Estuarine, Coastal and Shelf Science 270 (2022) 107850
Available online 10 April 2022
0272-7714/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).
Life history and reproductive traits of the East Atlantic deep-sea quill worm Hyalinoecia robusta Southward, 1977 (Annelida: Onuphidae)
Andr´es Arias
a,*, Hannelore Paxton
b,caDepartamento de Biología de Organismos y Sistemas (Zoología), Universidad de Oviedo, Oviedo, 33071, Spain
bSchool of Natural Sciences, Macquarie University, Sydney, NSW, 2109, Australia
cAustralian Museum Research Institute, 1 William Street, Sydney, NSW, 2010, Australia
A R T I C L E I N F O Keywords:
Polychaetes
Invertebrate reproduction Simultaneous hermaphroditism Protandry
Developmental patterns Spain
Iberian Peninsula Bay of Biscay
A B S T R A C T
Polychaetes or bristle worms (Annelida) are one of the best represented invertebrate groups in marine envi- ronments worldwide. Although the reproductive biology and life histories of polychaetes from many marine and estuarine habitats are well documented, our knowledge of the reproduction and life cycles of deep-sea poly- chaetes is still far from comprehensive. Hyalinoeciinae, a subfamily of Onuphidae, one of the most dominant and successful tubicolous deep-sea polychaete families, are characterised by their peculiar lifestyle as free-living epibenthic species, as they protrude from their tubes in a caterpillar-like fashion known as epibenthic crawl- ing. The reproductive biology, sexual strategies and developmental modes of Hyalinoecia spp. are largely un- known. We have studied quarterly samples of the East Atlantic deep-sea quill worm Hyalinoecia robusta Southward, 1977, collected during 2012–2013, from the adjacent slope of the Avil´es submarine Canyons System (Bay of Biscay) at 1500 m depth. Here we report on the annual cycle, revealing that H. robusta is iteroparous, reproducing annually during a breeding season from early spring to summer. The performed histological study revealed that the species is a simultaneous hermaphrodite with a previous adolescent male phase. This is the first evidence of the occurrence of hermaphroditism in the subfamily Hyalinoeciinae. Mature sperm was of the ent- aquasperm type and was stored in modified nephridial chambers that develop into external papillae that may detach from the worm and act as spermatophores at the time of reproduction. In H. robusta, the egg fertilisation seems to occur within intersegmental dorsal openings of the worm body wall or inside the worm tube. This hypothesis is consistent with the finding of fertilised eggs and developing embryos attached to the body wall and/or inside the worm tube in some specimens. The herein reported H. robusta reproductive traits do not support their conspecificity with populations from outside North Atlantic Ocean. Thus, the name H. robusta may be hiding other deep-water Hyalinoecia species.
1. Introduction
Polychaetes or bristle worms (Annelida) are one of the best repre- sented invertebrate groups in terms of species richness, abundance and biomass in marine environments worldwide (Fauchald, 1977). The reproductive biology and life histories of polychaetes from many marine and estuarine habitats are well documented in the literature (Cazaux, 1970; Giangrande, 1997; Rouse and Pleijel, 2006). However, our knowledge of the reproduction and life cycles of deep-sea polychaetes is still far from comprehensive, representing a noteworthy exception when compared to other marine habitats.
Sexual strategies and developmental modes of most deep-sea
polychaetes, mainly of species from non-chemosynthetic habitats, are also understudied (Rouse and Pleijel, 2006; Mercier et al., 2014). Among deep-sea polychaetes living at the limits or outside chemosynthetic en- vironments, Onuphidae is one of the most dominant and successful families, inhabiting tubes of their own construction (Surugiu et al., 2008; Louzao et al., 2010; Paxton and Arias, 2014; Meyer et al., 2016).
Yet, knowledge of their reproductive biology is scant, particularly that of deep-water species, as its discovery depends on the fortuitous occasions when reproductively active animals are sampled. Reproductive strate- gies of onuphids range from free-spawning to various forms of brooding, including brooding inside the body –viviparity- (Wilson, 1991; Paxton and Arias, 2014; Arias and Paxton, 2015). Egg size ranges from 175 to
* Corresponding author.
E-mail address: [email protected] (A. Arias).
Contents lists available at ScienceDirect
Estuarine, Coastal and Shelf Science
journal homepage: www.elsevier.com/locate/ecss
https://doi.org/10.1016/j.ecss.2022.107850
Received 30 December 2021; Received in revised form 25 March 2022; Accepted 5 April 2022
1400 μm in diameter, and while some species exist as free trochophores for a short period, none are planktotrophic; they soon settle and build their tubes (Rouse and Pleijel, 2006; Paxton and Arias, 2014). Brooding species commonly undergo direct development in the parental tube, in attached egg cases, or in jelly masses (Paxton, 1986; Budaeva and Fauchald, 2010; Paxton and Arias, 2014; Arias and Paxton, 2015).
Hermaphroditism has been registered in four species of Diopatra Audouin
& Milne Edwards, 1833 (Lieber, 1931; Arias et al., 2013, 2016; Arias and Paxton, 2015) and suggested for Paradiopatra antarctica (Monro, 1930) and Rhamphobrachium (Spinigerium) ehlersi Monro, 1930 (Hartman, 1967 and Paxton, 1986 respectively), and there is a growing awareness that the hermaphroditic condition may be widespread among this family (Arias et al., 2016). More hermaphroditic species may remain still un- reported since sex determination was usually accomplished only by external observation of the colour of ripe segments and partial macro- scopic dissections, without confirmation by histological sectioning (Arias and Paxton, 2015; Arias et al., 2016). The colour of ripe segments only indicates the presence of the dominant gamete within the coelom, masking hermaphroditic individuals (Arias et al., 2013, 2016).
Within Onuphidae, members of the subfamily Hyalinoeciinae are characterised by their peculiar lifestyle; unlike the majority of the group (i.e. Onuphinae spp.), they are mobile epibenthic species. Their first parapodia are enlarged and have thick chaetae, allowing the animal to protrude from the tube and move in a caterpillar-like fashion known as epibenthic crawling (Paxton, 1986; Meyer et al., 2016). Hyalinoecia Malmgrem, 1867, commonly known as ‘quill worm’, besides naming and objectively defining this subfamily, represents one of the most intriguing genera of deep-sea polychaetes. This genus, which comprises 20 accepted species (Read and Fauchald, 2020), secretes hardened trans- lucent quill-like tubes lacking the typical outer layer of foreign particles of most onuphid species. The chemical composition of the Hyalinoecia tubes was found to be a combination of onuphic acid and a mucoprotein (Defretin, 1971); besides, they are unique in having an internal double or triple valve system that seals the ends of the tube (Paxton, 1986).
The reproductive biology, sexual strategies and developmental modes of Hyalinoecia spp. are largely unknown. Up to date, only a few studies have dealt with reproductive aspects and most of them reported partial or very specific information on a single aspect such as the egg- size, sperm-type or presence of brooded embryos and juveniles
(Table 1). To our knowledge, no Hyalinoecia species from the deep sea, or any other deep-water onuphid polychaete, has ever been studied in order to assess the gametogenesis, sexuality nor sperm-transfer method.
Among Hyalinoecia spp., the Atlantic deep-water quill worm, H. robusta Southward, 1977, is one of the rarest and most restrictedly distributed species. Hyalinoecia robusta was described from the soft mud bottoms of the southern Bay of Biscay, eastern North Atlantic, in depths of 1500–2300 m. It is interesting to note that bottom photographs of the area where this species was originally found showed that its tubes were projected nearly vertically from the sediment (Southward, 1977). This observation contrasts with the habits of other Hyalinoecia spp. that are often found crawling over sediment. Hyalinoecia robusta was found from a small area in low densities. Only 10 specimens were collected from five R.V. SARSIA stations between 1968 and 1974 (Southward, 1977).
Southward (1977) also confirmed the occurrence of the species from off La Gomera (Canary Islands, NW Africa) at depths of 1100 m, also in such scarce density, of only two specimens (Southward, 1977; Nú˜nez, 1990).
Later, the species was reported in Northwest Africa from the continental slope up to depths of 2800 m (Hartmann-Schr¨oder, 1982; Rosenfeldt, 1982; Kirkegaard, 1988, 1995, 2001).
We have studied quarterly samples of H. robusta collected in the northern Winter and Autumn seasons 2012 and the Spring season 2013 respectively, from the adjacent slope of the Avil´es submarine Canyons System (Bay of Biscay) at 1500 m depth. The main aim of the present paper is to report for this period the annual cycle and other significant findings of the reproductive biology of H. robusta from the study site, with emphasis on gametogenesis and development, in order to elucidate the sexuality and sexual strategy of this intriguing species. This study aims as well to provide new data about its ecology and faunal associates.
2. Materials and methods 2.1. Study area and sampling
The Avil´es Canyons System (ACS) is an intricate network of sub- marine canyons and valleys within the Central Cantabrian Sea, Bay of Biscay. Its bathymetric range extends from 128 m depth at its head, located only at 12 km off the Asturian coastline, down to 4766 m depth when it reaches the abyssal plain (G´omez-Ballesteros et al., 2014;
Table 1
Summary of the different reproductive patterns in the genus Hyalinoecia.
Max. size (mm)
Species Locality Depth (m) Length
(mm) Width
(mm) Observation Developmental mode Reference
H. araucana Carrasco,
1983 SE Pacific, off Chile 600 48 2 Up to 69 young (3- to 13-chaetigers)
surrounding adult in tube Brooding in parental tube Carrasco (1983) H. artifex Verrill, 1880 N Atlantic, US
Atlantic margin 330–520 180 7,5 Numerous eggs (300–400 μm diameter) in body cavity; in vitro fertilisation - no development
?Broadcast spawning,
lecithotrophic larvae Meyer et al.
(2016) H. bermudensis (
Hartman, 1965) as Paronuphis bermudensis
N Atlantic, off
Bermuda 2.500 8 0,6 Anterior half of body with large
eggs, posterior half with two well developed young
viviparity Hartman
(1965)
H. incubans Orensanz,
1990 Pacific, off New
Zealand 128–146 36 2 Up to 70 young (10-chaetigers) in
internal pouches of tube Brooding in parental tube Orensanz (1990) H. tubicola (O.F. Müller,
1776) as Onuphis tubicola
Mediterrranean,
Naples ? 65 1,5 Female gametogenesis; first report
of cluster of nurse cells associated with oocyte and pitted egg envelope
? Broadcast spawning,
lecithotrophic larvae Bergmann (1903)
H. tubicola Mediterranean,
Naples ? ? ? Ultrastructure of mature
spermatozoa ?Broadcast spawning,
lecithotrophic larvae Cotelli and Lora Lamia Donin (1975)
H. tubicola Sperm morphology classified as ect-
aquasperm or putative ent- aquasperm
Jamieson and Rouse (1989) H. robusta Southward,
1977 Bay of Biscay 1500–2300 >80 5–6 hermaphroditism; spermatophores;
fertilisation in tube/body cavity Spermatophores; semi- internal fertilisation; ? brooding in parental tube
present study
Romero-Romero et al., 2016, Fig. 1).
Samples were collected during the BIOCANT Oceanographic Campaign. This project consisted of three phases/oceanographic cruises:
BIOCANT 1, taking place from 3 to 13 March 2012 (Winter season) BIOCANT 2, between 27 September and 6 October 2012 (Autumn sea- son) and BIOCANT 3, from 24 April to 4 May 2013 (late Spring season) onboard the R/V Sarmiento de Gamboa. At each cruise samples were taken at eleven different stations distributed on the main axis of the ACS (stations C2, C3, C4A, C4B, C5, C6, C8, P4 and P5) and on the adjacent continental slope (stations P3 and TP) (Fig. 1). At each station, benthic samples were collected with a 5 m wide Agassiz dredge hauled at the bottom floor during 1 h (for additional details see Romero-Romero et al.
(2016)).
2.2. Treatment of specimens: general morphology and taxonomic procedures
Collected H. robusta specimens (within their tubes) were anaes- thetised in 7% MgCl2 and later fixed and stored in 10% neutral buffered formalin. Specimens (N = 42) were examined under a dissecting ste- reomicroscope to study their overall morphology. Body width (without parapodia) was measured at chaetiger 10 as size reference. Terminology of general and prostomial appendages follows Paxton (1986; 1998 respectively). Voucher specimens from the study sites (fixed in 10%
buffered formalin, preserved in 70% ethanol) have been deposited at the Museo Nacional de Ciencias Naturales, Madrid (MNCN). Specimens were stained with methylene blue solution and examined under a dis- secting stereomicroscope. Methylene blue staining increased the contrast of some morphological structures, such as branchiae, para- podial lobes and cirri. Glycerol slides of parapodia were prepared to examine chaetal morphology and distribution and examined under a compound light microscope. For a detailed ultrastructural analysis, selected specimens were prepared for scanning electron microscopy (SEM). Specimens were dehydrated in an ascending series of ethanol, critical point dried using acetone as transition liquid, mounted on aluminium stubs and sputter-coated with gold. Samples were then imaged using a JEOL 6610 LV scanning electron microscope.
2.3. Treatment of specimens: gametogenesis and development
After measuring, all specimens (N = 42) were classified by size classes. Also, when possible, the total length of complete specimens was measured and correlated with the width of the 10th chaetigerous segment. Then, a sample of the coelomic fluid (~1 ml) of all worms per season was extracted with a Pasteur pipette after making a short incision in their body wall at its median region. The extracted coelomic fluid was then mounted on a cavity slide and examined under a compound mi- croscope. We also estimated the mean number of coelomic eggs in mature ovigerous complete specimens. For each ovigerous individual (N
=38), the mean oocyte size from forty oocytes was calculated using the average value of the longest and shortest oocyte diameter as estimate of oocyte size. All measurements were done on mounted oocytes using a calibrated eyepiece graticule. The oocyte diameter was used as an index of the stage of maturation. All tubes were carefully studied to assess the presence/absence of brooding eggs, embryos or attached egg masses.
For studies on gut content and presence of endosymbionts in the coelom, specimens of H. robusta were dissected ventrally under the stereomicroscope.
In order to study the gametogenesis and sexual strategy, serial his- tological sections were examined after treating individuals as follows.
Specimens were post-fixed in Bouin’s solution for 24–48 h, washed thereafter in 70% ethanol for two days prior to dehydration, dehydrated following standard methods, transferred to xylene and embedded in paraffin wax. Serial, 6 μm thin sections were cut with a Leica 2045 microtome and double stained with haematoxylin and eosin and Para- pak ® (Meridian Biosciences, Inc., Cincinnati, Ohio) trichrome method. A total of 42 individuals collected from all sampling seasons and distributed among all size classes established were histologically examined.
Selected specimens and gamete samples were prepared for SEM for the study of gamete ultrastructure and their external structures. Samples were prepared for SEM examination as described above.
3. Results
3.1. Species description and ecological features Family ONUPHIDAE Kinberg, 1865
Fig. 1. The study area in the central Cantabrian Sea. The red asterisk shows the sampling station (BIOCANT-TP) where H. robusta was found. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Subfamily HYALINOECIINAE Paxton, 1986 Hyalinoecia Malmgren, 1867
Hyalinoecia robusta Southward, 1977
Type locality: R. S. SARSIA ST87 Station, Bay of Biscay 3.1.1. Description (based on Southward (1977) and studied topotype specimens)
Hyalinoecia robusta is a moderately robust, medium-sized species measuring up to 80–120 mm in length and 5–6 mm width for complete specimens of 80–90 segments. This species can be characterised as fol- lows: Prostomium anteriorly rounded with two globose frontal lips (Fig. 2A and B). Nuchal grooves straight to slightly curved. Median antenna to chaetiger 10–16. Eyes absent. Peristomium with middorsal anterior fold, ventral lip without median section; peristomial cirri ab- sent. Chaetiger 1 to 3 longer than following ones (Fig. 2C). Anterior three pairs of parapodia modified and directed anteroventrally (Fig. 2C);
parapodia 1 greatly prolonged, extended beyond anterior margin of prostomium (Fig. 2B and C). Prechaetal lobes of modified parapodia auricular, as long as postchaetal lobes. Ventral cirri subulate on anterior three chaetigers, fourth usually short and triangular, replaced by ventral pads from chaetiger 5. Dorsal cirri without basal swelling or process;
reduced to absent in posterior parapodia. Branchiae as single filaments from chaetiger 18–22 to almost body end. First two pairs of parapodia with simple bidentate hooded hooks; hooks absent from chaetiger 3, limbate and pectinate chaetae present from chaetiger 2 up to end of body. Pectinate chaeta with 19–21 teeth and rolled lateral margins (Fig. 2D and E). Subacicular hooks from chaetigers 19–37.
Preserved specimens were pinkish and slightly iridescent, without pigment marks or colour pattern, except for pigment spot on anterior part of prostomium (Fig. 2A). All specimens were found inside curved, horny and translucent tubes varying in colour from pale yellow to amber/clear brown colour (Fig. 2F). Tubes are curved and cylindrical
Fig. 2. Hyalinoecia robusta, photographs of preserved specimens (A, C, F, G) and scanning electron micrographs (B, D, E). A, B – detail of prostomium and peri- stomium, dorsal view; C – anterior end, dorsal view; D, E − pectinate chaetae from anterior chaetigers; F – tubes; G – fertilised eggs attached to parapodia.
with a smooth or slightly corrugated surface with growth ridges, con- taining flap valves at both ends but lacking whitish crescent-shaped marks. Smallest tubes were thin and almost transparent, while medium-sized and large ones were pale yellow to amber and thicker- walled (Fig. 2F).
3.1.2. Remarks
In the NE Atlantic, H. robusta overlaps geographically with H. tubicola (O.F. Müller, 1776) but they are partitioned along depth gradients. Hyalinoecia robusta is a much deeper and scarce species, while H. tubicola occurs at shallower depths, from the continental shelf to the upper slope, commonly in high densities. Morphologically, both species can be differentiated by the following features: i) tube type (robust and thicker in H. robusta, thin and less curved in H. tubicola); ii) shape of frontal lips (globose on H. robusta, pear-shaped or subulate on H. tubicola); iii) prostomial eyespots (absent on H. robusta, present on H. tubicola) and iv) origin of branchial filaments (from chaetiger 18–22 in H. robusta, from chaetiger 22–26 in H. tubicola).
3.1.3. Ecology, habitat and distribution
Parasitic apicomplexan gregarines were observed at different developmental stages (sporozoite, earlier and mature trophozoite) in the coelomic cavity and gut of several studied worms. The prevalence of host infected worms was 45% (N = 37). Epibiosis was present on tubes of specimens of all sizes. Globigerinid foraminiferans, lepetellid molluscs and sponges of the order Poecilosclerida were the most common epi- bionts found. In more than half of the studied specimens, the gut was filled with partly digested stomach content and fecal pellets in the posterior end of the body; otherwise mainly sediment, either alone or combined with mucus or different types of organic material, like fora- miniferan protozoans.
Hyalinoecia robusta presented a very restricted distribution within the Aviles Canyons System (ACS) and its surroundings. The species was
only found in one sampling station, BIOCANT station TP (44◦02.09′N, 5◦53.98′W), located at 1500 m depth from the lower continental slope in the boundaries of La Gaviera and El Corbiro Canyons (secondary canyons of the ACS) (Fig. 1). This station was located over deep-sea coral outcrops, outstanding white corals, Desmophyllum pertusum (Linnaeus, 1758) and Madrepora oculata Linnaeus, 1758, and lithistid sponges.
3.2. Sexual strategy, gametogenesis and development
Collected specimens of Hyalinoecia robusta ranged in length from 55 to 120 mm and the width of the 10th chaetiger ranged from 2.0 to 5.0 mm. The allometric relationship between the length and the width was positive (R2 =0.91; P ˂ 0.001). The smallest complete ovigerous spec- imen was 64 mm long and 2.4 mm wide; the smallest worm with coelomic sperm was 55 mm long with a width of 2.0 mm. The study of gametogenesis of 37 H. robusta specimens through histological sections revealed that the specimens of the smallest size class (width 1.5–2.0 mm) were exclusively males and the specimens from 2.1 to 5.0 mm in width were all simultaneous hermaphrodites (Fig. 3), presenting at the same time coelomic male and female gametes at different develop- mental stages. The relative quantity of sperm decreased with the size of worms, with low sperm production in specimens of larger size classes.
The smallest simultaneous hermaphrodite found (width 2.4 mm) was larger than the smallest male (width 2.0 mm). Pure females were not encountered during the study period. The estimated mean number of oocytes (fecundity) in a mature average-sized specimen with a volume near 0.8 ml (60 mm length -excluding anterior end and posterior region-, 4.0 mm wide) was 289.5 (range: 240–330; SD = 27.43; N = 10). The overall male: hermaphrodite ratio was 1:9.
The mean oocyte size variation and the size-frequency distribution of oocytes observed in the coelomic cavity of seasonal collected specimens (Fig. 4) showed that in the Avil´es Canyon System the species displayed an annual breeding season with a spawning period from late spring to
Fig. 3. Distribution sizes of males and hermaphrodites of H. robusta in the ACS population during the study period.
summer. The mean width of ovigerous worms was 3.8 mm, with in- dividuals in the largest size class (4.6–5 mm) only present in the Autumn sample (Fig. 3); however, individuals in the smaller classes (between 3.1 and 3.5 mm) were found in all sampling seasons (Fig. 3). According to the degree of the oocyte development (Fig. 4), the reproductive cycle of H. robusta can be divided into the following three stages: i) proliferating and growing stage, ii) maturing and iii) spawning stage. The prolifer- ating and growing stage occurred from October to the following March, the maturing and spawning stage took place during a reproductive season presumably extending from early Spring to Summer when ripe eggs of the largest size-classes (450–500 μm) represented more than 70% of oocytes filling the coelomic cavity. Within the same individual oogenesis was asynchronous and oocytes of various size-classes and different stages of oogenesis can be observed within the coelomic cavity (Fig. 5). Previtellogenesis and vitellogenesis occurred free in the coelom (Fig. 5). Oocyte-associated nurse cells in a single cap-like cluster were observed in oocytes with a diameter up to 150–200 μm (Fig. 5D), oo- cytes in larger sizes were always encountered without attached nurse cells (Figs. 5C and 6B, D, 7A).
Mature oocytes and eggs in advanced cleavage phases were found on dorsal intersegmental openings (Fig. 6B, C, D, 8B, C) and/or attached to parapodia of eight studied specimens (Figs. 2G and 6C). These eggs and early embryos were covered in a mucus-like substance (Fig. 8C). This observation indicates that the dorsal intersegmental openings of the body wall act as an exit route for ripe eggs and also as a site of fertilisation.
The spermatogonial cells were observed throughout the whole annual cycle associated with the small blood vessels that supply the lateral coelomic cavity and the nephridia (Fig. 5C and D). In spring, the spermatogonial cells developed spermatogonia which proliferated and gave rise to nests of spermatocytes. Shortly thereafter, the spermatocytes became spermatids that were detached from the clusters and continued the spermiogenesis free in the coelom close to the nephridia or inside
modified nephridial chambers (Figs. 5C and 7A). Spermatids resulted in mature sperm that formed aggregates in the coelom and into nephridial chambers. The histological study also showed that the metanephridia of H. robusta are not clearly separated into gonadal and excretory ducts, developing interconnected chambers, which store the sperm (Figs. 5C and 7A). These modified nephridial chambers filled by sperm were observed in all ovigerous specimens studied. At beginning of maturity, when the sperm start to accumulate in aggregates in the coelom/
nephridial chambers, additional ducts to nephridial-ducts (herein considered as gonoducts) are formed. In specimens larger than 2.4 mm in width, these gonoducts develop into a large funnel that opens dorsally (Figs. 6A and 7B, C, D) through a more or less globular-shaped papilla with a diameter that varied from 200 to 700 μm (Figs. 6A and 8A).
However, their morphology and size are largely a consequence of the quantity of the stored sperm (Fig. 7C and D). The papillae had a whitish colour in preserved condition (Fig. 6A) and appeared in pairs that were located on both sides of the segment midline from chaetiger 14–19 to chaetiger 20–30 (Figs. 6A, 8A and 9A). The papillae can detach from the worm body and thus they were also found loosely within the H. robusta tubes, causing the worm to have only one papilla or none in the corre- sponding segment midline (Fig. 8A).
The external morphology of the spermatozoon of H. robusta is characterised by having an elongated, high conical head topped by a narrow tubular acrosome and a long tail (Fig. 9B–D). The base of the head is slightly wider than the short ring-shaped mid-piece (Fig. 9C).
The mean length of the head, from the acrosome tip to the posterior mid- piece, is 3.30 μm (N = 20, SD = 0.13). The entire spermatozoon surface is surrounded by the plasmalemma (Fig. 9C and D), which appears coarse in the acrosomal and nuclear regions, and becomes smooth at the mitochondrial and the flagellar regions. The acrosomal complex is formed by a relatively large acrosomal vesicle, elongate in shape. The middle piece consists of five almost spherical mitochondria encircling a collar formation (Fig. 9C and D). A ring is absent around the initial Fig. 4. Quarterly size-frequency histograms of coelomic oocyte diameter of H. robusta from the ACS from March 2012 to May 2013. N: number of individuals of each sample.
portion of flagellum (Fig. 9C and D).
4. Discussion
Onuphid polychaetes are one of the most successful deep-water families in terms of species richness, abundance and biomass contribu- tors (Paxton, 1986; Paxton and Arias, 2014; Gillet and Dauvin, 2003;
Surugiu et al., 2008; Meyer et al., 2016). Species of the genus Hyalinoecia are characterised by their peculiar lifestyle; unlike most of the group, they are free-living epibenthic species that built distinctive quill-like tubes (Fig. 2F).
The ecological habits and the interspecific relationships of the spe- cies of the genus Hyalinoecia, even the well-known H. tubicola, are practically unknown. To date, the parasite gregarine apicomplexans had not been described on H. robusta or on other Hyalinoecia spp., consti- tuting a newly reported symbiotic association. The presence of Fora- minifera in the gut content was probably due to the unselective ingestion of sediment, and that of trophozoites or cysts of gregarines to accidental ingestion of sporozoites with sediment. Our results suggest the sediment as the major food item. This may be consistent with the general
observations in onuphids, that are primarily considered as omnivorous scavengers (Fauchald and Jumars, 1979). However, the apparently detritivorous habit of H. robusta contrasts with the carnivorous or scavenging carnivorous lifestyle reported in other Hyalinoecia spp., such as H. tubicola longibranchiata McIntosh, 1885 and H. artifex Verrill, 1880 (Cummings et al., 2013).
The reproductive biology, sexual strategies and developmental modes of Hyalinoecia spp. are also largely unknown. Table 1 summarises the scarce studies that have dealt with reproductive aspects of Hyali- noecia spp. Most of them reported partial or very specific information on a single aspect such as the egg-size, sperm-type or presence of brooded embryos and juveniles. Until the present work, no Hyalinoecia species from the deep sea, or any other deep-water onuphid polychaete, had ever been studied to assess the gametogenesis, sexuality nor sperm- transfer method. Our knowledge of their reproductive biology is scarce due to the rarity of material. We were able to find a sufficient number of H. robusta specimens in quarterly samples (winter, spring, and autumn of 2012–2013), from the adjacent slope of the Avil´es sub- marine Canyons System. Thus, we have been able to sketch the annual cycle of H. robusta in the Bay of Biscay, the type locality of the species.
Fig. 5. Hyalinoecia robusta, transverse histological sections. A – gravid adult, overall view; B – gravid adult showing a dorsal opening of the body wall; C – overall view to show the nephridium with associated blood vessels and sperm cells, and vitellogenic oo- cytes; D – clusters of oogonial cells and spermato- gonia associated with blood vessels of lateral coelomic cavity and early previtellogenic oocytes associated with nurse cells. (c) coelomic cavity; (do) dorsal opening; (g) gut; (m) muscle; (nc) nerve cord;
(ne) nephridium; (nec) nephridial chamber; (nuc) nurse cells; (o) oocyte; (oo) oogonial cells; (po) pre- vitellogenic; (s) sperm; (so) spermatogonia; (v) blood vessel.
The studied population showed an annual periodicity in breeding with the spawning season taking place in the summer months. Individuals of H. robusta in the largest size classes were present in all samples throughout the annual cycle, indicating that the species is iteroparous with a discrete reproduction with only one reproductive period per year.
At the time of maturity, the nephridia of H. robusta develop into modi- fied nephridial chambers that store the sperm and take on the function of genital ducts. Nephridial ducts with both urinary and genital functions were also observed in mature specimens of several Diopatra spp. and one Onuphis species Audouin & Milne Edwards, 1833 (Arias et al., 2013, 2016; Krishnamoorthi, 1963; Arias and Paxton, 2015).
The results of the present study demonstrate that male and female gametes develop at the same time within the same individual, providing the first-time evidence of the occurrence of simultaneous hermaphro- ditism in H. robusta and also within the subfamily Hyalinoeciinae.
Within Onuphidae, hermaphroditism has been previously reported only
in four species of the genus Diopatra (Lieber, 1931; Arias et al., 2013, 2016; Arias and Paxton, 2015) and suggested for R. (Spinigerium) ehlersi (Paxton, 1986). Furthermore, our study has also shown that this species passes through a protandrous or adolescent male phase before reaching the simultaneous hermaphrodite condition. Specimens of larger size classes have a reduced sperm production and this is commonly associ- ated with hermaphroditic polychaetes which are able to self-fertilise (Sella, 2006), therefore, it would not be surprising that the phenome- non of self-fertilisation may occur in isolated specimens.
The occurrence of a previous adolescent male phase is widespread in simultaneous hermaphrodite polychaetes (Sella, 2006) and may be justified by the “size advantage hypothesis” (Charnov, 1982; Ghiselin, 1987). This hypothesis is based upon two assumptions: i) the repro- ductive success of an individual as male or as female is closely linked to body size, and ii) reproductive success and size is different for each sex.
Therefore, protandry, or a previous protandric phase before Fig. 6. Hyalinoecia robusta, photographs of gravid specimens. A – anterior end showing dorsal openings and papillae; B – median region in lateral view, showing eggs and embryos by transparency; C – detailed view of an embryo attached to parapodia; D – median region in dorsal view, showing eggs and embryos by transparency.
(ch) chaetiger; (e) embryo; (o) oocyte; (op) dorsal openings (arrows pointing to them); (s) spermaducal papillae.
simultaneous hermaphroditism, is expected if a larger body size in- creases female fecundity more than male fertility (Schroeder and Her- mans, 1975; Sella, 2006; Arias and Paxton, 2015). Hyalinoecia robusta shows a strong correlation between body size and egg number, with larger specimens producing more eggs than smaller ones. As eggs are more costly (in energy terms) to produce than sperm, a small individual with limited resources cannot produce a profitable number of the costly eggs. Nevertheless, it can produce a significant quantity of less-expensive sperm (increasing fitness) and instead use its remaining resources to rapidly grow to achieve a larger size capable of producing the necessary number of eggs. Simultaneous hermaphroditism is typi- cally correlated with brooding behaviour, sedentary habits and living at low population densities (Heath, 1977; Ghiselin, 1987; Giangrande, 1997; Sella, 2006). Although these relationships are not very strict, members of the H. robusta population from the Avil´es Canyons System fulfill all these requirements.
During the present study we documented the spermiogenesis of H. robusta and showed that mature spermatozoa (Fig. 9B–D) have elongated heads and a modified ring-shaped mid-piece resembling the ent-aquasperm type as defined by Jamieson and Rouse (1989). Very similar sperm have been reported for H. tubicola (Cotelli and Lora Lamia Donin, 1975). Hyalinoecia robusta sperm may not fertilise the eggs directly in seawater in the classical manner. While the ent-aquasperm is also shed into the ambient water like the ect-aquasperm, it differs in that it reaches the female/hermaphrodite and may be stored in spermathe- cae, or, in the case of sedentary polychaetes, in the tube (Jamieson and Rouse, 1989). Thus, in H. robusta, the egg fertilisation may occur within the dorsal openings of the body wall or inside the worm tube. This possibility is consistent with the finding of fertilised eggs and developing embryos attached to the body wall and/or inside the worm tube in some specimens (Figs. 6C and 8B, C). Moreover, the ent-aquasperm mode of
fertilisation appears to be related to the production of a small number of large-yolked eggs, and proved effective in worms which are not, or are poorly, motile (Jamieson and Rouse, 1989). Sperm of the same kind as that found in H. robusta occur in three other onuphid genera: Diopatra, Kinbergonuphis Fauchald, 1982 and Rhamphobrachium Ehlers, 1887; in all cases it has been reported from brooding species (Rouse, 1999; Hsieh and Simon, 1990; Arias et al., 2013; Paxton and Arias, 2014). This apparent correlation between the ent-aquasperm type and the brooding behaviour in onuphids has been previously suggested by Jamieson and Rouse (1989). These same authors postulated as well that the fertilisa- tion by ent-aquasperm requires fewer eggs than in external fertilisation and that it is accompanied by a tendency to lecithotrophy, two condi- tioning factors that fit with the biology of H. robusta.
Papillated H. robusta specimens (Figs. 6A and 8A) were found among all size classes larger than 2.4 mm in width of the 10th chaetiger. These specimens were examined histologically and all found to be simulta- neous hermaphrodites. Furthermore, the conducted histological and ultra-structural studies have shown that these papillae are filled by mature sperm. On the basis of these findings, we considered that the papillae act as seminal vesicles that store the own spermatozoa and also as an aggregate transfer mode, in which the sperm are packing together in a multilayered structure (Fig. 9A). These hollow capsules can be de- tached from the worm and were also found free within H. robusta tubes, consequently we considered that they may act as spermatophores. In the same way, Paxton and Arias (2014) reported similar sperm-filled fibrous capsules, acting as spermatophores, in another deep-water onuphid, Rhamphobrachium (Spinigerum) brevibrachiatum (Ehlers, 1875). Brooding in polychaetes is usually coincident with sperm transfer between in- dividuals in the form of spermatophores or spermatozeugmata that are transferred during copulation/pseudocopulation or float through water to be collected by the females (Rouse, 1999). In polychaetes, aggregate Fig. 7. Hyalinoecia robusta, transverse histological sections. A – overall view to show the nephridium with associated blood vessels and sperm cells; B – gravid adult showing the connection of the lateral coelomic cavity with the dorsal openings and the papillae; C – overall view of the spermaducal papillae;
D – detailed view of the spermatozoa packages inside the papilla. (c) coelomic cavity; (do) dorsal opening;
(g) gut; (m) muscle; (ne) nephridium; (nec) nephridial chamber; (o) oocyte; (p) parapodium; (pa) papilla; (s) sperm.
sperm transfer by means of spermatophores has been considered as an adaptive character in sessile tubicolous worms, outstanding families the Spionidae and Siboglinidae. Many spionids have buoyant spermato- phores and all known siboglinids release their sperm through bundles or spermatophores (Blake and Arnofsky, 1999; Young, 2003). Hsieh and Simon (1990) reported free transfer of demersal spermatophores in Kinbergonuphis simoni (Santos, Day & Rice, 1981). In laboratory exper- iments, male worms of this species were observed to release mushroom-shaped spermatophores as clumps from the tube openings.
When spermatophores were placed near the tube openings of females, within a minute the females extended part of their body from the tube and picked up the spermatophores with their anterior parapodia and palps, pulling them into their tubes (Hsieh and Simon, 1990). The tube dwelling H. robusta lifestyle limits direct bodily contact and its low population number reduce the individual encounters between mature specimens. Thus, free transfer spermatophores may be the only way for H. robusta, since the alternative (i.e. broadcast spawning) requires large numbers of both type of gametes and synchronous reproduction of the Fig. 8. Hyalinoecia robusta, scanning electron micrographs. A – anterior body region showing papillae of different sizes; B – median body region showing eggs and embryos protruding from the dorsal intersegmental openings; C – detailed view of embryos protruding from the dorsal opening.
population. Free spermatophore transfer with spermatozoa storage has been proposed as an efficient low risk mode of sexual reproduction with high fertilisation efficiency (e.g. 98.9% in K. simoni (Hsieh and Simon, 1990)).
An accurate knowledge of the reproductive biology is essential when assessing the zoogeography of the species involved. In polychaetes, brooding behaviour and lecithotrophy tend to limit larval dispersal and are usually correlated with species with narrow distribution ranges or more restricted distribution. Kirkegaard (1995) reported H. robusta from several locations of Indonesia based upon specimens collected at shelf depths. These specimens were collected at densities of up to 20 speci- mens per sampling station and also, some of them differed from the original description by having up to three branchial filaments (Kirke- gaard, 1995). The herein reported reproductive features of the species are not consequential with a great larval spreading and populations connectivity that may explain such a wide distribution of this species.
Thus, the H. robusta reproductive traits together with the ecological and morphological differences between the Indonesian and the Northeast Atlantic specimens make their conspecificity very unlikely. Accordingly, the name H. robusta may be hiding other deep-water Hyalinoecia species and hence we think that it should be considered as a species complex, until proven otherwise.
Further research and detailed histological studies, involving other Hyalinoecia species that remain poorly known biologically, are needed to fully understand the sexual strategies within the genus. A
comprehensive knowledge of reproductive biology of the organisms involved is necessary for ecological forecasting and even hindcasting biogeographic distributions of deep-water species.
CRediT authorship contribution statement
Andr´es Arias: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Hannelore Paxton: Writing – review &
editing, Writing – original draft, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We would like to thank Maria Jo˜ao Santos and Pilar Ríos for their help with the identification of the gregarines and sponges found, respectively. We also thank Julio Parapar and two anonymous reviewers for helpful comments. We are grateful to technicians of the Scientific- Technical Services of University of Oviedo for assistance with SEM mi- croscopy. This is a contribution from the Fauna Ib´erica Project, Fig. 9. Hyalinoecia robusta, scanning electron micrographs. A – two broken papillae showing their internal multilayered structure; B – number of spermatozoa.; C, D – enlarged view of spermatozoa, showing their spindle-shaped head and the spherical mid-piece.
subproject Annelida-Polychaeta VII: Palpata-Canalipalpata II (ref.
PGC2018-095851-B-C64) and the Marine Observatory of Asturias (OMA).
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