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Liriomyza spp.

There are 376 species currently recognized in the genus Liriomyza (David Henshaw, pers. comm., 2000), of which 136 are found naturally in Europe (Seymour, 1994). The adult flies of all these species look very similar. They are all small (1–3 mm in length) and, from above, are seen to be mostly black, with in most species a bright yellow scutellum. As a result, separating these species can be difficult. Close examination reveals small external differences that can be used to separate the species, such as the relative length of sections along particular wing veins, the presence, position and size of certain setae or the colour of the cuticle at the point where particular head setae arise. However, considerable variation in these character states is seen in the polyphagous pest species. As a consequence, for the pest species concerned, the ranges of variation of these characters often overlap, limiting their diagnostic value. Four species, Liriomyza bryoniae , Liriomyza huidobrensis , Liriomyza sativae and Liriomyza trifolii are listed in EU Plant Health Directive 2000/29 (EU, 2000). L. bryoniae is indigenous to Europe, while the other three all originated in the New World. All are polyphagous pests of ornamental and vegetable crops. Because of the different phytosanitary measures applied when the various Liriomyza spp. are detected on plant material, precise species identification is required. To identify these species, the diagnostician has not only to distinguish between them, but also to distinguish them from the background fauna of indigenous Liriomyza spp. (which are mostly not pests). This composition of this background fauna varies across Europe and no one morphological dichotomous key has been produced that will separate each of the four species both from each other and from the European fauna. This protocol presents methodologies by which the identity of these four species can be confirmed, whether the material available for examination consists of larvae or pupae (Fig. 1), or adult flies (Fig. 2). Since larvae and puparia possess few distinguishing morphological characters, isozyme analysis (Appendix I) and PCR-RFLP analysis (Appendix III) are recommended as additional methods. PCR-RFLP may also be useful to confirm morphological analysis of adults or to identify damaged specimens. A further species, L. strigata (Meigen, 1830), is a common, polyphagous species, indigenous to Europe. Because it is sometimes a minor pest itself and because it can be found in close proximity with the four listed species, the species is included in this protocol. L. cocculi (Frick, 1953) is a species from Hawaii whose close relationship to L. huidobrensis is indicated by the structure of the male genitalia (Spencer, 1990). However, it has a dark scutellum, is unlikely to be encountered in Europe or in association with imported commodities and is not discussed further here.


Introduction
There are 376 species currently recognized in the genus Liriomyza (David Henshaw, pers. comm., 2000), of which 136 are found naturally in Europe (Seymour, 1994). The adult flies of all these species look very similar. They are all small (1-3 mm in length) and, from above, are seen to be mostly black, with in most species a bright yellow scutellum. As a result, separating these species can be difficult. Close examination reveals small external differences that can be used to separate the species, such as the relative length of sections along particular wing veins, the presence, position and size of certain setae or the colour of the cuticle at the point where particular head setae arise. However, considerable variation in these character states is seen in the polyphagous pest species. As a consequence, for the pest species concerned, the ranges of variation of these characters often overlap, limiting their diagnostic value.
Four species, Liriomyza bryoniae , Liriomyza huidobrensis , Liriomyza sativae and Liriomyza trifolii are listed in EU Plant Health Directive 2000/29 (EU, 2000. L. bryoniae is indigenous to Europe, while the other three all originated in the New World. All are polyphagous pests of ornamental and vegetable crops. Because of the different phytosanitary measures applied when the various Liriomyza spp. are detected on plant material, precise species identification is required.
To identify these species, the diagnostician has not only to distinguish between them, but also to distinguish them from the background fauna of indigenous Liriomyza spp.
(which are mostly not pests). This composition of this background fauna varies across Europe and no one morphological dichotomous key has been produced that will separate each of the four species both from each other and from the European fauna.
This protocol presents methodologies by which the identity of these four species can be confirmed, whether the material available for examination consists of larvae or pupae (Fig. 1), or adult flies (Fig. 2). Since larvae and puparia possess few distinguishing morphological characters, isozyme analysis (Appendix I) and PCR-RFLP analysis (Appendix III) are recommended as additional methods. PCR-RFLP may also be useful to confirm morphological analysis of adults or to identify damaged specimens. A further species, L. strigata (Meigen, 1830), is a common, polyphagous species, indigenous to Europe. Because it is sometimes a minor pest itself and because it can be found in close proximity with the four listed species, the species is included in this protocol. L. cocculi (Frick, 1953) is a species from Hawaii whose close relationship to L. huidobrensis is indicated by the structure of the male genitalia (Spencer, 1990). However, it has a dark scutellum, is unlikely to be encountered in Europe or in association with imported commodities and is not discussed further here.  (Blanchard, 1926); Liriomyza cucumifoliae (Blanchard, 1938); Liriomyza langei (Frick, 1951); Liriomyza dianthi (Frick, 1958) (Frick, 1952); Liriomyza canomarginis (Frick, 1952); Liriomyza minutiseta (Frick, 1952); Liriomyza propepusilla (Frost, 1954); Liriomyza munda (Frick, 1957); Liriomyza guytona (Freeman, 1958)  Feeding punctures and leaf mines are usually the first and most obvious sign of the presence of Liriomyza spp. They remain intact and relatively unchanged over a period of weeks. Mine configuration is often considered a reliable guide to the identification of agromyzid species of no economic importance (as in many such cases the species are host-specific). However, with the polyphagous pest species, mine configuration is 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 range of variation means that identification from mine patterns alone should be treated with caution.

Feeding punctures
Feeding punctures of Liriomyza spp. are rounded and usually about 0.2 mm in diameter. They appear as white speckles on the upper leaf surface. The appearance of the punctures does not differ between species, nor can the pattern of their distribution on the leaf be used to separate species.

Leaf mines
The larvae feed mostly in the upper part of the leaf, mining through the green palisade tissue. Mines are usually off-white, with trails of frass appearing as broken black strips along their length. Repeated convolutions in the same small part of the leaf will often result in discoloration of the mine with dampened black and dried brown areas appearing, usually as the result of plant-induced reactions to the leaf miner. The typical appearances of mines (Web Figs 9 and 10) of these species are: • a tightly coiled, almost blotch-like mine -L. trifolii • a looser, irregular serpentine mine -L. bryoniae and L. sativae • an irregular serpentine mine tending to be restricted by veins within segments of the leaf and undulating between upper and lower leaf surface -L. huidobrensis • a mine closely following the main vein toward (and occasionally into) the petiole -L. strigata. Larvae exit the fully developed mines in order to pupariate (usually in the soil, sometimes on the surface of the leaf). The exit hole characteristically takes the form of a semicircular slit.
The mines of other species of agromyzids may look similar to those described above. Nevertheless, the feeding punctures and mines of Chromatomyia syngenesiae can usually be separated from those described above (Web Fig. 11). The feeding punctures of C. syngenesiae are larger (up to 1.0 mm in diameter) and distinctly oval in shape. The mines appear cleaner, uniformly white, with less convolutions and the frass appearing as distinctly separated black dots. As with L. huidobrensis , the mines can undulate between the upper and lower leaf surfaces. The larvae of C. syngenesiae , and of C. horticola , pupariate within the mine with the anterior spiracles usually projecting out from the lower surface of the leaf.

Identification of family and genus
Morphological terminology used in this protocol is based on that of McAlpine et al . (1981).

Family: Agromyzidae
Agromyzids are small flies whose larvae are leaf miners, stem borers or gall-makers.
2 Note: it has recently been proposed that L. huidobrensis is in fact a complex of two cryptic species. This follows a study of specific sequences in mitochondrial and nuclear genomes (Scheffer, 2000;Scheffer & Lewis, 2001). The name Liriomyza langei has been applied to North American populations, and the name L. huidobrensis to Central and South American populations. All invasive populations were found to belong to L. huidobrensis as so defined. L. langei and L. huidobrensis could not be separated morphologically, but a PCR-RFLP protocol for separating them has been published (Scheffer et al ., 2001). This can only distinguish between these two taxa, and L. bryoniae would produce as false a result as L. huidobrensis . The authors do not comment on other species such as L. strigata . They also note that, potentially, the primers used would also amplify parasitoid DNA and therefore recommend restricting use of the protocol to adult material. For the purposes of this Standard, the name L. huidobrensis will continue to be applied to all the specimens originating from the trans-American populations that cannot currently be separated by morphological means.

Formal description (of the adult)
The following combination of characters (Web Fig. 3), which define the family Agromyzidae , follows Hennig (1958) (as quoted in Spencer, 1987). Vibrissae present; 1-7 frontal bristles present; costal break present at the apex of Sc; cell cup small; A1 not reaching wing margin; pregenital sclerites of male with a simple (fused) tergal complex (tergites 6 -8) with only two spiracles between tergite 5 and the genital segment; and anterior part of abdominal segment 7 in female forming an oviscape.

Practical diagnosis (based on the larval stages)
In practice, agromyzids are recognizable because their larvae feed in the living tissue of plants (three-quarters of them are leaf miners). There are leaf miners in other Dipteran families. Typically, agromyzid larvae are cylindrical in shape, tapering anteriorly; with projections bearing the anterior and posterior spiracles, the former positioned on the dorsal surface of the prothorax, the latter backwardly directed at the rear; prominent, strongly sclerotized mouthparts, the mandibles with its longitudinal axis at oblique or right angles to the rest of the cephalopharyngeal skeleton and usually bearing two or more pairs of equally sized teeth, directed anteriorly, the ventral cornua (the posteriorly directed 'arms') commonly shorter than the dorsal ones. For a summary of information on the morphology and biology of the immature stages of agromyzids, with a large bibliography and illustrations of the cephalopharyngeal skeleton and posterior spiracles for a number of species, see Ferrar (1987).

Genus: Liriomyza
Formal description (of the adult) Small flies, 1-3 mm in length; fronto-orbital setulae reclinate; usually with a dark prescutellar area concolorous with the scutum, rarely yellow; scutellum yellow in most species, rarely dark; costa extends to vein M1; discal cell small; dm-cu crossvein present in most species; 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).

Practical diagnosis
The economically important species discussed in this protocol are seen from above to be mostly black with a yellow frons and a bright yellow scutellum. The legs are variably yellow. They possess the typical wing venation for the genus (Web Fig. 4).

Natural species groups
The Liriomyza spp. considered here separate into two distinct natural groups, based on the structure of the male genitalia, and the colour and the structure of the posterior spiracles of the larvae. However, the external characters of the adult flies useful for identification (Table 1), particularly those based on colour, do not fall neatly into these two groupings: Group 1 ( L. bryoniae , L. huidobrensis , L. strigata, L. cocculi); Group 2 (L. sativae, L. trifolii).

Identification of the different life stages Eggs
The eggs are laid into the leaf tissue. They are white and oval, about 0.25 mm in length. Neither genus nor species identification is possible.

Larvae and pupae
There are three larval instars, which feed as they tunnel through the leaf tissue. The newly emerged larvae are about 0.5 mm long but reach 3.0 mm when full-grown. They are typical of agromyzids in gross form (see above, and Web Fig. 12a). Pupae are oval, about 2.0 mm in length, very slightly flattened ventrally, with projecting anterior and posterior spiracles. In practice, for larvae and pupae, the two natural groups can be distinguished from each other morphologically but not the species within the groups. Species determination requires electrophoretic analysis (see Appendix I) or PCR-RFLP (Appendix III).

Group 1
Larvae are cream-coloured but in the final instar additionally develop a yellow-orange patch dorsally at the anterior end, which can extend right around to the ventral surface. Each posterior spiracle consists of an ellipse with pores along the margin (Web Fig. 12b). It can be difficult to make out the number of pores, which according to Spencer (1973), are: L. bryoniae 7-12 pores; L. huidobrensis about 6 -9 pores and L. strigata 10 -12 pores. Puparia are variable in coloration, from yellow-orange to dark brown. In L. bryoniae and L. strigata, they are mostly, but not exclusively, at the lighter end of the colour range. Mostly the colour of L. huidobrensis puparia tends to anthracite. The form of the larval spiracles is retained in the puparium although the pores are less clearly discernible.

Group 2
Larvae are translucent when newly emerged, yellow-orange later. Each posterior spiracle is tricorn-shaped with three pores, each on a distinct projection, the outer two elongate (Web Fig. 12c). Puparia are yellowish-orange, sometimes a darker golden-brown. Again the form of the larval spiracles is retained but the detail is less obvious.

External characters
Important morphological characters are shown in Table 1. For morphological keys, descriptions of species and illustrations of the male aedeagus of a number of European species of Liriomyza (and other agromyzids), see Spencer (1972Spencer ( , 1976. For species descriptions and illustrations of species worldwide, including economically important species, see Spencer (1973,1990).

Identification based on distiphallic structure
The distiphallus is the terminal part of the aedeagus (the intromittent organ, part of the male genitalia) (Web Fig. 14a,d; Web Fig. 18 (Plate 1)) and its complex three-dimensional structure is here of considerable diagnostic value. Indeed, the distiphallus provides a single character by which all five species can be reliably identified. In other words, all other species of Liriomyza, including those not discussed here, can be eliminated. The distiphallus is a very small, fragile structure enclosed by membranes and requires careful dissection and subsequent examination under a high power microscope. The basic structure of the distiphallus differs in the two natural species groups: in Group 1, there are two distal bulbs side by side (Web Fig. 14b), while in Group 2 there is only one distal bulb with a medial constriction dividing distinct lower and upper sections (Web Fig. 14c). Separation of the five species using the distiphallus is described in Appendix II. Brief summary descriptions of the five species are provided below.
Group 1 -distiphallus with two distal bulbs L. bryoniae: bulb rims of distiphallus circular; relatively yellow, medium-size fly with both vertical setae on yellow. L. huidobrensis: bulbs of distiphallus meet only at their rims; a larger and darker fly with both vertical setae on black and the black extending forward along the upper orbits; third antennal segment usually darkened. L. strigata: bulbs of distiphallus meet along their length; medium to large, moderately dark fly with at least the outer vertical seta on black.
Group 2 -distiphallus with one distal bulb L. sativae: slight medial constriction on the distiphallus bulb; smaller, moderately dark fly with at least the outer vertical seta on black; section a of wing vein Cu1A much longer relative to section b than in Group 1 species. L. trifolii: marked medial constriction on the distiphallus bulb; relatively yellow, smaller fly with both vertical setae on yellow; section a of wing vein Cu1A much longer relative to section b than in Group 1 species.

Reporting and documentation
Guidance on reporting and documentation is given in EPPO Standard PM7/-(in preparation).

Sample preparation
Individual larvae or puparia are homogenized in either 10 µL of NADP solution in a microtube using a moulded plastic crusher (with the homogenate then being transferred to the well of the sample plate) (Protocol A) or in 5 µL of NADP solution in situ in the well of the sample plate using a heat-sealed Pasteur pipette (Protocol B). Samples taken from the freezer should be kept below 4°C (e.g. in melting ice) until immediately before use.

Electrophoresis
Each of the outside chambers of the electrophoresis tank is filled with 100 mL 25 mm Tris Glycine, pH 8.5 buffer solution.
Paper wicks are soaked in this solution and then attached to the inner walls of these two chambers along their length so that in each case one side drops into the solution and the other just overhangs into the next chamber. Each gel in turn is removed from the buffer solution, blotted between sheets of filter paper, in order to remove excess liquid, and placed onto the aligning base. The homogenates are then applied from the sample plate to the gel using the applicator. Three to four applications per gel may be required to ensure sufficient homogenate on the gel. The gel is then placed across the middle two chambers of the electrophoretic tank with the cellulose side down so that good contact is made between the cellulose and the wicks. Protocol A: the gels are run simultaneously for 18 min at 200 V (1 mA per gel). Protocol B: the gels are initially run simultaneously for 18 min at 200 V (1 mA per gel). Electrophoresis is then interrupted and the first plate removed (to be stained for glucose-6-phosphate dehydrogenase). The second plate is then run for a further 20 min, still at 200 V.

Staining
Staining schedules essentially follow those outlined by Hebert & Beaton (1989). Staining solutions are prepared fresh from stock solutions while the electrophoresis is in progress. Note that PMS and l-amino acid oxidase are light-sensitive and should only be added to the relevant staining solutions (Table 2) immediately before they are used. The gels are removed from the electrophoresis tank and placed on a plexiglass plate. The staining solution is mixed with approximately 2 mL molten agar and gently and evenly poured over the gel. Bands are usually visible within a minute or two but, if this proves not to be the case, the staining reactions may be incubated in the dark for up to 45 min at 37°C. The staining reaction may be brought to a halt at any time by placing the agar-overlain gel plate in a 7% (v/ v) solution of acetic acid.
Protocol A: the three gels are, respectively, stained for glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehyrogenase (IDH) and malic enzyme (ME). Protocol B: the first gel to be removed from the electrophoresis tank is stained for G6PDH, the second for leucine-glycine peptidase (PEP).

Interpretation of band patterns
Interpretation of the band patterns is achieved using the biochemical keys presented in Tables 3 and 4.

Appendix II Identification to species using the male distiphallus
A diagrammatic representation of the successive steps undertaken in this procedure is presented as Fig. 2. The distiphallus of male Liriomyza spp. is a very small, fragile structure enclosed by membranes and requires careful dissection before examination under a high power microscope. Evidence of distiphallic structure should be correlated with evidence of external morphology (Table 1) in order to confirm the identification. G6PDH IDH ME PEP Tris-HCl, 0.1 m, pH 8.0 0.6 0.6 0.6 0.6 mL NADP (2 mg mL −1 ) 1.5 1.5 1.5 -

Determining the sex of flies
In the male, the lobes of the epandrium, which are dark and pubescent and not so heavily sclerotized as the female tube, curve around and down at the rear of the abdomen, from the dorsal to the ventral sides (Web Fig. 13a). A slit-like opening is seen between the lobes, triangular when more fully open, through which the rest of the male genitalia can be viewed. The lobes hardly extend beyond the last tergite. In the female, the abdominal segments beyond segment 6 form a black, heavily sclerotized tube which extends out beyond the 6th tergite (Web Fig. 13b) with a circular opening visible in posterior view at the end of the tube. The 6th tergite covers the basal half of the tube from above, though it is visible in lateral and ventral views.

Preparation and examination of the distiphallus
Using fine mounted needles, carefully separate the abdomen from the rest of the fly. Briefly wet in absolute ethanol, and bring to the boil in 10% KOH (or NaOH) and boil for 60 -90 s. Transfer to cold glacial acetic acid and leave for 3 min. Blot off excess glacial acetic acid and transfer to a drop of Heinz mounting medium (or a similar semiviscous mounting fluid such as Berlese solution or Hoyer's solution) on a cavity slide. Under a binocular stereoscopic microscope and using fine mounted needles, carefully dissect out the genital complex from the cuticle and the immediate, surrounding membranes (see Web Fig. 18 [Plate 1]). Using fine mounted needles, position the genital complex for lateral viewing under a compound light microscope (recommended at 400 × magnification).
Re-position the genital complex for ventral viewing of the distiphallus (again at 400 × magnification). Use the key in Table 5 for diagnostic determination of the species.