Robin M. Giblin-Davis

Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises

Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well‐acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)‐based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

Evaluating high‐throughput sequencing as a method for metagenomic analysis of nematode diversity

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time‐consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400‐bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.

Chemical and Behavioral Ecology of Palm Weevils (Curculionidae: Rhynchophorinae)

Palm weevils in the subfamily Rhynchophorinae (Curculionidae) (Rhynchophorus spp., Dynamis borassi, Metamasius hemipterus, Rhabdoscelus obscurus, and Paramasius distortus) use male-produced aggregation pheromones for intraspecific chemical communication. Pheromones comprise 8, 9, or 10 carbon, methyl-branched, secondary alcohols. (4S,5S)-4-Methyl-5-nonanol (ferrugineol) is the major aggregation pheromone for R. ferrugineus, R. vulneratus, R. bilineatus, M. hemipterus, and D. borassi and a minor component for R. palmarum. (5S,4S)-5-Methyl-4-octanol (cruentol), (3S,4S)-3-methyl-4-octanol (phoenicol), and (4S,2E)-6-methyl-2-hepten-4-ol (rhynchophorol) are the main aggregation pheromones for R. cruentatus, R. phoenicis, and R. palmarum, respectively. Plant kairomones strongly enhance pheromone attractiveness but none of the identified volatiles, such as ethyl acetate, ethyl propionate, or ethyl butyrate are as synergistic as fermenting plant (palm or sugarcane) tissue. Studying orientation behavior of foraging weevils to semiochemical devices helped to design and test traps for weevil capture. Generally, 3 mg per day of synthetic pheromone (with non-natural stereoisomers being benign) plus insecticide-treated plant tissue constitute highly attractive trap baits. Potential exists for pheromone-based mass-trapping of weevils to reduce their populations and the spread of the weevil-vectored red ring disease, for monitoring their population dynamics to facilitate pest management decisions, and for detection and possible interception of non-native weevils at ports of entry.

FIELD RESPONSE OF RHYNCHOPHORUS CRUENTATUS (COLEOPTERA: CURCULIONIDAE) TO ITS AGGREGATION PHEROMONE AND FERMENTING PLANT VOLATILES

Semiochemicals from 2.5 kg of chopped stem tissue from cabbage palmetto, Sabal palmetto (Walter), frozen or fresh stem tissue from sugarcane, Saccharum officinarum L., or syncarp tissue from pineapple, Anana comosus (L.), were equally suitable for field attraction of Rhynchophorus cruentatus (F.) when used with 0.4 mg/d of its aggregation pheromone, 5-methyl-4-octanol (cruentol). Twenty-eight different chemicals known to be fermentation products from palm sap were screened with 0.4 mg/d cruentol for field attraction of R. cruentatus adults. Good chemically-mediated field trapping of R. cruentatus was achieved with cruentol plus ethyl acetate (852 mg/d) and to a lesser degree with each of the following: (S)-(-)-ethyl lactate (release rate not determined; ND), ethyl isobutyrate (40 mg/d), ethyl butyrate (255 mg/d), or ethanol (51 mg/d). However, none of the test chemicals with cruentol were as effective as 1.5 kg of fermenting sugarcane or S. palmetto tissue plus cruentol. Also, none of these chemicals were attractive by themselves at the rates tested. A combination of individually released ethanol (48 mg/d), ethyl acetate (131 mg/d), ethyl butyrate (34 mg/d), ethyl isobutyrate (40 mg/d), and (S)-(-)-ethyl lactate (ND) with cruentol was as effective for the capture of R. cruentatus as cruentol plus any of the individual components at the rates tested. Several trap designs were evaluated for future research and implementation of semiochemically-mediated monitoring and management of R. cruentatus.

Reproducibility of read numbers in high-throughput sequencing analysis of nematode community composition and structure

Although nematodes are the most abundant metazoan animals on Earth, their diversity is largely unknown. To overcome limitations of traditional approaches (labour, time, and cost) for assessing biodiversity of nematode species in environmental samples, we have previously examined the suitability of high‐throughput sequencing for assessing species level diversity with a set of control experiments employing a mixture of nematodes of known number and with known sequences for target diagnostic loci. Those initial experiments clearly demonstrated the suitability of the approach for identification of nematode taxa but lacked the replicate experiments necessary to evaluate reproducibility of the approach. Here, we analyze reads generated from three different PCR amplifications and three different sequencing reactions to examine the differential PCR amplification, the possibility of emulsion PCR artefacts, and differences between sequencing reactions. Our results suggest that both qualitative and quantitative data are consistent and highly reproducible. Variation associated with in‐house PCR amplification or emPCR and sequencing are present but the representation of each nematode is very consistent from experiment to experiment and supports the use of read counts to estimate relative abundance of taxa in a metagenetic sample.

Tropical nematode diversity: vertical stratification of nematode communities in a Costa Rican humid lowland rainforest

Comparisons of nematode communities among ecosystems have indicated that, unlike many organisms, nematode communities have less diversity in the tropics than in temperate ecosystems. There are, however, few studies of tropical nematode diversity on which to base conclusions of global patterns of diversity. This study reports an attempt to estimate nematode diversity in the lowland tropical rainforest of La Selva Biological Research Station in Costa Rica. We suggest one reason that previous estimates of tropical nematode diversity were low is because habitats above the mineral soil are seldom sampled. As much as 62% of the overall genetic diversity, measured by an 18S ribosomal barcode, existed in litter and understorey habitats and not in soil. A maximum‐likelihood tree of barcodes from 360 individual nematodes indicated most major terrestrial nematode lineages were represented in the samples. Estimated ‘species’ richness ranged from 464 to 502 within the four 40 × 40 m plots. Directed sampling of insects and their associated nematodes produced a second set of barcodes that were not recovered by habitat sampling, yet may constitute a major class of tropical nematode diversity. While the generation of novel nematode barcodes proved relatively easy, their identity remains obscure due to deficiencies in existing taxonomic databases. Specimens of Criconematina, a monophyletic group of soil‐dwelling plant‐parasitic nematodes were examined in detail to assess the steps necessary for associating barcodes with nominal species. Our results highlight the difficulties associated with studying poorly understood organisms in an understudied ecosystem using a destructive (i.e. barcode) sampling method.

Aggregation pheromones and host kairomones of West Indian sugarcane weevil, Metamasius hemipterus sericeus

Coupled gas chromatographic–electroantennographic detection (GC-EAD) analyses and coupled GC-mass spectrometry (MS) of volatiles produced by male and female West Indian sugarcane weevils (WISW), Metamasius hemipterus sericeus (Oliv.), revealed eight male specific, EAD-active compounds: 3-pentanol (1), 2-methyl-4-heptanol (2), 2-methyl-4-octanol (3), 4-methyl-5-nonanol (4), and the corresponding ketones. In field experiments in Florida, alcohols 1–4 in combination with sugarcane were most attractive, whereas addition of the ketones or replacement of alcohols with ketones significantly reduced attraction. In Costa Rica field experiments testing alcohols 1–4 singly and in all binary, ternary, and quaternary combinations revealed 4 in combination with 2 was the major aggregation pheromone, equally attracting male and female WISW. Stereoisomeric 4 and (4S,5S)-4, the only isomer produced by WISW, were equally attractive. Addition of 4S-, 4R- or (±)-2 to (4S,5S)-4 significantly enhanced attraction. Sugarcane stalks in combination with 2 plus 4 (ratio of 1:8) were highly synergistic, whereas EAD-active sugarcane volatiles ethyl acetate, ethyl propionate, or ethyl butyrate only moderately increased attractiveness of the pheromone lure.

Ecometagenetics confirm high tropical rainforest nematode diversity.

The general patterns of increasing biodiversity from the poles to the equator have been well documented for large terrestrial organisms such as plants and vertebrates but are largely unknown for microbiota. In contrast to macrobiota, microbiota have long been assumed to exhibit cosmopolitan, random distributions and a lack of spatial patterns. To evaluate the assumption, we conducted a survey of nematode diversity within the soil, litter and canopy habitats of the humid lowland tropical rainforest of Costa Rica using an ultrasequencing ecometagenetic approach at a species‐equivalent taxonomic level. Our data indicate that both richness and diversity of nematode communities in the tropical rainforests of Costa Rica are high and exceed observed values from temperate ecosystems. The majority of nematode species were unknown to science, providing evidence for the presence of highly endemic (not cosmopolitan) species of still completely undiscovered biodiversity. Most importantly, the greater taxonomic resolution used here allowed us to reveal predictable habitat associations for specific taxa and thus gain insights into their nonrandom distribution patterns.

OPTIMIZATION OF SEMIOCHEMICAL-BASED TRAPPING OF Metamasius hemipterus sericeus (OLIVIER) (COLEOPTERA: CURCULIONIDAE)

Response of adults of the West Indian sugarcane weevil,Metamasius hemipterus sericeus, to various semiochemical treatments and physical trap designs was studied in southern Florida in field-grown banana and Canary Island date palms. Ethyl acetate released alone at 860–1007 mg/day was as effective for the capture ofM. h. sericeus as a combination of ethyl acetate (844–919 mg/day), ethyl propionate (348–362 mg/day), and ethyl butyrate (117–137 mg/day) and in one trial was more effective than fermenting sugarcane (250 g), ethyl propionate alone (353–384 mg/day), ethyl butyrate alone (123–174 mg/day), or no treatment. Ethyl acetate released alone at 675–683 mg/day was as attractive as 250 g of fermenting sugarcane or the racemic blend of the male-produced aggregation pheromones [(±)-5-methyl-nonan-4-ol and (±)-2-methyl-heptan-4-ol (8:1 ratio) “metalure”] at 3 mg/day. Weevil counts increased with binary combinations of ethyl acetate, sugarcane, and/or metalure over these treatments alone and the ternary combination was two to three times more effective than any of the binary treatments. Attraction to ethyl acetate released alone at 777 mg/day with metalure was greater than to the hydrolysis products of ethyl acetate (ethanol and/or acetic acid each released at about 6–8 mg/day) with metalure. Weevil counts from traps baited with 250 g of sugarcane and metalure increased with increasing dose of ethyl acetate to about 400 mg/day and then appeared to plateau. Ethyl acetate (about 700 mg/day) and metalure increased weevil counts in traps with increasing amounts of sugarcane (0–2 kg). Molasses (45 g) + water (158 ml) substitutes for sugarcane were about as effective for capturingM. h. sericeus as 250 g of fermenting sugarcane [all treatments with ethyl acetate (847 mg/day) and metalure]. Early experiments used a lethal pitfall trap. We demonstrated that several alternative trap designs were more effective than the lethal pitfall trap for capturing weevils. Color and height (on ground vs. 1 m on pole) did not affect trap efficacy when baited with the ternary combination of ethyl acetate (672–825 mg/day), sugarcane (250 g), and metalure.

Aggregation pheromone of palmetto weevil, Rhynchophorus cruentatus (F.) (Coleoptera: Curculionidae)

Abstract5-Methyl-4-octanol is the major aggregation pheromone of the palmetto weevil,Rhynchophorus cruentatus (F.). The pheromone (cruentol) was identified by coupled gas chromatographic-electroantennographic (GC-EAD) analysis of male-produced volatiles, coupled GC-mass spectrometry (MS) in electron impact and chemical ionization mode, and coupled GC-high resolution MS. In laboratory and field assays, a diastereomeric mixture of synthetic cruentol greatly enhanced attraction of weevils to cabbage palmetto,Sabal palmetto (Walter), stem tissue, indicating that cruentol and host volatiles are synergistically attractive. An attractive lure in combination with efficient traps should facilitate development of semiochemical-based management forR. cruentatus.