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Bemisia tabaci

2004; Wiley; Volume: 34; Issue: 2 Linguagem: Inglês

10.1111/j.1365-2338.2004.00729.x

1365-2338

There are or pairs of minute anterior submarginal setae and pairs of minute posterior submarginal …, the th pair may be well developed . There are pairs of dorsal setae, which are highly variable in size and may be asymmetrical. They may all be minute or very well …, arising from enlarged bases.,

Nematode management and characterization studies

EPPO BulletinVolume 34, Issue 2 p. 281-288 Diagnostic protocols for regulated pests†Free Access Bemisia tabaci First published: 10 September 2004 https://doi.org/10.1111/j.1365-2338.2004.00729.xCitations: 9 European and Mediterranean Plant Protection Organization PM 7/35(1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF 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 Bemisia tabaci. Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003-09. Introduction Bemisia tabaci is a plant sap-sucking insect in the family Aleyrodidae of superfamily Aleyrodoidea (whiteflies). It is broadly polyphagous, feeding on an estimated 600 plant species. Since the early 1980s, it has caused escalating problems to both field and protected agricultural crops and ornamental plants. Heavy infestations of B. tabaci may reduce host vigour and growth, cause chlorosis and uneven ripening, and induce physiological disorders. The larvae produce honeydew on which sooty moulds grow, reducing the photosynthetic capabilities of the plant, resulting in defoliation and stunting. B. tabaci is also a vector of over 100 plant viruses in the genera Begomovirus (Geminiviridae), Crinivirus (Closteroviridae) and Carlavirus or Ipomovirus (Potyviridae) (Jones, 2003). Begomoviruses are the most numerous of the B. tabaci-transmitted viruses and can cause crop yield losses of between 20% and 100% (Brown & Bird, 1992). B. tabaci possibly originated in India (Fishpool & Burban, 1994) and as a result of widespread dispersal, particularly during the last 15 years, is now distributed nearly worldwide. Identity Name: Bemisia tabaci (Gennadius, 1889–Aleurodes). Synonyms: Bemisia inconspicua (Quaintance, 1900 –Aleurodes), and many others. A complete list of synonyms and type data is given by Mound & Halsey (1978) and Perring (2001). Taxonomic position: Insecta: Hemiptera: Homoptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae: Aleyrodinae. Notes on taxonomy and nomenclature: B. tabaci was first described in 1889 as a pest of tobacco in Greece, as Aleurodes tabaci (Gennadius, 1889). It was subsequently described under numerous names before its morphological variability was recognized (Mound, 1963; Mound & Halsey, 1978; Russell, 1957). The existence of host races or biotypes was proposed in the 1950s to describe distinct populations of B. tabaci with specific host associations and virus-vector capabilities. In the mid-1980s, reports emerged of a newly evolved 'B biotype', a highly polyphagous variant that was almost twice as fecund as previously recorded populations (Brown et al., 1995). The B biotype has become a major pest of world agriculture and been described as a separate species, B. argentifolii Bellows & Perring, based on RAPD-PCR banding patterns, isoelectric focusing electrophoresis, crossing experiments, mating behaviour and morphological evaluation (Bellows et al., 1994). It can induce phytotoxic disorders in certain plant species, for example silvering of leaves in Cucurbita spp., hence the common name 'silverleaf whitefly'. To date, 41 distinct populations of B. tabaci have been characterized using a variety of techniques and 24 of these populations given a specific biotype designation (Perring, 2001). Molecular (Frohlich et al., 1999) and allozyme (Brown et al., 2000) data from these studies supports the idea that B. tabaci is a suite of highly cryptic sibling species that cannot currently be distinguished morphologically. Perring (2001) recently reviewed the species complex and proposed the existence of seven distinct groups, based on comparison of populations from various locations. However, for the purposes of this protocol, the name B. tabaci refers to all described variants in the B. tabaci species complex. Bayer computer code: BEMITA (BEMIAR has been used for the B biotype or B. argentifolii). Phytosanitary categorization: EPPO A2 list: no. 178; EU Annex designation: I/A1 (non-European populations); I/B (European populations). Detection Whiteflies are usually detected by close examination of the undersides of leaves to search for the tiny yellow/cream, scale-like larval instars. They also occasionally occur on the upper surfaces of the leaves and vary from being widely scattered to forming dense clusters. Shaking the plant may disturb the small white adults, which flutter out and quickly resettle. Adults may also be found on sticky traps placed above infested plants. Samples of larvae should be collected while still attached to the leaves and stored dry or in phials of 70% ethanol for examination in the laboratory. Infested plants may exhibit a range of symptoms due to direct feeding damage, contamination with honeydew and associated sooty moulds, whitefly-transmitted viruses and phytotoxic responses. There may be one or a combination of the following symptoms: chlorotic spotting, vein yellowing, intervein yellowing, leaf yellowing, yellow blotching of leaves, yellow mosaic of leaves, leaf curling, leaf crumpling, leaf vein thickening, leaf enations, leaf cupping, stem twisting, plant stunting, wilting and leaf loss. Phytotoxic responses such as a severe silvering of courgette and melon leaves usually indicate the presence of a B biotype infestation. Identification The Taxonomy Of The Aleyrodidae Is Based Almost Entirely On The Final (Fourth) Larval Instar Or 'puparial Stage'. The Exuvium Is Often Referred To As The 'pupal Case'. With Very Few Exceptions, Accurate Whitefly Identification Is Only Possible From Microscopic Examination Of A Slide-mounted Puparium Or Pupal Case. The Following Descriptions In This Section All Refer To This Stage. Slide Preparation Methods For Whitefly Puparia Are Presented In Appendix I. A High Power Microscope (× 100 To × 400) Is Required To See All The Diagnostic Characters. The Morphological Terminology Used Here Follows That Of Bellows Et Al. (1994). It is often difficult to distinguish between third larval instars and puparia until the specimens are slide-mounted, although puparia are generally larger. The antennae of the puparia are straight or gently curved, uniform in width and overlap with the front legs. The antennae of the third instars are strongly curved forming a U shape, broad at the base and apically narrow, and do not overlap with the front legs. Whitefly puparia are notorious for exhibiting considerable environmentally induced morphological variation. This phenotypic variation is largely dependent on the tactile experience of the first instar before settling to feed, which is determined by leaf surface topography and population density (Neal & Bentz, 1999). Characters such as size, body shape, colour, length of dorsal setae and tubercle development are highly variable. This variation has been studied in detail for B. tabaci by numerous authors including Azab et al. (1969), Bethke et al. (1991), David & Ananthakrishnan (1976), Harakly (1973), Mohanty & Basu (1986), Mound (1963) and Rossell et al. (1997). This variation needs to be taken into account during the identification process. Identification of slide-mounted puparia Family Aleyrodidae The family is easily recognizable by the presence of a vasiform orifice, operculum and lingula (see Web Figs 1, 4, 6, 8). Genus Bemisia The type-species of Bemisia Quaintance & Baker, 1914 is Aleurodes inconspicua, a synonym of B. tabaci, by original designation. Some 40 species are assigned to the genus Bemisia (Martin, 1999) but there is currently no comprehensive key available for their identification. The majority of Bemisia species have a limited geographical distribution and host-plant range (Mound & Halsey, 1978) and are unlikely to be encountered in plant trade. B. tabaci is the most geographically widespread, polyphagous and economically important species assigned to the genus. According to Martin (1999), wide phenotypic variation is a particular generic trait of Bemisia. The genus may be identified using the following combination of characters: • cuticle usually completely pale, occasionally brownish pigmentation • margin irregular crenulate, often modified at caudal and/or tracheal openings at margin to form ill-defined combs of fine teeth, with margin often shallowly indented at these points • transverse moulting sutures not reaching margin • medial length of abdominal segment VII less than half that of VI • vasiform orifice acute-triangular, sometimes laterally sinuous, posteroapically often ill-defined and usually leading into a pronounced caudal furrow; operculum occupying basal half of orifice; head of lingula typically elongate-triangular, finely spinulose, bearing a pair of apical setae, always exposed but included within vasiform orifice • chaetotaxy and presence/absence of dorsal sculpturing and tubercles may be highly variable within species • ventrally, caudal and thoracic tracheal folds marked, usually finely stippled. A simple key to separate Bemisia from other plant-pest genera present in Europe and the Mediterranean area is given in Table 1. It is advisable to also refer to Martin et al. (2000) for a comprehensive key to the whiteflies of this region and Martin (1987) for a key to common whitefly pests of the World (both works include B. tabaci). Table 1. Key to puparia of some pest genera of Aleyrodidae found in Europe and the Mediterranean. For general morphology of a whitefly puparium see Web Fig. 1 1. With 6 pairs of abdominal compound wax-producing pores, on segments III–VIII, the anterior two pairs much smaller than the posterior four pairs Paraleyrodes – Without abdominal compound pores 2 2. Pupal case dark brown to black 3 – Pupal case colourless, with or without brownish patches 4 3. Less than half of total length of vasiform orifice occupied by operculum and lingula together Acaudaleyrodes – More than half of total length of vasiform orifice occupied by operculum alone or by operculum and lingula together Aleurolobus 4. Dorsal disc and/or submargin with a pattern of stout, acute or tubiform spines 5 – Dorsal surface without a pattern of stout spines, although sometimes with a submarginal row of conspicuous hairs or setae, or with a few stout setae on the dorsal disc 6 5. Dorsal spines tubiform, siphon-like Siphoninus – Dorsal spines acute Aleurocanthus 6. Submargin with a regular row of normally 14 fine, acute setae. Lingula basally bilobed Parabemisia Submargin without a regular row of fine, acute setae and lingula not basally bilobed 7 7. Submargin with a row of papillae and lingula lobulate Trialeurodes – Submargin without a row of papillae; lingula not lobulate 8 8. Thoracic and caudal tracheal openings at margin marked by invaginated pores Dialeurodes – Thoracic and caudal tracheal openings at margin marked by combs of differentiated teeth or not marked 9 9. Wide submarginal area separated from dorsal disc by a suture-like fold Aleurothrixus – Submarginal area not separated from dorsal disc by a suture-like fold 10 10. Lengths of abdominal segments I–VIII similar medially. Vasiform orifice subcordate, hardly longer than wide, sides slightly convex. Caudal furrow present but not pronounced. Margin regularly crenulate Aleyrodes – Length of abdominal segments VII much reduced medially. Vasiform orifice triangular, much longer than wide, sides straight to concave. Caudal furrow pronounced Bemisia Bemisia tabaci See Web Figs 1–6. In life, the puparium appears translucent, cream to distinctly yellow, without evident adorning wax secretion. Dorsum with thin, transparent wax layer. Size, 0.55–0.87 mm long, 0.35–0.64 mm wide. Shape suboval, often strongly tapered to posterior. When slide-mounted, cuticle evenly pale. Margin finely crenulate, thoracic tracheal opening slightly indented and without tracheal combs; caudal opening may also be slightly indented and without comb. Minute anterior and posterior marginal setae present. Caudal setae long and stout. Dorsum Generally smooth. Up to 5 median tubercles and 8 pairs of subdorsal abdominal papillae may be present or tubercles and papillae absent. Small discoidal pores, with associated smaller porette (often difficult to detect) are aligned in four serially arranged groups. Pores may be duplicated or apparently missing. Longitudinal moulting suture reaching margin; transverse moulting sutures each with obtuse angle halfway to lateral margin, not reaching margin. Vasiform orifice triangular, inset from puparial margin by less than its own length, the orifice leading to a distinct narrow caudal furrow; operculum covering anterior half of orifice; lingula spatulate, with two stout terminal setae, distal portion covered in minute acanthae. Chaetotaxy There are 3 or 4 pairs of minute anterior submarginal setae and 5 pairs of minute posterior submarginal setae, the 5th pair may be well developed (these minute setae are often very difficult to detect). There are 6 pairs of dorsal setae, which are highly variable in size and may be asymmetrical. They may all be minute (12 µm) (Web Fig. 2) or very well developed (up to 140 µm) (Web Fig. 3), arising from enlarged bases. Venter Thoracic tracheal folds usually with numerous minute spinules; spinules sometimes lacking in individuals with enlarged dorsal setae. Variation Phenotypic variation is largely dependent on the tactile experience of the first instar, which is determined by leaf surface topography and population density (Neal & Bentz, 1999). For example, puparia collected from hirsute leaves are often smaller, more pointed posteriorly, show dorsal setal enlargement, have dorsal tubercles and papillae, and often have their outlines indented by stout plant hairs (Web Fig. 3). Puparia collected from glabrous leaves are generally larger, rounded posteriorly, show little or no setal enlargement, have no dorsal tubercles or papillae and are oval (Web Fig. 2). Male puparia are usually slightly smaller than females. Detailed morphological descriptions and illustrations of puparia are given by Mound (1963), Hill (1969) and Bellows et al. (1994) (as B. argentifolia). The latter includes a detailed drawing of a syntype specimen of B. tabaci. Possible confusion with similar species Separation of all life stages of Bemisia tabaci from Trialeurodes vaporariorum Whiteflies are frequently detected on imported plants in the EU. Apart from B. tabaci, the species most commonly encountered is the glasshouse whitefly Trialeurodesvaporariorum and it is important for an NPPO to be able to distinguish all life stages of these two species. It is always preferable to make an identification from the puparium stage and those of B. tabaci can usually easily be separated from T. vaporariorum in the field with a × 20 hand lens. The appearance of the adults and empty eggs can also help to indicate which species is present but should not be used in isolation. The main characters that can be used to separate B. tabaci from T. vaporariorum in the field are listed in Table 2. Characters that can be used to separate all stages, except the egg, when examined under a high power microscope (× 100–400), are listed in Table 3. Hill (1969) gives a more detailed morphological comparison although he does not indicate the range of variation that may be encountered. Table 2. Comparison of some morphological and behavioural characters of Bemisia tabaci and Trialeurodes vaporariorum seen with low magnification (× 20) Bemisia tabaci Trialeurodes vaporariorum Egg Oviposition pattern Eggs usually scattered or grouped in small clusters; may form semicircles on smooth leaves. Eggs usually laid in neat circles or semicircles; may be scattered on very hairy leaves. Colour Yellowish-white when laid, becoming pale brown. Yellowish-white when laid, becoming dark Semitransparent and golden brown after hatching. brown to almost black. Smoky black after hatching Shape after hatching Often remains erect and maintains shape. Often flattened or bent double. Puparium Colour Often distinctly yellow; may be cream. Usually cream. Brown when parasitized. Black when parasitized. Shape Oval or elliptical, often pointed posteriorly. Outline often distorted by plant hairs. Oval or elliptical, rounded posteriorly. Surroundedby palisade of wax giving a 'pill-box' appearance. Outline not distorted by plant hairs. Dorsum 1–7 pairs of well developed dorsal setae present; longer on plants with hirsute leaves. Glassy wax rods and submarginal papillae absent. Dorsal setae absent. Dorsal and submarginal papillae present. Distinct glassy wax rods usually present. Excretory apparatus Vasiform orifice triangular and lingula swollen and pointed distally. Vasiform orifice subcordate and lingula lobed. Distribution of puparia Often scattered and density per leaf usually low, except on smooth leaves. Frequently grouped and the density per leaf high. Adult Colour Body dark yellow. Body pale yellow. Wing shape Forewings with anterior margin straight. Forewings with anterior margin curved. Position at rest Appears narrower and more pointed posteriorly with the wings held at a sharper angle ('tent-like'). Appears broader and more rounded posteriorly with the wings held more flatly. Flight pattern Often direct. Haphazard. Table 3. Comparison of morphological characters of slide-mounted Bemisia tabaci and Trialeurodes vaporariorum seen under high magnification (× 100–400) Bemisia tabaci Trialeurodes vaporariorum 1st instar Marginal setae 16 pairs. 17 pairs. Cephalic tubercles Weakly developed. Well developed, subrectangular, mesad. Vasiform orifice Closed posteriorly. Open posteriorly. 2nd instar Cephalic and 8th abdominal dorsal setae Usually minute. Well developed. Vasiform orifice Subcordate. Open posteriorly. 3rd instar Marginal crenulations Irregular. Uniform. Vasiform orifice Subcordate. Triangular, open posteriorly. Lingula Swollen and pointed distally. Lobed distally. Puparium Web Figs 2, 3 and 4(i), 5 and 6 Web Fig. 4(i)i(i) Submarginal papillae Absent. Present. Marginal crenulations Irregular. Uniform. Vasiform orifice Subcordate. Triangular, open posteriorly. Lingula Swollen and pointed distally. Distinctly lobed distally. Adult Upper and lower compound eyes Connected by a single ommatidium (or very small gap less than the width of an ommatidium). Separate. Mesotibia Opposite tufts of 2–3 stout setae may be present. Conspicuous opposite tufts (combs) of 4–7 stout setae present. 4th antennal segment Sensorial cone absent. Stout sensorial cone near apex. 7th antennal segment 1 sensorial cone present. 2 sensorial cones present; 1 is small, slender and difficult to see. Aedeagus Slender with smooth ventral base. Generally thicker and more robust with spiculate ventral base. Male collar Clear. Pigmented. Male abdominal dorsal surface Distinct pores absent. Distinct pores present. Female cement gland Usually distinctly sinuous; without bands and small head. Not sinuous; with transverse bands (seen under phase contrast); large disc shaped head. Female abdominal wax plates Reticulate. Striated. Other whiteflies While B. tabaci and T. vaporariorum account for the majority of detection of whiteflies in imported consignments, it should not be assumed that these are the only species of Bemisia and Trialeurodes to be encountered in plant trade as there is the risk of overlooking species such as B. afer (Priesner & Hosny) (= B. hancocki Corbett), T. lauri (Signoret), T. packardi (Morrill), T. ricini (Misra) and T. variabilis (Quaintance). B. afer has been detected on numerous occasions on imported plant material in the UK, most frequently on fresh vegetables from West Africa and bay (Laurus nobilis) plants from Europe. In the puparial stage, B. afer is the whitefly species most likely to be confused with B. tabaci during phytosanitary inspection. B. afer is broadly polyphagous and widespread in the tropics and subtropics and has recently been found breeding under glass and in restricted areas outdoors in the UK (Malumphy, 2003). Mixed populations of B. afer and B. tabaci have been intercepted on cassava (Manihot esculenta) leaves imported from Africa. The puparia of B. afer and B. tabaci may be separated by comparing the morphological characters listed in Table 4. Table 4. Comparison of some morphological characters of Bemisia afer and B. tabaci puparia Morphological character Bemisia tabaci Bemisia afer (Web Figs 2, 3 and 4i, 5 and 6) (Web Figs 4ii, 7 and 8) Caudal setae Always stout and usually as long or longer than vasiform orifice. Little variation between individuals. Usually less than half the length of the vasiform orifice and often minute; highly variable between individuals. Vasiform orifice Slightly longer than the length of the caudal furrow; with straight sides. Usually shorter than length of the caudal furrow; with sides often distinctly concave. Lingula Shorter and slightly wider. Highly variable, generally longer and narrower. Dorsal surface Distinct stippling absent; small tubercles and papillae may be present. Occasionally with distinct stippling and well developed tubercles and papillae. Dorsal setae Up to seven pairs of enlarged, well developed setae present; longer on plants with hirsute leaves. Highly variable; often minute and difficult to detect but may be well developed. Dorsal pore/porette pairs Single pair between median line and first abdominal setae. Most puparia with two pairs between median line and first abdominal setae; they are often difficult to detect. Outline Variable but may be strongly tapered to posterior, particularly on plants with hirsute leaves. Variable but usually oval and rounded or slightly tapered to posterior. Colour Often distinctly yellow. Highly variable, yellow, cream or almost transparent. Morphological descriptions, illustrations and keys to the puparia of B. afer (and B. tabaci) are given by Bink-Moenen (1983), Bink-Moenen & Gerling (1992), Mound (1965), Martin (1987, 1999) and Martin et al. (2000). B. afer shows considerable variation in size, position of the vasiform orifice (Web Fig. 8) with respect to the posterior margin, extent of dorsal sculpturing, length of caudal setae and shape of the lingula. Mound (1965) discussed the phenotypic variation displayed by the puparia of the B. afer species group (Web Fig. 7) which according to Martin (1999), is even more complex than that found in B. tabaci. According to Martin et al. (2000), there are only two species of Bemisia recorded in Europe and the Mediterranean, B. afer and B. tabaci. However, there are three other species names available, B. citricola Gomez-Menor, B. ovata (Goux) and B. spiraeoides Mound & Halsey. These three nominal species belong to the afer species group and may be separated from B. tabaci using the characters given in Table 4. Parabemisia myricae (Kuwana) is superficially similar to B. tabaci but is easily separated by the characters given above for the genus Bemisia. It may also be separated by the transverse moulting sutures reaching the margin and the absence of the caudal furrow. In B. tabaci the transverse moulting sutures do not reach the margin and the caudal furrow is distinct. Requirements for a positive diagnosis This protocol distinguishes B. tabaci from the species most likely to be confused with it (B. afer) and from the whitefly species most frequently encountered in phytosanitary inspections (T. vaporariorum). With experience, it is possible to identify B. tabaci puparia with the use of a low power microscope without making a slide preparation. The requirements for a positive identification vary according to the experience of the diagnostician. Figure 9 provides a general scheme. A good slide preparation of a pupal case is generally needed and the specimen should match the morphological description and illustrations in this protocol. The specimen should be compared with authoritatively identified specimens and/or checked by an experienced whitefly specialist. However, in practice, an experienced diagnostician can identify poorly mounted and damaged specimens and will not need to examine every character described above. Voucher specimens from each interception should be kept for a period in case they require re-examination or further investigation. Figure 9Open in figure viewerPowerPoint Decision scheme for the detection and morphological identification of Bemisia tabaci. Identifications made from stages other than the puparia should be recorded as provisional. Eggs and early instars can be reared to the puparial stage to confirm the identification. In practice, experience of past interceptions, hosts and country of origin, mean that one can strongly suspect nonpuparial stages to be B. tabaci. Report on the diagnosis A report on execution of the protocol should include: • results obtained by the recommended procedures • information and documentation on the origin of the infested material • a description of the symptoms (including if possible photographs) • measurements, drawings or photographs of the morphological features required for a positive diagnosis • an indication of the magnitude of the infestation • comments, as appropriate, on the certainty or uncertainty of the identification. Further information Further information on this organism can be obtained from: Dr C. Malumphy, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK. Footnotes 1 The Figures in this Standard marked 'Web Fig.' are published on the EPPO website http://www.eppo.org. Acknowledgements This protocol was originally drafted by: Dr C. Malumphy, Central Science Laboratory, York (GB). Appendix Appendix 1 Preparation of immature and adult whiteflies for microscopic examination Specimens usually need to be macerated, de-waxed, dehydrated, cleared and in some cases, bleached or stained, before mounting on microscope slides. Voucher specimens and those required for future study are mounted in Canada balsam, which Brown (1997) concluded was one of the best mediums for permanent preparations. Specimens that need to be processed rapidly are mounted in Heinz, either directly or by using the temporary method described below. Post-emergence pupal cases and early larval instars are particularly suitable for temporary quick-mounting. The permanent preparation technique given below is modified from Martin (1987). The procedures are not rigid and can be readily modified to suit particular samples. Specimens are manipulated and mounted on microscope slides with the aid of a binocular dissecting microscope. Heat is supplied, where necessary, by a heating block. Square-based watch glasses and glass slides should be accurately labelled with a waterproof marker throughout the procedure. The permanent preparation technique requires a minimum time of just over 1 h and the temporary technique 20 min. Materials Ammonium solution 35%; acid fuchsin stain (0.5 g acid fuchsin; 25 mL 10% HCL; 300 mL distilled water); Canada balsam; chloral phenol (160 g chloral hydrate crystals; 20 mL glucose syrup 50% ww; 160 g phenol crystals); clove oil; distilled water; ethanol 70–95%; glacial acetic acid; glucose syrup; Heinz (10 g Polyvinyl Alcohol (PVA); 40–60 mL distilled water, depending on viscosity required; 10 mL glycerol; 25 mL 11/2% phenol solution; 100 g chloral hydrate crystals; 35 mL 85–92% lactic acid); hydrogen peroxide 30-volume; potassium hydroxide (KOH) 10%; xylene. Procedures Permanent slide preparation This method is suitable for larval instars and adults. The best mounts are usually made from 'pupal cases' from which the adults have recently emerged although good results can be obtained from puparia with adequate maceration. Parasitized specimens should be avoided as they are often morphologically atypical. Parasitism can cause the puparium to become melanic, induce morphological variation, damage the puparium with the parasitoid exit hole and obscure diagnostic characters with the black fragments of ecdysed parasitoid larval cuticle. Obtaining good preparations of adults with this method is difficult (but not impossible) due to their fragile nature. Their bodies may collapse during dehydration in glacial acetic acid and the delicate wings are easily damaged. Gently remove specimens from the leaf surface using a mounted blunt needle taking care not to puncture the specimen. Place about 10 specimens into 70–90% ethanol in a watch glass, cover with a glass square and heat gently to around 80 °C for 5–10 min. Fixation in hot alcohol hardens specimens and makes them less fragile, so they lose fewer setae during mounting. Add a few drops of cold 10% KOH to cool the alcohol. Pipette off the alcohol and KOH using a fine glass teat pipette, taking care not to accidentally suck up the specimens. Add approximately 1 mL of KOH and heat to around 80 °C for 5–10 min, or until the specimens lose most of their colour. The length of time required varies consi