Nudibranchs out of water: long-term temporal variations in the abundance of two Dendrodoris species under emersion

Study site

This study was carried out in an oyster bank located in the Sado Estuary Natural Reserve, on the West coast of Portugal (38.474857°N, 8.775255°W; Fig. 1). Given its elevation, this bank is submerged during high tides and emerged during low tides (< 0.8 m relative to Mean Lower Low Water), presenting a total emerged area of 1050 m². The predominant substrate is sand and mud with a great amount of oyster and cockle shelves (Fig. 1).

Nudibranch abundance

Adult nudibranchs of the species D. herytra and D. grandiflora were visually counted monthly, between January 2011 and June 2015. Surveys were carried out during the lowest tide of the month (< 0.8 m) and covered the entire emerged oyster bank. While looking for nudibranchs, we also looked for egg masses and sponges, the natural prey of Dendrodoris.

To evaluate if nudibranchs were sexually mature and if they die after spawning, 10 nudibranchs of each species were collected during the peak of abundance in April 2015 and transported to the aquaculture facilities of Laboratório Marítimo da Guia (Cascais, Portugal). They were kept in two separate groups aquaria, in recirculating systems under the same conditions as those observed in the study area (i.e. pH 8.0; salinity 35, temperature 21 °C). Individuals were not fed and were maintained for 15 days in those conditions. The occurrence of mating, spawning and death after spawning was assessed. Organisms considered dead where normally found at the bottom upside down. Death was confirmed after loss of mobility, increase of mucus release, high loss of colour, and loss of muscular flexibility.

Environmental conditions

To establish a connection with the abundance of nudibranchs, environmental data were collected between January 2011 and June 2015. Water temperature, turbidity and dissolved oxygen were measured with a multiparameter probe (CTD YSI 6600 V2, Tropical Marine Centre, Portugal) and salinity was measured with a refractometer (V² Refractometer, Tropical Marine Centre, Portugal) with a precision of ± 1. Measurements were made in the surroundings of the emerged bank during low tide when nudibranchs were observed and collected.

Statistical analyses

To investigate the relation between D. herytra and D. grandiflora abundance and environmental variables, a generalized additive model (GAM) with a zero-inflated Poisson (ZIP) family was used for each species. The ZIP model is especially useful to analyse count data with many zero observations, such as our data [25]. Prior to statistical analyses, the independency of environmental variables was checked using a correlation matrix.

The method used to choose the best model was an “all possible subsets” method of analysis, in which several models were created for all possible combinations of variables. Akaike Information Criterion (AIC) was used to select the most parsimonious model. Based on these results, the final model included water temperature, salinity, turbidity and dissolved oxygen as independent variables. A mix of smooth and parametric model components was used [26]. For D. grandiflora, a thin plate regression spline with “shrinkage” was fitted [26] to water temperature, turbidity and dissolved oxygen, while for D. herytra the same smooth parameter was fitted only to turbidity and dissolved oxygen.

All statistical analyses were implemented in R (version 3.1.1), using the mgcv package [26].

Nudibranch abundance

Nudibranchs were mostly encountered emerged, but some were occasionally found in small tidal pools or under oyster shelves. Most of the individuals were found alone, static, and some upside down. No dead animals, egg masses or sponges (their natural prey) were encountered in any of the monthly surveys during the 5 years. The highest abundance of both species was observed during the first year of observations (i.e. 2011), followed by the year 2013 (Fig. 2). An accentuated decrease in nudibranch abundance was observed in 2012 and 2015. In general, D. grandiflora presented higher abundances than D. herytra (Fig. 2).

The appearance of emerged nudibranchs presented a marked seasonal pattern (Fig. 2). Nudibranchs were observed from March to June, mainly during April and May, and disappeared completely from July onwards. This pattern was consistent during the five consecutive years, with just a few small changes observed over time. For instance, in 2011 nudibranchs of both species started to appear emerged in April (Fig. 2), while in the following years (i.e. from 2012 to 2015) they started to appear earlier in March (Fig. 2), although in very low numbers. The maximum abundance was always reached in April (Fig. 2), except for 2014 when the peak was reached later in May (Fig. 2). Afterwards, nudibranchs’ abundance started to decrease until a complete disappearance in July (Fig. 2), although in 2014 they disappeared abruptly in June (Fig. 2).

Nudibranchs captured in April 2015 and kept under laboratory conditions were seen copulating and spawning as soon as they arrived at the lab (Fig. 1), which indicates that the organisms from the study population were sexually mature during the peak of abundance of that year. Moreover, we found no evidence of death after spawning, with some individuals spawning more than once.

Environmental conditions

Variation in the environmental parameters of the Sado estuary, from January 2011 to June 2015, is presented in Fig. 3. Water temperature, as well as turbidity, showed consistent patterns throughout the five years of observations. As expected, water temperature showed an increasing trend from winter to summer months, almost like a bell-shaped graph (Fig. 3A1–A6). Maximum water temperatures occurred between June and August, depending on the year. Salinity showed no clear pattern (Fig. 3B1–B6). Turbidity varied generally between 1 and 5 NTU during most of the year, except for March and April, when it could reach values as high as 75 NTU (Fig. 3C1–C6). Dissolved oxygen varied between 60% (4.5 mg l⁻¹ O₂ around 26 °C) and 150% (12.7 mg l⁻¹ O₂ around 13 °C), with a sharp increase occurring in the beginning of the spring season and reaching a peak in April or May depending on the year (Fig. 3).

Relationship between nudibranch abundance and environmental conditions

Emersion and microhabitat selection

Nudibranchs are known for their sudden appearance and disappearance in intertidal areas (see Table 1), but reports of emerged nudibranchs are scarce [10, 11, 27]. The present work constitutes the first comprehensive report of the seasonal occurrence of emerged nudibranchs during low tides. In contrast to other molluscs, nudibranchs do not possess a shell that protects them from desiccation and allows them to create a high humidity environment within their shells. Instead, they usually seek areas of great humidity and refuge beneath rocks or other shelters (see more about microhabitat selection in [28]). Although nudibranchs are generally not found in unsheltered and open areas, in the present study we showed that dozens of individuals were emerged at low tides during spring months. Nevertheless, it is worth mentioning that the texture of the substratum (i.e. oyster shell bank) seems to play an important role in microhabitat selection by Dendrodoris species, since all the other intertidal banks in the surrounding area—without oyster shells coverage and mostly constituted by mud substrate with little sand—never revealed any emerged specimens. From this, we argue that oyster shells must provide some crypsis by providing a disrupted visual background that might contribute to a reduction in visual predation. Also, this could mean that oyster shells protect nudibranchs from dehydration. However, the fact that most nudibranchs were not beneath the shells makes this microhabitat selection argument incomplete.

Horizontal migrations and spawning

There is a great diversity of theories that explain the sudden appearance and disappearance of nudibranchs in intertidal areas (as synthesized in Table 1). However, it is still unknown if these events are related with horizontal migrations, and what are the causes that motivate these movements. Some authors state that, as a result of these horizontal migrations, nudibranchs seasonally appear as mass aggregations in intertidal areas to mate and spawn [4, 6]. In our surveys, organisms were mostly seen isolated and no egg masses were seen in the study site. This way, it seems unlikely that the nudibranchs were in the study area to mate or to spawn. Nonetheless, the individuals that were brought to the laboratory immediately started to copulate and spawned, indicating that they were sexually mature. Thus, the hypothesis of a horizontal migration to intertidal areas to mate should not be set aside and further studies are necessary to clear this out.

The sudden disappearance of nudibranchs from intertidal areas has often been attributed to death after spawning [4, 6]. During our surveys, we did not observe dead individuals in the field or any evidence of death after spawning in the specimens kept in the laboratory, with some nudibranchs spawning more than once. This way, our results with Dendrodoris spp. do not support this hypothesis. However, and like other molluscs, death after spawning may be a species-specific trait. While a similar pattern was observed for Onchidoris bilamellata, a species that spawns several times before dying [29], other nudibranchs seem to experience death after spawning (e.g. Archidoris montereyensis, in [11]).

Environmental determinants

The abundance of D. herytra and D. grandiflora was correlated with most of the assessed environmental factors, namely water temperature, turbidity and dissolved oxygen. Our long-term survey consistently showed that both species appeared emerged during the low tides of the spring months. They started to appear in March and their abundance was highest between April and May, when water temperatures were generally between 17 and 21 °C (except for 2014, when it reached 24 °C). Both species completely disappear in July, before the high summer temperatures. A study conducted in the western Atlantic revealed that most nudibranchs have high thermal sensitivity and that some species disappear with rising summer temperatures [30]. A negative impact of high temperatures on D. herytra and D. grandiflora may in part explain their disappearance from the intertidal area at the end of the spring. Nevertheless, temperature alone cannot explain their presence only during spring, since no nudibranchs were observed at similar temperatures in autumn.

The presence of D. herytra and D. grandiflora in the intertidal area was consistently related with a peak in turbidity and dissolved oxygen. Elevated turbidity and oxygen super saturation (values exceeding 100%) can be related with spring blooms that result from an increase in phytoplankton biomass and photosynthesis during spring [31–33]. As a result, secondary productivity also increases, including zooplankton grazers [34], which benefits D. grandiflora’s planktotrophic larvae [22]. In addition, high turbidity and dissolved oxygen may be a result of turbulence which in turn will help the distribution of planktonic larvae in the water column [35]. This way, Dendrodoris planktonic life stages may benefit from increasing phytoplankton concentration. However, it is noteworthy that no sponges (the prey of the adults of Dendrodoris spp. [24]) were found in the study location during the 5 years of survey. It is also important to notice that elevated turbidity consistently preceded an increase in nudibranchs abundance (also accompanied by the peak of dissolved oxygen). Thus, we hypothesize that the increase in turbidity is the key environmental factor that triggers the migration of nudibranchs.

In conclusion, the present work constitutes an additional example of a nudibranch population that seasonally appears in intertidal areas, but the first comprehensive report of nudibranchs under tidal emersion. The sudden appearance and disappearance of Dendrodoris nudibranchs in intertidal areas may result from a seasonal horizontal movement of adult nudibranchs from subtidal areas to mate in intertidal areas during spring, when phytoplankton production is enhanced and planktotrophic larvae may benefit from greater food availability. However, further studies are required to test this hypothesis.