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Gonadal development and sexuality of Larkinia grandis (Arcida: Arcidae) inhabiting southeastern Gulf of California

ABSTRACT

Larkinia grandis (Broderip & G.B. Sowerby I, 1829), an important fishing resource for Mexican communities, is an Arcidae clam. It is also considered a species with aquaculture potential. In this work we investigated the gonadal phases and sexuality in a population of L. grandis in the Gulf of California. Our findings support the hypothesis that there is one male per female in the population studied. It also documents that the shape, position and color of the gonads of L. grandis are consistent with observations in other Arcidae species. Additionally, five gonadal phases are differentiated and described in males and females (development, mature, spawning, post-spawning and resting), with a noticeable presence of brown cells during post-spawning and the onset of the resting phase, suggesting that those cells are involved in the reabsorption of remnants. Additionally, asynchronous gametogenesis in males, synchronic gametogenesis in females and batch spawning are defined. The results of this contribution can be used in the efforts to protect this bivalve.

KEY WORDS:
Bivalve; estuary; gametogenesis; gonad; mangrove cockle; sex cells

INTRODUCTION

Larkinia grandis (Broderip & G.B. Sowerby I, 1829) (Mollusca: Bivalvia: Arcida: Arcidae) is a clam distributed from the Ballena Lagoon (Baja California, Mexico) to Tumbes (Northern Peru) (Coan and Valentich-Scott 2012Coan EV, Valentich-Scott P (2012) Bivalve seashells of tropical west America. Marine bivalve mollusks from Baja California to Peru. Santa Barbara, Santa Barbara Museum of Natural History, 2157 pp.), where it is known by the common name of “mangrove cockle” (García-Domínguez et al. 2008García-Domínguez FA, de Haro-Hernández A, García-Cuellar A, Villalejo-Fuerte M, Rodríguez-Astudillo S (2008) Ciclo reproductivo de Anadara tuberculosa (Sowerby, 1833) (Arcidae) en Bahía Magdalena, México. Revista de Biología Marina y Oceanografía 43:143-152. https://doi.org/10.4067/S0718-19572008000100015
https://doi.org/10.4067/S0718-1957200800...
) or “pianguas” (Lucero-Rincón et al. 2012Lucero C, Cantera J, Neira R (2012) Pesquería y crecimiento de la piangua (Arcoida: Arcidae) Anadara tuberculosa en la Bahía de Málaga del Pacífico colombiano, 2007-2007. Revista de Biología Tropical 60: 203-217. https://doi.org/10.15517/rbt.v60i1.2754
https://doi.org/10.15517/rbt.v60i1.2754...
), respectively. This bivalve is found near the coastline, living in close relationship with the roots of the Rhizophora spp. mangrove, buried in the muddy sediment, or very rarely, half-buried or exposed (Fischer et al. 1995Fischer W, Krupp F, Schneider W, Sommer C, Carpenter K, Niem N (1995) Guía FAO para la identificación de especies para los fines de la pesca, Pacífico Centro Oriental. FAO, Rome, vol. 1, 646 pp.). Several members of the family Arcidae, including L. grandis, are commercially exploited on the Pacific coast (Cruz and Palacios 1983Cruz RA, Palacios J (1983) Biometría del molusco Anadara tuberculosa en Punta Morales, Puntarenas, Costa Rica. Revista de Biología Tropical 31: 175-179., Cruz 1987a). The clam L. grandis is caught along with other species of the same genus [Anadara mazatlanica (Hertlein & A.M. Strong, 1943), Anadara multicostata (G.B. Sowerby I, 1833), Anadara similis (C.B. Adams, 1852), and Anadara tuberculosa (G.B. Sowerby I, 1833)] in Mexico (CONAPESCA 2018CONAPESCA (2018) Anuario Estadístico de Acuacultura y Pesca. Comisión Nacional de Acuacultura y Pesca, Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación, CDMX. https://www.conapesca.gob.mx/work/sites/cona/dgppe/2017/ANUARIO_ESTADISTICO_2017.pdf [Accessed: 21/09/2019]
https://www.conapesca.gob.mx/work/sites/...
). Generally, the data provided by the Mexican government does not distinguish among mangrove cockle species and fishery management does not consider the biological characteristics of each species when exploiting them commercially in Mexico. Interest in these clams has grown in recent years due to their use in handicrafts and for direct human consumption. Additionally, mangrove cockles have aquaculture potential in Mexico (Sotelo-Gonzalez et al. 2019Sotelo-Gonzalez MI, Sanchez-Cardenas R, García-Ulloa M, Góngora-Gómez AM, Salcido-Guevara LA, Arzola-González JF, Sepúlveda CH (2019) Las almejas pata de mula (Arcidae) de Sinaloa, México. Ciencia y Mar 23 (69): 45-50.), following the example of other countries (Broom 1985Broom MJ (1985) The biology and culture of marine bivalve molluscs of the genus Anadara. Manila, ICLARM Studies and Reviews, vol. 12, 37 pp., Galdámez-Castillo et al. 2007Galdámez-Castillo AM, Pacheco-Reyes SP, Pérez-García IM, Kino S (2007) Guía para la producción de Anadara spp. 2006-2007: producción artificial de semillas, cultivo intermedio y cultivo de Anadara tuberculosa y A. grandis. Proyecto de Moluscos. CENDEPESCA, JICA, San Salvador.).

Studying the gonadal development and sexuality of wild mollusk populations of commercial importance helps to reveal their reproductive phases and to define their reproductive patterns (Bricelj et al. 2017Bricelj VM, Kraeuter JM, Flimlin G (2017) Status and trends of hard clam, Mercenaria mercenaria, populations in a coastal lagoon ecosystem, Barnegat Bay-Little Egg Harbor, New Jersey. In: Buchanan GA, Belton TJ, Paudel B (Eds) A comprehensive assessment of Barnegat Bay-Little Egg Harbor, New Jersey. Coconut Creek, Florida, Journal of Coastal Research, 205-253.). At the same time, it is a standard procedure to describe the changes in the tissues and cells of the gonad generated by the accumulation of energy and expulsion of gametes (Karray et al. 2015Karray S, Smaoui-Damak W, Rebai T, Hamza-Chaffai A (2015) The reproductive cycle, condition index, and glycogen reserves of the cockles Cerastoderma glaucum from the Gulf of Gabès (Tunisia). Environmental Science and Pollution Research 22: 17317-17329. https://doi.org/10.1007/s11356-015-4337-6
https://doi.org/10.1007/s11356-015-4337-...
), and to explain the way in which organisms modulate the use of their reserves in relation to environmental variables (Boulais et al. 2017Boulais M, Chenevert KJ, Demey AT, Darrow ES, Robison MR, Roberts JP, Volety A (2017) Oyster production is compromised by acidification experienced seasonally in coastal regions. Scientific Reports 7: 13276. https://doi.org/10.1038/s41598-017-13480-3
https://doi.org/10.1038/s41598-017-13480...
), under normal or anomalous conditions. Furthermore, research on the sexual behavior of bivalves helps to understand the interactions between them and their environment and can provide information for the development of selective breeding programs (Breton et al. 2018Breton S, Capt C, Guerra D, Stewart D (2018) Sex-Determining Mechanisms in Bivalves. In: Leonard J (Eds) Transitions Between Sexual Systems. Springer, Cham, 165-192.).

Previous studies on the gonadal development of L. grandis arrived at conflicting results. Four gonadal phases were identified in a population in Costa Rica (Cruz 1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8.) and six phases in Nicaragua (Aguirre-Rubí 2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
). Cruz (1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8.) did not describe the resting phase and Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) generalized the gonadal phases for three species [L. grandis, A. tuberculosa and Polymesoda arctata (Deshayes, 1855)]. In addition, Cruz (1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8., 1987bCruz RA (1987b) Tamaño y madurez sexual en Anadara grandis (Pelecypoda: Arcidae). Brenesia 27: 9-12.) stated that L. grandis is a gonochoristic (dioecious) species, while Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) did not define the sexuality of the three species, but documented one hermaphrodite organism (female with intersex) among 40 analyzed individuals in which the male and female acini were separate.

Additional information for other species of Arcidae is mentioned below. Broom (1983Broom MJ (1983) Gonad development and spawning in Anadara granosa (L.) (Bivalvia: Arcidae). Aquaculture 30: 211-219. https://doi.org/10.1016/0044-8486(83)90163-1
https://doi.org/10.1016/0044-8486(83)901...
) identified six gonadal phases and one hermaphrodite individual in Anadara granosa (Linnaeus, 1758) from the west coast of West Malaysia; Broom (1985Broom MJ (1985) The biology and culture of marine bivalve molluscs of the genus Anadara. Manila, ICLARM Studies and Reviews, vol. 12, 37 pp.) defined that in A. granosa the sexes are undoubtedly separate; the gonadal status of A. granosa and Anadara antiquata (Linnaeus, 1758) were studied in central Java (Afiati 2007aAfiati N (2007a) Gonad maturation of two intertidal blood clams Anadara granosa (L.) and Anadara antiquata (L.) (Bivalvia: Arcidae) in central Java. Journal of Coastal Development 10: 105-113., 2007bAfiati N (2007b) Hermaphroditism in Anadara granosa (L.) and Anadara antiquata (L.) (Bivalvia: Arcidae) from central Java. Journal of Coastal Development 10: 171-179.) and one study suggests that both species could be protandrous sequential hermaphrodites (Afiati 2007bAfiati N (2007b) Hermaphroditism in Anadara granosa (L.) and Anadara antiquata (L.) (Bivalvia: Arcidae) from central Java. Journal of Coastal Development 10: 171-179.); Jahangir et al. (2014Jahangir S, Siddiqui G, Ayub Z (2014) Temporal variation in the reproductive pattern of blood cockle Anadara antiquata from Pakistan (northern Arabian Sea). Turkish Journal of Zoology 38: 263-272. https://doi.org/10.3906/zoo-1302-7
https://doi.org/10.3906/zoo-1302-7...
) published four gonadal phases for A. antiquata from Pakistan and only individuals with separate sexes were described. In Colombia, Manjarrés-Villamil et al. (2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.) described the five gonadal phases and found 15 hermaphrodite individuals in A. similis, and mentioned that hermaphroditism in the species needs to be further studied. Ghribi et al. (2017Ghribi F, Bello G, Zupa R, Passantino L, Santamaria N, El Cafsi M, Corriero A (2017) Reproductive and tissue plasticity in Arca noae (Bivalvia: Arcidae). The European Zoological Journal 84: 473-487. https://doi.org/10.1080/24750263.2017.1368725
https://doi.org/10.1080/24750263.2017.13...
) observed four phases in the gonads of 142 females and 42 males, and documented five cases of protandric hermaphroditism in Arca noae Linnaeus, 1758.

The gonadal status of A. tuberculosa has been studied in the Pacific Coast of Costa Rica (Cruz 1984Cruz RA (1984) Algunos aspectos de la reproducción en Anadara tuberculosa (Pelecypoda: Arcidae) de Punta Arenas, Puntarenas, Costa Rica. Revista de Biología Tropical 32: 45-50.), Mexico (Pérez-Medina 2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
, García-Domínguez et al. 2008García-Domínguez FA, de Haro-Hernández A, García-Cuellar A, Villalejo-Fuerte M, Rodríguez-Astudillo S (2008) Ciclo reproductivo de Anadara tuberculosa (Sowerby, 1833) (Arcidae) en Bahía Magdalena, México. Revista de Biología Marina y Oceanografía 43:143-152. https://doi.org/10.4067/S0718-19572008000100015
https://doi.org/10.4067/S0718-1957200800...
) and Colombia (Lucero-Rincón et al. 2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
). These studies revealed differences in the number of gonadal phases and used different nomenclature. In Costa Rica, Cruz (1984Cruz RA (1984) Algunos aspectos de la reproducción en Anadara tuberculosa (Pelecypoda: Arcidae) de Punta Arenas, Puntarenas, Costa Rica. Revista de Biología Tropical 32: 45-50.) did not find evidence of sexual reversal in any specimen of A. tuberculosa. In Mexico, Pérez-Medina (2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) documented two hermaphroditic organisms of A. tuberculosa and defined that it is gonochoric, but with casual hermaphroditism. In contrast, Lucero-Rincón et al. (2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
) determined that A. tuberculosa is a protandric hermaphrodite in Colombian Pacific.

This contribution describes the gonadal phases and sexuality in a wild population of L. grandis in the southeastern Gulf of California and provides additional observations about the sex ratio and the color and anatomy of the gonads.

MATERIAL AND METHODS

The clams were collected in the El Cohui estuary (25°26’-19°38’N; 105°48’-43°90’W) within the San Ignacio-Navachiste-Macapule lagoon system in the state of Sinaloa, Mexico (Fig. 1), where an important area for fishing of L. grandis is located. In total, 240 clams were collected by free divers, from August 2017 to July 2018. Each sample (n = 20 per month) was placed in a container with sea water and transported to the laboratory (30 minutes) for processing purposes.

Figure 1
El Cohui estuary (ê), San Ignacio-Navachiste-Macapule lagoon system, Sinaloa, southeastern Gulf of California, Mexico.

First, the length (mm) and weight (g) of each clam were registered; next, the shells were opened, and the soft tissues were removed. The soft tissues were macroscopically analyzed to observe external alterations, the appearance of the gonads and their location within the visceral cavity (Álvarez-Dagnino et al. 2017Álvarez-Dagnino E, Santamaría-Miranda A, García-Ulloa M, Góngora-Gómez AM (2017) Reproduction of Megapitaria squalida (Bivalvia: Veneridae) in the Southeast Gulf of California, Mexico. Revista de Biología Tropical 65: 881-889. https://doi: 10.15517/rbt.v65i3.26371
https://doi: 10.15517/rbt.v65i3.26371...
).

The soft tissues were fixed with Davidson solution, rinsed with distilled water to remove excess fixative and placed in 70% alcohol until dehydration (Álvarez-Dagnino et al. 2017Álvarez-Dagnino E, Santamaría-Miranda A, García-Ulloa M, Góngora-Gómez AM (2017) Reproduction of Megapitaria squalida (Bivalvia: Veneridae) in the Southeast Gulf of California, Mexico. Revista de Biología Tropical 65: 881-889. https://doi: 10.15517/rbt.v65i3.26371
https://doi: 10.15517/rbt.v65i3.26371...
). Then, a longitudinal section of the gonad tissue was dehydrated and embedded in paraffin (Buesa and Peshkov 2009Buesa RJ, Peshkov MV (2009) Histology without xylene. Annals of Diagnostic Pathology 13: 246-256. https://doi.org/10.1016/j.anndiagpath.2008.12.005
https://doi.org/10.1016/j.anndiagpath.20...
). Histological sections of 3 µm thickness were obtained and stained using the Hematoxylin-Eosin-Floxin (HEF) technique (Humason 1972Humason GL (1972) Animal tissue techniques. W.H. Freeman and Company, San Francisco, 572 pp.).

The sex of individuals was identified from the histological sections. The sex ratio (number of males per female, n:1) was calculated dividing the number of males by the number of females (Álvarez-Dagnino et al. 2017Álvarez-Dagnino E, Santamaría-Miranda A, García-Ulloa M, Góngora-Gómez AM (2017) Reproduction of Megapitaria squalida (Bivalvia: Veneridae) in the Southeast Gulf of California, Mexico. Revista de Biología Tropical 65: 881-889. https://doi: 10.15517/rbt.v65i3.26371
https://doi: 10.15517/rbt.v65i3.26371...
). The Yates-corrected Chi-squared test was used to compared the observed sex ratio with the expected value 1:1, using χ2 with n-2 degree of freedom and a significance level α = 0.05 (Zar 1996Zar JH (1996) Biostatistical Analysis. Third edition. Prentice-Hall. Inc, Upper Saddle River, 662 pp.).

The histological sections were analyzed qualitatively using an optical microscope (Zeiss model Axiostar 10x, 40x and 100x) to identify the gonadal phases (development, maturity, spawning, post-spawning and resting) based on cellular and tissue characteristics and stages of gametogenesis, considering the criteria of Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) for L. grandis, Pérez-Medina (2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) and García-Domínguez et al. (2008García-Domínguez FA, de Haro-Hernández A, García-Cuellar A, Villalejo-Fuerte M, Rodríguez-Astudillo S (2008) Ciclo reproductivo de Anadara tuberculosa (Sowerby, 1833) (Arcidae) en Bahía Magdalena, México. Revista de Biología Marina y Oceanografía 43:143-152. https://doi.org/10.4067/S0718-19572008000100015
https://doi.org/10.4067/S0718-1957200800...
) for A. tuberculosa, Manjarrés-Villamil et al. (2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.) for A. similis, Broom (1983Broom MJ (1983) Gonad development and spawning in Anadara granosa (L.) (Bivalvia: Arcidae). Aquaculture 30: 211-219. https://doi.org/10.1016/0044-8486(83)90163-1
https://doi.org/10.1016/0044-8486(83)901...
) for A. granosa, Afiati (2007aAfiati N (2007a) Gonad maturation of two intertidal blood clams Anadara granosa (L.) and Anadara antiquata (L.) (Bivalvia: Arcidae) in central Java. Journal of Coastal Development 10: 105-113.) for A. granosa and A. antiquata, Ghribi et al. (2017Ghribi F, Bello G, Zupa R, Passantino L, Santamaria N, El Cafsi M, Corriero A (2017) Reproductive and tissue plasticity in Arca noae (Bivalvia: Arcidae). The European Zoological Journal 84: 473-487. https://doi.org/10.1080/24750263.2017.1368725
https://doi.org/10.1080/24750263.2017.13...
) for A. noae. Additionally we found sub-phases of gonadic resting and described them based on differences in the dispositions and thickness of the connective tissue and in the number of brown cells. The browns cells were identified according to Ghribi et al. (2017Ghribi F, Bello G, Zupa R, Passantino L, Santamaria N, El Cafsi M, Corriero A (2017) Reproductive and tissue plasticity in Arca noae (Bivalvia: Arcidae). The European Zoological Journal 84: 473-487. https://doi.org/10.1080/24750263.2017.1368725
https://doi.org/10.1080/24750263.2017.13...
). These data were documented with a Nikon D5200 camera adapted to a microscope. The images were transferred and processed on a computer with the Sigma Scan Pro program (version 5.0 Systat Software, Inc., Richmon, CA, United States) (Álvarez-Dagnino et al. 2017Álvarez-Dagnino E, Santamaría-Miranda A, García-Ulloa M, Góngora-Gómez AM (2017) Reproduction of Megapitaria squalida (Bivalvia: Veneridae) in the Southeast Gulf of California, Mexico. Revista de Biología Tropical 65: 881-889. https://doi: 10.15517/rbt.v65i3.26371
https://doi: 10.15517/rbt.v65i3.26371...
) in order to measure the diameter of the oocytes (mean±standard deviation µm) of L. grandis. Only oocytes with a visible nucleus were measured, and due to their irregular shape, first, the area was measured and then the diameter (D) was calculated assuming a circular form of each oocyte. An amount of 1200 oocytes at different oogenesis stages were measured from 84 individuals.

RESULTS

All clams collected were adults. Their length and weight ranged from: 44.57 to 142 mm and 41.90 to 337 g, respectively. The male (n=95) and female (n=108) gonads were in different individuals in L. grandis, as in gonochoristic species, and the sex ratio was 0.88 males per female (0.88:1). This sexual proportion was not statistically different from the expected value 1:1 (χ2 203-2 = 12.07, p = 0.36), and neither were the sex ratios estimated for most months (χ2 ⁓20-2 = 0.07-2.57, p = 0.1-0.81), with the exception of April 2018, when there were more males than females (3:1; χ2 20-2 = 7.14, p = 0.02) (Fig. 4). Also, 37 indeterminate individuals (sexually) were identified. The external appearance of the gonads on L. grandis showed sexual dichromatism when gametogenesis occurs. This is more noticeable in their maturity phase. The gonad of males was cream-beige, and the gonad of females was orange-brown. The color tonality intensified as the gonad matured. The gonads were located within the visceral mass and occupied 20-40% of it (Figs 2, 3).

Figures 2-3
Internal morphology of Larkinia grandis: (2) male; (3) female. (g) Gonad, (dg) digestive gland, (m) mantle, (am) adductor muscle, (gi) gills. Scale bar: 1.0 cm.

Figure 4
Monthly sexual proportion of Larkinia grandis during the 2017-2018 period. The dotted line marks one female as a reference for the estimated number of males by month. The asterisk (*) indicates that the sexual proportion is statistically different (p < 0.05) from the expected value (one male per one female, 1:1).

In females, the oogenesis cell stages distinguished were: oogonia (4.58 ± 0.80 µm D), previtellogenic oocyte (9.77±1.68 µm D), vitellogenic oocyte (17.65 ± 2.30 µm D) and yolked oocyte (33.08±2.83 µm D). In males, the spermatogenic cell stages observed were: spermatogonia, spermatocytes, spermatids, as well as spermatozoa. Females presented synchronic gametogenesis due to the presence of one or two gametogenic cell stages in a single acinus. While males presented an asynchronous gametogenesis because the simultaneous presence of all spermatogenic cell stages in a single acinus.

Five gonadal phases were differentiated for males and females: development, mature, spawning, post-spawning and resting. For the latter, three sub-phases were identified. Immature gonads (no previous ripening) were not defined. At the beginning of the progression of the gonadal phases, the acini are roughly round or elliptical, increase in size and their walls become thinner (development and mature phases), then the acini acquire irregular shape (after spawning phase) and finally the acini recover their roughly rounded or elliptical regular form (resting phase). Also the cell types and their quantity change due mainly to gametogenesis and reabsorption processes. The detailed description of gonadal phases for males and females of L grandis is in Table 1.

Table 1
Description of the gonadal phases of males and females of Larkinia grandis in southeastern Gulf of California.

Figures 5-8
Histological sections of Larkinia grandis male’s gonads: (5) development; (6) maturity; (7) spawning; (8) post-spawning. (al) Acinus lumen, (aw) acinus wall, (bv) blood vessel, (bc) brown cells, (dt) digestive tissue, (ma) mature acinus, (rsz) residual spermatozoa, (scll) spermatogenic cells, (sz) spermatozoa. Black scale bars: 100 µm, white scale bars: 50 µm.

Figures 9-12
Histological sections of Larkinia grandis female’s gonads: (9) development; (10) maturity; (11) spawning; (12) post-spawning. (al) acinus lumen, (aw) acinus wall, (bc) brown cells, (dt) digestive, (oo) oogonia, (pvo) previtellogenic, (ryo) residual yolked oocyte, (yko) yolked oocyte. Black scale bars: 100 µm, white scale bars: 50 µm.

Figures 13-15
Histological sections of the resting sub-phases of Larkinia grandis: (13) sub-phase one; (14) sub-phase two; (15) sub-phase three. (al) acinus lumen, (aw) acinus wall, (bc) brown cells. Black scale bars: 100 µm, white scale bars: 50 µm.

DISCUSSION

Among the bivalves studied, the majority were gonochoric (Gosling 2015Gosling E (2015) Marine bivalve molluscs. Second edition. John Wiley & Sons, London, 524 pp.). This is consistent with the findings for the population of L. grandis in southeastern Gulf of California, and also in the Pacific coast of Costa Rica, where hermaphrodite organisms were not found and the sexual proportion was close to one male per female (1:1) (Cruz 1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8., 1987b). Only on April 2018, the studied population of L. grandis had more males than females, but the sex ratios estimated for the other months and the absence of hermaphrodites suggest that the deviation from the 1:1 sex ratio was casual. Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) found one hermaphrodite specimen of L. grandis (2.5% of the total sample) on the Nicaraguan coast. In that publication, however, the sex ratio was not specified. Other Arcidae species have been defined as gonochoric, even though they have a low percentage of hermaphroditic organisms in the population, such as A. granosa (0.33%; Broom 1985Broom MJ (1985) The biology and culture of marine bivalve molluscs of the genus Anadara. Manila, ICLARM Studies and Reviews, vol. 12, 37 pp.) and A. tuberculosa (0.98%; Pérez-Medina 2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) because the percentage of hermaphroditism was considered low and the sexual proportion was close to 1:1. In these cases, hermaphroditism can be considered a casual phenomenon (Pérez-Medina 2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
), and according with the observation of Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
), casual hermaphroditism could also occur in L. grandis. However, more research is needed on hermaphroditism in bivalves to improve our understanding of it (Breton et al. 2018Breton S, Capt C, Guerra D, Stewart D (2018) Sex-Determining Mechanisms in Bivalves. In: Leonard J (Eds) Transitions Between Sexual Systems. Springer, Cham, 165-192.). It is necessary to understand sexuality in species such as A. tuberculosa, which in different populations has been classified as gonochoric (Cruz 1984Cruz RA (1984) Algunos aspectos de la reproducción en Anadara tuberculosa (Pelecypoda: Arcidae) de Punta Arenas, Puntarenas, Costa Rica. Revista de Biología Tropical 32: 45-50.), gonochoric with casual hermaphroditism (Pérez-Medina 2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) and protandric hermaphroditism (Lucero-Rincón et al. 2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
). These different sexualities have been defined according with different percentage of hermaphroditism, and some estimates of sex ratio and size differences between males and females that suggest sex change. Some questions that remain to be answered are, what percentage of hermaphroditism should be in the population to rule out gonochoric sexuality? How could we define whether disparity in sex ratio and size between sexes is due to different longevity of females and males as suggested by Cruz (1987bCruz RA (1987b) Tamaño y madurez sexual en Anadara grandis (Pelecypoda: Arcidae). Brenesia 27: 9-12.)? The criteria to define sexuality need to be improved and clarified.

In this work, the shape and position of the gonads of L. grandis at the different gonadal phases are similar to those reported by Pérez-Medina (2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) and García-Domínguez et al. (2008García-Domínguez FA, de Haro-Hernández A, García-Cuellar A, Villalejo-Fuerte M, Rodríguez-Astudillo S (2008) Ciclo reproductivo de Anadara tuberculosa (Sowerby, 1833) (Arcidae) en Bahía Magdalena, México. Revista de Biología Marina y Oceanografía 43:143-152. https://doi.org/10.4067/S0718-19572008000100015
https://doi.org/10.4067/S0718-1957200800...
) for A. tuberculosa, and by Manjarrés-Villamil et al. (2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.) for A. similis. The gonad, regardless of sex, was located along the digestive gland. Karray et al. (2015Karray S, Smaoui-Damak W, Rebai T, Hamza-Chaffai A (2015) The reproductive cycle, condition index, and glycogen reserves of the cockles Cerastoderma glaucum from the Gulf of Gabès (Tunisia). Environmental Science and Pollution Research 22: 17317-17329. https://doi.org/10.1007/s11356-015-4337-6
https://doi.org/10.1007/s11356-015-4337-...
) mentioned that gonad and digestive tissues in bivalve mollusks such as the sand clam Cerastoderma glaucum (Bruguière, 1789), are intertwined to facilitate the flow of nutrients according to the energy demand during the reproductive phases, while Menezes-Tunholi et al. (2016Menezes-Tunholi V, Menezes-Tunholi A, Teixera-Santos A, da Silva-Garcia J, Maldonado A Jr, da-Silva WS, de Azevedo-Rodrigues ML, Pinheiro J (2016) Evaluation of the mitocondrial system in the gonad-digestive gland complex of Biomphalaria glabrata (Mollusca, Gastropoda) after infection by Echinostoma paraensei (Trematoda, Echinostomatidae). Journal of Invertebrate Pathology 136: 136-14. https://doi.org/10.1016/j.exppara.2016.05.008
https://doi.org/10.1016/j.exppara.2016.0...
) highlighted the metabolic role of the gonad-digestive gland complex of the Biomphalaria glabrata (Say, 1818) gastropod. The tissue fusion described by both authors coincides with the conformation of the gonad-digestive gland complex in L. grandis.

In bivalve mollusks, the color of the gonad is considered a characteristic of sexual differentiation; frequently, female gonads are orange, while male gonads are creamy white (Mikhailov et al. 1995Mikhailov AT, Torrado M, Méndez J (1995) Sexual differentiation of reproductive tissue in bivalve molluscs: identification of male associated polypeptide in the mantle of Mytilus galloprovincialis Lmk. The International Journal of Developmental Biology 39: 545-548.). However, there are species in which the gonads are of the same color. For instance, Meléndez-Galicia et al. (2015Meléndez-Galicia C, Estrada-Navarrete FD, Hernández-Covarrubias V, Arellano-Torres AA, Hernández-Montaño D (2015) Madurez gonádica del ostión de roca Crassostrea iridescens, de la costa de Michoacán, México. Ciencia Pesquera 23: 25-36.) and Álvarez-Dagnino et al. (2017Álvarez-Dagnino E, Santamaría-Miranda A, García-Ulloa M, Góngora-Gómez AM (2017) Reproduction of Megapitaria squalida (Bivalvia: Veneridae) in the Southeast Gulf of California, Mexico. Revista de Biología Tropical 65: 881-889. https://doi: 10.15517/rbt.v65i3.26371
https://doi: 10.15517/rbt.v65i3.26371...
) observed that in the rock oyster [Crassostrea iridescens (Hanley, 1854)] and the callista clam [Megapitaria squalida (G. B. Sowerby I, 1835)], respectively, gonads between sexes were of the same color throughout their reproductive cycle. In the present study, the gonads of L. grandis presented sexual dichromatism when gametogenesis occurs and the color tonality intensified as the gonad matured, similar to what has been observed in other bivalve species (Morriconi et al. 2002Morriconi E, BJ Lomovasky, Calvo J, Brey T (2002) The reproductive cycle of Eurhomalea exalbida (Chemnitz, 1795) (Bivalvia: Veneridae) in Ushuaia Bay (50°50’S), Beagle Channel (Argentina). Invertebrate Reproduction and Development. 42: 61-68., Aragón-Noriega et al. 2007Aragón-Noriega EA, Chávez-Villalba J, Gribben PE, Alcántara-Razo E, Maeda-Martínez AN, Arámbula-Pujol EM, García-Juárez AR, Maldonado-Amparo R (2007) Morphometric relationships, gametogenic development and spawning of the geoduck clam Panopea globosa (Bivalvia: Hiatellidae) in the Central Gulf of California. Journal of Shellfish Research 26(2): 423-431. http://hdl.handle.net/10453/573, Góngora-Gómez et al. 2016Góngora-Gómez AM, García-Ulloa M, Arellano-Martínez M, Abad-Rosales S, Domínguez A, Puente-Palafox J (2016) Annual reproductive cycle and growth of the pen shell Atrina maura (Pterioidea: Pinnidae) on sand-bottom culture in the Ensenada Pabellones lagoon system, Gulf of California, Mexico. Invertebrate Reproduction & Development 60: 28-38. https://doi.org/10.1080/07924259.2015.1126535
https://doi.org/10.1080/07924259.2015.11...
). The macroscopic dissection in L. grandis was not enough to distinguish their sex when the gonad is not mature, mainly due to the gonadal tissue being very small and difficult to examine with a naked eye. Histological analysis was necessary to adequately describe the sex of L. grandis, as well as in other clams (Cruz 1987bCruz RA (1987b) Tamaño y madurez sexual en Anadara grandis (Pelecypoda: Arcidae). Brenesia 27: 9-12., Juhel et al. 2003Juhel G, Culloty SC, O’Riordan RM, Connor JO, de Faoite L, McNamara RA (2003) Histological study of the gametogenic cycle of the freshwater mussel Dreissena polymorpha (Pallas, 1771) in Lough Derg, Ireland. Journal Molluscan Studies 69: 365-373. https://doi.org/10.1093/mollus/69.4.365
https://doi.org/10.1093/mollus/69.4.365...
, Lucero-Rincón et al. 2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
), and the development of the gonads.

Five gonadal phases were differentiated in females and males of L. grandis (development, mature, spawning, post-spawning and resting). The resting phase was not previously described by Cruz (1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8.), but in a complementary work, Cruz (1987bCruz RA (1987b) Tamaño y madurez sexual en Anadara grandis (Pelecypoda: Arcidae). Brenesia 27: 9-12.) mentioned one finding of an individual that could have been in the resting phase since it was different from the immature ones, but the tissue characteristics of both phases were not described. Meanwhile, Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) grouped rest and spent gonads in one phase and described them as inactive and undifferentiated, but tissue details were not documented because his work was focused on other objectives. Immature individuals were not found in the sample of the present work. The smallest organism was 44.57 mm in length, and according with Cruz (1987bCruz RA (1987b) Tamaño y madurez sexual en Anadara grandis (Pelecypoda: Arcidae). Brenesia 27: 9-12.), the immature specimens are smaller than that, between 16.5 and 20 mm in length. Unlike the contributions mentioned above, this work describes the development process in only one phase, due to an overlap between the presence of previtellogenic and vitellogenic oocytes. And while Cruz (1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8.) documented the maximum maturity phase and grouped attributes of the spawning and post-spawning process in one phase (spent), Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) documented the mature, spawning and post-spawning phases, similar to the present work, but without describing the tissue details and only mentioning general features.

Despite the differences outlined above, the gonadal phases in L. grandis described by the present work are similar to the observations of Cruz (1987aCruz RA (1987a) The reproductive cycle of the mangrove cockle Anadara grandis (Bivalvia: Arcidae) in Costa Rica. Brenesia 27: 1-8.) and Aguirre-Rubí (2017Aguirre-Rubí JR (2017) Contribution to the development of biomonitoring programmes for the assessment of chemical pollution and ecosystem health disturbance in mangrove-line caribbean coastal systems using bivalves as biomonitors and sentinels. PhD Thesis, Universidad del País Vasco, España, https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
) for the species. The difference is that the present work gives more detailed descriptions and uses slightly different nomenclature (Table 2). Additionally, the findings of this work are consistent with the descriptions of the gonadal phases of other species of Arcidae such as A. tuberculosa (Cruz 1984Cruz RA (1984) Algunos aspectos de la reproducción en Anadara tuberculosa (Pelecypoda: Arcidae) de Punta Arenas, Puntarenas, Costa Rica. Revista de Biología Tropical 32: 45-50., Pérez-Medina 2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
, García-Domínguez et al. 2008García-Domínguez FA, de Haro-Hernández A, García-Cuellar A, Villalejo-Fuerte M, Rodríguez-Astudillo S (2008) Ciclo reproductivo de Anadara tuberculosa (Sowerby, 1833) (Arcidae) en Bahía Magdalena, México. Revista de Biología Marina y Oceanografía 43:143-152. https://doi.org/10.4067/S0718-19572008000100015
https://doi.org/10.4067/S0718-1957200800...
, Lucero-Rincón et al. 2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
), A. antiquata and A. granosa (Jahangir et al. 2014Jahangir S, Siddiqui G, Ayub Z (2014) Temporal variation in the reproductive pattern of blood cockle Anadara antiquata from Pakistan (northern Arabian Sea). Turkish Journal of Zoology 38: 263-272. https://doi.org/10.3906/zoo-1302-7
https://doi.org/10.3906/zoo-1302-7...
), A. similis (Manjarrés-Villamil et al. 2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.) and A. noae (Ghribi et al. 2017Ghribi F, Bello G, Zupa R, Passantino L, Santamaria N, El Cafsi M, Corriero A (2017) Reproductive and tissue plasticity in Arca noae (Bivalvia: Arcidae). The European Zoological Journal 84: 473-487. https://doi.org/10.1080/24750263.2017.1368725
https://doi.org/10.1080/24750263.2017.13...
). It is important to note that there are different classifications of the gonadal phases even for the same species (Table 2), mainly due to the criteria applied by each researcher.

Table 2
Gonadal phases defined in different Arcidae species.

During the post-spawning phase and the onset of the resting phase of L. grandis (sub-phase 1), the presence of brown cells was noticeable, and their amount was reduced towards the end of the resting phase (sub-phases 2 and 3). Also, brown cells have been observed in the acini of ripe and partially spawned gonads of A. noae (Ghribi et al. 2017Ghribi F, Bello G, Zupa R, Passantino L, Santamaria N, El Cafsi M, Corriero A (2017) Reproductive and tissue plasticity in Arca noae (Bivalvia: Arcidae). The European Zoological Journal 84: 473-487. https://doi.org/10.1080/24750263.2017.1368725
https://doi.org/10.1080/24750263.2017.13...
), in ripe and spawning gonads of A. tuberculosa (Lucero-Rincón et al. 2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
) and in spawning and post-spawning gonads of A. similis (Manjarrés-Villamil et al. 2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.). These cells were named as brown secretion granules in A. tuberculosa and A. similis (Lucero-Rincón et al. 2013Lucero-Rincón CH, Cantera JR, Gil-Agudelo DL, Muñoz O, Zapata LA, Cortes N, Gualteros WO, Manjarres A (2013) Análisis espacio temporal de la biología reproductiva y el reclutamiento del molusco bivalvo Anadara tuberculosa en la costa del Pacífico colombiano. Revista de Biología Marina y Oceanografía 48: 312-334. https://doi.org/10.4067/S0718-19572013000200011
https://doi.org/10.4067/S0718-1957201300...
, Manjarrés-Villamil et al. 2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.). In mollusks, brown cells (also known as rhogocytes) are characterized by phagocytosis (endocytosis), role in metal ion metabolism, transport or storage of nutrients, synthesis or breakdown of respiratory pigments, supportive cell, selective reabsorption, and others (reviewed by Haszprunar 1996Haszprunar G (1996) The molluscan rhogocyte (pore-cell, blasenzelle, cellule nucale), and its significance for ideas on nephridial evolution. Journal of Molluscan Studies 62: 185-211.). In L. grandis, their presence and abundance after spawning suggests that brown cells are involved in the reabsorption of remnants during gonad recovery (post-spawning phase), and after the gonad is recovered, the abundance of brown cells decreases (resting phase).

Different stages of sex cells were distinguished in the same acini of male gonads of L. grandis (spermatogonia, spermatocytes, spermatids and spermatozoa), as reported by Ma et al. (2017Ma X Ji A, Zhang Z, Yang D, Liang S, Wang Y, Qin Z (2017). Piwi1 is essential for gametogenesis in mollusk Chalmys farreri. Journal of Life and Environmental Sciences 5: e3412. https://doi.org/10.7717/peerj.3412
https://doi.org/10.7717/peerj.3412...
) for the scallop Chlamys farreri (Johnes & Preston, 1904) and by Chatchavalvanich et al. (2006Chatchavalvanich K, Jindamongkon P, Kovitvadhi U, Thongpan A, Kovitvadhi S (2006) Histological structure of gonads in the fresh water pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900. Invertebrate Reproduction & Development 49: 245-253. https://doi.org/10.1080/07924259.2006.9652214
https://doi.org/10.1080/07924259.2006.96...
) for the freshwater mussel Hyriopsis bialatus Simpson, 1900. This asynchronous gametogenesis in males of L. grandis occurs because different cohorts of gametes that are forming occur simultaneously, and this indicates that spermatozoa production is continuous during the development and maturity of the gonadal phases in males. In contrast, female gonads presented synchronic gametogenesis due to the presence of one or two gametogenic cell stages in a single acinus, suggesting that oocyte production is discrete, and therefore it takes time for the next cohort to be ready to be released.

Since partially or totally empty acinus during the spawning phase of L. grandis were observed, and in some cases gamete-filled acinus, we suggest that this clam spawns in batches, a condition also reported by Pérez-Medina (2005Pérez-Medina BP (2005) Biología reproductiva de Anadara tuberculosa (Bivalvia: Arcidae) en el estero Santo Domingo, B.C.S., México. Master Thesis, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California Sur, México. Available online at: https://addi.ehu.es/handle/10810/29964?show=full
https://addi.ehu.es/handle/10810/29964?s...
), Manjarrés-Villamil et al. (2013Manjarrés-Villamil AL, Lucero-Rincón CH, Gualteros WO, Cantera-Kintz JR, Gil-Agudelo DL (2013) Abundancia y madurez sexual de Anadara similis en el manglar de Luisico, Bahía Málaga, Pacífico Colombiano. Boletín de Investigaciones Marinas y Costeras 42: 215-231.) and Hernández-Hernández (2014Hernández-Hernández G (2014) Aspectos reproductivos de la almeja pata de mula Anadara multicostata (Sowerby, 1833) en la laguna de Guerrero Negro, B.C.S., México. Tesis de Licenciatura. Departamento Académico de Biología Marina, Universidad Autónoma de Baja California Sur, México.) for A. tuberculosa, A. similis and A. multicostata, respectively.

ACKNOWLEDGEMENTS

The authors thank Instituto Politécnico Nacional for the logistical and economic support provided for the development of this research through the projects: “Morfometría y fisiología de la almeja venus Chione undatella de Bahía La Paz, y el puerto minero de Santa Rosalía, Baja California Sur, México (IPNSIP 20195442) and “Incidencia estacional de nematodos y Perkinsus marinus de la almeja venus Chione undatella de Bahía La Paz y del puerto minero de Santa Rosalía, Baja California Sur, México (IPNSIP 20190071). Also, we are thankful for the support of project PRODEP “Forzamiento ambiental en especies clave de los ecosistemas acuáticos” (Convenio DSA 511-6/17-7679 Folio UAS-PTC-131). Special acknowledgements for the laboratories and other support received from the Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional of the Instituto Politécnico Nacional and the Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa. M. I. Sotelo-Gonzalez thanks to CONACYT (Grant 714818). Special thanks to Jorge Manuel Elenes Lizarraga for the English language review.

LITERATURE CITED

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Publication Notes

  • Available online:

    March 9, 2021
  • Zoobank Register:

    http://zoobank.org/85853685-58E7-4323-9EF8-6A9BCE1B8220
  • Publisher:

    © 2021 Sociedade Brasileira de Zoologia. Published by Pensoft Publishers at https://zoologia.pensoft.net

Edited by

Editorial responsibility:

Carlos Eduardo Belz

Publication Dates

  • Publication in this collection
    15 Mar 2021
  • Date of issue
    2021

History

  • Received
    15 July 2020
  • Accepted
    23 Nov 2020
  • Published
    09 Mar 2021
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