Co-obligate symbioses have repeatedly evolved across aphids, but partner identity and nutritional contributions vary across lineages

Aphids are a large family of phloem-sap feeders. They typically rely on a single bacterial endosymbiont, Buchnera aphidicola, to supply them with essential nutrients lacking in their diet. This association with Buchnera was described in model aphid species from the Aphidinae subfamily and has been assumed to be representative of most aphids. However, in two lineages, Buchnera has lost some essential symbiotic functions and is now complemented by additional symbionts. Though these cases break our view of aphids harbouring a single obligate endosymbiont, we know little about the extent, nature, and evolution of these associations across aphid subfamilies. Here, using metagenomics on 25 aphid species from nine subfamilies, re-assembly and re-annotation of 20 aphid symbionts previously sequenced, and 16S rRNA amplicon sequencing on 223 aphid samples (147 species from 12 subfamilies), we show that dual symbioses have evolved anew at least six times. We also show that these secondary co-obligate symbionts have typically evolved from facultative symbiotic taxa. Genome-based metabolic inference confirms interdependencies between Buchnera and its partners for the production of essential nutrients but shows contributions vary across pairs of co-obligate associates. Fluorescent in situ hybridisation microscopy shows a common bacteriocyte localisation of two newly acquired symbionts. Lastly, patterns of Buchnera genome evolution reveal that small losses affecting a few key genes can be the onset of these dual systems, while large gene losses can occur without any co-obligate symbiont acquisition. Hence, the Buchnera-aphid association, often thought of as exclusive, seems more flexible, with a few metabolic losses having recurrently promoted the establishment of a new co-obligate symbiotic partner

Deciphering host-parasitoid interactions and parasitism rates of crop pests using DNA metabarcoding

Abstract An accurate estimation of parasitism rates and diversity of parasitoids of crop insect pests is a prerequisite for exploring processes leading to efficient natural biocontrol. Traditional methods such as rearing have been often limited by taxonomic identification, insect mortality and intensive work, but the advent of high-throughput sequencing (HTS) techniques, such as DNA metabarcoding, is increasingly seen as a reliable and powerful alternative approach. Little has been done to explore the benefits of such an approach for estimating parasitism rates and parasitoid diversity in an agricultural context. In this study, we compared the composition of parasitoid species and parasitism rates between rearing and DNA metabarcoding of host eggs and larvae of the millet head miner, Heliocheilus albipunctella De Joannis (Lepidoptera, Noctuidae), collected from millet fields in Senegal. We first assessed the detection threshold for the main ten endoparasitoids, by sequencing PCR products obtained from artificial dilution gradients of the parasitoid DNAs in the host moth. We then assessed the potential of DNA metabarcoding for diagnosing parasitism rates in samples collected from the field. Under controlled conditions, our results showed that relatively small quantities of parasitoid DNA (0.07 ng) were successfully detected within an eight-fold larger quantity of host DNA. Parasitoid diversity and parasitism rate estimates were always higher for DNA metabarcoding than for host rearing. Furthermore, metabarcoding detected multi-parasitism, cryptic parasitoid species and differences in parasitism rates between two different sampling sites. Metabarcoding shows promise for gaining a clearer understanding of the importance and complexity of host-parasitoid interactions in agro-ecosystems, with a view to improving pest biocontrol strategies

Representativeness of the sampling analyzed in our study: Numbers of genera and species included in our dataset, known in Europe and occurring worldwide are reported for each aphid subfamily.

<p>*A full list of the materials analyzed and associated data are available in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097620#pone.0097620.s002" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097620#pone.0097620.s003" target="_blank">Table S2</a>. Classification is as for Remaudière and Remaudière <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097620#pone.0097620-Remaudire1" target="_blank">[28]</a> and Nieto Nafria <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097620#pone.0097620-NietoNafria2" target="_blank">[29]</a>. European data were provided by Fauna Europea (<a href="http://www.faunaeur.org/" target="_blank">http://www.faunaeur.org/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097620#pone.0097620-NietoNafria3" target="_blank">[37]</a>, and world data were provided by Foottit <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097620#pone.0097620-Foottit1" target="_blank">[1]</a>.</p

Intraspecific representativeness of our dataset.

<p>Frequency histograms of specimen numbers (A), number of haplotypes per species (B) and changes in the number of haplotypes with respect to the number of specimens sampled per species (C; box and whisker plot with the bottom and top of the boxes representing the 25th and 75th percentiles, respectively, bands near the middle of the boxes representing the medians and the ends of the whiskers representing the 10th and 90th percentiles).</p

Taxonomic representativeness of our dataset.

<p>Linear regression of the number of species per genus sampled in this study against the number of known species per genus in Europe.</p

Distribution of pairwise K2P distances among 1020 specimens of aphids, based on <i>COI</i> sequences.

<p>Graphs A and B include all pairwise comparisons, graph C includes each pair of haplotypes only once. On the box and whisker plots in B and C, the bottom and top of the boxes represent the 25th and 75th percentiles, respectively, the bands near the middle of the boxes represent the median, the ends of the whiskers represent the 2.5^th and 97.5^th percentiles and dots represent the outliers beyond 95% of the distribution.</p

Haplotype accumulation curves.

<p>The curves represent the mean number of haplotypes accumulated through random permutations (subsampling of sequences) for <i>Aphis fabae</i> (dotted line), <i>A. craccivora</i> (dashed line) and <i>Brachycaudus helichrysi</i> (solid line).</p

Patterns of <i>COI</i> divergence for 155 species represented by at least two individuals.

<p>For each nominal species, minimum between-species divergence (Min-BSD) is plotted against maximum within-species divergence (Max-WSD). Points above the diagonal correspond to cases in which species identification is straightforward. Colored dots represent nominal species detected as outliers in the species divergence distribution. Green dots represent the species with high levels of intraspecific divergence; red dots represent species with exceptionally low levels of interspecific genetic divergence. Distances are calculated with a K2P model of base substitution.</p

Focus on some problematic clades for barcode assignment.

<p>See Figure S4 for the complete NJ tree. Identification numbers of each clade are reported on the tree silhouette. Bootstrap support values >50 are provided. Note that the scale of genetic K2P divergence differs between subtrees.</p