A new species, Bursaphelenchus ulmophilus sp. n., from the hofmanni group is described morphologically and molecularly. This nematode species was found associated with Dutch elm disease of Ulmus glabra in parks of St Petersburg, Russia, and is vectored by adults and larvae of the bark beetles Scolytus multistriatus and S. scolytus. Bursaphelenchus ulmophilus sp. n. is characterised by the following features: body length 600-850 μm, stylet 12-14 μm long with base slightly and smoothly expanded, but lacking knobs, median bulb almost spherical in female and slightly ovoid in male, pharyngeal gland lobe dorsal, 4-5 body diam. long. This species has an oval spermatheca filled with spherical nucleic sperm 4-5 μm diam. Female post-uterine sac ca 0.5 of the vulva-anus distance and ca 3 vulval body diam. long, female tail reflexed, strongly hooked ventrally with a digitate or conically rounded tip. The male has seven caudal papillae arranged as 1 + 2 + 2 + 2, P1 is unpaired, anterior to cloacal opening, paired P2 at cloacal aperture, paired P3 and paired pore-like 'gland papilla' P4 at the lateral edges of the bursa which has the posterior border rounded to truncate. Phylogenetic analyses of the D2-D3 of 28S rRNA, partial 18S rRNA and ITS rRNA gene sequences revealed that B. ulmophilus sp. n. formed a clade with species of the hofmanni group and shared close relationships with B. hofmanni and B. pinasteri.
Summary The copulatory organs and pharynx are important for nematode classification. This research aimed to study the musculature of the wood-inhabiting nematode, Rhabditolaimus ulmi, using confocal microscopy. The lip region is hexagonal with enlarged lateral lips; the pharynx is diplogastrid type with two bulbs. The corpus comprises the fused stegostoma, procorpus and metacorpus, with six glands opening in the stegostoma and the corpus limen. The stegostoma has a large dorsal bulge and the posterior bulb is devoid of valve, with two circles of dilators and a basal sphincter. The female genital muscles have a four-radial symmetry around the vaginal opening to the uterus. Two vaginal constrictors and four pairs of dilators of two types are connected to the vagina-uterus opening; four dilators run perpendicular to the uterus axis and are connected to the uterine muscular corset. The sphincter between the intestine and cloaca consists of the middle ring with two pairs of processes connected ventrally with intestinal fasciculi and dorsally with the body wall. The ring contains the inner biconical X-structure. In the male, the gubernaculum has the dorsal erector and a pair of transverse muscles. The wide gubernacular spicule protractor envelops spicules anteriorly. The ventral tail muscle is wide, unpaired, and nine pairs of serial diagonal muscles are located beneath the body wall muscles, followed by three pairs of postcloacal oblique muscles of the tail. Anal depressors comprise a pair of transverse muscles with expanded middle globular parts. The copulatory muscles are of the Rhabditidae and Cephalobidae type modified because of spicules elongation. The muscle characters code of the previously published tabular key for 15 muscle characters is 73422-22144-42223; it can be used for phylogenetic reconstructions.
Summary To develop models of forest ecosystem functioning, it is necessary to estimate parameters of the population dynamics of nematode species. Parameters of ontogenesis of Bursaphelenchus willibaldi from oak in culture of the fungus Botrytis cinerea were determined in vitro: oviposition began 2.7 (1-3) days after inoculation with adult nematodes; timelines of juvenile formation were: second-stage juveniles: 3.2 (3-4) days, third-stage juveniles: 3.6 (3-5) days, fourth-stage juveniles: 4.8 (3-6) days, first generation (1G) ended with a J4 moulting to adult after 5.8 (4-6) days. One day after the formation of the first generation, nematodes left the 2% potato-sugar agar (PSA) medium with the feed fungus and moved upwards, forming a migratory group (MG). In PSA the total nematode abundance reached peaks on day 11 (2G) and in the MG on day 27 (4.5G). The fecundity of females during population growth was determined as 3.4 (2.1-5.9) eggs day−1 and a model of exponential population growth was developed for total nematodes , ; and for females , . Under conditions of unrestricted reproduction and temperature of 21-23°C, the nematode and fungus can colonise 1 m3 of PSA-like medium in 36.8 days. The survival stage in the propagative generation was presumably J4. The female lifespan consists of a feeding and reproductive phase in the PSA (6 days) and a survival phase in the MG (1 month to 1 year). In MG, nematodes formed swarming groups of 20-200 individuals.
The parameters of individual development and population cycle in in vitro nematodes Panagrolaimus detritophagus were revealed. The nematodes are bacterial feeders and commensals of the cerambycid Monochamus galloprovincialis from the pine Pinus sylvestris; nematodes use beetles as vectors. Mean development time (T) from egg to juvenile is 1–2 days for J2, 3–4 days for J3, and 4–7 days for J4; to adults (G, generation) 7 (6–8) days. In vitro the population cycle is equal to 4 generations and ends with 90% of survival juveniles (J3, day 34). In the growth phase of the population, the proportion of eggs exceeds the proportion of other stages of the developmental cycle: 39±11% for 7 days; 53±10% for 21 days. The average oviposition rate of females is 4.5±1.3/day and only 56±12% of eggs proceed to immediate development (hatching and molting of juveniles). The remaining mass of eggs enter development only after 27 days (4 individual generations). This feature may be considered as a form of delay or a brief diapause at the egg stage. Individual females may accumulate up to 4 synchronous eggs in the body and lay them simultaneously. The average life span of an adult female is 13–20 days. Formulas for the exponential growth of the number of females and the total nematode population have been developed.
The endoparasitic nematode, Bursaphelenchus crenati, in beetle tunnels of Hylesinus crenatus from the ash Fraxinus excelsior L. with signs of ash dieback, is recorded for the first time in Central Russia and Belarus. Third‐stage dauer juveniles were extracted from the bark beetle, H. crenatus, and cultured on Botrytis cinerea. Morphological, morphometric and molecular analyses of these populations are presented here. Some morphological differences of dauers and adults were found between Belarusian and Russian populations, which may be attributed to seasonal variations. For description of new Bursaphelenchus species, it is recommended to indicate not only the number of lateral incisures but also the number of bands. Recommendations are made to classify the male caudal papillae of Bursaphelenchus into two groups: papilliform and gland papillae with separate numerals. Emended diagnosis, tabular diagnostic polytomous key for the species of Sexdentati group with lists of their vectors and plants, is given. Phylogenetic relationships between some species of the Sexdentati group are also presented based on the analysis of partial 18S, ITS and 28S rRNA gene sequences.
In this study juvenile stages of Bursaphelenchus crenati (Sexdentati group) were distinguished based mainly on the genital primordium structure. Dauer stage juveniles were sampled under elytra of Hylesinus crenatus in galleries in bark of the wilted Fraxinus excelsior in the Voronezh region of Russia and were multiplied in laboratory cultures on the fungus Botrytis cinerea. Individual development included five stages (J1-J4 and adults) separated by molts. The first molt J1 to J2 occurred inside the egg and the J2 was at the hatching stage. Sex of juvenile stages can be identified using the morphology and size of the genital primordia and a body size of nematodes. Sex of juveniles may be identified from the J3 stage by the presence of the cloacal primordium in male juvenile and orientation of the germinal zone of the genital primordium. A tabular key to developmental stages of B. crenati is given. The body grows during molts and within each stage. The body increases rapidly after J3 stage combined with the most active cellular differentiation and elongation of the genital primordium. The dauer juveniles collected from elytra of vector beetles were similar to the third juvenile stage of the propagative generation by genital primordium structure and body size.
The nematode Rhabditolaimus ulmi was found in galleries, adults, and larvae of Scolytus multistriatus, the vector of the Dutch elm disease, in St. Petersburg parks. This nematode cooccurred with Bursaphelenchus ulmophilus, which is another phoretic partner of S. multistriatus. Nematodes were cultured on the fungus Botryotinia fuckeliana in potato sugar agar (PA) and used for morphological analyses of adults, juveniles, eggs, and dauers. Nematode females showed a didelphic female genital tract rather than a monoprodelphic gonad as reported in the original description. Male bursa peloderan, caudal papillae include three preanal pairs and one precloacal unpaired papillae; seven postanal papilla pairs, among which one is pore-like and possibly the phasmid homolog, one subdorsal, and a pair of three closely situated posteriorly at bursa alae. The juvenile stages differ in size and structure of their sexual primordia. Sex of juveniles may be identified from the third stage. The dauer juvenile is a phoretic third juvenile stage (DJ3), which enters and remains localized in the buccal cavity of beetle adults and last-instar larvae and also under the elytra and in the ovipositor's cavity of pupae and imagoes. The first molt J1-J2 occurred inside the eggshell. Adult females laid eggs in early stages of embryonic development or containing molted J2. The propagative non-phoretic J2 inside the egg and J3 have a long and well-developed median bulb. The phoretic dauer DJ3 has a small spherical bulb like the J1 juvenile within the egg. In a sterile fungal culture, the nematodes feed on both mycelium and their unidentified ecto-symbiotic bacteria, located on nematode surface coat and multiplying in PA. Diagnosis and tabular key to the Rhabditolaimus species are given. Phylogenetic analysis of the D2-D3 of 28S rRNA gene sequences resulted in the Bayesian consensus tree with the highly supported clade of the Rhabditolaimus species.
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