Trialeurodes vaporariorum (Westwood)

Malumphy, Christopher, Walsh, Katherine, Suarez, Belen, Collins, Dominique W. & Boonham, Niel, 2009, Morphological and molecular identification of all developmental stages of four whitefly species (Hemiptera: Aleyrodidae) commonly intercepted in quarantine, Zootaxa 2118, pp. 1-29 : 18-23

Trialeurodes vaporariorum (Westwood)

( Figs 36–47 )

T. vaporariorum was originally described by Westwood (1856) in the genus Aleyrodes , from specimens collected from Gonolobus sp., Tecoma velutina , Bignonia spp., Aphelandra spp., Solanum spp. and other similar soft-leaved plants in botanical collections in the UK. It is one of the most polyphagous and cosmopolitan whitefly species ( Mound & Halsey, 1978) and is a serious pest of glasshouse crops and ornamental plants. It is a vector of plant-pathogenic viruses.

Specimens measured

CHINA: (intercepted in UK), on penjing Ulmus parvifolia , 15.i.2001, PHSI, CSL2010177 (1 third instar); (intercepted in UK), on penjing U. parvifolia , 6.ii.2001, PHSI, CSL2010478 (2 first and 1 second instar); (intercepted in UK), on penjing U. parvifolia , 3.xii.1999, PHSI, CSL997451 (1 third instar). GUERNSEY: (intercepted in UK) on Fuchsia sp., 13.xii.2000, PHSI, CSL2007985 (3 first instars). KENYA: (intercepted in UK), on Hypericum androsaemum , 7.ii.2001, PHSI, CSL2010493 (2 first, 5 second and 1 third instars). NETHERLANDS: (intercepted in UK), on Lantana camara , 28.ii.2003, PHSI, CSL201741 (5 eggs and 1 third instar); (intercepted in France, Coquelles), on Aster sp., 12.viii.2003, PHSI, CSL20308761 (10 eggs and 9 first instars). PORTUGAL: (intercepted in UK), on Fuchsia hybrida , 23.xi.2000, PHSI, CSL2007424 (3 first instars and 1 second instar), CSL2007421 (5 first instars), CSL2007425 (2 first instars). TANZANIA: (intercepted in UK), on Sutera sp., 26.i.2001, PHSI, CSL2010333 (7 second and 1 third instar). UNITED KINGDOM: Avon, Bristol, botanical garden, on Solanum quitense , 5.xii.2000, PHSI, CSL2007668 (3 adult females); Berkshire, Windsor, on E. pulcherrima , 25.ix.2001, PHSI, CSL2014873 (2 second and 5 third instars); Essex, Wickford, on Symphytum officinale , 24.iii.2006, PHSI, CSL 206035536 (2 adult males); North Yorkshire, Nunnington, on Rhodochiton atrosanguineum , 25.vi.2001, R. Hammon, CSL2011894 (1 first instar); Cheshire, Macclesfield, on E. pulcherrima , 3.vii.2003, PHSI, CSL20308408 (2 first and 8 second instars), 25.vi.2004, PHSI, CSL20408585 (5 adult males and 19 females); Merseyside, Wirral, on Fuchsia sp., 15.viii.2003, PHSI, 20308958 (10 eggs and 8 third instars).

OVUM ( Fig. 36 )

Habitus. Cream coloured when first laid, becoming yellow and usually a dark smokey colour. Red eyes and yellow fat bodies of the first instar clearly visible prior to hatching. The eggs are usually laid in partial or complete circles but may be scattered on hirsute leaves or when populations occur at high densities. Elliptical, chorion smooth and shiny with little wax evident. The area surrounded the eggs is often lightly dusted with powdery wax by the adult female but this soon disappears. Each egg is erect and firmly attached to the leaf surface by a slender peduncle extending from the base of the egg, inserted into the leaf. The egg often collapses after hatching and is a distinct, dark smoky or black colour. Length 249 microns (204–288 microns), width 131 microns (112–144 microns), 1.91 (1.69–2.12) times longer than wide. Peduncle length 34 microns (28–39 microns), width is 9 microns (8–12 microns).

FIRST-INSTAR LARVA ( Fig. 37 )

Habitus. Scale-like, pale translucent yellow, becoming darker as they mature. A narrow band of white powdery wax present around the margin. Reddish eye spots, two yellow abdominal mycetomes and abdominal segmentation visible. Clumped distribution, forming distinct feeding groups. The first instars are usually located within a few mm’s of the hatched eggs.

Margin. Outline ovoid, lozenge-shaped; usually widest across mesothoracic coxae; length 314 microns (276–364 microns), width 190 microns (164–224 microns), 1.66 (1.54–1.95) times longer than wide. With 17 pairs of well-developed, fine, acute setae: MS14 length 54 microns (44–76 microns); CS length 134 microns (96–156 microns). Ratio CS/MS14 length = 2.49 (1.84–3.13). Margin relatively smooth, not modified at thoracic and abdominal tracheal openings.

Dorsum. Chaetotaxy comprises paired ASS, length 25 microns (14–32 microns); CeS length 6 microns (3–8 microns); 1 AS length 6 microns (3–8 microns); and 8 AS length 9 microns (4–14 microns). Meso-/ metathoracic and abdominal segmentation faintly marked, becoming less distinct with maturity. Cephalic tubercles well developed, oval to sub-rectangular. Single pair of geminate pore/porettes on 4th abdominal subdorsal area. Vasiform orifice sub-oval to almost rectangle, open behind, length 29 microns (26–36 microns), inset from posterior margin by less than its length, half-occupied by the operculum. The operculum is quadrate with the posterior margin spiculate. The posterior corners may be pronounced forming triangular points. Lingula spatulate, head exposed and spiculate with 2 pairs of stout setae.

Ven t er. Legs well developed. Mid and hind coxae and tibio-tarsi with long spine-like setae. Claw digitules long. Antennae long and slender, length 82 microns (72–92 microns). The solenidia are variable in size and the antennae may be asymmetrical. Abdominal setae bases correspond approximately with the middle of the vasiform orifice. Cuticle fine, diaphanous.

SECOND-INSTAR LARVA ( Fig. 38 )

Habitus. Distribution the same as the first instars. Pale translucent yellow, becoming darker with maturity.

Margin. Outline ovoid, usually widest across mesothoracic coxae; length 407 microns (360–444 microns), width 265 microns (228–292 microns), 1.55 (1.41–1.64) times longer than wide. Marginal setae, fine, acute. AMS length 4 microns (1–6 microns), PMS length 16 microns (12–20 microns), CS length 50 microns (46–56 microns), ratio CS/PMS = 3.16 (2.30–4.33). Margin crenulate, not modified at thoracic tracheal openings.

Dorsum. Chaetotaxy comprises paired CeS, length 62 microns (28–76 microns); 1 AS length 6 microns (3–8 microns); and 8 AS length 44 microns (10–58 microns). There is a single longitudinal row of approximately eight pairs of close-set geminate pore/porettes on each side. Meso-/metathoracic and abdominal segmentation marked. Abdominal segment VII reduced medially, pockets distinct. Vasiform orifice cordate to rhomboid, length 36 microns (32–41 microns), inset from posterior margin by slightly less than its length, closed posteriorly, half-occupied by the operculum, lingula head exposed, distinctly lobed, spiculate with 2 pairs of stout setae. Operculum quadrate to semi-circular with spiculate posterior margin.

Ven t er. Legs roughly triangular, each with a subcircular apical pad. Antennae short and slender, length 25 microns (20–32 microns). Abdominal setae placed mid vasiform orifice. Cuticle fine, diaphanous.

THIRD-INSTAR LARVA ( Fig. 39 )

Habitus. Distribution similar to first instars. Larger and darker yellow than first and second instars.

Margin. Outline ovoid, widest across mesothoracic legs; length 528 microns (488–612 microns), width 338 microns (276–406 microns), 1.57 (1.47–1.78) times longer than wide. Marginal setae, fine, acute. AMS minute and can be difficult to detect, length 5 microns (2–7 microns); PMS length 21 microns (14–28 microns). CS length 65 microns (48–84 microns). Ratio CS/PMS = 3.20 (2.58–4.38). Margin distinctly and evenly crenulate; not modified at thoracic and abdominal tracheal openings.

Dorsum. Chaetotaxy comprises paired CeS, length 66 microns (2–84 microns); 1 AS, length 5 microns (4–11 microns); and 8 AS, length 64 microns (6–78 microns). The CeS and 8 AS exist in two states: minute or well developed. It is possible that 1 AS may also be found well developed under the right environmental conditions but they were not observed in this state during this study. Approximately 12 pairs of close-set geminate pore/porettes distributed longitudinally from the head to rear of abdomen as shown in Fig. 39 . Abdominal segmentation rather faintly marked, medially and submedially. Abdominal segment VII reduced medially, pockets distinct. Vasiform orifice cordate to rectangular, closed behind, length 46 microns (42–52 microns), inset from posterior margin by about its own length, just over half occupied by the operculum with only part of lingula head visible. Lingula head lobed, spinulose with pair of stout setae.

Ven ter. Legs roughly triangular, each with a subcircular apical pad. Antennae, mesad but apex strongly curved back on itself; length 17 microns (12–22 microns). Abdominal setae placed anterior to vasiform orifice. Cuticle fine, diaphanous.

TABLE 3. C t values obtained following TaqMan PCR using the primers and probes designed for the identification of B. tabaci , B. afer , T. ricini and T. vaporariorum in simplex.

Species Name Simplexassays

Standard deviations are presented for duplicate reactions in the real-time PCR.

ADULT ( Figs 41–47 )

Body pale yellow, wings hyaline, covered with sparse, powdery wax. Antennae 7-segmented. Antennal segment II about half as long as antennal segment III; antennal segment III about as long as segments IV–VII combined; segments IV–VII subequal, although segment V may be longer. Segment III with one sensorium located on the proximal portion, and three sensoria (a cone and two rhinarial-types) close together on the distal portion; segment IV with a sensorial cone; segment V with an distal rhinarial-type sensorial; segment VI with a subapical sensorial cone; segment VII with two sensorial cones and a rhinarial sensorium, and terminating in a narrow conical sensorium. Upper eye composed of 52–55 ommatidia, each 8.7 (8.0–9.5) microns in diameter; lower compound eye composed of about 30–31 ommatidia, each 12.8 (11.2–13.9) microns in diameter, arranged in interconnected groups of 6 pigmented ommatidia surrounding a clear, smaller ommatidium. Upper and lower eyes separate. Metatibial combs consisting of 17 setae, all tibial brushes consisting of 4–6 adjacent setae. Male claspers paired, with about 10 long setae. Aedeagus thick, robust, ventral base spiculate; distal portion tapered and strongly curved near tip. Male collar and female gonapophysis and supragenital plate strongly pigmented. Female cement gland not sinuous, with transverse bands and with a large disc shaped-head.

Primer and probe design

The alignment of COI sequences revealed many polymorphisms between the whitefly species; Fig. 48 details these differences in the region of the primer and probe sequences. Suitable sites for primer and probe sequences for B. tabaci had the fewest number of mismatches with other species, between 0–7 in the forward primer (with a maximum of 2 in the last 8 bases at the 3′ end) and between 3 and 5 mismatches in the reverse primer (with a maximum of 3 in the last 8 bases at the 3 ′ end). When compared with T. vaporariorum the B. tabaci sequences had the least mismatches overall, 5 in total. The T. ricini assay had the greatest number of mismatches, between 16 and 19 mismatches in the forward and reverse primers.

TABLE 4. C t values obtained following TaqMan PCR using the primers and probes designed for the identification of B.

tabaci , B. afer , T. ricini and T. vaporariorum performed in two multiplex reactions for the Bemisa species and the

Trialeurodes species.

Standard deviations are presented for duplicate reactions in the real-time PCR Real-time PCR assay

DNA extracts from B. tabaci individuals produced Ct values of between 18.2–27.0 when tested with the B. tabaci -specific TaqMan assay; individuals of B. afer , T. ricini and T. vaporariorum tested with the appropriate assays produced Ct values of 20.2–21.3, 17.5–20.0 and 16.5–18.3 respectively (Table 3). Where an assay was used to test non-matching DNA no amplification was seen in 35 cycles. In addition, individuals of T. lauri from Turkey, a species very closely related to T. ricini ( Malumphy et al., 2007) were tested producing no amplification (data not shown). Three populations each of B. tabaci , T. ricini and T.

vaporariorum , as well as two of B. afer and one mixed population of both B. afer and B. tabaci , were tested with the TaqMan assays in multiplex (Table 4). Testing in multiplex produced comparable results with the simplex assays.

Figures

FIGURE 48. CO 1 sequence alignments showing sequence polymorphisms between species in the annealing sites of primers and probes. Mismatches are shown in bold, forward primer, probe and reverse primer annealing sites are boxed, left to right respectively.

FIGURES 31 – 38. Trialeurodes spp. 31, T. ricini, diagrammatic representation of left compound eye of adult female; T. ricini, 32, upper and lower compound eyes linked by two to four ommatidia; T. ricini, 33, male antenna; T. ricini, 34, aedeagus; T. ricini, 35, female cement gland; 36, T. vaporariorum, ovum on Euphorbia pulcherrima from Italy; 37, T. vaporariorum, first instar on E. pulcherrima from the UK; 38, T. vaporariorum, second instar on E. pulcherrima from Italy.

FIGURES 39 – 47. Trialeurodes vaporariorum. 39, third instar on E. pulcherrima from Italy; 40, fourth instar on E. pulcherrima from Italy; 41, diagrammatic representation of left compound eye of adult female; 42, upper and lower compound eyes separate; 43, mesotibia combs; 44, male abdomen with dorsal discoidal pores; 45, female antenna; 46, aedeagus; 47, female cement gland.