Description of the female, nymph and larva and mitochondrial genome, and redescription of the male of Ixodes barkeri Barker, 2019 (Acari: Ixodidae), from the short-beaked echidna, Tachyglossus aculeatus, with a consideration of the most suitable subgenus for this tick Author Barker, Dayana Author Kelava, Samuel Author Shao, Renfu Author Seeman, Owen D. Author Jones, Malcolm K. Author Nakao, Ryo Author Barker, Stephen C. Author Apanaskevich, Dmitry A. text Parasites & Vectors 2022 117 2022-04-01 15 1 1 18 http://dx.doi.org/10.1186/s13071-022-05165-2 journal article 298676 10.1186/s13071-022-05165-2 c260cad4-49c0-4827-8e69-bf597c8a47ae 1756-3305 11552076 Ixodes barkeri Barker, 2019 Male . (Based on 6 specimens , 3 measured—refer to Table 1 ; Figs. 2 , 3 , 8 ) Idiosoma ( Fig. 2 ) broadly elongate-oval with broadly rounded posterior margin, widest slightly posterior to mid-length; length from apices of scapulae to posterior body margin 2.17–2.59 mm ( 2.42 mm ), width 1.53–1.72 (1.61), ratio 1.42–1.56 (1.49). Lateral groove distinct. Conscutum ( Figs. 2 , 8 ) length 2.12–2.56 (2.38), width 1.37–1.48 (1.44), ratio 1.54–1.73 (1.65); laterally and postero-laterally with a distinct narrow non-sclerotised band; scapulae short, blunt; lateral carinae absent; cervical grooves indistinct; dense moderately large punctations evenly distributed over conscutum, except for pseudoscutum area; pseudoscutum with indistinct punctations; setae moderately dense, very short ( c. 0.01) and indistinct ( Fig. 2 ). Venter plate outlines as illustrated ( Fig. 2 ); median plate: length 1.06–1.18 (1.10), width 0.74–0.84 (0.81), ratio 1.26–1.43 (1.37); adanal plate: length 0.74–0.82 (0.79), width 0.42–0.48 (0.45), ratio 1.67–1.95 (1.77); anal plate: 0.54–0.60 (0.58), width 0.32–0.36 (0.34), ratio 1.67–1.71 (1.69). All ventral plates with dense, moderately large punctations ( Figs. 1 , 2 ). Genital aperture ( Fig. 2C ) located at level of posterior margin of coxae II; posterior margin of genital apron deeply concave. Ventral setae ( Fig. 2 ) moderately dense, very short, evenly distributed on all plates; length of setae on median plate c. 0.01. Anal groove ( Fig. 2C ) straight anteriorly and open posteriorly. Spiracular plate ( Fig. 2A ) broadly oval, longer than wide, length 0.34–0.42 (0.38), width 0.28–0.34 (0.31), ratio 1.21–1.25 (1.23). Fig. 2 Ixodes barkeri Barker, 2019 , scanning electron micrographs of idiosoma of male. A Dorsal view; B dorsolateral view; C ventral view. Scale bars : 0.5 mm Gnathosoma ( Figs. 3 , 8 ) length from palpal apices to cornual apices dorsally 0.45–0.48 (0.46), width between lateral projection of palpal segments I 0.43–0.48 (0.46), ratio 0.98–1.04 (1.01). Dorsal basis capituli ( Fig. 3A ) length from medial insertion of palpal segment I to cornual apices 0.21–0.25 (0.23), width 0.32–0.35 (0.33), ratio width to length 1.41–1.52 (1.46), subrectangular, posterior margin nearly straight; cornua long, ratio total length of basis capituli, including cornua, to cornual length 4.83–5.78 (5.39), triangular with narrowly rounded apex. Ventral basis capituli ( Fig. 3C ) subrectangular; lateral margins with slight constrictions at mid-length; auriculae absent; short converging ridges in auricular areas. Palpi ( Fig. 3B ) short, length dorsally (segments II and III) 0.23–0.24 (0.23), maximum width (in dorsolateral plane) 0.18–0.19 (0.19), ratio 1.22–1.29 (1.26), length of palpal segment I ventrally 0.16 ( n = 1), maximum width ventrally 0.17 ( n = 1), ratio 0.94 ( n = 1); segment I greatly enlarged, greatest dimension in anteromedian-posterolateral direction; dorsally segment I subrectangular; ventrally segment I subtriangular, posterior margin with very long and narrow spur with sharply pointed apex; segments II and III fused together with indistinct suture between them, narrower proximally and abruptly widening to broadly rounded apex. Hypostome ( Fig. 3C ) length 0.18–0.19 (0.18), width 0.08–0.10 (0.09), ratio 1.75–2.10 (1.95); club-shaped, widening to broadly rounded apex with medial indentation; base of hypostome at level of base of palpal segment I; dental formula 3/3, basal half of hypostome without denticles, denticles sharply pointed. Fig. 3 Ixodes barkeri Barker, 2019 , scanning electron micrographs of male. A Spiracular plate ( arrows show orientation of spiracular plate: a, anterior; d, dorsal). B Gnathosoma,dorsal view. C Gnathosoma, ventral view. D Gnathosoma,anteroventral view. E Coxae. F Trochanter I, dorsal view. Scale bars : A–D , F 0.1 mm; E,0.2 mm Fig. 4 Ixodes barkeri Barker,2019 ,scanning electron micrographs of female. A Idiosoma, dorsal view. B Scutum,dorsal view. C Scutum, dorsolateral view. D Idiosoma showing scutum and alloscutum with punctations and setae, dorsal centrolateral portion. E Idiosoma, ventral view. Scale bars : A , E 0.5 mm; B , C 0.2 mm; D 0.1 mm Fig. 5 Ixodes barkeri Barker,2019 ,scanning electron micrographs of female. A Spiracular plate ( arrows show orientation of spiracular plate: a, anterior; d, dorsal). B Gnathosoma, dorsal view. C Gnathosoma, ventral view ( I , palpal article 1; II , palpal article 2; ss the strongly salient part of palpal article 1). D Gnathosoma, anteroventral view. E Coxae. F Trochanter I , dorsal view. Scale bars : A, F,0.1 mm; B–E, 0.2 mm Legs moderately long, slender. Coxae ( Figs. 2E , 9 ): coxae I–IV with long and narrow external spur with narrowly rounded to sharply pointed apex; spur on coxae I–III subequal, spur on coxa IV nearly twice shorter those on coxae I–III; coxae I–IV without syncoxae. Trochanter I with long, triangular spur with sharply pointed apex; trochanters I–IV with long, narrow, with sharply pointed apex spur ventrally. Tarsus I: length 0.56–0.60 (0.58); tarsus IV length 0.53–0.58 (0.55); tarsi only slightly humped subapically. Fig. 6 Ixodes barkeri Barker,2019 ,scanning electron micrographs of nymph. A Scutum. B Spiracular plate ( arrows show orientation of spiracular plate: a, anterior; d, dorsal). C Gnathosoma, dorsal view. D Gnathosoma,ventral view. E Gnathosoma, anteroventral view. F Coxae. Scale bars : A, C–F, 0.1 mm; B, 0.05 mm Fig. 7 Ixodes barkeri Barker,2019 ,scanning electron micrographs of larva. A Scutum. B Gnathosoma, dorsal view. C Gnathosoma, ventral view. D Gnathosoma, anteroventral view. E Coxae. Scale bars : A , E 0.1 mm; B–D , 0.05 mm Female. (Based on 1 to 5 specimens —refer to Table 1 ; Figs. 4 , 5 , 9 ) Idiosoma ( Figs. 4A , 9 ) length from scapular apices to posterior body margin in moderately engorged specimen 4.1 ( n = 1), width in moderately engorged 2.5 ( n = 1), ratio 0.16 ( n = 1), broadly suboval, widest approximately at mid-length. Scutum ( Figs. 4 , 9 ) length 1.05–1.18 (1.13; n = 4), width 1.40–1.58 (1.49; n = 4), ratio 0.73–0.79 (0.75; n = 4); lateral margins diverging for approximately 2/3 of scutum length, broadly rounded posteriorly; lateral carinae lacking; cervical grooves shallow; dense, small punctations evenly distributed throughout scutum; setae ( Fig. 4 ) relatively sparse, very short ( c. 0.005), indistinct and nearly equal to those on alloscutum, distributed as figured. Alloscutum ( Fig. 4 ) as illustrated; setae of alloscutum ( Fig. 4C ) numerous, evenly distributed, very short, length of setae in central field c. 0.01, indistinct. Venter ( Fig. 4E ) as illustrated; genital aperture ( Fig. 4E ) medial to coxae III; genital groove ( Fig. 4E ) well developed; anal groove ( Fig. 4E ) oval with open posterior margin; ventral setae numerous, length of preanal setae c. 0.01, evenly distributed. Spiracular plates ( Fig. 5A ) length 0.27–0.32 (0.30; n = 4), width 0.39–0.47 (0.44; n = 4), ratio 0.68–0.70 (0.69; n = 4); broadly oval; marginal row of perforations in groove anteriorly. Gnathosoma ( Fig. 5B ) length from palpal apices to cornual apices dorsally 0.60–0.70 (0.65; n = 4), width between lateral projection of palpal segments I 0.66–0.74 (0.71; n = 4), ratio 0.89–0.95 (0.92; n = 4). Dorsal basis capituli ( Fig. 5B ) length from medial insertion of palpal segment I to cornual apices 0.22–0.25 (0.24; n = 4), width 0.51–0.55 (0.54; n = 4), ratio width to length 2.20–2.32 (2.26; n = 4), subrectangular, posterior margin nearly straight; cornua short, ratio of total length of basis capituli (including cornua) to cornual length 17.50–20.00 (19.12; n = 4), triangular with broadly rounded apex; subcircular porose areas poorly distinct, not indented, separated by distance nearly equal to their own width, several openings arranged in discrete punctations. Ventral basis capituli ( Fig. 5C ) subrectangular; lateral margins with slight constrictions at mid-length; auriculae absent; short converging ridges in auricular areas. Palpi ( Fig. 5B ) short, length dorsally (segments II and III) 0.41–0.50 (0.46; n = 4), maximum width (in dorsolateral plane) 0.21–0.25 (0.23; n = 4), ratio 1.85–2.10 (1.95; n = 4), length of palpal segment I ventrally 0.31–0.36 (0.34; n = 4), maximum width ventrally 0.19–0.20 (0.20; n = 4), ratio 1.64–1.75 (1.68; n = 4); segment I greatly enlarged, greatest dimension in anteromedian-posterolateral direction; dorsally segment I subrectangular with convex medial margin; ventrally segment I subtriangular, posterior margin with long moderately narrow spur with narrowly rounded apex; segments II and III fused together with indistinct suture between them, narrower proximally and abruptly widening to broadly rounded apex. Hypostome ( Fig. 5C ) length 0.36–0.40 (0.38; n = 4), width 0.12–0.16 (0.14; n = 4), ratio 2.46–2.85 (2.68; n = 4); club-shaped, widening to broadly rounded apex with medial indentation; base of hypostome approximately at level of base of palpal segment II; dental formula 4/4 (few rows may be 3/3), basal half of hypostome without denticles, denticles sharply pointed. Fig. 9 Mitochondrial genomes of Ixodes ( Endopalpiger ) australiensis , I. ( Endo. ) barkeri , I. ( Endo. ) woyliei and I. ( Exopalpiger ) fecialis . Protein-coding genes are shown in green, tRNAs are in yellow, rRNAs are in red, and the two control regions are in blue. Protein-coding genes are labelled by their four-character abbreviations,tRNAs are labelled by their one-letter amino acid abbreviations, and the two control regions are labelled as CR1 and CR2. Mitochondrial genome size variation is indicated in parentheses.The arrangement of genes in these four species is identical except that the main cluster of tRNA genes has the arrangement ARN SE F in the three species of Endopalpiger [ I. ( Endo. ) australiensis , I. ( End. ) barkeri and I. ( End. ) woyliei ], whereas in the one species of Exopalpiger [ I. ( Exo. ) fecialis ] the arrangement is ARN ES F.The arrangement in I. ( Exo. ) fecialis is the first known arrangement in an Ixodidae tick that is different from ARNSEF.Thus, ARN ES F might be a synapomorphy for the subgenus Exopalpiger Fig. 8 Ixodes barkeri Barker,2019 ,light microscopy image of female (Barker & Barker Collection reference #B5321), male (# B4994), nymph (#B5321) and larva (# B5321). Horizontal broken scale bars: 1 mm; vertical scale bars also in mm Fig. 10 Maximum likelihood (ML) phylogenetic tree from entire mt genomes (14,935 bps).The sequence alignment was put though Gblocks to remove regions with alignment gaps.Tip labels indicate NCBI accession numbers and (Barker & Barker Collection reference nos.). Numbers above branches show maximum likelihood bootstrap support, whereas numbers below branches show the Bayesian posterior probability support. Ixodes pavlovskyi Pomerantzev, 1946 , one of the species “Other Ixodes ” (sensu Barker & Murrell, 2004), for which an entire mitochondrial (mt) genome was available in GenBank, was set as the outgroup.The scale bar indicates 0.06 nucleotide substitutions per nucleotide site for the 14,935 nucleotide sites in our alignment of theses entire mt genomes. So, for example, there were about 896 nucleotide substitutions along the branch that leads to I. ( Ceratixodes ) uriae plus I. ( Sternalixodes ) holocyclus plus I. ( Exopalpiger ) fecialis , which is marked with an asterisk [i.e. 0.06 nucleotide substitutions per nucleotide site × 14,935 nucleotide sites (bps) =1896 nucleotide substitutions].Ticks in bold were sequenced in the present study Legs moderately long, slender. Coxae ( Figs. 5E , 9 ): coxae I–IV with moderately long and narrow external spur with narrowly rounded apex; spur on coxae I– III subequal, spur on coxa IV nearly twice shorter than those on coxae I–III; coxae I–IV without syncoxae. Trochanter I with moderately long, triangular spur with sharply pointed apex; trochanters I–IV without spur ventrally. Tarsus I: length 0.66–0.76 (0.73; n = 4); tarsus IV length 0.57–0.66 (0.63; n = 4); tarsi only slightly humped subapically. Nymph. (Based on 34 specimens —refer to Table; Fig. 6 ) Scutum ( Fig. 6A ) length 515–520 (518; n = 2), width 690–700 (695; n = 2), ratio 0.74–0.75 (0.75; n = 2); lateral margins diverging for approximately half of scutum length, broadly rounded posteriorly; posterolateral margin with slight indentations; lateral carinae lacking; cervical grooves shallow; moderately dense, small punctations evenly distributed throughout scutum; setae ( Fig. 6A ) relatively sparse, very short: length in central field of scutum 10 ( n = 2), indistinct and nearly equal to those on alloscutum, distributed as figured. Setae of alloscutum numerous, evenly distributed, very short, length of setae in central field 10–13 (11; n = 2), indistinct. Anal groove oval with open posterior margin; ventral setae numerous, evenly distributed. Spiracular plates ( Fig. 6B ) broadly oval; marginal row of perforations in groove anteriorly. Gnathosoma ( Fig. 6C ) length from palpal apices to cornual apices dorsally 285 ( n = 2), width between lateral projection of palpal segments I 375–380 (378; n = 2), ratio 0.75–0.76 (0.76; n = 2). Dorsal basis capituli ( Fig. 6C ) length from medial insertion of palpal segment I to cornual apices 108 ( n = 2), width 238–240 (239; n = 2), ratio width to length 2.21–2.23 (2.22; n = 2), subrectangular, posterior margin nearly straight; cornua moderately long, triangular with narrowly rounded apex. Ventral basis capituli ( Fig. 6D ) subrectangular; lateral margins with slight constrictions at mid-length; auriculae absent; short converging ridges in auricular areas. Palpi ( Fig. 6C ) short, length dorsally (segments II and III) 190–193 (191; n = 2), width 73–85 (79; n = 2), ratio 2.24–2.66 (2.45; n = 2), length of palpal segment I ventrally 140–150 (145; n = 2), maximum width ventrally 125–130 (128; n = 2), ratio 1.12–1.15 (1.14; n = 2); segment I greatly enlarged, greatest dimension in anteromedian-posterolateral direction; dorsally segment I subrectangular with convex medial margin; ventrally segment I subtriangular, posterior margin with long moderately broad spur with narrowly rounded apex; segments II and III fused together with indistinct suture between them, narrower proximally and abruptly widening to broadly rounded apex. Hypostome ( Fig. 6D ) length 150 ( n = 1), width 73 ( n = 1), ratio 2.07 ( n = 1); club-shaped, widening to broadly rounded apex with medial indentation; base of hypostome approximately at level of base of palpal segment II; dental formula 3/3 (few basal rows 2/2), basal half of hypostome without denticles, denticles sharply pointed. Legs moderately long, slender. Coxae ( Fig. 6F ): coxae I–IV with external spur; spur on coxae I–III moderately long, nearly subequal; spur on coxa I with broadly rounded apex, spur on coxae II and III with narrowly rounded to sharply pointed apex; spur on coxa IV very short; coxae I–IV with syncoxae occupying approximately 1/3, 1/4, 1/5 and 1/6 respectively of coxal width. Trochanters I–IV without spur ventrally. Tarsus I: length 320–335 (328; n = 2); tarsus IV length 298–310 (304; n = 2); tarsi only slightly humped subapically. Larva. (Based on 2 specimens —refer to Table 1 ; Figs. 7 , 10 ) Scutum ( Fig. 7A ) length 238 ( n = 1), width 360 ( n = 1), ratio 0.66 ( n = 1); lateral margins diverging for approximately half of scutum length, broadly rounded posteriorly; posterolateral margin with slight indentations; lateral carinae lacking; cervical grooves shallow; setae 3 pairs, length of Sc 1 11 ( n = 1); length of Sc 4 14 ( n = 1). Dorsal setae of alloscutum undetermined in number since we only had larvae that were engorged to examine: it was impossible to confidently count and associate setae of the idiosoma dorsally and ventrally. Length of Cd 1 17 ( n = 1), length of Md 1 23 ( n = 1). Ventral setae undetermined number; 1 pair on anal valves; 3 pairs of sternals, length of St 1 19 ( n = 1); 2 pairs of preanals, length of Pa 1 22 ( n = 1), length of Pa 2 32 ( n = 1) . Gnathosoma ( Fig. 7B–D ) length from palpal apices to cornual apices dorsally 123 ( n = 1), width between lateral projection of palpal segments I 155 ( n = 1), ratio 0.79 ( n = 1). Dorsal basis capituli ( Fig. 7B ) width 135 ( n = 1), subrectangular, posterior margin nearly straight; cornua practically indistinct. Ventral basis capituli ( Fig. 7C ) subrectangular; lateral margins with slight constrictions at mid-length; auriculae absent. Post-hypostomal setae two pairs, length of Ph 1 4 ( n = 1), length of Ph 2 4 ( n = 1); distance between Ph 1 37 ( n = 1), distance between Ph 2 27 ( n = 1). Palpi ( Fig. 7B ) short, length dorsally (segments II and III) 77 ( n = 1), maximum width (in dorsolateral plane) 42 ( n = 1), ratio 1.83 ( n = 1), maximum width of palpal segment I ventrally 50 ( n = 1); segment I greatly enlarged, greatest dimension in anteromedian-posterolateral direction; dorsally segment I subrectangular with convex medial margin; ventrally segment I subtriangular, posterior margin with long moderately broad spur with narrowly rounded apex; segments II and III fused together with indistinct suture between them, narrower proximally and abruptly widening to broadly rounded apex; segment I lacking setae, segments II and III combined with nine dorsal and three ventral setae. Hypostome ( Fig. 7C ) length 83 ( n = 1), width 34 ( n = 1), ratio 2.44 ( n = 1); club-shaped, widening to broadly rounded apex with medial indentation; base of hypostome approximately at level of mid-length of palpal segment I; dental formula 2/2, approximately five denticles in files; basal half of hypostome without denticles, denticles sharply pointed. Legs moderately long, slender. Coxae ( Fig. 7E ): coxae I–III with external spur; spur on coxa I moderately long, on coxa II and III short to very short; spur on coxae I– III with broadly rounded apex; coxae I–III with syncoxae occupying approximately 1/3, 1/4 and 1/5 respectively posteromedian portion of coxal width. Trochanters I– IV without spur ventrally. Tarsus I: length 183 ( n = 1); tarsus III length 166 ( n = 1); tarsi only slightly humped subapically. Remarks By having a greatly enlarged palpal segment I that extends inwardly and anteriorly, all active life stages of I. barkeri most closely resemble those of the Endopalpiger species of Australasia: I. acer , I. australiensis , I. giluwensis , I. hydromyidis , I. luxuriosus , I. mirzai , I. planiscutatus , I. steini , I. stellae , I. tasmani , I. victoriensis , I . woyliei and I. zaglossi (refer to [ 8 , 19 , 20 ]). Te males of I. australiensis , I. tasmani , I. victoriensis and I. zaglossi have been described [ 19 , 21 – 24 ]. Te male of I. barkeri is easily distinguished from the males of the Endopalpiger species by the absence of the syncoxal areas on all coxae (vs. well-developed syncoxae on coxae I–IV in all those species). Te female of I. barkeri resembles only that of I. woyliei by the absence of syncoxal areas on coxae (vs. females of all other Endopalpiger with well-developed syncoxae). Te female of I. barkeri can be differentiated from I. woyliei by the scutum and basis capituli dorsally and ventrally without lateral carinae and other longitudinal ridges (vs. lateral carinae and longitudinal ridges present in I. woyliei ), the considerably smaller palpal segment I with a long spur on its posterior margin (vs. greatly enlarged palpal segment I with shorter spur on its posterior margin in I. woyliei ), 4/4 dental formula on hypostome (vs. 6/ 6 in I. woyliei ) and the long spur on trochanter I dorsally (vs. indistinct spur in I. woyliei ). Te nymph of I. australiensis , I. hydromyidis , I. luxuriosus ([ 25 ] wrote that the nymph of I. luxuriosus had not been described, although there is a brief description of it in [ 26 ]), I. steini , I. tasmani , I. victoriensis and I. woyliei have been described [ 8 , 19 , 22 , 24 , 26 ]. Unfortunately, all of these published descriptions and illustrations are too brief for confident comparison. Nonetheless, we note that the nymph of I. barkeri has a scutum without lateral carinae (vs. distinct carinae in I. victoriensis ), a scutum and basis capituli dorsally and ventrally without distinct longitudinal ridges (vs. with distinct, sharp ridges in I. woyliei ), a distinct cornua (vs. no cornua in I. australiensis , I. hydromyidis , I. tasmani and I. woyliei ), mostly 3/3 dental formula on the hypostome (vs. 2/2 dental formula in I. hydromyidis and I. tasmani , 4/ 4 in I. australiensis ), external spurs on coxae I–IV (vs. apparently no spurs on coxae in I. hydromyidis , I. luxuriosus , I. steini and I. tasmani ) and tarsi I–IV slightly humped subapically, without a notch (vs. strongly humped tarsi with distinct notch in I. victoriensis ). Te larvae of I. hydromyidis , I. tasmani and I. victoriensis have been described [ 22 , 24 , 27 , 28 ]. Unfortunately, as with the nymphs, all of these published descriptions and illustrations of larvae are too brief for confident comparison. Nonetheless, we note that the larva of I. barkeri has indistinct cornua on the basis capituli dorsally (vs. distinct cornua in I. victoriensis ) and has external spurs on coxae I–III (vs. no spurs on coxae in I. hydromyidis and I. tasmani ). Our diagnoses may be broadened and improved once the nymphs and larvae of the other Australasian species of Endopalpiger are redescribed and illustrated accurately.