Understanding the Symbiotic Relationship between the Sea Urchin Loxechinus albus (Molina, 1782) and the Pea Crab Pinnaxodes chilensis (H. Milne Edwards, 1837): a Potential Parasitism

© 2023 Academia Sinica, Taiwan Open Access Understanding the Symbiotic Relationship between the Sea Urchin Loxechinus albus (Molina, 1782) and the Pea Crab Pinnaxodes chilensis (H. Milne Edwards, 1837): a Potential Parasitism Hans N. Jaramillo1, Luis P. Salas-Yanquin1, Joseline A. Büchner-Miranda1, Víctor M. Cubillos1, Jaime A. Montory2, Jan A. Pechenik3, Luis M. Pardo1, and Oscar R. Chaparro1,* 1Universidad Austral de Chile, Instituto de Ciencias Marinas y Limnológicas, Valdivia, Chile. *Correspondence: E-mail: [email protected] (Chaparro). E-mail: [email protected] (Jaramillo); [email protected] (Salas-Yanquin); [email protected] (Büchner-Miranda); victor[email protected] (Cubillos); [email protected] (Pardo) 2Universidad de Los Lagos, Centro i~mar, Camino a Chinquihue km 6, Puerto Montt, Chile. E-mail: [email protected] (Montory) 3Biology Department, Tufts University, Medford, MA 02155, USA. E-mail: [email protected] (Pechenik) Received 1 July 2022 / Accepted 27 January 2023 / Published 25 April 2023 Communicated by James D. Reimer The echinoderm Loxechinus albus has a symbiotic relationship with the pinnotherid crustacean Pinnaxodes chilensis. Females of the crustacean develop in the terminal section of the sea urchin’s digestive system, remaining there for life. This relationship has been suggested as commensalism. However, a potential negative impact on gonadal development and on the morphology of the sea urchin’s digestive system suggest that it is instead parasitic. To study if there is a negative impact of the crustacean symbiont on the host, specimens of L. albus of all sizes were collected from a rocky shore in southern Chile. The gonadal and somatic tissues of sea urchins that were and were not harboring the pinnotherid were weighed and compared. Our results show that the presence of the pinnotherid was related to sea urchin gonads of lower biomass, decreased gonadosomatic index levels, and alterations in the morphology of the terminal portion of the host digestive system. The lower gonadal biomass suggests a negative impact on gamete production as well as a diversion of energy due to changes of the digestive system tissues and the potential consumption of algal food by the resident crustacean. These results suggest that the prolonged relationship between these two species is one of parasitism rather than one of commensalism. Key words: Parasitism, Symbiosis, Sea urchins, Pinnotherid, Crustacean. Citation: Jaramillo HN, Salas-Yanquin LP, Büchner-Miranda JA, Cubillos VM, Montory JA, Pechenik JA, Pardo LM, Chaparro OR. 2023. Understanding the symbiotic relationship between the sea urchin Loxechinus albus (Molina, 1782) and the pea crab Pinnaxodes chilensis (H. Milne Edwards, 1837): a potential parasitism. Zool Stud 62:18. doi:10.6620/ZS.2023.62-18. BACKGROUND Marine invertebrates have a wide variety of lifestyles. Many species become adults as independent, free-living individuals, either in the water column or as members of the benthos. Other species, however, spend at least part of their lives—compulsorily or deliberately—living in close association with other organisms (Waide et al. 1999; Thébault and Loreau 2005; Thiebot and Weimerskirch 2012; Khandeparker and Anil 2013). Such “symbiotic” associations are usually long-term, can be of different types, and can occur at different stages of development (Das and Varma 2009), and can include a diverse range of interactions, Zoological Studies 62:18 (2023) doi:10.6620/ZS.2023.62-18 1 © 2023 Academia Sinica, Taiwan behaviors, and physical associations. Although several of these relationships favor the guest organism, that organism must adapt to the physical limitations and to the physiological and mobility characteristics of its hosts. Guest organisms can not only modify their own morphology, physiology, reproduction and behavior to fit in better with the life-style of the host, but can often impact the physical characteristics of the host to create a better living space for themselves (Patton 1965; Bell and Stancyk 1983; Weber and Apprill 2020). This lifestyle is quite common in marine invertebrates, and members of several crustacean taxa have been identified that adopt the symbiotic lifestyle (Douglas 1994; Thiel and Baeza 2001; Narvarte and Saiz 2004; de Bruyn et al. 2009). In some cases, the between-species relationship provides an advantage for the guest at the expense of the host that must be considered as parasitism (Dales 1957). Some species of crustaceans in the Pinnotheridae family can be symbionts, while others can be freeliving (Schmitt et al. 1973). Pinnotherids belong to the little-studied cryptofauna due to their small size, their symbiotic habits, and the taxonomic problems derived from their substantial sexual dimorphism and morphological changes during their life cycle (Martínez 2014). Many members of this family associate symbiotically with their host (McDermott 2009), either optionally or compulsorily, usually as adults (Schmitt et al. 1973; Stevens 1990; Becker and Türkay 2010). They are able to colonize multiple classes of hosts of different morphologies and habitats, such as molluscs (Geiger and Martin 1999; da Cruz-Kaled et al. 2006; Ahyong 2020), annelids (Komai et al. 2014; Britayev et al. 2017), arthropods (Feldman et al. 1996; McDermott 2009), or echinoderms (Bell and Stancyk 1983; de Bruyn et al. 2009; Tresnati et al. 2021). Associations between pinnotherids and their hosts can sometimes be either mutualistic or commensal (Reeves and Brooks 2001), but in some cases they can also have a relationship that negatively affects their hosts (Hamel et al. 1999; Bologna and Heck 2000; Ocampo et al. 2014; 2021; Becker and Türkay 2017; Gajbhiye and Khandeparker 2017). In the case of the symbiotic relationship of pinnotherids with species of echinoderms, the pinnotherids generally obtain their food through water recirculated by the host, as seen with some holothurian species (Wells and Wells 1961), or by feeding on the pieces of macroalgae accumulated in the host digestive system, as seen in some sea urchin symbionts (Gutiérrez-Martínez 1971). Along the Chilean coastline, an interesting interaction has been identified between the sea urchin Loxechinus albus (the host) and the pinnotherid crustacean Pinnaxodes chilensis (the guest) (Baez and Martínez 1976; Lardies and Castilla 2001; Vásquez and Bay-Schmith 2010–2011; Gonzalez-Canales et al. 2018). L. albus is a species of high economic importance in Chilean waters. Its range extends from northern Peru (6°S) to Tierra del Fuego in southern Chile (55°S), while its bathymetric range goes from shallow coastal water down to depths of several hundred meters (Larraín 1975). It is also one of the most important consumers of macroalgae in rocky intertidal and subtidal environments (Dayton 1985; Gebauer and Moreno 1995; Vásquez 2007). The crustacean P. chilensis also has a large distribution, with a range extending from the Chincha Islands, Peru (approximately 13°S) to southern Chile (approximately 53°S) (Retamal and Moyano 2010). Whereas the males of this crustacean species are free-living, the females are adapted for a symbiotic life inhabiting the final part of the sea urchin’s intestine, which provides it with great environmental homogeneity (Baez and Martínez 1976) and enough food to increase its likelihood of survival and reproductive success (Lardies and Castilla 2001). Males of this species are smaller than females (Gutiérrez-Martínez 1971). During the reproductive season, the male enters the intestine of the host sea urchin, via the sea urchin’s anal orifice, in search of a mate, and then leaves the sea urchin after copulation (Gutiérrez-Martínez 1971; Gonzalez-Canales et al. 2018). The embryos of this and some related species then have a free-living larval period in the plankton (Hamel et al. 1999; Hsueh 2001; Gonzalez-Canales et al. 2018). However, as soon as these larvae settle and metamorphose, they acquire the appearance of an adult juvenile (Gutiérrez-Martínez 1971). After this stage, females necessarily look for a sea urchin host to grow inside, while males either remain in the benthos or enter a host for a short time (Gutiérrez-Martínez 1971). The symbiotic life of the pinnotherids and the wide diversity of the echinoderm species that house them (Bell 1988; Lardies and Castilla 2001; de Bruyn et al. 2009; Vásquez and Bay-Schmith 2010–2011) are very interesting subjects for understanding the relationship between species and the ecological role that their lifestyle represents. The association between the host sea urchin L. albus and the guest pinnotherid P. chilensis has sometimes been considered as commensalism (e.g., Gutiérrez-Martínez 1971); however, other authors, such as Fenucci (1967), have identified this relationship as parasitism. The growth of the P. chilensis female in the terminal portion of the host’s digestive system forces the sea urchin to gradually increase the size of that digestive region under stress; the host’s gonads also become deformed (Vásquez and Bay-Schmith 2010–2011). For example, it has been observed that the presence of the pinnotherid page 2 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan Pinnotheres maculatus was associated with a decline in the GSI of the scallop Argopecten irradians, thereby reducing its reproductive potential (Bologna and Heck 2000). An equivalent situation has been identified in specimens of Mytilus galloprovincialis when the bivalve hosted females of the parasite Pinnotheres sinensis, which negatively impacted the condition index of the host mussels (Sun et al. 2006). Similarly, Takeda et al. (1997) found that the symbiont Pinnixa tumida reduced the growth of its host, the holothurian Paracaudina chilensis, due to consumption of the mucus secreted in the host’s alimentary canal. Likewise, specimens of the oyster Crassostrea virginica from populations with a high rate of infestation by the pinnotherid Pinnotheres ostreum developed a smaller than normal gonadal area, impacting the host’s reproductive capacities (O'Beirn and Walker 1999). To date, it has not been investigated whether the deformations of the intestine and gonads of L. albus generated by the guest P. chilensis weaken the sea urchin or even cause its death (Vásquez and BaySchmith 2010–2011), but if so, then the relationship should be viewed as parasitic rather than as commensal. In the present study, we sought to characterize in detail the impacts of the symbiotic crustacean on the sea urchin host’s tissues and on its gamete production to determine whether the relationship is truly commensal or in fact parasitic. MATERIALS AND METHODS Specimen collection Specimens (73 individuals, with test diameters ranging from 2.6 to 9.5 cm) of the sea urchin Loxechinus albus were collected during January and February 2021 in the lower rocky intertidal of Calfuco Beach (39°46'50"S, 73°23'34"W), southern Chile (Fig. 1) and then maintained in the laboratory in a 30 L aquarium with circulating seawater (10°C and 33 salinity) and constant aeration. The individuals were kept under these conditions for a maximum of 2 days, until all of the specimens collected in each sampling event were processed, as described below. Morpho-gravimetric quantifications of sea urchin The maximum exoskeleton diameter of each sea urchin was measured using digital vernier calipers. The wet weight of each specimen was then obtained to the nearest 0.01 g using an analytical balance. Before weighing, the specimens were kept out of the seawater for 1 h to equalize water loss by aerial exposure among all individuals. The soft tissues were then separated from each sea urchin’s exoskeleton and the gonadal tissue was then separated from the rest of the soft tissues. The sex of each individual was determined using a compound microscope (Olympus BX 41) to identify the presence of eggs or sperm. These gonadal tissues were then deposited, separately, into pre-labeled and pre-weighed aluminum foil containers. The samples were then maintained at 60°C for 48 hours, until they reached a constant dry weight, and then weighed to the nearest 0.01 g to determine dry tissue weights. The Gonadosomatic Index (GSI) was then estimated using the following equation: GSI = (gonad dry weight  (total soft dry tissue weight)) * 100. Pinnotherid processing To verify the presence/absence of the pinnotherid P. chilensis, we dissected the digestive tissues of 73 individual sea urchins using surgical forceps. Specimens were identified following the information reported by Takeda and Masahito (2000) and Campos (2017). All of the pinnotherids were examined using a magnifying glass, to determine their sex, based on the shape of the abdomen and the presence of incubated embryos (Thatje and Calcagno 2014). In each pinnotherid, the maximum length of the carapace (Lardies and Castilla 2001; McDermott 2006) was measured using digital vernier calipers. Subsequently, the wet weight of each pinnotherid was obtained to the nearest 0.01 g. All specimens were kept for 1 h out of the water before being weighed to equalize water loss by aerial exposure among all individuals. Impact of the pinnotherid on sea urchin digestive system morphology To identify the impact of symbiotic pinnotherids on the morphology of the host’s digestive system, we photographed the terminal portion of the digestive tract of infected and non-infected sea urchins. Each digestive system was placed in a Petri dish with seawater and photographed at 10X using a stereomicroscope coupled with a digital camera. All photographs also included a reference scale, allowing us to determine the area of the sea urchin’s digestive system in which the pinnotherid symbiont was located. The images were later processed using Image J software. Subsequently, these pinnotherids were also photographed as previously described and the area of the carapace (excluding the thoracic appendages) was measured. The pinnotherid carapace area was page 3 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan subsequently compared with the surface area of the host sea urchin’s digestive system, in order to identify whether the host’s digestive system had expanded in response to the presence of the symbiotic crustacean. Data analyses The normality and homoscedasticity of the data were identified using the Kolmogorov-Smirnov test and the Levene test, respectively. When these criteria were Fig. 1. A, Sea urchin without the upper part of the testa, showing the parasitic pinnotherid. P = pinnotherid, G = sea urchin gonads. B, Intertidal environment from which sea urchins were obtained. C, Map of South America, indicating (arrow) the sampling site of the sea urchin used in the present study. page 4 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan not met, we used a non-parametric test (e.g., ANCOVA with permutations). Linear or non-linear regressions were performed as required in each case. Potential adjustment regressions were used for the ratios of total wet weight, gonadal dry weight and dry weight of the other sea urchins' tissues with its diameter as an independent variable. Linear regressions were used to assess the relationships of the pinnotherid carapace length and the dry weight of the sea urchin gonads, with the sea urchin diameter serving as the independent variable in both cases. The same analysis was used for the data of wet weight, dry weight of other tissues and the area of the sea urchin intestine, with the length of the pinnotherid carapace serving as the independent variable. Logistic regression was used to determine the relationship between the length of the carapace and the pinnotherid tissue weight, with the reproductive condition of the female serving as the dichotomous variable (ovigerous – non ovigerous). Comparisons of total wet weight between sea urchins with and without pinnotherid symbionts, as well as between male and female sea urchins were made using ANCOVA analysis. The same analysis was used for comparisons of the terminal area of the intestine in parasitized and non-parasitized sea urchins, the carapace area of the pinnotherid with the expansion of the sea urchin’s digestive system, and the gonadal weight with the “other tissues” of male and female sea urchins. The covariate for the first two cases was the diameter of the sea urchin test and, for the last two, the length of the pinnotherid carapace. ANCOVA with permutations (5000 permutations) was used to analyze the sea urchin gonadal dry weight and gonad somatic index, according to the presence or absence of pinnotherids. In addition, GSI was analyzed in relation to the sea urchin sex (male and female); in each of these cases, the test diameter of the sea urchin was used as the covariate. The same test was used to analyze by sex (males and female sea urchins), the wet weight data and dry weight of other sea urchin tissues according to the presence or absence of the pinnotherids, with the sea urchin diameter serving as the covariate. Statistical analyses were carried out using Sigmaplot, STATISTICA 7.0 software and R statistical package (Team RC 2021). RESULTS Size and frequency distribution of sea urchins with symbiont pinnotherids The test diameters of the sea urchins collected from the population at Calfuco ranged between 2.6 and 9.5 cm, with most of the tests being between 6.0 and 8.0 cm (Fig. 2A). Of the 73 sea urchins that were collected, 85.7% were infested with pinnotherids (Fig. 2B); 47% of the infected sea urchins were females and 53% were males. Gravimetry of sea urchin and the impact of infesting crab Sea urchin total wet weight Sea urchin total wet weight was strongly related to the sea urchin’s test diameter (Nonlinear regression: F(1,72) 1429.5; P < 0.001, Fig. 3A). Sea urchins harboring pinnotherids had significantly greater total wet weights than those without pinnotherids (ANCOVA: F(1,70) 5.222; P = 0.002, Fig. 3B), and the impact was similar for infested sea urchins of both sexes (ANCOVA with permutations: F(1,58) 5000; P = 0.078, Fig. S1A). Larger parasitized sea urchins typically harbored larger pinnotherid symbionts (Linear Regression: F(1,60) 77.43; P < 0.001, Fig. 3C), and the relationship was similar regardless of the sex of the sea urchin host (ANCOVA: F(1,59) 1.361; P = 0.267, Fig. S1B). Sea urchin gonads Larger sea urchins tended to have heavier gonads (Non linear Regression: F(1,71) 85.64; P < 0.001, Fig. 4A). The relationship between sea urchin dry gonadal weight and the sea urchin’s test diameter was significantly altered by the presence of the pinnotherid, with parasitized individuals generally having smaller gonads (ANCOVA with permutations: F(1,69) 5000; P < 0.001, Fig. 4B). A significant interaction between the sex and diameter of the sea urchin was also identified for the dry weight of the gonad of parasitized males and females (ANOVA with permutations: F(1,58) 5000; P = 0.022, Fig. S2A). On the other hand, sea urchins with larger gonads tended to harbor significantly larger pinnotherids (Linear Regression: F(1, 61) 32.911; P < 0.001, Fig. 4C). Finally, no significant differences were identified in the gonadal dry weight of sea urchin between males and females depending on the size of the hosted pinnotherid (ANCOVA: F(1,59) 2.262; P = 0.138, Fig. S2B). “Other soft tissues” of the sea urchin Larger sea urchins had more non-gonadal soft tissues than smaller sea urchins (Nonlinear Regressión: F(1, 64) 105.1; P < 0.001, Fig. 5A). The presence of page 5 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan the pinnotherid was not associated with the weight of non-gonadal sea urchin tissues (ANCOVA with permutations: F(1,62) 5000; P = 0.5, Fig. 5B), for both male and female sea urchins (ANCOVA with permutations: F(1,62) 5000; P = 1, Fig. S3A). On the other hand, sea urchins with the largest weight of the ‘other tissues’ tended to harbor significantly larger pea crabs (Linear Regression: F(1,55) 47.00; P < 0.05, Fig. 5C), but no differences between sexes were identified regarding the size of the pinnotherid (ANCOVA: F(1,63) 3.169; P = 0.07, Fig. S3B). Sea urchin gonadosomatic index There was no significant relationship between sea urchin size and the gonadosomatic index (GSI) (ANCOVA with permutations: F(1,62) 5000; P = 0.103). However, higher values were seen in sea urchins that were not hosting pinnotherids (ANCOVA with permutations: F(1,62) 5000; P = 0.011), with Fig. 2. A, Frequency distribution of sea urchin test diameter for the specimens sampled from Calfuco beach in January and February 2021. B, Relationship between sea urchin test diameter and the proportion of infested and non-infested sea urchins. N = 73. Sea urchin test diameter (cm) 2.6-3 3.1-3.5 3.6-4 4.1-4.5 4.6-5.5 5.6-6 6.1-6.5 6.6-7 7.1-7.5 7,6-8 8.1-8.5 8.6-9 9.1-9.5 Number of urchin 0 2 4 6 8 10 12 14 A Sea urchin test diameter (cm) 2.6-3 3.1-3.5 3.6-4 4.1-4.5 4.6-5.5 5.6-6 6.1-6.5 6.6-7 7.1-7.5 7,6-8 8.1-8.5 8.6-9 9.1-9.5 Infested sea urchin (%) 0 20 40 60 80 100 120 With pinnotherid Without pinnotherid B page 6 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan parasitized sea urchins presenting the lowest values of gonadosomatic index (Fig. 6). Sea urchin sizes and pinnotherid size Larger sea urchins tended to harbor larger pinnotherids (Linear Regression: F(1,60) 87.97; P < 0.001). The smallest infested sea urchin identified during this research had a test diameter of 3.1 cm, while the largest had a test diameter of 9.5 cm (Fig. 7A). All pinnotherids collected from sampled sea urchins were females. The most extreme carapace Fig. 3. A, Relationship between sea urchin size and its total wet weight for the specimens collected from Calfuco Beach. N = 73. B, Sea urchin wet weight with pinnotherid and without pinnotherids, and C, Relationship between total wet weight of the sea urchin host and the carapace length of the harbored pinnotherid. N = 62. Sea urchin test diameter (cm) 23456789 10 Sea urchin total wet wt. (g) 0 50 100 150 200 250 300 350 With pinnotherid Without pinnotherid y = 34.84 x - 100.42 R2= 0.92 y = 37.71 x - 134 R2 = 0.88 B Sea urchin wet tissue wt. (g) 0 50 100150 200 250 300350 Pinnotherid carapace length (cm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 y = 0.006 x + 0.656 R² = 0.56 C Sea urchin test diameter (cm) 2 3 4 5 6 7 8 9 10 Sea urchin wet tissue wt. (g) 0 50 100 150 200 250 300 350 y = 0.5341x 2.7854 R² = 0.95 A page 7 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan lengths for the pinnotherid females found inside the sea urchin hosts were 0.5 and 2.7 cm (Fig. 7B). Whether the pinnotherid females were ovigerous or non-ovigerous was significantly related to their wet weight (Logistic regression: Wald = 9.139; d.f. = 1; P = 0.002) and size of the female host crab (Logistic regression: Wald = 4.360; d.f. = 1; P = 0.004). In the study population, the smallest gravid female recorded was 1.2 cm (Fig. 7B). Fig. 4. A, Relationship between the dry gonadal weight and the test diameter of the sea urchins collected on Calfuco Beach in 2021. N = 73. B, sea urchin test diameter and dry gonadal weight in sea urchins with pinnotherid and without pinnotherid symbionts, and C, Relationship between symbiont-harboring sea urchin dry gonadal weight and the length of the pinnotherid carapace. N = 62. Sea urchin test diameter (cm) 23456789 10 Sea urchin dry gonad wt. (g) 0 2 4 6 8 10 With pinnotherid Without pinnotherid y = 0.72 X - 1.63 R 2 = 0.62 y = 0.62 X - 1.84 R 2 = 0.51 B Sea urchin test diameter (cm) 2345678 9 10 Sea urchiun gonad dry wt. (g) 0 2 4 6 8 10 y = 0.0654x 1.8344 R² = 0.54 A Sea urchin dry gonadal wt. (g) 01234567 Pinnotherid carapace length (cm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 y = 0.2483 x + 0.8728 R² = 0.35 C page 8 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan Presence of pinnotherid and morphology of the sea urchin’s digestive system The diameter of the terminal section of the sea urchin’s intestine was significantly larger for sea urchins that were harboring pinnotherids than for sea urchins that were not hosting pinnotherids (ANCOVA: F(1,11) 7.387; P = 0.02, Fig. 8A). The expanded area of an Fig. 5. A, Relationship between the test diameter and dry weight of the non-gonadal tissues of the sea urchins collected at Calfuco Beach. N = 73. B, Relationship between presence or absence of pinnotherid symbionts and the weight of non-gonadal sea urchin tissues. C, Influence of the size of the hosted pinnotherid on the dry non-gonadal tissue weight of the sea urchin specimens collected in the Calfuco Beach. N = 62. Sea urchin test diameter (cm) 23456789 10 Sea urchin non-gonadal dry tissue wt. (g) 0 1 2 3 4 5 6 7 y = 0.0746x 1.9461 R² = 0.71 A Non-gonadal tissue dry wt. (g) 0 1 2 345 6 Pinnotherid carapace length (cm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 y = 0.2374 x + 0.6725 R² = 0.47 C 7 Sea urchin test diameter (cm) 2345678 9 10 Sea urchin other tissue dry wt. (g) 0 2 4 6 8 With pinnotherid Without pinnotherid B y = 0.70 x - 1.33 R 2 = 0.76 y = 0.85 X - 2.45 R 2 = 0.70 10 page 9 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan Gebauer P, Moreno CA. 1995. Experimental validation of the growth rings of Loxechinus albus (Molina, 1782) in southern Chile (Echinodermata: Echinoidea). Fish Res 21:423–435. doi:10.1016/0165-7836(94)00292-5. Geiger DL, Martin JW. 1999. The pea crab Orthotheres haliotidis new species (Decapoda: Brachyura: Pinnotheridae) in the Australian abalone Haliotis asinina Linnaeus,1758 and Haliotis squamata Reeve, 1846 (Gastropoda: Vetigastropoda: Haliotidae). Bull Mar Sci 64:269–280. Gonzalez-Canales ME, Marco-Herrero E, Andreu-Cazenave M, González-Gordillo JI. 2018. Larvae development of the symbiotic pea crab Pinnaxodes chilensis (H. Milne Edwards, 1837) (Decapoda, Brachyura, Pinnotheridae) reared in laboratory. Arthropod Struc Dev 47:91–103. doi:10.1016/j.asd.2017.11.003. Gutiérrez-Martínez J. 1971. Notas biológicas sobre Pinnaxodes chilensis (M. Edwards) y descripción de su primera Zoea (Crustacea, Decapoda, Brachyura). Mus Nac Hist Nat, Santiago 15:3–10. Hamel J-F, Ng PKL, Mercier A. 1999. Life cycle of the pea crab Pinnotheres halingi sp. nov., an obligate symbiont of the sea cucumber Holothuria scabra Jaeger. Ophelia 50:149–175. doi:1 0.1080/00785326.1999.10409393. Hsueh P-W. 2001. Intertidal distribution, symbiotic association and reproduction of Pinnotheres bidentatus (Brachyura: Pinnotheridae) from Taiwan. J Nat Hist 35:1681–1692. doi:10.1080/002229301317092397. Jangoux M. 1987. Diseases of Echinodermata. III. Agents metazoans (Annelida to Pisces). Dis Aquat Org 3:59–83. doi:10.3354/ DAO003059. Khandeparker L, Anil AC. 2013. Association of bacteria with marine invertebrates: Implications for ballast water management. Ecohealth 10:268–276. doi:10.1007/s10393-013-0857-z. Komai T, Nishi E, Taru M. 2014. A new species of Pinnixa (Crustacea: Decapoda: Brachyura: Pinnotheridae) associated with a tube worm, Chaetopterus cautus (Annelida: Polychaeta), from Tokyo Bay, Japan. Zootaxa 3793:119–132. doi:10.11646/zootaxa.3793. 1.5. Kuo A-L, Lin F-J, Hsu J-T, Chan Y-S, Ueng Y-T. 2018. The population structure and parasitic relationships of oyster (Crassostrea angulata), Arcotheres sinensis (Pinnotheridae), and Rhopalione sinensis (Bopyridae) at the oyster reefs of western Taiwan. Crustaceana 91:1433–1451. doi:10.1163/15685403-00003842. Lardies MA, Castilla JC. 2001. Latitudinal variation in the reproductive biology of the commensal crab Pinnaxodes chilensis (Decapoda: Pinnotheridae) along the Chilean coast. Mar Biol 139:1125–1133. doi:10.1007/s002270100661. Larraín AP. 1975. Los equinoideos regulares fósiles y recientes de Chile. Gayana, Zool 35:5–189. Martínez M. 2014. Cangrejos pinoteridos (Brachyura: Pinnotheridae) de aguas someras de la costa Atlántica mexicana. PhD dissertation, Universidad Nacional Autónoma de México, Mexico. McDermott JJ. 2009. Hypersymbioses in the pinnotherid crabs (Decapoda: Brachyura: Pinnotheridae): a review. J Nat Hist 43:785–805. doi:10.1080/00222930802702480. McDermott JJ. 2006. The biology of Austinixa gorei (Manning & Felder, 1989) (Decapoda, Brachyura, Pinnotheridae) symbiotic in the burrows of intertidal ghost shrimp (Decapoda, Thalassinidea, Callianassidae) in Miami, Florida. Crustaceana 79:345–361. doi:10.1163/156854006776759608. Mena S, Salas-Moya C, Wehrtmann IS. 2014. Living with a crab: effect of Austinotheres angelicus (Brachyura, Pinnotheridae) infestation on the condition of Saccostrea palmula (Ostreoida, Ostreidae). Nauplius 22:151–158. doi:10.1590/S010464972014000200009. Miller A, Inglis GJ, Poulin R. 2008. Use of the introduced bivalve, Musculista senhousia, by generalist parasites of native New Zealand bivalves. NZ J Mar Freshw Res 42:143–151. doi:10.1080/00288330809509944. Miyake S. 1939. Note on crabs of the genus Echinoecus Rathbun living commensally with echinoids (Parthenopidae, Eumedoniidae). Ann Zool Jap 18:83–94. Narvarte MA, Saiz MN. 2004. Effects of the pinnotherid crab Tumidotheres maculatus on the Tehuelche scallop Aequipecten tehuelchus in the San Matías Gulf, Argentina. Fish Res 67:207– 214. doi:10.1016/j.fishres.2003.09.041. Ng PKL, Manning RB. 2003. On two new genera of pea crabs parasitic in holothurians (Crustacea: Decapoda: Brachyura: Pinnotheridae) from the Indo-West Pacific, with notes on allied genera. Proc Biol Soc Wash 116:901–919. O'Beirn FX, Walker RL. 1999. Pea crab, Pinnotheres ostreum Say, 1817, in the Eastern oyster, Crassostrea virginica (Gmelin, 1791): Prevalence and apparent adverse effects on oyster gonad development. Veliger 42:17–20. Ocampo EH, Nuñez JD, Cledón M, Baeza JA. 2014. Parasitic castration in slipper limpets infested by the symbiotic crab Calyptraeotheres garthi. Mar Biol 161:2107–2120. doi:10.1007/ s00227-014-2490-y. Ocampo EH, García MP, Nuñez JD, Luppi TA. 2021. Impact on reproductive performance and body condition in a small limpet parasitized by a large castrator pea crab. J Morphol 282:1604– 1615. doi:10.1002/jmor.21409. Patton WK. 1965. Commensal Crustacea. Mar Biol Assoc India., Proc. Symposium Ernakuleam 1965, Part III, Symposium 2:1228–1243. Reeves MN, Brooks WR. 2001. Host selection, chemical detection, and protection of the symbiotic pinnotherid crabs Dissodactylus crinitichelis and Clypeasterophilus rugatus associated with echinoderms. Symbiosis 30:239–256. Retamal MA, Moyano HI. 2010. Zoogeografía de los crustáceos decápodos chilenos marinos y dulceacuícolas. Lat Amer J Aquat Res 38:302–328. Runil F. 2014. Caracterización genético-poblacional y variabilidad morfológica de Pinnaxodes chilensis (Decápoda: Pinnotheridae) en la costa oriental del Pacífico Sur. Marine Biologist Dissertation, Universidad Austral de Chile, Chile. Schmitt WL, McCain JC, Davidson ES. 1973. Crustaceorum Catalogus 3. Decapoda I Brachyura I: Fam. Pinnotheridae. In: Gruner HE, Holthuis LB (eds). Den Haag (The Netherlands), pp. 1–160. Silliman BR, Layman CA, Altieri AH. 2003. Symbiosis between an alpheid shrimp and a xanthoid crab in salt marshes of midAtlantic states, USA. J Crustac Biol 23:876–879. doi:10.1651/ C-2410. Stevens PM. 1990. Specificity of host recognition of individuals from different host races of symbiotic pea crabs (Decapoda: Pinnotheridae). J Exp Mar Biol Ecol 143:193–207. doi:10.1016/0022-0981(90)90070-S. Sun W, Sun S, Yuqi W, Baowen Y, Weibo S. 2006. The prevalence of the pea crab, Pinnotheres sinensis, and its impact on the condition of the cultured mussel, Mytilus galloprovincialis, in Jiaonan waters (Shandong province, China). Aquaculture 253:57–63. doi:10.1016/j.aquaculture.2005.07.037. Takeda M, Masahito P. 2000. Notes on the pinnotherid crabs of the genus Pinnaxodes (Crustacea: Decapoda: Brachyura). Bull Natn Sci Mus, Tokyo, Ser A 26:99–112. Takeda S, Tamura S, Washio M. 1997. Relationship between the pea crab Pinnixa tumida and its endobenthic holothurian host Paracaudina chilensis. Mar Ecol Prog Ser 149:143–154. doi:10.3354/meps149143. Team RC. 2021. R: A language and environment for statistical page 16 of 17Zoological Studies 62:18 (2023) © 2023 Academia Sinica, Taiwan computing. R Foundation for Statistical Computing. Vienna, Austria. Thatje S, Calcagno JA. 2014. Brachyura. In: JA Calcagno (Ed.) Los invertebrados marinos. Buenos Aires. Argentina. Thébault E, Loreau M. 2005. Trophic interactions and the relationship between species diversity and ecosystem stability. Am Nat 166:95–114. doi:10.1086/444403. Thiebot J-B, Weimerskirch H. 2012. Contrasted associations between seabird and marine mammals across four biomes of the southern Indian Ocean. J Ornith 154:441–453. doi:10.1007/s10336-0120909-0. Thiel M, Baeza JA. 2001. Factors affecting the social behaviour of Crustaceans living symbiotically with other marine invertebrates: A modelling approach. Symbiosis 30:163–190. Tresnati J, Yasir I, Aprianto R, Yanti A, Bestari AD, Tuwo A. 2021. Peanut crab Pinnotheres halingi, a symbiotic commensal or parasite of sandfish Holothuria scabra? IOP Conf. Ser.: Earth Environ Sci 763:012004. doi:10.1088/1755-1315/763/1/012004. Trottier O, Jeffs AG. 2015. Recruitment of the parasitic pea crab Nepinnotheres novaezelandiae into green-lipped mussels Perna canaliculus. Dis Aquatic Org 112:199–205. doi:10.3354/ dao02809. Vásquez JA. 2007. Ecology of Loxechinus albus. Dev Aquatic Fish Sci 37:227–241. doi:10.1016/S0167-9309(01)80012-0. Vásquez P, Bay-Schmith E. 2010–2011. Efecto de la expansión rectal por el comensal Pinnaxodes chilensis (Crustacea: Decapoda) en el erizo de mar Loxechinus albus (Echinodermata: Echinoidea). Bol Soc Biol Concepción, Chile 80:83–88. Waide RB, Willig MR, Steiner CF, Mittelbach G, Gough L, Dodson SI, Juday GP, Parmenter R. 1999. The relationship between productivity and species richness. Ann Rev Ecol Syst 30:257– 300. doi:10.1146/annurev.ecolsys.30.1.257. Watts JC, Carroll JM, Munroe DM, Finelli CM. 2018. Examination of the potential relationship between boring sponges and pea crabs and their effects on eastern oyster condition. Dis Aquat Organ 130:25–36. doi:10.3354/dao03257. Weber L, Apprill A. 2020. Diel, daily, and spatial variation of coral reef seawater microbial communities. PLoS ONE 15(3):e0229442. doi:10.1371/journal.pone.0229442. Wells HW, Wells MJ. 1961. Observations on Pinnaxodes floridensis, a new species of pinnotherid Crustacean commensal in Holothurians. Bull Mar Sci 11:267–279. Yasuoka N, Yusa Y. 2017. Effects of a crustacean parasite and hyperparasite on the Japanese spiny oyster Saccostrea kegaki. Mar Biol 164:217. doi:10.1007/s00227-017-3250-6. Zaixso HE, Stoyanoff P, Gil DG. 2009. Detrimental effects of the isopod, Edotia doellojuradoi, on gill morphology and host condition of the mussel, Mytilus edulis platensis. Mar Biol 156:2369–2378. doi:10.1007/s00227-009-1265-3. Supplementary materials Fig. S1. A) Relationship between sea urchin test diameter and total wet weight for parasitized sea urchin males (red circles) and females (blue circles). B) Relationship between the total wet weight of male (red circles) and females (blue circles) host sea urchins and the carapace length of the pinnotherid. N = 62. (download) Fig. S2. A) Relationship between sea urchin test diameter and dry gonadal weight in males (red circles) and females (blue circles) of parasitized sea urchins. B) Relationship between the gonad dry weight of male (blue circles) and female (red circles) sea urchin and the carapace length of the hosted pinnotherid. N = 62. (download) Fig. S3. A) Relationship between sex (red circles: male, blue circles: female) of the sea urchin host on nongonadal tissue weight. B) Influence of the size of the hosted pinnotherid on the dry non-gonadal tissue weight of the male (read circles) and females (blue circles) of sea urchin specimens collected in the Calfuco Becah. N = 62. (download) page 17 of 17Zoological Studies 62:18 (2023)