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A morphological, molecular, and behavioural characterization of Rhionaeschna galapagoensis is presented, based on a series of specimens and observations from San Cristóbal Island, Galápagos, including both adults and larvae. Several of the characters proposed earlier to distinguish between the adults of this species and its closest relative, R. elsia, are found to be variable, but the presence of a black band over the fronto–clypeal suture is confirmed as a good diagnostic character. The ultimate instar larvae of R. galapagoensis is described for the first time, and diagnosed from its closest relatives by a combination of characters, including the acute angle between the prothoracic apophyses, absence of lateral spines on abdominal segment 6, and length of cerci relative to paraprocts. Molecular analysis confirmed that R. galapagoensis and R. elsia are sister species, and showed that their genetic distance is the closest among the analyzed species, which is to be expected given the young age of the Galápagos Islands. The larvae of R. galapagoensis were very common and widespread in the mountain streams and a pond in the southwest of San Cristóbal. Swarms of tens of individuals formed at sunrise in the coastal vegetation, together with adults of Tramea cf. cophysa, feeding on small flying insects. Males showed patrolling behaviour on small sections of the streams and at a pond. Only one copulation was observed, lasting 10 minutes. Females oviposited alone on floating vegetation in running and standing waters. Our observations corroborate that R. galapagoensis and R. elsia are two parapatric species, morphologically and genetically close. In San Cristóbal, R. galapagoensis had large populations, apparently not threatened.

In this work we explored the female mating frequency of the quasi-gregarious egg parasitoid Anaphes nitens and its consequences on offspring sex-ratio. In a previous study we have shown that sex-ratio (proportion of males) increases with maternal age, suggesting that sperm depletion could occur if females mate only once. Here, we tested: (1) whether A. nitens females are polyandrous; (2) whether polyandry increases daughter production; and (3) the relationship between polyandry, fecundity and longevity. Virgin females were mated and provided with host egg-capsules and drops of honey as food. Afterward, they had the opportunity to re-mate daily during the rest of their life. About 55% of females were polyandrous, but multiple mating significantly increased the proportion of males in the offspring. In particular, daughter production of multi-mated females crashed at the 9th day of life, despite having obtained 2-3 matings. Hence, this suggests that females did not replenish their spermatheca by additional mating additional and that the amount of sperm received in a single copulation could be enough to produce an unconstrained offspring sex allocation. It is possible that the observed sex-ratio could depend on a limited storage capacity of the spermatheca or on the onset of physiological problems such as difficulty of sperm transfer, sperm plugs or displacement. Up to 52% of the multi-mated females did not produce any progeny after the first copulation. This suggests that a second mating was useful to obtain viable sperm. Furthermore, polyandry did not affect fecundity or longevity. Polyandry may be adopted to prevent matings with sperm depleted or incompatible males or to reduce the male harassment frequently experienced under laboratory conditions. [ABSTRACT FROM AUTHOR]

We investigated, by means of topical, residual and field assays, the toxicity of four insecticides (Bacillus thuringiensis, azadirachtin, flufenoxuron and ethofenprox) to adult, larvae and eggs of theEucalyptussnout-beetleGonipterus scutellatusand their side-effects on the adults of the solitary egg parasitoidAnaphes nitens, its natural enemy. In the topical toxicity experiment, both flufenoxuron and ethofenprox severely affectedG. scutellatus, causing 100% adult mortality after 7 days, and 75 and 100% larval mortality, respectively. Ethofenprox also caused 93% larval pre-eclosion mortality and 100% parasitoid mortality. In the residual experiment, azadirachtin acted as an effective larvicide when ingested (73% mortality after 7 days). Ethofenprox showed the highest toxicity, with 100% mortality of adults and larvae ofG. scutellatusafter 24 h. On the day 7 of observation, flufenoxuron resulted in 93% adult mortality and 100% larval mortality. After contact with residuals, only ethofenprox and flufenoxuron had adversely affectedA. nitens(98% mortality after 48 h).Bacillus thuringiensisexhibited only a slight activity against the pest or the parasitoid. The field experiment was carried out on three localities, with a preliminary sampling, and three samplings after the applications of the products, on days 7, 14 and 30. We evaluated the parasitism rate, parasitoid sex ratio, parasitoid pre-eclosion mortality, the abundance ofG. scutellatusadults, larvae and egg capsules, the pre-eclosion mortality rate of weevil eggs and larvae and the foliar damage. Only fluenoxuron and ethofenprox reduced the pest population and the foliar damages, but ethofenprox also significantly decreased the rate of parasitism (17%) after 7 days. Given our experimental results, we suggest that flufenoxuron and azadirachtin could be used in association with the natural enemy. [ABSTRACT FROM AUTHOR]

Mating in Gonipterus scutellatus is very long and lasts on average 6.98 ± 0.49 hr. This could be interpreted as a mechanism of male guarding, but males neither increase copulation duration nor ejaculate volume when sperm competition risk increases. We studied paternity patterns using RAPDs, and the influence of multiple matings on fertility, fecundity and sperm supply. Using twicemated females, we compared the percentage of polymorphic bands shared between first male/offspring, second male/offspring, mother/offspring, unrelated adults (males). In agreement with a previous study on paternity with the sterilemale technique, we found that sperm mixing seems the prevalent sperm competition mechanism. In a second experiment, adults were randomly assigned to three treatments: females mated only once; females allowed to mate ad libitum with one male; and females that could mate with seven different males. Females of the last group showed a significant increase in fertility. Additionally, we compared the stored sperm volumes among four groups: female whose copulation was interrupted after 15 min, those that mated once, twice or three times. This experiment indicates that sperm supply increases with the number of matings, excluding the possibility of sperm removal. These facts and the occurrence of sperm mixing might explain the lack of male response to increased male density in previous studies. [ABSTRACT FROM PUBLISHER]

Ectomycorrhizal (ECM) fungal community of the European chestnut has been poorly investigated, and mostly by sporocarp sampling. We proposed the study of the ECM fungal community of 2-year-old chestnut hybrids Castanea × coudercii (Castanea sativa × Castanea crenata) using molecular approaches. By using the chestnut hybrid clones 111 and 125, we assessed the impact of grafting on ECM colonization rate, species diversity, and fungal community composition. The clone type did not have an impact on the studied variables; however, grafting significantly influenced ECM colonization rate in clone 111. Species diversity and richness did not vary between the experimental groups. Grafted and ungrafted plants of clone 111 had a different ECM fungal species composition. Sequence data from ITS regions of rDNA revealed the presence of 9 orders, 15 families, 19 genera, and 27 species of ECM fungi, most of them generalist, early-stage species. Thirteen new taxa were described in association with chestnuts. The basidiomycetes Agaricales (13 taxa) and Boletales (11 taxa) represented 36% and 31%, of the total sampled ECM fungal taxa, respectively. Scleroderma citrinum, S. areolatum, and S. polyrhizum (Boletales) were found in 86% of the trees and represented 39% of total ECM root tips. The ascomycete Cenococcum geophilum (Mytilinidiales) was found in 80% of the trees but accounted only for 6% of the colonized root tips. These results could help to unveil the impact of grafting on fungal symbionts, improving management of chestnut agro-ecosystems and production of edible fungal species.

The identity and relevance of the ectomycorrhizal (ECM) fungal partners of Eucalyptus globulus was investigated in NW Spain, to detect which symbionts mainly support its invasiveness. Root tips of E. globulus and of three common native plant species (Quercus robur, Pinus pinaster and Halimium lasianthum) were collected in eucalypt plantations, Q. robur forests, P. pinaster plantations and shrublands. Fungal taxonomical identity was ascertained by use of rDNA and direct sequencing. We studied diversity, composition and colonization rate of the ECM fungal communities of E. globulus to determine if fungal assemblages are host specific (i.e. similar in different habitats) or more dependent on the neighbourhood context. We also identified the type of associations formed (i.e. co-introductions, familiar or novel associations). Twenty-six ECM taxa were associated with E. globulus. Most of them engaged in novel associations with eucalypts, whereas only three fungal species were co-introduced Australian aliens. Eucalypt fungal richness, diversity and colonization rate differed between habitats, being higher in native oak forests, whereas in shrublands E. globulus showed the lowest colonization rate and diversity. The Australian fungus Descolea maculata dominated the eucalypt fungal assemblage and also spread to the native host plants, in all the habitats, posing the risk of further co-invasion.

The Panel on Plant health performed a pest categorisation of the Australian Eucalyptus snout-beetle Gonipterus scutellatus (Coleoptera: Curculionidae), for the EU. G. scutellatus should be referred as the G. scutellatus species complex because it includes several cryptic species. A complete nomenclature of the species present in the EU is still pending. It is a quarantine pest listed in Annex IIB of Council Directive 2000/29/EC. Protected zones are in place in Greece and Portugal (Azores). In the EU, it has been found in Italy, France, Spain and Portugal. It only consumes Eucalyptus species leaves. The main pathways of spread are the trade of Eucalyptus timber, hitchhiking in various commodities, trade of apple fruit as well as of plants for planting or plant parts. Spread by flight is also possible. The climate of the EU protected zones is similar to that of the Member States (MS) where the G. scutellatus complex is established, and the pest's main host plants are present. The damaged trees suffer die-back and the development of epicormics shoots. Severe attacks may provoke massive amounts of tree death. Biological control by using the egg parasitoid wasp Anaphes nitens is the most effective control measure. Some species within the G. scutellatus complex are not yet present in the EU (including G. scutellatus sensu stricto) and might therefore be considered as potential union quarantine pests for the EU territory. At least two species within the G. scutellatus complex (most likely G. platensis and Gonipterus species no. 2) meet the criteria assessed by EFSA for consideration as potential protected zone quarantine pests for the territory of the protected zones: Greece and Portugal (Azores). The criteria for considering the G. scutellatus complex as a potential regulated non-quarantine pest for the EU are not met since plants for planting are not the main pathway., (© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Quantitative differences in plant defence metabolites, such as glucosinolates, may directly affect herbivore preference and performance, and indirectly affect natural enemy pressure. By assessing insect abundance and leaf damage rate, we studied the responses of insect herbivores to six genotypes of Brassica oleracea var. acephala, selected from the same cultivar for having high or low foliar content of sinigrin, glucoiberin and glucobrassicin. We also investigated whether the natural parasitism rate was affected by glucosinolates. Finally, we assessed the relative importance of plant chemistry (bottom-up control) and natural enemy performance (top-down control) in shaping insect abundance, the ratio of generalist/specialist herbivores and levels of leaf damage. We found that high sinigrin content decreased the abundance of the generalist Mamestra brassicae (Lepidoptera, Noctuidae) and the specialist Plutella xylostella (Lepidoptera, Yponomeutidae), but increased the load of the specialist Eurydema ornatum (Hemiptera, Pentatomidae). Plants with high sinigrin content suffered less leaf injury. The specialist Brevicoryne brassicae (Hemiptera, Aphididae) increased in plants with low glucobrassicin content, whereas the specialists Pieris rapae (Lepidoptera, Pieridae), Aleyrodes brassicae (Hemiptera, Aleyrodidae) and Phyllotreta cruciferae (Coleoptera, Chrysomelidae) were not affected by the plant genotype. Parasitism rates of M. brassicae larvae and E. ornatum eggs were affected by plant genotype. The ratio of generalist/specialist herbivores was positively correlated with parasitism rate. Although both top-down and bottom-up forces were seen to be contributing, the key factor in shaping both herbivore performance and parasitism rate was the glucosinolate concentration, which highlights the impact of bottom-up forces on the trophic cascades in crop habitats.

In this work, by artificially reproducing severe (75%) and moderate (25%) defoliation on maritime pines Pinus pinaster in NW Spain, we investigated, under natural conditions, the consequences of foliage loss on reproduction, abundance, diversity and richness of the fungal symbionts growing belowground and aboveground. The effect of defoliation on tree growth was also assessed. Mature needles were clipped during April 2007 and 2008. Root samples were collected in June-July 2007 and 2008. Collection of sporocarps was performed weekly from April 2007 to April 2009. Taxonomic identity of ectomycorrhizal fungi was assessed by using the internal transcribed spacer (ITS) regions of rDNA through the polymerase chain reaction (PCR) method, subsequent direct sequencing and BLAST search. Ectomycorrhizal colonization was significantly reduced (from 54 to 42%) in 2008 by 75% defoliation, accompanied with a decline in species richness and diversity. On the other hand, sporocarp abundance, richness and diversity were not affected by foliage loss. Some ECM fungal symbionts, which are assumed to have a higher carbon cost according to the morphotypes structure, were reduced due to severe (75%) defoliation. Furthermore, 75% foliage loss consistently depressed tree growth, which in turn affected the ectomycorrhizal growth pattern. Defoliation impact on ECM symbionts largely depends on the percentage of foliage removal and on the number of defoliation bouts. Severe defoliation (75%) in the short term (2 years) changed the composition of the ECM community likely because root biomass would be adjusted to lower levels in parallel with the depletion of the aboveground plant biomass, which probably promoted the competition among mycorrhizal types for host resources. The persistence of fungal biomass in mycorrhizal roots would be crucial for nutrient up-take and recovery from defoliation stress of the host plants.

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification), molecular analysis (internal transcribed spacer region amplification and sequencing), and fruitbody sampling, we assessed diversity and composition of the ectomycorrhizal fungal community colonizing juvenile Pinus pinaster Ait. under natural conditions in NW Spain. Overall, we found 15 Basidiomycetes and two Ascomycetes. Members of the family Thelephoraceae represented up to 59.4% of the samples. The most frequent species was Tomentella sublilacina followed by Thelephora terrestris, Russula drimeia, Suillus bovinus, and Paxillus involutus, while the less frequent were Pseudotomentella tristis, Lactarius subdulcis, Russula ochroleuca, and Entoloma conferendum. From October 2007 to June 2008, we sampled 208 sporocarps belonging to seven genera and nine species: Thelephora terrestris, Paxillus involutus, Suillus bovinus, Xerocomus badius, Scleroderma verrucosum, Amanita gemmata, A. rubescens, Amanita sp., and Russula sp. The species belonging to the genus Amanita, X. badius and S. verrucosum were not found on root samples. By comparing our results with a bibliographic review of papers published from 1922 to 2006, we found five genera and six species which have not been previously reported in symbiosis with P. pinaster. This is the first time that the diversity of the ectomycorrhizal fungal community associated with P. pinaster was investigated using molecular techniques. Considering that only 38% of the genera found by sequencing were found as fruitbodies, we conclude that integrating morphotyping and sporocarps surveys with molecular analysis of ectomycorrhizas is important to documenting the ectomycorrhizal fungus community.

Anaphes nitens is a solitary parasitoid of the egg capsules of the Eucalyptus snout beetle, Gonipterus scutellatus. Some traits of its natural history suggest that Local Mate Competition (LMC) could account for sex ratio adjustment in this species. We tested whether males emerged early, a prerequisite for fully local mating, and investigated the occurrence and effect of superparasitism on adult size and pre-emergence mortality, factors that might influence sex ratio adjustment. We found in field-collected egg capsules that males emerged first. To investigate the effects of superparasitism on adult size, we compared the sizes of parasitoids that emerged early and late from egg capsules collected in the field, and from egg capsules parasitized and superparasitized in the laboratory. Superparasitism reduced parasitoid size, affecting females more strongly than males, and increased pre-emergence mortality. We estimated A. nitens sex ratio and parasitism rate in the field during 2 years in five localities and during 4 years in a sixth. Following LMC we expected an increase in sex ratio (proportion of males) with increasing parasitism rate (assumed to reflect parasitoid density). We found that sex ratio decreased from 0.38 when the parasitism rate was low (0-20%) to 0.21 when parasitism was high (80-100%). In contrast with field results, a laboratory experiment showed that: (1) at a low parasitism level sex ratio was clearly female biased (0.28+/-0.04), (2) at a high parasitism level sex ratio increased (0.40+/-0.07), (3) male larval survivorship was not lower than female survivorship, and (4) low-quality hosts (i.e. superparasitized) were allocated more males. We conclude that LMC cannot explain the sex ratio adjustment observed in the field, even at low parasitism rates, and alternative explications for highly female-biased sex ratios must be found. One such alternative is female-biased dispersal.