Description of immature stages of Rhinusa species (Coleoptera, Curculionidae, Mecinini) with a focus on diagnostic morphological characters at the species and genus levels.

The mature larvae of the following fourteen Rhinusa species are described and illustrated: Rhinusaantirrhini (Paykull, 1800), R.asellus (Gravenhorst, 1807), R.collina (Gyllenhal, 1813), R.eversmanni (Rosenschoeld, 1838), R.florum (Rubsaamen, 1895), R.herbarum (H. Brisout de Barneville, 1862), R.incana (Kirsch, 1881), R.linariae (Panzer, 1796), R.melas (Boheman, 1838), R.neta (Germar, 1821), R.pilosa (Gyllenhal, 1838), R.rara Tosevski & Caldara, 2015, R.tetra (Fabricius, 1792), and R.vestita (Germar, 1821). The pupae of thirteen of them (except R.incana) were also described. The comparison of larval morphological characters and plant preferences provides evidence supporting the existence of different species groups previously established according to a phylogenetic analysis based on adult morphological characters. The following diagnostic attributes distinguishing the genus Rhinusa are highlighted. For the larvae: (1) pronotal shield indistinct; (2) thoracic prodorsal fold small or even vestigial; (3) abdominal postdorsal folds (especially of segments III-VII) high or even in the form of conical protuberances; (4) cuticle of abdominal segments densely covered with asperities; (5) cuticle without dark spots or dark pigmentation; (6) head suboval, rarely round; (7) labrum usually with 2 als; (8) des1 short or absent, rarely elongated; and (9) fs1-3 usually absent or minute. For the pupae: (1) body stout; (2) head protuberances always present; (3) pronotal protuberances (if present), separated at bases of the pronotum, always wider than higher; (4) abdominal protuberance usually present, wide or round; (5) femora usually with a single fes; and (6) urogomphi short or vestigial. Keys to the larvae and pupae described here are provided. All the characters used for identification are illustrated by photographs or drawings. Biological and distribution data, including new information, are provided for all the species studied.

Developmental Biology and Identification of a Garden Pest, Otiorhynchus (Podoropelmus) smreczynskii Cmoluch, 1968 (Coleoptera, Curculionidae, Entiminae), with Comments on Its Origin and Distribution.

The mature larva and pupa of Otiorhynchus smreczynskii are described and illustrated with complete chaetotaxy for the first time. Five larval instars and the factors for larval growth are determined, and the larval development of this species is comprehensively described. In order to confirm species affiliation, selected larvae were subjected to genetic analysis (mtCOI). Host plants and unique feeding signs of some Entiminae species are presented, and all available data on development are documented and interpreted. Additionally, the morphometry of 78 specimens (48 of O. smreczynskii and 30 of O. rotundus) was examined in order to verify the usefulness of morphological features in distinguishing both species. The female genitalia of both species are illustrated, described and compared with each other for the first time. Finally, the updated distribution of O. smreczynskii is given, and a possible origin of O. smreczynskii and O. rotundus is proposed.

Description of immature stages of Gymnetron species (Coleoptera, Curculionidae, Curculioninae), with particular emphasis on the diagnostic morphological characters at the generic and specific levels.

The immature stages of the following five Palaearctic Gymnetron species are described for the first time: G.tibiellum Desbrochers des Loges, 1900, G.veronicae (Germar, 1821), G.rotundicolle Gyllenhal, 1838, G.melanarium (Germar, 1821), and G.villosulum Gyllenhal, 1838. These species belong to four different groups previously established according to a phylogenetic analysis: the first two belong to the G.veronicae group and the other three to groups respectively bearing their name (G.rotundicolle, G.melanarium, and G.villosulum groups). All these species exhibit several diagnostic characters distinguishing them from each other. Some characters that can be used to separate Gymnetron from other genera in the tribe are also suggested. Three highly significant characters for the larvae and three for the pupae were identified. For the larvae they are: (1) labial palpi with single palpomeres, (2) all spiracles unicameral, and (3) epipharynx with a single pair of mes or none at all. For the pupae they are: (1) the pronotum with prominent pronotal protuberances, (2) abdominal segment VIII with a conical abdominal protuberance dorsally, and (3) very short or even reduced urogomphi. The species studied here are compared with those Gymnetron species already known and with other genera in the tribe Mecinini. Keys to the larvae and pupae described here are provided. All the characters used for identification are illustrated by photographs or drawings.

Description of two new species of Rhamphus related to R. oxyacanthae (Curculionidae, Curculioninae, Rhamphini) from Italy based on a morphological study supported by molecular data.

Two new species of the weevil genus Rhamphus from Italy are herein described: R. bavierai n. sp. (Sicily) and R. hampsicora n. sp. (Sardinia). Both are morphologically and from a molecular perspective close to R. oxyacanthae (Marsham, 1802) and R. monzinii Pesarini Diotti, 2012. Aside from a diagnostic description and a synoptic key, distribution data and notes on the host plants of the four species are reported. Whereas R. monzinii can be distinguished by several morphological characters, the other three species are morphologically very similar to each other and separable only by the combination of a few subtle characters. On the contrary, a preliminary molecular study revealed substantial divergences of mtCOI from 6.2 to 14.9% between the species, confirming the importance of an integrative taxonomy.

Symptomatology, (Co)occurrence and Differential Diagnostic PCR Identification of 'Ca. Phytoplasma solani' and 'Ca. Phytoplasma convolvuli' in Field Bindweed.

Field bindweed (Convolvulus arvensis) is one of the major natural plant hosts and reservoirs of 'Candidatus Phytoplasma solani' ('Ca. P. solani'), the causal agent of plant diseases in diverse agricultural crops, including Bois noir (BN) disease of grapevine. Phylogenetically, the most closely related phytoplasma to 'Ca. P. solani', the 'Ca. P. convolvuli', induces disease in field bindweed that is known by its symptoms as bindweed yellows (BY). The occurrence, coinfection and symptoms association of the two phytoplasmas in shared host plants were the subject of this study. Specific primers for the amplification of the elongation factor Tu gene (tuf) were developed for the identification of 'Ca. P. convolvuli' (by conventional nested PCR), as well as primers for simultaneous detection of 'Ca. P. solani' and 'Ca. P. convolvuli' by duplex SYBR Green real-time PCR. Among symptomatic bindweed plants, 25 and 41% were infected with a single phytoplasma species, 'Ca. P. solani' and 'Ca. P. convolvuli', respectively, while 34% were infected with both phytoplasmas. None of the non-symptomatic control plants carried phytoplasma, while non-symptomatic plants from our previous epidemiological studies in BN-affected vineyards were confirmed to be infected solely with 'Ca. P. solani'. Stamp gene typing revealed Rqg50 and Rqg31 'Ca. P. solani' genotypes in plants coinfected with 'Ca. P. convolvuli', while three diverse genotypes (Rqg50, GGY and Rpm35) were identified in a single locality with symptomatic bindweeds infected solely with 'Ca. P. solani'. Variations in symptoms and their association with each of the phytoplasmas are described and documented. The symptom of bushy appearance could be single out as specific for 'Ca. P. convolvuli' infection, while occurrence of 'Ca. P. solani' could not be unequivocally associated with specific alterations in infected bindweeds. The results are discussed in the context of the epidemiological and ecological complexity of 'Ca. P. solani'-induced diseases and the relationship between the two phytoplasma relatives in shared host plant.

Evidence for a cryptic parasitoid species reveals its suitability as a biological control agent.

Uncertainty about the taxonomic status and the specificity of a species commonly prevent its consideration as a candidate for biological control of pest organisms. Here we use a combination of molecular analysis and crossing experiments to gather evidence that the parasitoid wasp Ganaspis brasiliensis, a candidate for biological control of the invasive spotted wing drosophila Drosophila suzukii, is a complex of at least two cryptic species. Complementary experiments demonstrate that individuals from one genetic group readily parasitize several drosophila species regardless of their food source while individuals from the other one are almost exclusively specific to larvae feeding in ripening fruits. Because only D. suzukii attacks ripening fruits in its area of invasion, parasitoids from this second group appear to be well suited as a biological control agent. Our study demonstrates the need for a combination of biosystematics with biological and ecological investigations for the development of safe and efficient biological control programs.

Immature stages of Palearctic Mecinus species (Coleoptera, Curculionidae, Curculioninae): morphological characters diagnostic at genus and species levels.

The immature stages of ten Mecinus species are described for the first time and those of two other species are redescribed, adding important chaetotaxy characters that were missing from previous descriptions. These species belong to six of the nine assemblages of Mecinus species previously established according to a phylogenetic analysis. All these groupings are confirmed on the basis of several characters of mature larvae and pupae. Moreover, all the species show several characters that are useful for distinguishing them from each other, including cryptic species that previously had few differential characters. Some characters that may be useful for separating Mecinus from other genera in the tribe are suggested. To confirm the taxonomic identification of some larvae, the mtCOII gene was obtained and compared with sequences from identified adult specimens. The most important characters for separating the immature stages of the genera and species groups in Mecinus are the number of palpomeres of the labial palpi (1 or 2), the number of air tubes of the thoracic and abdominal spiracles (unicameral or bicameral), and the number of epipharyngeal setae. The species studied herein were compared with those known from other genera in the tribe Mecinini. Two keys, one to the described larvae and the other to the pupae, are provided. Detailed biological data, several of which are new, on some species are reported.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Rhinusa Stephens: a taxonomic revision of the species belonging to the R. linariae, R. herbarum, R. melas, and R. mauritii groups (Coleoptera Curculionidae).

The species of Rhinusa Stephens, 1829 (Curculionidae, Curculioninae, Mecinini) belonging to the R. linariae, R. herbarum, R. melas and R. mauritii groups are revised. The R. linariae group includes three species: R. linariae (Panzer, 1795), R. brisouti (Faust, 1891) and R. kumatschevi (Bajtenov, 1977). The R. herbarum group also includes three species: R. herbarum (H. Brisout de Barneville, 1863), R. mateui (Hoffmann, 1965) and R. vulpeculus (Reitter, 1907). The R. melas group includes two species: R. melas (Boheman, 1838) and R. korotyaevi sp. n. (Israel, Jordan). Lastly, the R. mauritii group is monobasic. The lectotypes of Gymnetron consors Desbrochers des Loges, 1898, Gymnetron herbarum H. Brisout de Barneville, 1863, Gymnetron heydeni Desbrochers des Loges, 1869, and Gymnetron melas Boheman, 1838 are designated. The following new synonymy is proposed: Rhinusa herbarum (H. Brisout de Barneville, 1863) = Gymnetron semirufum Desbrochers des Loges, 1898 (syn. n.). For each species treated, description or redescription, notes on type specimens, synonymies, comparative notes, distribution, bionomics when available, photographs of the habitus and drawings of the rostra, genitalia and other structures useful for taxonomy are provided. A key including all the groups of Rhinusa as currently considered is provided for the first time.

Evolution of host plant use and diversification in a species complex of parasitic weevils (Coleoptera: Curculionidae).

Weevils (Coleoptera: Curculionoidea) represent one of the most diverse groups of organisms on Earth; interactions with their host plants have been recognized to play a central role in their remarkable diversity, yet the exact mechanisms and factors still remain poorly understood. Using phylogenetic comparative analyses, here we investigate the evolution of host use and its possible role in diversification processes of Rhinusa and Gymnetron, two closely related groups of weevils that feed and develop inside plant tissues of hosts within the families Scrophulariaceae and Plantaginaceae. We found strong evidence for phylogenetic conservatism of host use at the plant family level, most likely due to substantial differences in the chemical composition of hosts, reducing the probability of shifts between host families. In contrast, the use of different plant organs represents a more labile ecological trait and ecological niche expansion that allows a finer partitioning of resources. Rhinusa and Gymnetron weevils initially specialized on plants within Scrophulariaceae and then shifted to the closely related Plantaginaceae; likewise, a gall inducing behavior evolved from non-galler weevils, possibly in response to resource competition, as galls facilitate larval development by providing enhanced nutrition and a favorable microhabitat. Results from trait-dependent diversification analyses suggest that both use of hosts within Plantaginaceae and parasitism on fruits and seed capsules are associated with enhanced diversification of Rhinusa and Gymnetron via low extinction rates. Our study provides quantitative evidence and insights on the ecological factors that can promote diversification in phytophagous insects that feed and develop inside plant tissues.

Resource allocation in response to herbivory and gall formation in Linaria vulgaris.

Trehalose and its precursor, trehalose 6-phosphate (T6P), are essential regulators of plant response to abiotic and biotic stress. Here we used the specific host-insect interaction between Linaria vulgaris (Plantaginaceae) and stem-galling weevil, Rhinusa pilosa (Mecinini, Curculionidae) with the aim to distinguish carbohydrate allocation patterns in response to herbivory, gall formation (G1, 24 h after oviposition), and gall development (G2, 7 days after oviposition) under controlled conditions. The hypothesis is that herbivory and galling induce distinct responses in both leaves and stems, and that shifts in carbon allocations are regulated by signaling sugars. Systemic response to herbivory was accumulation of T6P and maltose. The main feature of G1 in the stems was accumulation of trehalose, accompanied by increased T6P, turanose and glucose content, oppositely to the leaves. In G2, galls had 3-folds higher weight than controls, with further accumulation of fructose, glucose, turanose, and total water-insoluble carbohydrates (TIC), while the sucrose/hexose ratio decreased. Analysis of fast chlorophyll fluorescence kinetic (OJIP) transients in G2 showed a slight decrease in quantum yield of electron transport flux from QA to QB, and towards photosystem I acceptor side, correlated with the decreased content of photosynthetic pigments and hexoses accumulation. Redistribution of photosynthates, and accumulation of T6P were induced in response to herbivory, indicating its signaling role. The results support the hypothesis that R. pilosa can induce plant reprogramming towards the accumulation of beneficial carbohydrates in developing gall by mechanisms which include both T6P and trehalose.

Widespread plant specialization in the polyphagous planthopper Hyalesthes obsoletus (Cixiidae), a major vector of stolbur phytoplasma: Evidence of cryptic speciation.

The stolbur phytoplasma vector Hyalesthes obsoletus is generally considered as a polyphagous species associated with numerous wild and cultivated plants. However, recent research in southeastern Europe, the distribution centre of H. obsoletus and the area of most stolbur-inflicted crop diseases, points toward specific host-plant associations of the vector, indicating specific vector-based transmission routes. Here, we study the specificity of populations associated with four host-plants using mitochondrial and nuclear genetic markers, and we evaluate the evolution of host-shifts in H. obsoletus. Host-plant use was confirmed for Convolvulus arvensis, Urtica dioica, Vitex agnus-castus and Crepis foetida. Mitochondrial genetic analysis showed sympatric occurrence of three phylogenetic lineages that were ecologically delineated by host-plant preference, but were morphologically inseparable. Nuclear data supported the existence of three genetic groups (Evanno's ΔK(3) = 803.72) with average genetic membership probabilities > 90%. While populations associated with C. arvensis and U. dioica form a homogenous group, populations affiliated with V. agnus-castus and C. foetida constitute two independent plant-associated lineages. The geographical signal permeating the surveyed populations indicated complex diversification processes associated with host-plant selection and likely derived from post-glacial refugia in the eastern Mediterranean. This study provides evidence for cryptic species diversification within H. obsoletus sensu lato: i) consistent mitochondrial differentiation (1.1-1.5%) among host-associated populations in syntopy and in geographically distant areas, ii) nuclear genetic variance supporting mitochondrial data, and iii) average mitochondrial genetic distances among host-associated meta-populations are comparable to the most closely related, morphologically distinguishable species, i.e., Hyalesthes thracicus (2.1-3.3%).

Morphological characters of immature stages of Palaearctic species of Cleopomiarus and Miarus and their systematic value in Mecinini (Coleoptera, Curculionidae, Curculioninae).

The relationship between the genera Cleopomiarus and Miarus of Mecinini (Curculionidae, Curculioninae) was tested on the basis of morphological characters from the immature stages. The mature larvae of five Cleopomiarus species (C.distinctus (Boheman, 1845), C.graminis (Gyllenhal, 1813), C.longirostris (Gyllenhal, 1838), C.medius (Desbrochers des Loges, 1893), and C.meridionalis (H. Brisout de Barneville, 1863)), three Miarus species (M.abnormis Solari, 1947, M.ajugae (Herbst, 1795), and M.campanulae (Linnaeus, 1767)), and the pupae of four Cleopomiarus species (C.distinctus, C.graminis, C.longirostris, and C.medius) and two Miarus species (M.abnormis and M.ajugae) are described in detail for the first time. To confirm the taxonomic identification of some larvae, DNA COI barcode was obtained and compared with those of adults. The immature stages of the species herein studied were compared with those known from other genera in tribe Mecinini. It is suggested that Miarus and Cleopomiarus may be monophyletic based on several shared distinctive characters. Larvae of Miarus have a characteristic maxillary mala with six finger-like dms of two sizes (one or two dms very long and the rest of medium length), this feature being apparently unique among weevils. Other genus-specific character states are observed in the pupae, such as the length of setae on the head, rostrum and pronotum, including the number of rs on the rostrum, ds on pronotum, and finally the shape of the urogomphi. A key to the described larvae and pupae were respectively presented. New biological and distributional data on some species are reported.

Morphology versus DNA barcoding: two sides of the same coin. A case study of Ceutorhynchus erysimi and C. contractus identification.

Genotyping of 2 well-known weevil species from the genus Ceutorhynchus (Coleoptera: Curculionidae) distributed in west Palearctic, C. erysimi and C. contractus, revealed phenotype versus genotype inconsistencies in a set of 56 specimens (25 C. erysimi and 31 C. contractus) collected from 25 locations in Serbia and Montenegro. An analysis of the mitochondrial cytochrome oxidase subunit I gene (COI), widely used as a barcoding region, and a nuclear gene, elongation factor-1α (EF-1α), revealed stable genetic divergence among these species. The average uncorrected pairwise distances for the COI and EF-1α genes were 3.8%, and 1.3%, respectively, indicating 2 genetically well-segregated species. However, the genetic data were not congruent with the phenotypic characteristics of the studied specimens. In the first place, C. erysimi genotypes were attached to specimens with phenotypic characteristics of C. contractus. Species-specific PCR-RFLP assays for the barcoding gene COI were applied for the molecular identification of 101 additional specimens of both morphospecies (33 C. erysimi and 68 C. contractus) and were found to confirm this incongruity. The discrepancy between the genetic and morphological data raises the question of the accuracy of using a barcoding approach, as it may result in misleading conclusions about the taxonomic position of the studied organism. Additionally, the typological species concept shows considerable weakness when genetic data are not supported with phenotypic characteristics as in case of asymmetric introgression, which may cause certain problems, especially in applied studies such as biological control programs in which the biological properties of the studied organisms are the main focus.

Host-associated genetic differentiation in a seed parasitic weevil Rhinusa antirrhini (Coleptera: Curculionidae) revealed by mitochondrial and nuclear sequence data.

Plant feeding insects and the plants they feed upon represent an ecological association that is thought to be a key factor for the diversification of many plant feeding insects, through differential adaptation to different plant selective pressures. While a number of studies have investigated diversification of plant feeding insects above the species level, relatively less attention has been given to patterns of diversification within species, particularly those that also require plants for oviposition and subsequent larval development. In the case of plant feeding insects that also require plant tissues for the completion of their reproductive cycle through larval development, the divergent selective pressure not only acts on adults, but on the full life history of the insect. Here we focus attention on Rhinusa antirrhini (Curculionidae), a species of weevil broadly distributed across Europe that both feeds on, and oviposits and develops within, species of the plant genus Linaria (Plantaginaceae). Using a combination of mtDNA (COII) and nuclear DNA (EF1-alpha) sequencing and copulation experiments we assess evidence for host associated genetic differentiation within R. antirrhini. We find substantial genetic variation within this species that is best explained by ecological specialisation on different host plant taxa. This genetic differentiation is most pronounced in the mtDNA marker, with patterns of genetic variation at the nuclear marker suggesting incomplete lineage sorting and/or gene flow between different host plant forms of R. antirrhini, whose origin is estimated to date to the mid-Pliocene (3.77 Mya; 2.91-4.80 Mya).

Anatomical injury induced by the eriophyid mite Aceria anthocoptes on the leaves of Cirsium arvense.

Anatomical injury of the leaves of the invasive species, Cirsium arvense (L.) Scop., caused by the eriophyid mite Aceria anthocoptes (Nal.), which is the only eriophyid mite that has been recorded on C. arvense worldwide, is described. The injury induced by the mite feeding on the leaves of C. arvense results in visible russeting and bronzing of the leaves. Other conspicuous deformations are folding and distortion of the leaf blade and curling of leaf edge, as well as gradual drying of leaves. The anatomical injury of the mature leaves of field-collected plants was limited to the epidermis of the lower leaf surface. However, on young leaves of experimentally infested plants, rust mite injuries extend to epidermal cells on both leaf surfaces and to those of deeper mesophyll layers. On these leaves, lesions on the lower leaf surface even affected the phloem of the vascular bundles. Leaf damage induced by A. anthocoptes is discussed with regard to the mite's potential as a biological control agent of C. arvense.