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Follicular apoptosis in the mussel (Mytella strigata) as potential indicator of environmental stress in coastal ecosystems Article in Journal of Environmental Science and Health Part A Toxic/Hazardous Substances & Environmental Engineering · January 2010 DOI: 10.1080/10934520903388806 · Source: PubMed
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Follicular apoptosis in the mussel (Mytella strigata) as potential indicator of environmental stress in coastal ecosystems Alejandra García-Gasca a; Beatriz Leal-Tarin a; Julián Ríos-Sicairos a; Rubí Hernández-Cornejo a; Gabriela Aguilar-Zárate a; Miguel Betancourt-Lozano a a Centro de Investigación en Alimentación y Desarrollo, Unidad Mazatlan, Mazatlan, Mexico Online publication date: 15 December 2009
To cite this Article García-Gasca, Alejandra, Leal-Tarin, Beatriz, Ríos-Sicairos, Julián, Hernández-Cornejo, Rubí, Aguilar-
Zárate, Gabriela and Betancourt-Lozano, Miguel(2010) 'Follicular apoptosis in the mussel (Mytella strigata) as potential indicator of environmental stress in coastal ecosystems', Journal of Environmental Science and Health, Part A, 45: 1, 56 — 61 To link to this Article: DOI: 10.1080/10934520903388806 URL: http://dx.doi.org/10.1080/10934520903388806
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Journal of Environmental Science and Health Part A (2010) 45, 56–61 C Taylor & Francis Group, LLC Copyright ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934520903388806
Follicular apoptosis in the mussel (Mytella strigata) as potential indicator of environmental stress in coastal ecosystems ´ RI´OS-SICAIROS, RUBI´ ALEJANDRA GARCI´A-GASCA, BEATRIZ LEAL-TARIN, JULIAN ´ ´ HERNANDEZ-CORNEJO, GABRIELA AGUILAR-ZARATE and MIGUEL BETANCOURT-LOZANO
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Centro de Investigaci´on en Alimentaci´on y Desarrollo, Unidad Mazatlan, Mazatlan, Mexico.
Follicular apoptosis in the tropical mussel Mytella strigata was assessed in three coastal lagoons located in the southern Gulf of California, Mexico. Mussels were collected from three coastal lagoons associated with different scenarios of anthropogenic stress during one year. The gonad of each mussel was dissected, weighed, and sampled for histology and apoptosis analysis by TUNEL labeling. Two apoptotic indices were used: the apoptotic index of cells (AIC) based on the number of follicular cells in apoptosis in one thousand cells counted per gonad, and the apoptotic index of follicles (AIF) based on the number of follicular cells per follicle per gonad. Both indices showed high association with each other for all developmental stages, although AIF seemed to better discriminate among sites. Higher AIF and AIC were observed at the Urias Estuary (1.6 and 1.5 respectively) ranked as highly polluted, followed by Ensenada del Pabellon (0.82 and 0.95 respectively), ranked as moderately polluted, and the Teacapan Estuary (0.57 and 0.76 respectively) ranked as slightly polluted. Our data indicate that the apoptotic index in tropical mussels could be a useful indicator of environmental stress in coastal ecosystems; however, the ecological relevance of follicular apoptosis in polluted environments needs further investigation. Keywords: Follicular apoptosis, Mytella strigata, apoptotic index, polluted environments.
Introduction In Mexico, many coastal ecosystems impacted by anthropogenic activities show signs of environmental degradation. Three coastal lagoons in the Gulf of California impacted by different anthropogenic activities (i.e., agriculture, aquaculture, cattle farming, mining and industry among others) were selected for a one year biomonitoring study using follicular apoptosis as a biomarker of environmental pollution. The tropical estuarine mussel Mytella strigata was chosen as a sentinel species based on the following characteristics: it is a sessile organism, it filters large amounts of water for feeding and breathing, it is a native species in Mexico, sex ratio is close to 1:1, it is easy to sample and transport[1] , and has shown the ability to bioaccumulate contaminants in the field.[2] This species also inhabits mangrove areas where Rizophora mangle is present.[2]
Address correspondence to Miguel Betancourt-Lozano, Cen´ en Alimentacion ´ y Desarrollo, Mazatlan, tro de Investigacion Mexico. E-mail:
[email protected]
Follicular atresia is a degenerative process to eliminate follicles when oocytes are not ovulated. It has been shown that in fish (and other vertebrates) one mechanism of follicular atresia is apoptosis.[3] Follicular atresia could be a normal event in female gonads in spawning and post-spawning stages, but not in gametogenic or mature gonads since these are proliferative stages. Different hypothesis have been proposed to explain the causes of follicular atresia: oocyte over-ripening, oocyte recycling as an energy source to restore gonadic tissue, environmental stress due to pollution, nutritional deficit, or low temperatures.[4] Follicular apoptosis has been documented in fish as a response to toxicants such as methylmercury[5] and complex mixtures contained in bleached kraft pulp mill effluents.[3] In mussels, apoptosis has been reported to occur in gills and hemocytes in response to toxicants[6–8] ; however, to our knowledge there are no records of follicular apoptosis in mussels as a response to environmental pollution. Therefore, in the present study apoptosis was evaluated as a mechanism of follicular atresia in Mytella strigata populations exposed to different scenarios of environmental stress from anthropogenic activities.
Follicular apoptosis in the mussel Materials and methods
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Study sites Three coastal lagoons in the southern Gulf of California, Mexico (Fig. 1) were selected to represent a pollution gradient based on their environmental scenarios: The Urias Estuary is a highly polluted system adjacent to Mazatlan, Sinaloa (between 23◦ 09 -23◦ 13 N and 106◦ 18 -106◦ 25 W). It is a 6.18 square mile estuarine system that constantly receives a complex variety of contaminants due to intensive industrial (i.e. seafood processing plants and oil storage facilities from the Mexican oil company), domestic (37,800 m3 d−1 of untreated sewage from approximately 300,000 inhabitants)[9] , agricultural and aquacultural activities.[10,11] The system also sustains considerable fishing vessel traffic, a diesel power station supplying electricity to the southern part of the state of Sinaloa, as well as symptoms of environmental degradation such as loss of biodiversity, eutrophication and organic enrichment.[12] Ensenada del Pabellon was selected as a moderately polluted system. It is a 48.65 square mile lagoon situated between 24◦ 19 -24◦ 35 N and 107◦ 28 -107◦ 45 W, close to the cities of Culiacan and Navolato, (populations of 745,532 and 145,622 respectively). In this area, agriculture is one of the most important activities, with 667,184.53 acres dedicated to the culture of
57 tomato, chili, cucumber, egg plant and rice amongst others. Agricultural run-off, along with sugar industry and shrimp farming waste, reaches the system via the Culiacan River.[13] Mussels were also collected from the Teacapan Estuary, which is part of the Teacapan-Agua Brava-Marismas Nacionales system. The whole system covers about 579.15 square miles[14] , located between 22◦ 04 -22◦ 35 N and 105◦ 20 -150◦ 50 W and receives inputs from different rivers. This system was regarded as slightly polluted with influence of several nearby, small-scale, anthropogenic activities, including: agriculture, cattle production, shrimp farming and shrimp and oyster fisheries. Mussel collection and sampling Mussels were collected in 2005 (July and October), and 2006 (January, April, and July) from two sites (A and B) in each system (Fig. 1). Site selection was based on the location of the effluents and the presence of the mussels. A total of 24 organisms per site (48 organisms per system) were sampled each time. Animals were measured and weighed with and without valves. The gonad of each animal was dissected, weighed, and fixed in 4% formalin. Samples were transported to the laboratory to perform histology and apoptosis analysis by TUNEL labeling. Histology Gonad samples were preserved in 4% formalin for 24 hours, then washed in running water, and dehydrated with increasing concentrations of ethanol. Samples were processed by conventional histology according to Lightner[15] , and stained with Hematoxylin-Eosin. Observations were performed by light microscopy to assess gonad development and sex identification. Apoptosis
Fig. 1. Study Area. The three systems (Ensenada del Pabellon, Urias Estuary, and Teacapan Estuary) are located in the state of Sinaloa, in the southern Gulf of California. From each system, two sites (A and B) were selected for mussel sampling based on the location of the effluents and the presence of the mussels.
Based on the histological characterization of gonadic development, the TDT-mediated dUTP nick and labeling (TUNEL assay) was randomly performed on 6 female gonads per site per month. Five-µm sections were obtained from each gonad and rehydrated with decreasing ethanol concentrations to phosphate buffer saline (PBS). R The TUNEL assay was performed with the TACS.XL DAB In Situ Apoptosis Detection Kit (Trevigen) following the manufacturer instructions. Sections were then washed with distilled water, dehydrated with increasing ethanol concentrations, and mounted with synthetic resin. Slides were analyzed by light microscopy, with TUNEL-positive follicular cells (AFC) stained brown (Fig. 2). Two apoptotic indices were calculated: the apoptotic index of cells (AIC) based on the number of follicular cells in apoptosis in 1,000 cells counted per gonad[16–18] , and the
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(gametogenesis) is the stage in which oocytes proliferate and grow. As oocytes grow, connective tissue decreases. Oocyte development begins as hemispherical stalked cells attached to the wall of the follicle, enlarging in diameter as maturity approaches. (b) Mature is the stage in which the follicle is filled with mature oocytes, and little or no connective tissue is present at this stage. (c) Partially spawned is the stage in which oocytes are released. Some follicles still contain oocytes, and others are empty presenting broken walls. (d) Spawned is the stage in which follicles are empty except
Fig. 2. Partially spawned gonad of Mytella strigata. The TUNEL assay was performed to quantify follicular cells in apoptosis. NFC) normal follicular cells; AFC) follicular cells in apoptosis; 400X.
apoptotic index of follicles (AIF) based on the number of follicular cells per follicle per gonad: AIC = number of TUNEL − positive cells/1000 follicular cells (1) AIF = number of TUNEL − positive cells/number of follicles counted∗ 100 (2)
Statistical analyses Linear regression analysis was used to determine the association between apoptotic indices (AIC and AIF). Apoptosis data was logarithmically transformed (ln x + 1) in order to perform a comparison of apoptotic levels between sites and gonadic stages by two-way ANOVA coupled with a Holm– Sidak multiple comparison test. The significance level was P < 0.05.
Results and discussion Morphometric characteristics from collected mussels were recorded: 4.48 ± 0.65 cm length, 2.11 ± 0.35 cm wide and 1.47 ± 0.23 cm height. The average weight with and without valves was 7.72 ± 3.06 g and 2.26 ± 1.14 g, respectively. The histological analysis in all sampled sites showed that overall sex ratios were generally close to 1:1, with only one site in Urias and another in Teacapan, showing sex ratios of 1.5 and 1.9 females per male respectively. For females, four developmental stages were identified in differentiated gonads according to Garc´ıa-Dom´ınguez et al.[19] : (a) Development
Fig. 3. Regression analysis for AIC and AIF. Regression coefficients are indicated for each stage: (A) Gametogenesis, (B) Mature, (C) Partially spawned, and (D) Spawned.
Follicular apoptosis in the mussel
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for atretic oocytes, whereas in some cases, new oocytes have started to develop. The regression analysis between AIC and AIF showed a strong relationship with each other (Fig.3). However, in the spatial comparison, the AIF index was able to detect more
Fig. 4. Apoptotic indices in each gonadic stage per site per system: (A) Gametogenesis, (B) Mature, (C) Partially spawned, and (D) Spawned. Different letters indicate significant differences in follicular apoptosis; lowercase letters refer to the apoptotic index of follicles (AIF) and capital letters refer to the apoptotic index of cells (AIC). Pab - Ensenada del Pabellon, Uri - Urias, Tea Teacapan. Sample sizes (n) are indicated.
59 significant differences between sites than AIC (Fig. 4), indicating that, although both were good indicators of apoptosis, AIF was more useful at discriminating among sites. This is relevant as apoptotic indices were calculated and compared separately for each gonadic developmental stage, since follicular atresia is a normal event in female spawned gonads (due to regression of residual gonadic tissue)[4,20] , but not in gametogenic or mature gonads. Data from year long mussel monitoring, indicate that populations from the Urias Estuary presented higher levels of follicular apoptosis, particularly in gametogenesis (Table 1). Based on AIF values, gametogenic gonads showed significantly higher apoptotic indices in both sampling sites within the Urias Estuary (P = 0.002) and site B of Ensenada del Pabellon (P < 0.05) compared to the other sites (Fig. 4A), whereas mature gonads also showed significantly higher levels (P = 0.043) at site A of Urias (Fig. 4B). This association could be related to the small Urias Estuary being impacted by numerous point and non-point contaminant sources, For example, site A is located near a shrimp fleet, and sewage and industrial effluents, whereas site B is close to a thermoelectric power plant, shipyards and a shrimp farm. Studies in this system have revealed the presence of high levels of heavy metals in both sediments[11] and organisms[2,21–23] , as well as polycyclic aromatic hydrocarbons (PAHs) and organochlorine compounds.[24] Significantly higher apoptotic indices were also observed in site A of Urias in partially spawned gonads (P < 0.05), whereas site B showed significantly higher apoptosis levels (P = 0.004) in post-spawned gonads (Figs. 4C and D, respectively). Overall, the Urias Estuary system had above AIF and AIC averages (1.6 and 1.5, respectively), followed by Ensenada del Pabellon (0.82 and 0.95, respectively) and Teacapan (0.57 and 0.76, respectively) (Table 1). Follicular apoptosis has been related to pollution in fish. Jans et al.[3] analyzed Hsp70 expression and follicular apoptosis in fish exposed to bleached kraft pulp mill effluent, associating them to reduced ovarian size, decreased plasmatic testosterone and increased plasmatic 17β-estradiol. In the current study, mussels from the Urias Estuary presented the highest reproductive effort, with a higher proportion of mature females (37.5%) compared to Ensenada del Pabellon (26%) and Teacapan (15%), and a higher gonadosomatic index (GSI) throughout the year (16.47 in Urias, 14.91 in Ensenada del Pabellon, and 7.73 in Teacapan), indicating that apoptotic indices were not related to a reduced ovarian size in the studied populations. Follicular apoptosis in mature organisms has also been related to ripe oocytes remaining for too long in the follicle causing overripening.[4] However, current results show apoptotic indices were higher in mussels from Urias during all developmental stages, indicating that follicular apoptosis could be due to a combination of biological and/or environmental factors rather than just over-ripening. Compared to Urias, follicular apoptosis occurred in a lower extent in mussels from Ensenada del Pabellon, which did not show elevated apoptotic indices, but interestingly
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Table 1. Apoptotic index of follicles (AIF) and apoptotic index of cells (AIC) for each developmental gonadic stage per system.
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Urias Estuary Ensenada del Pabellon Teacapan Estuary
Gametogenesis AIF AIC
Mature AIF AIC
Partially-spawned AIF AIC
Spawned AIF AIC
Global average AIF AIC
1.15 0.69 0.01
0.53 0.26 0
1.88 0.7 1.55
2.84 1.63 0.71
1.6 0.82 0.57
1.58 1.19 0.27
0.39 0.25 0
TUNEL-positive cells were recorded in all gonadic stages. This site was ranked as moderately polluted due to its agricultural nature and reports of agrochemicals such as DDT, endosulfan, lindane, methyl-parathion in mussel tissues.[13] In contrast, mussels from Teacapan only showed TUNELpositive cells in partially-spawned and spawned gonads (when apoptosis is naturally induced by atresia), indicating this could be related to the notably lower anthropogenic impact in this system (although some pesticides, such as lindane, have been reported in clam and shrimp tissues[24,25] , as well as Hg in fish tissues[26,27] ). Overall results suggest that pollution could be related to the elevated and consistent apoptotic signal through the gonadic developmental cycle. Considering that in most natural environmental scenarios, genotoxicity occurs as a consequence of exposure to complex mixtures[7] , the risk to natural populations, and its ecological significance becomes difficult to establish. However, as the effects are directly elicited on germinal cells, we hypothesize that abnormal follicular apoptosis, particularly in proliferative stages, could be an indicator of chemical stress which may have an impact in the reproductive fitness of the overall population.
Conclusion Our data indicate that apoptosis is a mechanism of follicular atresia in the mussel Mytella strigata, and the apoptotic index could be a useful indicator of environmental stress in coastal ecosystems, although further investigation of the ecological relevance of follicular apoptosis in mussels inhabiting polluted environments is required.
Acknowledgments The authors would like to thank S. Abad-Rosales, I. Mart´ınez-Rodriguez, and R. Hern´andez-Guzm´an for technical assistance, as well as C. Bastidas and D. Herrera for sampling assistance. We also extend a special thanks to ´ fishermen Hipolito Holague, Juan Sarmiento, In´es Pardo, Willy, Arturo Jim´enez, and Delfino for assistance in the field. This work was funded by FOSEMARNAT-2004-01˜ 199 grant awarded to AGG, and Fomix Conacyt-Espana J200-142 grant awarded to LMGP.
1.48 0.64 1.83
2.57 1.73 0.95
1.5 0.95 0.76
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