The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps.

Chalcidoidea are mostly parasitoid wasps that include as many as 500 000 estimated species. Capturing phylogenetic signal from such a massive radiation can be daunting. Chalcidoidea is an excellent example of a hyperdiverse group that has remained recalcitrant to phylogenetic resolution. We combined 1007 exons obtained with Anchored Hybrid Enrichment with 1048 ultra-conserved elements (UCEs) for 433 taxa including all extant families, >95% of all subfamilies, and 356 genera chosen to represent the vast diversity of the superfamily. Going back and forth between the molecular results and our collective knowledge of morphology and biology, we detected bias in the analyses that was driven by the saturation of nucleotide data. Our final results are based on a concatenated analysis of the least saturated exons and UCE datasets (2054 loci, 284 106 sites). Our analyses support an expected sister relationship with Mymarommatoidea. Seven previously recognized families were not monophyletic, so support for a new classification is discussed. Natural history in some cases would appear to be more informative than morphology, as illustrated by the elucidation of a clade of plant gall associates and a clade of taxa with planidial first-instar larvae. The phylogeny suggests a transition from smaller soft-bodied wasps to larger and more heavily sclerotized wasps, with egg parasitism as potentially ancestral for the entire superfamily. Deep divergences in Chalcidoidea coincide with an increase in insect families in the fossil record, and an early shift to phytophagy corresponds with the beginning of the "Angiosperm Terrestrial Revolution". Our dating analyses suggest a middle Jurassic origin of 174 Ma (167.3-180.5 Ma) and a crown age of 162.2 Ma (153.9-169.8 Ma) for Chalcidoidea. During the Cretaceous, Chalcidoidea may have undergone a rapid radiation in southern Gondwana with subsequent dispersals to the Northern Hemisphere. This scenario is discussed with regard to knowledge about the host taxa of chalcid wasps, their fossil record and Earth's palaeogeographic history.

L-DOPA functions as a plant pheromone for belowground anti-herbivory communication.

While mechanisms of plant-plant communication for alerting neighbouring plants of an imminent insect herbivore attack have been described aboveground via the production of volatile organic compounds (VOCs), we are yet to decipher the specific components of plant-plant signalling belowground. Using bioassay-guided fractionation, we isolated and identified the non-protein amino acid l-DOPA, released from roots of Acyrtosiphon pisum aphid-infested Vicia faba plants, as an active compound in triggering the production of VOCs released aboveground in uninfested plants. In behavioural assays, we show that after contact with l-DOPA, healthy plants become highly attractive to the aphid parasitoid (Aphidius ervi), as if they were infested by aphids. We conclude that l-DOPA, originally described as a brain neurotransmitter precursor, can also enhance immunity in plants.

Genomic and metabolic profiling of two tomato contrasting cultivars for tolerance to Tuta absoluta.

MAIN CONCLUSION: Dissimilar patterns of variants affecting genes involved in response to herbivory, including those leading to difference in VOC production, were identified in tomato lines with contrasting response to Tuta absoluta. Tuta absoluta is one of the most destructive insect pest affecting tomato production, causing important yield losses both in open field and greenhouse. The selection of tolerant varieties to T. absoluta is one of the sustainable approaches to control this invasive leafminer. In this study, the genomic diversity of two tomato varieties, one tolerant and the other susceptible to T. absoluta infestation was explored, allowing us to identify chromosome regions with highly dissimilar pattern. Genes affected by potential functional variants were involved in several processes, including response to herbivory and secondary metabolism. A metabolic analysis for volatile organic compounds (VOCs) was also performed, highlighting a difference in several classes of chemicals in the two genotypes. Taken together, these findings can aid tomato breeding programs aiming to develop tolerant plants to T. absoluta.

TPS Genes Silencing Alters Constitutive Indirect and Direct Defense in Tomato.

Following herbivore attacks, plants modify a blend of volatiles organic compounds (VOCs) released, resulting in the attraction of their antagonists. However, volatiles released constitutively may affect herbivores and natural enemies' fitness too. In tomato there is still a lack of information on the genetic bases responsible for the constitutive release of VOC involved in direct and indirect defenses. Here we studied the constitutive emissions related to the two most abundant sesquiterpene synthase genes expressed in tomato and their functional role in plant defense. Using an RNA interference approach, we silenced the expression of TPS9 and TPS12 genes and assessed the effect of this transformation on herbivores and parasitoids. We found that silenced plants displayed a different constitutive volatiles emission from controls, resulting in reduced attractiveness for the aphid parasitoid Aphidius ervi and in an impaired development of Spodoptera exigua larvae. We discussed these data considering the transcriptional regulation of key-genes involved in the pathway of VOC metabolism. We provide several lines of evidence on the metabolic flux from terpenoids to phenylpropanoids. Our results shed more light on constitutive defenses mediated by plant volatiles and on the molecular mechanisms involved in their metabolic regulation.

Insights on the Impact of Arbuscular Mycorrhizal Symbiosis on Tomato Tolerance to Water Stress.

Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers, whose exploitation could represent a promising avenue for the development in the future of a more sustainable next-generation agriculture. The best understood function in symbiosis is an improvement in plant mineral nutrient acquisition, as exchange for carbon compounds derived from the photosynthetic process: this can enhance host growth and tolerance to environmental stresses, such as water stress (WS). However, physiological and molecular mechanisms occurring in arbuscular mycorrhiza-colonized plants and directly involved in the mitigation of WS effects need to be further investigated. The main goal of this work is to verify the potential impact of AM symbiosis on the plant response to WS To this aim, the effect of two AM fungi (Funneliformis mosseae and Rhizophagus intraradices) on tomato (Solanum lycopersicum) under the WS condition was studied. A combined approach, involving ecophysiological, morphometric, biochemical, and molecular analyses, has been used to highlight the mechanisms involved in plant response to WS during AM symbiosis. Gene expression analyses focused on a set of target genes putatively involved in the plant response to drought, and in parallel, we considered the expression changes induced by the imposed stress on a group of fungal genes playing a key role in the water-transport process. Taken together, the results show that AM symbiosis positively affects the tolerance to WS in tomato, with a different plant response depending on the AM fungi species involved.

Exposing belowground plant communication.

Root exudation could be harnessed for ecological and applied research.

Using Age-Stage Two-Sex Life Tables to Assess the Suitability of Three Solanaceous Host Plants for the Invasive Cotton Mealybug Phenacoccus solenopsis Tinsley.

Phenacoccus solenopsis Tinsley (Hemiptera: Coccomorpha: Pseudococcidae), the cotton mealybug, is an invasive polyphagous species that has been extending its geographic range, posing a conspicuous threat to many Mediterranean crops of economic importance. These include three species of Solanaceae, namely Solanum lycopersicum L. (tomato), Solanum tuberosum L. (potato) and Solanum melongena L. (eggplant) all of which are economically important worldwide. In this study, we used age-stage two-sex life tables to investigate the suitability of these three plant species as hosts for P. solenopsis and to calculate pest fitness, life history parameters and population projection parameters. All tested host plants that were suitable for the pest and eggplant host plant induced a higher fecundity (276.50 ± 10.78 eggs/female), net reproductive rate (R0) (243.32 ± 15.83 offspring/female) and finite rate of increase (λ) (1.18 ± 0.0043 day-1) and more extended adult longevity (males: 6.50 ± 0.34 days and females: 24.15 ± 0.50 days). Population growth predictions over a period of 90 days of infestation, commencing with an initial population of 10 eggs showed that adult population size was 674,551 on tomato, 826,717 on potato and 355,139 on eggplant. Our data on plant host preference of P. solenopsis will aid the development of appropriate management strategies and achieve successful control of this invasive pest in key Mediterranean crop systems.

Tomato below ground-above ground interactions: Trichoderma longibrachiatum affects the performance of Macrosiphum euphorbiae and its natural antagonists.

Below ground and above ground plant-insect-microorganism interactions are complex and regulate most of the developmental responses of important crop plants such as tomato. We investigated the influence of root colonization by a nonmycorrhizal plant-growth-promoting fungus on direct and indirect defenses of tomato plant against aphids. The multitrophic system included the plant Solanum lycopersicum ('San Marzano nano'), the root-associated biocontrol fungus Trichoderma longibrachiatum strain MK1, the aphid Macrosiphum euphorbiae (a tomato pest), the aphid parasitoid Aphidius ervi, and the aphid predator Macrolophus pygmaeus. Laboratory bioassays were performed to assess the effect of T. longibrachiatum MK1, interacting with the tomato plant, on quantity and quality of volatile organic compounds (VOC) released by tomato plant, aphid development and reproduction, parasitoid behavior, and predator behavior and development. When compared with the uncolonized controls, plants whose roots were colonized by T. longibrachiatum MK1 showed quantitative differences in the release of specific VOC, better aphid population growth indices, a higher attractiveness toward the aphid parasitoid and the aphid predator, and a quicker development of aphid predator. These findings support the development of novel strategies of integrated control of aphid pests. The species-specific or strain-specific characteristics of these below ground-above ground interactions remain to be assessed.

The Norwegian species of Copidosoma Ratzeburg (Hymenoptera: Chalcidoidea: Encyrtidae).

The species of Copidosoma Ratzeburg (Hymenoptera: Encyrtidae) are reviewed for Norway. Sixteen species are recognized, of which one, Copidosoma longicaudata sp. nov., is newly described, illustrated and compared with closely related species. Seven species represent new distribution records for Norway--C. aithyia (Walker), C. genale (Thomson), C. herbaceum Mercet, C. primulum (Mercet), C. radnense Erdos, C. thebe (Walker) and C. truncatellum (Dalman). An illustrated dichotomous key for the identification of both sexes of Norwegian Copidosoma is presented.

Small volatile lipophilic molecules induced belowground by aphid attack elicit a defensive response in neighbouring un-infested plants.

In pioneering studies on plant-aphid interactions, we have observed that Vicia faba plants infested by aphids can transmit signals via the rhizosphere that induce aboveground defence in intact, neighbouring plants. The aphid parasitoid Aphidius ervi is significantly attracted towards intact broad bean plants grown in a hydroponic solution previously harbouring Acyrtosiphon pisum-infested plants. To identify the rhizosphere signal(s) possibly mediating this belowground plant-plant communication, root exudates were collected using Solid-Phase Extraction (SPE) from 10-day old A. pisum-infested and un-infested Vicia faba plants hydroponically grown. To verify the ability of these root exudates to trigger defence mechanisms against the aphids we added them to V. fabae plants grown in hydroponic solution, and tested these plants in the wind-tunnel bioassay to assess their attractiveness towards the aphids' parasitoids A. ervi. We identified three small volatile lipophilic molecules as plant defence elicitors: 1-octen-3-ol, sulcatone and sulcatol, in SPE extracts of A. pisum-infested broad bean plants. In wind tunnel assays, we recorded a significant increase in the attractiveness towards A. ervi of V. faba plants grown in hydroponic solution treated with these compounds, compared to plants grown in hydroponic treated with ethanol (control). Both 1-octen-3-ol and sulcatol have asymmetrically substituted carbon atoms at positions 3 and 2, respectively. Hence, we tested both their enantiomers alone or in mixture. We highlighted a synergistic effect on the level of attractiveness towards the parasitoid when testing the three compounds together in respect to the response recorded against them singly tested. These behavioural responses were supported by the characterization of headspace volatiles released by tested plants. These results shed new light on the mechanisms underlying plant-plant communication belowground and prompt the use of bio-derived semiochemicals for a sustainable protection of agricultural crops.

Not All Field Margins Are Equally Useful: Effects of the Vegetation Structure of Margins on Cereal Aphids and Their Natural Enemies.

Differences in the semi-natural vegetation of field margins will affect the biological control services derived from the presence of these semi-natural habitats adjacent to fields. Of the plant functional traits that are most relevant for insects, plant life forms reflect different aspects of plant structure and functioning that can help predict the value of marginal vegetation for arthropods in agricultural systems. The aim of this study was to determine the effect of the vegetation structure of field margins on cereal aphids and on some of their natural enemies (parasitoids, hoverflies and ladybugs) in terms of plant life forms. We characterized margin vegetation using the relative cover of each life form and sampled insects in crops along transects parallel to field margins. Our results show that in the studied areas, the abundance of natural enemies was greater near margins dominated by annual plants than in margins dominated by perennial plants. On the other hand, the abundances of aphids and parasitism rates were higher near margins dominated by perennial woody plants than near margins dominated by perennial herbaceous plants. By promoting specific life forms in existing margins, farmers can enhance the conservation biological control and relieve aphid pressure on their crops.

Wolbachia infection and genetic diversity of Italian populations of Philaenus spumarius, the main vector of Xylella fastidiosa in Europe.

Philaenus spumarius is a cosmopolitan species that has become a major threat to European agriculture being recognized as the main vector of the introduced plant pathogen Xylella fastidiosa, the agent of the "olive quick decline syndrome", a disease which is devastating olive orchards in southern Italy. Wolbachia are bacterial symbionts of many insects, frequently as reproductive parasites, sometime by establishing mutualistic relationships, able to spread within host populations. Philaenus spumarius harbors Wolbachia, but the role played by this symbiont is unknown and data on the infection prevalence within host populations are limited. Here, the Wolbachia infection rate was analyzed in relation to the geographic distribution and the genetic diversity of the Italian populations of P. spumarius. Analysis of the COI gene sequences revealed a geographically structured distribution of the three main mitochondrial lineages of P. spumarius. Wolbachia was detected in half of the populations sampled in northern Italy where most individuals belonged to the western-Mediterranean lineage. All populations sampled in southern and central Italy, where the individuals of the eastern-Mediterranean lineage were largely prevalent, were uninfected. Individuals of the north-eastern lineage were found only in populations from the Alps in the northernmost part of Italy, at high altitudes. In this area, Wolbachia infection reached the highest prevalence, with no difference between north-eastern and western-Mediterranean lineage. Analysis of molecular diversity of COI sequences suggested no significant effect of Wolbachia on population genetics of P. spumarius. Using the MLST approach, six new Wolbachia sequence types were identified. Using FISH, Wolbachia were observed within the host's reproductive tissues and salivary glands. Results obtained led us to discuss the role of Wolbachia in P. spumarius, the factors influencing the geographic distribution of the infection, and the exploitation of Wolbachia for the control of the vector insect to reduce the spread of X. fastidiosa.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Molecular and chemical mechanisms involved in aphid resistance in cultivated tomato.

*An integrated approach has been used to obtain an understanding of the molecular and chemical mechanisms underlying resistance to aphids in cherry-like tomato (Solanum lycopersicum) landraces from the Campania region (southern Italy). The aphid-parasitoid system Macrosiphum euphorbiae-Aphidius ervi was used to describe the levels of resistance against aphids in two tomato accessions (AN5, AN7) exhibiting high yield and quality traits and lacking the tomato Mi gene. *Aphid development and reproduction, flight response by the aphid parasitoid A. ervi, gas chromatography-mass spectrometry headspace analysis of plant volatile organic compounds and transcriptional analysis of aphid responsive genes were performed on selected tomato accessions and on a susceptible commercial variety (M82). *When compared with the cultivated variety, M82, AN5 and AN7 showed a significant reduction of M. euphorbiae fitness, the release of larger amounts of specific volatile organic compounds that are attractive to the aphid parasitoid A. ervi, a constitutively higher level of expression of plant defence genes and differential enhancement of plant indirect resistance induced by aphid feeding. *These results provide new insights on how local selection can offer the possibility of the development of innovative genetic strategies to increase tomato resistance against aphids.

Electrophysiological and behavioural response of Philaenus spumarius to essential oils and aromatic plants.

The meadow spittlebug, Philaenus spumarius, is a highly polyphagous widespread species, playing a major role in the transmission of the bacterium Xylella fastidiosa subspecies pauca, the agent of the "Olive Quick Decline Syndrome". Essential oils (EOs) are an important source of bio-active volatile compounds that could interfere with basic metabolic, biochemical, physiological, and behavioural functions of insects. Here, we report the electrophysiological and behavioural responses of adult P. spumarius towards some EOs and related plants. Electroantennographic tests demonstrated that the peripheral olfactory system of P. spumarius females and males perceives volatile organic compounds present in the EOs of Pelargonium graveolens, Cymbopogon nardus and Lavandula officinalis in a dose-dependent manner. In behavioral bioassays, evaluating the adult responses towards EOs and related plants, both at close (Y-tube) and long range (wind tunnel), males and females responded differently to the same odorant. Using EOs, a clear attraction was noted only for males towards lavender EO. Conversely, plants elicited responses that varied upon the plant species, testing device and adult sex. Both lavender and geranium repelled females at any distance range. On the contrary, males were attracted by geranium and repelled by citronella. Finally, at close distance, lavender and citronella were repellent for females and males, respectively. Our results contribute to the development of innovative tools and approaches, alternative to the use of synthetic pesticides, for the sustainable control of P. spumarius aiming to contrasting the expansion of X. fastidiosa.

Natural enemies of armored scales (Hemiptera: Diaspididae) and soft scales (Hemiptera: Coccidae) in Chile: Molecular and morphological identification.

Scale insects (Hemiptera: Sternorrhyncha: Coccomorpha) are key pests of agricultural crops and ornamental plants worldwide. Their populations are difficult to control, even with insecticides, due to their cryptic habits. Moreover, there is growing concern over the use of synthetic pesticides for their control, due to deleterious environmental effects and the emergence of resistant populations of target pests. In this context, biological control may be an effective and sustainable approach. Hymenoptera Chalcidoidea includes natural enemies of scale insects that have been successfully used in many biological control programs. However, the correct identification of pest scale species and their natural enemies is particularly challenging because these insects are very small and highly specialized. Integrative taxonomy, coupling DNA barcoding and morphological analysis, has been successfully used to characterize pests and natural enemy species. In this study, we performed a survey of parasitoids and predators of armored and soft scales in Chile, based on 28S and COI barcodes. Fifty-three populations of Diaspididae and 79 populations of Coccidae were sampled over the entire length of the country, from Arica (18°S) to Frutillar (41°S), between January 2015 and February 2016. The phylogenetic relationships obtained by Bayesian inference from multilocus haplotypes revealed 41 putative species of Chalcidoidea, five Coccinellidae and three Neuroptera. Species delimitation was confirmed using ABGD, GMYC and PTP model. In Chalcidoidea, 23 species were identified morphologically, resulting in new COI barcodes for 12 species and new 28S barcodes for 14 species. Two predator species (Rhyzobius lophantae and Coccidophilus transandinus) were identified morphologically, and two parasitoid species, Chartocerus niger and Signiphora bifasciata, were recorded for the first time in Chile.

Trichoderma atroviride P1 Colonization of Tomato Plants Enhances Both Direct and Indirect Defense Barriers Against Insects.

Numerous microbial root symbionts are known to induce different levels of enhanced plant protection against a variety of pathogens. However, more recent studies have demonstrated that beneficial microbes are able to induce plant systemic resistance that confers some degree of protection against insects. Here, we report how treatments with the fungal biocontrol agent Trichoderma atroviride strain P1 in tomato plants induce responses that affect pest insects with different feeding habits: the noctuid moth Spodoptera littoralis (Boisduval) and the aphid Macrosiphum euphorbiae (Thomas). We observed that the tomato plant-Trichoderma P1 interaction had a negative impact on the development of moth larvae and on aphid longevity. These effects were attributed to a plant response induced by Trichoderma that was associated with transcriptional changes of a wide array of defense-related genes. While the impact on aphids could be related to the up-regulation of genes involved in the oxidative burst reaction, which occur early in the defense reaction, the negative performance of moth larvae was associated with the enhanced expression of genes encoding for protective enzymes (i.e., Proteinase inhibitor I (PI), Threonine deaminase, Leucine aminopeptidase A1, Arginase 2, and Polyphenol oxidase) that are activated downstream in the defense cascade. In addition, Trichoderma P1 produced alterations in plant metabolic pathways leading to the production and release of volatile organic compounds (VOCs) that are involved in the attraction of the aphid parasitoid Aphidius ervi, thus reinforcing the indirect plant defense barriers. Our findings, along with the evidence available in the literature, indicate that the outcome of the tripartite interaction among plant, Trichoderma, and pests is highly specific and only a comprehensive approach, integrating both insect phenotypic changes and plant transcriptomic alterations, can allow a reliable prediction of its potential for plant protection.

Tomato Plants Treated with Systemin Peptide Show Enhanced Levels of Direct and Indirect Defense Associated with Increased Expression of Defense-Related Genes.

Plant defense peptides represent an important class of compounds active against pathogens and insects. These molecules controlling immune barriers can potentially be used as novel tools for plant protection, which mimic natural defense mechanisms against invaders. The constitutive expression in tomato plants of the precursor of the defense peptide systemin was previously demonstrated to increase tolerance against moth larvae and aphids and to hamper the colonization by phytopathogenic fungi, through the expression of a wealth of defense-related genes. In this work we studied the impact of the exogenous supply of systemin to tomato plants on pests to evaluate the use of the peptide as a tool for crop protection in non-transgenic approaches. By combining gene expression studies and bioassays with different pests we demonstrate that the exogenous supply of systemin to tomato plants enhances both direct and indirect defense barriers. Experimental plants, exposed to this peptide by foliar spotting or root uptake through hydroponic culture, impaired larval growth and development of the noctuid moth Spodoptera littoralis, even across generations, reduced the leaf colonization by the fungal pathogen Botrytis cinerea and were more attractive towards natural herbivore antagonists. The induction of these defense responses was found to be associated with molecular and biochemical changes under control of the systemin signalling cascade. Our results indicate that the direct delivery of systemin, likely characterized by a null effect on non-target organisms, represents an interesting tool for the sustainable protection of tomato plants.

Anagyrus Howard (Hymenoptera: Encyrtidae) parasitoids of the invasive Delottococcus aberiae (De Lotto) (Hemiptera: Pseudococcidae) from South Africa, with description of two new species.

Delottococcus aberiae is an invasive mealybug native to South Africa that has been accidentally introduced into Spain. A survey of natural enemies in its native area to potentially be used for biological control of this pest resulted in a number of species, among which four belonged to the genus Anagyrus. Following an integrative approach using morphological and molecular data, two species new to science are here described and compared with closely related ones: Anagyrus aberiae sp.n. and Anagyrus antoniae sp.n. A dichotomous key to separate the species of Anagyrus attacking D. aberiae in South Africa is provided. [Zoobank LSID: Anagyrus aberiae Guerrieri sp. nov. (Fig. 1-7): LSID urn:lsid:zoobank.org:pub:8CF8983B-93DC-4ECF-A8FB-CF76E94319B3 Anagyrus antoniae Guerrieri sp. nov. (Fig. 8-12): LSID urn:lsid:zoobank.org:pub:8CF8983B-93DC-4ECF-A8FB-CF76E94319B3].

New records, descriptions and notes on Encyrtidae (Hymenoptera: Chalcidoidea) from Iran.

New additions to the Iranian fauna are reported. Three new species of Encyrtidae, Anagyrus osmoi sp. nov., Metaphycus prengoi sp. nov., and Microterys obricoi sp. nov., are described. Three new records, Anagyrus saccharicola Timberlake, 1932, Copidosoma filicorne (Dalman, 1820) and Paranathrix acanthococci (Myartseva, 1977) and one note are reported.