Fast cycling culture of the annelid model Platynereis dumerilii

Abstract

Platynereis dumerilii, a marine annelid, is a model animal that has gained popularity in various fields such as developmental biology, biological rhythms, nervous system organization and physiology, behaviour, reproductive biology, and epigenetic regulation. The transparency of P. dumerilii tissues at all developmental stages makes it easy to perform live microscopic imaging of all cell types. In addition, the slow-evolving genome of P. dumerilii and its phylogenetic position as a representative of the vast branch of Lophotrochozoans add to its evolutionary significance. Although P. dumerilii is amenable to transgenesis and CRISPR-Cas9 knockouts, its relatively long and indefinite life cycle, as well as its semelparous reproduction have been hindrances to its adoption as a reverse genetics model. To overcome this limitation, an adapted culturing method has been developed allowing much faster life cycling, with median reproductive age at 13-14 weeks instead of 25-35 weeks using the traditional protocol. A low worm density in boxes and a strictly controlled feeding regime are important factors for the rapid growth and health of the worms. This culture method has several advantages, such as being much more compact, not requiring air bubbling or an artificial moonlight regime for synchronized sexual maturation and necessitating only limited water change. A full protocol for worm care and handling is provided.

Fig 1. The life cycle of Platynereis dumerilii.

The various life stages are depicted and classified based on the worm’s pelagic/benthic behavior. The mentioned time points correspond to those obtained at a thermostated temperature of 18°C [18]. The organism has a diameter of approximately 160 μm from the fertilized egg to 48 hpf. By 72hpf, the swimming larva measures around 250 μm in length. Around 6 dpf, upon starting feeding, it begins elongating through posterior segment addition. Immature worms reach 3–5 cm length. At the end of sexual metamorphosis, the worms become bulkier, contracting longitudinally by almost 50%.

Fig 2. Protocol overview for Fast Forward strain culturing in Platynereis dumerilii.

The Results section provides a detailed description of the overall progression towards selecting FF individuals and achieving the proposed protocol.

Fig 3. Commensals commonly found in Platynereis dumerilii’s culture boxes.

Cyanobacteria and filamentous green algae are constitutive of the mat. Dimorphilus, nematodes and ciliates feed at the surface of the mat. Dinoflagellates are free swimming above the mat.

Fig 4. Cumulative plots of mature worm count in each generation of the FF strain selection.

The columns are named as in Table 2. The density, food regime, and general statistics for each generation are described in Table 2. The dashed lines and red numbers indicate when 50% of the initially transplanted worms have matured (median maturation age). Blue numbers indicate the average age of worms selected to spawn the entire next generation.

Fig 5. Plots of biological controls and tests on the FF strain.

(A) Graph showing the effects of the density of transplanted young worms per box (at 10 dpf) on survival (green curve) and maturation. The maturation data is presented considering only the matures collected alive (blue curve) or including the dead matures found after weekends (red curve). (B) Comparison of the growth rate of FF culture worms when fed frozen algae (green) versus fresh algae (purple). To estimate growth, the length of tubes, covering the bottom of the boxes, and closely corresponding to worm length [21] was measured after two months using box photographs and ImageJ. The two populations do not conform to a normal distribution (Shapiro test). A Mann-Whitney test rejects their dissimilarity (p = 0.99). (C) Comparison of the weights of mature individuals between the FF and control (traditional culture) strains. Mature individuals were extracted from tubes before the release of gametes, gently wiped of excess water with a paper towel, and weighed on a precision balance. Each sex was compared separately as males are slimmer than females. The populations are not normally distributed (Shapiro test). The Mann-Whitney test rejects the similarity of FF and control weights for each sex (P < 0.001). (D) Comparison of the number of segments in mature individuals between the FF strain and control strain. Mature males and females have a fixed number of “thoracic” segments (15 and 22 respectively) and a highly variable number of “abdominal” segments. Animals were reversibly immobilized in a mix of 50% seawater-50% MgCl₂ 7.5% for 20 minutes to count segments. The populations are not normally distributed (Shapiro test). The Chi-square goodness of fit test rejects the similarity of FF and control segment numbers for each sex (p < 0.001). (E) Comparison of egg size between the FF and control strain. Several batches of eggs were sampled, and the diameter of the eggs was measured immediately after fertilization using micrographs and ImageJ. Specifically, only the largest diameter at the equatorial level, orthogonal to the animal-vegetal pole, was considered. The two populations appeared to be normally distributed, and a t-test was performed (p = 0.19) rejecting dissimilarity. (F) Regeneration capacity of the FF strain. FF and control strains were assessed by amputating a group of polymorphic and FF worms at the half-body level and allowing them to regenerate for 6 days. The number of new segment anlagen observed at the end of the 6-day period was used as an indicator of the worms’ regeneration capacity and speed. The two populations fit a Poisson distribution better and were compared with a Chi-square goodness of fit test (p = 0.68), rejecting dissimilarity.