First Account of Epibiotic Diatoms Taxa from the Shells of Green Swimming Crabs Callinectes bellicosus (Stimpson 1859) (Decapoda, Portunidae)

1. Introduction

The interaction between an organism (basibiont) and those that live on it (epibionts) is known as epibiosis. This type of relationship has been documented as a common phenomenon in aquatic ecosystems, especially in marine environments [1], and the phenomenon of adhesion of microalgae (including diatoms) to living substrata is well studied [2]. Diatoms represent a principal constituent of microphytobenthos, and a common habitat of theirs are living substrata, sometimes plants and algae (epiphytic diatoms) and other times animals (epizoic diatoms) [2,3]. In this vein, the importance of epizoic growth of diatoms has been described for many types of hosts including insects, turtles, diving birds, marine mammals [4,5,6,7,8,9], and crustaceans such as amphipoda, cladocerans, copepods, decapods, and hoplocarids [10,11,12,13,14]. Studies on crabs’ epibiota have focused on the macro-epibionts, seeking to elucidate their masking behavior [1,15]. In the Gulf of California, the type and quantity of pieces used by decorator crabs (Majidae) was studied [16], observing the dominance of macroalgae and sponges. Others have focused on the adherence capability of epibionts such as diatoms. However, little was hitherto known of the species composition, seasonality, or the types of habitats in which epizoic diatoms occur on green swimming crabs in any area. Moreover, further ecological research on the interactive relations between epibiotic diatoms and their basibionts requires basic floristic information in order to carry out formal biodiversity or ecological studies related to the consequences of using animal surfaces as substrate by benthic diatoms. Given the challenge that this presents, in general, there is scarce information on diatom epibiosis with crabs [17,18] inasmuch the species composition and richness of the epibiotic diatoms has received very little attention.

The green swimming crab Callinectes bellicosus Stimpson 1859 (Decapoda: Portunidae) is distributed along the Eastern Pacific, ranging from California, USA, to the Tehuantepec Gulf, Oaxaca, Mexico. It inhabits estuaries, coastal lagoons, and soft bottoms up to 90 m deep [19,20], and adults can live for 3–4 years [19,21]. The species is omnivorous, as well as an opportunistic predator, often burrowing down superficially in the sediments to wait for its prey [22]. This behavior makes green swimming crabs not only accessible but suitable as a substrate for a variety of small marine biota, which they may carry on their carapaces, such as various algal forms [23,24], including benthic diatoms. However, up to now, the species composition of epibiotic diatoms of green swimming crabs had remained unexplored. Thus, it is unknown how many diatom species or forms may be found that are either truly epizoic or just opportunistic, and the differential benefits that these may have in terms of survival, reproduction, or dispersal. The basic floristics of epibiotic diatoms will surely provide a reliable platform to undertake further ecological studies between them and this basibiont.

According to the above, in the present study the epibiotic diatom flora living on carapaces of C. bellicosus from the subtropical coastal lagoon of La Paz, BCS, Mexico was investigated. The related background on this shows that benthic diatom flora from the northwest region of Mexico has been previously studied in several localities including mangrove environments [25]. In these and later investigations around 1500 benthic diatom taxa have been recorded, which provide a reliable reference for our work. Moreover, the structure of the benthic diatom assemblages has been described based on the characteristic taxa in a way that a typical benthic diatom flora can be recognized for the region. So, considering the observed distribution of the benthic diatom taxa on the various substrates inspected in said works we asked if the diatom flora living on the shells of green swimming crabs would be similar to that recorded for mangrove sediments, its typical habitat in the northwest region of Mexico. Thus, we tested the null hypothesis that the diatom flora living on the carapaces of green swimming crabs would not be similar to that recorded historically for the mangrove sediments in the southern Baja California Peninsula (northwest region of Mexico). This abduction (Ho) responds to the influence that mobility of the basibiont and constant exposure of the colonized surface, plus the discrete temporal sampling that could exhibit a seasonal variation in species composition of the colonizing diatoms, that may result in a difference in species composition between the typical diatom flora and that living on the inspected green crab specimens.

2. Materials and Methods

The La Paz coastal lagoon forms part of Bahia de La Paz, a shallow-bottom water body that reaches 10 m deep with a dominant soft bottom. In the northwest part of the lagoon the mangrove swamp of Zacatecas Estuary is located (24°10′27″ N, 110° 26′ 06″ W) (Figure 1). There, in September 2022, two male specimens of C. bellicosus (Figure 2) were captured using a hand net and identified according to [20].

Diatoms were separated from the cephalothoraxes and chelae of the swimming crabs using a toothbrush to generate a compound sample of the two specimens. The brushed-off material was placed in a 250 mL flask and preserved in commercial 70% ethanol. Afterward, to eliminate organic matter that would obstruct visibility of the diatom frustules, the compound sample was oxidized by adding 3 mL of 70% nitric acid to 2 mL of sample and then heating with a burner to the boiling point, where it was held until the emission of gas subsided, indicating the end of the reaction (ca. 3 min). The oxidized sample was rinsed repeatedly with deionized water until reaching a circumneutral pH. Then, six mounted slides were inspected under a Zeiss® Axio Lab A1 (Zeiss, Germany) compound microscope equipped with phase contrast and a Canon 5D Mark II camera (Canon, Japan). Diatoms were identified based on frustule morphology using classic and recent references: [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45].

A systematic list of the diatom taxa was constructed following practical formats [46,47,48,49]. Nomenclatural updates for the identified taxa were performed based on www.algaebase.org [50] and www.marinespecies.org [51]. This is complemented by an iconographic catalog of all the recorded taxa (Table 1).

3. Results

The inspection of the brushed-off material from the green-crab´s shell yielded 106 diatom taxa belonging to 46 genera (Table 1; Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17 and Figure 18). At class level, 91% (97 taxa) were Bacillariophyceae, 5% (5) Coscinodiscophyceae, and 4% (4) Mediophyceae. The taxonomic families with most species were Bacillariaceae (with 17), Mastogloiaceae (9), Naviculaceae (9), and Diploneidaceae (8). Meanwhile, the highest-represented genera in terms of the number of species were Nitzschia (10), Mastogloia (9), Diploneis (7), Navicula (7), Amphora (5), Cocconeis (5), Tryblionella (4) and Gyrosigma (4), which accounted for 49% of all taxa. We found that 24 genera were singletons, i.e., they included a single taxon, representing 23% of all the records (Table 1). The most frequent species were Navicula normaloides (Figure 13E) and N. platyventris (Figure 13H–K). Meanwhile, Biddulphia californica (Figure 3A,B), Campylodiscus subangularis (Figure 18L,O), Melosira westii var. quadrata (Figure 3H,I), Nitzschia ligowskii (Figure 17M,N), Petroneis besarensis (Figure 9E–G) and Tryblionella pararostrata (Figure 16N) are new records for the study area and the northwestern Mexican region. This means that 93 % of the diatom taxa that we found on the shell of the green crabs are also characteristic or typical of the local epipelic diatom flora recorded for mangrove sediments in the region. This constitutes evidence that refutes the posed null hypothesis, thus supporting an alternate hypothesis.

4. Discussion

The study area consists mainly of mangrove swamps, thus soft sediments dominate the bottom substrate, with low availability of hard substrate. This means that hard-shell organisms (such as the green swimming crabs) may serve as an alternate substrate for the colonization and settlement of multiple forms of benthic diatoms [17]. In fact, our species richness estimation bears the closest resemblance to the estimated values in previous studies of benthic diatom taxocoenoses from mangrove environments in the La Paz coastal lagoon [52,53,54]; the latter study yielded 150 taxa recorded overall on several pro-thrombolytic platforms, of which 42% were also found on the inspected green swimming crabs’ carapaces. That is, at 53 taxa per crab-specimen, the species richness falls within the interval estimated for samples of benthic diatom assemblages from mangrove (productive) environments [55].

Although studies on benthic diatoms in the northwest Mexican region are scarce and recent, several have been carried out expressly in the mangrove environments and have yielded a high species richness of diatoms thriving in the sediments [55]. Epipelic diatoms from these environments may constitute a reserve of potential colonizers for various basibionts, including the highly motile green crabs, which may also represent a small-scale dispersion vehicle for benthic diatoms.

When comparing the species richness value estimated in our work (106 taxa) with those of similar studies with other crab species, we found that species richness estimations in said studies were reported to be lower. However, no comparison can be made between this diatom flora and those from other studies with the green swimming crab (or even other crabs from the same genus) because none have been conducted. The closest reference is a study by [18] that inspected basibiotic diatoms on a quite different marine arthropod, albeit with what seems a similar adhesion surface, the horseshoe crab (Tachypleus gigas O. F. Müller 1785), a species that dwells in moderately deep waters and migrates nearshore for breeding. These authors recorded 17 and 20 diatom taxa living on female and male specimens, respectively. Whilst, considering studies with crabs in general, 65 diatom taxa were recorded as epibionts from 25 genera living on spider crabs (Schizophrys dahlak Griffin & Tranter 1986), confirming that the number of epizoic diatom taxa are normally much lower than in our study (less than half, in this case). Although, life habits between these crab species are quite different and the comparison may just be related to the composition of the shell.

Beyond this, there is great potential to be added to the literature. For instance, eventhough the floristic reference for the northwestern Mexican region is considerable, the epibiotic taxocenosis on green swimming crabs rendered six new records. This does not necessarily mean that these are epizoic forms, but it supports the premise that continuous and extensive sampling—including various substrates and unexplored areas—along with exhaustive floristic inspection of the samples will enrich the benthic diatom species list, even at a global scale. This was earlier observed by [56] who recorded thirty diatom taxa epizoic on specimens of stone fish (Scorpaena mystes Jordan and Starks 1895), from the central Gulf of California that were new for Mexican littorals, and out of which twelve had not hitherto been recorded for American coasts. As such, evidently floristic records of benthic diatoms for the La Paz lagoon are far from being complete. Furthermore, the records that are currently available are the products of discrete sampling, both spatially and temporally, as is the case for taxa epibiotic on green swimming crabs. They are also limited by sampling size; for instance [56] inspected twenty specimens of stone fish, while for the present study, only two specimens of Callinectes bellicosus were used.

In this study 93% of the diatom taxa found on the shell of the green crabs are typical of the local epipelic diatom flora, a degree of similarity that refuted our null hypothesis, i.e., supporting the alternate hypothesis that the epibiotic diatom assemblages living on the shells of green crabs are constituted mainly by typical or characteristic taxa found in mangrove sediments or from neighbor sites in northwestern Mexico. With respect to the significance of said similarity, in our own experience even replicas or repetitions of samples may vary in similarity from 70-80% vs. the main sample when using similarity indices [57]. But because difference in species richness affect the similarity value the approach that we used was direct.

Further hypothesis-driven studies about the ecological relations and small-scale dispersion of benthic diatoms by means of green crab transportation will provide fertile grounds for improving our understanding of the interactive dynamics of epibionts and basibionts. In the case of Callinectes bellicosus and diatoms, this research should also help to complete our knowledge of those diatom taxa that are either epizoic or opportunistic, by working to overcome the evident spatial and temporal limitations of the existing contributions to the floristic records for the Mexican coasts. In this manner, it may be explored if the species composition or the structure of the epibiotic assemblages abides by the theoretical patchy distribution typical of benthic diatoms. Furthermore, considering the limited displacement capability of this basibiont, the inspection of specimens of green crab collected in other locations of the La Paz lagoon could broaden our knowledge by yielding distinct epibiotic diatom taxa.