Serratia montpellierensis sp. nov., Isolated from Laboratory-Reared Parasitic Wasps Psyttalia lounsburyii Silvestri and Psyttalia ponerophaga Silvestri (Hymenoptera: Braconidae).

Two strains of bacteria, PsyLou2AT and PsyPon4B, were isolated from adult braconid wasps Psyttalia lounsburyii and Psyttalia ponerophaga, respectively. These laboratory-reared wasps were investigated as agents for biological control of the olive fruit fly, Bactrocera oleae. Analysis of 16S rRNA genes of the two isolates demonstrated that they were highly related and belonged to the genus Serratia. Genomic sequencing of these isolates revealed genomes of 5,152,551 bp and 5,154,385 bp for PsyLou2AT and PsyPon4B, respectively, and both genomes had a mol% G+C content of 59.6%. Phylogenetic analyses using BLAST-based average nucleotide identity (ANIb), and digital DNA-DNA hybridization methods indicated that PsyLou2AT was most closely related to Serratia nevei S15T, producing ANIb and dDDH values of 96.11% and 70.2%, respectively. Since these values were literally on the species cutoff threshold, additional S. nevei genome assemblies were analyzed using ANIb and dDDH calculations. This revealed that among assemblies that were clearly identifiable as S. nevei, S. nevei S15T was the most closely related to PsyLou2AT, and that a majority of assemblies produced dDDH values of 68.3-68.7% relative to PsyLou2AT. Additionally, PsyLou2AT differed biochemically from S. nevei S15T in that it produced positive Voges Proskauer tests, produced protease, lacked arginine dihydrolase, and did not utilize D-lactose. Hence, PsyLou2AT represents a novel taxon within the Serratia, for which we propose the name Serratia montpellierensis sp. nov. The type strain is PsyLou2AT (=LMG 32817T =NRRL B-65689T).

Integrative Taxonomy and Synonymization of Aculus mosoniensis (Acari: Eriophyidae), a Potential Biological Control Agent for Tree of Heaven (Ailanthus altissima).

The taxonomy of Aculus mosoniensis appears to be an unresolved question and its clarification is required, owing to the potential relevance of this mite species as a biological control agent of the tree of heaven. This paper is aimed at giving accurate details on a previously and shortly announced synonymization with Aculops taihangensis, using a morphological and molecular approach. A fusiform morph of A. mosoniensis was distinguished from a vermiform morph and this latter was recognized as deutogyne, which was herein documented. Phylogenetic relationships between Chinese Ac. taihangensis and all A. mosoniensis mites collected in twenty localities in Europe were examined through the analysis of the mitochondrial cytochrome c subunit I (CO1) protein and the nuclear ribosomal internal transcribed spacer 1 region (ITS1). CO1 sequences of Ac. taihangensis from the Shandong province in China and those from mites collected in Austria and Slovenia were 100% identical; the ITS1 sequence of an Ac. taihangensis paratype matched for 99.8% with those obtained from protogynes and deutogynes of A. mosoniensis collected in Italy. All these data supported the announced synonymization of A. mosoniensis with Ac. taihangensis. Aculusmosoniensis was found genetically variable, with five CO1 haplotypes in Europe (becoming eight along with those of Ac. taihangensis) clustering in two highly supported maternal lineages and eight ITS1 haplotypes (becoming nine along with those of Ac. taihangensis) distributed in four supported clades. No overlap between intra- and interspecies distances was observed for both markers and all studied A. mosoniensis populations clustered in one monophyletic mitochondrial clade, suggesting that only one single species might occur in Europe. However, more mite clades may be related to more tree of heaven biotypes with potential ecological differences, which might have potential effects on the biological control and should be investigated.

Gross Morphology of Diseased Tissues in Nezara viridula (Hemiptera: Pentatomidae) and Molecular Characterization of an Associated Microsporidian.

Nezara viridula (L.) (Hemiptera: Pentatomidae), commonly known in the U.S. as the southern green stink bug (SGSB), is a cosmopolitan, highly polyphagous feeder that causes severe damage to a wide range of agronomically important crops such as fruit, vegetable, grain, tobacco, and cotton, throughout much of the United States, and is a global pest of considerable ecological, agricultural, and economical interest. During dissection of female Nz. viridula, conspicuous black and brown spots or lesions were observed on various internal organs. To determine the cause of these spots or lesions, tissues of fat body, spermatheca, ovaries, and ovulated eggs were collected from healthy and infected individuals. The gross morphology of the spots was characterized, and the microorganisms associated with the infection were identified by amplicon sequencing of the V4 region of the small subunit rRNA gene. The presence of a microsporidian pathogen Nosema maddoxi, Becnel, Solter, Hajek, Huang, Sanscrainte, & Estep (Microsporidia: Nosematidae) which has been observed on other species of stink bug, was evidenced for the first time. The characterization of the gross morphology of this associated microsporidian may enable more rapid determination of microsporidia infection in stink bug colonies and field populations.

Field Assessment of the Host Range of Aculus mosoniensis (Acari: Eriophyidae), a Biological Control Agent of the Tree of Heaven (Ailanthus altissima).

Tree of heaven (Ailanthus altissima) is a fast-growing deciduous tree native to China, considered a serious invasive species worldwide, with several socio-economic and ecological impacts attributed to it. Chemical and mechanical methods have limited efficacy in its management, and biological controls may offer a suitable and sustainable option. Aculus mosoniensis (Ripka) is an eriophyid mite that has been recorded to attack tree of heaven in 13 European countries. This study aims to explore the host range of this mite by exposing 13 plant species, selected either for their phylogenetic and ecological similarity to the target weed or their economic importance. Shortly after inoculation with the mite, we recorded a quick decrease in mite number on all nontarget species and no sign of mite reproduction. Whereas, after just one month, the population of mites on tree of heaven numbered in the thousands, irrespective of the starting population, and included both adults and juveniles. Significantly, we observed evidence of damage due to the mite only on target plants. Due to the specificity, strong impact on the target, and the ability to increase its population to high levels in a relatively short amount of time, we find A. mosoniensis to be a very promising candidate for the biological control of tree of heaven.

Taxonomic Description of Stenodiplosis tectori n. sp. (Diptera: Cecidomyiidae), a Seed Parasite of Cheatgrass, Anisantha tectorum, Based on Morphological and Mitochondrial DNA Data.

Cheatgrass is an annual grass species from Eurasia that has become invasive in much of western North America. It has been implicated in recent increases in the frequency, size, and intensity of wildfires, contributing to severe economic, environmental, and social destruction. In order to reduce this damage, the USDA-ARS established a classical biological control program against cheatgrass. In 2018 and 2019, adult gall midges were collected emerging from cheatgrass seed heads collected at several sites in Bulgaria and Greece; this is the first gall midge ever recorded from cheatgrass. Morphological comparisons with related midge species recorded from other plant hosts revealed that this midge from cheatgrass is a new species, described here as Stenodiplosis tectori n. sp. This status was supported by sequence comparisons of a barcode region of the gene encoding the mitochondrial cytochrome c subunit I (CO1) protein in Stenodiplosis tectori n. sp. and three congeners. The present study is the first to report MT-CO1 data in the genus Stenodiplosis. The ingroup Stenodiplosis tectori n. sp. collected in the Balkans grouped in one phylogenetic supported clade, with an average K2P-distance from its closest related congener, S. sorghicola, of 7.73% (SD = 1.10). The findings indicated relatively high year-to-year within-population diversity. Implications for this gall midge's utility as a biological control agent of cheatgrass are discussed.

Molecular Identification of Trissolcus japonicus, Parasitoid of the Brown Marmorated Stink Bug, by Species-Specific PCR.

The samurai wasp, Trissolcus japonicus (Ashmead), has been proposed as a biocontrol agent against brown marmorated stink bugs (BMSB), due to its ability to parasitize and kill BMSB eggs. However, the wasps' small size makes it challenging for those untrained in morphological identification to determine the wasps' species. To circumvent this problem, a molecular method was created to identify T. japonicus. The method uses species-specific primers, designed in this study, which target the variable region of the mitochondrial Cytochrome Oxidase 1 (CO1) locus. After confirming successful DNA extraction from samples, the PCR amplification using our primers produced 227-bp PCR products for all T. japonicus specimens and no amplification in other microhymenoptera candidates. Additionally, DNA from BMSB-parasitized eggs gave positive PCR amplification, while the control BMSB samples showed no amplification. This indicates that PCR with our primers specifically and sensitively differentiates T. japonicus specimens from other similar wasp species and discriminates between T. japonicus-parasitized and non-parasitized BMSB eggs. Finally, an in silico analysis of CO1 sequences demonstrated that our primers match the sequences of four different haplotypes of T. japonicus, indicating that our diagnostic method could potentially be applied to analyze T. japonicus populations throughout North America, Europe, and parts of Asia.

Gryon ancinla Kozlov & Lê (Hymenoptera: Scelionidae): host association, expanded distribution, redescription and a new synonymy.

BACKGROUND: Gryon Haliday (Platygastroidea: Scelionidae) is a cosmopolitan genus of egg-parasitoid wasps primarily associated with Heteroptera. NEW INFORMATION: Gryon ancinla Kozlov & Lê is reported for the first time outside of Vietnam, in China and Cambodia, and as an egg parasitoid of the pestiferous leaf-footed bug, Acanthocoris scaber (L.). Gryon ancinla is redescribed based on recently collected specimens and compared to closely related species of Gryon in the region. Gryon clavaerus Kozlov & Lê is treated as a junior synonym and some characters found in the charon species group are discussed.

Assemblage of the Egg Parasitoids of the Invasive Stink Bug Halyomorpha halys: Insights on Plant Host Associations.

Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) is an invasive alien species and a key agricultural pest. Its native parasitoids (Trissolcus japonicus Ashmead and Tr. mitsukurii Ashmead) have been registered in several countries where H. halys brought dramatic economic losses and where biological control is considered to be the most effective long-term solution. By searching for stink bug egg masses and exposing sentinel egg masses, we monitored the distribution of native and exotic egg parasitoids in Trentino-Alto Adige (Italy), an area where both the host and parasitoids are in expansion. We recorded ten pentatomids, seven parasitoid species, with the first report of Tr. japonicus in this area and a hyperparasitoid. In the assemblage, Anastatus bifasciatus (Geoffroy) and Tr. mitsukurii were the dominant parasitoids, with a different distribution in terms of context and host plants. Sycamore was the host plant where the highest number of naturally laid parasitized egg masses (26%) were recorded. Trissolcus mitsukurii showed the highest parasitism rate, and was often found in apple orchards. The emergence of exotic parasitoids showed a temporal delay compared to native ones. Sequence analysis of 823 bp of the CO1 mitochondrial gene revealed that the recovered Tr. japonicus and Tr. mitsukurii harbored one single haplotype each. These haplotypes were previously found in 2018 in Northern Italy. While sentinel egg masses proved to be very effective in tracking the arrival of exotic Trissolcus species, the collection of stink bug egg masses provided fundamental data on the plant host species. The results lend strong support to the adaptation of exotic Trissolcus species to the environmental conditions of the range of introduction, providing new information on plant host-associations, fundamental for the development of biological control programs.

A modified DNA barcode approach to define trophic interactions between native and exotic pentatomids and their parasitoids.

The establishment of invasive Halyomorpha halys (Stål) outside of its native range may impact native species assemblages, including other pentatomids and their scelionid parasitoids. This has generated interest in defining species diversity and host-parasitoid associations in this system to better understand the impact of invasive alien species on trophic interactions in invaded regions. Information on scelionid-pentatomid associations in natural habitats is lacking, and species-level identification of these associations can be tenuous using rearing and dissection techniques. Naturally occurring pentatomid eggs were collected in areas where H. halys has established in Canada and were analysed using a modified DNA barcoding approach to define species-level trophic interactions. Identification was possible for >90% of egg masses. Eleven pentatomid and five scelionid species were identified, and trophic links were established. Approximately 70% of egg masses were parasitized; parasitism and parasitoid species composition were described for each species. Telenomus podisi Ashmead was the dominant parasitoid and was detected in all host species. Trissolcus euschisti Ashmead was detected in several host species, but was significantly more prevalent in Chinavia hilaris (Say) and Brochymena quadripustulata (Fabricius). Trissolcus brochymenae Ashmead and Tr. thyantae Ashmead were recorded sporadically. Parasitism of H. halys was 55%, and this species was significantly less likely to be parasitized than native pentatomids. The scelionid species composition of H. halys consisted of Te. podisi, Tr. euschisti and Tr. thyantae. Although these species cannot develop in fresh H. halys eggs, we demonstrate that parasitoids attempt to exploit this host under field conditions.

Phylogeography of the Wheat Stem Sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae): Implications for Pest Management.

The wheat stem sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae), is a key pest of wheat in the northern Great Plains of North America, and damage resulting from this species has recently expanded southward. Current pest management practices are inadequate and uncertainty regarding geographic origin, as well as limited data on population structure and dynamics across North America impede progress towards more informed management. We examined the genetic divergence between samples collected in North America and northeastern Asia, the assumed native range of C. cinctus using two mitochondrial regions (COI and 16S). Subsequently, we characterized the structure of genetic diversity in the main wheat producing areas in North America using a combination of mtDNA marker and microsatellites in samples collected both in wheat fields and in grasses in wildlands. The strong genetic divergence observed between North American samples and Asian congeners, in particular the synonimized C. hyalinatus, did not support the hypothesis of a recent American colonization by C. cinctus. Furthermore, the relatively high genetic diversity both with mtDNA and microsatellite markers offered additional evidence in favor of the native American origin of this pest. The genetic diversity of North American populations is structured into three genetic clusters and these are highly correlated with geography. Regarding the recent southern outbreaks in North America, the results tend to exclude the hypothesis of recent movement of damaging wheat stem sawfly populations from the northern area. The shift in host plant use by local populations appears to be the most likely scenario. Finally, the significance of these findings is discussed in the context of pest management.

Species composition, activity patterns and blood meal analysis of sand fly populations (Diptera: Psychodidae) in the metropolitan region of Thessaloniki, an endemic focus of canine leishmaniasis.

Species composition, activity patterns and blood meal analysis of sand fly populations were investigated in the metropolitan region of Thessaloniki, North Greece from May to October 2011. Sampling was conducted weekly in 3 different environments (animal facilities, open fields, residential areas) along the outskirts of the city in areas of increased canine leishmania transmission. Six sand fly species (Phlebotomus perfiliewi, Phlebotomus tobbi, Phlebotomus simici, Plebotomus papatasi, Sergentomya minuta and Sergentomya dentata) were identified using both classical and molecular techniques. DNA barcodes were characterized for the first time for two (P. simici and S. dentata) of the six recorded species. Phylogenetic analysis based on the COI gene sequences confirmed the grouping of P. tobbi, P. perniciosus and P. perfiliewi (subgenus Larrousius) and the monophyly of P. simici (subgenus Adlerius). By far the most prevalent species was P. perfiliewi, followed by P. simici and P. tobbi. The largest populations of sand flies were collected from animal facilities, followed by residential areas and open agricultural fields. Peak activity of sand flies overall occurred mid-August to mid-September and then declined sharply in October. Blood meal analysis showed that P. perfiliewi and P. simici feed preferentially on humans (88% & 95%, respectively) but also feed on chickens and goats. When designing a control strategy to alleviate sand fly nuisance in the region of Thessaloniki the following conclusions can be reached from this study: a) August and September are high risk months due to increased sand fly activity levels, b) animal facilities within or adjacent to urban settlements are high risk areas and may act as a maintenance and amplification foci for the vector as well as the parasite, and c) the abundance, ubiquity and feeding behavior of P. perfiliewi and P. simici establishes them as potentially important vectors of Leishmania in the region.

Mild-Vectolysis: A Nondestructive DNA Extraction Method for Vouchering Sand Flies and Mosquitoes.

Nondestructive techniques allow the isolation of genomic DNA, without damaging the morphological features of the specimens. Though such techniques are available for numerous insect groups, they have not been applied to any member of the medically important families of mosquitoes (Diptera: Culicidae) and phlebotomine sand flies (Diptera: Psychodidae). This study presents Mild-Vectolysis, the first nondestructive DNA extraction methodology for vouchering taxa of mosquitoes and sand flies, which provided sufficient amounts of DNA, tested in a verified barcode (cytochrome oxidase I gene), while preserving their morphological integrity. Application of the method to sand flies allowed successful insect identification post DNA extraction, as all basic taxonomical structures necessary for identification (pharynx, cybarium, and genitalia) remained intact. The development of the methodology was more challenging in mosquitoes, due to the fragility of key morphological characters (scales and color). A small modification of the lysis buffer concentration, in combination with the adjustment of the incubation time, a postlysis freezing stage, and the avoidance of ethanol, achieved the extraction of sufficient DNA quantity, while preserving the integument of the mosquitoes, although a small proportion of the scales and the color still appeared to have been lost. In addition to the practicality and efficiency of our methodology, preserving of the original insect specimen post DNA extraction is highly advantageous, as it allows for 1) utilization of the specimen for further analysis and 2) storage for vouchering.

Trichoderma asperellum sensu lato consists of two cryptic species.

Analysis of a worldwide collection of strains of Trichoderma asperellum sensu lato using multilocus genealogies of four genomic regions (tef1, rpb2, act, ITS1, 2 and 5.8s rRNA), sequence polymorphism-derived (SPD) markers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) of the proteome and classical mycological techniques revealed two morphologically cryptic sister species within T. asperellum, T. asperellum, T. asperelloides sp. nov. and a third closely related but morphologically distinct species. T. yunnanense. Trichoderma asperellum and T. asperelloides have wide sympatric distribution on multiple continents; T. yunnanense is represented by a single strain from China. Several strains reported in the literature or represented in GenBank as T. asperellum are re-identified as T. asperelloides. Four molecular SPD typing patterns (I-IV) were found over a large geographic range. Patterns I-III were produced only by T. asperellum and pattern IV by T. asperelloides and T. yunnanense. Pattern I was found in North America, South America, Africa and Europe and Asia (Saudi Arabia). Pattern III was found in Africa, North America, South America and Asia, not in Europe. Pattern II was found only in Cameroon (central Africa) and Peru. Pattern IV was found in all continents. All SPD II pattern strains formed a strongly supported subclade within the T. asperellum clade in the phylogenetic tree based on rpb2 and MLS (combined multilocus sequence). The diversity of DNA sequences, SPD markers and polypeptides in T. asperellum suggests that further speciation is under way within T. asperellum. MALDI-TOF MS distinguished T. yunnanense from related taxa by UPGMA clustering, but separation between T. asperellum and T. asperelloides was less clear.

Microsatellite variability in the entomopathogenic fungus Paecilomyces fumosoroseus: genetic diversity and population structure.

The hyphomycete Paecilomyces fumosoroseus (Pfr) is a geographically widespread fungus capable of infecting various insect hosts. The fungus has been used for the biological control of several important insect pests of agriculture. However knowledge of the fungus' genetic diversity and population structure is required for its sustainable use as a biological control agent. We investigated length and sequence polymorphisms of nine microsatellite loci for 33 Pfr accessions sampled from various host species and geographical locations, and our results reveal complex mutational processes for these molecular markers. Only Pfr isolates from Bemisia tabaci were amplified successfully, indicating the existence of Pfr genotypes specifically associated with B. tabaci. Genetic relationships among the 25 Pfr isolates from B. tabaci were inferred from allelic variability data at eight microsatellite loci that were polymorphic and subsequently from sequence data from the flanking regions of three selected loci. Maximum parsimony and neighbor joining analyses partitioned Pfr genetic diversity in two major lineages. One lineage included genotypes from the B-biotype of B. tabaci distributed across the Americas and was strongly supported in both analyses. Another lineage was distributed across Asia and consisted of four distinct clusters. Allele size homoplasy was found at the three microsatellite loci. We obtained better discrimination and resolution of the relationships among isolates with sequence data, although not all isolates could be typed. Thus sequencing of microsatellite flanking regions and repeats is a promising approach for the identification of Pfr isolates that specifically infect certain B. tabaci biotypes and phylogeographic studies.

Invasion of Lepidium draba (Brassicaceae) in the western United States: distributions and origins of chloroplast DNA haplotypes.

Advances in phylogeography are of great value for understanding the population structure and origins of invasive genotypes. Such insights provide constructive information for current or future biological control research efforts. In this study, we investigated a highly variable chloroplast DNA (cpDNA) marker for populations of the weed Lepidium draba (Brassicaceae) in its native Eurasian and invasive US ranges. We sequenced DNA from 684 individuals from Eurasia and the US and found 41 different haplotypes. Our comparative study between the native and invasive ranges showed a 33% reduction in allelic richness (A) and a 7% reduction in haplotype diversity (h) since introduction into the US. Most genetic variation in the native range was observed within geographical regions and populations, not between regions, and this result was similar for the invasive range. Assignment tests indicated the most likely origins of many invasive haplotypes. Some of these occurred in western Europe, supporting an expanded native range that had been proposed for the species. Exact locations were identified for a diverse set of invasive haplotypes which can be used in ongoing host-specificity tests of potential biological control agents.

Coffee (Coffea arabica L.) genes early expressed during infection by the rust fungus (Hemileia vastatrix).

SUMMARY The beverage cash crop coffee (Coffea arabica L.) is subject to severe losses caused by the rust fungus Hemileia vastatrix. In naturally resistant coffee plants, a specific hypersensitive reaction (HR) may be elicited early to stop fungal infection. To isolate host genes involved in HR, we undertook an expressed sequence tags (ESTs) analysis. Two cDNA libraries were constructed using suppression subtractive hybridization (SSH) and 527 non-redundant ESTs were generated from 784 randomly picked clones. Classification of the ESTs into several functional categories showed that more than one-quarter of the predicted proteins might encode disease resistance (R) proteins, stress- and defence-proteins, and components of signal transduction pathways. Twenty-eight differentially screened sequences (DSSs) were selected after differential hybridization of 1000 cDNA clones from each library. Investigation of the expression patterns of a subset of 13 DSSs showed higher levels of gene expression in inoculated plants compared with control plants. HR-up-regulation of transcript accumulation occurred for 9 out of the 13 genes 24 and 48 h after H. vastatrix challenge. Two genes encoded homologues of the Arabidopsis DND1 and NDR1 proteins, suggesting conservation of resistance signalling pathways in perennial plants. Other HR-regulated sequences matched receptor kinases, AP2 domain- and WRKY transcription factors, cytochromes P450, heat shock 70 proteins, glucosyltransferases and proteins of unknown function. The ESTs reported here provide a useful resource for studying coffee resistance responses and for improving C. arabica for durable disease resistance.