Molecular diagnostic tests

Because only adult agromyzid adult males can be authoritatively identified to species using morphological features, definitive diagnosis of immature stages (egg, larva and pupa) and adult females requires other techniques.  These techniques are important for rapid diagnosis of leafminers in quarantine situations. Initially, electrophoretic techniques were developed and used routinely in Great Britain and Europe to distinguish Liromyza huidobrensis from other polyphagous Liriomyza spp. such as L. trifolii, L. sativae and L. bryoniae (Collins 1996; EPPO 2005; Menken & Ulenberg 1983, 1986; Oudman 1992; Oudman et al. 1995; Zehnder et al. 1983). A major drawback of these tests was the requirement for using live material.

In recent years, molecular tests (which can be used on appropriately preserved specimens) have been developed and used for research in phylogeny of Agromyzidae (Scheffer 2005; Scheffer et al. 2007), investigation of possible cryptic species and dispersal pathways (Scheffer 2000; Scheffer & Lewis 2001, 2005, 2006; Scheffer et al. 2001) and for diagnostic tests (Chiu et al. 2000; Feng et al. 2007; Kox et al. 2005; Masetti et al. 2006; Miura et al. 2004; Morgan et al. 2000). 

Initially, RAPD-PCR (random amplified polymorphic DNA polymerase chain reaction) tests were employed (Chiu et al. 2000; Morgan et al. 2000), but more recently polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) tests have been developed targeting either cytochrome oxidase I or II regions in the mitochondral genome (Kox et al. 2005; Masetti et al. 2006; Miura et al. 2004; Scheffer 2000). The PCR-RFLP tests are more reproducible than RAPDs and are cost-effective (Masetti et al. 2006).

The development of multiplex real-time PCR assays based on the COI gene, which allow rapid detection of all life stages of L. huidobrensis, L. sativae and L. trifolii in a single test is a major advance for routine diagnostic use (Nakamura et al. 2013; Sooda et al. 2017). Specimens of immature leafminers can be collected in ethanol or on FTA® filter paper cards (Blacket et al. 2015). Squashing specimens on FTA filter paper cards can overcome logistical challenges when collecting on surveys. Samples on cards stored for up to 2 years at room temperature could be successfully amplified and sequenced (Blacket et al. 2015).

References
Blacket MJ, Rice AD, Semeraro L & Malipatil MB (2015). DNA-based identifications reveal multiple introductions of the vegetable leafminer Liriomyza sativae (Diptera: Agromyzidae) into the Torres Strait Islands and Papua New Guinea. Bulletin of Entomological Research 105: 533-544.

Chiu YC, Wu WJ, Shiao SF & Shih CJ (2000). The application of RAPD-PCR to develop rapid diagnostic technique for identification of 6 species of Liriomyza spp. Chinese Journal of Entomology 20: 293-309 (in Chinese). pdf

Collins DW (1996). The separation of Liriomyza huidobrensis (Diptera: Agromyzidae) from related indigenous and non-indigenous species encountered in the United Kingdom using cellulose acetate electrophoresis. Annals of Applied Biology 128: 387-398.

EPPO (2005). Liriomyza spp. EPPO Bulletin 35: 335-344.

Feng X, Chen N-Z, Ma J, Zhu S-F & Hu X-N (2007). Molecular identification of Liriomyza trifolii (Burgess) (Dipt., Agromyzidae) based on real-time PCR. Journal of Applied Entomology 131: 548-552

Kox LFF, van den Beld HE, Lindhout BL & de Goffau LJW (2005). Identification of economically important Liriomyza species by PCR-RFLP analysis. EPPO Bulletin 35: 79-85.

Masetti A, Luchetti A, Mantovani B & Burgio G (2006). Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis, (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy. Journal of Economic Entomology 99: 1268-1272.

Menken SBJ & Ulenberg SA (1983). Diagnosis of the agromyzids Liriomyza bryoniae and L. trifolii by means of starch gel electrophoresis. Entomologica Experimentalis et Applicata 34: 205-208.

Menken SBJ & Ulenberg SA (1986). Allozymatic diagnosis of four economically important Liriomyza species (Diptera, Agromyzidae). Annals of Applied Biology 109: 41-47.

Miura K, Tagami Y, Ohtaishi M & Iwasaki A (2004). Application of molecular techniques to distinguish Liriomyza trifolii from L. sativae on tomato cultivation in Japan. Journal of Economic Entomology 97: 964-969.

Morgan DJW, Reitz SR, Atkinson PW & Trumble JT (2000). The resolution of Californian populations of Liriomyza huidobrensis and Liriomyza trifolii (Diptera : Agromyzidae) using PCR. Heredity 85: 53-61.

Nakamura S, Masuda T, Mochizuk A, Konishi K, Tokumaru S, Ueno K & Yamaguchi T (2013). Primer design for identifying economically important Liriomyza species (Diptera: Agromyzidae) by multiplex PCR. Molecular Ecology Resources 13, 96-102.

Oudman L (1992). Identification of economically important Liriomyza species (Diptera: Agromyzidae) and their parasitoids using enzyme electrophoresis. Proceedings of the Section Experimental and Applied Entomology of the Netherlands Entomological Society 3: 135-139.

Oudman L, Aukema B, Menken SBJ. & Ulenberg SA (1995). A procedure for identification of polyphagous Liriomyza species using enzyme electrophoresis. Bulletin OEPP/EPPO Bulletin 25: 349-355.

Scheffer SJ (2000). Molecular evidence of cryptic species within the Liriomyza huidobrensis (Diptera: Agromyzidae). Journal of Economic Entomology 93: 1146-1151.

Scheffer SJ (2005). Invasive Diptera: using molecular markers to investigate cryptic species and the global spread of flies, pp. 371-387. In The evolutionary biology of flies. (eds Wiegmann BM & Yeates DK), Columbia University Press, New York.

Scheffer SJ & Lewis ML (2001). Two nuclear genes confirm mitochondrial evidence of cryptic species within Liriomyza huidobrensis (Diptera: Agromyzidae). Annals of the Entomological Society of America 94: 648-653.

Scheffer SJ & Lewis ML (2005). Mitochondrial phylogeography of the vegetable pest Liriomyza sativae (Diptera: Agromyzidae): divergent clades and invasive populations. Annals of the Entomological Society of America 98: 181-186.

Scheffer SJ & Lewis ML (2006). Mitochondrial phylogeography of the vegetable pest Liriomyza trifolii (Diptera: Agromyzidae): diverged clades and invasive populations. Annals of the Entomological Society of America 99: 991-998.

Scheffer SJ, Wijesekara A, Visser D & Hallett RH (2001). Polymerase chain reaction-restriction fragment-length polymorphism method to distinguish Liriomyza huidobrensis from L. langei (Diptera: Agromyzidae) applied to three recent leafminer invasions. Journal of Economic Entomology 94: 1177-1182.

Scheffer SJ, Winkler IS & Weigmann BM (2007). Phylogenetic relationships within the leaf-mining flies (Diptera:  Agromyzidae) inferred from sequence data from multiple genes. Molecular Phylogenetics and Evolution 42: 756-775.

Sooda A, Gunawardana D, Li D & Kumarasinghe K (2017). Multiplex real-time PCR assay for the detection of three invasive leafminer species: Liriomyza huidobrensis, L. sativae and L. trifolii (Diptera: Agromyzidae). Austral Entomology 56, 153-159.

Zehnder GW, Trumble JT & White WR (1983). Discrimination of Liriomyza species (Diptera: Agromyzidae) using electrophoresis and scanning electron microscopy. Proceedings of the Entomological Society of Washington 85: 564-574.

Updated
2021-01-01

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