Rezende Ventura, C.R., & Pinto de Oliveira, M.S. (2021). How can an infaunal brooding echinoid be maintained in the laboratory? A case study with Cassidulus mitis (Echinoidea: Cassiduloida). Revista de Biología Tropical, 69(S1), 550-557. DOI 10.15517/rbt. v69iSuppl.1.46394 DOI 10.15517/rbt.v69iSuppl.1.46394 How can an infaunal brooding echinoid be maintained in the laboratory? A case study with Cassidulus mitis (Echinoidea: Cassiduloida) Carlos Renato Rezende Ventura1* Monalisa Sousa Pinto de Oliveira1 1. Museu Nacional, Universidade Federal do Rio de Janeiro. Departamento de Invertebrados, Laboratório de Echinodermata. [email protected] (*Correspondence), [email protected] Received 01-IX-2020. Corrected 20-X-2020. Accepted 28-XI-2020. ABSTRACT Introduction: Cassiduloids play a prominent role in echinoid evolutionary history because they probably are the ancestral group of clypeasteroids. Some extant species are brooding and rare in the environment. Consequently, there are no studies on their maintenance in the laboratory. Objective: Establish an efficient aquarium system for C. mitis, endemic to Brazil, for ontogenetic studies. Methods: Four aquarium systems were built, with 3 replicates each one: (1) with seawater flow [F]; (2) with seawater flow and air injection into sediment [FA]; (3) without seawater flow but with air injection into the sediment [A]; and (4) without both seawater flow and air injection into the sediment [C]. Each experimental aquarium (three per treatment) had two adults. Each of the two sets of experiments lasted about 60 days. Results: We observed low mortality in the first 30 days in all systems and, after 30 days, it was higher in those with air-pumped into the sediment (system A in the first set of experiments, and system FA in the second one). Conclusions: For experiments lasting 30 days, our four systems are suitable. For longer periods, we recommend aquaria with seawater flow and without air-pumps into the sediment. Key words: experimental model-species, closed aquarium system, survival rate, sand-bottom, echinoid deposit feeder. In general, brooding marine invertebrates laboratory for enough time to record valuable have a rapid direct development or an abbrevi- changes in morphology. ate indirect development with a non-feeding The echinoid Cassidulus mitis Krau, 1954, larval stage followed by a quick metamorpho- belongs to the order Cassiduloida Claus, 1880, sis. In particular, echinoderms have these pat- which comprises about 68 genera and 800 spe- terns and are recognized as useful models for cies, most of them extinct (Mooi, 1990a; Kroh studies on embryology, life history, evolution, & Mooi, 2020). The first records of cassidu- and development (Evo-Devo), among other loids appear in the Lower Jurassic (about 200 fields of knowledge (Raff & Byrne, 2006; Hey- m.y.a). This order reached its highest diversity land, Schuh & Rast, 2018). However, it is not in Eocene (about 55 m.y.a.) when it represented easy to find coastal marine species considered 60% of all echinoids (Kier, 1962). The decline that are suitable good experimental models. of cassiduloids occurred between the Late The most significant difficulties are their avail- Tertiary and Quaternary (about 30 m.y.a.). ability in the field and their maintenance in the This diversity drop was probably related to 550 Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 550-557, March 2021 (Published Mar. 10, 2021) global cooling and the increase of competi- Therefore, the maintenance of adults in the tion with other echinoids. The decline of cas- laboratory is crucial. siduloids coincided with the rise of the sand Cassidulus mitis is an endemic infaunal dollars (Clyperasteroida) and heart urchins species from Brazil (Tommasi & Lima-Verde, (Spatangoida), considered as sister-groups. The 1970). It occurs in a relatively high abun- fossil records corroborate the hypothesis of dance in one locality, and it has a continu- competition, causing the diversity decrease of ous reproductive cycle, develops yolky eggs, cassiduloids. Mooi (1990a) states that there embryos, and lecithotrophic larvae. Cassidulus are 30 extant species within the order Cassidu- mitis broods its offspring among the female loida. The morphological similarity between spines for 18 days, when the post-metamorphic the fossil and extant species is evident (Brito juveniles (that have the Aristotle’s lantern) & Ramires, 1974; Brito, 1981; Smith & Bengs- leave their parent to the coarse-sandy environ- ton, 1991; Squires & Demetrion, 1995), which ment (MacCord & Ventura, 2004; Contins & means that extant cassiduloids have a highly Ventura, 2011). After this period, juveniles conserved morphology. In other words, no continue their development, assuming an ellip- significant morphological novelty has occurred soidal shape, and lose their lantern. As adults, along their lineage since the Upper Creta- they start to consume the organic matter from cean (Smith, 2001). Therefore, the extant spe- the sediment. cies show signs of “character exhaustion,” as Therefore, Cassidulus mitis is a crucial defined by Wagner (2000). For these reasons, species of evolutionary relevance. Some previ- the cassiduloids fit in the concept of “living ous studies have already gathered important fossil” (Schopf, 1984; Mooi, 1990b). information about its biology. Its ready avail- Cassiduloids play a prominent role in ability in a coastal area gives it the potential echinoids’ evolutionary history because they to be a good species model. However, it is still probably are the ancestral group of the sand necessary to establish methods to keep it in dollar lineage (clypeasteroids). Among all echi- the laboratory. This study aims to establish an noids, the species of Cassiduloida are unique efficient aquarium system to maintain C. mitis that have the Aristotle’s lantern only during the in laboratory conditions for enough time for developmental studies. early young stage (post-metamorphosis). The morphological similarity between the lanterns of small cassiduloids and large clypeasteroids MATERIALS AND METHODS is the primary support for this hypothesis. In other words, the retention of ancestral young Study Area characters in the adult descendant species (neoteny) supports this hypothesis. Despite The population of C. mitis occurs at the its evolutionary relevance, the loss of the beach Praia Vermelha (22º 57’ 18” S; 43º 9’ 48” Aristotle’s lantern of extant cassiduloids still W) located at the entrance of the Guanabara has not been well-studied (Gladfelter, 1978; Bay (Rio de Janeiro State, Brazil). It is a short Märkel, 1978; Contins & Ventura, 2011). The beach with a sharp decline in the bottom caused principal cause for this knowledge gap is the by waves and periodic storms. The substrate at difficulty of finding dense populations in the the Praia Vermelha consists of homogeneous field and the ability to maintain cassiduloids coarse sand of rounded grains of quartz (Freire, in the laboratory for enough time to record Santos, Fontoura, Magalhães & Grohamann, all significant morphological changes during 1992). The climate in this region is typically the loss of Aristotle’s lantern. The best way tropical, warm, and rainy, with two distinct to keep young post-metamorphic cassiduloids seasons, dry in autumn and winter, and rainy in alive is on adult females, among their spines. spring and summer. Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 550-557, March 2021 (Published Mar. 10, 2021) 551 Collection of specimens, seawater, and sediment Specimens of C. mitis were collected from the infralittoral zone at depths between 2 and 4 meters by apnea free-diving in May and August 2019. The temperature of the seawater was about 22oC. The salinity was about 35 ppm. We Fig. 1. Experimental design of the four aquarium systems. collected seawater and sediment just after the (FA) Aquaria with seawater flow and air-pumped in collection of the sea urchins. sediment bottom; (F) Aquaria with seawater flow; (A) Aquaria without seawater flow and with air-pumped in the Aquarium systems, monitoring of survival, sediment; and (C) Aquaria without both seawater flow and seawater temperature, and salinity air-pumped in sediment. Adult sea urchins were kept in four aquar- ium systems, simulating somewhat of natural the sediment – [C] (Fig. 1 and Fig. 2). The air conditions. These were four sets of experimen- into sediment was provided by an air-pump tal aquaria (plastic box of 33.5cm x 24,0cm connected to porous-stone sticks (12 cm long) x 16,0cm) with seawater and a layer of 5 cm buried in each aquarium’s sediment layer. of sediment from the Praia Vermelha. Each Each experimental aquarium had two adults system had one treatment with three replicates: (a total of 12 females and 12 males, ranging (1) Aquaria with seawater flow – [F]; (2) from 2.2cm to 4.6cm and 2.2cm to 3.9cm Aquaria with seawater flow and air-pumped of the longest test diameter, respectively). into the sediment bottom – [FA]; (3) Aquaria Sexual differences in C. mitis can be distin- without seawater flow and with air-pumped guished by their dimorphic genital papillae in into the sediment – [A]; and (4) Aquaria with- the apical region of the test (Tripneustes-type out both seawater flow and air-pumped into papillae) (Tahara, Okada & Kobayashi, 1958; Fig. 2. Arrangement of the four experimental aquarium systems. A - Aquaria with seawater flow and air-pumped into the sediment (FA); Aquaria with seawater flow (F); Aquaria without seawater flow and with air-pumped into the sediment (A); Aquaria without both seawater flow and air-pumped into the sediment (C); Tanks (30L) (R). Numbers (1, 2, 3) represent replicates in each treatment. B – A single flow-aquarium system. Immersed pump (P); Filters (F). Arrows represent the flow directions. Aquaria systems developed by Renato R. Ventura. 552 Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 550-557, March 2021 (Published Mar. 10, 2021) Pearse & Cameron, 1991). These experimental Linear Mixed Model to compare statistical aquaria systems are original and were devel- differences of the survival rates of C.