Syphacia ethiopiana sp. n. (Nematoda: Oxyurida: Syphaciinae) from the endemic Ethiopian rodent Stenocephalemys albipes Rüppell, 1842 Текст научной статьи по специальности «Биологические науки»

Russian Journal of Nematology, 2023, 31 (2), 161 - 169

Syphacia ethiopiana sp. n. (Nematoda: Oxyurida:

Syphaciinae) from the endemic Ethiopian rodent

Stenocephalemys albipes Rüppell, 1842

1 12 Anton R. Gromov , Leonid A. Lavrenchenko and Sergei E. Spiridonov

'Laboratory of Mammalian Microevolution, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 119071, Moscow, Russia

2Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 119071, Moscow, Russia e-mail: s_e_spiridonov@rambler. ru

Accepted for publication 20 November 2023

Summary. Oxyurid nematodes from the hindgut of the Ethiopian endemic rodent, Stenocephalemys albipes Ruppell, 1842, were examined and found to represent a new species of the genus Syphacia Seurat, 1916, Syphacia ethiopiana sp. n. The new species is morphologically similar to the common intestinal nematode of mice, Syphacia obvelata (Rudolphi, 1802) Seurat, 1916, but differs significantly in the nucleotide sequences of ITS rDNA and LSU rDNA. SEM images of the cuticular surface of the anterior end and other structures are provided.

Key words: African continent, pinworms, rodent host, Syphaciinae.

The nematodes of the genus Syphacia Seurat, 1916 inhabit the posterior parts of the intestines of rodents (Hugot, 1988). While the Syphacia fauna of some regions of the world (British Isles, Indonesia, Japan) is quite well studied (Okamoto et al., 2007, 2009; Dewi et al., 2016; Stewart et al., 2018), it remains understudied in other areas especially with regard to the use of modern techniques of molecular taxonomy. On the African continent, more data are available for the western part, while these nematodes remain almost unstudied in an East African country such as Ethiopia. At the same time, this part of the African continent appears to be a promising region for the study of rodent parasites, due to the presence of a number of local endemic rodent species. One such endemic is the white-

footed mouse (Stenocephalemys albipes Ruppell,

1842), found in the Ethiopian highlands on both sides of the Great Rift Valley. An evaluation of the phylogenetic relationships of parasitic nematodes from an endemic rodent can provide insight into the evolution of these widespread nematodes and information on the evolution of their rodent hosts.

MATERIAL AND METHODS

The field studies were carried out at two sites near Addis Ababa (Menagesha Forest: 8.965898° N, 38.549031° E, 2523 m a.s.l.; Mount Entoto: 9.085885° N, 38.711802° E, 2858 m a.s.l.) during 15-29 May 2023. Sixteen specimens of the white-

footed mice Stenocephalemys albipes were captured using Sherman live traps (23 * 9.5 * 8 cm) baited with sliced carrot and vegetable oil. All the fieldwork complied with laws and regulations of Ethiopia, and sampling was conducted in accordance with local laws and regulations (see Acknowledgements). The intestines of the white-footed mice were removed and dissected in physiological saline. Nematodes were collected with a needle and preserved in the 6% formalin heated up to 60-70°C for morphological studies and in 80% ethanol for molecular studies. The material was further processed at the A.N. Severtsov Institute of Ecology and Evolution RAS (SIEE).

For light microscopy, the nematodes were removed from the fixative, rinsed in water and transferred to a water solution of glycerol and ethanol. After evaporation of the liquids, the nematodes were mounted on slides in a drop of pure glycerol and sealed with paraffin rings (Seinhorst, 1959). The drawings and measurements were made with camera lucida. For SEM studies, several males and females were dehydrated through a graded ethanol series and acetone and dried in a critical point using HCP-2 Hitachi drier (Hitachi Ltd, Tokyo, Japan). After sputter coating the nematodes with gold using BIO-RAD SC502 sputter coater (Bio-Rad Laboratories Inc., Hercules, USA), they were examined in a Mira 3 Tescan electron microscope (Tescan Orsay Holding, a.s., Brno, Czech Republic) at 10.0 kV.

© Russian Society of Nematologists, 2023; doi: 10.24412/0869-6918-2023-2-161-169

Fig. 1. Syphacia ethiopiana sp. n. A - male, paratype, total view, lateral view; B - male, paratype, posterior end, lateral; C-E - variability of spicule and gubernaculum shape (C - holotype); F - female, paratype, total view, lateral; G - immature female (fourth-stage juvenile?), total view, lateral; H - egg with juvenile near the vulva opening and operculum position on the eggshell, lateral. Scales in micrometers.

The DNA was extracted from two males and two females from each of two sites where these nematodes were collected by digestion in proteinase K in the presence of mercapoethanol according to the procedure of Holterman et al. (2006). PCR reactions were performed using Encyclo Plus PCR kit (Evrogen , Russia) according to the manufacturer's instructions. Three pairs of primers were used to amplify three regions of ribosomal DNA: Nem18Sf (CGC GAA TRG CTC ATT ACA ACA GC) and Nem18Sr (GGG CGG TAT CTG ATC GCC) to amplify about 900 bp of SSU rDNA; NC5 (GTA GGT GAA CCT GCG GAA GGA TCA

T) and NC2 (TTA GTT TCT TTT CCT CCG CT) to obtain a portion of ITS rDNA and C1F (ACC CGC TGA ATT TAA GCA T) and D2R (TCC GTG TTT CAA GAC GG) to amplify partial LSU rDNA. PCR protocols included primary denaturation at 94°C for 3 min followed by 34 cycles 94°C for 30 s, annealing for 30-60 s and elongation at 72°C for 1 min, followed by post-amplification extension at 72°C for 7 min. Annealing temperature was 52°C for SSU and ITS primers, and 54°C for LSU primers. Attempts to amplify the partial sequences of cytochrome c oxidase subunit I with primers SyphaCOIF (TGG TCT GCT TTT GTT GGT AGT T)

Fig. 2. Syphacia ethiopiana sp. n. male, structure of surface. A - anterior end, sub-apical view; B - cephalic plate, sub-apical view; C - posterior end, lateral view.

and SyphaCOIR (AAC CAC CCA ACG TAA ACA TAA A) proposed by Okamoto et al. (2007) were not successful. Obtained sequences were deposited in GenBank NCBI under accession numbers: OR832774 for SSU rDNA, OR832775 for ITS rDNA, and OR832776 for LSU rDNA. The sequences of other Syphacia species were searched in GenBank with the Basic Local Alignment Search Tool (BLAST) (Altshul et al., 1990) and downloaded for comparative analysis. Sequence alignments were generated using CLUSTAL_X (Thompson et al., 1997) under default values for gap opening and gap extension penalties. Obtained alignments were analysed with MEGA X (Kumar et al., 2018) and PAUP4.0b10 (Swofford, 1998) to

estimate the phylogenetic relationships and calculate the distances between new and known species of the genus.

DESCRIPTION Syphacia ethiopiana sp. n.

(Fig. 1-3; Table 1)

Male. Holotype and 11 paratypes (Table 1). Body ventrally curved in all specimens (Fig. 1A). Three prominent mamelons, each 90-95 ^m long, present along midventral line (Fig. 1A). First mamelon located in 500-540 ^m from apex, second in 80-100 ^m from first one, and third in 160-190 (im from second one. Cephalic vesicle simply structured.

Fig. 3. Syphacia ethiopiana sp. n. female, fine structure of surface. A - anterior end, apical view; B - cephalic plate, apical view; C - cephalic plate, apical view; D - mouth opening; E - lateral differentiation of cuticle (lateral ala); F -eggshell surface and operculum seam.

Table 1. Syphacia ethiopiana sp. n. Measurements. Format: mean ± SD (range).

Characters Holotype, male Males (n = 11) Females (n = 4)

Body length 1727 1542±189 (1132-1820) 4434±924 (3613-5525)

Maximum body diameter 120 107±20 (82-140) 168±23 (133-181)

Pharynx length 218 199±26 (168-245) 324±6.0 (320-333)

Pharynx corpus length 143 132±19 (110-168) 212±4.8 (208-219)

Basal bulb length 58 55±6 (48-63) 86±3 (82-88)

Basal bulb width 45 45±3 (40-53) 67±2.2 (65-70)

Distance: apex - nerve ring 75 82±7 (69-93) 110-±4.7 (104-115)

apex - excretory pore (EP) 324 293±25 (235-333) 287±4.7 (282-291)

apex - vulva - - 550±22 (512-583)

Tail length 88 92±9.7 (78-107) 551±31 (523-571)

Tail filament 63 62±8.6 (49-73) -

Spicule length 79 72±7.3 (58-80) -

Gubernaculum length 30 31±4.5 (23-37) -

A 14.4 14.6±2.3 (11.7-19.6) 27.4±9.9 (20.2-41.5)

B 7.9 7.8±0.7 (6.7-8.8) 13.6±2.6 (11.2-16.6)

C 19.6 16.9±1.8 (13.3-19.6) 8.1±1.9 (6.4-9.2)

V% - ± 12.7±1.9 (10.5-14.9)

Pharynx length / EP ratio 0.67 0.68±0.1 (0.57-0.77) 1.13±0.03 (1.10-1.17)

Spicule/gubernaculum ratio 2.6 2.4±0.2 (2.0-2.7) -

Tail/filament ratio 1.4 1.5±0.1 (1.4-1.6) -

Eggshell (length-width) - 90-94 x 29-31

Cervical alae absent. Lateral ala in the shape of a singular ridge (Fig. 2A). Anterior end blunt, ending in cephalic plate (Fig. 2A, B). Cephalic plate laterally elongated, separated from posterior cuticle by visible rim (Fig. 2B). Amphid openings slit-like, located on cephalic plate. Poorly visible traces of varicose ornamentation on median parts of cephalic plate present. Mouth opening triangular, with three protruding sectors (Fig. 2B). Caudal region with three pairs of papillae and protruding accessory hook. Internal structures of accessory hook not decorated. A pair of protruding papillae situated in precloacal region and a pair of smaller ones located in adanal position. Another pair of protruding papillae flanking the base of the tail filament. Spicule thin, distal tip pointed. Gubernaculum optically more transparent than spicule. Accessory hook shape variable (Fig. 1B-E).

Females. Four paratypes (Table 1). Body more or less curved ventrally. Body of gravid females with numerous eggs always strongly curved (Fig. 1F). Simple cephalic vesicle. Lateral ala as single 2 ^m thick and 5 ^m high ridge with two fields of smooth cuticle on both sides (Fig. 3E). Cervical alae absent. Intestine appears as non-transparent dark cord. Intestinal lumen visible only near rectum. Huge ventral cell, associated with excretory vesicle occupies main space between basal bulb and vagina. Cephalic plate laterally elongated, bearing four protruding cephalic papillae and slit-like amphidial openings (Fig. 3A, C). Prominent varicose ornamentation on cephalic end median surfaces

present. Mouth opening triradiate, surrounded by three protrusions of body and pharynx cuticle. Anterior margin of pharyngeal cuticle with minuscule teeth (Fig. 3B, D). Vulva aperture anteriorly displaced (Table 1). Majority of females without eggs. Specimens with eggs contain approx. 100 with eggshells fully formed. Eggs nearing vulva with a juvenile inside. Eggs 'D-shaped' with operculum at arched side. Eggshell covered with numerous tiny pits; operculum seam present (Fig. 3F). Tail long, pointed.

Immature female juveniles. Body shorter (L = 1075-2117 ^m), without formed reproductive system. Cephalic vesicle pronounced. Cell associated with excretory pore proportionally larger than in mature female (Fig. 1G). Intestine well developed, with thick walls filled with granules and prominent lumen.

Diagnosis. An analysis of the relationships of Syphacia ethiopiana sp. n. with congeners is facilitated by the in-depth analysis of cephalic and copulatory structures of the subfamily Syphaciinae by Quentin (1971) and comprehensive cladistic analysis of this subfamily by Hugot (1988). The genus Syphacia was divided into three subgenera by Hugot (1988), with subgenera Cricetoxyuris Hugot, 1988 and Seuratoxyuris Hugot, 1988 established to include the Syphacia species with several synapomorphies (developed lateral alae, ornamentation of the male accessory piece, cephalic plate extremely elongated in the lateral plane). Syphacia species with reduced or absent lateral alae,

100/100/92

100/100/96

100/100/100

100/100/100

100/100/100

Syphacia nigeriana Poland OKI 43567 Syphacia nigeriana Poland OK 143554 Syphacia nigeriana Russia MN652171 Syphacia nigeriana Portugal OK143553 Syphacia nigeriana UK OK 143552 Syphacia nigeriana UK OK143589 Syphacia nigeriana UK OKI43532 Syphacia obvelata MF142452 Syphacia obvelata Senegal OKI43549 Syphacia obvelata Senegal OK 143 548 Syphacia ethiopiana sp. n. Syphacia sp. Senegal OK 143586 Syphacia sp. Senegal OKI 43585 Syphacia sp. Mali OKI43590 Syphacia sp. Senegal OKI43584 Syphacia sp. Senegal OK 143583 Syphacia frederici Russia MN652172

Fig. 4. Phylogeny of closely related Syphacia species as inferred from analysis of ITS rDNA with three methods. Bootstrap support values over 60% are given in the format Maximum Parsimony / Neighbor Joining / Maximum Likelihood (T92 + G model).

Table 2. Pairwise nucleotide differences in the sequence of ITS rDNA between Syphacia spp. - beneath diagonal -absolute difference in bp, over the diagonal - as percentage (rounded value).

1. 2. 3. 4.

1. S. ethiopiana sp. n. - 6.0-6.7% 13.9-14.1% 13.0-13.8%

2. Syphacia sp. Senegal 40-42 - 13.0-14.7% 13.0-14.5%

3. S. obvelata 86-87 89-92 - 6.9-7.5%

4. S. nigeriana 81-86 81-88 43-47 -

laterally displaced cephalic papillae, three mamellons in males and a comparatively simple

organisation of the accessory piece were placed by

Hugot (1988) in the subgenus Syphacia with S. obvelata Seurat, 1916 as the type species. The differentiation of S. ethiopiana sp. n. from the species included by Hugot (1988) in this subgenus and those described since his revision of the subgenus is presented here along the biogeographical groups. Two species of the subgenus have a cosmopolitan distribution: S. obvelata (Rudolphi, 1802) from the common mouse Mus musculus L. and S. muris (Yamaguti, 1935) from Rattus spp. The eggshells of S. obvelata are significantly larger compared to S. ethiopiana sp. n. (118-153 pm x 33-54 pm vs 90-94 pm x 29-31 pm). Conversely, S. muris eggshells are smaller than those of S. ethiopiana sp. n. (72-82 pm x 25-36 pm vs 90-94 pm x 29-31 pm). A spicule is shorter in S. muris (42-54 pm) compared to S. ethiopiana sp. n. (58-80 pm). Lateral alae are absent in S. muris females but present in S. ethiopiana sp. n. (Fig. 3E).

Syphacia nigeriana Baylis, 1928 was originally described from various rodents in Nigeria and the Central African Republic. Recently, ribosomal and mitochondrial DNA analyses have confirmed reports of this species from various European countries (Behnke at al., 2022). These authors provided the data on eggshell size for different populations of this species, showing that eggshells of S. ethiopiana sp. n. are smaller than those of all populations of S. nigeriana studied. The other two African species of the subgenus Syphacia Hugot, 1988 are S. megaloon Quentin, 1966 and S. lophuromyos Quentin, 1966 from the Congolese Mus minutoides setulosus (Peters) and the Central African Lophuromys sikapusi Temminck, respectively (Quentin, 1966). The first of these African species is characterised by a very short body (< 2 mm) in females, very large eggshells (150 pm x 60 pm) and the absence of lateral alae, which are distinguishing features from S. ethiopiana sp. n. Syphacia lophuromyos is characterised by an extremely long tail in males (240 pm), a 90 pm wide

pharynx bulb in females and larger eggshells (110 * 48 pm). Syphacia ohtaorum Hasegawa, 1991 was described from Mus caroli in Okinawa, Japan (Hasegawa, 1991) and was considered to be morphologically closest to S. megaloon. This Asian species can be distinguished from S. ethiopiana sp. n. by its much larger eggshells 128-139 pm * 34-40 pm and shorter tail in females.

Several Syphacia species have been described from the Palearctic region. Syphacia stroma (von Linstow, 1884) from Apodemus hosts differs from S. ethiopiana sp. n. in having much larger eggshells (123-150 pm * 41-64 pm). Conversely, S. emileromani Chabaud, Rausch et Desset, 1963 from the Japanese endemic Apodemus sylvaticus argenteus Tem. is characterised by smaller eggshells (85 pm * 38 pm). The most prominent diagnostic feature of S. frederici Roman, 1945, another Apodemus pinworm, is wide lateral alae (up to 14 pm), not reported for congeners. Another species described from Japan, S. montana Yamaguti, 1943, which was later reported throughout the Palearctic, is also characterised (Ishimoto, 1974) by larger eggshells (100-110 pm * 29-37 pm).

The morphology of several Syphacia species has been studied by SEM (Wiger et al., 1978), which provides additional distinguishing features for S. ethiopiana sp. n. Prominent differences between this latter and S. stroma can be seen in the organisation of the cephalic plate and the structure of the cuticle surface in the neck region. The characteristic shape of the mouth opening in S. nigeriana (the presence of longitudinal median thickenings) is the additional distinguishing feature. In S. stroma triradial mouth opening is nearly reaching the cuticular collar, encircling cephalic plate (Wiger et al., 1978), whereas in other Syphacia species, including S. ethiopiana sp. n., the edges of the triradial mouth are separated from the cuticular collar with a strip of cephalic plate cuticle. The cuticle surface in the neck region of S. frederici is covered with typical small longitudinal ridges, which are not present in S. ethiopiana sp. n.

Syphacia arctica Tiner et Rausch, 1950 was described from the Alaskan Dicrostonyx groenlandicus Trail (Nearctic region). Syphacia arctica resembles S. ethiopiana sp. n. in the similar structure of the gubernaculum (simple, cuneiform, without internal seam) but differs in the longer spicules (80-94 pm vs 58-80 pm) and wider eggshells (38-39 pm vs 29-31 pm in S. ethiopiana sp. n.).

Of the three Syphacia species described from the Neotropics, S. ethiopiana sp. n. can be distinguished by a combination of features related to male copulatory structures and eggshell size. Thus,

S. alata Quentin, 1968 from Colombian rodents differs from the present species in the structure of the accessory piece (dentate vs non-dentate), the gubernaculum with prominent internal seam and eggshells 95-97 pm * 34-40 pm in size. Syphacia venteli Travassos, 1937 from the Brazilian Nectomys squamipes (Brandt) is characterised by lateral alae 10-11 pm wide and eggshells 78-82 pm * 32-33 pm in size, which distinguish it from S. ethiopiana sp. n. Another neotropical species, S. odilbainae Hugot et Quentin, 1985, was described from a cricetid rodent Zygodontomys brevicauda (Allen et Chapman) captured in Colombia (Hugot & Quentin, 1985). This species is characterised by a very short spicule (50 pm), and a long tail and tail filament (130 pm and 102 pm, respectively), vs 78107 pm and 49-73 pm in S. ethiopiana sp. n.

Syphacia darwini Hugot et Quentin, 1985, described from an Australian rodent Melomys cervinipes (Gould), is characterised by the presence of only two mamelons in males. It can be additionally distinguished from S. ethiopiana sp. n. by its wide basal bulb (100 pm), the long gubernaculum (40 pm) and males with a 245 pm long tail and 215 pm long tail filament (Hugot & Quentin, 1985).

Molecular characterisation. The partial sequence of SSU rDNA (705 bp long) of Syphacia ethiopiana sp. n. was found to be completely identical to one sequence (KY462826) of the type species of the genus - Syphacia obvelata (Rudolphi, 1802) Seurat, 1916. The latter corresponds to S. obvelata material, originating from Mastomys coucha, collected in South Africa (Julius, R.S., Zoology & Entomology, University of Pretoria). Two other rDNA sequences from Syphacia ethiopiana sp. n. were more informative and were identical between two localities (Menagesha Forest and Entoto Hill). The difference between this species and S. obvelata in the sequence of the LSU rDNA was at the level of 39 bp in the 686 bp long alignment (approx. 6%). Syphacia ethiopiana sp. n. clustered with strong bootstrap support (> 90%) in the LSU rDNA tree with S. obvelata, S. montana and S. frederici under all methods of analysis (data not shown). In comparison, the difference between such morphologically distinct species as S. obvelata, S. montana and S. frederici is 22-23 bp. The differences between S. ethiopiana sp. n. and S. montana in the same alignment were at the level of 44-45 bp. The differences in the ITS rDNA sequences were obvious (Table 2, Fig. 4). The closest species to S. ethiopiana sp. n. in this 630 bp long alignment was an unidentified species from Senegal and Mali, differing by 40-42 bp (Stewart et

al., 2022). However, the difference between these two Syphacia was at the interspecific level. Two other species that were compared (S. obvelata and S. nigeriana) differed from S. ethiopiana sp. n. in 86-87 and 81-86 bp, respectively.

Type locality. Ethiopia, Menagesha National Forest.

Another locality. Entoto Hill, Gullele Botanical Garden.

Type material. Holotype male, no. 1390, paratype male no. 1391, paratype female no. 1392 in the collection of the Centre of Parasitology SIEE. http://zoobank.org/urn:lsid:zoobank.org:pub:AB BC637B-2647-4917-BA73-B8EACE910599.

Taxonomic remarks. Syphacia ethiopiana sp. n. is very similar in general morphology, anterior end structure of both sexes (Quentin, 1971), eggshell and male posterior end to the type species of the genus, S. obvelata. Several dozens of nominal species have been described in the genus Syphacia (Hugot, 1988), but only thirteen species have at least one nucleotide sequence. Syphacia taxonomy and species identification can be greatly improved by the application of nucleotide sequence analysis (Stewart et al., 2018). Several loci are informative for species delimitation, but the set of known sequences is highly variable and differs between species studied. In our analysis, these are sequences of both LSU rDNA and ITS rDNA that differ significantly from those of other studied species of Syphacia. The level of these differences are on the level of reported intraspecific in the genus and support the establishment of separate species for S. ethiopiana sp. n. Syphacia populations from Senegal and Mali (Behnke et al., 2022), which were found to be the closest to S. ethiopiana sp. n. in ITS rDNA sequences, most likely represent another independent species. Compared to the average level of intraspecific differences in ITS rDNA among Syphacia species, these Senegalese and Malian populations of Syphacia sp. are not conspecific with S. ethiopiana sp. n. It should also be noted that these former Syphacia have been found in multimammate mice of the genus Mastomys (M. erythroleucus, M. huberti and M. natalensis) from West Africa. Considering that the allied genera Stenocephalemys and Mastomys belong to the same tribe Praomyini (Nicolas et al., 2021) we can assume some coevolutionary processes between representatives of this clade of African Syphacia and their hosts. Further studies are needed to support this hypothesis.

ACKNOWLEDGEMENTS

For help in the field and with logistics, we acknowledge A.M. Martynov, E.D. Zemlemerova, Mengistu Walle, and all local collaborators. We are

indebted to the Oromia Forest and Wildlife Enterprise (OFWE) for the permission to work in the Suba Menagesha National Park (permission no. OFWE 247333 from 15/05/2023) and to the Gullele Botanic Garden (GBG) for the permission to work at the Mount Entoto (permission from 23/05/2023).

Financial support of this this study was provided by the Ministry of Science and Higher Education of the Russian Federation in the frames Agreement no. 075-15-2023-591.

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А.Р. Громов, Л.А. Лавренченко и С.Э. Спиридонов. Syphacia ethiopiana sp. n. (Nematoda: Oxyurida: Syphaciinae) от эндемичного грызуна Stenocephalemys albipes Ruppell, 1842 из Эфиопии. Резюме. Оксиуридные нематоды из задних отделов кишечника эфиопского эндемичного грызуна - белоногой мыши Stenocephalemys albipes Ruppell, 1842 были исследованы в световом и сканирующем микроскопе и отнесены к новому виду рода Syphacia Seurat, 1916 - S. ethiopiana sp. n. У представителей нового виды выявлены морфологические признаки, сближающие их с Syphacia obvelata (Rudolphi, 1802) Seurat, 1916, типовым видом рода, паразитирующими у мышей рода Mus Clerck, 1757. Анализ нуклеотидных последовательностей ITS rDNA и LSU rDNA выявил различия межвидового уровня. Приводятся морфологические признаки, отличающие новый вид от ранее описанных видов подрода Syphacia (Syphacia). Приводятся результаты изучения тонкого строения поверхности кутикулы в сканирующем электронном микроскопе.