The yellowfoot limpet Cellana sandwicensis, ‘opihi ‘alinalina, is a significant cultural resource and seafood for Hawaiians. With wild stocks greatly depleted and under significant commercial pressures, research efforts are focused on understanding their reproductive biology to improve management and ensure survival of this species.

During spawning season, wild aquaculture-housed limpets were examined on a monthly basis for sexual maturation, via gonadosomatic index and histological examination. Spawn events, which coincided with peak maturation, were observed. Furthermore, a single specimen was observed to be a hermaphrodite. While hermaphroditism has been a documented reproduction strategy for other Palletid spp., it has never been documented in any of the Hawaiian limpets (Cellana spp.).

Previously, Cellana sandwicensis was assumed to be gonochoristic with males and females. And despite sex ratios shifting across time and geographic location, no clear biological explanation existed for this phenomenon.

From this finding, we propose that the yellowfoot limpet may be a sequential hermaphrodite, which has, until now, gone undetected. This hermaphrodite, although carrying both gonads simultaneously, was most likely caught at an inter-sex level. It is then plausible to consider environmental factors (i.e. presence of conspecifics) playing a role in sex determination for this species.

In the future, defining sex ratios at various class sizes, time points, and geographic locations will improve our understanding of reproductive biology for yellow foot limpets. Moreover, monitoring individuals across multiple spawning events/seasons will clarify any speculation regarding their ability/inability to switch sexes.

Mau A, Fox K, Bingham JP (2017) The Reported Occurrence of Hermaphroditism in the Yellowfoot Limpet (Cellana sandwicensis Pease, 1981). Ann Aquac Res 4(4): 1045.

•    Hermaphroditism
•    Invertebrate
•    Mollusk
•    Sexual reproduction

Nearly all forms of sexual reproduction have been documented for mollusks. In terms of hermaphroditism, some mollusks exhibit simultaneous hermaphroditism, sequential hermaphroditism, or alternating hermaphroditism reproductive strategies [1]. In other cases, the ovo-testis, a small hermaphroditic duct containing both male and female gonad characteristics, is common [2]. Despite this plastic reproductive molluscan nature, only 3% of known species of Eogastropods (formerly Prostobranchs) [3] are functional hermaphrodites [4]. Moreover, hermaphroditism occurs in less than 0.29% of all Patellid species, which is considerably rare [5]. For Patella ferruginea, a well-documented limpet, the minimum size at which sex change occurs is 40-60 mm shell length, and in the male to female direction. For protandrous species like this, size selecting (during harvesting) may negatively impact reproductive output and threaten species survival [6]. Additionally, selecting larger individuals has been reported to cause mediated sex change in smaller sized individuals, which shifts population dynamics [7]. These aspects of hermaphroditism are considerably important for conservation and management efforts for at-risk Patellid species.

The yellowfoot limpet, Cellana sandwicensis, ‘opihi ‘alinalina an at-risk Patellid species, may also exhibit a form of hermaphroditism. Previous research reports that mature limpets maintain separate sexes, gonochoristic. The typical spawning season coincides with the peak of winter, however, occasional spawning events have been recorded during other times of the year [8]. The peak of this spawning season is thought to shift year-to-year depending on the hydrodynamic environment (i.e. temperature, tide, and waves). The sex ratio shifts from being dominated by an unidentifiable sex during resting periods to a near 1:1 male to female ratio in spawning periods [8,9]. However, this interesting phenomenon still lacks a biological explanation. Hence forth, authors aim to show evidence of a bi-directional form of sequential hermaphroditism, and to explain how hermaphroditism may be a significant reproduction strategy for the survival of this Hawaiian limpet.

Yellowfoot limpets (Cellana sandwicensis) were collected from Rabbit Island, Oahu, Hawaii (21°19′52″N 157°39′32″W) to monitor sexual reproduction. Animals (n=45) were stocked at random into a circular flow-through aquaculture system (total tank surface area of 0.6 m2 ) and reared at ambient conditions.

Air temperature (18.51-33.57°C) was recorded in 4 second intervals using HOBO MX1101 Temperature/Relative Humidity Data Logger (Accuracy ± 0.2°C, Onset Computers, Bourne, MA). Seawater temperature (24.65-26.93°C) and dissolved oxygen was measured twice daily and averaged using an YSI Pro20 Dissolved Oxygen Meter (Accuracy ± 0.3°C and ± 0.2 mg L-1, YSI Inc., Yellow Springs, OH). Seawater pH (8.24) was measured once daily using an EcoSense pH10A instrument (YSI Inc., Yellow Springs, OH), and salinity (30 g L-1) was measured once daily using a Vital Sine Refractometer (Accuracy ±1.0%, Pentair, Apopka, FL).

Animals were offered a formulated diet (ad libitum) with access to feed between 16:00 and 08:00 hours. Animals (n=3) were sacrificed monthly to determine maturation status, and at the end of the trial. The reported hermaphrodite specimen was sacrificed at the end of the trial (May), and dissection of this individual revealed the presence of both ovary and testis.

Measurements for shell length and shell width (mm) were done using an electronic caliper (0.01 mm), and measurements for total weight (g), soft-body weight (g), and gonad weight (g) used an electronic scale (0.0001 g, AG104 Mettler-Toledo International, Columbus, OH). The gonadosomatic index (including both ovary and testis) was calculated as follows: GSI = [total gonad weight / total soft body weight] x 100.

Gonad tissue was fixed in 10% v/vneutral buffered formalin for 24 hours at room temperature and rinsed with 70% v/v ethanol. Samples were wax embedded, and 6 µm horizontal sections across the entire length of the gonad were stained with haematoxylin and eosin. Images were captured with Infinity Analyze V 6.5.4 Lumenera Software and Infinity3S-1UR microscopy camera (Lumenera Corporation, Ottawa, ON), and compound microscope Olympus BX43 (Olympus, Center Valley, PA).

The sex ratio during the current spawning season was 1:3 male to female—an egg-rich population. The sex ratio during this study differs from previous literature, which has previously been reported to be 1:1 male to female in two separate studies [8,9]. This may be due to different determinants of sex: environment (i.e. conspecific interaction, temperature and pollution/ pollutants) and/or genetics found across different time scales and geographic locations.

Recently, we surveyed yellowfoot limpets in the Papahanaumokuakea Marine National Monument (a marine reserve in the Northwestern Hawaiian Islands), which appeared to be another egg-rich population (unpublished data); and we postulate that the presence of larger individuals and/or high density aggregates may be factoring in to this phenomenon. More specifically, the sperm to egg ratio may play a role in controlling proper fertilization [10], and therefore influence sex ratios.

During this study, only 1 of 45 samples was found to be hermaphroditic. The specimen’s shell (Figure 1A/2B) was 62.08 mm long and 49.54 mm wide, it is considered a relatively large individual (the average shell length and shell width for this collected group was 37.96 ± 3.76 mm and 29.91 ± 3.24 mm, respectively). The specimen’s total weight was 35.54 g and soft body tissue weight was 10.75 g. The total gonad weight, including both ovary and testis, was 0.68 g; and the GSI was 6.33% at the time of dissection. In March 2017, this individual was observed spawning in the tank environment, naturally. We determined this specimen to be a functioning male based on the release of white colored gametes. However, the presence of female reproductive organs found during dissection provided evidence that this specimen should be classified as a hermaphrodite.

The small sample size and low rate of hermaphroditism (2.22%) may simply be a result of random, genetic mutation acting on this single specimen. However, we must also consider hermaphroditism as a viable reproduction strategy for this species. When considering the classification of hermaphrodites, yellowfoot limpets might be recognized as simultaneous hermaphrodites since both ovary and testis were observed in this specimen. However, histology indicates that this specimen was not a functioning simultaneous hermaphroditism. The testis of this specimen contained mature spermatozoa with gaps in the gonadal cavity indicating a mature/post-spawn stage of gonad development (Figure 3A/3B). The ovary contained primordial germ cell proliferation, indicating an early stage of gonad development for future spawning events (Figure 2A/2B). Because this individual had an immature ovary and functioned as a male during spawning season, it would not be physiologically capable of selfing (self-fertilization) or cross-fertilizing with a mate—a recognizable characteristic of simultaneous hermaphrodites [4].

The apparent sex switch occurring in this individual specimen is most likely a form of sequential hermaphroditism. However, based on current data for yellowfoot limpets, there is no evident bias in sex ratio at any given size class that would indicate this to be a protandrous or protogynous species (unidirectional sex change) [11]. A more feasible theory is that this species maintains a bi-directional form of sequential hermaphroditism—a reproductive strategy found in fish [12], oysters [13], and limpets [14]—that allows switching of sexes in either direction based on environmental influences.

During sex change of sequential hermaphrodites, it is theorized that gonad development is arrested and the animal is not reproductively viable [5]. We explain that the discovery of a specimen carrying both male and female gonads may illustrate a rarely observed intermediate stage during the transition between sexes—where an individual would usually transition into an unidentifiable sex (reduced gonad cavity). We also consider that this specimen appears to sex-switch in a single spawning season, and may have continued developing into a functioning female without a resting phase between the transitions. This would mean that yellowfoot limpets can potentially sex-switch at any given time of the year. However, it appears that the “normal” period where we could expect the sex switching to occur would be between spawning seasons (resting period/offseason).

Interestingly enough, hermaphrodites that switch sex back and forth are only found in broadcast spawning species of marine snails [1]. From an evolutionary perspective, the ability to switch sexes is crucial for the survival of the species when lacking copulatory organs. For yellowfoot limpet, an at-risk species, individuals capable of switching sex based on their available partners allows them to reproduce under adverse conditions (i.e. low densities). We therefore suggest these limpets most likely undergo environmental sex determination—where individuals can influence the sexual status of another conspecific via pheromones— as opposed to genetic sex determination associated with other sequential hermaphrodites [4].

To test this speculative theory, researchers would need to monitor broodstock across multiple spawning seasons to determine if functional sex-switching occurs. And based on the low rate of hermaphroditism, a relatively large number of individuals would be required. Based on our postulated theory, altering the size class and/or densities in the tank environment over time may increase the rate of hermaphroditism. Additionally, frequently sampling throughout the year would potentially increase detectability of direct sex-change (intermediate stage) within a single spawning season.

The yellowfoot limpet (Cellana sandwicensis), is an important traditional cultural and food resource, and a candidate aquaculture species. As part of proper management, conservation, and production, it is imperative to understand the life cycle and mode of reproduction for these marine snails. In this current study, we report a paradigm-shifting discovery, a hermaphrodite individual carrying an ovary and testis. Previous to this study, it has been reported that individuals are of separate sexes, male or female, but hermaphroditism was never observed. Although there were reports of annual shifts in sex ratios, this phenomenon could not be explained. We propose a theory that the yellowfoot limpet is a sequential hermaphrodite and is capable of switching sex in a bi-directional manner dependent on environmental cues.

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