Three new genera, two new species and one new combination of family Hystrignathidae (Nematoda: Thelastomatoidea) from Ceracupes fronticornis (Westwood) (Insecta: Passalidae) in China

Two new genera and two new species of family Hystrignathidae were collected from Ceracupes fronticornis (Westwood) from the Yunnan Province, China. Pseudoxyo yunnanensisgen. et sp. nov. differs from the related genera by having the cervical region armed with alternating rows of spines, with 22 spines in the first row, and by lacking the first cephalic annule. Sinospinata chitwoodigen. et sp. nov. can be easily distinguished from the related genera by having the cervical region of females armed with irregularly arranged spines, two or three spines clustered together in their roots in some spines. Meanwhile, Huntinemagen. nov. was proposed to replace Huntia Zhang, Yin, Carreno & Zhang, 2021 because the name of this genus was preoccupied by Huntia Gray & Thompson, 2001 for placement of two new species of spiders. In addition, the 18S and 28S rDNA partial sequences of two new species were obtained.


Introduction
The members of Hystrignathidae Travassos, 1920 (Nematoda: Thelastomatoidea) are only parasitic in passalid beetles. To date, 36 genera have been described with more than 100 species (Zhang et al. 2021(Zhang et al. , 2022. The species are mainly distributed in North and South America, Africa, and Australia (Adamson & Van Waerebeke 1992;Morffe & García 2011, 2013aGarduño-Montes de Oca & Oceguera-Figueroa 2020). However, recent studies on thelastomatoid nematodes from two Chinese passalid beetles have been found 4 new genera and 5 new species of hystrignathid nematodes (Zhang et al. 2021(Zhang et al. , 2022, which indicated it is highly probable that there is a great richness of nematodes to be discovered in passalid beetles in China. In the present study, nematode specimens were collected from Ceracupes fronticornis (Westwood) from the Yunnan Province, China. Two new genera and two new species of Hystrignathidae were confirmed and described here. In addition, Huntinema was proposed to replace Huntia Zhang, Yin, Carreno & Zhang, 2021 because the name of Huntia was preoccupied. Moreover, the 18S and 28S rDNA partial sequences for the new species were also generated.

Light and scanning electron microscopy
The passalid beetle, Ceracupes fronticornis (Westwood), were collected from the Yunnan Province, China. They were dissected and examined for nematodes. The collected nematode specimens were killed with hot water (60-70°C) and then fixed in 80% ethanol. For light microscopical examination, preserved nematodes were placed in a 5% solution of glycerin in 95% ethanol. These were left uncovered for 48 h to allow the ethanol to evaporate, thereby leaving the specimens in 100% glycerin. This was done to limit any damage to the worms caused by rapid transfer to pure glycerin. Measurements were taken with the aid of a calibrated eyepiece micrometer. De Man's ratios a, b, c, V% and V'% were calculated. All measurements are given in micrometers as range values followed by mean values in parentheses. Drawings were made with the aid of a Nikon microscope drawing attachment. For scanning electron microscopy studies, specimens were fixed in 7.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated through ethanol and acetone, and then subjected to critical point drying. The specimens were coated with gold and examined with a S-4800 Field Emission scanning electron microscope at an accelerating voltage of 15 kV. The specimens have been deposited in the College of Life Sciences, Hebei Normal University (HBNU), Hebei Province, China.

Molecular Procedures
Randomly selected samples were used for further molecular analysis. Genomic DNA from each sample was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer's instructions. The 18S sequence was amplified by PCR using the forward primer 18SF (5 0 -CCCGATTGATTCTGTCGGC-3 0 ) and the reverse primer 18SR (5 0 -TGATCCTTCTGCAGGTTCACC-TAC-3 0 ) (Floyd et al. 2005). The 28S sequence was amplified by PCR using the forward primer D2A (5 0 -ACAAGTACCGTGAGGG AAAGTTG-3 0 ) and the reverse primer D3B (5 0 -TCGAAGGAACCAGC-TACTA-3 0 ) ( Morffe et al. 2019). The PCR reactions for both 18S rDNA and 28S rDNA were performed in a total volume of 25 lL, containing 2 lL of template, 0.5 lL each of forward and reverse primers, and 12.5 lL of 29Taq MasterMix (Beijing Bio-Lab, China). The 28S rDNA PCR cycling parameters were as follows: an initial enaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s, and 2°C for 70 s, followed by a final extension step at 72°C for 7 min. The 18S rDNA PCR cycling parameters were as follows: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 70 s, followed by a final extension step at 72°C for 7 min. PCR products were checked on GoldView-stained 1% agarose gels. Samples were sent to Shanghai Sangon, China for sequencing. Sequencing for each sample was carried out for both strands. Sequences were aligned using ClustalX and adjusted manually. The 18S rDNA and 28S rDNA sequences determined were compared (using the algorithm BLASTn) with those available in the National Center for Biotechnology Information (NCBI) database. Type-material: Holotype female (HBNU-I-2021023); paratypes: 9 females (HBNU-I-2021024-2021032). Prevalence: 6.1% (7 infected out of 115 examined). Intensity: 2-10 (mean 5) specimens. Site in host: Hindgut. Representative DNA sequences: One partial 28S and one partial 18S rDNA sequence of the new species are deposited in the GenBank database under the accession numbers ON751930 and ON751935, respectively. Etymology: The new species is named for its occurrence in Yunnan Province, China.

Systematics
Description. Female: Body relatively stout. Cervical cuticle bearing alternating rows of spines. Spines originating 20 lm behind head, ending at level of anus ( Fig. 2A, E). First row with 22 spines, about 8 lm long; second row of spines longer than the first, about 13 lm long. Size of spines gradually smaller after nerve ring (Fig. 1A). Oral opening rounded, surrounded by a cuticular ring (Fig. 2C). Head bearing eight papillae arranged in 4 pairs, a pair of amphids (Fig. 2C). Length of stoma about 5 times of head (Fig. 1A). Oesophagus consisting of a muscular, clavate corpus, short isthmus and basal bulb. Bulb rounded, valve-plate well-developed. Nerve ring encircling corpus in middle of its length (Fig. 1A). Excretory pore located posterior to basal bulb (Fig. 1D). Intestine simple, its anterior region slightly dilated. Reproductive system amphidelphic. Vulva located near mid-body (Fig. 1D). Vagina slightly extending anteriorly, connecting with two opposite uteri. Anterior ovary reflexed at excretory pore; posterior ovary reflexed at about two times of body width before anus (Fig. 1C, D). Eggs ovoid, smoothshelled (Fig. 1E). Tail conical, attenuated, sharply pointed. Male not observed.
Measurements. Female (n = 10): a = 9.8-15.6 (12.8); b = 4.8-7.0 (5.9); c = 6.2-8.0 (7.5); V  opposite rows of spines, by having the first row with 22 spines instead of 16 spines, and by lacking the first cephalic annule. Pseudoxyo gen. nov. is different from Urbanonema and Xyo by having the first row with 22 spines instead of 32 spines, and by lacking the first cephalic annule. In addition, Urbanonema differs from Pseudoxyo gen. nov. by having the stoma with a dilated anterior end.
The new genus is very similar to Parahystrignathus by having females with the cervical region armed with alternating rows of pointed spines, clavate procorpus, and similar cephalic structure, however, it can be distinguished from the latter by having the first row with 22 spines instead of 16 spines.
Sinospinata gen. nov. Diagnosis: Female body relatively stout. Cervical cuticle bearing irregularly arranged spines, spines originating just behind first cephalic annule, extending to level of excretory pore. Two or three spines clustered together in their roots in some spines. First row of spines with 44 elements. Lateral alae absent. Oral opening rounded, surrounded by a cuticular ring.
Head bearing 8 papillae, and a pair of amphids. First cephalic annule larger than head. Oesophagus consisting of a muscular, clavate corpus, short isthmus and basal bulb. Reproductive system amphidelphic. Vulva located near mid-body. Eggs ovoid, smoothshelled. Tail conical, attenuated, sharply pointed. Male unknown. Description. Female body relatively stout. Cervical cuticle bearing irregularly arranged spines, spines originating just behind first cephalic annule, extending to level of excretory pore (Fig. 3A). Two or three spines clustered together in their roots in some spines (Figs. 3C,F;4D,E). First row of spines with 44 elements. There are a few of small spines sparsely distributed before first row (Figs. 3C, 4A). After fifth row, number of spines gradually decreased. Lateral alae absent. Oral opening rounded, surrounded by a cuticular ring. Head bearing 8 papillae, and a pair of amphids (Fig. 4B, C). First cephalic annule cone-like, truncated, larger than head. Length of stoma is about twice of the first cephalic annule. Anterior end of stoma dilated and spherical (Fig. 3G). Oesophagus consisting of a muscular, clavate procorpus, short isthmus and basal bulb (Fig. 3D). Bulb rounded, valveplate well-developed. Nerve ring encircling corpus at 40% of its length. Excretory pore located just posterior to basal bulb. Intestine simple, its anterior region slightly dilated. Reproductive system amphidelphic.
Vulva located near mid-body. Vagina slightly extending anteriorly, connecting with two opposite uteri. Anterior ovary reflexed at excretory pore, posterior ovary reflexes forward at the mid-region between vulva and anus. Eggs ovoid, smooth-shelled. Tail Discussion: Sinospinata gen. nov. is similar to Carlosia, Hystrignathus, Parahystrignathus, Pseudoxyo gen. nov., Urbanonema and Xyo by having females with the cervical region armed with pointed spines, by having clavate procorpus and didelphic reproductive system. However, it can be easily distinguished from above related genera by having the cervical region of females armed with irregularly arranged spines, two or three spines clustered together in their roots in some spines.
In addition, Carlosia differs from the new genus in the cervical region in having only two longitudinal rows of spines. Sinospinata gen. nov. differs from Hystrignathus, Parahystrignathus, Pseudoxyo gen. nov. and Xyo by having the stoma with a dilated vs. a narrow anterior end.
Sinospinata gen. nov. resembles Urbanonema because both genera share the presence of a dilated anterior end of stoma. However, the new genus can be separated from Urbanonema by having the cervical cuticle armed with irregular vs. alternating rows of spines, and by the first row with 44 spines instead of 32 spines.