Tetranychus turkestani is a serious pest of cotton, corn, vegetables, fruit trees and forests in Xinjiang. All life stages of T. turkestani were observed using both light and scanning electron microscope. The egg is smooth and spherical. The eupathidial spinnerets of larva and protonymph are different from that of the deutonymph and adult in shape. The spinneret of adult female is obviously thicker and larger than that in male. The palpfemoral seta of adult male is a short, stout, spine-like process. T. turkestani has fourteen pairs of dorsal setae. The number of ventral setae increases regularly in number in each molt. The leg chaetotaxy has a great change from larva to deutonymph.
Tetranychus turkestani Ugarov et Nikolskii (Acari: Tetranychidae) is a serious pest on many plants, including cotton (Gossypium hirsutum L.), corn (Zea mays L.), sorghum (Sorghum bicolor L.), medlar (Achras sapota L.), tomato (Lycopersicon esculentum Mill.), bean (Phaseolus vulgaris L.), and other vegetables, fruit trees, and forest trees (Ugarov & Nikolskii 1937; Yu et al. 2000; Sohrabi & Shishehbor 2008; Guo et al. 2013; Zhang et al. 2016). It is mainly distributed in Russia, Kazakhstan, the United States, the Middle East and Xinjiang of China (Hill & Donnell 1991; Ros & Breeuwer 2007; Imani et al. 2009; Li et al. 2015). Previous research on T. turkestani focused on its biology and control (Yuan et al. 2008; Yang et al. 2012; Yang et al. 2013; Li et al. 2014; Duan et al. 2015; Liu et al. 2015). To our knowledge, its morphology has not been well studied, although all life stages of the mite have been simply described (Wang 1981; Lu 1990).
Morphological characters, such as the peritreme, aedeagus, tarsal claws and empodium, have often been used in identifying the species of Tetranychidae, and resolving the phylogenetic relationships between species (Gutierrez & Helle 1985; Lindquist 1985). Jeppson et al. (1975), Mollet & Sevacherian (1984) and Hong et al. (1994) studied the morphological characters and the density of the integumentary lobes and considered that the integumentary lobes were involved with thermal regulation. Some scholars have described the morphological characteristics of the mouthparts, setae and pedipalps, and inferred their functions from the morphology (Bostanian & Morrison 1973; Hislop & Jeppson 1976; Razaq et al. 2000).
The purpose of this study is to describe the morphological characteristics of all life stages of T. turkestani using light and scanning electron microscope, add ultrastructural details to the characteristics of T. turkestani.
Materials and Methods
The stock colony of T. turkestani was initiated from individuals collected from a cotton field near Huayuan, Shihezi City, Xinjiang Uygur Autonomous Region in 2010. This colony was maintained on potted sword bean (Semen canavaliae Gladiatae) in a growth chamber (FLI-2000H) at 26 ± 1 °C, 60% RH, and a 16:8 h (L:D) photoperiod.
Mite eggs of different ages, 24 hr, 48 hr and 72 hr were photographed and measured using a stereo microscope (Zeiss Discovery V20). The post embryonic stages including larva, protonymph, deutonymph, and adult (female and male) were slide mounted, photographed and measured under a compound microscope (Olympus BX53).
Scanning electron microscope
The samples were fixed overnight in fixative (2.5% glutaraldehyde and 10% paraformaldehyde in 0.2 M phosphate buffer pH 7.2) at 4 °C. The fixed samples were rinsed three times with the same buffer, and dehydrated in a graded series of acetone. Samples were then dried under natural conditions for 12 h. The dried samples were mounted on specimen stubs, coated with gold using ion sputter (E-1045), and observed under a scanning electron microscope (LEO 1430VP) at 20 kV and photographed.
Morphological measurements and statistical analyses
Body length and width, and idiosomal setal length were measured under the light microscope (LM) for 10 specimens, respectively. The data are presented as mean ± s.e. and analysed using SPSS 17.0 (SPSS, Chicago, IL).
Both sexes of T. turkestani have five stages, including egg, larva, protonymph, deutonymph and adult (Figs. 1A–1F).
Adult female (n=10)
Chelicera consist of a pair of whip-like stylet (St) (Fig. 2C) and a cystiform stylophore (Sp) (Figs. 2D, 3A). Palpus (Pa) (Fig. 3A) has 5 segments: trochanter (Tr), femur (Fe), genu (Ge), tibia (Ti) and tarsus (Ta) (Fig. 3B). Palpfemoral seta (d) is setiform (Fig. 3A), palptibial claw (CI) is well developed (Fig. 3D), palptarsus has striated cuticle and comprises seven smooth projections (suζ, ω, ul′ζ ul″ζ, a, b and c) on its tip (Fig. 3D). Eupathidial spinneret (suζ) is cylindrical and its length is approximately two times as long as its width. Solenidion (ω) is peg-shaped. Eupathidia (ul′ζ and ul″ζ are similar in form, elongate with a blunt apex. Three hair-like setae (a, b, c) are attenuate. Subcapitulum (Sub) bears a pair of subcapitular setae (m) (Fig. 3C). The apex of rostrum (Ro) is a large opening surrounded by soft flaps (Fig. 3C). Peritremes (Pe) are embedded in part of the membranous cuticle of stylophore. The last few segments of the peritreme are hooked (Fig. 2D).
Dorsum: Fourteen pairs of dorsal setae (v2, sc1 sc2, c1, c2, c3, d1, d2, e1 e2, f1, f2, h2 and h3) present (Figs. 2A, 3E). Eyes (Eye) on either side are consistently present between setae sc1 and sc2 surrounded by striated integument (Fig. 3A). The striae between the setae v2 and sc1 is semiorbicalar, and the integumentary lobes are semi-oblong (Fig. 4A). The striae between the setae c1, d1 and e1 is transverse, and the integumentary lobes are semi-oblong or triangular (Figs. 4B, 4C). The striae between the seta e1 is longitudinal and slanting, the integumentary lobes are semi-oblong (Fig. 4D). There is a diamond-shaped area between the setae e1 and f1, the integumentary lobes are semi-oblong or triangular (Fig. 4E). There are three lyrifissure (Ly) on the outside of the setae c2, d2 and e2, and surrounded by semi-oblong integumentary lobes (Fig. 4F).
Venter: The chaetotaxy includes six pairs of coxisternal setae (1b, 1c, 2b, 2c, 3b and 4b) (Fig. 2B), three pairs of intercoxal setae (1a, 3a and 4a) (Fig. 2B), one pair of aggenital setae (ag) (Figs. 2B, 3E), two pairs of genital setae (g1 and g2) and two pairs of pseudanal setae (ps1, and ps2) (Fig. 3E). The genital opening (GO) and anal opening (AO) are on the ventral side of the opisthosoma (Fig. 3E). The genital opening is transverse and surrounded by characteristically wrinkled membranous cuticle, and the Semi-circular shaped genital flap (GF) is in front of it (Fig. 3E). The striae of the genital flap is transverse, without integumentary lobes (Fig. 3F). The striae in the upper area of the genital flap is longitudinal and have a small number of granules (Fig. 3F).
Legs: Each leg includes coxa (Cx), trochanter (Tr), femur (Fe), genu (Ge), tibia (Ti), tarsus (Ta) and pretarsus (Pr) (Fig. 5A). There are nine tactile setae and one solenidion on tibia I (Fig. 5B). Two pairs of duplex setae (ω′, ft′ and ω″, ft″) are found on the dorsum of tarsus I (Fig. 5C). The longer seta (ω′ and ω″) is elongate and tapered, and the shorter seta (ft′ and ft″) is barbed and apically forked. Tarsus I also has other kinds of setae, such as tectal setae (tc′), prorals (p′ζ) and primiventrals (pv) (Fig. 5D). The barbed tectal setae have well-developed sockets, while prorals and primiventrals are smooth. The empodium (Em) has three pairs of minute, attenuate hair-like processes (Fig. 5D). The claws of tarsus I are strongly reduced to two pairs of tenent hairs (TH) (Fig. 5D).
Adult male (n=10)
Chelicera: The shape of the stylophore (Sp) and stylet (St) is similar to that of adult female (Figs. 6C, 7A). Palpus (Pa) (Fig. 7A) also has 5 segments (Fig. 7B). The palpfemoral seta (d) is a short, stout, spine-like process (Fig. 7B). The palptarsus also has seven smooth-surfaced projections (suζ, ω, ul′ζ, ul′, a, b and c) (Fig. 7C). Eupathidial spinneret (suζ) is obviously shortened and smaller than that in female. Subcapitulum (Sub), rostrum (Ro) and peritreme (Pe) are similar to those of adult female (Figs. 6C, 7C). The rostral gutter (RG) is located on the dorsal surface of the rostrum (Fig. 7B).
Venter: The chaetotaxy includes six pairs of coxisternal setae (1b, 1c, 2b, 2c, 3b and 4b), three pairs of intercoxal setae (1a, 3a and 4a), one pair of aggenital setae (ag) (Fig. 6B), two pairs of genital setae (g1 and g2) and two pairs of pseudanal setae (ps1 and ps2) (Fig. 7D).
Aedeagus: The shaft (Sh) of aedeagus is bent back to form a large terminal knob (TK) with an anterior projection that is blunt and a posterior projection that is acute. There is an obvious angle on the posterior projection near the 1/3 region (Fig. 6D).
Legs also have 7 segments. There are nine tactile setae and four solenidion on tibia I (Fig. 8B). The shape and situation of the duplex setae (ω′, ft′ and ω″, ft″) are similar to those of adult female (Figs. 8A, 8C). The primilaterals (pl′, pl″) are located laterally and apically on tarsus I (Fig. 8D). The primiventrals (pv) is located ventrally (Fig. 8D). Empodium I (Em) consists of a claw-like structure with a strong mediodorsal spur about one half of the appendage (Fig. 8D). The claws of tarsus I are reduced to two pairs of tenent hairs (TH) (Fig. 8D).
It is smooth and spherical in shape with a diameter of about 129.55 ± 1.86 µm (Figs. 1A, 9D). The freshly laid eggs are colorless and transparent (Fig. 9A) and then gradually become canary yellow to dark yellow within one or two days (Figs. 9B, 9C). Eye spots appear redon the third day (Fig. 9C).
Palptarsus has seven smoothly surfaced projections (suζ, ω, ul′ζ, ul″ζ, a, b and c) (Fig. 10C). Eupathidial spinneret (suζ) is a slender cylinder structure, and its length is about three times its width. The shapes of the solenidion (ω), two eupathidia (ul′ζ and ul″ζ) and three setae (a, b and c) are similar to those of adult female (Fig. 10C).
Dorsum: Larvae have fourteen pairs of dorsal setae (v2, sc1, sc2, c1, c2, c3, d1, d2, e1, e2, f1, f2, h2 and h3) (Figs. 10A, 10D).
Venter: The chaetotaxy includes one pair of coxisternal setae (1b) (Fig. 10B), two pairs of intercoxal setae (1a and 3a) (Fig. 10B) and two pairs of pseudanal setae (ps1 and ps2) (Fig. 10D). Legs: Leg chaetotaxy as follows: I-1-0-3-4-6-7 ± 1 duplex; II- 0-0-3-4-5-7 ± 1 duplex; III-0-0-2-2-5-6 (Figs. 11A–11C).
Palptarsus has seven smooth projections (suζ, ω, ul′, ul″, a, b and c) which are similar to those of larvae (Fig. 12C).
Dorsum: Protonymph has fourteen pairs of dorsal setae (v2, sc1, sc2, c1, c2, c3, d1, d2, e1, e2, f1, f2, h2 and h3) (Figs. 12A, 12D).
Legs: Leg chaetotaxy as follows: I-2-0-3-4-6-9 + 2 duplexes; II- 1-0-3-4-5-9 + 1 duplex; III-1-0-2-2-5-8; IV-0-0-2-2-5-6 (Figs. 13A–13D).
Palptarsus has seven smooth projections (suζ, Omega;, ul′ζ, ul″ζ, a, b and c) which are similar to those in adult female (Fig. 14C).
Dorsum bears fourteen pairs of dorsal setae (v2, sc1, sc2, c1, c2, c3, d1, d2, e1, e2, f1, f2, h2 and h3) (Figs. 14A, 14D).
Venter: In addition to those setae in protonymph, two pairs of coxisternal setae (2c and 4b) (Fig. 14B), one pair of intercoxal setae (4a) (Fig. 14B) and one pair of genital setae (g1) (Fig. 14D) are added. Genital folds begin to form.
Legs: Leg chaetotaxy as follows: I-2-1-5-5-10-13 + 2 duplexes; II-2-1-4-4-6-10 + 1 duplex; III-1-1-2-3-5-9; IV-1-0-2-3-5-8 (Figs. 15A–15D).
Comparison of the lengths of dorsal setae of different development stages
In female the lengths of the dorsal setae increase significantly as the mites grow from larva to adult (Table 1). Seta sc1 is the longest at the adult stage and shortest at the larval stage. All dorsal setae of adult female are significantly longer than those of male. Lengths of v2, sc2, c2, d1, d2 and e2 don't change significantly in deutonymph and adult male. The length of h3 shows no significant differences at the stages of larva, protonymph and adult male.
The length of dorsal setae of T. turkestani at different development stages (n=10).
The shapes of the stylophore, stylets, palpi and rostral gutter of T. turkestani are similar to other spider mites, such as T. atlanticus, T. urticae, Oligonychus punicae and Panonychus citri (Baker & Connell 1963; Bostanian & Morrison 1973; Hislop & Jeppson 1976; Razaq et al. 2000). The stylophore and rostral gutter have the function of protecting the stylets, and the rostral gutter serves as a vessel for stylets to protract and retract (Razaq et al. 2000). In comparison to Eriophyoidea which has short stylets and are capable of very shallow penetration into a plant (Jeppson et al. 1975), Tetranychoidea possesses long stylets which could easily penetrate plant tissues, so it may kill the plant cells (Evans et al. 1961).
The pattern of the striae between setae e1 and f1 may be of diagnostic value in adult females in Tetranychidae (Lindquist 1985). The ultrastructure of the striae and integumentary lobes are also of diagnostic importance in distinguishing diapausing and non-diapausing populations of the same species or between closely related species (Boudreaux & Dosse 1963; Dosse & Boudreaux 1963; Jeppson et al. 1975). The morphological characters of the striae and integumentary lobes of T. urticae have been reported (Brandenburg & Kennedy 1981; Mollet & Sevacherian 1984). There is also a diamond-shaped area between the setae e1 and f1 in T. urticae, and the shape of integumentary lobe is different in different regions (Carbonnelle & Hance 2004). The shape of integumentary lobes of T. turkestani is similar to that of feeding green females of T. urticae collected in Lattes, France (Carbonnelle & Hance 2004).
Spider mites have the habit of spinning webs (Saito 1977, 1983). Adult female of T. turkestani covers its eggs and lavae with webs to protect them from predators and other external factors. The eupathidial spinneret is more developed in adult female than in male. The shape of the eupathidial spinneret of T. turkestani adults is similar to that of T. urticae and T. truncatus (Bostanian & Morrison 1973; Sakunwarin et al. 2004), but different from that of T. bunda and T. musae (Flechtmann & Knihinicki 2002; Auger et al. 2008). It is much slender, about three times as long as wide in T. bunda (Flechtmann & Knihinicki 2002) and as long as wide in T. musae (Auger et al. 2008).
Many sensory receptors are found on the tip of the mouthparts and legs of mites (Alberti & Coons 1999; Walter & Proctor 1999). Some scholars have studied the sensilla of T. urticae and T. truncatus, and they concluded that the solenidion and two eupathidia on palptarsus are chemoreceptors, the three setae are mechanosensitive sensilla, and the solenidia (ω′ and ω″) of duplex setae are chemosensitive sensilla (Bostanian & Morrison 1973; Sakunwarin et al. 2004). The six sensilla on the palptarsus of T. turkestani adults are similar in shape and position to those of T. urticae and T. truncatus.
Most of the articles mainly describe the leg chaetotaxy of female and male mites (Stone 1986; Ehara & Gotoh 1992; Ehara 1995; Flechtmann & Knihinicki 2002), and we studied the leg chaetotaxy of all stages. There is a great change in the leg chaetotaxy of tibia and tarsus from larva to deutonymph, and the chaetotaxy of leg III of larva is consistent with the chaetotaxy of leg IV of protonymph. The shapes of the tarsal claw and empodium in Tetranychidae are important taxonomic characters (Wang 1981). The shape of the empodium I of T. turkestani is different from that of T. musae (Auger et al. 2008). The empodium I of female adult T. turkestani have three pairs of minute, attenuate hair-like processes, while that of T. musae bear three pairs of proximoventral hairs and a large mediodorsal spurs (Auger et al. 2008). The empodium I of male adult T. turkestani has a similar shape as the empodium II of that of T. lintearius, which consists of three pairs of distally fused proximoventral spurs and possesses a strong mediodorsal spur (Stone 1986). However, the empodium I of male T. musae adults consists of a double claw-like structure with a strong mediodorsal spur (Auger et al. 2008). To summarize, this paper increases understanding of the external morphological characteristics of T. turkestani.
We thank Dr. John Richard Schrock from Emporia State University, USA for his generous help with revising the manuscript. We also thank Professor Jeff Geer from College of Agriculture, Shihezi University, Xinjiang for reading the manuscript. We are grateful to Professor Zhaotian Fan from Xinjiang University who helped us take the SEM photos. We appreciate Professor Jianzhen Lin from Institute of Plant Protection, Fujian Academy of Agricultural Sciences and Dr Qing-Hai Fan from Plant Health & Environment Laboratory, Ministry for Primary Industries, New Zealand for sending some literatures to us. This research was supported by Public Welfare Industry (Agriculture) Research Project (No. 201103020).