Two independent LAMP assays for rapid identification of the serpentine leafminer, Liriomyza huidobrensis (Blanchard, 1926) (Diptera: Agromyzidae) in Australia.

Liriomyza huidobrensis is a leafminer fly and significant horticultural pest. It is a quarantine listed species in many countries and is now present as an established pest in Australia. Liriomyza huidobrensis uses a broad range of host plants and has potential for spread into various horticultural systems and regions of Australia. Rapid in-field identification of the pest is critically needed to assist efforts to manage this pest. Morphological identification of the pest is effectively limited to specialist examinations of adult males. Generally, molecular methods such as qPCR and DNA barcoding for identification of Liriomyza species require costly laboratory-based hardware. Herein, we developed two independent and rapid LAMP assays targeted to independently inherited mitochondrial and nuclear genes. Both assays are highly sensitive and specific to L. huidobrensis. Positive signals can be detected within 10 min on laboratory and portable real-time amplification fluorometers. Further, we adapted these assays for use with colorimetric master mixes, to allow fluorometer free in-field diagnostics of L. huidobrensis. Our LAMP assays can be used for stand-alone testing of query specimens and are likely to be essential tools used for rapid identification and monitoring of L. huidobrensis.

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.

Meschia brevirostris sp. nov., from New Caledonia (Hemiptera: Heteroptera: Lygaeoidea: Meschiidae).

A new species, Meschia brevirostris sp. nov. (Hemiptera: Heteroptera: Lygaeoidea: Meschiidae), is described from New Caledonia. Photographs and SEM micrographs of the male and female habitus, genital structures and selected morphological structures are presented.

Revision of a unique Australian leafhopper genus Stenopsoides Evans (Hemiptera: Cicadellidae: Idiocerinae: Macropsini).

The Australian genus Stenopsoides Evans (Idiocerinae: Macropsini) is revised. The type species, S. turneri Evans, is redescribed and three new species are added: S. newi Semeraro sp. nov., S. punctatus Semeraro sp. nov. and S. truncatus Semeraro sp. nov.. Species photographic plates, a distribution map and an illustrated key to the four species are provided. The possibility of parthenogenetic reproduction occuring in this genus is discussed.

Barcoding Queensland Fruit Flies (Bactrocera tryoni): impediments and improvements.

Identification of adult fruit flies primarily involves microscopic examination of diagnostic morphological characters, while immature stages, such as larvae, can be more problematic. One of the Australia's most serious horticultural pests, the Queensland Fruit Fly (Bactrocera tryoni: Tephritidae), is of particular biosecurity/quarantine concern as the immature life stages occur within food produce and can be difficult to identify using morphological characteristics. DNA barcoding of the mitochondrial Cytochrome Oxidase I (COI) gene could be employed to increase the accuracy of fruit fly species identifications. In our study, we tested the utility of standard DNA barcoding techniques and found them to be problematic for Queensland Fruit Flies, which (i) possess a nuclear copy (a numt pseudogene) of the barcoding region of COI that can be co-amplified; and (ii) as in previous COI phylogenetic analyses closely related B. tryoni complex species appear polyphyletic. We found that the presence of a large deletion in the numt copy of COI allowed an alternative primer to be designed to only amplify the mitochondrial COI locus in tephritid fruit flies. Comparisons of alternative commonly utilized mitochondrial genes, Cytochrome Oxidase II and Cytochrome b, revealed a similar level of variation to COI; however, COI is the most informative for DNA barcoding, given the large number of sequences from other tephritid fruit fly species available for comparison. Adopting DNA barcoding for the identification of problematic fly specimens provides a powerful tool to distinguish serious quarantine fruit fly pests (Tephritidae) from endemic fly species of lesser concern.