Breeding of the sea spider Phoxichildium femoratum (Rathke, 1799): functional anatomy perspective

Breeding of the sea spider Phoxichildium femoratum (Rathke, 1799): functional anatomy perspective | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Breeding of the sea spider Phoxichildium femoratum (Rathke, 1799): functional anatomy perspective Maria Petrova, Ekaterina Bogomolova This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5804077/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Aug, 2025 Read the published version in Marine Biology → Version 1 posted 4 You are reading this latest preprint version Abstract Sea spiders (Pycnogonida) are one of two arthropod taxa that exhibit external fertilization, making them interesting subjects for the reconstruction of plesiomorphic arthropod breeding features. However, data on pycnogonid breeding are limited, particularly their fertilisation was not observed at all. Most knowledge on of their breeding is mere assumptions based on occasional observations. In this work, we observed and photo- and video- recorded the breeding behaviour (courtship, oviposition, fertilization, and eggmass formation) of Phoxichilidium femoratum (Rathke, 1799). Additionally, we studied eggmasses for the presence of sperm and additional cement. The male initiates mating by climbing onto the female’s back and courts her until oviposition. During oviposition, the male gonopores are in proximity to the eggmass, and sperm is injected into the swollen vitelline envelope surrounding the laid eggs. Once oviposition is complete, the male leaves the female hooking the eggmass with one of his ovigers. No additional shaping of the eggmass by the male was recorded, nor was there any fastening of the eggs with additional cement. The eggs in the eggmass are secured solely by swollen vitelline envelopes. Thus, fertilization in a mucous eggmass is undoubtfully demonstrated for the species (and likely characteristic of most sea spiders). Our observation questions the cementing function of male femoral glands, widely accepted in review literature. Also, basing on comparison of P. femoratum with other sea spiders we suggest a hypothesis on eggmasses evolution. Pycnogonida reproduction behaviour mating fertilization oviposition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Sea spiders (Pycnogonida) are one of two arthropod taxa that exhibit external fertilization. They are a sister group to Euchelicerata (Ballesteros et al. 2021; Sabroux et al. 2023; Sharma and Gavish-Regev 2025). The combination of their basal phylogenetic position and plesiomorphic fertilization mode makes them interesting for the reconstruction of ancestral chelicerate and arthropod breeding features. In review literature on Pycnogonida, it is claimed that males take oviposited eggs from the female, form eggmasses of various shapes, and carry these eggmasses with their specialized ovigerous legs until the hatching of larvae or even longer (Helfer and Schlottke 1935; Arnaud and Bamber 1988). Based on this information, femoral glands characteristic of most pycnogonid males were supposed to be cement glands that secrete glue for fastening the eggs in eggmasses. This assumption, proposed by Dohrn (1881), was later reproduced in subsequent works (Helfer and Schlottke 1935; Arnaud and Bamber 1988). However, the idea was supported only by a single observation (Nakamura and Sekiguchi 1980), while other studies describing pycnogonid mating do not mention the glands at all (Prell 1910; Burris 2011b). For most pycnogonids, motile flagellate sperm with an elongated head has been described (Arnaud and Bamber 1988). This sperm morphology is considered characteristic of fertilization in a mucous medium (Pitnick et al. 2009); however, the exact moment of fertilization in Pycnogonida has not been observed. There was an attempt to relate known details of pycnogonid breeding behaviour to pycnogonid phylogeny (Sabroux et al. 2023). However, the data on this aspect of their biology remain incomplete and insufficient for comparative analysis. Comprehensive descriptions of pycnogonid breeding behaviour, including the moment of oviposition, have been made only for three families, with a single species per family (Prell 1910; Nakamura and Sekiguchi 1980; Burris 2011b), and only one of these was documented with photographs (Nakamura and Sekiguchi 1980), while other works were limited to verbal descriptions or drawings. Despite the paucity of data, some generalizations about pycnogonid breeding behaviour have been attempted. Bain and Govedich (2004) suggested a classification of pycnogonid breeding behaviour types based on oviger morphology. Three morphological types of ovigers were identified: the unmodified “nymphonid” type, the slightly modified “ammotheid” type, and the highly modified “phoxichilidiid” type. For each of these types, “characteristic” breeding behavior is inferred based on a single detailed description, combined with occasional mentions made for other species (Bain and Govedich 2004a). However, ovigers of the same morphological type can occur in very different pycnogonid taxa with apparently different breeding behaviours and habitus. For example, “nymphonid type” ovigers with fully developed armature are characteristic of both P. longiceps , which makes egg-bracelets glued with cement (Nakamura and Sekiguchi 1980), and Colossendeidae, which fasten an amorphous eggmass to stones (Moran et al. 2024). “Phoxichilidiid type” ovigers are represented in both gracile Phoxichilidiidae and robust Pycnogonidae. In addition to anatomical differences (overall habitus, gonopore position), sperm structure in Phoxichilidiidae and Pycnogonidae also differs (King and El-Hawawi 1978; Petrova and Bogomolova 2024b). This implies differences in fertilization between these two families. While the breeding behaviour of Pycnogonum litorale (Strøm, 1762) has been satisfactorily described in Pycnogonidae (Prell 1910), reports of breeding in Phoxichilidiidae are merely occasional mentions (Hoek 1881; Cole 1901, 1906; Loman 1907). For this work, Phoxichilidium femoratum (Rathke, 1799) was chosen as a widely distributed and easily obtained phoxichilidiid species, whose reproductive system was studied during our previous work (Petrova and Bogomolova 2024a, b). We aim to describe and thoroughly document the breeding behaviour of this species, particularly the moment of fertilization and eggmass formation, with a focus on the functional explanation of reproductive system morphology (glands, gonopore position). Materials and methods 2.1 Collecting and keeping of animals Both male and female specimens of P. femoratum (Rathke, 1799) were procured from the sublittoral zone near the White Sea Biological Station of Moscow State University (66° 34′ N, 33° 08′ E). Animals were collected manually during low tide or through scuba diving. Specimens were obtained throughout their breeding season from June to August, 2021–2023. Upon arrival at the laboratory, sea spiders were examined for eggmasses or eggs in the femora and sorted by sex. Only males carrying eggmasses and females with mature eggs in their femora were chosen for the study. Eggmasses were peeled from the males' ovigers before subsequent observations. Animals were housed in 500 ml containers filled with filtered seawater, which was changed every 2–3 days, at a temperature of 8–12°C, with a shred of veiling fabric as a substrate. Males and females were kept separately, with no more than 30 specimens per container. Clava multicornis (Forsskål, 1775) was offered as food. 2.2 Morphometry To collect data on sexual dimorphism, 17 females and 15 males were relaxed in a 7% MgCl₂ solution mixed with filtered seawater at a 1:1 ratio. The specimens were straightened in a drop of the same solution on the bottom of a plastic Petri dish and fixed in position with a cover glass and pieces of plasticine as spacers. The animals were then photographed from the ventral side using an encoded stereomicroscope (Leica M165C) with a digital camera (Leica DFC420), washed in fresh seawater, and used for further experiments. The manipulations did not show any noticeable effect on the animals’ health or behaviour in our earlier observations. In the photographs, trunk length, coxae 2, femur, and tibia 1 lengths and widths were measured using Photoshop CS6. Trunk length was measured from the base of the proboscis to the posterior edge of the fourth segment, while the lengths of leg segments were measured from joint to joint, and widths at their widest points (Fig. 1b). Additionally, width-to-length ratios were calculated for each measured leg segment, as well as ratios of each leg segment’s length to trunk length and tibia 1 to femur length and width. For calculations and data visualisation MS Excel was used. 2.3 Observation of breeding bechaviour. Obtaining of freshly laid eggs. Observation of eggs development For the observation of the mating process and the collection of freshly laid eggs, animals were transferred into 200 ml containers with filtered seawater placed on ice, with 2-3 specimens of each sex per container and a piece of veiling fabric as a substrate. These groups were checked approximately every five minutes and kept together until the formed pairs finished mating or for 2-3 days. The mating process was observed using a stereomicroscope. For documentation, the animals were placed into a small (approximately 1 litre) aquarium filled with cold seawater. Photographs and videos of the process were taken with a Nikon D3300 camera equipped with a Nikkor Micro 60 mm lens and a Nikon SB700 speedlight. The aquarium was cooled with ice placed underneath, and the water temperature was maintained at about 10-12°C. To assess the possible frequency of mates for one male and one female, groups consisting of a single male/female with four specimens of the opposite sex were placed into 50 ml containers and checked for mating pairs and new eggmasses twice a day. Seven groups for each sex were formed, and the groups were observed for three weeks. Freshly laid eggs were collected using a plastic pipette as soon as the eggs appeared to check for the presence of spermatozoa under a microscope, to fix them for TEM observation of cement, and to observe whether the unformed eggmasses developed successfully. "Formed" eggmasses, one day post-fertilisation, were peeled from male ovigers for TEM observation of cement. Some males with eggmasses were left intact and kept in separate 50 ml containers until larval hatching to observe the condition of the eggmasses and to record the normal timing of development. The conditions for the males were the same as for the other adult animals. Eggmasses taken from males were kept in 20 ml containers filled with filtered seawater at 8-12°C and checked daily for development and general condition (presence of overgrowth, embryo mortality); water was replaced after each check. 2.4 Expirement on male femoral glands function To examine whether the male femoral glands have a reproductive function, an attempt was made to block their functioning. For the experiment, three groups of five males and five females were formed. In the first group, the dorsal side of the males’ femurs (where the glands open) was covered with cyanoacrylate glue. In the second group, the lateral side of the males’ femurs was covered with the glue, leaving the gland openings unblocked to test whether the glue layer on the femurs affects the rate of matings. The males in the third group were left intact. All three groups were kept under the conditions described above (section 2.2) and checked for new eggmasses and breeding pairs twice a day for three weeks. 2.5 Microscopy of egmasses Fragments of eggmasses were examined for sperm and matrix using a Leica DM2500 microscope equipped with a Leica DFC420С (5.0MP) digital camera. 2.6 TEM of eggmasses matrix For transmission electron microscopy (TEM), eggmasses were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer, which was diluted with filtered seawater in a 1:1 ratio, and left overnight at 4°C. After fixation, the samples were rinsed in the same buffer and postfixed with a 1% osmium tetroxide solution buffered with the same buffer for 1 hour at room temperature. They were then dehydrated using a series of graded ethanol solutions and acetone, and embedded in Epon (Epoxy Embedding Medium Kit – Fluka). Ultra-thin sections measuring 70-80 nm were cut with a diamond knife using a Leica UC6 ultratome, contrasted with uranyl acetate (4% aqueous solution) and lead citrate (1% aqueous solution), and examined using a JEOL JEM-1011 TEM equipped with an ORIUS SC1000W digital camera or a JEOL JEM-1400 Flash. 2.7 Artwork preparation Photographs of breeding animals, eggs and eggmasses was edited to improve contrast and brightness in Adobe Photoshop CS6. Individuals in breeding pairs were countered using Adobe Illustrator CC 2017. Schematic drawings were also prepared in Adobe Illustrator CC 2017. Data on morphometry and eggmasses per male number was visualized using MS Excel and then edited for better appearance in Adobe Illustrator CC 2017. Video recordings were sped up and converted using Adobe Premier Pro CC 2017. Results 3.1. Sex ratio, sexual dimorphism and fecundity in natural population The sex ratio in the population is approximately 1:1. P. femoratum exhibits clear sexual dimorphism. The absence of ovigers in males is characteristic of the family, and females are significantly larger than males (the difference is visually noticeable). The body proportions of males and females differ. The average body length is similar in both sexes; the larger size of females is attributed solely to their longer legs with larger femurs. However, males have longer and thinner second coxae than females (Fig. 1a). Breeding animals (gravid females and egg-carrying males) are observed from May to June. In June, a single male can carry up to 14 eggmasses, and up to 9 eggmasses on one oviger (as recorded in a male with only a single oviger), but this number decreases by the end of summer. In June, the average number of eggmasses per male is 6, although this number varies significantly (standard deviation 4). The distribution of the number of eggmasses per male is shown in Fig. 2. Eggmasses are roughly evenly distributed between the two ovigers. 3.2 Breeding behaviour, oviposition and fertilization, eggmasses formation Being placed in a common container animals pair within 0,5-1 h. No preceding specific behaviour was observed, males quickly detect females and crawl toward them. Then the male climbs on the back of the female and tries to position on the top of it. From the start of the courtship, the male sits head-to-head on the female and holds her first trunk processes by his ovigers (Fig. 3a, a’). Initially, male holds by its legs on female legs in random order and at random places or does not hold at all (Fig. 3a, a’). Then, male tries to fix his legs of 2nd – 4th pairs on the coxae3 of female’s 1st – 3rd legs (Fig. 3b, b’). Thus, the male sits on the back of the female so, that his 2nd – 4th legs over spaces between female’s legs (Fig. 3b–c’). The first pair of male’s legs remains free (Fig. 3c, c’). Female can counteract and try to push male’s legs in case she is not ready to spawn or disturbed (for instance, by too bright light) (Video S1). As the male successes to assume the right posture he stays in the position and about once per half a minute strains coxae squatting down (Video S2). The courtship can continue from 2-3 h to a couple of days. However, in case the courtship lasts more than 5-7 hours, it usually does not finish by spawning: the couple just breaks up. Sometimes, two males climb on the back of one female one under other. In such “stacks”, one of males remains and pairs with the female after a while (0,5-3 hours), however, no obviously aggressive actions between two males was observed. Sometimes the “competence” becomes too long (up to a couple of days) and finishes with unsuccessful mating (the “stack” breaks up). If the “compenetence” was won by one of males the other one can pair with the rest “free” female, but sometimes it apparently, is completely unattractive and do not pairs at all. After some time of courtship in the femurs of the female, peristaltic contractions of the vitellarium become noticeable (Fig. 4b, Video S3). Soon after that, the female strains her legs so that they form a dome and lays eggs inside the dome (Fig. 4b, b’; 5). During oviposition, the male remains in the same position as during courtship (Fig. 4a–b’): his legs are strained so that the coxae of the 2 nd to 4 th pairs of legs incline between the coxae of the female’s legs and press into the forming eggmass (Fig. 4a–c’). Thus, the male gonopores contact the eggmass but are widely spaced from the female ones (Fig. 5). The first pair of the male’s legs remains free, with its coxae far from the eggmass (Fig. 4a–b’). In the eggmass, taken at any moment during oviposition, spermatozoa can be found (Fig. 6); eggs develop being taken from the pair at any moment of oviposition. As oviposition finishes, the male releases his ovigers and leans forward (Fig. 7a, a’). He then hooks the eggmass with one of the ovigers (Fig. 7b, b’) and pulls it forward (Fig. 7b–d). At the same time, the female pulls backward (Fig. 7C–D). The eggmass separates from the female and remains on the male’s oviger (Fig. 7d). In this way, the most recent eggmasses appear on the male’s ovigers distally to older ones. After spawning, the male takes no further action with the eggmass. The day after oviposition, the mucous vitelline envelopes gluing the eggs slightly condense; the eggmass compacts but remains loose (Fig. 8a). Some eggs from the edge of the eggmass fall off and remain on the bottom of the container. In eggmasses taken from the pair either during oviposition or after it, as well as a day after oviposition, the matrix surrounding the eggs is uniform and composed of swollen vitelline envelopes (Fig. 8b, c). No additional matrix was found; the eggs are widely spaced and arranged irregularly (Fig. 8a, b). The eggmasses are roughly teardrop-shaped, with a thin “handle” where they are threaded onto the oviger (Fig. 8a). It appeared impossible to video record the departure of the male, as the constant bright light necessary for the recording frightened the animals, causing the female to try to crawl away from the light spot instead of pulling in the direction opposite to the male’s pulling. On the other hand, the flashlights used during time-lapse photography (one every 1-1.5 minutes) did not affect their behaviour. For the same reason (constant bright light disturbs animals), video recordings of the entire breeding behaviour are impossible; however, small video fragments of oviposition and courtship can be captured without disrupting the process. During the entire process of oviposition and afterwards, the male’s femurs are widely spaced from the eggmass being formed (Fig. 4, 5, 7). No manipulations that could be interpreted as transferring the secretions from the femoral glands to the eggmass were observed. Males with freshly laid eggmasses can regularly be found courting a new female in a tank containing both sexes. Obviously, males are ready for the next spawning almost immediately after finishing the previous one and can obtain several (in our observations, 2-3) eggmasses a day if enough females are available. However, in a common container with numerous specimens of both sexes, the distribution of obtained eggmasses varies greatly between males (0-3 per day), and it is unclear whether this is due to the need for rest for the male or its unattractiveness to females (or the absence of females that are attractive to the male). In containers with one female and several males, new eggmasses appeared 5-8 days after the previous spawning. Unsuccessful courtships were not detected in such containers between eggmass formations. Coating the male’s femoral glands with cyanoacrylate glue demonstrated no effect on breeding activity and eggmasses shape. Discussion 4.1. The mechanism of oviposition In our video recordings of courted P. femoratum females, peristaltic contractions of the vitellarium wall are undoubtedly discernible before and during oviposition. This observation supports our earlier suggestion regarding the transport of mature eggs by the muscles of the vitellarium wall (Petrova and Bogomolova 2024b). The midgut diverticulum remains motionless during oviposition and, evidently, does not participate in the protraction of the eggs being laid. The opposite was described for Propallene longiceps (Bohm, 1879) (Nakamura and Sekiguchi 1980), but the source of the discrepancy (whether it is due to interspecies variation or observational error) is unclear (elaborated in Petrova and Bogomolova 2024a). 4.2. When fertilization happens? In P. femoratum , fertilisation undoubtedly occurs while oviposition is in progress, as can be concluded from the presence of spermatozoa in an eggmass taken from a breeding pair at any moment during oviposition, and the fact that eggs develop when removed at any time during the formation of the eggmass. The same is most likely true for P. longiceps , whose eggs also develop when removed during oviposition (Nakamura and Sekiguchi 1980). In both cases, the male's coxae 2 with gonopores are in contact with the forming eggmass during oviposition, thus allowing sperm to be injected into the swollen mucous vitelline envelopes gluing the eggs. The structure of the spermatozoon in P. femoratum also corresponds to those proposed for fertilisation in the mucous eggmass (Petrova and Bogomolova 2024b). The mobile flagellate spermatozoon with an elongated head is most commonly described for Pycnogonida (van Deurs 1974; El‐Hawawi and King 1978, 1983), suggesting that fertilisation in a forming mucous eggmass also occurs in Nymphonidae and Ammotheidae. Despite the similar spermatozoon morphology and likely the same mode of fertilisation in Ammotheidae, it has been shown for Achelia simplissima (Hilton, 1939) that eggmasses taken from a mating pair during oviposition fail to develop, implying that fertilisation occurs after oviposition (Burris 2011b). However, this conclusion regarding later fertilisation in the species may be due to observational error, as eggmasses taken from males quickly become overgrown by fungi and bacteria and die before larvae hatch (personal observation on P. femoratum , Nymphon spp., Pseudopallene spinipes (Fabricius, 1780)). Direct observation of spermatozoa in the eggmass and developing eggs is necessary for an accurate conclusion on the timing of fertilisation. In P. litorale , a divergent filiform aflagellate spermatozoon has been described (King and El-Hawawi 1978), suggesting differences in fertilisation in this species. The position of gonopores in a mating pair of P. litorale differs from those in P. femoratum . In P. litorale , the female's gonopores are positioned only on the fourth walking legs and are shifted dorsally; the male's gonopores are located on the ventral side of the fourth walking legs. In a mating pair, the male gonopores touch the female ones, making fertilisation as close to internal as possible (Prell 1910). Unfortunately, data on spermatozoon structure are available only for a small part of pycnogonid diversity, making hypotheses about the evolution of their fertilisation biology speculative. 4.3. Eggmasses formation In our observations on P. femoratum , neither the male nor the female shapes the eggmasses. For A. simplissima , males were observed tightening eggmasses taken from the female with their ovigers (Burris 2011b), and in P. longiceps , males cement eggs into egg bracelets (Nakamura and Sekiguchi 1980). Additionally, a male grooming and compacting an eggmass was recently reported for Colossendeis megalonyx Hoek, 1881 (Moran et al. 2024). On the other hand, males of P. litorale also do not shape their eggmasses with ovigers (Schmidt and Bückmann 1971). In Ammotheidae, Callipallenidae, and Colossendeidae, ovigers are present in both sexes and have rows of spines (“combs”) on their distal segments, while the “phoxichilidiid type” ovigers characteristic of P. femoratum and P. litorale are present only in males and lack comb spines (Bain and Govedich 2004a). Thus, as suggested by Bain and Govedich (2004a), the shaping of the eggmass may be functionally related to the morphology of the ovigers. However, Pycnogonidae and Phoxichilidiidae are not sister clades according to current pycnogonid phylogeny (Ballesteros et al. 2021; Sabroux et al. 2023), and other details related to their breeding (gonopore position, sperm structure) differ drastically (discussed in section 4.2.). Freshly laid eggs of P. litorale appear on the dorsal side of the female; their transition and eggmass formation are apparently facilitated by the cuticle structure (Schmidt and Bückmann 1971). In contrast, in P. femoratum , eggs are oviposited on the ventral side of the female, and the eggmass is formed by pressure from the proximal segments of the legs, composing a “dome.” Thus, upon closer inspection, the mechanisms of eggmass formation in P. femoratum and P. litorale also differ. Consequently, either the unification of Phoxichilidium ’s and Pycnogonum ’s ovigers into the same type is inaccurate, or oviger morphology does not strictly correlate with breeding behaviour. In the first case, the classification of ovigers needs to be refined, considering not only the number of segments and the presence/absence of spines but also the size of the ovigers relative to the whole body. Clarifying the probability of the second case requires observations of the breeding behaviour of other Pycnogonida. Eggmasses of irregular shape have been described in different pycnogonid families distributed across the entire pycnogonid tree: Pycnogonidae (Prell 1910; Dogiel 1913; Schmidt and Bückmann,1971), Phoxichilidiidae (Dogiel 1913; present work), Ammotheidae (Berry 1980; Burris 2011b), and Colossendeidae (Moran et al. 2024), while a regular arrangement of eggs in eggmasses has been mentioned only for Nymphonoidea (Dogiel 1913; Nakamura and Sekiguchi 1980), which are nested within the crown of the pycnogonid tree. In all pycnogonids with described oviposition, eggs are primarily laid as an amorphous mass (Prell, 1910; Schmidt and Bückmann, 1971; Nakamura and Sekiguchi, 1980; Brenneis and Wagner, 2023; Moran et al., 2024; present work). Thus, it is likely that an amorphous eggmass is a plesiomorphic state for Pycnogonida, while its compaction is apomorphic. 4.4. Male femoral glands function In males of most sea spiders, glands positioned in the femora and, sometimes, in the tibiae of the walking legs were described. These glands are commonly believed to produce a cement that fastens eggs into an eggmass (Helfer and Schlottke 1935; Arnaud and Bamber 1988). However, actions that can be unambiguously interpreted as cementing eggs into the eggmass have only been described once for P. longiceps (Nakamura and Sekiguchi 1980), while for the other species, the function of the femoral glands remains merely an assumption. In P. femoratum , femoral glands are also present (Loman 1907; Petrova and Bogomolova 2024b). Nonetheless, no actions that could be interpreted as the transfer of the femoral glands’ secretion to the forming eggmass were observed during matings of P. femoratum . Moreover, the glands open on the dorsal side of the femur, which is far from the eggmass throughout the oviposition process. Additionally, it is difficult to imagine that the openings of the femoral glands could touch the eggmass or be reached by the male’s ovigers to transfer their secretion to the forming eggmass due to pycnogonid anatomy; furthermore, no substance apart from swollen vitelline envelopes was found in the formed eggmass. Thus, the cementing function of the male’s femoral glands is unlikely in P. femoratum . However, the exact function of the male’s femoral glands remains unclear. As sexually dimorphic structures, they are probably related to reproduction. However, covering the gland openings with cyanoacrylate glue does not affect mating rates and eggmasses shape. Two alternative explanations for the absence of this effect exist: 1) the gland secretion is unnecessary for successful breeding (at least under the conditions of the experiment); 2) the secretion of the glands is a substance with a molecular weight low enough to penetrate through polymerised cyanoacrylate, thus covering the gland openings does not block them. In the first case, a fungicidal and bactericidal function can be suggested because eggmasses carried by the male are not overgrown by bacteria and fungi, in contrast to those taken from the male and kept separately. However, these two explanations are not mutually exclusive, leaving us with a demand for further study of the composition of the gland secretion and partner search in P. femoratum to clarify the role of the male’s femoral glands. Additionally, the ambiguity of the function of male glands in P. femoratum and the diversity of “cement” gland positions among pycnogonids (Dohrn 1881), combined with the variety of eggmass shapes, call into question the generally accepted cement function of these glands and necessitate functional research on different species to draw accurate conclusions. 4.5. Who initiates the mating? Initially, the observation of mating choices was not intended in this work; thus, experiments revealing mating biases were not planned. However, this issue is the main focus of most other studies on sea spider breeding. Therefore, some thoughts on the topic, induced by the literature and based on our qualitative observations, are elaborated below. Under laboratory conditions, sea spiders often crawl over one another without regard to the sex of the individuals and apparently without any mating implications. No specific behaviour that could be interpreted as initiation of mating by a female was detected. No obvious female-female competition or aggression was observed. Unsuccessful courtships involving a resisting female or two males courting the same female imply that the initial movement could not have been made by the female. Signs of male initiative were also noted for P. longiceps (Nakamura and Sekiguchi 1980). In contrast, the initiation of contact by the female was reported for Propallene saengeri Staples, 1979 (Bain and Govedich 2004b), A. simplissima (Burris 2011b), and Achelia sawayai Marcus, 1940 (Berry 1980). For the P. saengeri , even fights between competing females were described. For Achelia , specific female actions interpreted as attracting male attention were recorded (Berry 1980; Burris 2011b). The only mention of female-female competition for mates is extremely vague and based on a single observation (Bain and Govedich 2004b). In all cases, the action that can be unambiguously interpreted as the start of mating is the climbing of the male onto the female’s back (Berry 1980; Nakamura and Sekiguchi 1980; Burris 2011b). Thus, the decision to mate is apparently made by the male. Genetic studies do not reveal higher competition among males or females for mates, demonstrating that the limiting factor for mate acquisition is encounter probability (Barreto and Avise 2008, 2010, 2011; Burris 2011b). Thus, in different species, one partner or another can refuse to mate. In our observations on P. femoratum , females can actively resist males’ courting attempts, while in Achelia , males may ignore females’ pursuits, and unsuccessful courting was not reported (Berry 1980; Burris 2011b). One possible interpretation is that the moment of female choice can vary between the studied species. However, there is a potential source of observational error. In our experiments, animals were maintained in the laboratory, and all observations were made at high population densities (100-200 animals per m²), whereas the approximated natural population density for the species is less than 20-30 animals per m². Although natural population densities are noted for both Achelia studied, those maintained for observation are not (Berry 1980; Burris 2011b). Nothing is known about the population density of P. longiceps , either natural or experimental (Nakamura and Sekiguchi 1980). Consequently, it cannot be excluded that the male initiative observed in our experiments and those by Nakamura and Sekiguchi is an effect of overpopulation and that at natural densities gravid females may seek out and follow mature males. Further observations considering natural population density are necessary to definitively clarify which sex initiates mating. 4.6. Sexual dimorphism in P. femoratum Sexual size dimorphism in P. femoratum is quite pronounced; females are significantly larger than males due to their longer legs with larger femurs. However, males have longer coxae 2. It seems very likely that the larger size of females provides extra space for vitellogenesis, while the longer coxae 2 in males are necessary to position the male’s gonopores in proximity to the forming eggmass. A very similar dimorphism has been described for Ammothea hilgendorfi (Böhm, 1879) (Ammotheidae); the main difference is that in this species, males have a wider trunk, while dimorphism of coxae 2 was not mentioned (Barreto and Avise 2008). Both A. hindeldorphi and P. femoratum exhibit a similar type of post-embryonic development. Both have endoparasitic larvae and produce numerous small eggs (Brenneis et al. 2017). Such breeding ecology implies high fecundity selection (Ramirez Llodra 2002). Consequently, the noticeably larger size of females, provided by reproductively significant parts, is most obviously explained by fecundity selection. However, considering the costs of male brood care demonstrated in A. simplissima (Burris 2011a), selection for smaller male size cannot be excluded either. Declarations Supplementary materials Video S1 Video S1 Female resisting the courting male. Female tries to peel off male’s legs with her walking legs. The video is 5 times accelerated. Video S2 Video S2 Male courting a female. Regular “squatting down” of a courting male is demonstrated. The video is 5 times accelerated. Video S3 Video S3 Peristaltic of the ovary. On the video is an oviposing pair, the femur of the female where the peristaltic waves are clearly discernible is encircled. The video is 5 times accelerated. Acknowledgements Authors are thankful to A. Semenov and other divers of Pertsov White Sea Biological Station for their assistance with sampling. We are grateful to A. Lavrov and F. Bolshakov for their technical support and opportunities at the Center of microscopy, WSBS, MSU. Also we want to thank the Electron Microscopy Laboratory of the Shared Facilities Center of Lomonosov Moscow State University, sponsored by the RF Ministry of Education, Science and Research. Author contributions All authors contributed to the study conception and design. Ekaterina Bogomolova supervised and discussed the work. Maria Petrova conceptualized and conducted the research. The first draft of the manuscript was written by Maria Petrova, Ekaterina Bogomolova commented the previous manuscript. All authors read and approved the final manuscript. Funding The study was supported by the scientific project of the state order of the government of Russian Federation to Lomonosov Moscow State University #121032300121–0 Data availability Data will be provided under request. Competing interests The authors have no relevant financial or nonfinancial interests to disclose. Ethics approval This work did not require ethical approval from an animal welfare committee. Generative AI and AI-assisted technologies in the writing process During the preparation of this work the author(s) used ChatGPT in order to improve language and readability. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication. References Arnaud F, Bamber RN (1988) The biology of Pycnogonida. Advances in marine biology 24:1–96. https://doi.org/10.1016/S0065-2881(08)60073-5 Bain BA, Govedich FR (2004a) Courtship and mating behavior in the Pycnogonida (Chelicerata: Class Pycnogonida): a summary. Invertebrate Reproduction & Development 46:63–79. https://doi.org/10.1080/07924259.2004.9652607 Bain BA, Govedich FR (2004b) Mating behaviour, female aggression and infanticide in Propallene saengeri (Pycnogonida: Callipallenidae). The Victorian Naturalist 121:168–171. Ballesteros JA, Setton EVW, Santibáñez-López CE, Arango CP, Brenneis G, Brix S, Corbett KF, Cano-Sánchez E, Dandouch M, Dilly GF, Eleaume MP, Gainett G, Gallut C, McAtee S, McIntyre L, Moran AL, Moran R, López-González PJ, Scholtz G, Williamson C, Woods HA, Zehms JT, Wheeler WC, Sharma PP (2021) Phylogenomic Resolution of Sea Spider Diversification through Integration of Multiple Data Classes. Molecular Biology and Evolution 38:686–701. https://doi.org/10.1093/molbev/msaa228 Barreto FS, Avise JC (2008) Polygynandry and sexual size dimorphism in the sea spider Ammothea hilgendorfi (Pycnogonida: Ammotheidae), a marine arthropod with brood-carrying males. Molecular Ecology 17:4164–4175. https://doi.org/10.1111/j.1365-294X.2008.03895.x Barreto FS, Avise JC (2010) Quantitative measures of sexual selection reveal no evidence for sex-role reversal in a sea spider with prolonged paternal care. Proc R Soc B 277:2951–2956. https://doi.org/10.1098/rspb.2010.0311 Barreto FS, Avise JC (2011) The genetic mating system of a sea spider with male-biased sexual size dimorphism: evidence for paternity skew despite random mating success. Behav Ecol Sociobiol 65:1595–1604. https://doi.org/10.1007/s00265-011-1170-x Berry MB (1980) The embryological development of Achelia sawayai (Ammotheidae, Pycnogonida), with notes on some phases of the behavior and anatomy of the adult, and on the phylogenetic position of the Pycnogonida. Duke University Brenneis G, Bogomolova EV, Arango CP, Krapp F (2017) From egg to “no-body”: an overview and revision of developmental pathways in the ancient arthropod lineage Pycnogonida. Front Zool 14:6. https://doi.org/10.1186/s12983-017-0192-2 Burris ZP (2011a) Costs of exclusive male parental care in the sea spider Achelia simplissima (Arthropoda: Pycnogonida). Mar Biol 158:381–390. https://doi.org/10.1007/s00227-010-1566-6 Burris ZP (2011b) The polygamous mating system of the sea spider Achelia simplissima . Invertebrate Reproduction & Development 55:162–167. https://doi.org/10.1080/07924259.2011.557555 Cole LJ (1901) Notes on the habits of Pycnogonids. The Biological Bulletin 2:195–207. https://doi.org/10.2307/1535715 Cole LJ (1906) Feeding habits of the pycnogonid Anoplodactylus lentus . Zool Anz 29:740–741. Dogiel V (1913) Embryologische Studien an Pantopoden. Zeitschrift für wissenschaftliche Zoologie 107:575–741. El‐Hawawi ASN, King PE (1978) Spermiogenesis in a pycnogonid Nymphon gracile (Leach). J Submicr Cytol 10:345–365. El‐Hawawi ASN, King PE (1983) Spermiogenesis in a pycnogonid Achelia echinata Hodge. Acta Zoologica 64:227–233. https://doi.org/10.1111/j.1463-6395.1983.tb00804.x Helfer H, Schlottke E (1935) Pantopoda. In: Klassen und Ordnungen des Tierreichs. Akademische Verlagsgesellschaft m.b.h, pp 1–314 Hoek PPC (1881) Report on the Pycnogonida, dredged by H.M.S. Challenger during the years 1873–76. Challenger Rep Zool 3:1–167. King PE, El-Hawawi ASN (1978) Spermiogenesis in the pycnogonid Pycnogonum littorale (Ström). Acta Zoologica 59:97–103. https://doi.org/10.1111/j.1463-6395.1978.tb00116.x Loman JCC (1907) Biologische Beobachtungen an einem Pantopoden. Tijdschrift der Nederlandsche Dierkundige Vereeniging 2:255–84. Moran AL, Lobert GT, Toh MWA (2024) Spawning and larval development of Colossendeis megalonyx , a giant A ntarctic sea spider. Ecology 105:e4258. https://doi.org/10.1002/ecy.4258 Nakamura K, Sekiguchi K (1980) Mating behavior and oviposition in the pycnogonid Propallene longiceps . Marine Ecology Progress Series 2:163–168. https://doi.org/10.3354/meps002163 Petrova M, Bogomolova E (2024a) The female reproductive system of the sea spider Phoxichilidium femoratum (Rathke, 1799). Arthropod Structure and Development 81:101370. https://doi.org/10.1016/j.asd.2024.101370 Petrova M, Bogomolova E (2024b) The male reproductive system of the sea spider Phoxichilidium femoratum (Rathke, 1799). Arthropod Structure & Development 83:101404. https://doi.org/10.1016/j.asd.2024.101404 Pitnick S, Hosken DJ, Birkhead TR (2009) 3 - Sperm morphological diversity. In: Birkhead TR, Hosken DJ, Pitnick S (eds) Sperm Biology. Academic Press, London, pp 69–149 https://doi.org/10.1016/B978-0-12-372568-4.00003-3 Prell H (1910) Beitrage zur Kenntnis der Lebensweise einiger Pantopoden. Bergens Museums Aarbok 11:1–30. Ramirez Llodra E (2002) Fecundity and life-history strategies in marine invertebrates. In: Advances in Marine Biology. Elsevier, pp 87–170 https://doi.org/10.1016/S0065-2881(02)43004-0 Sabroux R, Corbari L, Hassanin A (2023) Phylogeny of sea spiders (Arthropoda: Pycnogonida) inferred from mitochondrial genome and 18S ribosomal RNA gene sequences. Molecular Phylogenetics and Evolution 182:107726. https://doi.org/10.1016/j.ympev.2023.107726 Schmidt H-W, Bückmann D (1971) Beobachtungen zur Lebensweise von Pycnogonum litorale (Ström) (Pantopoda). Oecologia 7:242–248. https://doi.org/10.1007/BF00345214 Sharma PP, Gavish-Regev E (2025) The Evolutionary Biology of Chelicerata. Annu Rev Entomol 70:144–63. https://doi.org/10.1146/annurev-ento-022024-011250 Tomaschko K-H, Wilhelm E, Bückmann D (1997) Growth and reproduction of Pycnogonum litorale (Pycnogonida) under laboratory conditions. Marine Biology 129:595–600. https://doi.org/10.1007/s002270050201 van Deurs B (1974) Spermatology of Some Pycnogonida (Arthropoda), with Special Reference to a Microtubule‐Nuclear Envelope Complex. Acta Zoologica 55:151–162. https://doi.org/10.1111/j.1463-6395.1974.tb00189.x Supplementary Files VideoS1.MP4.mp4 href="https://drive.google.com/file/d/1fy7NnsGij8IRJcCc880pul5f-M-NNyaw/view?usp=sharing"> Video S1 Video S1 Female resisting the courting male. Female tries to peel off male’s legs with her walking legs. The video is 5 times accelerated. VideoS2.MP4.mp4 href="https://drive.google.com/file/d/1c7bf1Gj7K9WJXgzv0FTESfJzyoTg3cI6/view?usp=sharing"> Video S2 Video S2 Male courting a female. Regular “squatting down” of a courting male is demonstrated. The video is 5 times accelerated. VideoS3.MP4.mp4 href="https://drive.google.com/file/d/1uq16GFb7g0d7NXvMJnmL7kdQpAsHp1wU/view?usp=sharing"> Video S3 Video S3 Peristaltic of the ovary. On the video is an oviposing pair, the femur of the female where the peristaltic waves are clearly discernible is encircled. The video is 5 times accelerated. Cite Share Download PDF Status: Published Journal Publication published 22 Aug, 2025 Read the published version in Marine Biology → Version 1 posted Reviewers agreed at journal 24 Jan, 2025 Reviewers invited by journal 24 Jan, 2025 Editor assigned by journal 13 Jan, 2025 First submitted to journal 10 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5804077","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":406838837,"identity":"394278d6-81bf-427d-8f2b-9c6e6b037891","order_by":0,"name":"Maria Petrova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYHACNsYGBMNGzr4dxDWwIFpLmrEBzwGQFgmitRxO3CCRAOLg1qLbfvzZwxk19+SgDGbG7ZLPr274USDBwN/enYBNi9mZHHPDDceKjaEMNmbL2TllN3uADpM4c3YDVi0HctgkH7AlJG6DMHjYGG7npN3gAWoxkMjFruX882eSD/4BtUAYEjwMN8+k3fyDT8uNBDPJjW1ALRCGgYTBDfZjt/HacuONmeTMvgRjGMNAsieH7baMgQQPTr+cT38m2fMtQQ7K+F/fz3782c03f2zk+Nt7sWrBBngMwCSxykGA/QEpqkfBKBgFo2D4AwBf0Wwh4TD4qgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0007-1272-2659","institution":"Lomonosov Moscow State University Faculty of Biology: Moskovskij gosudarstvennyj universitet imeni M V Lomonosova Biologiceskij fakul'tet","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"","lastName":"Petrova","suffix":""},{"id":406838838,"identity":"03a81b50-0e07-4917-bfe0-64b07b65e44b","order_by":1,"name":"Ekaterina Bogomolova","email":"","orcid":"","institution":"Lomonosov Moscow State University Faculty of Biology: Moskovskij gosudarstvennyj universitet imeni M V Lomonosova Biologiceskij fakul'tet","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Bogomolova","suffix":""}],"badges":[],"createdAt":"2025-01-10 13:31:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5804077/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5804077/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00227-025-04707-3","type":"published","date":"2025-08-22T16:29:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74949149,"identity":"af5076e1-a1a6-4fcc-a5cc-6f9ffe8519ea","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSexual size dimorphism.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e – body parameters measured and proportions of both sexes. Proportions are on grey background, body parameters on white. Error bars show standard error. While the trunk length is the same in males and females, males have longer coxae2, and females have larger legs. \u003cstrong\u003eb\u003c/strong\u003e – anatomy of \u003cem\u003eP. femoratum\u003c/em\u003e and measures made. The same parameters were measured for both sexes, but females have no ovigers and different gonopores position. Gonopores are marked with solid black circles. Abbreviations: c1, c2, c2 – coxae 1, 2, 3 correspondingly; ch – cheliphore; fem – femur; ov – ovigers; pb – proboscis; pr – propodus; ta – tarsus; ti1, 2 – tibiae 1,2 correspondingly; wl – walking legs\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/bbb068571e7a2ec0fe823d66.png"},{"id":74949586,"identity":"58ed96af-23f8-42e6-a4b7-967912d5ecac","added_by":"auto","created_at":"2025-01-28 16:05:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of eggmasses among males collected in June. \u003c/strong\u003eNumber of eggmasses per male varies between 1 and 14, the most popular number is 6. Sample size 50 males\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/6ad41faf10a07fbca5f978d2.png"},{"id":74949587,"identity":"1ba1868f-a752-498c-9eec-3f34f4683225","added_by":"auto","created_at":"2025-01-28 16:05:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8314623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCourtship.\u003c/strong\u003e \u003cstrong\u003ea, b, c \u003c/strong\u003e– live photos of courting males on the female’s back; \u003cstrong\u003ea’, b’, c’ \u003c/strong\u003e– colored male and female from photos \u003cstrong\u003ea, b, c \u003c/strong\u003ecorrespondingly; males are coloured yellow, females are purple. \u003cstrong\u003ea\u003c/strong\u003e – the beginning of courtship, male holds female by ovigers, but not walking legs; \u003cstrong\u003eb, c\u003c/strong\u003e – advanced stages of courting, male positioned in the final position holding the female with ovigers and walking legs, the 1\u003csup\u003est\u003c/sup\u003e pair of male;s walking legs remains free; \u003cstrong\u003eb\u003c/strong\u003e – side view; \u003cstrong\u003ec \u003c/strong\u003e– dorsal view. Abbreviations: f1–4 – female walking legs 1–4 correspondingly; fch – female cheliphore; fp – female proboscis; m1–4 – male walking legs 1–4 correspondingly; ov – oviger. Scale bars 1 mm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/497155a58a37fdc4c6434fc9.png"},{"id":74949157,"identity":"1193bdca-de89-4ca7-8f27-428bebb19109","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9696212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOviposition. a, b, c\u003c/strong\u003e – live photos of oviposing pairs; \u003cstrong\u003ea’, b’, c’\u003c/strong\u003e – colored male and female from photos \u003cstrong\u003ea, b, c\u003c/strong\u003e correspondingly; males are coloured yellow, females are purple. Female folds her legs forming a dome of its proximal parts and lays eggs, male presses his coxae 2 of legs 2–4 into the forming eggmass, 1\u003csup\u003est\u003c/sup\u003e walking legs remain free. \u003cstrong\u003ea\u003c/strong\u003e – dorsolateral view; \u003cstrong\u003eb\u003c/strong\u003e – lateral view, male’s 2\u003csup\u003end\u003c/sup\u003e walking leg is amputated; \u003cstrong\u003ec\u003c/strong\u003e – ventrolateral view. Green dots mark approximate position of male femoral gland’s openings. Abbreviations: em – eggmass; f1–4 – female walking legs 1–4 correspondingly; fch – female cheliphore; fp – female proboscis; m1–4 – male walking legs 1–4 correspondingly; ov – oviger; pw – peristaltic waves. Scale bars 1 mm\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/01c8152ca7df12616b67a3a6.png"},{"id":74949154,"identity":"9d7526ec-2a88-4e91-88cf-9db94232b871","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOviposition, schematic drawing.\u003c/strong\u003e A pair of animals during oviposition and fertilization is shown with a half removed revealing gonopores position. Male gonopores are in the proximity of the eggmass, but fare from the female ones. Male is yellow, female is purple male gonopores are shown as small solid black circles, female gonopores – as big solid black circles. Green dots mark approximate position of male femoral gland’s openings. Abbreviations: ch – cheliphore; em – eggmass; f1–4 – female walking legs 1–4 correspondingly; m1–4 – male walking legs 1–4 correspondingly; ov – oviger; p – proboscis\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/f09ee31bdf08f05e1a80d05c.png"},{"id":74949588,"identity":"d96f7692-4c15-46c0-ba65-e6d8e7b62fb2","added_by":"auto","created_at":"2025-01-28 16:05:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14000308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFertilisation.\u003c/strong\u003e A micrograph of a fragmeng of an eggmass, taken from an oviposing pair. Spermatozoa in the mucous matrix are encircled with dash lines; white arrowheads mark the edge of vitelline envelopes on the edge of the fragment. Abbreviarions: egg – egg. Scale bar 50 µm\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/d60eaad13430c8eb3e35ad38.png"},{"id":74949160,"identity":"61d3b180-35b0-4b6c-9c15-87ba45b67925","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10658707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMale’s leaving.\u003c/strong\u003e \u003cstrong\u003ea, b, c, d\u003c/strong\u003e– live photos from a time-lapse series of a breaking pair; \u003cstrong\u003ea’, b’\u003c/strong\u003e – colored male and female from photos \u003cstrong\u003ea, b\u003c/strong\u003e correspondingly; males are coloured yellow, females are purple. \u003cstrong\u003ea\u003c/strong\u003e – the beginning of the leaving, male frees his ovigers and leans forward; \u003cstrong\u003eb\u003c/strong\u003e – male hooks the eggmass with his left oviger; inset demonstrates enlarged fragment with the oviger hooking the eggmass; \u003cstrong\u003ec\u003c/strong\u003e – male pulls the eggmass forward, female in the opposite direction; \u003cstrong\u003ed\u003c/strong\u003e – eggmass separates from the female and the male leaves; inset demonstrates enlarged fragment with the oviger hooking the eggmass. Green dots mark approximate position of male femoral gland’s openings. Abbreviations: em – eggmass; f1-4 – female walking legs 1-4 correspondingly; fch – female cheliphore; m1-4 – male walking legs 1-4 correspondingly; ov – oviger. Scale bars 1 mm\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/656ad970a15609450102e73a.png"},{"id":74949156,"identity":"21243026-2067-4f9a-b0ee-a4a5fef12c73","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11910477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFormed eggmass. a\u003c/strong\u003e – an eggmass a day after oviposition; white arrow shows the hole made by oviger; \u003cstrong\u003eb\u003c/strong\u003e – live micrograph of a fragment of an egmass 9 hours after oviposition; homogenous matrix surrounds the eggs; the edge of the eggmass is marked with white arrowheads; \u003cstrong\u003ec\u003c/strong\u003e – TEM micrograph of the matrix surrounding eggs in an eggmass; black arrowheads mark the contact between vitelline envelopes of two eggs. Abbeviations: egg – egg; ven – vitelline envelope. Scale bars: \u003cstrong\u003ea\u003c/strong\u003e – 500 µm; \u003cstrong\u003eb\u003c/strong\u003e – 50 µm; \u003cstrong\u003ec\u003c/strong\u003e – 1 µm\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/b04dee4391296f33296bc9e8.png"},{"id":89847240,"identity":"f4166689-0a12-4a5c-b8df-bbeb438aa5d5","added_by":"auto","created_at":"2025-08-25 16:42:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":50586297,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/e3ca05ac-68a0-45d8-b178-bc00131dbd1a.pdf"},{"id":74949152,"identity":"a1590341-19b0-4b35-a51d-dd85d5d7ba55","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17366968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003ca href=\"https://drive.google.com/file/d/1fy7NnsGij8IRJcCc880pul5f-M-NNyaw/view?usp=sharing\"\u003e\u003cem\u003e\u003cstrong\u003eVideo S1\u003c/strong\u003e\u003c/em\u003e\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S1 Female resisting the courting male.\u003c/strong\u003e Female tries to peel off male’s legs with her walking legs. The video is 5 times accelerated.\u003c/p\u003e","description":"","filename":"VideoS1.MP4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/8f64a9e2ee21c8d5702b8cbc.mp4"},{"id":74949158,"identity":"bb67662c-cae8-438a-bc26-8ccde40b0c33","added_by":"auto","created_at":"2025-01-28 15:57:28","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31611328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003ca href=\"https://drive.google.com/file/d/1c7bf1Gj7K9WJXgzv0FTESfJzyoTg3cI6/view?usp=sharing\"\u003e\u003cem\u003e\u003cstrong\u003eVideo S2\u003c/strong\u003e\u003c/em\u003e\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S2 Male courting a female.\u003c/strong\u003e Regular “squatting down” of a courting male is demonstrated. The video is 5 times accelerated.\u003c/p\u003e","description":"","filename":"VideoS2.MP4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/f6e5a64574be6e3bc0214986.mp4"},{"id":74949161,"identity":"b410e62b-5ea7-430b-9a3a-e4af5d750d5d","added_by":"auto","created_at":"2025-01-28 15:57:29","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18252927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003ca href=\"https://drive.google.com/file/d/1uq16GFb7g0d7NXvMJnmL7kdQpAsHp1wU/view?usp=sharing\"\u003e\u003cem\u003e\u003cstrong\u003eVideo S3\u003c/strong\u003e\u003c/em\u003e\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S3 Peristaltic of the ovary.\u003c/strong\u003e On the video is an oviposing pair, the femur of the female where the peristaltic waves are clearly discernible is encircled. The video is 5 times accelerated.\u003c/p\u003e","description":"","filename":"VideoS3.MP4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804077/v1/ef06cf332f9f7433e45c8437.mp4"}],"financialInterests":"","formattedTitle":"Breeding of the sea spider Phoxichildium femoratum (Rathke, 1799): functional anatomy perspective","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSea spiders (Pycnogonida) are one of two arthropod taxa that exhibit external fertilization. They are a sister group to Euchelicerata (Ballesteros et al. 2021; Sabroux et al. 2023; Sharma and Gavish-Regev 2025). The combination of their basal phylogenetic position and plesiomorphic fertilization mode makes them interesting for the reconstruction of ancestral chelicerate and arthropod breeding features. In review literature on Pycnogonida, it is claimed that males take oviposited eggs from the female, form eggmasses of various shapes, and carry these eggmasses with their specialized ovigerous legs until the hatching of larvae or even longer (Helfer and Schlottke 1935; Arnaud and Bamber 1988). Based on this information, femoral glands characteristic of most pycnogonid males were supposed to be cement glands that secrete glue for fastening the eggs in eggmasses. This assumption, proposed by Dohrn (1881), was later reproduced in subsequent works (Helfer and Schlottke 1935; Arnaud and Bamber 1988). However, the idea was supported only by a single observation \u0026nbsp;(Nakamura and Sekiguchi 1980), while other studies describing pycnogonid mating do not mention the glands at all (Prell 1910; Burris 2011b). For most pycnogonids, motile flagellate sperm with an elongated head has been described (Arnaud and Bamber 1988). This sperm morphology is considered characteristic of fertilization in a mucous medium (Pitnick et al. 2009); however, the exact moment of fertilization in Pycnogonida has not been observed.\u003c/p\u003e\n\u003cp\u003eThere was an attempt to relate known details of pycnogonid breeding behaviour to pycnogonid phylogeny (Sabroux et al. 2023). However, the data on this aspect of their biology remain incomplete and insufficient for comparative analysis. Comprehensive descriptions of pycnogonid breeding behaviour, including the moment of oviposition, have been made only for three families, with a single species per family (Prell 1910; Nakamura and Sekiguchi 1980; Burris 2011b), and only one of these was documented with photographs (Nakamura and Sekiguchi 1980), while other works were limited to verbal descriptions or drawings.\u003c/p\u003e\n\u003cp\u003eDespite the paucity of data, some generalizations about pycnogonid breeding behaviour have been attempted. Bain and Govedich (2004) suggested a classification of pycnogonid breeding behaviour types based on oviger morphology. Three morphological types of ovigers were identified: the unmodified \u0026ldquo;nymphonid\u0026rdquo; type, the slightly modified \u0026ldquo;ammotheid\u0026rdquo; type, and the highly modified \u0026ldquo;phoxichilidiid\u0026rdquo; type. For each of these types, \u0026ldquo;characteristic\u0026rdquo; breeding behavior is inferred based on a single detailed description, combined with occasional mentions made for other species (Bain and Govedich 2004a). However, ovigers of the same morphological type can occur in very different pycnogonid taxa with apparently different breeding behaviours and habitus. For example, \u0026ldquo;nymphonid type\u0026rdquo; ovigers with fully developed armature are characteristic of both \u003cem\u003eP. longiceps\u003c/em\u003e, which makes egg-bracelets glued with cement (Nakamura and Sekiguchi 1980), and Colossendeidae, which fasten an amorphous eggmass to stones (Moran et al. 2024). \u0026ldquo;Phoxichilidiid type\u0026rdquo; ovigers are represented in both gracile Phoxichilidiidae and robust Pycnogonidae. In addition to anatomical differences (overall habitus, gonopore position), sperm structure in Phoxichilidiidae and Pycnogonidae also differs (King and El-Hawawi 1978; Petrova and Bogomolova 2024b). This implies differences in fertilization between these two families. While the breeding behaviour of \u003cem\u003ePycnogonum litorale\u003c/em\u003e (Str\u0026oslash;m, 1762) has been satisfactorily described in Pycnogonidae (Prell 1910), reports of breeding in Phoxichilidiidae are merely occasional mentions (Hoek 1881; Cole 1901, 1906; Loman 1907).\u003c/p\u003e\n\u003cp\u003eFor this work, \u003cem\u003ePhoxichilidium femoratum\u003c/em\u003e (Rathke, 1799) was chosen as a widely distributed and easily obtained phoxichilidiid species, whose reproductive system was studied during our previous work (Petrova and Bogomolova 2024a, b). We aim to describe and thoroughly document the breeding behaviour of this species, particularly the moment of fertilization and eggmass formation, with a focus on the functional explanation of reproductive system morphology (glands, gonopore position).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Collecting and keeping of animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth male and female specimens of \u003cem\u003eP. femoratum\u003c/em\u003e (Rathke, 1799) were procured from the sublittoral zone near the White Sea Biological Station of Moscow State University (66\u0026deg; 34\u0026prime; N, 33\u0026deg; 08\u0026prime; E). Animals were collected manually during low tide or through scuba diving. Specimens were obtained throughout their breeding season from June to August, 2021\u0026ndash;2023.\u003c/p\u003e\n\u003cp\u003eUpon arrival at the laboratory, sea spiders were examined for eggmasses or eggs in the femora and sorted by sex. Only males carrying eggmasses and females with mature eggs in their femora were chosen for the study. Eggmasses were peeled from the males\u0026apos; ovigers before subsequent observations. Animals were housed in 500 ml containers filled with filtered seawater, which was changed every 2\u0026ndash;3 days, at a temperature of 8\u0026ndash;12\u0026deg;C, with a shred of veiling fabric as a substrate. Males and females were kept separately, with no more than 30 specimens per container. \u003cem\u003eClava multicornis\u003c/em\u003e (Forssk\u0026aring;l, 1775) was offered as food.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Morphometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo collect data on sexual dimorphism, 17 females and 15 males were relaxed in a 7% MgCl₂ solution mixed with filtered seawater at a 1:1 ratio. The specimens were straightened in a drop of the same solution on the bottom of a plastic Petri dish and fixed in position with a cover glass and pieces of plasticine as spacers. The animals were then photographed from the ventral side using an encoded stereomicroscope (Leica M165C) with a digital camera (Leica DFC420), washed in fresh seawater, and used for further experiments. The manipulations did not show any noticeable effect on the animals\u0026rsquo; health or behaviour in our earlier observations.\u003c/p\u003e\n\u003cp\u003eIn the photographs, trunk length, coxae 2, femur, and tibia 1 lengths and widths were measured using Photoshop CS6. Trunk length was measured from the base of the proboscis to the posterior edge of the fourth segment, while the lengths of leg segments were measured from joint to joint, and widths at their widest points (Fig. 1b). Additionally, width-to-length ratios were calculated for each measured leg segment, as well as ratios of each leg segment\u0026rsquo;s length to trunk length and tibia 1 to femur length and width. For calculations and data visualisation MS Excel was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Observation of breeding bechaviour. Obtaining of freshly laid eggs. Observation of eggs development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the observation of the mating process and the collection of freshly laid eggs, animals were transferred into 200 ml containers with filtered seawater placed on ice, with 2-3 specimens of each sex per container and a piece of veiling fabric as a substrate. These groups were checked approximately every five minutes and kept together until the formed pairs finished mating or for 2-3 days. The mating process was observed using a stereomicroscope. For documentation, the animals were placed into a small (approximately 1 litre) aquarium filled with cold seawater. Photographs and videos of the process were taken with a Nikon D3300 camera equipped with a Nikkor Micro 60 mm lens and a Nikon SB700 speedlight. The aquarium was cooled with ice placed underneath, and the water temperature was maintained at about 10-12\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eTo assess the possible frequency of mates for one male and one female, groups consisting of a single male/female with four specimens of the opposite sex were placed into 50 ml containers and checked for mating pairs and new eggmasses twice a day. Seven groups for each sex were formed, and the groups were observed for three weeks.\u003c/p\u003e\n\u003cp\u003eFreshly laid eggs were collected using a plastic pipette as soon as the eggs appeared to check for the presence of spermatozoa under a microscope, to fix them for TEM observation of cement, and to observe whether the unformed eggmasses developed successfully. \u0026quot;Formed\u0026quot; eggmasses, one day post-fertilisation, were peeled from male ovigers for TEM observation of cement. Some males with eggmasses were left intact and kept in separate 50 ml containers until larval hatching to observe the condition of the eggmasses and to record the normal timing of development. The conditions for the males were the same as for the other adult animals.\u003c/p\u003e\n\u003cp\u003eEggmasses taken from males were kept in 20 ml containers filled with filtered seawater at 8-12\u0026deg;C and checked daily for development and general condition (presence of overgrowth, embryo mortality); water was replaced after each check.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Expirement on male femoral glands function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine whether the male femoral glands have a reproductive function, an attempt was made to block their functioning. For the experiment, three groups of five males and five females were formed. In the first group, the dorsal side of the males\u0026rsquo; femurs (where the glands open) was covered with cyanoacrylate glue. In the second group, the lateral side of the males\u0026rsquo; femurs was covered with the glue, leaving the gland openings unblocked to test whether the glue layer on the femurs affects the rate of matings. The males in the third group were left intact. All three groups were kept under the conditions described above (section 2.2) and checked for new eggmasses and breeding pairs twice a day for three weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Microscopy of egmasses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFragments of eggmasses were examined for sperm and matrix\u0026nbsp;using a Leica DM2500 microscope equipped with a Leica DFC420С (5.0MP) digital camera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 TEM of eggmasses matrix\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transmission electron microscopy (TEM), eggmasses were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer, which was diluted with filtered seawater in a 1:1 ratio, and left overnight at 4\u0026deg;C. After fixation, the samples were rinsed in the same buffer and postfixed with a 1% osmium tetroxide solution buffered with the same buffer for 1 hour at room temperature. They were then dehydrated using a series of graded ethanol solutions and acetone, and embedded in Epon (Epoxy Embedding Medium Kit \u0026ndash; Fluka). Ultra-thin sections measuring 70-80 nm were cut with a diamond knife using a Leica UC6 ultratome, contrasted with uranyl acetate (4% aqueous solution) and lead citrate (1% aqueous solution), and examined using a JEOL JEM-1011 TEM equipped with an ORIUS SC1000W digital camera or a JEOL JEM-1400 Flash.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Artwork preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotographs of breeding animals, eggs and eggmasses was edited to improve contrast and brightness in Adobe Photoshop CS6. Individuals in breeding pairs were countered using Adobe Illustrator CC 2017. Schematic drawings were also prepared in Adobe Illustrator CC 2017. Data on morphometry and eggmasses per male number was visualized using MS Excel and then edited for better appearance in Adobe Illustrator CC 2017. Video recordings were sped up and converted using Adobe Premier Pro CC 2017.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Sex ratio, sexual dimorphism and fecundity in natural population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sex ratio in the population is approximately 1:1. \u003cem\u003eP. femoratum\u003c/em\u003e exhibits clear sexual dimorphism. The absence of ovigers in males is characteristic of the family, and females are significantly larger than males (the difference is visually noticeable). The body proportions of males and females differ. The average body length is similar in both sexes; the larger size of females is attributed solely to their longer legs with larger femurs. However, males have longer and thinner second coxae than females (Fig. 1a).\u003c/p\u003e\n\u003cp\u003eBreeding animals (gravid females and egg-carrying males) are observed from May to June. In June, a single male can carry up to 14 eggmasses, and up to 9 eggmasses on one oviger (as recorded in a male with only a single oviger), but this number decreases by the end of summer. In June, the average number of eggmasses per male is 6, although this number varies significantly (standard deviation 4). The distribution of the number of eggmasses per male is shown in Fig. 2. Eggmasses are roughly evenly distributed between the two ovigers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Breeding behaviour, oviposition and fertilization, eggmasses formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeing placed in a common container animals pair within 0,5-1 h. No preceding specific behaviour was observed, males quickly detect females and crawl toward them. Then the male climbs on the back of the female and tries to position on the top of it. From the start of the courtship, the male sits head-to-head on the female and holds her first trunk processes by his ovigers (Fig. 3a, a\u0026rsquo;). Initially, male holds by its legs on female legs in random order and at random places or does not hold at all (Fig. 3a, a\u0026rsquo;). Then, male tries to fix his legs of 2nd \u0026ndash; 4th pairs on the coxae3 of female\u0026rsquo;s 1st \u0026ndash; 3rd legs (Fig. 3b, b\u0026rsquo;). Thus, the male sits on the back of the female so, that his 2nd \u0026ndash; 4th legs over spaces between female\u0026rsquo;s legs (Fig. 3b\u0026ndash;c\u0026rsquo;). The first pair of male\u0026rsquo;s legs remains free (Fig. 3c, c\u0026rsquo;). Female can counteract and try to push male\u0026rsquo;s legs in case she is not ready to spawn or disturbed (for instance, by too bright light) (Video S1). As the male successes to assume the right posture he stays in the position and about once per half a minute strains coxae squatting down (Video S2). The courtship can continue from 2-3 h to a couple of days. However, in case the courtship lasts more than 5-7 hours, it usually does not finish by spawning: the couple just breaks up.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSometimes, two males climb on the back of one female one under other. In such \u0026ldquo;stacks\u0026rdquo;, one of males remains and pairs with the female after a while (0,5-3 hours), however, no obviously aggressive actions between two males was observed. Sometimes the \u0026ldquo;competence\u0026rdquo; becomes too long (up to a couple of days) and finishes with unsuccessful mating (the \u0026ldquo;stack\u0026rdquo; breaks up). If the \u0026ldquo;compenetence\u0026rdquo; was won by one of males the other one can pair with the rest \u0026ldquo;free\u0026rdquo; female, but sometimes it apparently, is completely unattractive and do not pairs at all.\u003c/p\u003e\n\u003cp\u003eAfter some time of courtship in the femurs of the female, peristaltic contractions of the vitellarium become noticeable (Fig. 4b, Video S3). Soon after that, the female strains her legs so that they form a dome and lays eggs inside the dome (Fig. 4b, b\u0026rsquo;; 5). During oviposition, the male remains in the same position as during courtship (Fig. 4a\u0026ndash;b\u0026rsquo;): his legs are strained so that the coxae of the 2\u003csup\u003end\u003c/sup\u003e to 4\u003csup\u003eth\u003c/sup\u003e pairs of legs incline between the coxae of the female\u0026rsquo;s legs and press into the forming eggmass (Fig. 4a\u0026ndash;c\u0026rsquo;). Thus, the male gonopores contact the eggmass but are widely spaced from the female ones (Fig. 5). The first pair of the male\u0026rsquo;s legs remains free, with its coxae far from the eggmass (Fig. 4a\u0026ndash;b\u0026rsquo;). In the eggmass, taken at any moment during oviposition, spermatozoa can be found (Fig. 6); eggs develop being taken from the pair at any moment of oviposition.\u003c/p\u003e\n\u003cp\u003eAs oviposition finishes, the male releases his ovigers and leans forward (Fig. 7a, a\u0026rsquo;). He then hooks the eggmass with one of the ovigers (Fig. 7b, b\u0026rsquo;) and pulls it forward (Fig. 7b\u0026ndash;d). At the same time, the female pulls backward (Fig. 7C\u0026ndash;D). The eggmass separates from the female and remains on the male\u0026rsquo;s oviger (Fig. 7d). In this way, the most recent eggmasses appear on the male\u0026rsquo;s ovigers distally to older ones. After spawning, the male takes no further action with the eggmass. The day after oviposition, the mucous vitelline envelopes gluing the eggs slightly condense; the eggmass compacts but remains loose (Fig. 8a). Some eggs from the edge of the eggmass fall off and remain on the bottom of the container. In eggmasses taken from the pair either during oviposition or after it, as well as a day after oviposition, the matrix surrounding the eggs is uniform and composed of swollen vitelline envelopes (Fig. 8b, c). No additional matrix was found; the eggs are widely spaced and arranged irregularly (Fig. 8a, b). The eggmasses are roughly teardrop-shaped, with a thin \u0026ldquo;handle\u0026rdquo; where they are threaded onto the oviger (Fig. 8a).\u003c/p\u003e\n\u003cp\u003eIt appeared impossible to video record the departure of the male, as the constant bright light necessary for the recording frightened the animals, causing the female to try to crawl away from the light spot instead of pulling in the direction opposite to the male\u0026rsquo;s pulling. On the other hand, the flashlights used during time-lapse photography (one every 1-1.5 minutes) did not affect their behaviour. For the same reason (constant bright light disturbs animals), video recordings of the entire breeding behaviour are impossible; however, small video fragments of oviposition and courtship can be captured without disrupting the process.\u003c/p\u003e\n\u003cp\u003eDuring the entire process of oviposition and afterwards, the male\u0026rsquo;s femurs are widely spaced from the eggmass being formed (Fig. 4, 5, 7). No manipulations that could be interpreted as transferring the secretions from the femoral glands to the eggmass were observed.\u003c/p\u003e\n\u003cp\u003eMales with freshly laid eggmasses can regularly be found courting a new female in a tank containing both sexes. Obviously, males are ready for the next spawning almost immediately after finishing the previous one and can obtain several (in our observations, 2-3) eggmasses a day if enough females are available. However, in a common container with numerous specimens of both sexes, the distribution of obtained eggmasses varies greatly between males (0-3 per day), and it is unclear whether this is due to the need for rest for the male or its unattractiveness to females (or the absence of females that are attractive to the male).\u003c/p\u003e\n\u003cp\u003eIn containers with one female and several males, new eggmasses appeared 5-8 days after the previous spawning. Unsuccessful courtships were not detected in such containers between eggmass formations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCoating the male\u0026rsquo;s femoral glands with cyanoacrylate glue demonstrated no effect on breeding activity and eggmasses shape.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003e4.1. The mechanism of oviposition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our video recordings of courted \u003cem\u003eP. femoratum\u003c/em\u003e females, peristaltic contractions of the vitellarium wall are undoubtedly discernible before and during oviposition. This observation supports our earlier suggestion regarding the transport of mature eggs by the muscles of the vitellarium wall (Petrova and Bogomolova 2024b). The midgut diverticulum remains motionless during oviposition and, evidently, does not participate in the protraction of the eggs being laid. The opposite was described for \u003cem\u003ePropallene longiceps\u003c/em\u003e (Bohm, 1879) (Nakamura and Sekiguchi 1980), but the source of the discrepancy (whether it is due to interspecies variation or observational error) is unclear (elaborated in Petrova and Bogomolova 2024a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. When fertilization happens?\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eP. femoratum\u003c/em\u003e, fertilisation undoubtedly occurs while oviposition is in progress, as can be concluded from the presence of spermatozoa in an eggmass taken from a breeding pair at any moment during oviposition, and the fact that eggs develop when removed at any time during the formation of the eggmass. The same is most likely true for \u003cem\u003eP. longiceps\u003c/em\u003e, whose eggs also develop when removed during oviposition (Nakamura and Sekiguchi 1980). In both cases, the male\u0026apos;s coxae 2 with gonopores are in contact with the forming eggmass during oviposition, thus allowing sperm to be injected into the swollen mucous vitelline envelopes gluing the eggs. The structure of the spermatozoon in \u003cem\u003eP. femoratum\u003c/em\u003e also corresponds to those proposed for fertilisation in the mucous eggmass (Petrova and Bogomolova 2024b). The mobile flagellate spermatozoon with an elongated head is most commonly described for Pycnogonida (van Deurs 1974; El‐Hawawi and King 1978, 1983), suggesting that fertilisation in a forming mucous eggmass also occurs in Nymphonidae and Ammotheidae.\u003c/p\u003e\n\u003cp\u003eDespite the similar spermatozoon morphology and likely the same mode of fertilisation in Ammotheidae, it has been shown for \u003cem\u003eAchelia simplissima\u003c/em\u003e (Hilton, 1939) that eggmasses taken from a mating pair during oviposition fail to develop, implying that fertilisation occurs after oviposition (Burris 2011b). However, this conclusion regarding later fertilisation in the species may be due to observational error, as eggmasses taken from males quickly become overgrown by fungi and bacteria and die before larvae hatch (personal observation on \u003cem\u003eP. femoratum\u003c/em\u003e, \u003cem\u003eNymphon\u003c/em\u003e spp., \u003cem\u003ePseudopallene spinipes\u0026nbsp;\u003c/em\u003e(Fabricius, 1780)). Direct observation of spermatozoa in the eggmass and developing eggs is necessary for an accurate conclusion on the timing of fertilisation.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eP. litorale\u003c/em\u003e, a divergent filiform aflagellate spermatozoon has been described (King and El-Hawawi 1978), suggesting differences in fertilisation in this species. The position of gonopores in a mating pair of \u003cem\u003eP. litorale\u003c/em\u003e differs from those in \u003cem\u003eP. femoratum\u003c/em\u003e. In \u003cem\u003eP. litorale\u003c/em\u003e, the female\u0026apos;s gonopores are positioned only on the fourth walking legs and are shifted dorsally; the male\u0026apos;s gonopores are located on the ventral side of the fourth walking legs. In a mating pair, the male gonopores touch the female ones, making fertilisation as close to internal as possible (Prell 1910). Unfortunately, data on spermatozoon structure are available only for a small part of pycnogonid diversity, making hypotheses about the evolution of their fertilisation biology speculative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Eggmasses formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our observations on \u003cem\u003eP. femoratum\u003c/em\u003e, neither the male nor the female shapes the eggmasses. For \u003cem\u003eA. simplissima\u003c/em\u003e, males were observed tightening eggmasses taken from the female with their ovigers (Burris 2011b), and in \u003cem\u003eP. longiceps\u003c/em\u003e, males cement eggs into egg bracelets (Nakamura and Sekiguchi 1980). Additionally, a male grooming and compacting an eggmass was recently reported for \u003cem\u003eColossendeis megalonyx\u003c/em\u003e Hoek, 1881 (Moran et al. 2024). On the other hand, males of \u003cem\u003eP. litorale\u003c/em\u003e also do not shape their eggmasses with ovigers (Schmidt and B\u0026uuml;ckmann 1971). In Ammotheidae, Callipallenidae, and Colossendeidae, ovigers are present in both sexes and have rows of spines (\u0026ldquo;combs\u0026rdquo;) on their distal segments, while the \u0026ldquo;phoxichilidiid type\u0026rdquo; ovigers characteristic of \u003cem\u003eP. femoratum\u003c/em\u003e and \u003cem\u003eP. litorale\u003c/em\u003e are present only in males and lack comb spines (Bain and Govedich 2004a). Thus, as suggested by Bain and Govedich (2004a), the shaping of the eggmass may be functionally related to the morphology of the ovigers. However, Pycnogonidae and Phoxichilidiidae are not sister clades according to current pycnogonid phylogeny (Ballesteros et al. 2021; Sabroux et al. 2023), and other details related to their breeding (gonopore position, sperm structure) differ drastically (discussed in section 4.2.). Freshly laid eggs of \u003cem\u003eP. litorale\u003c/em\u003e appear on the dorsal side of the female; their transition and eggmass formation are apparently facilitated by the cuticle structure (Schmidt and B\u0026uuml;ckmann 1971). In contrast, in \u003cem\u003eP. femoratum\u003c/em\u003e, eggs are oviposited on the ventral side of the female, and the eggmass is formed by pressure from the proximal segments of the legs, composing a \u0026ldquo;dome.\u0026rdquo; Thus, upon closer inspection, the mechanisms of eggmass formation in \u003cem\u003eP. femoratum\u003c/em\u003e and \u003cem\u003eP. litorale\u003c/em\u003e also differ. Consequently, either the unification of \u003cem\u003ePhoxichilidium\u003c/em\u003e\u0026rsquo;s and \u003cem\u003ePycnogonum\u003c/em\u003e\u0026rsquo;s ovigers into the same type is inaccurate, or oviger morphology does not strictly correlate with breeding behaviour. In the first case, the classification of ovigers needs to be refined, considering not only the number of segments and the presence/absence of spines but also the size of the ovigers relative to the whole body. Clarifying the probability of the second case requires observations of the breeding behaviour of other Pycnogonida.\u003c/p\u003e\n\u003cp\u003eEggmasses of irregular shape have been described in different pycnogonid families distributed across the entire pycnogonid tree: Pycnogonidae (Prell 1910; Dogiel 1913; Schmidt and B\u0026uuml;ckmann,1971), Phoxichilidiidae (Dogiel 1913; present work), Ammotheidae (Berry 1980; Burris 2011b), and Colossendeidae (Moran et al. 2024), while a regular arrangement of eggs in eggmasses has been mentioned only for Nymphonoidea (Dogiel 1913; Nakamura and Sekiguchi 1980), which are nested within the crown of the pycnogonid tree. In all pycnogonids with described oviposition, eggs are primarily laid as an amorphous mass (Prell, 1910; Schmidt and B\u0026uuml;ckmann, 1971; Nakamura and Sekiguchi, 1980; Brenneis and Wagner, 2023; Moran et al., 2024; present work). Thus, it is likely that an amorphous eggmass is a plesiomorphic state for Pycnogonida, while its compaction is apomorphic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Male femoral glands function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn males of most sea spiders, glands positioned in the femora and, sometimes, in the tibiae of the walking legs were described. These glands are commonly believed to produce a cement that fastens eggs into an eggmass (Helfer and Schlottke 1935; Arnaud and Bamber 1988). However, actions that can be unambiguously interpreted as cementing eggs into the eggmass have only been described once for \u003cem\u003eP. longiceps\u003c/em\u003e (Nakamura and Sekiguchi 1980), while for the other species, the function of the femoral glands remains merely an assumption. In \u003cem\u003eP. femoratum\u003c/em\u003e, femoral glands are also present (Loman 1907; Petrova and Bogomolova 2024b). Nonetheless, no actions that could be interpreted as the transfer of the femoral glands\u0026rsquo; secretion to the forming eggmass were observed during matings of \u003cem\u003eP. femoratum\u003c/em\u003e. Moreover, the glands open on the dorsal side of the femur, which is far from the eggmass throughout the oviposition process. Additionally, it is difficult to imagine that the openings of the femoral glands could touch the eggmass or be reached by the male\u0026rsquo;s ovigers to transfer their secretion to the forming eggmass due to pycnogonid anatomy; furthermore, no substance apart from swollen vitelline envelopes was found in the formed eggmass. Thus, the cementing function of the male\u0026rsquo;s femoral glands is unlikely in \u003cem\u003eP. femoratum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, the exact function of the male\u0026rsquo;s femoral glands remains unclear. As sexually dimorphic structures, they are probably related to reproduction. However, covering the gland openings with cyanoacrylate glue does not affect mating rates and eggmasses shape. Two alternative explanations for the absence of this effect exist: 1) the gland secretion is unnecessary for successful breeding (at least under the conditions of the experiment); 2) the secretion of the glands is a substance with a molecular weight low enough to penetrate through polymerised cyanoacrylate, thus covering the gland openings does not block them. In the first case, a fungicidal and bactericidal function can be suggested because eggmasses carried by the male are not overgrown by bacteria and fungi, in contrast to those taken from the male and kept separately. However, these two explanations are not mutually exclusive, leaving us with a demand for further study of the composition of the gland secretion and partner search in \u003cem\u003eP. femoratum\u003c/em\u003e to clarify the role of the male\u0026rsquo;s femoral glands. Additionally, the ambiguity of the function of male glands in \u003cem\u003eP. femoratum\u003c/em\u003e and the diversity of \u0026ldquo;cement\u0026rdquo; gland positions among pycnogonids (Dohrn 1881), combined with the variety of eggmass shapes, call into question the generally accepted cement function of these glands and necessitate functional research on different species to draw accurate conclusions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5. Who initiates the mating?\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, the observation of mating choices was not intended in this work; thus, experiments revealing mating biases were not planned. However, this issue is the main focus of most other studies on sea spider breeding. Therefore, some thoughts on the topic, induced by the literature and based on our qualitative observations, are elaborated below.\u003c/p\u003e\n\u003cp\u003eUnder laboratory conditions, sea spiders often crawl over one another without regard to the sex of the individuals and apparently without any mating implications. No specific behaviour that could be interpreted as initiation of mating by a female was detected. No obvious female-female competition or aggression was observed. Unsuccessful courtships involving a resisting female or two males courting the same female imply that the initial movement could not have been made by the female. Signs of male initiative were also noted for \u003cem\u003eP. longiceps\u003c/em\u003e (Nakamura and Sekiguchi 1980). In contrast, the initiation of contact by the female was reported for \u003cem\u003ePropallene saengeri\u003c/em\u003e Staples, 1979 (Bain and Govedich 2004b), \u003cem\u003eA. simplissima\u003c/em\u003e (Burris 2011b), and \u003cem\u003eAchelia sawayai\u003c/em\u003e Marcus, 1940 (Berry 1980). For the \u003cem\u003eP. saengeri\u003c/em\u003e, even fights between competing females were described. For \u003cem\u003eAchelia\u003c/em\u003e, specific female actions interpreted as attracting male attention were recorded (Berry 1980; Burris 2011b). The only mention of female-female competition for mates is extremely vague and based on a single observation (Bain and Govedich 2004b). In all cases, the action that can be unambiguously interpreted as the start of mating is the climbing of the male onto the female\u0026rsquo;s back (Berry 1980; Nakamura and Sekiguchi 1980; Burris 2011b). Thus, the decision to mate is apparently made by the male.\u003c/p\u003e\n\u003cp\u003eGenetic studies do not reveal higher competition among males or females for mates, demonstrating that the limiting factor for mate acquisition is encounter probability (Barreto and Avise 2008, 2010, 2011; Burris 2011b). Thus, in different species, one partner or another can refuse to mate. In our observations on \u003cem\u003eP. femoratum\u003c/em\u003e, females can actively resist males\u0026rsquo; courting attempts, while in \u003cem\u003eAchelia\u003c/em\u003e, males may ignore females\u0026rsquo; pursuits, and unsuccessful courting was not reported (Berry 1980; Burris 2011b). One possible interpretation is that the moment of female choice can vary between the studied species. However, there is a potential source of observational error. In our experiments, animals were maintained in the laboratory, and all observations were made at high population densities (100-200 animals per m\u0026sup2;), whereas the approximated natural population density for the species is less than 20-30 animals per m\u0026sup2;. Although natural population densities are noted for both \u003cem\u003eAchelia\u0026nbsp;\u003c/em\u003estudied, those maintained for observation are not (Berry 1980; Burris 2011b). Nothing is known about the population density of \u003cem\u003eP. longiceps\u003c/em\u003e, either natural or experimental (Nakamura and Sekiguchi 1980). Consequently, it cannot be excluded that the male initiative observed in our experiments and those by Nakamura and Sekiguchi is an effect of overpopulation and that at natural densities gravid females may seek out and follow mature males. Further observations considering natural population density are necessary to definitively clarify which sex initiates mating.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6. Sexual dimorphism in \u003cem\u003eP. femoratum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSexual size dimorphism in \u003cem\u003eP. femoratum\u003c/em\u003e is quite pronounced; females are significantly larger than males due to their longer legs with larger femurs. However, males have longer coxae 2. It seems very likely that the larger size of females provides extra space for vitellogenesis, while the longer coxae 2 in males are necessary to position the male\u0026rsquo;s gonopores in proximity to the forming eggmass.\u003c/p\u003e\n\u003cp\u003eA very similar dimorphism has been described for \u003cem\u003eAmmothea hilgendorfi\u0026nbsp;\u003c/em\u003e(B\u0026ouml;hm, 1879) (Ammotheidae); the main difference is that in this species, males have a wider trunk, while dimorphism of coxae 2 was not mentioned (Barreto and Avise 2008). Both \u003cem\u003eA. hindeldorphi\u003c/em\u003e and \u003cem\u003eP. femoratum\u003c/em\u003e exhibit a similar type of post-embryonic development. Both have endoparasitic larvae and produce numerous small eggs (Brenneis et al. 2017). Such breeding ecology implies high fecundity selection (Ramirez Llodra 2002). Consequently, the noticeably larger size of females, provided by reproductively significant parts, is most obviously explained by fecundity selection. However, considering the costs of male brood care demonstrated in \u003cem\u003eA. simplissima\u003c/em\u003e (Burris 2011a), selection for smaller male size cannot be excluded either.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVideo S1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S1 Female resisting the courting male.\u003c/strong\u003e Female tries to peel off male\u0026rsquo;s legs with her walking legs. The video is 5 times accelerated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVideo S2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S2 Male courting a female.\u003c/strong\u003e Regular \u0026ldquo;squatting down\u0026rdquo; of a courting male is demonstrated. The video is 5 times accelerated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVideo S3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S3 Peristaltic of the ovary.\u003c/strong\u003e On the video is an oviposing pair, the femur of the female where the peristaltic waves are clearly discernible is encircled. The video is 5 times accelerated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are thankful to A. Semenov and other divers of Pertsov White Sea Biological Station for their assistance with sampling. We are grateful to A. Lavrov and F. Bolshakov for their technical support and opportunities at the Center of microscopy, WSBS, MSU. Also we want to thank the Electron Microscopy Laboratory of the Shared Facilities Center of Lomonosov Moscow State University, sponsored by the RF Ministry of Education, Science and Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Ekaterina Bogomolova supervised and discussed the work. Maria Petrova conceptualized and conducted the research. The first draft of the manuscript was written by Maria Petrova, Ekaterina Bogomolova commented the previous manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the scientific project of the state order of the government of Russian Federation to Lomonosov Moscow State University #121032300121\u0026ndash;0\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be provided under request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or nonfinancial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work did not require ethical approval from an animal welfare committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenerative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used ChatGPT in order to improve language and readability. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArnaud F, Bamber RN (1988) The biology of Pycnogonida. Advances in marine biology 24:1\u0026ndash;96. https://doi.org/10.1016/S0065-2881(08)60073-5\u003c/li\u003e\n\u003cli\u003eBain BA, Govedich FR (2004a) Courtship and mating behavior in the Pycnogonida (Chelicerata: Class Pycnogonida): a summary. Invertebrate Reproduction \u0026amp; Development 46:63\u0026ndash;79. https://doi.org/10.1080/07924259.2004.9652607\u003c/li\u003e\n\u003cli\u003eBain BA, Govedich FR (2004b) Mating behaviour, female aggression and infanticide in \u003cem\u003ePropallene saengeri \u003c/em\u003e(Pycnogonida: Callipallenidae). 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Akademische Verlagsgesellschaft m.b.h, pp 1\u0026ndash;314\u003c/li\u003e\n\u003cli\u003eHoek PPC (1881) Report on the Pycnogonida, dredged by H.M.S. Challenger during the years 1873\u0026ndash;76. Challenger Rep Zool 3:1\u0026ndash;167.\u003c/li\u003e\n\u003cli\u003eKing PE, El-Hawawi ASN (1978) Spermiogenesis in the pycnogonid \u003cem\u003ePycnogonum littorale\u003c/em\u003e (Str\u0026ouml;m). Acta Zoologica 59:97\u0026ndash;103. https://doi.org/10.1111/j.1463-6395.1978.tb00116.x\u003c/li\u003e\n\u003cli\u003eLoman JCC (1907) Biologische Beobachtungen an einem Pantopoden. Tijdschrift der Nederlandsche Dierkundige Vereeniging 2:255\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eMoran AL, Lobert GT, Toh MWA (2024) Spawning and larval development of \u003cem\u003eColossendeis megalonyx \u003c/em\u003e, a giant A ntarctic sea spider. Ecology 105:e4258. https://doi.org/10.1002/ecy.4258\u003c/li\u003e\n\u003cli\u003eNakamura K, Sekiguchi K (1980) Mating behavior and oviposition in the pycnogonid \u003cem\u003ePropallene longiceps\u003c/em\u003e. Marine Ecology Progress Series 2:163\u0026ndash;168. https://doi.org/10.3354/meps002163\u003c/li\u003e\n\u003cli\u003ePetrova M, Bogomolova E (2024a) The female reproductive system of the sea spider \u003cem\u003ePhoxichilidium femoratum\u003c/em\u003e (Rathke, 1799). Arthropod Structure and Development 81:101370. https://doi.org/10.1016/j.asd.2024.101370\u003c/li\u003e\n\u003cli\u003ePetrova M, Bogomolova E (2024b) The male reproductive system of the sea spider \u003cem\u003ePhoxichilidium femoratum \u003c/em\u003e(Rathke, 1799). Arthropod Structure \u0026amp; Development 83:101404. https://doi.org/10.1016/j.asd.2024.101404\u003c/li\u003e\n\u003cli\u003ePitnick S, Hosken DJ, Birkhead TR (2009) 3 - Sperm morphological diversity. In: Birkhead TR, Hosken DJ, Pitnick S (eds) Sperm Biology. Academic Press, London, pp 69\u0026ndash;149 https://doi.org/10.1016/B978-0-12-372568-4.00003-3\u003c/li\u003e\n\u003cli\u003ePrell H (1910) Beitrage zur Kenntnis der Lebensweise einiger Pantopoden. Bergens Museums Aarbok 11:1\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eRamirez Llodra E (2002) Fecundity and life-history strategies in marine invertebrates. In: Advances in Marine Biology. Elsevier, pp 87\u0026ndash;170 https://doi.org/10.1016/S0065-2881(02)43004-0\u003c/li\u003e\n\u003cli\u003eSabroux R, Corbari L, Hassanin A (2023) Phylogeny of sea spiders (Arthropoda: Pycnogonida) inferred from mitochondrial genome and 18S ribosomal RNA gene sequences. Molecular Phylogenetics and Evolution 182:107726. https://doi.org/10.1016/j.ympev.2023.107726\u003c/li\u003e\n\u003cli\u003eSchmidt H-W, B\u0026uuml;ckmann D (1971) Beobachtungen zur Lebensweise von \u003cem\u003ePycnogonum litorale\u003c/em\u003e (Str\u0026ouml;m) (Pantopoda). Oecologia 7:242\u0026ndash;248. https://doi.org/10.1007/BF00345214\u003c/li\u003e\n\u003cli\u003eSharma PP, Gavish-Regev E (2025) The Evolutionary Biology of Chelicerata. Annu Rev Entomol 70:144\u0026ndash;63. https://doi.org/10.1146/annurev-ento-022024-011250\u003c/li\u003e\n\u003cli\u003eTomaschko K-H, Wilhelm E, B\u0026uuml;ckmann D (1997) Growth and reproduction of \u003cem\u003ePycnogonum litorale\u003c/em\u003e (Pycnogonida) under laboratory conditions. Marine Biology 129:595\u0026ndash;600. https://doi.org/10.1007/s002270050201\u003c/li\u003e\n\u003cli\u003evan Deurs B (1974) Spermatology of Some Pycnogonida (Arthropoda), with Special Reference to a Microtubule‐Nuclear Envelope Complex. Acta Zoologica 55:151\u0026ndash;162. https://doi.org/10.1111/j.1463-6395.1974.tb00189.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pycnogonida, reproduction, behaviour, mating, fertilization, oviposition","lastPublishedDoi":"10.21203/rs.3.rs-5804077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5804077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Sea spiders (Pycnogonida) are one of two arthropod taxa that exhibit external fertilization, making them interesting subjects for the reconstruction of plesiomorphic arthropod breeding features. However, data on pycnogonid breeding are limited, particularly their fertilisation was not observed at all. Most knowledge on of their breeding is mere assumptions based on occasional observations. In this work, we observed and photo- and video- recorded the breeding behaviour (courtship, oviposition, fertilization, and eggmass formation) of Phoxichilidium femoratum (Rathke, 1799). Additionally, we studied eggmasses for the presence of sperm and additional cement. The male initiates mating by climbing onto the female’s back and courts her until oviposition. During oviposition, the male gonopores are in proximity to the eggmass, and sperm is injected into the swollen vitelline envelope surrounding the laid eggs. Once oviposition is complete, the male leaves the female hooking the eggmass with one of his ovigers. No additional shaping of the eggmass by the male was recorded, nor was there any fastening of the eggs with additional cement. The eggs in the eggmass are secured solely by swollen vitelline envelopes. Thus, fertilization in a mucous eggmass is undoubtfully demonstrated for the species (and likely characteristic of most sea spiders). Our observation questions the cementing function of male femoral glands, widely accepted in review literature. Also, basing on comparison of P. femoratum with other sea spiders we suggest a hypothesis on eggmasses evolution.","manuscriptTitle":"Breeding of the sea spider Phoxichildium femoratum (Rathke, 1799): functional anatomy perspective","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 15:57:23","doi":"10.21203/rs.3.rs-5804077/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-01-24T20:40:27+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-24T19:57:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-13T08:10:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biology","date":"2025-01-10T08:29:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"02477914-5ab4-4eeb-b9e9-9012b50239e6","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:35:57+00:00","versionOfRecord":{"articleIdentity":"rs-5804077","link":"https://doi.org/10.1007/s00227-025-04707-3","journal":{"identity":"marine-biology","isVorOnly":false,"title":"Marine Biology"},"publishedOn":"2025-08-22 16:29:30","publishedOnDateReadable":"August 22nd, 2025"},"versionCreatedAt":"2025-01-28 15:57:23","video":"","vorDoi":"10.1007/s00227-025-04707-3","vorDoiUrl":"https://doi.org/10.1007/s00227-025-04707-3","workflowStages":[]},"version":"v1","identity":"rs-5804077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5804077","identity":"rs-5804077","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}