Piñol J., Senar M.A., Symondson W.O.C. (2018) The choice of universal primers and the characteristics of the species mixture determine when DNA metabarcoding can be quantitative. Molecular Ecology. : 0-0.LinkDoi: 10.1111/mec.14776
DNA metabarcoding is a technique used to survey biodiversity in many ecological settings, but there are doubts about whether it can provide quantitative results, that is, the proportions of each species in the mixture as opposed to a species list. While there are several experimental studies that report quantitative metabarcoding results, there are a similar number that fail to do so. Here, we provide the rationale to understand under what circumstances the technique can be quantitative. In essence, we simulate a mixture of DNA of S species with a defined initial abundance distribution. In the simulated PCR, each species increases its concentration following a certain amplification efficiency. The final DNA concentration will reflect the initial one when the efficiency is similar for all species; otherwise, the initial and final DNA concentrations would be poorly related. Although there are many known factors that modulate amplification efficiency, we focused on the number of primer-template mismatches, arguably the most important one. We used 15 common primers pairs targeting the mitochondrial COI region and the mitogenomes of ca. 1,200 insect species. The results showed that some primers pairs produced quantitative results under most circumstances, whereas some other primers failed to do so. In conclusion, depending on the primer pair used in the PCR amplification and on the characteristics of the mixture analysed (i.e., high species richness, low evenness), DNA metabarcoding can provide a quantitative estimate of the relative abundances of different species. © 2018 John Wiley & Sons Ltd.
Bañol, C., Pérez, N., Piñol, J., Barrientos, J.A., Ventura, D. (2017) Interaction of the aphid-parasitoid-ant network in plants associated with an organic citrus grove [Interacción de la red áfido-parasitoide-hormiga en plantas asociadas a un cultivo ecológico de cítricos]. Ecosistemas. 26: 67-79.LinkDoi: 10.7818/ECOS.2017.26-3.09
Mestre L., Piñol J., Barrientos J.A., Espadaler X. (2016) Differential ant exclusion from canopies shows contrasting top-down effects on community structure. Oecologia. 180: 193-203.LinkDoi: 10.1007/s00442-015-3442-z
Predators have far-reaching effects on communities by triggering top-down trophic cascades that influence ecosystem functioning. Omnivory and intraguild interactions between predators give rise to reticulate food webs and may either strengthen or dampen trophic cascades depending on context. Disentangling the effects of multiple predator species is therefore crucial for predicting the influence of predators on community structure. We focused on ants as dominant generalist predators in arthropod communities and set up a differential ant exclusion from canopies to examine its effects on assemblage species composition and densities of five arthropod groups (psocopterans, aphids, spiders, heteropterans and beetles). We coupled a glue band with tubes allowing only the ant Lasius grandis to reach the canopies to isolate its effect from the rest of crawling predators (ants, earwigs) and compared it against a full exclusion and a control. L. grandis alone had widespread effects on assemblage species composition, with contrasting species-specific responses within groups, where some species affected by L. grandis presence were not further affected by the presence of the whole crawling predator assemblage, and vice versa. Overall, L. grandis caused two- to threefold decreases of generalist predators and a threefold increase of aphids. However, it lacked further top-down effects on primary consumers, which only emerged when all crawling predators were present. This differential exclusion demonstrates the distinctive and widespread intraguild effects on community structure of a single ant species that contrast with the top-down effects exerted by the whole crawling predator assemblage. © 2015, Springer-Verlag Berlin Heidelberg.
Romeu-Dalmau C., Espadaler X., Piñol J. (2016) Management implications of earwigs' overwintering sites in a Mediterranean citrus grove. International Journal of Pest Management. : 1-6.LinkDoi: 10.1080/09670874.2015.1129079
To promote earwigs as natural enemies of pests, or to control their populations if they damage crops, earwigs can be managed during their overwintering period on the ground. Here, we obtained more than a ton of soil to study earwigs' overwintering sites in a citrus grove. We found four species of earwigs: Forficula pubescens, Euborellia annulipes, Euborellia moesta, and Nala lividipes. Surprisingly, and although the European earwig Forficula auricularia is abundant in the citrus canopies the rest of the year, we did not find any F. auricularia, indicating that this species spends the winter outside the citrus grove. Therefore, farmers willing to manage European earwig populations in citrus orchards need to consider the possibility that earwigs may spend the winter outside the field. Earwigs that were overwintering in the citrus grove were more abundant at the south side beneath the canopies than at the north side or between rows, indicating that management practices such as soil tillage can impact overwintering earwigs only beneath the canopies, but not between citrus rows. Overall, our results provide insights into how earwig populations can be successfully managed during winter in citrus orchards. © 2016 Taylor & Francis
Gomez-Polo P., Alomar O., Castane C., Aznar-Fernandez T., Lundgren J.G., Pinol J., Agusti N. (2015) Understanding trophic interactions of Orius spp. (Hemiptera: Anthocoridae) in lettuce crops by molecular methods. Pest Management Science. : 0-0.LinkDoi: 10.1002/ps.3989
BACKGROUND: The aphid Nasonovia ribisnigri (Mosley) (Hemiptera: Aphididae) and the thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) are common pests in Mediterranean lettuce crops, where Orius spp. are common generalist predators. Predation by Orius spp. was studied in a lettuce plot by conventional polymerase chain reaction (PCR) and real-time PCR analyses using specific primers of both main pests. Also, high-throughput sequencing was used to have a wider approach of the diet of these predators in natural field conditions. RESULTS: Molecular analyses indicated a higher predation on N. ribisnigri in spring and on F. occidentalis in summer. Predation on alternative prey, like Collembola, was also found in both seasons. Real-time PCR was more sensitive than conventional PCR in showing the target trophic links, whereas high-throughput sequencing revealed predation on other natural enemies - intraguild predation (IGP), showing other trophic interactions of Orius majusculus within the studied ecosystem. CONCLUSIONS: This study gives important information about the trophic relationships present in Mediterranean lettuce crops in different periods of the year. The detected predation by Orius spp. on alternative prey, as well as on other natural enemies, should be further investigated to clarify whether it adds or detracts to the biological control of N. ribisnigri and F. occidentalis. © 2015 Society of Chemical Industry.
Gomez-Polo P., Alomar O., Castane C., Lundgren J.G., Pinol J., Agusti N. (2015) Molecular assessment of predation by hoverflies (Diptera: Syrphidae) in Mediterranean lettuce crops. Pest Management Science. 71: 1219-1227.LinkDoi: 10.1002/ps.3910
BACKGROUND: Hoverflies (Diptera: Syrphidae) are generalist predators of a great variety of pests. Nasonovia ribisnigri (Hemiptera: Aphididae) and Frankliniella occidentalis (Thysanoptera: Thripidae) are two common pests in Mediterranean lettuce crops, where they occur alongside alternative prey (e.g. Collembola). A semi-field experiment was conducted in an experimental lettuce plot where hoverfly predation on N. ribisnigri, F. occidentalis and Collembola was studied by conventional PCR and qPCR using specific primers, as well as by next-generation sequencing (NGS) in order to reveal other potential trophic interactions. RESULTS: Trophic linkages between hoverflies and N. ribisnigri were the strongest both in spring and summer. F. occidentalis and Collembolans were also detected in both seasons, but with less frequency. qPCR detected a higher frequency of consumption than conventional PCR when both tests were run at optimal conditions. NGS analyses showed intraguild predation on other hoverfly species, as well as on anthocorids, spiders and even aphid parasitoids. CONCLUSIONS: Conventional PCR and qPCR provided important insights into Mediterranean hoverfly species predation on target pest and non-pest prey. NGS gave a complementary approach revealing a broader diet of these predators within the studied ecosystem. © 2014 Society of Chemical Industry.
Pinol J., Mir G., Gomez-Polo P., Agusti N. (2015) Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods. Molecular Ecology Resources. : 0-0.LinkDoi: 10.1111/1755-0998.12355
The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer-template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer-template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer-template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region. © 2014 John Wiley & Sons Ltd.
Brewitt K., Pinol J., Werner C., Beyschlag W., Espadaler X., Perez Hidalgo N., Platner C. (2014) Evaluating the importance of trophobiosis in a Mediterranean ant community: a stable isotope analysis. Insectes Sociaux. : 0-0.LinkDoi: 10.1007/s00040-014-0375-1
Trophobiosis between aphids (Aphididae, Hemiptera) and ants (Formicidae, Hymenoptera) is considered to provide an important source of nutrition for ants by aphid honeydew and aphids themselves used as prey. However, little is known about nutrient fluxes and the relative importance of trophobiosis for different ant species. Combining direct contact observations between ants and aphids with stable isotope analyses of distinct multitrophic sample sets (soil, plant, aphid, and ant), we aimed at disentangling the importance of trophobiosis in a Mediterranean food web and possible feedbacks on the functional diversity of ants in a species-rich organic Citrus plantation. We analyzed δ13C- and δ15N-values of sample sets for fertilized and natural soil, using the fertilizer as natural isotope label. The results showed trophic relationships between 18 host plant species, 22 aphid species, and 7 ant species. Direct observation revealed at least 40 different plant–aphid combinations and 25 aphid–ant combinations with a marked range of δ15N-values. However, the δ13C and δ15N isotope ratios still reflected the trophic levels. A significant correlation occurred between the isotope ratios of aphids and their host plants. However, no relationship was found between aphids and ants or between plants and ants revealing that many ant species do not exhibit a close relationship with their trophobiotic partners. Isotopic data allowed us to separate ant species into trophic functional groups and showed the relevance of other food resources. The applied fertilizer shifted the isotopic baseline for the whole trophic system. By combining the stable isotope analysis with the exact origin of the samples, we avoided a misleading interpretation of the high isotopic range of species. Thus, we emphasize the importance of considering a baseline in stable isotope food web studies.
Pinol J., San Andres V., Clare E.L., Mir G., Symondson W.O.C. (2014) A pragmatic approach to the analysis of diets of generalist predators: The use of next-generation sequencing with no blocking probes. Molecular Ecology Resources. 14: 18-26.LinkDoi: 10.1111/1755-0998.12156
Predicting whether a predator is capable of affecting the dynamics of a prey species in the field implies the analysis of the complete diet of the predator, not simply rates of predation on a target taxon. Here, we employed the Ion Torrent next-generation sequencing technology to investigate the diet of a generalist arthropod predator. A complete dietary analysis requires the use of general primers, but these will also amplify the predator unless suppressed using a blocking probe. However, blocking probes can potentially block other species, particularly if they are phylogenetically close. Here, we aimed to demonstrate that enough prey sequence could be obtained without blocking probes. In communities with many predators, this approach obviates the need to design and test numerous blocking primers, thus making analysis of complex community food webs a viable proposition. We applied this approach to the analysis of predation by the linyphiid spider Oedothorax fuscus in an arable field. We obtained over two million raw reads. After discarding the low-quality and predator reads, the libraries still contained over 61 000 prey reads (3% of the raw reads; 6% of reads passing quality control). The libraries were rich in Collembola, Lepidoptera, Diptera and Nematoda. They also contained sequences derived from several spider species and from horticultural pests (aphids). Oedothorax fuscus is common in UK cereal fields, and the results showed that it is exploiting a wide range of prey. Next-generation sequencing using general primers but without blocking probes provided ample sequences for analysis of the prey range of this spider and proved to be a simple and inexpensive approach. © 2013 John Wiley & Sons Ltd.
Mestre L., Garcia N., Barrientos J.A., Espadaler X., Piñol J. (2013) Bird predation affects diurnal and nocturnal web-building spiders in a Mediterranean citrus grove. Acta Oecologica. 47: 74-80.LinkDoi: 10.1016/j.actao.2013.01.001
Spiders and birds can greatly decrease insect populations, but birds also limit spider densities in some habitats. Bird predation is thought to be one of the causes behind nocturnal activity in spiders, so night-active spiders that hide in retreats during the day should be less affected by bird foraging than day-active spiders. However, this hypothesis has not yet been tested. We investigated the importance of bird predation on the spider community of a Mediterranean organic citrus grove. We excluded birds by placing net cages over the trees and we conducted visual searches in the canopies to sample web-building spiders. As there are many nocturnal species in the family Araneidae, we conducted searches both by day and by night to compare the abundance of active araneids in these two time periods. We sampled the tree trunks with cardboard bands to collect hunting spiders. In bird-excluded canopies there were more spiders of the families Araneidae and Theridiidae. There were higher numbers of active Araneidae at night, but these were just as negatively affected by bird predation as day-active Araneidae, so there was no evidence of nocturnal activity serving as an anti-predator strategy. We did not find any negative impact of birds on hunting spiders. Our results contrast with other studies reporting a negative effect of birds on hunting but not on web-building spiders. © 2013.
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