**3. Results**

In the area investigated, a total of 1755 sponges belonging to the class of Demospongiae were counted and classified in 14 families belonging to 16 different genera, with *Scopalina* (26.67%), *Aplysina* (14.30%), *Agelas* (13.16%), and *Aiolochroia* (9.46%) emerging as the more abundant, while the remaining genera only represented under 7% of the sponges found in the area (Table 2).


**Table 2.** Genera of sponges considered for the analyses in the study area with values of relative abundance, maximum and mean prevalence of association with *Nemalecium lighti*.

The genus *Scopalina* resulted as being the most abundant and prevalent group in both the shallow and the deep, with values of, respectively, 27.82% and 26.51% (Table 2). The genus *Agelas* showed a completely different distribution in relation to depth with a relative abundance of 5.45% in the shallow stations and 19.34% in the deep ones (Table 2). A similar trend was observed for *Niphates*, with an increase of the relative abundance from 4.66% in the shallow stations to 8.92% in the deep stations. The genera *Clathria*, *Cliona*, *Halisarca*, *Monanchora*, and *Phorbas* were extremely poorly represented in the study area at both depths, with *Halisarca* resulting as being the only genus completely absent in the shallow stations. Moreover, the spatial distribution of sponges between deep and shallow stations showed different trends for *Agelas*, *Desmapsamma*, *Ectyoplasia*, and *Niphates* at the two depths considered for the study, even if they were not statistically significant (Kruskal–Wallis test, *p* > 0.05).

Regarding the sponge-hydroid interactions, *Nemalecium lighti* has been found in association with 9 out of 16 genera of sponges and in 15 out of 16 of the dive sites surveyed. A total of 115 sponge individuals hosted at least one colony of *N. lighti*. The prevalence of the occurrence of the hydroid–sponge association in the Bonaire reef was 6.55%, with a maximum value of 33.33%. The mean prevalence of the association on the analyzed reef sites was 5.06 ± 1.91%. Among them, "Bari Reef" (BR), "Alice in Wonderland" (AiW), and "La Machaca" (LM) showed higher values with, respectively, 20.82 ± 3.56%, 17.59 ± 2.31%, and 8.67 ± 2.72%, with the others showing less than 6% (Table 1). However, the differences of prevalence among the tested sites were not statistically significant (Kruskal–Wallis test, *p* > 0.05). With regards to the depths considered, the mean prevalence for the deep stations resulted in being higher compared to the shallow stations with, respectively, 5.86 ± 2.77% and 4.27 ± 1.91% (Figure 3a), even if the differences between the stations were not statistically significant both in relation to the site and genera (Mann–Whitney Test, *p* > 0.05).

**Figure 3.** Prevalence of *Nemalecium lighti*–sponges association in the study area: (**a**) Mean association prevalence for all the genera considered and (**b**) for the genus *Aplysina* in relation to depth.

Furthermore, the taxon-specific prevalence was calculated for each genus of sponge that was found to be the host of the association (Table 2). Sponges belonging to the genus *Aplysina* were the most involved in the association with *N. lighti*, with a prevalence of 21.76 ± 4.72% in shallow stations and 27.22 ± 3.44% in the deep stations (Figure 3b). Similarly, *Verongula* and *Aiolochroia* were the second most involved in association with *N. lighti*, even if not notably in the shallow stations. By contrast, *Cliona* showed an elevated prevalence in the shallow station and no association in the deep station. There were seven genera (*Callyspongia*, *Clathria*, *Ectyoplasia*, *Halisarca*, *Monanchora*, *Niphates*, and *Phorbas*) that did not show an association in either the shallow stations or the deep stations, whereas the *Iotrochota*–*Nemalecium lighti* association was found only in deep stations. The differences in the prevalences among the analyzed genera were tested as being statistically significant (Kruskal–Wallis Test, *p* < 0.05).

When correlating the sponge dimension with the *N. lighti* association, most of the sponges belonged to the smaller size classes of 5–30 cm (~70%), whereas only a minor part of the sponges was comprised in the larger size classes of 40–50 cm (Figure 4a). Despite this, a positive increase in prevalence was recorded, with values of 1.41 ± 0.66%, 5.75 ± 0.79%, 8.09 ± 1.62%, 13.78 ± 4.40%, and 15.04 ± 1.96% for C1 to C5 size classes, respectively (Figure 4b). Furthermore, the sponges belonging to the genus *Aplysina* showed a particular behavior in this regard, being the only genus with association cases in all the size classes, in both shallow and deep stations.

**Figure 4.** Hosts' size class distribution and prevalence analyses in the study area: (**a**) Number of sponges for each class size; (**b**) Prevalence of *Nemalecium lighti*–sponges association in relation to the host class size. The bold line in the middle of the boxes is the median value, the bottom part of the boxes is the lower quartile, the top part of the boxes is the upper quartile, the lines departing from the boxes are the lower and upper extremes, and the circle is an outlier value.

## **4. Discussion**

Over the years, multiple studies have addressed the role of coral reef biodiversity and its overall impact on the health of these ecosystems [1,4,9]. More specifically, Bonaire coral reef ecosystems emerged as an incredible source of rare or previously unreported reef organism associations. In this perspective, this study assessed for the first time the distribution, host range, and prevalence of the sponges–*Nemalecium lighti* association in this region, aiming to partially fill the knowledge deficiency about cryptofauna associations involving sponges and hydrozoans. The surveys revealed associations between host sponges and *N. lighti* at all the explored sites except one, suggesting a widespread distribution for the association all along the west coast of Bonaire, with some sites showing notably high values of prevalence (BR, AiW, and LM). The host range of this association accounts for nine genera belonging to the class Demospongiae, suggesting that *N. lighti* can be considered as a generalist, since it appears to not target a specific sponge species, at least within the depth ranges we conducted our surveys in. The spatial distribution of the hydroids along the reef zonation has been addressed in previous studies, revealing that different environmental conditions and depths may have an impact on the development of associations and the peculiar assemblage of hydroid species [36–38]. In particular, it has been observed that the maximum diversity of hydrozoans and related associations with sponges was reached on the reef slope, with a continued increase with the depth until reaching 30 m [29]. Similarly, in the present study, the prevalence of associations is slightly higher in the deep stations compared to the shallow ones. However, this result needs to be confirmed in future studies by considering not only the shallower part of the coral reefs but extending the surveys to mesophotic depths. In addition, considering that *N. lighti* is a small cryptic hydroid species growing to a maximum 2 cm that settles on the surface of sponges but sometimes also in protected and shaded parts of their structure, it cannot be excluded that some of the interactions may have been overlooked during our surveys, resulting in an underestimation of its actual prevalence.

The taxon-specific prevalence revealed that the highest prevalence of occurrence of the *N. lighti*–sponge association was observed with the genus *Aplysina* (26%). This prevalence value was almost four times higher than for the second genus involved in the association (*Verongula* with 6.98 ± 2.34%) and extremely more significant than for all the other species, which showed prevalence values lower than 5%. Sponges of the genus *Aplysina* represent an abundant and important component of the marine coastal ecosystems in tropical and subtropical waters [39], where they contribute to the three-dimensionality of the reef structure, which is fundamental for hosting and sustaining several species of associated fauna. The preference of *N. lighti* for this genus may be the result of the complex tubular growth morphology that exposes the epibiont hydroid to a strong current and water flow, characteristics usually exploited by this species [16,40].

In addition, sponges are known to produce an array of chemicals and metabolic products that are ecologically important for different purposes (e.g., growth, protection, competition) [41]. Among them, sponges belonging to the genus *Aplysina* are known to produce high concentrations of brominated alkaloids metabolites (up to 13% of the dry weight) related to antimicrobial activity and cytotoxic activity [42]. These sponges probably produce them as a chemical defense, biofouling, and deterrent against fish predators, as tested on *Thalassoma bifasciatum* and *Blennius sphinx* [43–49]. This peculiar characteristic of the *Aplysina* sponges may be one of the factors that enhances the association with *N. lighti*, as it may take advantage of this defense mechanism of the host to protect itself from predators or microbial offense [50]. However, future investigations to test this hypothesis and to elucidate the nature of this association in the reefs of Bonaire are needed.

Finally, the observed increase in prevalence related to the dimension of the sponges suggests that other factors pertaining to the host also determine the ability of *N. lighti* to settle on sponges. In particular, an increase in sponge (species-dependent) size may correspond to an increase of the favorable surface on which the hydroids can establish the association. In addition, the amount of time necessary for the sponge to grow may also be

a contributing factor for the settlement of larvae, increasing the probability of growth on the sponge surface. Further studies are needed to test both of the scenarios proposed and to clearly understand the effect of host size on this particular association.
