PM 7/53 (2) Liriomyza spp.

Abstract

EPPO BulletinEarly View EPPO STANDARD ON DIAGNOSTICSFree Access PM 7/53 (2) Liriomyza spp. First published: 21 April 2022 https://doi.org/10.1111/epp.12832AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Specific scope This Standard describes a diagnostic protocol for Liriomyza bryoniae, Liriomyza huidobrensis, Liriomyza sativae and Liriomyza trifolii. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.11 Use of brand names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may also be suitable. Specific approval and amendment This Standard was initially developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by a partnership of contractor laboratories and intercomparison laboratories in European countries. This revision was prepared on the basis of the IPPC Diagnostic Protocol adopted in 2016 (Appendix 16 to ISPM 27 Genus Liriomyza, IPPC (2016)). Molecular tests are described according to the EPPO format. First approved in 2004–09. Revision approved in 2021–10. Authors and contributors are given in the Acknowledgements section. 1 INTRODUCTION Agromyzidae is a family of small flies whose larvae feed on the internal tissue of plants, often as leafminers and stem miners. The majority of agromyzid species are either host specific or restricted to a small group of plants that are related to each other (Benavent-Corai et al., 2005). However, a few highly polyphagous species have become agricultural and horticultural pests in many parts of the world. These include four species of Liriomyza that are listed in plant quarantine legislation in various countries: L. bryoniae, L. huidobrensis, L. sativae and L. trifolii. These are all polyphagous pests of both ornamental and vegetable crops. The species-level identification in this protocol is restricted to these four species. Liriomyza is predominantly found in the north temperate zone but species are also found in the Afrotropical, Neotropical and Oriental regions. The adult flies of the 300-plus species of Liriomyza (including 130-plus in Europe) look very similar: they are all small (1–3 in length) and, from above, appear largely black with, in most species, a yellow frons and scutellum (e.g. Figure 1). As a result, separating the species of the genus can be difficult. Furthermore, in order to identify the four species, a diagnostician should not only recognize these four species, but also be able to identify them against the background fauna of indigenous Liriomyza species. FIGURE 1Open in figure viewerPowerPoint Adult Liriomyza bryoniae. Courtesy of Department of Environment, Food and Rural Affairs, GB Liriomyza bryoniae is essentially a Palaearctic species with records from across Europe and Asia, and from Egypt and Morocco in North Africa (EPPO, 2020). It is highly polyphagous and has been recorded from 16 plant families (Spencer, 1990). It is a pest of tomatoes, cucurbits (particularly melons, watermelon and cucumber) and glasshouse-grown lettuce, beans and lupins (Spencer, 1989, 1990). Liriomyza huidobrensis is thought to have originated in South America and has now spread throughout much of the world, including parts of North America, Europe, Africa, Asia and the Pacific (Lonsdale, 2011; EPPO, 2020). However, the species as formerly taxonomically defined was recently split into two morphocryptic species – L. huidobrensis and L. langei – and there is some uncertainty about the precise delineation of their relative distribution. Currently, L. langei has been confirmed only from the USA and it is highly likely that all invasive populations outside the USA are L. huidobrensis as now taxonomically defined (Scheffer & Lewis, 2001; Scheffer et al., 2001; Takano et al., 2008; Lonsdale, 2011). Liriomyza huidobrensis is highly polyphagous and has been recorded from 15 plant families (CABI, 2021). The major host families and species listed by EFSA (2012) are: Apiaceae (Apium graveolens), Asteraceae (Aster spp., Chrysanthemum spp., Gerbera spp., Dahlia spp., Lactuca sativa, Lactuca spp.), Brassicaceae (Brassica spp.), Caryophyllaceae (Gypsophila spp.), Chenopodiaceae (Spinacia oleracea, Beta vulgaris), Cucurbitaceae (Cucumis spp., Cucurbita spp.), Fabaceae (Medicago sativa, Phaseolus vulgaris, Pisum sativum, Pisum spp., Trifolium spp., Vicia faba), Liliaceae (Allium cepa, Allium sativum) and Solanaceae (Capsicum annuum, Capsicum frutescens, Petunia spp., Solanum lycopersicum, Solanum spp.). Liriomyza sativae originated in North, Central and South America and has now spread to many parts of Asia, Africa, Australia and the Pacific, but not to Europe (Lonsdale, 2011; EPPO, 2020). However, distributional notes on L. sativae are likely to be incomplete as there is evidence to indicate that the species is continuing to expand its range rapidly. It is another highly polyphagous pest of many vegetable and flower crops (Spencer, 1973, 1990). It has been recorded from nine plant families, although it is mainly found on hosts in the Cucurbitaceae, Fabaceae and Solanaceae (Spencer, 1973, 1990). Liriomyza trifolii, also originally from North, Central and South America, has been spread to large parts of Europe, Africa, Asia and the Pacific, most likely as the result of trade in chrysanthemum cuttings (Martinez & Etienne, 2002; Lonsdale, 2011; EPPO, 2020). It is highly polyphagous and has been recorded from 25 plant families (Spencer, 1990). The most economically important crops it attacks are beans, celery, chrysanthemums, cucumbers, gerberas, Gypsophila, lettuce, onions, potatoes and tomatoes (Spencer, 1989). A further (fifth) species, L. strigata, is included in the diagnostic protocol because it is closely related to both L. bryoniae and L. huidobrensis, and as such, a diagnostician must be able to eliminate this species when seeking to positively identify L. bryoniae, L. huidobrensis, L. sativae or L. trifolii. Liriomyza strigata is an Eurasian species (Pitkin et al. (n.d.) quoting Spencer (1976), Dempewolf (2001), Ellis (n.d.) and Martinez (2013)). The eastern borders of its distribution are not clearly defined, but the range extends beyond the Ural Mountains (Spencer, 1976) and it has been doubtfully recorded in Southeast Asia (Dempewolf, 2004). It is highly polyphagous, having been recorded from 29 plant families worldwide (Spencer, 1990). For more detailed information on the distribution and hosts plants of the-above mentioned Liriomyza species, see EPPO Global Database (EPPO, 2020). A flow diagram describing the diagnostic procedure for the detection and identification of Liriomyza spp. is presented in Figure 2. FIGURE 2Open in figure viewerPowerPoint Flow diagram describing the diagnostic procedure for the detection and identification of Liriomyza spp. *Tests described in Appendices 2 and 3 and morphological identification cannot be used to distinguish L. huidobrensis and L. langei (1) In cases where adult specimens are atypical or damaged 2 IDENTITY Name: Liriomyza bryoniae (Kaltenbach, 1858) Other scientific name: Agromyza bryoniae (Kaltenbach, 1858); Liriomyza solani (Hering, 1927); Liriomyza hydrocotylae Hering, 1930; Liriomyza mercurialis Hering, 1932; Liriomyza triton Frey, 1945; Liriomyza citrulli (Rohdendorf, 1950); Liriomyza nipponallia Sasakawa, 1961 Taxonomic position: Insecta: Diptera: Agromyzidae EPPO Code: LIRIBO Phytosanitary categorization: EU Protected Zone Quarantine Pest (Annex III) Name: Liriomyza huidobrensis (Blanchard, 1926) Other scientific name: Agromyza huidobrensis (Blanchard, 1926); Liriomyza cucumifoliae (Blanchard, 1938); Liriomyza dianthi (Frick, 1958) Taxonomic position: Insecta: Diptera: Agromyzidae EPPO Code: LIRIHU Phytosanitary categorization: EPPO A2 list no. 283, EU Protected Zone Quarantine Pest (Annex III). The taxonomic relationship between L. huidobrensis (Blanchard) and L. langei (Frick) is complex. Liriomyza huidobrensis was originally described from specimens taken from Cineraria in Argentina by Blanchard (1926). Frick (1951) described L. langei from California as a species that he noted was primarily a pest of peas, although it had also damaged Aster. In 1973, Spencer then synonymized the two species as they were (and de facto remain) morphologically indistinguishable. Following a study of their mitochondrial and nuclear DNA sequences (Scheffer, 2000; Scheffer & Lewis, 2001), and supported by later rearing experiments (Takano et al., 2008), the two species were formally separated as two cryptic species (Lonsdale, 2011). The name L. langei Frick was resurrected and applied to the cryptic species from California, and the name L. huidobrensis (Blanchard) was applied to the cryptic species from South and Central America. Lonsdale (2011) attempted to delineate diagnostic morphological characters that could differentiate “most” specimens of the two species, but found the characters “subtle and sometimes overlapping” so he recommended the use of molecular data to support identification whenever possible. Scheffer and her collaborators consider that the ranges of the two species do not overlap (although Lonsdale (2011) recorded L. huidobrensis from California, once in 1968 and once in 2008, he states that it is unknown if the populations established), and that all of the invasive populations that they had studied were L. huidobrensis as so defined (Scheffer & Lewis, 2001; Scheffer et al., 2001). This means that reports from California in the literature predating Scheffer’s papers should be considered as applying to L. langei (with low uncertainty). Liriomyza langei is predominantly a Californian species although it has apparently been introduced into Hawaii, Oregon and Washington; populations found in Florida, Utah and Virginia in the mid-1990s did not establish (Lonsdale, 2011). Only L. huidobrensis has been confirmed in Mexico (Lonsdale, 2011), but Takano et al. (2005) reported that specimens of L. langei (described as the Californian clade) were intercepted at a Japanese inspection site on fresh vegetables originating from Mexico. Name: Liriomyza sativae (Blanchard, 1938) Other scientific name: Liriomyza verbenicola (Hering, 1951); Lemurimyza lycopersicae (Pla and de la Cruz, 1981); Liriomyza pullata (Frick, 1952); Liriomyza canomarginis (Frick, 1952); Liriomyza minutiseta (Frick, 1952); Liriomyza propepusilla (Frost, 1954); Liriomyza munda (Frick, 1957); Liriomyza guytona (Freeman, 1958): Liriomyza subpusilla (Frost) Taxonomic position: Insecta: Diptera: Agromyzidae EPPO Code: LIRISA Phytosanitary categorization: EPPO A2 list no. 282, EU A1 Quarantine pest (Annex II A) Name: Liriomyza trifolii (Burgess, 1880) Other scientific name: Liriomyza alliovora (Frick, 1955); Liriomyza phaseolunata (Frost) Taxonomic position: Insecta: Diptera: Agromyzidae EPPO Code: LIRITR Phytosanitary categorization: EPPO A2 list no. 131, EU Protected Zone Quarantine Pest (Annex III) 3 DETECTION 3.1 General information In practice, agromyzids are recognizable because their larvae feed in the living tissue of plants (three-quarters of them are leafminers). However, there are leafminers in other Dipteran families such as Anthomyiidae and Drosophilidae. Leafminer species are also present in Lepidoptera families; however, excrements of Agromyzidae larvae are located in the margin of the mines whereas they are located in the center for Lepidoptera larvae. Feeding punctures and leaf mines are usually the first and most obvious signs of the presence of Liriomyza. While fully formed mines should be readily visible to quarantine officials, early signs of infestation are much less obvious and are easily overlooked (Spencer, 1989). Mines remain intact and relatively unchanged over a period of weeks. Mine configuration may be used to guide to the identification of agromyzid species (as in many such cases the species are host specific). However, considering the broad host range, the pattern of mining may be affected by the host, by the physical and physiological condition of each leaf, and by the number of larvae mining the same leaf. This wider variability means that identification from mine pattern alone is not recommended. Examples of mine pattern for the four quarantine species and L. strigata are provided in Figures 3-5. FIGURE 3Open in figure viewerPowerPoint Typical characteristics of mines of (a) Liriomyza bryoniae, (b) Liriomyza huidobrensis and (c) Liriomyza strigata FIGURE 4Open in figure viewerPowerPoint Typical characteristics of mines of (a) Liriomyza sativae and (b) Liriomyza trifolii FIGURE 5Open in figure viewerPowerPoint Typical mines of Liriomyza spp.: (a) L. bryoniae on tomato; (b) L. huidobrensis on chrysanthemum; (c) L. trifolii on chrysanthemum; (d) L. sativae on pepper; and (e) L. strigata on an unidentified host. Courtesy of Department of Environment, Food and Rural Affairs, GB 3.1.1 Feeding and oviposition punctures Female flies use their ovipositor to puncture the leaves of the host plants, causing wounds that serve as sites for feeding (by both female and male flies) or for oviposition. Feeding punctures of Liriomyza species are rounded, usually about 0.2 mm in diameter, and appear as white speckles on the upper surface of the leaf. Oviposition punctures are usually smaller (0.05 mm) and more uniformly round. Feeding punctures made by the polyphagous agromyzid pest species Chromatomyia horticola and Chromatomyia syngenesiae are distinctly larger and more oval than those made by Liriomyza flies. The appearance of feeding and oviposition punctures does not differ among Liriomyza species, and the pattern of their distribution on the leaf cannot be used to identify species. Feeding punctures cause the destruction of a large number of cells and are clearly visible to the naked eye. 3.1.2 Leaf mines The larvae feed mostly just below the upper surface of the leaf, mining through the green palisade tissue. Mines are usually off-white, with trails of frass appearing as broken black lines along the length of the leaf. Repeated convolutions in the same small area of the leaf will often result in discolouration of the mine, with dampened black and dried brown areas appearing, usually as the result of plant-induced reactions to the leafminer. There are three larval stages, all of which feed within the leaves. The larvae predominantly feed on the plant in which the eggs are laid. The larvae of Liriomyza spp. leave the leaf when ready to pupariate (Parrella & Bethke, 1984), and their exit hole characteristically takes the form of a semi-circular slit; in contrast, the larvae of C. horticola and C. syngenesiae pupate inside the leaf at the end of the larval mine, with the anterior spiracles usually projecting from the lower surface of the leaf. Liriomyza puparia, therefore, may be found in crop debris, in the soil or sometimes on the leaf surface. Specimens may be found in different locations of the plant and surrounds depending on the life stages present, as follows: Eggs – inserted just below the leaf surface; Larvae – inside mines on leaves; Pupae – in crop debris, in the soil or sometimes on the leaf surface; Adult – free-flying, or on leaf surfaces while producing feeding and oviposition punctures. 3.2 Collection and preservation of specimens Liriomyza flies can be collected as immature life stages in association with mined leaf samples or as adults. Because the morphological characters used to diagnose species are based on male genitalia, adult males are needed in order to confirm species identification (see Section 4). Adult females are often identifiable with certainty only to genus level. Collecting multiple specimens (ideally 20) from a plant or a location will increase the likelihood of obtaining male flies, which is important unless molecular tests are to be used for diagnosis. 3.2.1 Collecting adults Adult flies are normally found on the foliage and can be collected by hand or with a sweep-net into glass vials or collected with an aspirator. Alternatively, they can be collected using yellow sticky traps, particularly in glasshouses. However, the most practical and reliable method for collecting leafminer flies such as Liriomyza species is to collect mined leaves containing living larvae. These can be placed in a large jar for rearing to adult flies in the laboratory. Techniques for rearing agromyzids are described in Griffiths (1962). For morphological identification adults and larvae can be boiled in water for a few minutes, transferred in 70% ethanol and stored indefinitely, although their colour fades over time. Dry storage of adults, for example as pinned specimens, is also possible. For molecular testing, specimens should be killed and preserved in 96–100% ethanol, stored frozen (at about −20 or −4.0°C) or preserved on FTA cards (Whatman) (Blacket et al., 2015). 3.2.2 Collecting immature life stages If the intention is to collect and preserve plant samples, leaves with suspect feeding punctures or mines should be picked and placed between sheets of newspaper to permit slow drying. Leaves with occupied mines from which it is intended to rear individuals can be safely removed for identification. They need to be packed in slightly damp, but not overly wet, laboratory tissue, and mailed in padded and sealed bags. In the laboratory, the mined leaves with living larvae can be placed in sealed Petri dishes with damp filter paper inserts and stored in an incubator at about 23°C (checking every two or three days to remove leaves that began to decay). 4 IDENTIFICATION Identification of leafminer species by morphological examination is restricted to adult male specimens because there are no adequate keys for the species-level identification of adult females or for eggs, larvae or pupae. Identification of adult material is possible by examination of morphological characters, in particular the genitalia of the male fly. The morphological characters of the male genitalia are examined under a high-power microscope (recommended at 400× magnification) able to perform biometric measurements and regularly calibrated using a micrometer. Using this protocol with good quality preparations should allow adults of the four quarantine species of Liriomyza to be identified with certainty by morphological examination alone (with the exception of L. huidobrensis and L. langei for the reasons discussed in Section 2). Molecular tests can be applied to all life stages, including the immature stages for which morphological identification to species level is not possible. Additionally, in cases where adult specimens are atypical or damaged, molecular tests may provide further relevant information about identity. However, the analytical specificity of molecular tests may be limited as they were developed for a purpose and evaluated against a restricted number of species. Therefore, the results from molecular tests need to be carefully interpreted. 4.1 Morphological identification Examination of the male genitalia (in particular, the distiphallus; Figure 6) is necessary in order to obtain a positive identification for any of the four target species of Liriomyza. Determination of flies’ sex and preparation of male distiphallus for examination (based on Malipatil & Ridland, 2008) are presented in Appendix 1. Evidence of distiphallic structure should be compared with characters of external morphology (Table 1) in order to confirm the species identification. FIGURE 6Open in figure viewerPowerPoint Male genitalia of Liriomyza huidobrensis (lateral view). Courtesy of Department of Environment, Food and Rural Affairs, GB TABLE 1. Adult morphological characters of selected Liriomyza speciesa a See also Figures 7, 8 and 10-14. L. bryoniae L. huidobrensis b b L. langei is morphologically indistinguishable from L. huidobrensis. L. sativae L. strigata L. trifolii Vertical setae Both vertical setae on yellow ground but black at hind margin of eye sometimes reaching base of outer verticle Both vertical setae on black (dark) ground Outer vertical setae on black ground. Inner vertical normally on dark ground or at margin with yellow Black colouration behind the eyes extending to at least the outer vertical setae Both vertical setae on yellow ground Frons and orbits Frons bright yellow, orbits slightly paler Frons yellow, generally more orange than pale lemon-yellow; upper orbits slightly darkened to upper orbital setae Frons and orbits bright yellow Frons bright yellow, orbits slightly paler Frons and orbits yellow Third antennal segment Small, bright yellow Normally brownish yellow. In palest specimens only faintly darkened (infuscated) Small, yellow Small, bright yellow Small, bright yellow Anepisternumc c Spencer refers to the mesopleura (nearly equivalent to Anepisternum) Predominantly yellow, small black mark at front lower margin Generally black on lower three-quarters but variable Predominantly yellow, with dark area, which in the palest specimens is limited to a small grey bar along the lower margin and in the darkest extends along the entire lower margin Yellow with black patch along lower margin. In darkest specimens entire lower half is black Yellow, small blackish grey grey on lower margin Mesonotum Black, largely shiny but with distinct matt undertone Semi shiny black Black, shiny Black, shiny but slightly matt Blackish grey, matt Femur Bright yellow with some brownish striations Yellow, variably darkened with black striations In darkened specimens appearing almost completely black but yellow always detectable on inner sides Bright yellow Yellow with some brownish striations Yellow with slight, variable brownish striation Wing length 1.75–2.1 mm 1.7–2.25 mm 1.3–1.7 mm 1.8–2.1 mm 1.3–1.7 mm Vein Cu 1A a twice length of b a 2–2.5 times length of b a 3–4 times length of b a 2–3 times length of b a 3–4 times length of b Male abdominal tergites Second and third visible tergites divided by a yellow medial furrow Only the second visible tergite divided by a yellow medial furrow Only the second visible tergite divided by a yellow medial furrow – Second to fifth visible tergites divided by a yellow medial furrow Male distiphallus Two distal bulbs; bulb rims circular Two distal bulbs, meeting only at their rims; bulb rims drawn out antero-ventrally One distal bulb with a slight constriction between upper and lower halves in dorso-ventral view; bulb appears more strongly sclerotized with a shorter basal stem Two distal bulbs, meeting from their rims to their bases; bulb rims drawn out antero-ventrally One distal bulb with marked constriction between lower and upper halves in dorso-ventral view; bulb appears less distinctly sclerotized with a longer basal stem Epiphallusd d Additional character compared with IPPC protocol. Apex of epiphallus is rounded Apex of epiphallus is rounded Apex of epiphallus is pointed Apex of epiphallus is rounded Apex of epiphallus is pointed Source: Mostly compiled from Spencer (1973, 1976), with information on the male abdominal tergites from Shiao (2004) (who did not include L. strigata in his analysis). Text in italics is for characters not found in the references cited before but mentioned in Appendix 16 to ISPM 27 Genus Liriomyza, IPPC (2016). a See also Figures 7, 8 and 10-14. b L. langei is morphologically indistinguishable from L. huidobrensis. c Spencer refers to the mesopleura (nearly equivalent to Anepisternum) d Additional character compared with IPPC protocol. 4.1.1 Identification of the family Agromyzidae Worldwide, the family Agromyzidae comprises about 2,500 species (Spencer, 1989, 1990). Detailed descriptions of agromyzid morphology are given by Spencer (1972, 1973, 1987), Dempewolf (2004) and Boucher (2010). Morphological nomenclature in this Standard follows Yeates et al. (2004). This online resource can also be consulted for clear illustrations of the anatomy of a typical acalyptrate fly (such as Agromyzidae). Adults The following combination of characters defines the family Agromyzidae (Hennig, 1958; Spencer, 1987; Boucher, 2010): Small in size, up to 1–6 mm, but usually 1–3 mm; Vibrissae present; One to seven frontal setae present; Wing with costal break present at the apex of Sc (Figures 7 and 8); Wing cell cup small – wing veins A1 + CuA2 not reaching wing margin (Figure 8); Male with pregenital sclerites with a fused tergal complex of tergites 6–8, with only two spiracles between tergite 5 and the genital segment; Female with the anterior part of abdominal segment 7 forming an oviscape (Figure 7 Q – ovipositor sheath). FIGURE 7Open in figure viewerPowerPoint Adult female morphology of Agromyzidae. Source: Spencer (1973). Side view of typical Agromyza sp. (after Sasakawa): A, arista; B, cheek; C, jowl; D, orbital bristles; E, orbital setulae; F, palp; G, proboscis; H, third antennal segment; I, vibrissa; J, acrostichals; K, dorso-central bristles; L, mesonotum; M, humerus; N, mesopleural area; O, notopleural area; P, haltere; Q, ovipositor sheath; R, scutellum; S, squama; T, squamal fringe; U, tergites; V, coxa; W, femur; X, tibia; Y, tarsi. 1, costa; 2, second costal section; 3, fourth costal section; 4, first cross-vein; 5, second cross-vein; 6, R1; 7, R 4+5; 8, M 1+2; 9, M 3+4; 10, sub-costa (Sc) FIGURE 8Open in figure viewerPowerPoint Wing venation of Liriomyza spp. Courtesy L. Gidron, PPIS, IL Larvae Generally the larvae (Figure 9a) are cylindrical in shape, tapering anteriorly, with projections bearing the anterior and posterior spiracles (Figure 9b and d), the former located on the dorsal surface of the prothorax, the latter posteriorly directed at the rear. The larvae also possess strongly sclerotized mouthparts; the mandibles with their longitudinal axis are at about right angles to the rest of the cephalopharyngeal skeleton (Figure 9c) and usually bear two or more pairs of equally sized anteriorly directed teeth, with the ventral cornua (the posteriorly directed paired “arms”) commonly shorter than the dorsal ones. FIGURE 9Open in figure viewerPowerPoint Larval morphology of Agromyzidae (Phytomyza chelonei): (a) lateral view; (b) anterior spiracle; (c) cephalopharyngeal skeleton; and (d) posterior spiracle. Source: Stehr (1991) For a summary of information on the morphology and biology of the immature stages of agromyzids, with an extensive bibliography and illustrations of the cephalopharyngeal skeleton and posterior spiracles for a number of species, see Ferrar (1987). 4.1.2 Identification of the genus Liriomyza Adult flies of the genus Liriomyza have the following morphological characters (Spencer, 1976): Fronto-orbital setulae reclinate (backward pointing); Dark pre-scutellar area concolorous with the scutum in most species, rarely yellow; Scutellum yellow in most species, rarely dark; Subcosta becomes a fold distally and ends in costa separately (Figure 8); Costa extends to vein M1+222 In most of the Liriomyza species the costa vein ends at the point where M1+2 reaches the wing margin as noted in Spencer (1976). However, there are a number of other Liriomyza species, including L. sativae and L. trifolii, that have slightly varied length costa, and for which the costa are fractionally extending beyond the point where M1+2 reaches the wing margin (Figure 9). (Figure 8); Discal cell (dm) small (Figure 8); Second (outer) crossvein (dm–cu) present in most species (Figure 8); Stridulating organ present in males (a “scraper”, a chitinized ridge on the hind femora; and a “file”, a line of low chitinized scales on the connecting membrane between the abdominal tergites and sternites). In practice, most species of Liriomyza (including the four target species included in this diagnostic protocol) are seen from above to be mostly black with a yellow frons and a bright yellow scutellum. The legs are variably yellow. The target species possess the typical wing venation (Figure 9) and the generalized male genitalia of the genus. There are several genera that may be confused with Liriomyza. The closely related genera Phytomyza, Chromatomyia and Phytoliriomyza can generally be separated from Liriomyza by their proclinate (forward pointing) fronto-orbital setulae (always reclinate or occasionally upright or missing in Liriomyza), and by the scutellum, which is generally grey or black but occasionally slightly yellowish centrally (entirely yellow in most Liriomyza). In Phytomyza and Chromatomyia, the costa extends only to R4+5, whereas in Phytoliriomyza and Liriomyza it extends to vein M1+22 (Spencer, 1977). Phytoliriomyza species are gall-forming (on a stem or leaf) internal