Methods |
Corresponding author: Stéphane Boyer ( stephane.boyer@univ-tours.fr ) Academic editor: Matt Hill
© 2020 Marie-Caroline Lefort, Jacqueline R. Beggs, Travis R. Glare, Thomas E. Saunders, Erin J. Doyle, Stéphane Boyer.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Lefort M-C, Beggs JR, Glare TR, Saunders TE, Doyle EJ, Boyer S (2020) A molecular approach to study Hymenoptera diets using wasp nests. NeoBiota 63: 57-79. https://doi.org/10.3897/neobiota.63.58640
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The study of animal diets has benefited from the rise of high-throughput DNA sequencing applied to stomach content or faecal samples. The latter can be fresh samples used to describe recent meals or older samples, which can provide information about past feeding activities. For most invertebrates, however, it is difficult to access ‘historical’ samples, due to the small size of the animals and the absence of permanent defecation sites. Therefore, sampling must be repeated to account for seasonal variation and to capture the overall diet of a species.
This study develops a method to describe the overall diet of nest-building Hymenoptera, based on a single sampling event, by analysing prey DNA from faeces accumulated in brood cells. We collected 48 nests from two species of introduced paper wasps (Polistes chinensis Fabricius and P. humilis Fabricius) in the urban and peri-urban areas of Auckland, New Zealand and selected two samples per nest. One from brood cells in the outer layer of the nest to represent the most recent diet and one from brood cells in an inner layer to represent older diet.
Diet differed between species, although both fed mainly on Thysanoptera, Lepidoptera and Acariformes. Prey taxa identified to species level included both agricultural pests and native species. Prey communities consumed were significantly different between inner and outer nest samples, suggesting seasonal variation in prey availability and/or a diversification of the wasps’ diet as the colony grows. We also show for the first time potential predation of marine organisms by Polistes wasps.
Our study provides field evidence that Polistes wasps feed on agricultural pests, supporting the hypothesis that some social wasp species could have a suppressing effect on agricultural pests. The proposed methodology is readily applicable to other nest-building Hymenoptera and has the potential to provide comprehensive knowledge about their diet with minimum sampling effort. Such knowledge is essential to measure the ecological impact of invasive Vespidae and support the conservation of native invertebrate biodiversity.
eDNA, frass, metabarcoding, paper wasps, social insects, trophic interactions
In recent years, the study of invertebrate diets has been improved through the application of molecular methods to detect trophic interactions (
Social wasp colonies are organised around a caste system containing a reproductive queen, male drones and female workers which construct a nest out of mud or wood fibres (
Social wasps are considered pests in many regions of the world (
The main aim of the study was to develop a new molecular method to study the diet of social wasps by retrieving prey DNA from faeces left by wasp larvae inside the nest. Historically, wasp diets have been studied by collecting and identifying the prey items carried by adult foraging wasps when they return to the nest to feed the larvae (
We collected Polistes nests in the Auckland region, New Zealand, during the peak of the 2017 summer season (i.e. March-May). As wasp nests are built every year and do not last more than one season, potential nesting sites were located with the aid of previous occurrence records on iNaturalistNZ (https://inaturalist.nz/), an open biodiversity observation platform built to record the occurrence of taxa across New Zealand. A total of 53 active nests, i.e. with live adults flying around and live larvae developing inside, were collected between 1 March and 15 May 2017 (Fig.
Location of samples collected in and around Auckland. Black dots correspond to areas where one or several nests were collected. See legend for colour code for land cover classification (from geofolio.org).
Amongst the collected nests, only the largest and best preserved were used for analyses (n = 44 for P. chinensis and n = 4 for P. humilis). Using bleached and sterilised tweezers, insect frass was sampled from one cell located on the outer ring (representing late season) and from one cell located on an inner ring (representing early season) of each nest. These two rings or layers represent different batches of brood and, therefore, they preserve a record of the diet of the colony at two different times. The older frass samples may have been produced by the earliest larvae, which develop between November and January (
As prey DNA was likely to be highly degraded, primers targeting a short (313 bp) gene region of the mitochondrial gene COI (Cytochrome Oxydase subunit 1) were used for amplification. We selected the pair of primers mICOIintF (5'-GGWACWGGWTGAACWGTWTAYCCYCC-3';
The bioinformatic workflow was performed by NGBS (Nextgen Bioinformatic Services, New Zealand), based on the vsearch pipeline (
To limit false positives and remove potential sequencing errors, only MOTUs, for which read abundance within a sample was superior to 0.5% of the total, were retained. To ensure reliable identification, only sequences for which the best hit had a query coverage over 70% were retained, which corresponded to an overlap of 250 bp or more between the query and the best hit. Retained MOTUs were identified to species level when the percentage identity was ≥ 98% or assigned to the order of the corresponding best hits when their percentage identity was between 80 and 98%. Reads with percentage identity < 80% were not retained for prey identification.
MOTUs, for which the best hit had more than 98% identity, but the species name was not available in Genbank (e.g. only genus or family name), were searched against the Barcode Of Life Database (BOLD). Only MOTUs identified as terrestrial invertebrates were considered as prey. Reads corresponding to Polistes wasp DNA were used to confirm species identification of the wasp to which the nest belonged. Prey MOTUs, confidently identified at the species level, were categorised as native or introduced species based on Scott (1984) and the New Zealand Organisms Register (http://www.nzor.org.nz/).
Due to an imbalance in the number of samples analysed for P. humilis (n = 8) and P. chinensis (n = 88), we used the non-parametric Kruskall-Wallis test to compare the number of reads, as well as prey richness between the two wasp species. To assess for potential DNA degradation in older samples, we compared the number of reads and the diversity of MOTUs detected in inner versus outer samples. The number of reads was compared using the non-parametric Wilcoxon test to account for the non-normality and paired nature of the data. With regards to diversity, the cumulative number of MOTUs detected from the inner and outer layer of P. chinensis’ nests was compared using a Koglomorov-Smirnov test. As the analysis of only eight samples from four nests for P. humilis led to a low coverage of its diet, results for this species are only considered as indicative. For this same reason, a detailed analysis of prey community was only conducted for P. chinensis. Differences in prey species assemblage between inner and outer samples of P. chinensis nests were investigated using an Analysis of Deviance on a multivariate generalised linear model. A negative binomial distribution was chosen for this analysis, based on the dispersion of the residuals. In nests where more than 50 reads of marine origin were detected, the number of reads from commercial seafood and non-commercial sea organisms were analysed in relation to the distance to the sea using the non-parametric Kruskall-Wallis test. This analysis aimed to determine whether marine DNA came from active predation, scavenging from human fishing activities or contamination from sea spray.
All analyses were performed in R (
DNA was successfully amplified and sequenced from all faecal samples. A total of 7,767,737 high quality merged reads were obtained after clean-up of the raw Illumina reads. Of these, 7,530,408 could be clustered at 97% identity in 1,436 MOTUs, which were then compared to the NCBI database and analysed thereafter (see Suppl. material
Detection of organisms in the faeces of wasp larvae a Proportion of MOTUs corresponding to the main categories (left hand-side) and detailed categories excluding fungi (right hand-side) b Number of reads per sample in relation to wasp species (left boxplots) and number of reads in inner versus outer samples (right boxplots) c Total number of reads corresponding to marine, predator and terrestrial invertebrate prey DNA after removal of fungal DNA in both wasp species (left) and in outer versus inner samples (right).
The number of reads per sample was not significantly different between the two species of wasps (KW, χ2 = 0.325, df = 1, p = 0.569) (Fig.
When comparing inner and outer samples, significantly more reads were obtained from outer samples (Wilcoxon, V = 376, p = 0.029) (Fig.
Only 11% of the MOTUs corresponding to terrestrial invertebrates could be identified to the species level, corresponding to 21 species of prey in the diet of P. chinensis and eight species in the diet of P. humilis. (Table
Using 88 samples from P. chinensis nests, 260 MOTUs were detected, corresponding to an estimated 91% of the species’ overall terrestrial invertebrate diet (Fig.
Detection of prey MOTUs in the faeces of wasp larvae. Cumulative curves of number of invertebrate prey MOTUs in relation to number of samples. Left: P. chinensis, right: P. humilis. Cumulative curves are in red, the area delimited by the dashed lines corresponds to the 95% confidence interval. On the left, the horizontal solid line represents the estimated total number of prey MOTUs in the diet of P. chinensis. The purple and green hatched areas correspond to cumulative curves obtained with inner and outer samples, respectively.
When considering only terrestrial invertebrates, a total of 15 different orders were detected in the diet of P. chinensis and nine orders in the diet of P. humilis.
For both wasp species, the most commonly detected prey belonged to the orders Thysanoptera, Lepidoptera and Acariformes. They were respectively detected in 100%, 74% and 53% of P. chinensis samples and in 100%, 88% and 63% of P. humilis samples (Fig.
Diet composition of two Polistes wasp species from urban and sub-urban areas of Auckland (New Zealand), based on DNA analyses of larval faeces in nests (n = 88 for P. chinensis; n = 8 for P. humilis) a Frequency of occurrence of different invertebrate orders in the diet of P. chinensis (left) and P. humilis (right) b Relative read abundance of prey taxa as measured from each individual faecal sample (i.e. for each individual wasp larvae) in inner and outer cells of the nest. c Occurrence of the different prey genera, in the diet of each individual wasp larvae. See a for colour code.
At the MOTU level, the prey community composition, based on relative read abundance of prey taxa as measured from each individual faecal sample (RRA), varied with species (Analysis of Deviance: Dev1,94 = 713.4, p = 0.002) and to a lesser extent with the location of the samples in the nest (Dev1,93 = 576.3 p = 0.032). Similar results were obtained at the order level, with significant differences in prey community composition between wasp species (Dev1,94 = 90.18, p = 0.002) (Fig.
A total of 32 MOTUs could be identified to the species level corresponding to 23 species, most of which were lepidopteran species (18/23) (Table
Prey taxa identified at the species level in the diet of Polistes humilis and P. chinensis.
Species | Family | Order | Native or Introduced | Agricultural pest | Number of samples tested positive | |
P. humilis (n = 8) | P. chinensis (n = 88) | |||||
Anarsia dryinopa | Gelechiidae | Lepidoptera | Introduced | 6 | ||
Anatrachyntis badia | Cosmopterigidae | Lepidoptera | Introduced | 2 | 14 | |
Caliroa cerasi | Tenthredinidae | Hymenoptera | Introduced | Pest | 1 | |
Chrysodeixis eriosoma | Noctuidae | Lepidoptera | Introduced | Pest | 6 | |
Ctenoplusia limbirena | Noctuidae | Lepidoptera | Introduced | 13 | ||
Ctenopseustis fraterna | Tortricidae | Lepidoptera | Native | 3 | 8 | |
Ctenopseustis obliquana | Tortricidae | Lepidoptera | Native | 5 | 17 | |
Declana floccosa | Geometridae | Lepidoptera | Native | 3 | 3 | |
Declana leptomera | Geometridae | Lepidoptera | Native | 2 | ||
Ectopatria umbrosa | Noctuidae | Lepidoptera | Introduced | 6 | ||
Ectopsocus meridionalis | Ectopsocidae | Psocoptera | Introduced | 2 | ||
Eressa strepsimeris | Erebidae | Lepidoptera | Introduced | 8 | ||
Graphania mutans | Noctuidae | Lepidoptera | Native | 7 | ||
Isotenes miserana | Tortricidae | Lepidoptera | Introduced | 1 | ||
Leucania stenographa | Noctuidae | Lepidoptera | Introduced | 11 | ||
Meteorus pulchricornis | Braconidae | Hymenoptera | Introduced | 4 | ||
Mythimna separata | Noctuidae | Lepidoptera | Introduced | Pest | 18 | |
Oxysarcodexia varia | Sarcophagidae | Diptera | Introduced | 1 | ||
Planotortrix notophaea | Tortricidae | Lepidoptera | Native | 2 | ||
Polistes humilis | Vespidae | Hymenoptera | Introduced | 12 | ||
Scopula rubraria | Geometridae | Lepidoptera | Native | 4 | ||
Tebenna micalis | Choreutidae | Lepidoptera | Native | 2 | ||
Thysanoplusia orichalcea | Noctuidae | Lepidoptera | Introduced | Pest | 7 |
Over 236,000 reads clustered in 62 MOTUs were identified as marine organisms, mainly corresponding to polychaetes, fish, molluscs and cephalopods (Fig.
Marine organisms detected in the faeces of paper wasp larvae. Blue bars correspond to potentially commercial sea products, green bars correspond to non-commercial taxa.
Number of reads (top) and number of MOTUS (bottom) of marine origin in relation to distance to the sea. Samples collected within 1 km of the coast were considered close to sea, while samples collected at more than 1 km inland were considered as distant from the sea. Only samples with more than 50 reads from marine origin are represented. Seafood include fish, molluscs, cephalopods, crustaceans and echinoderms, while non-seafood include Polychaetes, Tunicates, Hydrozoa and Amoeba.
By analysing prey DNA from faeces accumulated in brood cells, we were able to describe the overall diet of two social Hymenoptera in the urban and peri-urban areas of Auckland, New Zealand. We detected both native species and agricultural pests in the diet of P. humilis and P. chinensis and our analysis showed variation between older and more recent faecal samples.
Our method led to the successful amplification of prey DNA from paper wasps’ nests and the description of the diet of two Polistes wasps in urban and peri-urban areas around Auckland, New Zealand. We used generalist degenerated PCR primers with the aim of amplifying DNA from a wide prey spectrum without a priori selection of particular taxa. However, wasp nests appeared to harbour a wide variety of fungi, which were strongly amplified by our primers (1,029 OTU detected). Some of these may be entomopathogenic fungi that are known to occur in wasp nests (
A large number of reads corresponding to species of marine origin were obtained from the faeces of wasps, in particular that of P. humilis. To our knowledge, predation of marine organisms by Polistes wasps has never been reported before. The great majority of reads belonged to marine worms, probably from the genus Dasybranchus, which includes species present in sand, rocky intertidal regions and shallow waters (e.g.
Such species could be exposed at low tide and may have been targeted by the wasps. However, we found no significant relationship between distance to the sea and number of reads from marine origin. Some of the other marine organisms detected in our samples, such as squid, are unlikely to have been captured by the wasps. While the presence of marine DNA may correspond with actual consumption, it may also reflect insects feeding on carrion washed up by the tide or feeding on the by-products of human activities (e.g. markets, fishing harbours, food waste). Many of the taxa detected include commercial marine products (fish, crustaceans, cephalopods, echinoderms), which would have been available near harbours or human habitation. Therefore, while these reads may not represent the natural diet of Polistes wasps, they do suggest that human activity could strongly influence the diet of some colonies by providing an alternative food source. The greater presence of DNA from marine origin in outer samples (five times more abundant than in inner samples) could suggest a seasonal effect of human fishing activity and/or external contamination by sea spray. Contamination is particularly likely for non-commercial species, mainly represented by polychaete worms, which release large amounts of DNA during swarming events.
The saturation of the MOTU accumulation curve (Fig.
Our results showed that the two species of wasps displayed a large overlap in their dietary niche (Fig.
Another important group, especially in the diet of P. chinensis, was Lepidoptera. Identification to the level of species was successful for about a third of the lepidopteran MOTUs (Suppl. material
Prey diversity in the diet of P. humiils and P. chinensis and diet overlap. The horizontal width of the rectangles corresponds to the number of MOTU for each order. Light grey areas correspond to MOTUs detected in the diet of each wasp species. Darker grey areas correspond to MOTUs detected in the diet of both wasp species. Most silhouettes were obtained from Phylopic.org.
Although mites were considered as potential prey in our analysis, it is likely that some of these taxa could be parasitic species that live on prey, on the wasps themselves or in the nest (
The sizes of collected nests varied over the eleven weeks of collection. Therefore, our samples were not suitable for a well-calibrated seasonal analysis of wasp diet. However, comparing samples taken from inner (older) and outer (younger) layers of the same nest could provide an estimate of how variable the colony diet was within the few weeks necessary to build one or more additional layers of brood cells. The composition of prey communities differed between inner and outer samples, which could reflect variation in prey availability at different times of the year. In addition, we detected greater prey richness in outer (younger) samples, which could indicate that the colony diet diversifies as the number of workers increases. However, this effect could also be explained by the higher degradation of specific prey DNA in inner (older) samples. Therefore, seasonal differences observed must be taken with caution as the rate of DNA degradation was not measured in our study. It is possible that a higher DNA degradation process in older samples conceals part of the early season prey diversity. We also recorded more contamination in samples located on the outer layers that were directly exposed to ambient air, than in samples located on inner layers that were somewhat sheltered inside the nest.
At the very least, the analysis of two faecal samples per nest (inner and outer samples) allowed for a greater coverage of each colony’s diet. However, the number of samples per nest required to accurately estimate the diet of a whole colony may be greater and could vary significantly, depending on the size of the nest and the species of interest. It is, for example, known that Polistes nests usually contain between 20 to 400 cells, while some Vespula species can build significantly larger nests. For example, Vespula germanica Fabricius, can build nests comprising over 450,000 cells (Scott 1984) with a colony biomass of up to 600 g (
Sampling multiple cells from each nest provides more insight into how the colony diet varies over time and it could identify predatory activity at a much higher resolution through time, potentially mirroring the phenology of the different prey species. However, in Vespula wasps, nests are often organised in multiple combs with little if any overlapping cell layers (
The methodology presented here has the potential to greatly assist in the study of social wasp ecology. Compared to traditional DNA recovery methods, we have developed a method which can provide an overview of the diet of a colony through time, based on a single sampling event. If nests are sampled after they are abandoned at the end of the season, it might be possible to uncover a comprehensive record of the colony diet, assuming the degradation of prey DNA remains limited. We hope that this method will be applied to study the ecology of other nest building Hymenoptera, including native and invasive species such as Asian hornets (Vespa velutina Lepeletier). Regarding the latter, better knowledge of their diet is essential to measure the ecological impact of their invasion and to ensure the conservation of native invertebrate biodiversity and the ecosystem services they provide (
This study was geographically limited to the urban and peri-urban regions of Auckland. Given this geographic restriction and the relatively low number of nests analysed for P. humilis, our results should not be regarded as a comprehensive description of the diet of Polistes wasps which are relatively widespread and abundant in many parts of New Zealand (
Raw DNA sequences will be made available on GenBank. The summarised data and R codes can be accessed at the following DOI addresses:
This study was funded by an internal early career research grant obtained my MCL in 2017 at the Unitec Institute of Technology (RI17004). Logistical support was provided by the Applied Molecular Solutions Research Focus at Unitec. University of Auckland provided financial support for JRB. We are grateful to the Environmental and Animal Science team at Unitec: Diane Fraser, Mel Galbraith, Dan Blanchon and Lorne Roberts for providing nests and Unitec students for providing first proof of concept during a Conservation Science tutorial (course NSCI 7732, year 2016).
Figure S1. Data processing and number of reads and MOTUs retained or discarded at each step of the bioinformatics analysis
Data type: statistical data
Figure S2. Percentage identity for all prey MOTUs and only Lepidoptera
Data type: statistical data
Explanation note: Percentage identity for all prey MOTUs (left) and only Lepidoptera (right). Red dots are MOTUs identified at the species level (i.e. percentage identity ≥ 98%).