Introduction

Most Hemiptera feed by injecting digestive enzymes into prey and then ingesting the liquefied tissues through a tube-like proboscis (extra-oral digestion) (Cohen 1995). This feeding mode presents a challenge to the study of their diet, largely eliminating the use of standard morphological identification of chitinous body parts in the gut. As a result, studies of hemipteran diets have typically used immunoassays employing prey-specific monoclonal antibodies (Greenstone 1996), PCR tests (Sheppard and Harwood 2005), or both (e.g., Fournier et al. 2008). One limitation of most antibody- and DNA-based applications is that some knowledge of the potential prey is required. Antibodies target epitopes that are specific to proteins from target prey species, and most DNA methods employ species- or taxon-specific primers in PCR tests to determine the presence/absence of target prey species or taxa (e.g., Agustí et al. 1999, 2000, 2003; Read 2002; Cuthbertson et al. 2003; Jarman et al. 2004; de León et al. 2006). While these methods can be powerful and have been verified for their accuracy using laboratory feeding experiments (Chen et al. 2000; Foltan et al. 2005; Harper et al. 2005, 2006; Sheppard et al. 2005; Harwood et al. 2007; McMillan et al. 2007), a major limitation arises when there is little or no prior knowledge of the prey in their natural habitat.

Buruli ulcer is a neglected emerging disease of skin and soft tissue that leads to scarring and disability (Johnson et al. 2005; Merritt et al. 2005). It is caused by Mycobacterium ulcerans (Actinomycetales: Mycobacteriaceae), an environmental pathogen that produces a destructive polyketide toxin (George et al. 1999). The disease has been reported in humans from at least 32 countries, with a large number of cases reported from West Africa (Duker et al. 2006; Walsh et al. 2008; Merritt et al. 2010). While transmission of the disease to human beings remains unclear, Buruli ulcer outbreaks have been associated with freshwater habitats (Thangaraj et al. 1999), particularly in areas where the landscape is disturbed by natural events such as flooding, or through deforestation, dam construction, agricultural diversion, or mining (Thangaraj et al. 1999; Johnson et al. 2005; Merritt et al. 2005; Duker et al. 2006; Wansbrough-Jones and Philips 2006).

A critical step in understanding M. ulcerans transmission is elucidating the diet of organisms that may potentially act as reservoirs and vectors of the pathogen in nature. Non-human mammals and reptiles have been tested in the environment without positive findings for the pathogen (Radford 1974), and several arthropod disease vectors (i.e., bedbugs, black flies, mosquitoes) tested negative in early studies (Revill and Barker 1972; Portaels et al. 2001). However, only a few organisms in each taxonomic group were tested in these early studies, and insect sampling methods were neither systematically employed nor standardized. Portaels et al. (1999) were the first to suggest that aquatic bugs (Hemiptera) might be reservoirs of M. ulcerans in nature, and recently they described the first isolation in pure culture of M. ulcerans from a water strider (Hemiptera: Gerridae, Gerris sp.) from Benin, West Africa (Portaels et al. 2008). A survey study (Portaels et al. 2001) based on detecting M. ulcerans DNA in aquatic insects (Hemiptera, Odonata, Coleoptera) in African Buruli ulcer-endemic swamps confirmed the earlier findings. More recent studies in Australia have suggested that mosquitoes may be involved in transmission (Johnson et al. 2007).

Figure 1.

Photograph of the study site near the village of Saduase, Ga East District, Ghana. High quality figures are available online.

Using experiments, it has been demonstrated that M. ulcerans could survive and multiply within the salivary glands of the aquatic bug Naucoris cimicoides (Hemiptera, Naucoridae), and that N. cimicoides could transmit the mycobacteria to mice (Marsollier et al. 2002, 2003). Naucoris spp. (Naucoridae, Hemiptera) live in freshwater ponds, lakes, and slow-flowing sections of streams and rivers. Naucoridae are predacious in both the immature (nymph) and adult stages, although little is known of the ecology and prey preferences of Naucoridae in nature. This is particularly true in tropical West Africa where the disease is prevalent. Most aquatic hemipterans are believed to be generalist predators on other aquatic invertebrates (Merritt et al. 2008), although some, including naucorid species, have mouthparts designed to aid in feeding on prey larger then themselves (e.g., Cohen 1995) such as tadpoles (Polhemus and Polhemus 1988) and larval fish (Louarn and Cloarec 1997).

Here we examine the diet of an abundant predator in Buruli ulcer-affected freshwater ecosystems in West Africa (Naucoris sp.) in a first attempt to understand its role in a tropical pond food web and potential sources of M. ulcerans. In the absence of any a priori knowledge of their prey, we used PCR to amplify all DNA in the Naucoris sp. gut using universal primers, clone the PCR product, and sequence a subset of clones. We then matched the resulting gut-content sequences to sequences obtained from potential prey collected from the same habitat, and to publically available sequence databases.

Materials and Methods

Results

PCR amplification of coxl and rrnL was equally successful for Naucoris sp. but rrnL was more consistently amplified for gut samples and potential prey. There were no Naucoris sp. coxl sequences available on the NCBI nucleotide database and the top hit of the blastn query was an unclassified Hemiptera (AAG5301 voucher ENT-OUBS-156, HM381306), whereas the database contained 8 rrnL sequences for Naucoridae collected from Madagascar, Europe, North and Central America, and the Philippines (Hebsgaard et al. 2004). Combining the newly generated rrnL sequences with blastn query results produced an aligned matrix of 62 taxa and 460 characters (sequence length 336–443 bp). In the maximum likelihood rrnL gene tree (In L = -8123.81514 ; Figure 2) our Naucoris sp. sequences were clearly nested within published data for the Hemiptera, the closest relative being Ambrysus sp. collected from North America (Figure 2). Ambrysus sp. was the second-ranked blastn hit, with Macrocoris sp. from Madagascar (Hebsgaard et al. 2004) the top hit but phylogenetically more distant in our analysis of the same sequences (Figure 2).

Table 1.

Results of comparisons of rrnL and coxl sequences that were PCR-amplified from Naucoris sp. guts and cloned (see Methods) to the NCBI nucleotide database using blastn queries. Taxa listed were, in each case, first on the hit table. Only full-length inserts (450 – 478 bp rrnL, 658 bp coxl) were used as query sequences. Other full-length inserts were identical to our sequences obtained from direct sequencing of sampled Naucoridae and resulted in a top blastn hit of Macrocoris sp. (rrnL) or Hemiptera sp. (coxl) (data not shown).

Figure 2.

Maximum likelihood rrnL gene tree reconstructed using a GTR model of evolution, including newly sequenced Naucoris sp. (terminals labeled FIELD), potential prey (PREY), cloned PCR products from Naucoris sp. guts / mouthparts (CLONES), and highly ranked sequences according to blastn queries (with GenBank alphanumeric accession codes, see text for criteria). High quality figures are available online.

Table 2.

Accession numbers of newly sequenced material including taxa (with closest match using blastn searches in parentheses) and sequence origin (directly sequenced organism, or sequenced clone from gut/mouthpart content).

Three of our cloned rrnL sequences from Naucoris sp. guts were identical to a field-caught Coleoptera species sampled at the study site as potential prey. The top database match (using blastn) to this sequence was Spercheus (Spercheidae) (Table 1) and the second-ranked match was Hydrobius sp. (Hydrophilidae) (not shown), but it is clear from our phylogenetic search that it is distantly related to both based on mtDNA rrnL (Figure 2). None of the other potential prey species that we collected from the sampling site and sequenced were recovered from gut sequence clones (Figure 2). Nonetheless, a number of interesting non-insect species were recovered from guts and identified with blastn queries (Table 1). These included Afrixalus sp. (Anura: Hyperoliidae), a sub-Saharan genus of frog for which we recovered one rrnL sequence from our clone library. Sequences from the coxl clone library included Embata and Floscularia (Rotifera), and Pythium (Oomycete fungi). All other fulllength clone sequences were identical to our Naucoridae sequences obtained using direct sequencing of PCR products (rrnL shown in Figure 2).

Discussion

Buruli ulcer (Mycobacterium ulcerans infection) causes severe morbidity in human populations associated with degraded freshwater habitats, but neither the reservoir nor the mode of transmission of M. ulcerans is known (Merritt et al. 2005, 2010). Here we investigated an abundant aquatic predator from a Buruli ulcer-endemic area in Ghana, Naucoris sp. water bugs (Hemiptera, Naucoridae). While Naucorids have been implicated in the transmission of M. ulcerans in laboratory studies, a limited knowledge of their place in aquatic food webs in nature makes it difficult assess the potential source and sinks of the pathogen. This is the first investigation of which we are aware to clone and sequence PCR products from universal primers to determine a Hemipteran diet without any prior knowledge of potential prey.

Our approach led to a broader perspective of the role of Naucoris sp. in the aquatic food web. Using a standard PCR-based method, we would have designed prey-specific primers for the 5 taxa of field-caught prey (e.g., Agustí et al. 2003). From this, we would have probably generated positive tests for the Coleoptera. In contrast, the universal primers, sequenced clones, and database queries used here allowed us to identify DNA in the guts that came from taxa that were not field-collected. These included fungi, rotifers, and an anuran, although larval anurans were collected from the field site and thus known to be present. A limitation of the database queries is clearly the fact that the extent of the database plays an important role. Using the newly generated Naucoris sp. sequences, none of our blastn query results gave a close match. Even our “barcode” coxl sequence gave a fairly meaningless match (Hemiptera sp.) to the Barcode of Life database ( www.barcodinglife.org).

Combining public databases and our own newly generated prey sequences was beneficial for confirming that the prey Coleoptera sequence cloned from the gut matched the field-caught Coleoptera species at the same habitat. Although neither the database nor our new sequence could provide identification, at least we could conclude with some confidence that the Naucoris sp. preys on the resident Coleoptera. These sequences were identical and, based on the phylogenetic gene tree, quite different from any species in GenBank. Interestingly, the blastn query and phylogenetic analysis gave different results. The second-ranked blastn result was phylogenetically closer to the query sequence than the top-ranked blastn query result. The more detailed phylogenetic analysis, using more of the available data and a GTR model of sequence evolution, likely revealed the closer relative. Although both query hits were relatively distant and neither is probably a good match, it does suggest that a phylogenetic approach is more accurate than a blast result in the absence of a complete database.

In conclusion, our approach provided the means to study an aquatic hemipteran diet without any prior knowledge of potential prey and despite the difficulties of extra-oral digestion. Naucoris sp. in this Buruli ulcerendemic area feeds on a wide range of prey and body sizes, including rotifers, insects, and anurans. Further work on M. ulcerans transmission will be aided by this food web information. Our results also suggest the approach could be successfully used to study the complex interactions within aquatic food webs, including even feeding on fungi. Our results corroborate previous suggestions that DNA-based approaches using universal primers and cloning provide an important tool for studying the prey spectrum of predators with unknown diets.