Microbiome of pear psyllids: A tale about closely related species sharing their endosymbionts.

Psyllids are phloem-feeding insects that can transmit plant pathogens such as phytoplasmas, intracellular bacteria causing numerous plant diseases worldwide. Their microbiomes are essential for insect physiology and may also influence the capacity of vectors to transmit pathogens. Using 16S rRNA gene metabarcoding, we compared the microbiomes of three sympatric psyllid species associated with pear trees in Central Europe. All three species are able to transmit 'Candidatus Phytoplasma pyri', albeit with different efficiencies. Our results revealed potential relationships between insect biology and microbiome composition that varied during psyllid ontogeny and between generations in Cacopsylla pyri and C. pyricola, as well as between localities in C. pyri. In contrast, no variations related to psyllid life cycle and geography were detected in C. pyrisuga. In addition to the primary endosymbiont Carsonella ruddii, we detected another highly abundant endosymbiont (unclassified Enterobacteriaceae). C. pyri and C. pyricola shared the same taxon of Enterobacteriaceae which is related to endosymbionts harboured by other psyllid species from various families. In contrast, C. pyrisuga carried a different Enterobacteriaceae taxon related to the genus Sodalis. Our study provides new insights into host-symbiont interactions in psyllids and highlights the importance of host biology and geography in shaping microbiome structure.

16S rRNA Sequencing Detected Profftella, Liberibacter, Wolbachia, and Diplorickettsia from Relatives of the Asian Citrus Psyllid.

The Asian citrus psyllid Diaphorina citri (Hemiptera: Psylloidea) is a serious pest of citrus species worldwide because it transmits Candidatus Liberibacter spp. (Alphaproteobacteria: Rhizobiales), the causative agents of the incurable citrus disease, huanglongbing or greening disease. Diaphorina citri possesses a specialized organ called a bacteriome, which harbors vertically transmitted intracellular mutualists, Ca. Carsonella ruddii (Gammaproteobacteria: Oceanospirillales) and Ca. Profftella armatura (Gammaproteobacteria: Betaproteobacteriales). Whereas Carsonella is a typical nutritional symbiont, Profftella is an unprecedented type of toxin-producing defensive symbiont, unusually sharing organelle-like features with nutritional symbionts. Additionally, many D. citri strains are infected with Wolbachia, which manipulate reproduction in various arthropod hosts. In the present study, in an effort to obtain insights into the evolution of symbioses between Diaphorina and bacteria, microbiomes of psyllids closely related to D. citri were investigated. Bacterial populations of Diaphorina cf. continua and Diaphorina lycii were analyzed using Illumina sequencing of 16S rRNA gene amplicons and compared with data obtained from D. citri. The analysis revealed that all three Diaphorina spp. harbor Profftella as well as Carsonella lineages, implying that Profftella is widespread within the genus Diaphorina. Moreover, the analysis identified Ca. Liberibacter europaeus and Diplorickettsia sp. (Gammaproteobacteria: Diplorickettsiales) in D. cf. continua, and a total of four Wolbachia (Alphaproteobacteria: Rickettsiales) lineages in the three psyllid species. These results provide deeper insights into the interactions among insects, bacteria, and plants, which would eventually help to better manage horticulture.

Wing buzzing as a mechanism for generating vibrational signals in psyllids (Hemiptera: Psylloidea).

Psyllids, or jumping plant lice (Hemiptera: Sternorrhyncha: Psylloidea), are a group of small phytophagous insects that include some important pests of crops worldwide. Sexual communication of psyllids occurs via vibrations transmitted through host plants, which play an important role in mate recognition and localization. The signals are species-specific and can be used to aid in psyllid taxonomy and pest control. Several hypotheses have been proposed for the mechanism that generates these vibrations, of which stridulation, that is, friction between parts of the forewing and thorax, has received the most attention. We have investigated vibrational communication in the European pear psyllid species Cacopsylla pyrisuga (Foerster, 1848) using laser vibrometry and high-speed video recording, to directly observe the movements associated with signal production. We describe for the first time the basic characteristics of the signals and signal emission of this species. Based on observations and analysis of the video recordings using a point-tracking algorithm, and their comparison with laser vibrometer recordings, we argue that males of C. pyrisuga produce the vibrations primarily by wing buzzing, that is, tremulation that does not involve friction between the wings and thorax. Comparing observed signal properties with previously published data, we predict that wing buzzing is the main mechanism of signal production in all vibrating psyllids.

Revision of the Neotropical genus Trigava O'Brien, 1999 (Hemiptera, Fulgoromorpha, Dictyopharidae, Nersiini), with descriptions of two new species from Peru and Brazil.

The Neotropical planthopper genus Trigava O'Brien, 1999 (Hemiptera, Fulgoromorpha, Dictyopharidae, Nersiini) is revised. Four species are included: T.brachycephala (Melichar, 1912) (the type species, from Peru), T.obrieni Song, Malenovský & Deckert, sp. nov. (from Brazil), T.peruensis Song, O'Brien & Bartlett, sp. nov. (from Peru), and T.recurva (Melichar, 1912) (from Bolivia and Peru). Lectotypes are designated for Igavabrachycephala Melichar, 1912 and Igavarecurva Melichar, 1912. All species are described, including habitus photographs and detailed illustrations of the male genitalia. Male and female genitalia are described for this genus for the first time. A key for identification of the species of Trigava and a distribution map are provided.

Taxonomic approach to the tachinid flies Dinera carinifrons (Fallén) (Diptera: Tachinidae) and Dinera fuscata Zhang and Shima using molecular and morphometric data.

Molecular phylogenetic and traditional morphometric methods were applied to examine six Palaearctic taxa of the taxonomically difficult tachinid fly genus Dinera Robineau-Desvoidy (Diptera: Tachinidae), with particular reference to D. carinifrons (Fallén) and D. fuscata Zhang and Shima. Results of a phylogenetic analysis based on the mitochondrial markers 12S and 16S rDNA and multivariate statistical analyses of 19 morphometric characters were used to delimit both species. A lectotype was designated for D. carinifrons to stabilize the nomenclature in the group. Dinera carinifrons has a transpalaearctic distribution and is present in Central Europe, especially in high altitudes of the Alps. It differs from the similar and closely related D. fuscata in that it has a slightly larger body size, a dense greyish microtrichosity on the body, and different head proportions. Dinera fuscata, as delimited here, is widespread in the Palaearctic region, including Europe. Slight differences in both molecular and morphometric characters were found between western (Europe and Iran) and eastern (China and Japan) populations of D. fuscata, which are interpreted as an intraspecific variation. Differential diagnosis between D. carinifrons and D. fuscata is provided in the form of a revised portion of the determination key to the Palaearctic Dinera by Zhang and Shima (2006).

Wolbachia infection dynamics in a natural population of the pear psyllid Cacopsylla pyri (Hemiptera: Psylloidea) across its seasonal generations.

Wolbachia is one of the most abundant intracellular symbionts of arthropods and has profound effects on host biology. Wolbachia transmission and host phenotypes often depend on its density within the host, which can be affected by multiple biotic and abiotic factors. However, very few studies measured Wolbachia density in natural host populations. Here, we describe Wolbachia in the pear psyllid Cacopsylla pyri from three populations in the Czech Republic. Using phylogenetic analyses based on wsp and multilocus sequence typing genes, we demonstrate that C. pyri harbours three new Wolbachia strains from supergroup B. A fourth Wolbachia strain from supergroup A was also detected in parasitised immatures of C. pyri, but likely came from a hymenopteran parasitoid. To obtain insights into natural Wolbachia infection dynamics, we quantified Wolbachia in psyllid individuals from the locality with the highest prevalence across an entire year, spanning several seasonal generations of the host. All tested females were infected and Wolbachia density remained stable across the entire period, suggesting a highly efficient vertical transmission and little influence from the environment and different host generations. In contrast, we observed a tendency towards reduced Wolbachia density in males which may suggest sex-related differences in Wolbachia-psyllid interactions.

Redescription of the Planthopper Bursinia genei (Dufour, 1849), with a New Record from Greece (Hemiptera: Fulgoromorpha: Dictyopharidae).

Bursinia genei (Dufour, 1849) is the most widespread species of the planthopper subfamily Orgeriinae (Hemiptera: Fulgoromorpha: Dictyopharidae) in Europe, found from the Iberian Peninsula to the Western Balkans. However, its diagnostically important genitalia and biology have been insufficiently described. We employ state-of-the-art synchrotron X-ray microtomography and photomicrography to re-describe B. genei, and to study the morphology of both its sexes in unprecedented detail. By examining specimens from across the distribution of B. genei, we find that they probably belong to a single, broadly distributed morphospecies. Our morphological examination allowed us to make inferences on its jumping mechanism and capacity for vibrational communication. We also record B. genei for the first time from Greece, further extending the range of this elusive species. Detailed information on the habitat of B. genei is also provided.

Myxobdella socotrensis sp. nov., a new parasitic leech from Socotra Island, with comments on the phylogeny of Praobdellidae (Hirudinida: Arhynchobdellida).

The Socotra Archipelago (Yemen) is remarkable for unique ecosystems and high endemism, for which it is often referred to as the "Galápagos of the Indian Ocean". Here we describe a new parasitic leech Myxobdella socotrensis sp. nov. from Socotra, the largest island of the archipelago. The new species was found in a freshwater spring attached to the endemic crab Socotrapotamon socotrensis (Hilgendorf, 1883) (Crustacea: Potamidae). Based on its morphology, ecology and a phylogenetic analysis of DNA sequence data (12S, 18S, 28S and COI gene markers), the new leech species is classified into the highly diversified family Praobdellidae (Hirudinida: Arhynchobdellida), distributed in tropical regions of Africa, Asia and Central and South America. The praobdellid leeches are known to infest mammalian mucous membranes, however, some taxa are associated with other hosts, namely crabs, amphibians and birds. By its morphology, the new species fits quite well in the current concept of the genus Myxobdella Oka, 1917. However, the monophyly of Myxobdella was not supported here by molecular data, pointing at the need of a more comprehensive systematic revision of the genus and family. As far as known, Myxobdella socotrensis sp. nov. is endemic to the Socotra Island, but more data are needed to understand its evolutionary origin, biology and distribution.

External structures of the metathoracic scent gland efferent system in the true bug superfamily Pyrrhocoroidea (Hemiptera: Heteroptera: Pentatomomorpha).

Pyrrhocoroidea represents an important group of true bugs (Insecta: Hemiptera: Heteroptera) which includes fire bugs, cotton stainers and other taxa widely used in experimental studies or known as pests. However, the morphology and phylogeny of Pyrrhocoroidea have been only poorly studied so far. Here, structures of the external scent efferent system of the metathoracic scent glands are examined in 64 out of 71 currently valid genera of Pyrrhocoroidea and scanning electron micrographs are provided for most taxa. Several characters are revealed which define each of the three higher taxa within Pyrrhocoroidea: Larginae (small auriculate peritreme lacking manubrium and median furrow; metathoracic spiracle never surrounded by evaporatorium), Physopeltinae (large, widely open ostiole; large peritremal disc with manubrium [new term], lacking median furrow; mace-like mycoid filter processes of equal shape and size on both anterior and posterior margins of metathoracic spiracle), and Pyrrhocoridae (elongate auriculate peritreme with deep median furrow). Within Pyrrhocoridae, three main types (A, B and C) of the external scent efferent system are distinguished, differring in the amount of reductions. The findings are interpreted in the context of phylogenetic hypotheses available for Pyrrhocoroidea and their close relatives, Coreoidea and Lygaeoidea. An updated identification key to the families and subfamilies of Pyrrhocoroidea applicable for both sexes is provided.

Comparative Genomics Underlines Multiple Roles of Profftella, an Obligate Symbiont of Psyllids: Providing Toxins, Vitamins, and Carotenoids.

The Asian citrus psyllid Diaphorina citri (Insecta: Hemiptera: Psylloidea), a serious pest of citrus species worldwide, harbors vertically transmitted intracellular mutualists, Candidatus Profftella armatura (Profftella_DC, Gammaproteobacteria: Burkholderiales) and Candidatus Carsonella ruddii (Carsonella_DC, Gammaproteobacteria: Oceanospirillales). Whereas Carsonella_DC is a typical nutritional symbiont, Profftella_DC is a unique defensive symbiont with organelle-like features, including intracellular localization within the host, perfect infection in host populations, vertical transmission over evolutionary time, and drastic genome reduction down to much less than 1 Mb. Large parts of the 460-kb genome of Profftella_DC are devoted to genes for synthesizing a polyketide toxin; diaphorin. To better understand the evolution of this unusual symbiont, the present study analyzed the genome of Profftella_Dco, a sister lineage to Profftella_DC, using Diaphorina cf. continua, a host psyllid congeneric with D. citri. The genome of coresiding Carsonella (Carsonella_Dco) was also analyzed. The analysis revealed nearly perfect synteny conservation in these genomes with their counterparts from D. citri. The substitution rate analysis further demonstrated genomic stability of Profftella which is comparable to that of Carsonella. Profftella_Dco and Profftella_DC shared all genes for the biosynthesis of diaphorin, hemolysin, riboflavin, biotin, and carotenoids, underlining multiple roles of Profftella, which may contribute to stabilizing symbiotic relationships with the host. However, acyl carrier proteins were extensively amplified in polyketide synthases DipP and DipT for diaphorin synthesis in Profftella_Dco. This level of acyl carrier protein augmentation, unprecedented in modular polyketide synthases of any known organism, is not thought to influence the polyketide structure but may improve the synthesis efficiency.

Effect of Daytime and Tree Canopy Height on Sampling of Cacopsylla melanoneura, a 'Candidatus Phytoplasma mali' Vector.

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus × domestica) and transmit the bacterium 'Candidatus Phytoplasma mali', the causative agent of apple proliferation. Adult psyllids were collected by the beating-tray method from lower and upper parts of the apple tree canopy in the morning and in the afternoon. There was a trend of catching more emigrant adults of C.melanoneura in the morning and in the lower part of the canopy. For C.melanoneura remigrants, no differences were observed. The findings regarding the distribution of adults were reflected by the number of nymphs collected by wash-down sampling. The density of C.picta was too low for a statistical analysis. The vector monitoring and how it is commonly performed, is suitable for estimating densities of C.melanoneura. Nevertheless, above a certain temperature threshold, prediction of C.melanoneura density might be skewed. No evidence was found that other relatively abundant psyllid species in the orchard, viz. Baeopelma colorata, Cacopsylla breviantennata, Cacopsylla brunneipennis, Cacopsylla pruni and Trioza urticae, were involved in 'Candidatus Phytoplasma mali' transmission. The results of our study contribute to an advanced understanding of insect vector behavior and thus have a practical impact for an improved field monitoring.

On the morphology and evolution of cicadomorphan tymbal organs.

Cicadas and many of their relatives (Hemiptera: Cicadomorpha) generate vibroacoustic signals using tymbal organs located on their first two abdominal segments. Although tymbals are well-studied in Cicadidae, their systematic distribution in other Cicadomorpha and their possible homologies to the vibroacoustic mechanisms of other Hemiptera have been debated for more than a century. In the present study, we re-examine the morphology of the musculoskeletal system of cicadomorphan vibroacoustic organs, and we document their systematic distribution in 78 species drawn from across the phylogeny of Cicadomorpha. We also compare their morphology to the recently-described snapping organ of planthoppers (Fulgoromorpha). Based on the structure and innervation of the metathoracic and abdominal musculoskeletal system, we find that several key elements of cicadomorphan vibroacoustic organs that have previously been assigned to the first abdominal segment in fact belong to the second. We find that tymbal organs are nearly ubiquitous in Cicadomorpha, and conclude based on their phylogenetic distribution, that they are likely to be synapomorphic. The unusual tymbal-like organs of the Deltocephalinae and Typhlocybinae, represent derived modifications. Finally, we propose a standardised terminology for sternal components of the cicadomorphan vibrational organs, which can be used in future taxonomic descriptions.

Overwintering of Culex modestus and Other Mosquito Species in a Reedbed Ecosystem, Including Arbovirus Findings.

The overwintering strategy of the mosquito Culex modestus, an important West Nile virus (WNV) vector in Europe, was explored under field conditions in reedbed (Phragmites australis) ecosystems in early 2019. A total of 30 Cx. modestus females were found in a BG-Sentinel trap placed in a plastic greenhouse as well as in a reference BG-Sentinel trap placed under the open sky, both set up within the reedbeds and inspected every 2-3 days from February 27 to April 10, 2019. Moreover, 186 females of Cx. pipiens, 3 females of Anopheles hyrcanus, and 3 females of Culiseta annulata were trapped in the monitored time span. While all Cx. modestus females tested negative for the presence of WNV and other arboviruses circulating in Central Europe, we confirmed WNV lineage 2 and Tahyna virus infection in several pools of the collected Cx. pipiens, demonstrating arbovirus overwintering. This pilot study highlights the need for large-scale monitoring activities covering different regions to identify the overwintering strategy of both mosquito-borne viruses and their vectors in Central Europe.

Species Separation within, and Preliminary Phylogeny for, the Leafhopper Genus Anoscopus with Particular Reference to the Putative British Endemic Anoscopus duffieldi (Hemiptera: Cicadellidae).

The subfamily Aphrodinae (Hemiptera: Cicadellidae) contains ~33 species in Europe within four genera. Species in two genera in particular, Aphrodes and Anoscopus, have proved to be difficult to distinguish morphologically. Our aim was to determine the status of the putative species Anoscopus duffieldi, found only on the RSPB Nature Reserve at Dungeness, Kent, a possible rare UK endemic. DNA from samples of all seven UK Anoscopus species (plus Anoscopusalpinus from the Czech Republic) were sequenced using parts of the mitochondrial cytochrome oxidase I and 16S rRNA genes. Bayesian inference phylogenies were created. Specimens of each species clustered into monophyletic groups, except for Anoscopusalbifrons, A. duffieldi and Anoscopuslimicola. Two A. albifrons specimens grouped with A. duffieldi repeatedly with strong support, and the remaining A. albifrons clustered within A. limicola. Genetic distances suggest that A. albifrons and A. limicola are a single interbreeding population (0% divergence), while A. albifrons and A. duffieldi diverged by only 0.28%. Shared haplotypes between A. albifrons, A. limicola and A. duffieldi strongly suggest interbreeding, although misidentification may also explain these topologies. However, all A. duffieldi clustered together in the trees. A conservative approach might be to treat A. duffieldi, until other evidence is forthcoming, as a possible endemic subspecies.

On the morphology and possible function of two putative vibroacoustic mechanisms in derbid planthoppers (Hemiptera: Fulgoromorpha: Derbidae).

A mechanism involving interaction of the metathoracic wing and third abdominal segment of derbid planthoppers was first discovered over a century ago, and interpreted as a stridulatory organ for sound production. Although referred to occasionally in later taxonomic works, the detailed morphology, systematic distribution, and behavioural significance of this structure have remained unknown, and its proposed use in sound production has never been corroborated. Here we examine the distribution and morphology of the supposed stridulatory organ of Derbidae and the recently-described vibratory mechanism of planthoppers - the snapping organ, across 168 species covering the entire taxonomic spectrum of the family. We find that many derbids possess snapping organs morphologically similar to those of other planthoppers, and find no evidence for the presence of tymbal organs, which were previously thought to generate vibrational signals in derbids. We find the supposed stridulatory mechanism to be widespread in Derbidae, and conclude that it provides several systematically and taxonomically important characters. Nevertheless, its morphology appears unsuitable for the production of sound, and we instead speculate that the mechanism plays a role in spreading chemical secretions or wax. Finally, we observe wax production by tergal glands in derbid larvae, and illustrate their external morphology in adults.

Socoflata gen. nov., described for two new planthopper species from the mountains in Socotra island (Hemiptera: Fulgoromorpha: Flatidae).

A new genus of flatid planthoppers (Hemiptera: Fulgoromorpha: Flatidae) is described from the island of Socotra (Yemen): Socoflata gen. nov., for S. aurolineata sp. nov. and S. histrionica sp. nov. (type species). Habitus, male and female external and internal genital structures of the new species are illustrated and diagnosed. Both Socoflata species are abundant and syntopic in the evergreen montane woodland and dwarf shrubland at high elevations in the Hagher mountains in central Socotra and are likely endemics of this area.

Revision of the Afrotropical genus Fernandea Melichar, 1912 (Hemiptera: Fulgoromorpha: Dictyopharidae), with description of a new species from Equatorial Guinea.

The Afrotropical planthopper genus Fernandea Melichar, 1912 (Hemiptera: Fulgoromorpha: Dictyopharidae: Dictyopharinae: Orthopagini) is revised to include two species: F. conradti Melichar, 1912 (the type species), with material studied from Cameroon, Equatorial Guinea (Bioko island) and Togo, and F. latifemorata sp. nov., described as new from mainland Equatorial Guinea. A lectotype is designated and a redescription is provided for F. conradti together with habitus photographs and detailed illustrations of the male and female terminalia which are published for the first time.

In search for a compromise between biodiversity conservation and human health protection in restoration of fly ash deposits: effect of anti-dust treatments on five groups of arthropods.

Recently, fly ash deposits have been revealed as a secondary refuge of critically endangered arthropods specialised on aeolian sands in Central Europe. Simultaneously, these anthropogenic habitats are well known for their negative impact on human health and the surrounding environment. The overwhelming majority of these risks are caused by wind erosion, the substantial decreasing of which is thus necessary. But, any effects of anti-dust treatments on endangered arthropods have never been studied. We surveyed communities of five arthropod groups (wild bees and wasps, leafhoppers, spiders, hoverflies and orthopteroid insects) colonising three fly ash deposits in the western Czech Republic. We focused on two different anti-dust treatments (~70 and 100 % cover of fly ash by barren soil) and their comparison with a control of bare fly ash. Altogether, we recorded 495 species, including 132 nationally threatened species (eight of them were considered to be extinct in the country) and/or 30 species strictly specialised to drift sands. Bees and wasps and leafhoppers contained the overwhelming majority of species of the highest conservation interest; a few other important records were also in spiders and orthopteroids. Total soil cover depleted the unique environment of fly ash and thus destroyed the high conservation potential of the deposits. On the other hand, partial coverage (with ~30 % of bare fly ash) still offered habitats for many of the most threatened species, as we showed by both regression and multivariate analyses, with a decrease of wind erosion. This topic still needs much more research interest, but we consider mosaic-like preservation of smaller spots of fly ash as one of the possible compromises between biodiversity and human health.