Histopathological Alterations in the Gonad of ...

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Journal of Aquatic Animal Health 0:1–11, 2018 © American Fisheries Society 2018 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1002/aah.10015

ARTICLE

Histopathological Alterations in the Gonad of Megapitaria squalida (Mollusca: Bivalvia) Inhabiting a Heavy Metals Polluted Environment 1

Josué Alonso Yee-Duarte, Bertha Patricia Ceballos-Vázquez, and Marcial Arellano-Martınez* Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas, Av. Instituto Politécnico Nacional s/n Col. Playa Palo de Santa Rita. C.P. 23096, La Paz, Baja California Sur, México

Marian Alejandra Camacho-Mondrag on Pesquera La Paz, Carretera a Pichilingue Instituto Nacional de Pesca y Acuacultura,Centro Regional de Investigacion Km 1, Col. Esterito. C.P. 23020 La Paz, Baja California Sur, México

Esther Urıa-Galicia 2

Instituto Politécnico Nacional,Escuela Nacional de Ciencias Biologicas, Unidad Profesional Lázaro Cárdenas, Manuel Carpio y Plan de Ayala s/n, Col. Santo Tomás. C.P. 11340 Delegacion Miguel Hidalgo, Prolongacion Ciudad de México, México

Abstract

The gonadal health status of the chocolate clam Megapitaria squalida collected from the Santa Rosalıa mining port and San Lucas beach (reference site), Gulf of California, Mexico, was assessed through histological analysis of the reproductive tissue, from which the histopathological alteration index (HAI) was determined. In addition, copper and iron accumulation in tissue was revealed using histochemical techniques. Our results showed a large presence of copper (30%) and iron (45%) only in the gonad tissue of clams from Santa Rosalıa, in which histopathological alterations observed were inflammatory responses, degenerative–progressive processes, cell death, and response to infectious agents. The HAI was significantly higher in Santa Rosalıa specimens (mean SE, 72.18 6.12) than in San Lucas clams (4.60 1.07). At San Lucas beach, a higher prevalence of histopathological alterations occurred in clams in the spent stage (43.2%) and in autumn (18.4%) and winter (17.8%) in concordance with the normal reproductive rest period, whereas at Santa Rosalıa a higher prevalence occurred in clams at the ripe stage (76.9%) and in spring (83.7%). In conclusion, our results showed the deteriorated health condition of gonads in M. squalida from the Santa Rosalıa mining port, which suggests there is a relationship of chronic exposure to local high levels of heavy metals. The high prevalence and intensity of histopathological alterations in the gonad suggest a strong adverse effect on gametogenesis, gamete quality, and, ultimately, in the reproductive potential of M. squalida at this site.

Marine coastal areas are the main zones of biological productivity, but are also the most severely affected zones due to the presence of pollutants derived from anthropogenic activities that impact the health of the local biota, hence raising significant environmental concerns (Ruiz

et al. 2011; Sheir et al. 2013). In Mexico, many coastal ecosystems affected by anthropogenic activities show signs of environmental degradation (Garcıa-Gasca et al. 2010). Several studies have shown that the marine surface sediments in the coastal area off the Santa Rosalıa mining

*Corresponding author: [email protected] Received June 16, 2017; accepted January 15, 2018 Published online xxx xx, 2017

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Dispatch: 12.2.18

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No. of pages: 11

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port, located on the east-central coast of the Gulf of California, Mexico, are highly contaminated with potentially toxic elements derived from mining and smelting activities that have been ongoing for more than a century (Shumilin et al. 2000; Huerta-Diaz et al. 2014). These activities have produced approximately 370 million tonnes of solid waste, which have been disposed on land and in the adjacent sea (Shumilin et al. 2011). As a result, coastal marine sediments contain abnormally high levels of heavy metals such as Fe (102,400 mg/kg), Cu (3,390 mg/kg), Zn (1,916 mg/kg), Co (166 mg/kg), Mn (6,770 mg/kg), Pb (226 mg/kg), and U (11.8 mg/kg) (Shumilin et al. 2011, 2013). In filter-feeding bivalves, chronic exposure to high heavy metal concentrations in water and sediments is typically associated with the impairment of physiological functions leading to a deterioration in health (Paul-Pont et al. 2010; Sacchi et al. 2013). Adverse effects may result either from indirect toxicity on homeostatic mechanisms, such as metabolism and immune response, or from alterations to specific tissues, which lead to an increased susceptibility to pathogens and the development of histopathological alterations (Pipe et al. 1999; Sheir et al. 2013). In particular, the deterioration of gonadal tissue and gamete viability is one of the most devastating consequences of marine pollution, as it can reduce the reproductive success and wellbeing of organisms (Vaschenko et al. 2013). In this sense, histopathological analysis has become a core tool in environmental toxicology and is regarded as one of the most representative approaches to assess the effects of pollution in various biomonitoring programs around the world (Costa et al. 2013; Cuevas et al. 2015). Despite the clear evidence of heavy metal pollution in the Santa Rosalıa coastal area, little is known about its effect on the local biota. Previous work revealed that the chocolate clam Megapitaria squalida showed a deteriorated physiological condition with small shell sizes and a negative allometric growth (Yee-Duarte et al. in press). In this context, this study conducted a comparative assessment of the health status of gonadal tissue in the chocolate clam from the Santa Rosalıa mining port and a nearby area that had neither mining nor port activities.

METHODS Sample collection.— Specimens of M. squalida were collected monthly between May 2012 and April 2013 in the “hot spot” area of the Santa Rosalıa mining port (27°200 N, 112°160 W), located within the Gulf of California, on the east-central coast of the Baja California peninsula, Mexico. At the same time, samples of clams were collected off San Lucas beach located 13 km south of Santa Rosalıa (27°130 N, 112°160 W); this site was used as reference site because there are neither mining nor port activities, and M. squalida has been reported to show a

good health status in this area (Yee-Duarte et al. in press). The weight and shell length of each clam were measured. A portion of the gonad was obtained from each specimen and fixed in a 10% solution of formaldehyde prepared with seawater. Histopathological analysis.— Each portion of gonadal tissue was dehydrated through a sequence of alcohols of increasing concentrations, then cleared with Hemo-De and embedded in Paraplast-Xtra. Using a microtome, 5-μmthick sections were obtained and stained with Harris hematoxylin and eosin stain (Humason 1979). In addition, 16 individual clams were randomly selected from each study area to collect gonad portions for staining with Perls’ Prussian Blue and Mallory’s hematein (Howard et al. 2004) to detect the presence of iron and copper in the tissue. These metals are found in high concentrations in sediments of Santa Rosalıa and are most commonly analyzed from the tissue of bivalve molluscs through conventional histochemistry. The microscopic anatomy of gonadal tissue was examined under a light microscope and, based on the observations, any alterations found were described according to Costa et al. (2013) and Cuevas et al. (2015). The prevalence of each histopathological alteration was calculated as the ratio between the number of affected individuals and the total number of individuals analyzed in each study area (Bush et al. 1997). Prevalence was compared between sexes, sampling site, stage of gonadal development, and season of the year. Histopathological alterations were categorized by severity level: (1) mild, (2) moderate, and (3) severe, as proposed by Costa et al. (2013) and Cuevas et al. (2015) for bivalve molluscs. Mild alterations involve changes that do not damage the tissues to any significant extent (e.g., brown cells, hemocytic infiltration, inflammatory responses). Moderate alterations involve changes that are more severe and lead to effects in tissues associated with organic functions (e.g., fibrosis, granulocytomas). Last, severe alterations involve irreversible tissue damage (e.g., severe atresia, loss of epithelium, necrosis, parasites, neoplasm). The health status of gonads was measured semiquantitatively by means of the histopathological alteration index (HAI), which is based on the severity of lesions (Poleksic and Mitrovic-Tutundzic 1994). The HAI was calculated for each individual as follows: HAI = (1 × SI) + (10 × SII) + (100 × SIII), where S represents the sum of the number of alterations for each severity level, and I, II, and III correspond to the number of alterations in severity categories 1 (mild), 2 (moderate), and 3 (severe), respectively. Values of HAI between 1 and 10 indicate a normal tissue function; between 11 and 20, mild alterations; between 21 and 50, moderate alterations; between 50 and 100, severe alterations; and values above 100 indicate

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GONAD HISTOPATHOLOGICAL ALTERATIONS IN MEGAPITARIA SQUALIDA

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irreversible tissue damage (Poleksic and MitrovicTutundzic 1994). In addition, the intensity of each alteration in both females and males was determined. To this end, two fields from each slide were taken at random (10 × ) using the Sigma Scan Pro software (version 5.0, Systat Software); the total area of gonadal tissue and the area of each alteration were measured. Coverage areas were calculated as the ratio between the area of the alteration and the total area of gonadal tissue and expressed as percent. The percent values of coverage areas calculated were categorized and, according to the modification of the scale proposed by Bacchetta and Mantecca (2009), the following levels were identified: 0 = normal structure of follicles and oocytes, light intensity; 1 = follicles with normal oocytes, 50% of the tissue. As a measure of iron and copper accumulation in gonadal tissue stained with Perls’ Prussian Blue or Mallory’s hematein, respectively, the area occupied by the metals was measured relative to total tissue area (%) in the 16 specimens selected for histochemistry (two random fields per specimen). Based on the scale proposed by Dove and Sammut (2007)—minor, moderate, and extensive—and on the calculation of the percentage of area occupied by the metal, an arbitrary value was assigned to each category; minor accumulation represents 35% of area occupied. Statistical analyses.— Comparative analyses of the percentage of area covered by iron and copper, HAI, and intensity levels of histopathological alterations were conducted using Kruskal–Wallis nonparametric tests followed by Duncan’s multiple-range comparison tests (Zar 1996). The percent coverage and HAI were compared between zones, while intensity levels were compared between each of the alterations. Differences in sex ratios were tested using a χ2 test (Zar 1996). The statistical analyses were performed using the software STATISTICA (StatSoft), version 6.0 for Windows. A significance level (α) of 0.05 was set for all tests.

RESULTS

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Histological Analysis of Metal Accumulation The microscopic analysis of the gonad of M. squalida from both study areas revealed the presence of iron and copper granules in the interfollicular connective tissue and, in some cases within follicles, they occupied the same space as gametes (Figure 1). These granules were positively stained in blue for iron and from dark red to black for copper. The accumulations in percentage of area of

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iron (v12 = 21.90, P = 0.0000) and copper (v12 = 7.44, P = 0.0064) (Table 1) were significantly higher in gonads of chocolate clams from Santa Rosalıa (Figure 1A, C, E, G) (moderate to extensive) than in those from San Lucas, where mainly a minor accumulation was observed (Figure 1B, D, F, H). Histopathological Alterations A total of 696 chocolate clams were analyzed: 370 (182 females and 188 males) from the Santa Rosalıa mining port and 326 (159 females and 167 males) from San Lucas beach. Morphometric variables of M. squalida and sex ratios by study site are shown in Table 2. The microscopic analysis of gonadal tissue of M. squalida from Santa Rosalıa revealed the presence of 10 histopathological alterations in the ovary and four in the testis. Specimens from San Lucas showed two alterations in the ovary and one in the testis. The histopathological alterations observed in the ovary of clams are shown in Figure 2. There was a high abundance of brown cells and atretic oocytes (Figure 2A). Brown cells were observed within the interfollicular connective tissue and, in some cases, inside follicles. Atretic oocytes were observed in different gametogenic development stages (mainly in postvitellogenic oocytes) and were deformed with a loss of structural integrity and degenerated cytoplasmic material. Numerous vacuoles in the cytoplasm and nuclei were also observed (Figure 2B), along with a loss of the nuclear chromatin in some postvitellogenic oocytes (Figure 2C), pyknosis characterized by condensation and basophilia of the nuclear chromatin (Figure 2D), and hypertrophied oocytes (Figure 2E), which are characterized by a large size (mean SE, 150.53 31.78 μm; n = 15) and, in some cases, occupied almost half of the follicular space attached to the follicle wall. Despite having the characteristics of a mature gamete cell, some oocytes were attached to the follicle wall by a long filamentary peduncle (82.73 7.59 μm, n = 15) (Figure 2F); in most cases, there were signs of oocyte degeneration. Recurring hemocytic infiltration in the connective tissue was also observed and, when it was very intense, granulocytomas were evident (Figure 2G). Some of these granulocytomas surrounded unidentified parasitic structures or oval to spherical inclusion bodies with dense staining margins, which are caused likely by bacterial colonies (Figure 2H) in the interfollicular connective tissue of the ovary. The histopathological alterations observed in the testis of clams from Santa Rosalıa are shown in Figure 3. As in the ovaries, testes showed a high abundance of brown cells within the interfollicular connective tissue, occasionally forming dense masses (Figure 3A). Hemocytic infiltration in the connective tissue was also observed, as well as granulocytomas in some cases (Figure 3B), which were

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encapsulating unidentified parasitic structures and some inclusion bodies formed by protein aggregates usually associated with bacterial colonies (Figure 3C). These bodies were observed in the connective tissue and within follicles, displacing sperm cells (Figure 3D). Prevalence The prevalence of each histopathological alteration by study site and sex are shown in Table 3. At Santa Rosalıa, the most prevalent alterations in female clams were brown cells (70.3%) and atretic oocytes (58.2%), while the least frequent alterations were filamentary peduncles (10.1%) and granulocytomas (6.4%). In male clams, a high prevalence of inclusion bodies (42%) and brown cells (41.5%) were

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observed, followed by hemocytic infiltration (28.7%) and granulocytomas (17%). In San Lucas beach samples, females only showed a low prevalence of atretic oocytes (14.5%) and hemocytic infiltration (10.7%), while males showed exclusively a low prevalence of hemocytic infiltration (10.2%). The total prevalence of histopathological alterations by sex, gonad developmental stages, and season are shown in Table 4. In female clams the prevalence was higher than in males at both sites. In Santa Rosalıa the higher prevalence occurred in the ripe stage (76.9%); in contrast, in San Lucas the higher prevalence occurred in the spent stage (43.2%). On the other hand, in Santa Rosalıa the prevalence of alterations was higher in spring (83.7%) and decreased concomitantly during the following seasons, to

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

FIGURE 1. Iron and copper granules in the gonad of Megapitaria squalida from (A, C, E, G) Santa Rosalıa and (B, D, F, H) San Lucas. Iron granules (arrows) are indicated in the testis (A and B) and ovary (E and F) (Perls’ Prussian Blue stain), and copper granules (arrows) are indicated in the testis (C and D) and ovary (G and H) (Mallory’s hematein stain). Scale bar: 50 μm.

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TABLE 1. Percentage of area occupied by copper and iron in the gonad of the 16 specimens of Megapitaria squalida selected for histochemistry from Santa Rosalıa and San Lucas Beach. Accumulation categories: *minor, **moderate, ***extensive.

Gonad area occupied (%) Specimen number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean SE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean SE

Santa Rosalıa Iron 22.04** 30.66** 40.54*** 41.49*** 41.89*** 42.67*** 43.61*** 45.57*** 45.76*** 46.11*** 46.46*** 49.99*** 51.72*** 56.48*** 57.53*** 57.70*** 45.01 2.33 Copper 12.36** 13.51** 14.11** 16.73** 17.60** 17.67** 22.48** 24.30** 24.58** 31.15** 31.82** 32.08** 38.26*** 50.06*** 52.03*** 61.39*** 28.75 3.74

San Lucas Beach 0.61* 1.13* 1.14* 1.47* 3.58* 4.00* 4.35* 4.54* 4.64* 5.16* 6.39* 7.74* 7.90* 10.46** 10.84** 14.34** 5.51 0.97 1.24* 1.34* 1.39* 1.49* 1.59* 1.65* 1.65* 2.45* 3.12* 3.45* 4.56* 5.09* 7.32* 9.28* 10.02** 10.57** 4.13 0.83

a minimum in winter (58.8%). In contrast, in clams from San Lucas beach, the prevalence was lowest in spring (3.2%) and highest in autumn and winter (18.4% and 17.8%, respectively). The HAI and Intensity Levels of Histopathological Alterations The HAI was significantly higher (v12 = 407.74, P = 0.000) in Santa Rosalıa specimens (72.18 6.12)

TABLE 2. Morphometric variables (mean SE; range) and sex ratios (no significant different from 1:1) of Megapitaria squalida from Santa Rosalıa and San Lucas Beach. N = sample size, M = male, F = female.

Variable Length (cm) Total weight (g) Sex ratio

Santa Rosalıa N = 370

San Lucas Beach N = 326

6.8 0.04 2.3–8.6 87.9 1.50 6.6–168 1.03M : 1F (χ2 = 0.04, P = 0.84)

7.7 0.05 5.5–12.5 140.3 3.36 38.7–480.4 0.80M : 1F (χ2 = 1.26, P = 0.26)

than in San Lucas beach clams (4.60 1.07). The intensity levels of each histopathological alteration in the ovary and testis of M. squalida from Santa Rosalıa are shown in Figure 4. Significant differences between the intensity levels (v29 = 104.19, P = 0.0000) of each histopathological alteration were observed, but only in ovaries. The pathologies observed with the highest intensity were atretic oocytes (2.48 0.10), followed by brown cells (2.34 0.07) and hemocytic infiltration (2.05 0.09). Pyknosis (1.37 0.25) and cytoplasmic and nuclear vacuolation (1.22 0.23) showed moderate intensity values, while granulocytomas (0.75 0.19), inclusion bodies (0.68 0.31), hypertrophied oocytes (0.24 0.08), loss of nuclear chromatin (0.45 0.18), and filamentary peduncles (0.12 0.08) were the pathologies with the lowest intensities. In the testis, no significant differences were observed between the intensity levels of the alterations (v23 = 6.6764, P = 0.0830). However, all the intensities occurred at high levels and were between 1.91 and 2.37.

DISCUSSION In this study, the high prevalence and intensity of histopathological alterations in the gonad of M. squalida from Santa Rosalıa mining port, as well as the absence of most of these changes in clams from San Lucas beach, suggest that tissue damage is correlated with the high concentrations of heavy metals previously reported in sediments from this site. Mu~ noz-Barbosa and Huerta-Dıaz (2013) reported that the concentrations of metals such as Cu, Pb, Mn, Co, and Zn at Santa Rosalıa are up to 10,000 times higher than at San Lucas beach (reference site 13 km away from Santa Rosalıa) and other parallel coastal areas within the Gulf of California. In addition, these heavy metal concentrations significantly exceed the reference values for natural levels in the earth’s crust and the ecotoxicological quality guidelines, suggesting they have a high possibility of being a toxicological hazard to marine biota (Shumilin et al. 2000, 2013). In the Santa

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FIGURE 2. Histopathological alterations in the ovary of Megapitaria squalida from Santa Rosalıa. (A) Presence of atretic oocytes undergoing degeneration. Arrows indicate high intensity of brown cells in the interfollicular connective tissue. (B) Numerous vacuoles in the cytoplasm and nuclei of oocytes. The box at the top left of the panel indicates an oocyte with condensed nuclear chromatin attached to the germinal portion of the follicle. (C) Loss of the nuclear chromatin (arrows) of some post-vitellogenic oocytes. (D) Presence of postvitellogenic oocytes with a pycnotic nucleus. (E) Hypertrophied oocyte attached to the germinal wall of the follicle. Note the difference in size between this and other oocytes in the follicle. (F) Oocytes attached to the follicular wall through a long and filamentary peduncle (arrows). The box at the top left of the panel indicates an oocyte with a long peduncle undergoing degeneration. (G) Intense hemocytic infiltration in the connective tissue. The dotted line indicates the formation of a granulocytoma surrounding an unidentified parasitic structure. (H) Granulocytoma surrounding an inclusion body (with dense staining margin) within the ovarian connective tissue. The box at the top right of the panel indicates the presence of an inclusion body (identified as suspected bacterial colony) surrounded by brown cells. Hematoxylin and eosin stain; scale bar: 50 μm.

Rosalıa area, Cu and Fe concentrations have only been reported in a few organisms, but invariably in high concentrations (Table 5). In this sense, the histochemical analysis in the present study revealed a higher accumulation of Cu and Fe granules (coverage area seven times larger) in chocolate clams from Santa Rosalıa than in those from San Lucas beach. This finding is in accord with the report of whole-body metal levels for M. squalida at two sites in Santa Rosalıa with different degrees of contamination; at

our collection site Cu was 14.6 1.8 mg/kg, which is in contrast with 9.5 0.9 mg/kg from a less contaminated site (Roldán-Wong et al. in press) that is similar to San Lucas beach. In fact, the mussels, Modiolus capax and Mytilus edulis, from Santa Rosalıa contain 11-fold higher concentrations of Cu and almost twofold higher levels of Fe than do mussels from other areas (Gutiérrez-Galindo et al. 1999; Cadena-Cárdenas et al. 2009; Mu~ noz-Barbosa and Huerta-Dıaz 2013).

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FIGURE 3. Histopathological alterations in the testis of Megapitaria squalida from Santa Rosalıa. (A) Accumulations of brown cells within interfollicular connective tissue. Note the presence of inclusion bodies (arrows; identified as suspected bacterial colonies) within the testis connective tissue. (B) Intense hemocytic infiltration in the testis connective tissue. The area enclosed by the dotted line indicates the formation of a granulocytoma surrounding an unidentified parasitic structure. (C) Inclusion body with dense-staining margin (arrow) in the interfollicular connective tissue surrounded by a granulocytoma. (D) Inclusion bodies within the follicle (area enclosed by dotted line) occupying the place of sperm cells. Hematoxylin and eosin stain; Scale bar: 50 μm.

TABLE 3. Prevalences (%) of histopathological alterations of the gonad (ovary and testis) of Megapitaria squalida from Santa Rosalıa and San Lucas Beach.

Prevalence (%) Histopathological alterations

Santa Rosalıa

San Lucas Beach

58.2 70.3 32.4 10.1 6.4 35.1 14.3 18.1 22.5 16.5

14.5 0 0 0 0 10.7 0 0 0 0

41.5 17.0 28.7 42.0

0 0 10.2 0

Ovary Atretic oocytes Brown cells Vacuolation Filamentary peduncule Granulocytoma Hemocytic infiltration Hypertrophied oocytes Loss of nuclear chromatin Inclusion bodies Pyknosis

TABLE 4. Total prevalence (%) of histopathological alterations in Megapitaria squalida by sex, gonad developmental stages, and season Santa Rosalıa and San Lucas Beach.

Prevalence (%) Variable Female Male Resting Developing Ripe Spawning Spent

Testis Brown cells Granulocytoma Hemocytic infiltration Inclusion bodies

Spring Summer Autumn Winter

Santa Rosalıa

San Lucas Beach

Sex 74.7 68.6 Gonad development stage 24.3 56 76.9 53.4 35 Season 83.7 77.2 76.3 58.8

15.7 12.5 0 0 5.7 17.8 43.2 3.2 9.1 18.4 17.8

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FIGURE 4. Intensity levels of each of the histopathological alterations in the ovary and testis of Megapitaria squalida from Santa Rosalıa. Mean values are significantly different for females only and are indicated by different letters. Error bars indicate SE.

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TABLE 5. Concentration (mean SD) of copper and iron previously reported in different organisms from Santa Rosalıa. Data are whole-body measurements except for octopus (mean and range).

Organism Megapitaria squalida Mytilus edulis Modiolus capax Modiolus capax Octopus hubbsorum Muscle Digestive gland Branchial hearts Gills Padina durvillaei

Cu (mg/kg) 14.6 1.8 49.6 9.8 89 (mean only) 58.7 2.3

Fe (mg/kg) Bivalve 193 5 369 125 No data 485 28 Octopus

Reference Roldán-Wong et al. (in press) Cadena-Cárdenas et al. (2009) Gutiérrez-Galindo et al.(1999) Mu~ noz-Barbosa and Huerta-Dıaz (2013) Roldán-Wong et al. (in press)

25 (6–37) 3,296 (726–5,880) 103 (26–166) 136 (84–187) 2,243 2,325

10 (1–20) 314 (162–644) 1,028 (156–5,340) 91 (16–529) Algae 53 38

Although copper and iron are important elements in the cellular composition of tissues and are essential cofactors for numerous enzymes, both can be toxic at levels above those required for the normal metabolic function (Soto et al. 1990; Gomes et al. 2011). Specifically, iron at high levels can cause oxidative injuries and abnormalities in iron metabolism, the effects of which may extend to various biological activities, such as growth, morphology (tissue abnormalities), behavior, and reproduction (González et al. 2010; Chandia et al. 2012), and toxicity may be apparent through degenerative tissue manifestations (Chandia et al. 2012). However, the cellular alterations observed in the exposed clams may not all be specifically

Rodrıguez-Figueroa et al. (2009)

due to high levels of copper and iron. Some of these changes could have been due to high levels of other heavy metals previously reported at abnormally high values in the sediments of Santa Rosalıa but were not analyzed in this study. The histopathological alterations observed in M. squalida from Santa Rosalıa are grouped in inflammatory responses (hemocytic infiltration, granulocytomas, brown cells), degenerative and progressive processes (atretic oocytes, vacuolization, hypertrophy, filamentous peduncle, pycnosis), death of cells and tissues (loss of nuclear chromatin), and response to infectious agents (inclusion bodies). Based on our HAI (72.18 6.12), these alterations


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represent serious lesions indicative of an impaired function of the gonadal tissue (Poleksic and Mitrovic-Tutundzic 1994). In contrast, the HAI values in chocolate clams from San Lucas beach (4.60 1.07) indicated that the gonad showed no evidence of serious damage, and therefore displayed a normal gonad function. Among the inflammatory responses, the most prevalent and of greater intensity was the presence of brown cells, followed by hemocytic infiltration and the formation of granulocytomas. Brown cells play a key role in the metabolism of metal ions, and they serve as a first line of defense for metal degradation and detoxification (Zaroogian and Yevich 1994). The presence of brown cells is considered to be an important indicator of stress caused by heavy metal pollution, as reported in Crassostrea angulata (Rodrıguez de la R ua et al. 2005; Vaschenko et al. 2013) and C. virginica (Guzmán-Garcıa et al. 2007). In addition, hemocytic infiltration and the formation of granulocytomas, although related to homeostatic processes (digestion, transport of metabolites, cellular repair, and the immunological activities of recognition, encapsulation, and phagocytosis) (Mayrand et al. 2005), are in some cases related to the effect of pollutants (De Vico and Carella 2012), as observed in Mytilus edulis chronically exposed to heavy metals (Sheir et al. 2013). In this sense, the highest prevalence of hemocytic infiltration in Santa Rosalıa clams and the absence of granulocytomas and brown cells in clams from San Lucas beach suggest that these inflammatory responses in Santa Rosalıa clams is mainly due to pollution, while hemocytic infiltration in clams from San Lucas beach is likely due to a normal clearance process, reabsorption of residual gametes, and postspawning cellular repair (postspawning stage). A high prevalence of degenerative processes in the gonadal tissue of clams from Santa Rosalıa was also found, which contrasts with the low prevalence in clams from San Lucas beach, especially in the case of atretic oocytes. Although the presence of this type of oocyte may be a result of a nutritional deficit, temperature anomalies, the presence of pathogens and/or parasites, and reproductive events (Bayne 1976), this has also been related to a typical response of bivalve molluscs to environmental pollution (Bacchetta and Mantecca 2009; Sheir et al. 2013). In this sense, Lowe and Pipe (1986) reported a high prevalence of atresia in M. edulis caused by the direct effect of pollutants, which led to the destabilization of lysosomes and yolk granules containing lysosomal enzymes. Additional evidence of the effect of pollution on the reproductive capacity of M. squalida in Santa Rosalıa is that in San Lucas beach, the higher prevalence (only hemocytic infiltration and atretic oocytes) occurs in the spent stage and in autumn and winter, in concordance with the normal reproductive rest period, whereas at Santa Rosalıa, the higher prevalence of effects occurs during the

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ripe stage (mainly brown cells and atretic oocytes) and in spring. Therefore, it seems likely that the high prevalence and intensity of atretic oocytes in clams from Santa Rosalıa is a consequence of chronic exposure to heavy metals, while in clams from San Lucas beach this is associated with a normal biological event, probably spawning and postspawning. This idea is supported by the observation that cytoplasmic and nuclei vacuolation, which indicates the onset of a cell degeneration process or autophagic cell death (Carella et al. 2015), occurred in Santa Rosalıa clams only. This alteration is commonly a cell response in aquatic animals suffering toxicity associated with organic and inorganic pollutants (Giri et al. 2000; Najle et al. 2000; Carella et al. 2015), as observed in the annelid, Limnatis nilotica (Khaled et al. 2016), the mussel, Dreissena polymorpha (Bacchetta and Mantecca 2009), and the limpet, Nacella concinna (Najle et al. 2000). Chocolate clams from Santa Rosalıa showed other significant alterations in the cell structure of female gametes in addition to atresia. Mature oocytes that had an abnormally elongated peduncle and were still attached to the follicular wall were observed, as were hypertrophied oocytes with an average diameter four times larger (150.5 μm) than postvitellogenic oocytes of clams from San Lucas beach (35.9 μm) and those reported previously (between 36 and 45.2 μm) for M. squalida from other areas (Arellano-Martınez et al. 2006). Both alterations indicate a pathology characterized by excessive cell growth and a delay in the maturation process—lesions typical of progressive changes (Carella et al. 2015). These hypertrophied oocytes should be distinguished as being different from the common viral gametogenic hypertrophy caused by a papilloma–polyoma virus observed in bivalves, a condition in which no host reaction, such as hemocytic infiltration, has been detected (Choi et al. 2004). Also found were oocytes in which the nuclear chromatin was condensed in a basophilic mass, which indicates imminent pyknosis (Usheva et al. 2006). Frequently, pyknosis is induced by pollutants in various mollusc tissues, as reported in the snail, Bellamya dissimilis, exposed to pesticides (Jonnalagadda and Rao 1996) and in the bivalve, Lamellidens marginalis, exposed to arsenic (Chakraborty and Ray 2009). The inclusion bodies observed in the gonad of M. squalida resembled bacterial colonies reported in a large number of species of bivalve molluscs (Paillard et al. 2004). Although in most cases these infections do not pose major pathological risks, their interaction with stressors (e.g., pollutants) can be synergistically harmful to the population affected (Morley 2010), as observed in M. edulis (Pipe and Coles 1995) and Crassostrea spp. (Kim et al. 1998) exposed to heavy metals. In this study, inclusion bodies do not seem to be associated with obvious damage

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YEE-DUARTE ET AL.

to gonad integrity. However, in the testis, some inclusions displaced sperm cells. In addition, the immune response was evident as the presence of granulocytomas encapsulating some inclusion bodies. In conclusion, our results show the deteriorated gonad health condition in M. squalida from the Santa Rosalıa mining port and suggest there is a correlation of chronic exposure with high local levels of heavy metals. The high prevalence and intensity of histopathological alterations in the gonad suggest a strong adverse effect on gametogenesis, gamete quality, and, ultimately, the reproductive potential of M. squalida from this site. However, to clarify the seasonal dynamics and the relationship to the normal gametogenic cycle, further study is necessary.

ACKNOWLEDGMENTS This research was funded by projects SIP 1535 and 1698, and CONACyT 284289. Josué Alonso Yee-Duarte is a fellow student of BEIFI and CONACyT, the results presented here are part of his Ph.D. thesis. Bertha Patricia Ceballos-Vázquez and Marcial Arellano-Martınez received grants from COFAA, EDI, and SNI-CONACyT. Marian Alejandra Camacho-Mondrag on received grants from SNI-CONACyT. We thank Marıa Elena Sánchez-Salazar for her editorial contribution to the English manuscript.

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