Molecular profiling of nematode associates with Rhynchophorus ferrugineus in southern Italy

Abstract A survey of nematodes associated with the red palm weevil Rhynchophorus ferrugineus was conducted in southern Italy in 2015 and 2016 in order to create a species inventory and obtain data about nematode biodiversity. A total of 70 insect samples (pupae and adults) were collected from infested Phoenix canariensis, Phoenix dactylifera, and Chamaerops humilis palms in three Italian Regions: sampling took place at 11 locations in Apulia, 1 in Basilicata, and 1 in Sardinia regions. Individual insects were dissected to determine nematode presence, and different nematode species were also recovered from red palm weevil cocoons collected at the sites in Apulia. Individual nematodes were molecularly identified by sequencing the ITS, D2‐D3 expansion domains of the 28SrRNA gene and the mitochondrial COI and inferring the phylogenetic relationships. The insect‐associated nematofauna identified belonged to the families Rhabditidae, Cephalobidae, and Diplogastridae. Just two nematode species, Teratorhabditis synpapillata and Mononchoides macrospiculum, were always found in association with adult insects and cocoons taken from all sampling sites. This paper reports on the biodiversity of the nematodes associated with R. ferrugineus and on current knowledge of the specific habitat of specialized and divergent entomophilic nematodes.

and other ornamental plants (Giovino et al., 2012). In Italy, C. humilis is largely cultivated along the coasts of the Tyrrhenian Sea, Sicily, and Sardinia, where most damage is caused by the South American palm borer Paysandisia archon (Burmeister) (Lepidoptera: Castniidae), which seems to reduce the plants' resistance to R. ferrugineus (EPPO, 2008b;Giovino et al., 2012).
Associations between Rhynchophorus species and nematodes are well documented for R. palmarum (L.) and R. cruentatus (F.), but very little is known about R. ferrugineus with regard to its fitness, evolution, coadaptation, speciation, defense, chemical communication, or pest management (Camerota et al., 2016;Esparza-Diaz, Olguin, Carta, Skantar, & Villanueva, 2013;Giblin-Davis et al., 2013;Kanzaki & Giblin-Davis, 2018). Nematodes are microscopic worms that are adapted to living in a variety of environments, and many can be associated with other organisms that provide them with shelter or transportation, for example, entomophilic, saprobiotic, phoretic, commensal, and parasitic nematodes (Kanzaki, 2017). The number of entomophilic nematodes associated with weevils is hard to determine because nematode identification at the species level is difficult. However, it is known that every insect species can be associated with 1-5 host-specific nematode species (Giblin-Davis et al., 2013;Kanzaki, Giblin-Davis, Gonzalez, & Manzoor, 2017). Two nematode species, Teratorhabditis palmarum Gerber & Giblin- Davis, 1990 and Acrostichus palmarum Kanzaki & Giblin-Davis, 2018 are naturally found associated with and distributed in R. palmarum, while the other nematode species observed, Bursaphelenchus cocophilus (Cobb) Baujard, B. gerberae, Caenorhabditis angariae, and Mononchoides sp., are occasionally carried by R. palmarum (Kanzaki et al., 2008;Kanzaki & Giblin-Davis, 2018;Kanzaki, Giblin-Davis, Zeng, Ye, & Center, 2009;Sudhaus, Kiontke, & Giblin-Davis, 2011). Teratorhabditis palmarum, Acrostichus rhynchophori Kanzaki et al., 2009, andMononchoides sp. Gerber &Giblin-Davis, 1990 were also found sympatrically associated with R. cruentatus, together with other nematode species. In R. ferrugineus, the only reported nematode associates were Teratorhabditis synpapillata and Praecocilenchus ferruginophorus (Rao & Reddy, 1980;Kanzaki et al., 2008). However, a close association of Mononchoides macrospiculum and T. synpapillata was recently found in R. ferrugineus from Apulia (Southern Italy) (Troccoli, Oreste, Tarasco, Fanelli, & Luca, 2015 Recent studies demonstrate that sequencing and phylogenetic analysis is a useful approach when dealing with small and/or scant specimens and taxon diversity cannot be determined with traditional approaches (Hazir, Kanzaki, Gulcu, Hazir, & Giblin-Davis, 2015;Kanzaki et al., 2012;Markmann & Tautz, 2005). Our study used sequencing and phylogenetic approaches to investigate the biodiversity of nematodes associated with R. ferrugineus samples collected in southern Italy. The main goals of the present study were as follows: (a) to collect RPW adults, pupae, and cocoons from different sampling sites in southern Italy; (b) to use sequencing and phylogenetic profiles to identify all nematodes associated with RPW; (c) to compare nematode associates among different geographical sites and palm hosts; and (d) to understand the evolution and phylogeny of nematode species and of their host.

| Insect collection and nematode isolation
In 2015 and 2016, RPW samples were collected from P. canariensis Chabaud, P. dactylifera L., and C. humilis L. palms in southern Italy presenting symptoms of RPW damage ( Figure 1) 11 sites in Apulia, 1 in Sardinia, and 1 in Basilicata (Table 1). The sample unit was defined as the weevils obtained from each infested tree. Cocoons, pupae, and adults were collected by debarking, placed individually in plastic bags and stored in a refrigerator at 8°C. Each RPW sample was dissected under a stereomicroscope (Figure 1f), and the external surface (including elytra), hemocoel, and reproductive system were examined separately to check for the presence of nematodes (Table 1; Figure 1). The isolated specimens were observed under a stereomicroscope to determine their feeding habits.

| DNA extraction, PCR amplification, and sequencing
Total DNA was extracted from individual nematodes obtained from dissected weevils and directly amplified as described by De Luca, Fanelli, Vito, Reyes, and Giorgi (2004).
D2-D3 and ITS-RFLP analyses were performed on PCR products from individual nematodes and digested using five units of the following restriction enzymes: Alu I, Ava II, Hinf I, and Rsa I (Roche Diagnostics, Manheim, Germany). The restricted fragments were then separated on a 2.5% agarose gel by electrophoresis. The gels were stained with gel red, visualized on a UV transilluminator, and photographed using a digital system.
The D2-D3 amplified products were purified using the protocol suggested by the manufacturer (High Pure PCR elution kit) and directly sequenced. Purified ITS and COI fragments were cloned and sent to MWG-Eurofins in Germany for sequencing in both directions. Several specimens were not sequenced successfully.

| Phylogenetic analysis
BLAST search at NCBI used all the new sequences obtained, which were submitted to the database and compared with the corresponding sequences to identify the closest matching nematode taxonomic and/or phylogenetic groups. Multi-alignment was performed using the MAFFT program (Katoh & Standley, 2013  In the RPW samples from Apulia, nematodes were found under the elytra and in the hemocoel of adults, and pupae and inside the cocoons and dead palm tissues of P. canariensis, P. dactylifera and C. humilis ( Figure 1). Two nematode species phoretically associated in the hemocoel and external surface of all RPW samples from P. canariensis and P. dactylifera were molecularly identified as T. synpapillata and M. macrospiculum by their 100% sequence identity with those in the database (Kanzaki et al., 2008;Troccoli et al., 2015), while only M. macrospiculum was detected in C. humilis. Apart from the two phoretic nematode species found in this study, other nematode species were also occasionally associated with R. ferrugineus. Of these, Oscheius tipulae (Lam & Webster, 1971), Sudhaus, 1993 (Camerota et al., 2016;Fanelli et al., 2017).

| Phylogenetic analyses
The newly obtained sequences of ITS and D2-D3 expansion segments of 28S rDNA, were multi-aligned with the closest sequences in GenBank and ML was used to reconstruct the phylogenetic trees (Figures 2 and 3).
With regard to the A. nanus population we found in Apulia, we recorded the first occurrence in R. ferrugineus of this nematode, which has previously been reported only in earthworm cocoons (Kraglund & Ekelund, 2002 The association patterns observed in our study suggest that the nematode species associated with a few Italian RPW may be incidentally associated, or else subject to host switching because nematodes and insects share similar environmental conditions. Furthermore, the low number of specimens recovered from each insect can be explained by competition for food sources, seasonal environmental changes, and competition with other microbes sharing the same palm habitat and insect host.
The presence of different nematodes associated with R. ferrugineus confirms the low association rate between nematodes and RPW, suggesting that RPW is probably not the typical or primary host for these nematodes.
In conclusion, the molecular approach allows us to assign anonymous sequences to taxon groups representing different trophic levels, and to determine taxon diversity in the context of ecological analysis. The present study demonstrates that most nematode associates are morphologically similar Rhabditidae and are thus difficult to identify at the species level. Furthermore, this study reveals a specific association of T. synpapillata and M. macrospiculum with RPW in southern Italy. Other nematode associations with the native RPW can occur incidentally because these new nematode associates share the same habitat as RPW, which acts as an occasional carrier. Although the origins of these associations are in most cases not clear, it appears that an important role in the evolution of these interesting entomophilic nematodes is played by associations with soil or possibly with other moist habitats, followed by host-carrier switching.

ACK N OWLED G M ENTS
The authors wish to thank Mrs Sarah Jane Christopher for helpful assistance in English revision of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.