Introduction
Thrips (Insecta: Thysanoptera) are among the most destructive piercing–sucking pests in agricultural systems worldwide. In addition to direct feeding damage, many thrips species are efficient vectors of tospoviruses, notably Tomato spotted wilt virus, which further amplifies their economic impact. Consequently, thrips have become a major constraint to vegetable and legume production globally. More than 100 thrips species have been reported from cucurbit and leguminous crops, with pest assemblages varying markedly among regions as a result of climatic conditions, cropping systems, and host plant availability (Mound Reference Mound2005; Reitz et al. Reference Reitz, Gao and Lei2011). Several species have attained global pest status, including Frankliniella occidentalis Pergande, Scirtothrips dorsalis Hood, Thrips palmi Karny, and Thrips tabaci Lindeman, which cause serious damage to vegetable production in both China and the United States (Reitz et al. Reference Reitz, Gao and Lei2011).
In China, the diversity and pest status of thrips on vegetable crops have increased markedly over the past two decades. More than 40 species have been recorded from cucurbit and legume systems, of which 8–10 cause regular economic losses. Megalurothrips usitatus Bagnall (Thripidae), the bean flower thrips, is one of the most destructive thrips species attacking leguminous crops in China and is listed as a Category I agricultural quarantine pest (Huang et al. Reference Huang, Zhang, Wang, Yuan, Wang, Sun and Lyu2025a; Shi et al. Reference Shi, Xie, Wu, Zou, Wu and Zhang2024). This species is widely distributed and highly polyphagous, feeding on at least 50 host plants in 12 families, including 32 species of Fabaceae (Huang et al. Reference Huang, Xu, Kong, Shi, Jiao, Zhang, Wu and Xie2025b). Both larvae and adults feed primarily within flowers using rasping–sucking mouthparts, causing floral deformation, reduced pod set, and characteristic symptoms such as blackened pod tips in cowpea (Vigna unguiculata) (Shi et al. Reference Shi, Xie, Wu, Zou, Wu and Zhang2024). Severe infestations can result in substantial yield and quality losses. Due to its small size, concealed feeding habits, short generation time, and high fecundity, M. usitatus is difficult to control, and excessive insecticide use has led to resistance development, increased pesticide residues, and environmental risks (Shi et al. Reference Shi, Xie, Wu, Zou, Wu and Zhang2024). Recent surveys indicate that this species has become increasingly dominant in southern China, particularly in Hainan province (Huang et al. Reference Huang, Zhang, Wang, Yuan, Wang, Sun and Lyu2025a, Reference Huang, Zhang, Wang, Yuan, Wang, Sun and Lyub).
These challenges have stimulated interest in natural enemies of thrips, including entomoparasitic nematodes. Species of the genus Thripinema Siddiqi, Reference Siddiqi1986 are obligate parasites of thrips and represent a highly specialized lineage within the family Allantonematidae (Lim and Driesche Reference Lim and Driesche2005; Sims et al. Reference Sims, Becnel and Funderburk2012). Members of this family are characterized by extreme morphological adaptations to parasitism, including obese parasitic females with an atrophied pharynx and reduced vulva, and males with a short or degenerate stylet (Siddiqi Reference Siddiqi2000). Within this group, Thripinema is distinctive in having small, oval parasitic females (approximately 0.15–0.23 mm long), sometimes with a terminal tail peg, and an ovary with multiple flexures occupying most of the body cavity (Siddiqi Reference Siddiqi2000).
To date, only five valid species of Thripinema have been described, i.e. T. aptini (Sharga Reference Sharga1932) Siddiqi, Reference Siddiqi1986, T. nicklewoodi (Nickle and Wood, Reference Nickle and Wood1964) Siddiqi, Reference Siddiqi1986 , T. reniraoi (Reddy, Nickle, and Rao Reference Reddy, Nickle and Rao1982), Siddiqi, Reference Siddiqi1986, T. khrustalevi Chizhov, Subbotin and Zakharenkova, Reference Chizhov, Subbotin and Zakharenkova1995, and T. fuscum Tipping, Nguyen, Funderburk and Smart, Reference Tipping, Nguyen, Funderburk and Smart1998. These species have been reported from a limited number of thrips hosts belonging to eight genera, including Frankliniella, Heliothrips, and Megalurothrips, and from several continents or countries (Lim and Driesche Reference Lim and Driesche2005).
Biologically, Thripinema species exhibit life cycles closely synchronized with their thrips hosts. Infective females penetrate the host cuticle and develop within the haemocoel, where they reproduce rapidly (Mason and Heinz Reference Mason and Heinz2002; Sims et al. Reference Sims, Becnel and Funderburk2012). Although parasitized thrips usually remain alive, their feeding activity, fecundity, and vector competence are substantially reduced (Arthurs and Heinz Reference Arthurs and Heinz2003; Sims et al. Reference Sims, Funderburk and Boucias2005, Reference Sims, Funderburk, Reitz and Boucias2009). Despite their ecological and applied significance, the taxonomy and diversity of Thripinema remain poorly understood. Host specialization, geographic isolation, and morphological differentiation suggest that additional undescribed species are likely to exist.
M. usitatus is a persistent pest in major cowpea production areas in southern China, especially in Hainan province in China. Its infestation has led to a serious reduction in cowpea yields and quality, and has also become a key biotic factor constraining the sustainable development of the industry. Occasionally, nematodes were observed coming out from thrips cultured in lab. Because no nematodes have been described previously extracted from these small insects in China, so natural thrips from cowpea flowers were collected from Sanya city, Hainan province, and nematodes were extracted from these thrips. Morphological characters supported by molecular sequences analysis based on conserved regions of ribosomal RNA and mitochondrial DNA were used to characterize the species, and the results showed that the species was an unknown species, here identified as Thripinema usitatum n. sp.
Materials and methods
Nematode extraction and fixative
All life stages of nematodes were dissected from the body cavity (haemocoel) of thrips (Megalurothrips usitatus) collected from cowpea (Vigna unguiculata) flowers in Sanya City, Hainan province, China. Nematodes were heat-killed in warm water (40°C) and fixed in FG fixative (formalin:glycerol:distilled water = 10:1:89). Fixed specimens were processed to anhydrous glycerine following standard procedures and mounted on permanent glass slides for morphological examination (Xie Reference Xie2005).
Morphological characterization
Observations, measurements, and photomicrographs were obtained using a ZEISS Scope A1 light microscope equipped with a digital imaging system. Morphometric data were collected following standard taxonomic practice for entomoparasitic nematodes, de Man formula were referenced in this paper (Siddiqi Reference Siddiqi2000).
For scanning electron microscopy (SEM), nematodes were prefixed in 2% glutaraldehyde buffered with phosphate-buffered saline (PBS) and post-fixed in 0.5% osmium tetroxide. Specimens were dehydrated through a graded ethanol series (10–100%), critical-point dried using CO₂, mounted on aluminium stubs, sputter-coated with gold–palladium, and examined at 10 kV using an FEI XL-30-ESEM scanning electron microscope at the Testing Center of South China Agricultural University (Chen et al. Reference Chen, Lu, Xie and Xu2024).
Molecular identification
Genomic DNA was extracted from single infective females using a proteinase K digestion protocol (Chen et al. Reference Chen, Lu, Xie and Xu2024). The D2–D3 expansion region of the 28S rRNA gene was amplified using primers D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al. Reference De Ley, Felix, Frisse, Nadler, Sternberg and Thomas1999). Partial 18S rRNA sequences were amplified using primer pairs 988F/1912R and 1813F/2646R (Holterman et al. Reference Holterman, Wurff, Elsen, Megen, Bongers, Holobachov, Bakker and Helder2006). The mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was amplified using primers JB3 and JB5 (Bowles et al. Reference Bowles, Blair and McManus1992).
PCR amplifications were performed in 25 μL reaction volumes containing 10 μL DNA template, 0.5 μL of each primer (10 μM), 12.5 μL 2× Rapid Taq Master Mix (with dye) (Vazyme, Beijing, China), and 1.5 μL sterile distilled water. Thermal cycling conditions consisted of an initial denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, annealing at 55°C for 28S or 53.5°C for 18S rRNA and mtCOI for 1 min, and extension at 72°C for 1 min; followed by a final extension at 72°C for 8 min.
PCR products were purified using a Gel Extraction Kit (CWBIO, Jiangsu, China), cloned into the pMD18-T vector (Takara, Japan), and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Newly obtained sequences were compared with those in GenBank using BLASTn and deposited under accession numbers provided in the phylogenetic analyses.
Phylogenetic analysis
Sequences of entomoparasitic nematodes and closely related taxa were retrieved from GenBank based on BLAST similarity. Phylogenetic analyses were conducted using Bayesian inference (BI) implemented in MrBayes v.3.2.1 based on partial 18S rRNA, 28S D2–D3 rRNA, and mtCOI sequences (Chen et al. Reference Chen, Lu, Xie and Xu2024).
For the 28S rRNA dataset, 19 sequences representing nine genera were analyzed, with Aglenchus agricola (de Man, 1884) Andrássy, 1954 (AY780979) and Basiria gracilis (Thorne, 1949) Siddiqi, 1963 (DQ328717) designated as outgroups. For mtCOI analysis, 19 sequences from four genera were included, using Heterodera schachtii Schmidt, 1871 (MK134702) and Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959 (MK798162) as outgroups. The 18S rRNA dataset comprised 31 sequences from six genera, with Cephalobus oryzae Karimova, 1957 (AF034390) and Acrobeloides bodenheimeri (Steiner, 1936) Thorne, 1937 (AF202159) as outgroups. Analyses were run under default settings.
Results
Morphological description
Thripinema usitatum n. sp. (Figs 1– 6).
Line drawings of Thripinema usitatum n. sp. Infective female: A. Entire body; D. Head region; G. Posterior part; P. tail; T. Vulval region and tail. Free living male: B–C. Entire body; E–F. Head region; Q–S. Tails. Parasitic female: H–N. Eggs: O. Scale bar: D–F, P–T = 20 μm; A–C, G, O = 50 μm; H–N = 100 μm.
Figure 1. Long description
There are twenty panels labeled A to T, arranged in rows from top left to bottom right. Panels A, B, and C show entire body outlines of infective female and free-living male. D, E, and F display head regions in detail. G shows the posterior part of the infective female. H to N depict parasitic female forms, each with internal structures and varying levels of granulation. O presents four eggs, each with visible internal content. L, M, and N show parasitic females with different internal patterns. P shows the tail of the infective female. Q, R, and S illustrate tails of free-living males, each with distinct internal lines. T shows the vulval region and tail. Scale bars are present: D to F, P to T equal 20 micrometers; A to C, G, O equal 50 micrometers; H to N equal 100 micrometers.
Photomicroscopy of infective female Thripinema usitatum n. sp. A–B. Head region; C–D. Anterior part; E. Posterior part of body with entire genital gland; F. Entire body; G. Tail; H–I. Posterior part of body. Scale bar: A–E, G–I = 20 μm; F = 50 μm.
Figure 2. Long description
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Photomicroscopy of free-living male Thripinema usitatum n. sp. A. Entire body; B–C. Anterior part; D, G–K. Tails in lateral view; E–G. Tails in ventral view; F. Posterior part of body with entire genital gland. Scale bar: B–E, G–K = 20 μm; A, F = 50 μm.
Figure 3. Long description
There are eleven panels labeled A to K. Panel A, top left, shows the entire curved body of the nematode in lateral view with a 50 micrometer scale bar. Panels B and C, right of A, display the anterior region in higher magnification, each with a 20 micrometer scale bar. Panel D, right of C, shows a lateral close-up of the tail. Panel F, far right, presents the posterior body with the genital gland visible, scale bar 50 micrometers. Panel E, inset on F, highlights the ventral tail. Panels G, H, I, J, and K, bottom row, show tails in lateral and ventral views, each with a 20 micrometer scale bar. Fine structural details such as cuticle texture, internal organs, and genital structures are visible in each region. Scale bars are consistently placed adjacent to each anatomical region for reference.
Photomicroscopy of parasitic female Thripinema usitatum n. sp. Scale bar: A–I, K–L = 50 μm; J = 100 μm.
Figure 4. Long description
Panel A at the top-left shows an elongated, oval-shaped nematode with a faint internal structure, scale bar 50 micrometers. Panel B displays a similar shape with more pronounced internal folds. Panel C presents a granular texture throughout the body. Panel D reveals internal convolutions. Panels E through I, in the second row, show close-ups of the anterior region, each with varying internal densities and granularities, all with scale bars of 50 micrometers. Panel J, at the bottom left, contains two nematodes of different sizes, scale bar 100 micrometers. Panel K, center bottom, shows a cluster of multiple nematodes, scale bar 50 micrometers. Panel L, bottom right, is a close-up of the anterior region with visible internal structures, scale bar 50 micrometers. The arrangement highlights morphological and internal structural variation among individuals and developmental stages.
SEM photos of infective female Thripinema usitatum n. sp. A. Lip region in overview; B–D, F. Lip region in lateral view, d. the arrow showed amphids; E. Tail terminal; G. Lateral field in middle body; H–I. Tails, arrow showed anus. Scale bar: A–G = 2 μm; H–I = 10 μm.
Figure 5. Long description
There are eight S E M photos labeled A to I, arranged in two rows. Panel A shows an overview of the lip region with a central protrusion. Panels B, C, D, and F display the lip region in lateral view, with D featuring an arrow pointing to the amphid. Panel E shows the tail terminal. Panel G presents the lateral field at mid-body. Panels H and I show tails, with an arrow in I indicating the anus. Scale bars for A to G represent 2 micrometers, while H and I represent 10 micrometers. Surface textures, annulations, and anatomical openings are visible in each respective panel.
SEM photos of free-living male and parasitic female Thripinema usitatum n. sp. Free-living male: A–B. Lip region; C. Tail in lateral side; D. Tail in ventral side. Parasitic female: E. Entire body; F. Lip region. Scale bar: A–D = 4 μm; E–F = 20 μm.
Figure 6. Long description
Panel A shows a close-up of the lip region of the free-living male, with the anterior end oriented toward the viewer. Panel B presents another view of the male lip region, highlighting surface texture. Panel C displays the lateral side of the male tail, showing its elongated structure. Panel D provides a ventral view of the male tail, with the cloacal opening visible. Panel E depicts the entire body of the parasitic female, oriented laterally, with surface debris present. Panel F focuses on the lip region of the parasitic female, showing surface details. Scale bars are present in each panel, with A through D equal to 4 micrometers and E through F equal to 20 micrometers.
Zoobank registration
urn:lsid:zoobank.org:pub:5C62AB13-945D-4116-97BA-4F845698A341.
Measurements
Holotype infective female: L = 283.2 μm; W = 11.3 μm; Stylet length = 11.3 μm; Tail length = 17.6 μm; Body width at anus (ABW) = 7.0 μm; Body width at vulva (VBW) = 9.6 μm; Distance from vulva to anus (VA) = 25.8 μm; a = 25.1; b = 1.6; c = 16.1; c′ = 2.5;V = 84.6; G = 28.1.
Paratype infective females (n = 29): L = 283.7 ± 17.9 (241.8–314.7) μm; W = 10.6 ± 0.6 (9.5–11.6) μm; Stylet length = 10.6 ± 0.8 (9.3–12.3) μm; Tail length = 20.4 ± 3.2 (15.5–28.3) μm; ABW = 7.5 ± 0.5 (6.3–8.1) μm; VBW = 9.1 ± 0.4 (8.5–9.8); a = 26.9 ± 2.1 (21.7–30.2); b = 1.6 ± 0.1 (1.4–1.8); c = 14.0 ± 2.3 (9.9–17.8); c′ = 2.7 ± 0.3 (2.2–3.5); V = 85.6 ± 2.0 (81.5–88.5); G = 25.4 ± 1.7 (22.9–28.5).
Paratype free-living males (n = 20): L = 281.0 ± 28.4 (247.0–384.5) μm; W = 13.0 ± 0.9 (11.3–14.7) μm; Spicule length = 12.5 ± 1.6 (10.0–15.2) μm; Tail length = 21.8 ± 1.6 (18.7–24.7) μm; ABW = 7.8 ± 0.4 (7–8.4) μm; Gubernaculum length = 4.8 ± 1.0 (3.1–6.3) μm; a = 21.6 ± 2.2 (19.0–27.5); c = 2.8 ± 0.3 (2.3–3.2).
Paratype parasitic females (n = 16): L = 232 ± 34.7 (174.8–276.74) μm; W = 76.7 ± 17.7 (52.9–108.4) μm; a = 3.1 ± 0.6 (2.4–4.6) μm.
Eggs (n = 30): L = 51.4 ± 4.0 (44.6–62.4) μm; W = 26.2 ± 4.0 (21.2–37.4) μm.
Description
Parasitic females. Parasitic females at different developmental stages were observed within thrips, ranging from slightly swollen young females to fully swollen, mature females with an oval body shape (Figs 1H–N, 3, and 6E). The body of mature parasitic females is broadly oval (Figs 1L and 4A–D). The lip region is depressed (Fig 4B), with cleft labial lobes (Fig 6F). The stylet and pharynx are indistinct or invisible in mature females but may occasionally be observed in young parasitic individuals (Fig 4L). The vulva is subterminal, with part of the uterus protruding outside the vulval opening (Figs 1H–K, M–N; 4F, J). The genital tube is spirally curved and occupies most of the body cavity in mature parasitic females (Fig 4B–D). The tail is rounded; however, a small terminal protrusion is present in young parasitic females (Fig 1M–N).
Infective female. The body is straight or slightly ventrally curved. The lateral field bears two incisures (Fig 5G). The lip region is continuous with the body contour (Figs 1D, 2A–D, and 5B–D, F). Amphids are inverted Y-shaped (Fig 5D). The stylet is weakly developed, with slight thickening in the mid-region (Figs 1D and 2A-B). The orifice of the dorsal oesophageal gland is not visible. The excretory pore is indistinct. The pharynx is long, with well-developed pharyngeal glands extending close to, and sometimes overlapping, the ovary. The vulva is located near the anus; both vulva and anus are difficult to observe after mounting on slides (Fig 2E, H–I). The spermatheca is small and rounded, containing a few small sperm. The post-vulval uterine sac is short or degenerated (Fig 2E, H–I). The tail is conoid with a blunt terminus.
Free-living males. The body is usually loosely L- or C-shaped, occasionally with the tail end dorsally curved. The head is high and swollen, distinctly wider than the adjoining body region. The lip region is depressed, with cleft labial lobes. The stylet and pharynx are degenerated. The testis is outstretched, extending into the pharyngeal region; the vas deferens contains individual sperm. Spicules are slender, straight, and slightly arcuate distally, resembling a chicken toenail at the posterior end on the ventral side, with swollen edges at the spicule base. Gubernaculum weakly developed, straight or slightly curved, with posterior end reflexed posteriorly a little, occasionally resembling a match head. The bursa extends nearly to the tail tip and is divided into two connected parts: a large, wide bursa with crenate margins surrounding the cloacal region, and a smaller, narrow bursa extending posteriorly. The tail is conoid to clavate, tapering distinctly to approximately one-third of its length and then narrowing further into a clavate terminal portion.
Eggs: elliptical.
Molecular description
Using conserved primers, partial rRNA and mtDNA sequences were successfully amplified from Thripinema usitatum n. sp., yielding two D2–D3 expansion region sequences of 28S rRNA, three identical mitochondrial cytochrome c oxidase subunit I (mtCOI) sequences, and one partial 18S rRNA sequence. BLASTn searches against the GenBank database revealed that no sequences of Thripinema species have been deposited to date. Nevertheless, all obtained sequences showed limited similarity to other entomoparasitic or entomophilic nematodes. The two 28S D2–D3 sequences differed from each other by a two-base insertion/deletion. BLAST analysis of these sequences yielded no closely related matches; the highest-scoring hit showed only 43% coverage and 88.95% identity to an unidentified Fergusobia species (FJ386990) associated with Melaleuca nodosa. The three mtCOI sequences obtained were identical and showed the highest BLAST match to a Strongylides sp. (LC534895) isolated from carnivore hosts, with 100% coverage and 86.59% sequence identity, indicating a relatively distant relationship. For the partial 18S rRNA sequence, T. usitatum n. sp. exhibited the highest similarity to Delatylus andersoni (KY907662), with 100% query coverage and 95.98% sequence identity. Similar levels of identity were observed with two additional Delatylus species, D. siricidicola (FJ004889; 95.78%) and D. posteroporus (KY098774; 95.57%).
In the Bayesian inference tree inferred from the 28S D2–D3 rRNA sequences, Thripinema usitatum n. sp. formed a well-supported monophyletic clade (posterior probability = 100) (Fig 7). This clade was embedded within a larger group composed predominantly of insect-parasitic nematodes of the genus Fergusobia, but was clearly separated from all described Fergusobia species by long branch lengths and high nodal support. Thripinema did not cluster with representatives of Howardula or Deladenus, which formed distinct lineages corresponding to their respective subfamilial affiliations. In the Bayesian inference tree inferred from mtCOI sequences, Thripinema usitatum n. sp. formed a highly supported monophyletic clade (posterior probability = 100) (Fig 8). This clade was clearly separated from other entomophilic nematodes, including Ektaphelenchoides spp., indicating a distinct mitochondrial lineage. The separation of insect-parasitic, animal-parasitic, and plant-parasitic nematodes was well resolved in the mtCOI phylogeny. In the Bayesian inference tree based on partial 18S rRNA sequences, Thripinema usitatum n. sp. clustered within the Allantonematinae, forming a lineage together with species of Howardula (Fig 9). However, the relationships among these genera were weakly supported, indicating limited resolution of 18S rRNA at the generic level. Nevertheless, Thripinema was clearly separated from members of Fergusobinae and Neotylenchinae with strong posterior probabilities.
Phylogenetic relationships of Thripinema usitatum n. sp within the species of entomoparasitic nematodes. The phylogenetic tree was constructed by Bayesian analysis using 28S rRNA D2-D3 expansion region sequences, with the optimal model GTR+ I + G under AIC standard. Posterior probability values exceeding 50% are given on appropriate clades.
Figure 7. Long description
The rooted phylogenetic tree starts at the bottom left with Basiria gracilis D Q 3 2 8 7 1 7 labeled as Boleodorinae and Aglenchus agricola A Y 7 8 0 9 7 as Atylenchinae. Moving upward, the next major branch splits into Deladenus tonekabonensis O R 2 3 8 9 2 5 and a clade containing Deladenus sp. noc14x J X 1 0 4 3 1 5, Deladenus sp. iuv172 J X 1 0 4 3 2 6, and Deladenus sp. R O H 0 1 1 L C 8 0 1 2 3 4, all labeled as Neotylenchinae. The next branch contains Howardula aoronymphium A Y 5 8 9 3 9 5, Rhabditida sp. F N 3 8 6 6 8 9, and Rhabditida sp. 3090ed F N 3 8 6 6 9 6. Above this, two sequences labeled Thripinema usitatum n. sp. P V 1 1 3 4 3 9 and Thripinema usitatum n. sp. P V 9 1 6 3 2 3 form a strongly supported clade (posterior probability 100) within Allantonematinae. The next major group is Fergusobiinae, containing multiple Fergusobia species and isolates, with support values at each node. The right margin annotates ‘Insect parasites’ for Fergusobiinae and Allantonematinae, and ‘Fungi feeders’ for the basal groups. The scale bar at the bottom left indicates 0.2 substitutions per site. Posterior probability values are shown at nodes, with values above 50 percent displayed. The tree structure reflects evolutionary relationships, with Thripinema usitatum n. sp. nested within Allantonematinae, distinct from Fergusobiinae and Neotylenchinae.
Phylogenetic relationships of Thripinema usitatum n. sp within the species of entomoparasitic nematodes. The phylogenetic tree was constructed by Bayesian analysis using 18S rRNA partial sequences, with the optimal model GTR+ I + G under AIC standard. Posterior probability values exceeding 50% are given on appropriate clades.
Figure 8. Long description
Starting at the bottom left root, the tree first branches to Heterodera schachtii MK134702 and Globodera rostochiensis MK798162, labeled as plant parasites. The next branch leads to Rhabditis sp. MF742407, labeled as bacteria feeders. Moving upward, Strongyloides sp. LC534895 and Strongyloides sp. LC533411 are grouped as animal parasites. The next major branch splits into two: one leads to Ektaphelenchoides sp. OM422674 and OM422671, labeled as potential insect parasites, and the other to three identical sequences of Thripinema usitatum n. sp., labeled as insect parasites. Above, a large clade contains Baylisascaris schroederi KJ587814, Ascaris suum AB591803, and four Ascaris lumbricoides sequences, all labeled as animal parasites. Further up, Toxascaris leonina OM867282, OM867283, OM867281, and Lagochilascaris minor PQ355500 are also grouped as animal parasites. At the top, Ophidascaris filaria OM867303, OM867304, OM867302, and MH285590 form the final animal parasite clade. Posterior probability values are shown at each node, with values such as 100, 99, 98, 87, 80, and 70. Host type labels are aligned to the right of each major clade.
Phylogenetic relationships of Thripinema usitatum n. sp with the species of entomoparasitic nematodes. The phylogenetic tree was constructed by Bayesian analysis using mtDNA COI gene partial sequences, with the optimal model GTR+ I + G under AIC standard. Posterior probability values exceeding 50% are given on appropriate clades.
Figure 9. Long description
The tree starts at the bottom left with Cephalobus oryzae and Acrobeloides bodenheimeri as outgroups, labeled as Cephaloboidea and Bacteria feeders. Moving upward, the tree splits into major clades. The main branch divides into Allantonematinae, Fergusobiinae, and Neotylenchinae. The Allantonematinae clade includes Howardula cf. neocosmis, Howardula neocosmis, Howardula aoronymphium, and Thripinema usitatum n. sp P V I 13499, with posterior probabilities labeled at nodes. Fergusobiinae contains several Fergusobia species, each with accession numbers. Neotylenchinae includes Deladenus siricidicola, Deladenus proximus, Deladenus posteroporus, and Tylenchomorpha, with further branching to Parasitelenchus bifurcatus and Hexatylus species. Lateral labels on the right indicate functional groups: Insect parasites, Allantonematinae, Fergusobiinae, Neotylenchinae, and Bacteria feeders. Posterior probability values are shown at nodes, with values above 50 percent. The scale bar at the bottom left represents 0.03 substitutions per site.
Type host and locality
The new species were dissected from thrips (Megalurothrips usitatus) isolated from flowers of cowpea, Vigna unguiculata (L.), in San-ya, Hainan province.
Type specimens
Holotype infective female, 12 paratype parasitic females, 26 infective females, and 19 free living males are deposited in the College of Plant Protection, South China Agriculture University, Guangzhou, Guangdong Province. Four parasitic females (Code:HN-Tu-p1) and three infective females and one free living males (Code: HN-Tu-p2) are deposited in the National Parasitic Resources Center in Shanghai, China.
Etymology
The species name ‘usitatum’ refers to its host name, Megalurothrips usitatus .
Diagnostic characters and relationships
Diagnosis: Free-living male body dorsally curved, forming a loose C shape; head high, spherical, wider than body contour, lip region retracted; stylet absent; spicule 12.5 (10.0–15.2) μm; bursa extending into tail terminal, the shape divided into two parts, one wider part around cloaca, other part narrowed extending into near tail terminal. Infective female straight, head low, not setoff with body contour, stylet base without knobs but slightly thickened, 10.6 (9.3–12.3) μm long; pharynx degenerate with obvious gland terminal, long and extending to genital ovaries or over; tail subconical with round tail terminal, anus and vulva hardly seen after fixed and mounted on slides. Parasitic female varied from bean ship to spindle shape, with a tiny tail terminal, and mostly seen genital residue around vulval region. Hyaline part visible in head of young parasitic females, stylet visible in live body, but position varied, after mounted, stylet invisible.
Relationships: The genus Thripinema currently comprises five valid species. Based on free-living male morphology, T. usitatum n. sp. is clearly distinguished from T. khrustalevi, T. fuscum, and T. aptini by the absence of a stylet. It is similar to T. nicklewoodi and T. reniraoi, which also lack a stylet, but can be differentiated. It differs from T. nicklewoodi by different parasitic female shape (spindle or bean-like without a conical head vs oval with a conical head), different tail shape of free living males (clavate tail vs non-clavate tail), and different shape of bursa (two parts vs one part). It differs from T. reniraoi by different shape and length of bursa of free living males (bursa extending to near terminus, two parts vs shorter bursa adanal, one part).
Biology
Nematodes were detected in the bodies of thrips at all life stages, including larvae, males, and females. The infection rates observed in Sanya, Haikou Province, in March 2025 were 8.3% in larvae, 15.6% in males, and 80% in females. Notably, a single female thrips could harbour multiple life stages of nematodes simultaneously, including eggs, males, infective females, parasitic females, and juveniles, alongside the thrips’ own eggs (Supplementary Figure 1). In thrips not parasitized by nematodes, the number of eggs per individual ranged from 2 to 9. In contrast, thrips parasitized by nematodes contained 0 to 8 thrips eggs and a total of 1 to 433 nematodes per individual. These nematodes included eggs (0–230), vermiform stages (0–321, including juveniles, infective females, and free-living males), and parasitic females (0–13). When all thrips were removed and flowers were dissected and either soaked or nematodes separated using the Baermann method, infective females could be successfully extracted. However, most of these infective females died within six hours after extraction.
Discussion
Nematodes parasitizing thrips have been known for more than a century, with the earliest record dating back to Uzel (1895), who observed an unnamed nematode inhabiting the haemocoel of Thrips physapus L. in Europe. Russell (1912) subsequently reported a similar nematode from Heliothrips fasciatus L. in California, although without formal description. The first taxonomic treatment of a thrips-parasitic nematode was provided by Sharga (Reference Sharga1932), who described Tylenchus aptini from Aptinothrips rufus Gmelin in England. This species was later transferred to Anguillulina by Lysaught (Reference Lysaught1936) and ultimately placed in the genus Thripinema by Siddiqi (Reference Siddiqi1986) during his revision of Howardula Cobb, 1921.
At present, the genus Thripinema comprises five valid species, namely T. aptini, T. nicklewoodi, T. reniraoi, T. khrustalevi, and T. fuscum, all of which parasitize thrips (Thysanoptera). These species have been recorded from twelve thrips species representing eight genera, including Aptinothrips, Frankliniella, Heliothrips, Megaluriothrips, and Taeniothrips (Table 1). The known geographical distribution of Thripinema includes England, Germany, India, Russia, New Brunswick (Canada), New Zealand, Chile, and the United States (Table 1) (Chizhov et al. Reference Chizhov, Subbotin and Zakharenkova1995; Funderburk et al. Reference Funderburk, Ripa, Espinosa and Rodriguez2002a; Nickle and Wood Reference Nickle and Wood1964; Sharga Reference Sharga1932; Tipping et al. Reference Tipping, Nguyen, Funderburk and Smart1998). No new species of Thripinema has been described since 1998, and molecular data for the genus remain extremely limited, with only two partial mtCOI sequences available for T. fuscum (OQ918270 from India and DQ915802 from the USA). The apparently restricted distribution of the genus likely reflects limited survey efforts rather than its true biogeographical range.
List of Thripinema species and their thrip hosts and potential relationships
Table 1. Long description
From the top row, columns are as follows: Thripinema nematodes, Thripi Hosts, Collection site and plant hosts, Parasitizing life stages of thrips, References. First row: T. aptini infects Aptinothrips rufus, Frankliniella schultzei, Microcephalothrips abdominalis; collected in UK and India; parasitizes adult females, rarely larvae and pupae, not males; Sharga Reference Sharga1932, Lysaght Reference Lysaught1936. Second row: T. nicklewoodi infects F. occidentalis, F. vaccinii, Taeniothrips vaccinophilus; USA, Canada, wide host range; females and males easily parasitized in lab; Nickel and Wood Reference Nickle and Wood1964, Wilson and Cooley Reference Wilson and Cooley1972. Third row: T. reniraoi infects Megalurothrips sp.; India, leguminous crop flowers; adult females; Reddy et al. Reference Reddy, Nickle and Rao1982. Fourth row: T. khrustalevi infects Thrips threhernei, T. physopus, F. australis; Russia, Chile, Compositae plants, Taraxacum officinale, Hieracium sp.; adult thrips; Chizhov et al. Reference Chizhov, Subbotin and Zakharenkova1995, Funderburk et al. Reference Funderburk, Ripa, Espinosa and Rodriguez2002a. Fifth row: Unknown species infects Thrips obscuratus, Thrips physopus, Heliothrips fasciatus; New Zealand, Germany, USA; no life stage listed; Teulon et al. 1997. Sixth row: T. fuscum infects F. fusca, F. occidentalis (occasionally), F. australis; USA, peanut flowers Arachis hypogaea; adult female thrips, rarely second instars and males in nature; Tipping et al. Reference Tipping, Nguyen, Funderburk and Smart1998, Funderburk et al. Reference Funderburk, Stavisky, Tipping, Gorbet, Momol and Berger2002b. Seventh row: T. ustatum n. sp. infects Megalurothrips usitatus; China; adult female, larvae, rare in male thrips; This paper. Superscript a indicates type hosts.
a Type hosts.
In the present study, Thripinema usitatum n. sp. is described based on an integrative approach combining detailed morphology, host association, life-history observations, and molecular phylogenetic analyses. Morphologically, the new species conforms to the diagnostic features of Thripinema, including pronounced sexual dimorphism, highly modified parasitic females, vermiform infective females, and free-living males with degenerate feeding structures. However, T. usitatum n. sp. can be readily distinguished from its congeners by a unique combination of characters, particularly the absence of a stylet in free-living males, the distinctive two-part bursa extending nearly to the tail terminus, and the characteristic shapes of parasitic females and male tails. These features clearly separate T. usitatum n. sp. from T. khrustalevi, T. fuscum, and T. aptini, which possess a stylet in males, as well as from T. nicklewoodi and T. reniraoi, which differ in parasitic female morphology and bursal configuration.
Molecular data generated in this study provide the first multilocus phylogenetic framework for the genus Thripinema. Sequences from three markers (18S rRNA, 28S D2–D3 rRNA, and mtCOI) were obtained and deposited in GenBank, representing the first comprehensive molecular characterization of a Thripinema species. Phylogenetic analyses based on the 28S D2–D3 region consistently placed T. usitatum n. sp. as a well-supported, monophyletic lineage embedded within a broader clade dominated by species of Fergusobia. Despite this association, Thripinema was clearly separated from all sampled Fergusobia Currie, 1937, species by long branch lengths and high posterior probabilities, supporting its status as a distinct evolutionary lineage. This pattern is congruent with morphological differences between the two genera, particularly in female body form, reproductive system organization, and host associations, and supports the continued recognition of Thripinema as a valid genus.
In contrast, phylogenetic inference based on partial 18S rRNA sequences placed T. usitatum n. sp. within Allantonematinae, clustering with species of Howardula. This result is consistent with previous studies demonstrating that the 18S rRNA gene is highly conserved and often lacks sufficient resolution for distinguishing genera within Allantonematinae. Therefore, while the 18S marker is useful for confirming subfamilial placement, it should not be relied upon alone for genus-level taxonomy in entomoparasitic nematodes. Among the three markers analyzed, mtCOI provided the highest level of resolution, clearly separating T. usitatum n. sp. from other entomophilic, animal-parasitic, and plant-parasitic nematodes. These results highlight the utility of mtCOI as a DNA barcode for insect-parasitic nematodes, although direct comparison with previously published T. fuscum mtCOI sequences was not possible due to the use of non-overlapping primer sets.
The life cycle of Thripinema species is highly specialized for parasitism of thrips. Infective females penetrate the host cuticle and develop within the haemocoel, where they mature into parasitic females and commence oviposition within four to five days post-infection; eggs hatch into juveniles within six to eight days (Mason and Heinz Reference Mason and Heinz2002; Sims et al. Reference Sims, Becnel and Funderburk2012). Juveniles eventually migrate to the hindgut, where mating occurs, and free-living stages emerge through the anus or ovipositor. Outside the host, infective females are thought to locate new thrips hosts in humid microhabitats such as flowers, leaf galls, and young foliage (Loomans et al. Reference Loomans, Murai, Greene and Lewis1997). Although parasitized thrips usually remain alive, their feeding activity, fecundity, and ability to transmit tospoviruses, including tomato spotted wilt virus, are often substantially reduced (Arthurs and Heinz Reference Arthurs and Heinz2003; Sims et al. Reference Sims, Funderburk and Boucias2005, Reference Sims, Funderburk, Reitz and Boucias2009). In the present study, no statistically significant differences in reproductive output were detected between parasitized and unparasitized thrips, suggesting that the effects of T. usitatum n. sp. on host reproduction may be context-dependent or influenced by host age, infection intensity, or environmental conditions.
Our biological observations revealed a complex within-host population structure of T. usitatum n. sp. Different developmental stages, including parasitic females, infective females, free-living males, juveniles, and eggs, frequently co-occurred within individual female thrips. Infection prevalence in Sanya, Haikou Province, in March 2025 was markedly higher in adult females (80%) than in larvae (8.3%) or males (15.6%), indicating a strong bias towards female hosts. The total nematode burden per parasitized thrips varied widely, ranging from 1 to 433 individuals, suggesting that T. usitatum n. sp. is capable of completing multiple generations within a single host. Such high infection intensities are consistent with previous reports on Thripinema species and underscore the intimate and persistent nature of their association with thrips.
From an applied perspective, species of Thripinema are of considerable interest as potential biological control agents of pest thrips. Notably, T. nicklewoodi parasitizes Frankliniella occidentalis Pergande, and T. fuscum parasitizes F. fusca Hinds, both of which are economically important pests and efficient vectors of tospoviruses. Field studies have demonstrated that T. fuscum can suppress F. fusca populations by more than 50% in Florida peanut agroecosystems (Funderburk and Latsha Reference Funderburk, Latsha, Grewal, Ehlers and Shapirollan2005; Tipping et al. Reference Tipping, Nguyen, Funderburk and Smart1998), while T. nicklewoodi significantly reduces virus transmission by F. occidentalis in greenhouse chrysanthemum production (Arthurs and Heinz Reference Arthurs and Heinz2003; Mason and Heinz Reference Mason and Heinz2002). Arthurs and Heinz (Reference Arthurs and Heinz2002) proposed strategies for mass production and augmentation of T. nicklewoodi, although no Thripinema species has yet been successfully cultured for commercial application.
In conclusion, the description of Thripinema usitatum n. sp. expands the known diversity of thrips-parasitic nematodes and provides the first integrative morphological and molecular framework for the genus. Morphological characters, multilocus phylogenetic analyses, and host associations collectively support its recognition as a valid new species. Future studies should focus on broader surveys of Thripinema diversity, population genetics, host specificity, and the ecological consequences of parasitism, as well as the development of rearing techniques to assess their potential role in integrated pest management.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26101515.
Acknowledgements
This research was supported by the National Key R&D Program of China (2024YFD1400100). The authors are grateful to the anonymous reviewer for the constructive comments, valuable suggestions, and careful reading of the manuscript. Their insightful feedback has greatly improved the quality of the present paper.
