Panagrolaimus detritophagus, Fuchs, 1930

Polyanina, K. S. & Ryss, A. Y., 2021, Parameters of ontogeny and population dynamics modeling of Panagrolaimus detritophagus (Nematoda: Rhabditida) in vitro, Proceedings of the Zoological Institute of the Russian Academy of Sciences 325 (1), pp. 91-98 : 92-95

Panagrolaimus detritophagus

Isolate origin: Russia, Nizhny Novgorod region, town Vyxa. GIS: 55.319100, 42.212830. Wood and bark of pine Pinus sylvestris L. Imagoes Monochamus galloprovincialis (Oliv., 1795) . Under elytrae and in folds between head and protorax. Sampling: July 2015; the laboratory nematode isolate was maintained in the Collection of living cultures of the Zoological Institute of the Russian Academy of Sciences (ZIN RAS) in sterile potato sugar agar medium (PSA) with an annual passage via multiplication in branches of the pine P. sylvestris .

Preparation of 2% potato sugar agar (PSA) medium

Forty g of peeled potatoes were cut into 1 cm 3 cubes, boiled for 40 min in 200 ml of distilled water, mashed with a press; the solution was filtered through two layers of gauze, poured into a heat-resistant container. Ten ml of glycerol, 4 g of sucrose and 4 g of agar were added to the hot solution, stirred until the reagents were dissolved, the volume of the solution was completed up to 200 ml, and sterilized for 40 min in an autoclave at 110°C and pressure of 3.5 atm. The autoclaved solution was poured at 80°C into 6 cm Petri dishes in a sterile microbiological box. The agar layer should be very thin (1–2 mm) for subsequent observation of nematodes.

Observations were carried out in a 1–2 mm PSA layer at room temperature of 21–23 (22)°C. The juvenile stages (2–4) of nematodes were identified by size ( Table 1). The morphology of juveniles and their molts was preliminarily studied; it was found that the first molt occurs in the egg shell, with three subsequent molts after hatching in the PSA medium. In addition to differences in body length, the juvenile stages differ in the size of the sexual primordium. The mature individuals were the largest, with external copulatory organs that were absent in juveniles: the spicules in males and the vulval structures in females.

Inoculation

Fifty most motile adults (females and males at a ratio of 30:20) were individually picked up with an entomological needle and transferred into a 20 µl drop of distilled water. They were washed twice in distilled water. In the sterile agar layer, a truncate pipette tip was used to make three depressions in the center of the Petri dish, then a 20 µl nematode suspension was introduced in these depressions. After 24 hours, bacteria from the surface of the nematode bodies multiplied and spread, forming white 1–3 mm spots on the surface of the agar medium; the nematodes fed at the periphery of the spots.

Observation of ontogenetic stages (from the egg)

After 1 to 3 days, the nematodes laid multiple eggs. Twenty eggs were pipetted into a new Petri dish with the PSA medium to monitor the course of individual development of juveniles and adult individuals. The experiment was performed in 10 replicates. When adult females were found, they were transferred to a new PSA medium to determine the timing of oviposition by new-generation females .

Observations on population dynamics

During the initial period (first generation), it is not difficult to count all nematodes and group them according to age-size stages, as well as to count the number of eggs in a thin layer of agar, for the entire Petri dish surface. However, when the nematode number increased significantly, accurate counting of subsequent generations becomes impossible. An alternative way to estimate the nematode densities using nematode extraction and counting in a sample of small partial volumes (see below) would disturb the microbiome conditions of copulation and egg production. Therefore, we used nematode counting in a random sample of 15 microscopic light fields, 3 cm 2 in size (10x microscope objective Mikmed-6 var-7., LOMO-Microsystems, https://lomo-microsystems. ru). Counting was performed through a thin layer of agar on the bottom lid, turning the Petri dish upside down. Thus we avoid opening the dish and disturbing the microbiome. The distribution of nematodes across the dish surface was characterized by aggregation around the spots of bacterial colonies. We delineated the areas of nematode concentration with a marker under a stereomicroscope Micromed MS-5- ZOOM LED and then counted nematodes in randomly selected 3 mm 2 light fields within those areas. The pattern of marked areas was photographed using a smartphone, and the total square value of the sum of areas was calculated using ImageJ 1.53e (https://imagej.nih.gov/ij/; National Institutes of Health , USA). Nematode numbers grouped by ontogenetic stage (eggs, J2, J3, J4, females and males) were summed for 15 randomly selected microscopic light fields and then these values were recalculated for the total surface of marked areas of aggregated nematode distribution. Population dynamics were traced in 13 Petri dishes at the following times starting from the moment of inoculation: 1, 2, 3, (4 or 5), 6, 7, 8, 9, 11, 14, 17, 21, 27 and 34 days .

Observations during the first generation

Introducing an inoculum synchronized to the adult stage and with a known number of females (30) makes it possible to calculate the time of one generation by the date of the first noticeable increase in the number of adult individuals. We denote the doubled number of adult females as an indicator of the end of one generation period; it means that all females of the inoculum, on average, laid at least one egg from which a female of a subsequent generation was developed. The date preceding the moment of adulthood of the subsequent generation makes it possible to calculate the total number of eggs laid for a single generation, an average egg production rate, as well as the proportion of eggs that proceed to hatching and a series of molts without delay (diapause).

Formulas for the day before the appearance

of females of the second generation (G-1)

The following formulas were used:

F(egg + jj) = [N(egg) + N(J2) + N(J3) + N(J4)]/ dd, where F (egg + jj) – mean fecundity of female per day; N(egg) – number of eggs on a counting date; N(J2)… N(J4) – number of juveniles of 2,3 and 4 stages on a counting date; dd – number of days from the moment of inoculation until the counting date (dd = G-1).

% Egg-Dev = [N(J2) + N(J3) + N(J4)]/[N(egg) + N(J2) + N(J3) + N(J4)], where % Egg-Dev – proportion of eggs, immediately starting to molt without a pause.

% Egg (diapause) = 1 − % Egg-Dev, where % Egg (diapause) – proportion of eggs with the arrested development (diapause).

Determination of the time of the end of the population cycle

When the number of females ceased to grow and the number of eggs decreased to rare finds, we considered that the population cycle was completed due to the exhaustion of food resources by the increased population. After counting in the light fields, we flushed the Petri dish with distilled water five times and poured the nematode suspension from a Petri dish into a 1.5 ml Eppendorf tube, then centrifuged the tube in a Microspin FV-2400 centrifuge-vortex (Biosan) at 2800 rpm; the supernatant was withdrawn with a syringe, the nematode suspension sediment was washed 5 times with distilled water and centrifuged in a vortex as described above, then fixed with hot TAF (4% formaldehyde with the addition of 2 ml triethanolamine per 100 ml solution as a buffer; in water bath with boiled water for 30 min) ( Ryss 2017a).

Two days after fixation date (time of hardening of the morphological structures of fixed worms), nematodes grouped by stage were counted in 10 drops of 20 µl volume to control the last count in the light fields in a Petri dish. The number of nematodes detected in 20 µl drop was recalculated to the entire 1.5 ml microtube volume, which corresponded to the number of nematodes in the Petri dish at the last count. Permanent collection slides were made from the nematode suspension by the express method of Ryss (2017b).

Statistical calculations were performed using the MS Excel software. Calculations of the mean and standard deviation of the sample were used; Student’s test (module “Student.test”) was used to assess the statistical significance values of difference between the samples.

Parameters and indices

Indices of individual development. G (egg-egg) is the duration of ontogeny (generation), time of individual development from egg to egg. Due to the developmental arrest of some (diapausing) eggs, this value varies greatly. Therefore, an equivalent parameter may be calculated: the average developmental time from a female of the previous generation to a female of a subsequent generation, G (fem-fem) (see below). T(J2), T (J3), T(J4), G-Female and G-Male is the timing of development from the egg to the corresponding subsequent stages: second, third, fourth stage juvenile and the nematode adult.

Indices of population cycle. Inoculum is the number of active adult females and males used to start the experimental culture in agar medium. The start of oviposition is the time (day) of the first egg laying by a group of inoculated females. H (J2..J4) is the time when the number of juveniles of the J2, J3 or J4 stage becomes equal to or exceeds the number of inoculat- ed females. G (fem–fem) is the day when the number of adult females exceeds at least twice the number of inoculated females. Pre-adult phase of the population cycle is the maximum period from the moment of inoculation when the number of females in the population is still equal to their number in the inoculum, but there are already numerous juveniles of different stages of the subsequent generation. Numbers of juveniles at this time were used to calculate the mean number of eggs produced by a female per day during the first generation as well as the percentage of eggs hatched without a developmental delay. Percentage of eggs proceeding to immediate reproduction (% Egg-Dev) and the residual index of % Egg-Dia = 1 − % Egg-Dev is the proportion of diapausing eggs.