kavanaugh, personal communication).

BTXs have been identified from five Pitohui species as well as I. kowaldi (13, 14). We examined the stomach contents of 11 I. kowaldi, 17 Pitohui dichrous, 12 Pitohui ferrugineus, 8 Pitohui kirhocephalus, and 1 Pitohui nigrescens, for a total of 49 individual birds. Stomach contents were classified by arthropod group and insect order, where recognizable, and plant and seed material. We verified that Pitohui and Ifrita do feed on small Choresine-sized insects (present in up to 30% of all individuals) (Table 2). Furthermore, one Pitohui did contain a Choresine in its stomach that was identified as Choresine nigroviolacea (Champion). Although we have not surveyed the toxin content in C. nigroviolacea, these studies demonstrate that toxic birds do feed on a variety of insects of the same size and even from the same genus, and we anticipate that they probably do feed on C. pulchra and other Choresines in areas where those species are present. The present results suggest that Choresine beetles are potentially a direct source of batrachotoxins for toxic New Guinea birds. An alternative hypothesis, that both the birds and the beetles may get their toxins from some common third source, such as a plant, seems unlikely, because these stomach contents and other reports (18) indicate that Ifrita is primarily, probably exclusively, insectivorous, although plant matter was also found in their stomachs, most likely arising from accidental ingestion of mosses, etc. while foraging. Only Pitohui species had seeds in their stomachs.

Over the last several years, we have used multiple methods to search for natural sources of BTXs, including radiotelemetry of Pitohui in New Guinea, examination of Pitohui and Ifrita stomach contents, survey of hundreds of insects and plants that might formulate Pitohui diets, and follow-up of leads provided by many local naturalists.

The present discovery emphasizes the value of traditional knowledge. Pitohui birds appear to be omnivorous and feed on a wide variety of insects, spiders, millipedes, other arthropods, fruit, and even smaller vertebrates. They do not appear to be diet specialists. In tropical rainforests, therefore, the diet of an omnivore, such as the Hooded Pitohui (P. dichrous), may contain literally thousands of species. Thus, the observations and folklore of traditional people can and did provide labor-saving leads and an incredible and essential store of knowledge and insights for scientists working in unfamiliar places.