Molecular identification of hymenopteran parasitoids and their endosymbionts from agromyzids.

Three polyphagous pest Liriomyza spp. (Diptera: Agromyzidae) have recently invaded Australia and are damaging horticultural crops. Parasitic wasps are recognized as effective natural enemies of leafmining species globally and are expected to become important biocontrol agents in Australia. However, the hymenopteran parasitoid complex of agromyzids in Australia is poorly known and its use hindered due to taxonomic challenges when based on morphological characters. Here, we identified 14 parasitoid species of leafminers based on molecular and morphological data. We linked DNA barcodes (5' end cytochrome c oxidase subunit I (COI) sequences) to five adventive eulophid wasp species (Chrysocharis pubicornis (Zetterstedt), Diglyphus isaea (Walker), Hemiptarsenus varicornis (Girault), Neochrysocharis formosa (Westwood), and Neochrysocharis okazakii Kamijo) and two braconid species (Dacnusa areolaris (Nees) and Opius cinerariae Fischer). We also provide the first DNA barcodes (5' end COI sequences) with linked morphological characters for seven wasp species, with three identified to species level (Closterocerus mirabilis Edwards & La Salle, Trigonogastrella parasitica (Girault), and Zagrammosoma latilineatum Ubaidillah) and four identified to genus (Aprostocetus sp., Asecodes sp., Opius sp. 1, and Opius sp. 2). Phylogenetic analyses suggest C. pubicornis, D. isaea, H. varicornis, and O. cinerariae are likely cryptic species complexes. Neochrysocharis formosa and Aprostocetus sp. specimens were infected with Rickettsia. Five other species (Cl. mirabilis, D. isaea, H. varicornis, Opius sp. 1, and Opius sp. 2) were infected with Wolbachia, while two endosymbionts (Rickettsia and Wolbachia) co-infected N. okazakii. These findings provide background information about the parasitoid fauna expected to help control the leafminers.

A new species of Diglyphus Walker (Hymenoptera, Eulophidae) from China, with morphological characterizations and molecular analysis.

Diglyphus Walker, 1844 (Hymenoptera: Eulophidae) is an economically important genus including species acting as biocontrol agents against agromyzid leafminer pests. A new species of Diglyphus, Diglyphusdifasciatus Liu, Hansson & Wan, sp. nov., was discovered during the identification of agromyzid leafminers and their associated parasitoid wasps collected from 2016 to 2022 in China, based on morphological characteristics and molecular analyses of COI, ITS2 and 28S genes. Diglyphusdifasciatus is similar to D.bimaculatus Zhu, LaSalle & Huang, distinguished by two interconnected infuscate vertical bands on the fore wing and the color of the scape. Molecular data support D.difasciatus and D.bimaculatus as two different species. The mean genetic distances between D.difasciatus and D.bimaculatus were 11.33%, 8.62%, and 0.18%, based on the COI, ITS2, and 28S genes, respectively.

Demographics and host-kill parameters of Diglyphus horticola Khan against Chromatomyia horticola (Goureau).

The pea leafminer, Chromatomyia horticola (Goureau) (Diptera: Agromyzidae) is a polyphagous and serious pest of peas. In India, this pest is attacked by many parasitoids and among them Diglyphus horticola Khan (Hymenoptera: Eulophidae) is an important one, however, demographics and pest-kill potential of this parasitoid has not been studied so far. This study presents the first report on its demographics and pest-kill potential on C. horticola. The parasitoid showed three modes of host-killing behaviour viz. host-feeding, parasitism and host-stinging. The parasitoid females killed more number of hosts by parasitism than host-feeding or host-stinging. The pre-adult survival, net reproductive rate, intrinsic rate of increase (rm) and finite rate of increase (λ) were higher on the 5-days old host larvae than those reared on the 3-days old larvae. Demographics and pest-kill parameters of D. horticola were also better on 5-days old host larvae than on 3-days old host larvae. Based on the study, D. horticola appeared to be a promising biocontrol agent for the suppression of C. horticola in peas and could be promoted through conservation biological control. Further studies are required to standardize the mass production protocol and release rates to use the parasitoid by augmentation.

Miocene Cupressinoxylon from Gökçeada (Imbros), Turkey with Protophytobia cambium mining and the study of ecological signals of wood anatomy.

Premise: The recognition of the Miocene Climate Optimum (MCO) in terrestrial palaeoenvironments of the Eastern Mediterranean is restricted to Lesbos and Lemnos Islands, Greece. This area is significant for its wood microfossils. A recently-discovered fossil wood assemblage from Gökçeada (Imbros) Island, Turkey, including tree species similar to the Greek findings, is thought to have an early Miocene age. Here, we revise the age of the latter plant fossiliferous locality, re-evaluate the area for the study of MCO for the terrestrial palaeoecosystems of the Eastern Mediterranean and the nomenclature errors referring to the occurrence of fossil wood. We present the plant-insect-environment interactions using detailed anatomical descriptions, of an extinct conifer and its extinct cambium miner feeding traces observed in its secondary xylem. Methods: Three thin sections were prepared with standard palaeoxylotomical techniques from a small section of the silicified wood; the sections were observed under a light microscope. The anatomy of the conifer and its damage patterns were compared with those of extant and fossil Cupressaceae and Agromyzidae, respectively. Pivotal results: The common anatomical features of the studied wood specimen and Hesperocyparis macrocarpa (Hartw.) Bartel and a shared characteristic (the number of the cross-field pits - a feature we consider of diagnostic value) with Xanthocyparis vietnamensis Farjon & T.H. Nguyên led to its assignment to the Hesperocyparis-Xanthocyparis-Callitropsis clade. The detailed study of the wound scars and anatomical abnormalities, the anatomical-environmental associations, and structural-functional reactions follow the identification of the wood's anatomy sensu Carlquist providing decisive results. Conclusions: Based on the distinctive characteristics presented, we identify our macrofossil as Cupressinoxylon matromnense Grambast, a stem or an extinct lineage of the Hesperocyparis-Xanthocyparis vietnamensis-Callitropsis nootkatensis clade with feeding traces of the fossil cambium miner of the genus Protophytobia Süss (Diptera: Agromyzidae), and anatomical damage and reaction tissue on adventitious shoots. The use of Protopinaceae and Pinoxylon F. H. Knowlton from the eastern Mediterranean are re-evaluated and corrections are provided. The age of the studied plant fossiliferous locality in Gökçeada is revised as middle Miocene, allowing the proposal of an eastern Mediterranean MCO hotspot, including Lesbos, Lemnos, and Gökçeada (Imbros) Islands.

DNA barcoding of the leaf-miner flies (Diptera, Agromyzidae) of Mitaraka, French Guiana.

Species level identification of Agromyzidae based on morphology is often challenging due to their small size and morphological homogeneity. DNA barcoding has been used regularly to assist with the identification of economically important species of Agromyzidae, but rarely as a tool for species delineation or identification in biodiversity surveys. The main objective of this study was to investigate whether DNA barcoding and the BIN (Barcoding Index) system could assist with species identification, species delineation, male/ female association, and diversity assessment of Agromyzidae material previously determined to morphospecies from Mitaraka, French Guiana. Amplification success was low, with sequences over 400 bp recovered for only 24 (48%) of the selected specimens. Sequences assigned to 17 morphospecies formed 16 distinct branches or clusters separated by very high (minimum of 10%) sequence divergence. Following the reassessment and subsequent reassignment of one specimen, congruence between morphology and DNA barcodes was high with a single instance of two morphospecies sharing identical sequences. While DNA barcoding did not assist with identification (none of our sequences matched those of named taxa in BOLD or GenBank), it did provide support for most of our morphospecies concepts, including male/female associations. The BIN system also provided access to information about the distribution and habitat preferences of several taxa. We conclude that DNA barcoding was a useful approach to study the species diversity of our samples but that much work remains to be done before it can be used as an identification tool for the Agromyzidae fauna of Mitaraka and the rest of the Neotropical region.

Diversity underfoot of agromyzids (Agromyzidae, Diptera) mining thalli of liverworts and hornworts.

Agromyzidae is a dipteran family that has diversified as internal plant feeders. Although most agromyzid species feed on herbaceous angiosperms, only a limited number of species has been recorded as miners of bryophytes. Extensive searches and rearing of bryophytivores in the Japanese Archipelago were made, resulting in that thallus-mining agromyzids are overwhelmingly widespread and diverse on thalloid liverworts and hornworts. By examining the morphology of adult flies, it was revealed that the agromyzid fauna comprise 39 species, of which 37 species are newly described. All the species are assigned to the genus Phytoliriomyza Hendel based on some shared morphological character states as follows: costa reaching M1; orbital setulae minute and erect (rarely proclinate); male epandrium with combs of fused tubercle-like setae and/or hypertrophied arms bearing tubercle-like setae; male distiphallus comprising a pair of stout, extended tubules; female cercus with two stout, apical, trichoid sensilla. Of the 39 agromyzid species in Japan, 36 species are associated with liverworts: 5 spp. on Marchantia (Marchantiaceae), 2 spp. on Dumortiera (Dumortieraceae), 3 spp. on Plagiochasma, 1 sp. on Asterella, 6 spp. on Reboulia (Aytoniaceae), 1 sp. on Wiesnerella (Wiesnerellaceae), 15 spp. on Conocephalum (Conocephalaceae), and 3 spp. on Riccia (Ricciaceae). Three species are associated with hornworts: 1 sp. on Folioceros (Anthocerotaceae), 1 sp. on Megaceros (Dendrocerotaceae), and 1 sp. on Notothylas,Phaeoceros (Notothyladaceae), and Anthoceros (Anthocerotaceae). The results suggest that 37 of the 39 species are host-specific at least to plant genus level, and that the inter-specific differences in male genitalia and color patterns of scutum, antenna, and maxillary palpus have contributed to reproductive isolation on the bryophytes that the flies share.

High Incidence of Related Wolbachia across Unrelated Leaf-Mining Diptera.

The maternally inherited endosymbiont, Wolbachia pipientis, plays an important role in the ecology and evolution of many of its hosts by affecting host reproduction and fitness. Here, we investigated 13 dipteran leaf-mining species to characterize Wolbachia infections and the potential for this endosymbiont in biocontrol. Wolbachia infections were present in 12 species, including 10 species where the Wolbachia infection was at or near fixation. A comparison of Wolbachia relatedness based on the wsp/MLST gene set showed that unrelated leaf-mining species often shared similar Wolbachia, suggesting common horizontal transfer. We established a colony of Liriomyza brassicae and found adult Wolbachia density was stable; although Wolbachia density differed between the sexes, with females having a 20-fold higher density than males. Wolbachia density increased during L. brassicae development, with higher densities in pupae than larvae. We removed Wolbachia using tetracycline and performed reciprocal crosses between Wolbachia-infected and uninfected individuals. Cured females crossed with infected males failed to produce offspring, indicating that Wolbachia induced complete cytoplasmic incompatibility in L. brassicae. The results highlight the potential of Wolbachia to suppress Liriomyza pests based on approaches such as the incompatible insect technique, where infected males are released into populations lacking Wolbachia or with a different incompatible infection.

Manual of North American Agromyzidae (Diptera, Schizophora), with revision of the fauna of the "Delmarva" states.

Тhe agromyzid (Diptera: Schizophora: Agromyzidae) fauna of America north of Mexico is described in the first part of this publication, including a genus key and discussions on morphology, life history and classification. The second part is a species-level revision of the family in the "Delmarva" states of the United States of America, that is, of the District of Columbia and the surrounding states of Delaware, Maryland, Virginia and West Virginia. The fauna of this region includes 156 species. This study presents 346 new state and provincial records and 23 new country records, two of which are new continental records (Agromyza abiens Zetterstedt and A. apfelbecki Strobl). Liriomyza endiviae Hering is no longer considered to occur in North America. Fifteen species are newly described: Agromyza echinalis sp. nov., Melanagromyza brunkei sp. nov., M. eoflacensis sp. nov., M. glyptos sp. nov., M. rutella sp. nov., Ophiomyia capitolia sp. nov., O. cuprea sp. nov., O. galiodes sp. nov., O. heleios sp. nov., O. kalia sp. nov., O. laticolis sp. nov., Cerodontha (Poemyza) ungulasp. nov., Phytomyza avicursa sp. nov., P. catenula sp. nov., and P. winkleri sp. nov. Four new species-level synonyms and one genus-level synonym are provided: Agromyza marmorensis Spencer syn. nov. is included as a synonym of A. aristata Malloch; Melanagromyza fastosa Spencer, syn. nov. is included as a junior synonym of Ophiomyia tiliae (Couden); Melanagromyza verbesinae Spencer is considered a synonym of M. vernoniana Steyskal; Phytomyza ranunculoides Spencer, syn. nov. is included as a junior synonym of Phytomyza loewii Hendel; the genus Liomycina Enderlein, syn. nov. is included as a junior synonym of Phytobia Lioy. Ophiomyia ultima (Spencer), comb. nov. is recombined from Melanagromyza. Euhexomyza albicula Spencer, stat. reinst., comb. nov. is resurrected from synonymy with E. winnemanae (Malloch). New host records are given.

Molecular Identification of Leafmining Flies From Australia Including New Liriomyza Outbreaks.

Some leafmining fly species are pests of agricultural and ornamental plants but they also include many species with little economic importance. The taxonomy of leafmining flies is often challenging because of putative cryptic species. Following new outbreaks of Liriomyza (Diptera:Agromyzidae) in Australia, we here characterize 13 dipteran leafminer species collected from Australia based on cytochrome c oxidase subunit 1 (COI) DNA barcodes, and we compare these with overseas data. We confirm barcodes of nine species from the Agromyzidae (Liriomyza sativae, L. huidobrensis, L. trifolii, L. bryoniae, L. chinensis, L. brassicae, L. chenopodii, Phytomyza plantaginis and P. syngenesiae) and one species from the Drosophilidae (Scaptomyza flava); we describe new haplotypes for some of these species. Furthermore, we provide the first DNA barcodes for two species (Cerodontha milleri and Phytoliriomyza praecellens) from the Agromyzidae and one species (Scaptomyza australis) from the Drosophilidae. These findings provide a baseline for DNA-based identification of pest Liriomyza incursions spreading across the Australian east coast in relation to other species already present in Australia.

A new species of Hemiptarsenus Westwood (Hymenoptera, Eulophidae) from China, with a key to Chinese species.

A new species, Hemiptarsenus jilinus Tao, sp. nov., is described and illustrated. All the type specimens were reared from Chromatomyia horticola (Goureau) (Diptera: Agromyzidae), a leafminer attacking the plants Ixeris polycephala Cass. and Pterocypsela indica (L.) Shih, in Jilin Province, north-eastern China. A key to Chinese species of the genus is provided.

Comparing Potential as Biocontrol Agents of Two Neotropical Parasitoids of Liriomyza sativae.

We evaluated the potential of two parasitoids as biocontrol agents of Liriomyza sativae Blanchard in northeastern Brazil. The two species were the koinobiont larval-pupal endoparasitoid Phaedrotoma scabriventris Nixon (Braconidae) and the idiobiont larval endoparasitoid Chrysocharis vonones (Walker) (Eulophidae). The biological parameters evaluated were survivorship, parasitism, and host-feeding, at 25 and 30°C. Differences between the species were observed at 25°C, but not at 30°C. At 25°C, the total parasitism for P. scabriventris (196.1 ± 17.7) and C. vonones (176.6 ± 7.24) was similar and with higher values compared to 30°C, 102.5 ± 8.81 and 89.1 ± 5 66 parasitized larvae, respectively. However, C. vonones showed a 3.97 lower survivorship as well as higher daily parasitism (1.4-fold) and host-feeding means (1.9-fold) than the braconid at 25°C. The results indicate a potential for both natural enemies to be used as biocontrol agents of L. sativae. The differences between species detected at 25°C suggest the best conditions for the application of each parasitoid.

Convergently Evolved Toxic Secondary Metabolites in Plants Drive the Parallel Molecular Evolution of Insect Resistance.

Natural selection imposed by natural toxins has led to striking levels of convergent evolution at the molecular level. Cardiac glycosides represent a group of plant toxins that block the Na,K-ATPase, a vital membrane protein in animals. Several herbivorous insects have convergently evolved resistant Na,K-ATPases, and in some species, convergent gene duplications have also arisen, likely to cope with pleiotropic costs of resistance. To understand the genetic basis and predictability of these adaptations, we studied five independent lineages of leaf-mining flies (Diptera: Agromyzidae). These flies have colonized host plants in four botanical families that convergently evolved cardiac glycosides of two structural types: cardenolides and bufadienolides. We compared each of six fly species feeding on such plants to a phylogenetically related but nonadapted species. Irrespective of the type of cardiac glycoside in the host plant, five out of six exposed species displayed substitutions in the cardiac glycoside-binding site of the Na,K-ATPase that were previously described in other insect orders; in only one species was the gene duplicated. In vitro assays of nervous tissue extractions confirmed that the substitutions lead to increased resistance of the Na,K-ATPase. Our results demonstrate that target site insensitivity of Na,K-ATPase is a common response to dietary cardiac glycosides leading to highly predictable amino acid changes; nonetheless, convergent evolution of gene duplication for this multifunctional enzyme appears more constrained.

Braconid wasps of subfamily Alysiinae (Hymenoptera: Braconidae) as endoparasitoids of Selachops flavocinctus Wahlberg, 1844 (Diptera: Agromyzidae) in the Central Urals, Russia.

The Agromyzidae fly Selachops flavocinctus Wahlberg, 1844 as host of the Alysiinae genera Asyntactus Marshall, 1898 (Alysiini) and Protochorebus Perepechaenko, 1997 (Dacnusini) is recorded for the first time. A new species from Central Urals, Protochorebus pervushini sp. nov., is described and illustrated. The new material for Asyntactus rhogaleus Marshall, 1898 is studied. A key to Protochorebus species is provided. Description of puparium of Selachops flavocinctus and new data on its life history are published for the first time.

New data on Marthiella Buffington (Hymenoptera, Cynipoidea, Figitidae), with description of a new species

New data on Marthiella Buffington (Hymenoptera, Cynipoidea, Figitidae), with description of a new species. A new species of Marthiella Buffington (Hymenoptera, Figitidae) from Brazil and Nicaragua is described and illustrated. Rhabdeucoela semirufa Kieffer is transferred to Marthiella and a new combination was established: Marthiella semirufa. A key for identifying species of Marthiella is provided in this study.

Nutrient stress, host plant quality and herbivore performance of a leaf-mining fly on grass.

Environmental stresses affect plant growth and performance in nature. Host plant quality in turn affects herbivore performance and population dynamics. In view of these interactions, two major hypotheses were formulated. The plant stress hypothesis proposes that physiologically stressed plants become more susceptible to herbivores. The plant vigour hypothesis proposes that plants that grow vigorously are favourable to herbivores. Here we test the plant stress/plant vigour hypotheses for a leaf miner, Agromyza nigripes (Diptera; Agromyzidae), on the grass Holcus lanatus. We assessed larval performance (survival, developmental time, pupal mass) on grasses growing under different levels of nutrients (Hoagland solution) and drought stress, under controlled field and greenhouse conditions. Plant vigour and nutrient content were high on soils with an intermediate nutrient concentration and lower under drought stress and soil nutrient shortage and overdose. Larval performance was also highest on wet soils with intermediate nutrient supply. The results of the mining flies support the plant vigour hypothesis (density, survival and development better on vigorous plants). Herbivore performance is higher on leaves with a higher protein content.